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Delfi ne Hallaert

‘From the cradle to the grave’

Novel therapeutic approaches to attack the microenvironment

in Chronic Lymphocytic Leukemia

Thesis_Final_2_01092008.indd 1Thesis_Final_2_01092008.indd 1 01-09-2008 11:08:0101-09-2008 11:08:01

Hallaert, Delfi ne

Novel therapeutic approaches to attack the microenvironment in Chronic

Lymphocytic Leukemia

Thesis, University of Amsterdam with references

© 2008 D.Y.H. Hallaert, Amsterdam, The Netherlands

Layout by Caroline Op de beeck, Wingene, Belgium

Cover design by Norbert Hallaert and Lindsay Vandenabeele, Brugge, Belgium

Printed by Buijten & Schipperheijn

Printing of this thesis was financially supported by:

University of Amsterdam

Ortho Biotech


‘From the cradle to the grave’

Novel therapeutic approaches to attack the

microenvironment in Chronic Lymphocytic Leukemia

ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad van doctor

aan de Universiteit van Amsterdam

op gezag van de Rector Magnifi cus

prof.dr. D.C. van den Boom

ten overstaan van een door het college voor promotie ingestelde

commissie, in het openbaar te verdedigen in de Aula der Universiteit

op woensdag 15 oktober 2008, te 14:00 uur

door

Delfi ne Yolande Hugo Hallaert

geboren te Brugge, België


PROMOTIECOMMISSIE

Promotor: Prof.dr. M.H.J. van Oers

Co-promotor: dr. E. Eldering

Overige leden: Prof.dr. J. Borst

Prof.dr. A. Hagenbeek

Prof.dr. M.H.H. Kramer

Prof.dr. J.P. Medema

Prof.dr. S.T. Pals

Faculteit der Geneeskunde


Aan wie ik lief heb


CONTENTS

Chapter 1 General introduction

Chapter 2 Crosstalk among Bcl-2 family members in B-CLL:

seliciclib acts via the Mcl-1/Noxa axis and gradual

exhaustion of Bcl-2 protection

Chapter 3 γ-secretase inhibitor (GSI)-1 induces apoptosis in CLL

cells via proteasome inhibition and Noxa upregulation

Chapter 4 Differential Noxa/Mcl-1 balance in peripheral versus

lymph node chronic lymphocytic leukemia cells

correlates with survival capacity

Chapter 5 Persistent Mcl-1/Bim protein signature after CD40

ligation in Chronic Lymphocytic Leukemia is associated

with specifi c drug sensitivity

Chapter 6 c-Abl Kinase Inhibitors Overcome CD40-Mediated Drug

Resistance in CLL; Implications for Therapeutic

Targeting of Chemoresistant Niches

Chapter 7 Concluding remarks

Chapter 8 Summary

Chapter 9 Nederlandse samenvatting

Dankwoord

Curriculum vitae

List of publications

Color fi gures

9

31

55

75

99

117

143

151

155

163

169

173

175


General introduction

1chapter


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11INTRODUCTION

CLL: CLINICAL AND PATHOLOGICAL FEATURES

Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disorder characterized

by monoclonal small, mature CD5+/CD19+ B cells which continue to accumulate in the

peripheral blood (PB), bone marrow (BM) and lymphoid organs1. There is a remarkable

clinical variability in patients with CLL2. Following diagnosis, some patients have

asymptomatic disease that may not progress for many years. Others are diagnosed

with advanced-stage disease, or have early-stage disease that progresses rapidly

and requires treatment. Clinical symptoms include peripheral blood lymhocytosis,

lymphadenopathy and hepatosplenomegaly. When the disease progresses many

patients develop hypogammaglobulinemia and neutropenia, resulting in an increased

susceptibility to bacterial infections. Furthermore, up to 25% of the patients develop

autoimmune cytopenias, mostly autoimmune hemolytic anemia and/or autoimmune

thrombocytopenia. The clinical variability is associated with the following molecular

markers: cytogenetic abnormalities3, immunoglobulin VH gene mutational status4;5,

ZAP-70 expression6;7 and CD38 expression8. How these factors infl uence the biology

of CLL is still subject of intensive research. Immunoglobulin gene mutational status

and ZAP-70 expression are associated with the capacity of CLL cells to respond

to signals delivered through the B-cell receptor (BCR)9. Loss of 17p10 or 11q11 may

cause dysfunction of p53 DNA damage pathways12.

Classically, CLL cells were thought to derive from naive B lymphocytes and to behave

as inert cells that passively accumulate13. However, mRNA studies showed that at the

level of gene expression both IgVH mutated and unmutated CLL cells are most similar

to memory B cells14;15. Moreover, stable isotope labeling studies with deuterated water

(D2O) have shown that CLL cells divide at a considerable rate, suggesting a more

dynamic disease than previously appreciated16. Survival and proliferation of the CLL

cells are infl uenced by interactions with non-leukemic cells in the microenvironment

of lymph nodes (LN), bone marrow and spleen1;17;18. Still, deregulated apoptosis

is considered to be a key factor and a major barrier to effective treatment of CLL.

Understanding apoptosis pathways and the impact of the microenvironment on

these pathways is therefore clinically relevant, because it may open new avenues to

effective treatment of CLL.

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12THERAPY IN CLL

Extensive discussion of the treatment of CLL is beyond the scope of this thesis.

Detailed reviews have recently been published19;20. Past and current therapeutic

approaches to CLL will be summarized here.

About twenty years ago, treatment consisted of alkylating agents, usually chlorambucil.

Remission rates were variable (50 – 70%). However, complete remission was rarely

obtained21. Combination treatment, such as CVP (cyclophosphamide, vincristine

and prednisone) and CHOP (cyclophosphamide, doxorubicine, vincristine and

prednisone), did not improve overall survival22. The introduction of the purine analog

fl udarabine has brought new impulse to the research of CLL treatments. Clinical trials

showed fl udarabine to induce higher complete remission rates and longer progression

free survival. Again, no survival advantage was achieved21. Until recently, the goal of

therapy has been palliation with minimal toxicity. Introduction of new therapies and

novel combinatorial approaches have initiated a paradigm shift and made potential

cure the goal of therapy. Three randomized trials, comparing FC (fl udarabine +

cyclophosphamide) with fl udarabine alone, have been published, all showing higher

response rates with prolongation of progression-free survival in the FC arm23-25.

Nevertheless, thus far no overall survival benefi t was obtained. Studies with the

most potent combination FC + Rituximab (anti-CD20 chimeric monoclonal antibody)

showed high overall and complete remission rate and prolonged progression-free

survival, in both previously untreated26 and relapsed27 CLL patients. Alemtuzumab

(anti-CD52 humanized antibody) has been shown to be effective also in the treatment

of p53 dysfunctional CLL patients28. Finally, Reduced Intensity allogeneic Stem cell

Transplantation (RIST) or ‘mini transplants’ have broadened the use of allogeneic stem

cell transplants in CLL patients. By this approach a long-term relapse-free survival

has been achieved 29, but the associated morbidity remains a serious obstacle for

wide application30;31. Thus, despite recent progress there remains a strong need for

novel effective and less toxic treatment options.

APOPTOSIS

Programmed cell death or apoptosis, is essential for tissue homeostasis, and disturbed

regulation of this process underlies many diseases, including cancer. Activation of

apoptosis is important for the removal of infected, transformed or damaged cells.

Apoptotic cells are defi ned according to morphological characteristics such as cellular


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13shrinkage, nuclear condensation, membrane blebbing and eventually fragmentation

into membrane bound apoptotic bodies32. During apoptosis, asymmetry of the cell

membrane results in exposure of phosphatidylserine (PS) on the cell surface. PS

functions as an ‘eat me’ signal on apoptotic cells which results in direct recognition,

engulfment and degradation by phagocytes33.

Caspases

Apoptosis involves a family of aspartate-specifi c cysteine proteases, called caspases34.

Caspases are synthesized as precursors (pro-caspases), and are converted into

mature enzymes upon apoptosis signals. Caspases can be divided into three groups

based on their structure and role in apoptosis. First, initiator caspases, consisting of

caspase-2, -8, -9 and -10 that cleave inactive pro-forms of effector caspases, leading

to activation. Second, effector caspases, consisting of caspase-3, -6 and -7, which in

turn cleave other protein substrates within the cell resulting in the apoptotic process35.

Finally, a third group of caspases is described, caspase-1, -4 and -5, which are not

directly involved in apoptosis execution, but play important roles in infl ammatory

cytokine activation and release36.

Pathways of apoptosis induction

Two major apoptotic signaling pathways are recognized in mammals: the extrinsic

or death receptor mediated pathway and the intrinsic or mitochondrial pathway. Key

players in the apoptosis signaling pathway are outlined in Figure 1.

The extrinsic pathway is activated upon ligand binding to the tumor necrosis receptor

(TNF-R) family members such as Fas (CD95/APO-1), TNF-R and TRAIL-R. This

results in receptor trimerization and recruitment of intracellular adaptor proteins,

TRADD or FADD, leading to the assembly of the death-inducing signaling complex

(DISC)37;38, and subsequent recruitment and assembly of initiator caspase-8. Caspase-

10 can also be recruited to the DISC in a similar manner, but it cannot functionally

substitute for caspase-839;40. Subsequently, activated caspase-8 is released into the

cytosol where it can activate effector caspases. In addition, caspase-8 can process

the BH3-only Bcl-2 family member Bid to the truncated form tBid41. Subsequently,

tBid can translocate to the mitochondria to exert its pro-apoptotic activity41;42.

The intrinsic pathway is initiated in response to cellular signals resulting from DNA

damage, cell cycle defects, growth factor withdrawal, hypoxia, or other types of severe

cell stress and is tightly regulated by Bcl-2 family of proteins. Progression through the


14pathway leads to mitochondrial outer membrane permeabilization (MOMP), resulting

in cytochrome c release and assembly of the apoptosome complex. The apoptosome

is a multimeric holoenzyme consisting of apoptotic protease-activating factor 1 (Apaf1),

pro-caspase-9 and dATP43. Other apoptosis promoting factors are also released

from the mitochondria, including Smac/DIABLO, Omi/HtrA2, endonuclease G and

apoptosis inducing factor (AIF). Smac/DIABLO and Omi/HtrA2 promote caspase

activation by interacting with inhibitors of apoptosis (IAP) family44;45. Endonuclease G

and AIF translocate to the nucleus and induce DNA degradation46;47.

Figure 1. See color fi gures. The two main pathways leading to apoptosis. The extrinsic pathway is

triggered by ligation of cell surface receptors, such as Fas, resulting in activation of caspase-8. The

intrinsic pathway is activated by cytotoxic stimuli, such as DNA damage, which leads to the release of

apoptosis promoting factors from the mitochondria. In the cytosol, cytochrome c results in the activation

of caspase-9. This pathway is regulated by the Bcl-2 family of proteins. Activated caspase-8 and -9 in

turn activate effector caspase-3, -6 and -7. Cross-talk between the pathways occurs through Bid, which is

cleaved by caspase-8 and then can activate the intrinsic pathway.


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15Apoptosis regulators

Bcl-2 family

The Bcl-2 (B cell lymphoma 2) family in mammals consists of pro- and anti-apoptotic

proteins. An overview of the Bcl-2 family protein members is presented in Table

1. They all share at least one conserved Bcl-2 homology (BH) domain and are

considered to act mainly on the mitochondria48. Based on structural and functional

features, they can be divided into three subfamilies: the anti-apoptotic subfamily

comprising of Bfl -1, Bcl-2, Bcl-W, Bcl-xL and Mcl-1 (Myeloid-cell leukemia sequence

1) and two other subfamilies which promote cell death: the Bax-like death family

and BH3-only family (in Table 1 also several ‘BH3-only contenders’ are mentioned).

Members of the Bax-like death family include Bax (Bcl-2-associated X protein), Bak

(Bcl-2-antagonist/killer) and Bok (Bcl-2-related ovarian killer), which contain three

BH domains. The BH3-only protein family includes at least 8 members including Bad

(Bcl-2-antagonist of cell death), Bik (Bcl-2-interacting killer), Bid (Bcl-2 interacting

domain), Hrk (Harakiri, also known as DP5), Bim (Bcl-2-interacting mediator of

cell death), Noxa, Puma (p53-upregulated modulator of apoptosis) and Bmf (Bcl-

2-modifying factor). They all have only a short BH3 motif. Biochemical studies

demonstrated that BH3-only proteins interact selectively and with varying affi nities

with anti-apoptotic counterparts. Whereas Bim and tBid bind avidly to all the pro-

survival proteins, the other BH3-only proteins associate only with selected subsets.

For example Noxa binds to Mcl-1 and Bfl -1, while Bad binds to Bcl-2, Bcl-XL and

Bcl-W49. For Noxa, however, others reported that Mcl-1 is the only binding partner50.

Promiscuous binders, like Bim, are much more potent killers than those that cannot

engage all the pro-survival proteins49. This indicates that the multiple anti-apoptotic

proteins all have a different function.

It is the complex interplay between the three Bcl-2 subfamilies that determines the

commitment of MOMP and subsequent apoptosis51. However, the detailed molecular

mechanism remains controversial. The BH3-only proteins act upstream of Bax and

Bak, because they cannot induce apoptosis in cells lacking both Bax and Bak52. How

they induce activation of Bax and Bak is addressed by two distinct models. The direct

activation model proposes that the BH3-only proteins termed ‘activators’ (Bid, Bim

and perhaps Puma) are capable of binding to and enabling the conformational change

and pore formation of Bax/Bak50;53-57. Other BH3-only proteins, termed ‘sensitizers’

(e.g. Noxa and Bad), can displace the activators from anti-apoptotic proteins. In

this model, which suggests the existence of a functional hierarchy within the BH3-

only subfamily, survival is the default. The indirect activation model (also referred as

displacement model), on the other hand, suggests that all BH3-only proteins bind to


16their specifi c anti-apoptotic relatives, which are inactivated, thus indirectly enabling

the Bax/Bak lethal function. Based on this model, in which death is the default, Bim,

Bid and Puma are the most potent apoptosis inducers simply because they are the

only ones able to engage all pro-survival proteins49;54;58;59. A strong argument for this

scenario was made using murine cells without Bim and Bid, and containing reduced

Puma. These cells were still capable of undergoing apoptosis55, something which is

hard to reconcile with the direct activation model.

So far most of this work has been done in artifi cial systems in vitro using isolated

mitochondria56, gene ablated murine cells, and transfected or virally transduced cell

lines49. These approaches leave open the important matter: how these proteins and

pathways function in human healthy tissues and primary cancer cells.

The Bcl-2 family is subdivided in three main categories: Anti-apoptotic Bcl-2-like proteins (A), pro-apoptotic

Bax-like proteins (B) and BH3-only proteins (C). Members for which solid evidence has been obtained are

reviewed in detail elsewhere58;60. In addition, other ‘candidates’, containing at least a conserved BH3-only

or BH3-like domain have been described and are presented in (D). (Adapted from Alves61)

Prosurvival Pro-apoptotic BH3-like contenders

(A) Bcl-2-like (B) Bax-like (C) BH3-only (D)

Bfl -1 Bak Bad Bcl-G(S)

Bcl-2 Bax Bik BRCC2

Bcl-W Bok Bid MAP-1

Bcl-XL

Hrk Mule

Mcl1 Bim NIP3

Noxa Nix

Puma Spike

Bmf

IAPs

The human IAP family has been implicated in cell division, cell cycle progression,

signal transduction62 and consists of at least 8 members, and includes XIAP, cIAP1,

cIAP2, NIAP, and survivin. Overexpression of several IAPs has been detected in

various cancers63. Of all IAPs, XIAP is most probably the only actual inhibitor of

caspases64. IAP1 and -2 associate with TNF-receptor family members and recent

data have demonstrated that their presence in fact blocks NF-κB signaling. When

unleashed, this pathway leads to TNF production which can kill cancer cells in an

autocrine fashion65. Survivin has been implicated in both apoptosis and cell division, but

compelling evidence now points to a specifi c role in chromosome segregation66;67.

Table 1. Overview of Bcl-2 family members and relatives


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17FLIP

c-FLIP (c-FLICE-inhibitory protein) is a family of alternatively spliced protein variants,

and primarily exists as long (c-FLIPL) and short (c-FLIP

S) isoforms in human cells.

c-FLIPL is homologous to caspase-8 but cannot become actived. Both FLIP variants

can be recruited to the DISC, where they can block pro-caspase-8 activation and

protect cells from death receptor mediated apoptosis68.

APOPTOSIS DEREGULATION IN CLL

Apoptosis regulatory proteins

In CLL several mechanisms contribute to resistance towards apoptosis. First,

Bcl-2 is expressed 1.7 – 25-fold higher in CLL than in normal lymphocytes, and

CLL cells isolated from patients refractory to standard chemotherapy show an

increased Bcl-2/Bax ratio69;70. The high expression of Bcl-2 has been postulated to

be related to deletions of two Bcl-2 suppressing miRNAs, miR-15a and miR-16-1,

located in a cluster at 13q14.3, which is deleted in ∼65% of the CLL patients71;72.

Furthermore, a polymorphism in the promoter region of the BCL-2 gene (-938C>A)

was reportedly associated with inferior clinical course and with increased expression

of Bcl-273. However, these initial fi ndings could not be corroborated in two follow-up

studies74;75. Secondly, high Mcl-1 levels were found to be associated with a failure to

achieve complete remission following chemotherapy in CLL76. Furthermore, Mcl-1

downregulation correlated with in vitro apoptosis induced by various therapeutics

in CLL77-79. Signifi cant Mcl-1 upregulation and subsequent protection against

spontaneous apoptosis was induced upon in vitro co-culture of CLL cells with CD40

ligand (CD154) expressed on fi broblasts80 and with follicular dendritic cells (FDC)81.

Thirdly, a nucleotide polymorphism, -248G>A, in the 5’ promoter region of Bax

was found in CLL, causing a reduced protein expression82. However, the outcome

of the disease did not seem to be infl uenced by this polymorphism, and, thus the

clinical impact was uncertain83. Finally, IAPs such as XIAP, cIAP1 and cIAP2 may

also be overexpressed in CLL84. XIAP inhibitors, which can potentially de-repress

caspase activity in malignant cells, are currently viewed as promising novel treatment

options85.

Death receptors

CLL cells express barely detectable levels of Fas on their surface, although transcripts

for both Fas and FasL (CD178) are commonly detected. Several stimuli have been

shown to up-regulate Fas expression, e.g. IFN and CD40-ligation, although CLL cells


18remain resistant to Fas-mediated apoptosis86. However, CLL cells become sensitive to

Fas-mediated apoptosis upon prolonged stimulation with CD40 ligand, concomitantly

with FLIP down-regulation and up-regulation of FADD87.

Death-inducing receptors for TNF-related apoptosis-inducing ligand (TRAIL), TRAIL-

R1, TRAIL-R2, TRAIL-R3 and TRAIL-R4 are expressed on primary CLL cells at

a low level. Nevertheless, CLL cells are resistant to TRAIL-induced apoptosis. In

correlation with low TRAIL receptor surface expression, DISC formation is hampered

and further caspase-8 activation is prevented88. Sensitivity to TRAIL was induced

when CLL cells were pretreated with nontoxic concentrations of histone deacetylase

(HDAC) inhibitors89. Furthermore, CD40 ligation induces expression of the pro-

apoptotic BH3-only protein Bid and TRAIL-R5. Death receptors CD95 and TRAIL-

R5 can act synergistically to induce caspase-dependent apoptosis of CLL cells and

Bid can facilitate cross-talk between mitochondrial-dependent and death receptor

inducing pathways90.

p53

p53 plays a protective role in normal somatic tissues by preventing division of damaged

cells91;92. The locus of the p53 gene is 17p13.1. The p53 protein acts in many cellular

processes, including cell-cycle checkpoints, DNA repair, senescence, apoptosis and

the surveillance of genomic integrity93;94. Stress stimuli such as DNA-damaging drugs

rapidly induce a transient increase in p53 protein95. Wild type (wt) p53 inhibits cancer

development by inducing transcription of a number of target genes involved in cell

cycle arrest and apoptosis. A p53-inducible gene involved in cell cycle arrest is p21

(also Cyclin-dependent kinase inhibitor 1A, CDKN1A). On the other hand, p53 can

trigger apoptosis via Bax96, Puma-97;98 and Noxa-99 gene transcription.

Deletions of p53 occur in about 10-15% of the CLL cases10;100. However, p53

dysfunction can also occur via alternative mechanisms, such as the inactivation of

ataxia telangiectasia mutated (ATM) gene12. ATM, encoded at chromosome 11q22.3,

is a kinase controlling p53 activation in response to DNA double-strand breaks.

Dysfunction of p53 by inactivation of ATM accounts for an additional abnormality in

15-20% of the patients101;102. In general, defects in p53 function occur in approximately

20% of CLL patients101. However, the frequency of p53 dysfunction increases to

nearly 50% as the disease progresses following initial therapy103;104. CLL patients with

p53 dysfunction do not respond to conventional therapy and tend to have a rapidly

progressive disease3.

Many therapeutic strategies require an active p53. Consequently, loss of p53 function

results in a selective resistance to DNA-damaging therapies, including alkylating

agents and fl udarabine103. Therefore, an important area of research is devoted to


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19the identifi cation of new treatment strategies that function independently of the p53

pathway.

ROLE OF THE MICROENVIRONMENT IN CLL

Although CLL cells show characteristics consistent with a defect in programmed

cell death and exhibit prolonged survival in vivo, during in vitro culture CLL cells

isolated from peripheral blood can rapidly undergo spontaneous apoptosis105;106.

This dichotomy highlights the relevance of an in vivo microenvironment capable of

delivering survival signals. Survival-supporting factors might rescue leukemia cells

from cytotoxic therapy107;18 and this may be the basis for subsequent relapse.

The CLL proliferating compartment is represented by focal aggregates of proliferating

lymphocytes that give rise to the so-called pseudofollicles or proliferation centers. In

the LN of CLL patients, the pseudo-follicles represent the histological hallmark, as

well as in the white pulp of the spleen and in the BM. Immunohistochemistry studies

have shown that pseudofollicles are clusters of CD5+ Ki67+ B cells surrounded by

new vessels108;109. In the LN and BM there are CD3+ T cells, most of them belonging to

the CD4+ T helper subset. The CD4+ T cells tend to concentrate around and within the

proliferating pseudofollicles and many of them express CD40L implying that they are

in an activated state. CLL cells retain the capacity to respond to CD40L, expressed

by the CD4+ T cells, thus resulting in their activation110. Furthermore, CLL cells

purifi ed from LN and PB constitutively express mRNA for T cell attracting chemokines

(namely CCL17 and CCL22) and the same holds true for CLL cells stimulated by in

vitro CD40-crosslinking110. These fi ndings indicate that physiological signals in the

tumor microenvironment, such as CD40L, give CLL cells chemo-attracting capacity to

activated CD4+ T cells, which in turn are able to deliver survival signals to the CLL cells.

Other accessory cells, such as bone marrow stromal cells111, FDCs81 and so-called

nurse-like cells112 that can be obtained from peripheral blood, may also be involved in

cross-talk between malignant cells and the microenvironment. Furthermore, various

pro-survival cytokines (IL-2, IL-4, IL-8, TNF-α, IFN-α, IFN-γ and VEGF) and their

respective receptors are found on CLL cells (reviewed in Kay 2002113). However, they

remain unresponsive to most mitogens that induce proliferation of normal B cells.

The pro-survival signaling pathways elicited in CLL appear to depend on both paracrine

and autocrine mechanisms. In some cases it is unclear whether the signal comes

entirely from the cell in the microenvironment or as a consequence of deregulation

in the CLL cells themselves. Alterations in several signaling pathways have been

suggested in CLL.

For example, NF-κB is a transcription factor that can promote survival through the


20induction of several anti-apoptotic proteins, such as Bfl -1, Bcl-X

L, IAPs and Flip114.

Higher levels of constitutive NF-κB are seen in unstimulated CLL cells compared to

healthy peripheral blood B cells115. Engagement with the TNF-superfamily members

CD40L, BAFF (B cell-activating factor of tumor necrosis factor (TNF) family, also

known as BlyS) and APRIL (A proliferation-inducing ligand), enhances this activity115;116.

However, whilst it has been shown that APRIL is overexpressed in some cases of

CLL117, the APRIL driving pro-survival signaling is also derived from nurse-like cells

in the tumor niche118. The activation of the canonical NF-κB pathway also contrasts

with the alternative pathway in response to BAFF116, which also suggests intrinsic

deregulation of the responses to certain cytokines. Additionally, recent studies

showed an association between the expression of NF-κB subunit Rel A and in vitro

survival of CLL cells119.

Also, the PI3K/AKT, Raf/MEK/ERK and JNK/STAT signaling pathways are essential

for cell survival, and are frequently deregulated in malignancy120. They control

expression and function of many proteins, including apoptosis regulatory proteins.

Several studies done in CLL suggested that also these pathways may contribute to

survival of the malignant cells121;122;123. Relatively new in the CLL fi eld is the nonreceptor

tyrosine kinase c-Abl. Oncogenic fusion versions of c-Abl drive malignancy in chronic

myeloid leukemia, and are the target for successful therapy with kinase inhibitors120. A

recent study highlighted the importance c-Abl itself in CLL survival124 and furthermore

it was reported that c-Abl becomes active upon CD40 triggering125. The relevance of

these signaling events for the in vivo biology of CLL, and especially in the context of

survival niches, is an important aspect that remains to be established.

THERAPEUTIC APROACH TO ATTACK THE

MICROENVIRONMENT

As mentioned above, despite improvements in treatment effects for CLL by a

combination of chemo- and immuno-therapy, the disease will invariably relapse.

Clearly, the clearance of the CLL peripheral blood pool which usually occurs is

not suffi cient. The likely hypothesis is that the proliferation centers in LN, BM and

spleen constitute “germinative” foci, afford resistance to drugs, and will replenish the

peripheral blood after a successful cycle of therapy.

Therefore, drugs that target bystander cells and/or their protective effects on CLL

cells in the microenvironmental “niche”, would provide more effi cacious options to

the treatment of CLL patients. Accordingly, the microenvironment may be targeted

by interfering with various cytokines or by modulating immune effector cells. Clinical


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21trials in refractory or relapsed CLL cases have reported encouraging results with

immunomodulating agents, i.e. thalidomide and its analog lenalidomide. Although the

exact antitumour activity of such compounds remains uncertain, they do not exert a

direct cytotoxic effect on CLL cells126;127. In addition, therapies aiming to target CLL

cells in the protective “niches” by counteracting the pro-survival changes provided at

these sites, could be a novel and potentially effective strategy. In this regard, small-

molecule BH3 mimetics such as ABT-737 which is a potent and specifi c Bcl-2/Bcl-XL/

Bcl-W inhibitor are now in preclinical or clinical development128.

To conclude, CLL cells possess and rely on various different ways to escape

apoptosis. Direct cell-to-cell contact between CLL cells and bystander cells creates a

microenvironment in which both membrane-bound and soluble factors collaborate in

protecting CLL cells from apoptosis. Learning how to manipulate the microenvironment,

or to specifi cally target the leukemic cells residing in these niches, may reveal new

strategies for restoring apoptosis sensitivity and improving therapeutic outcome.

SCOPE

The aims of this thesis are:

1. To gain insight into the mechanism of action of potential novel drugs with activity

independent of the p53 pathway, with emphasis on effects on apoptosis regulatory

molecules.

2. To obtain insight into the molecular basis of apoptosis (dys-) regulation and survival

of CLL cells both in peripheral blood and in the lymph node microenvironment in

connection with drug sensitivity.

In Chapters 2 and 3 the apoptosis pathway of two p53-independent drugs are

investigated; the cyclin dependent kinase (CDK) inhibitor roscovitine (Seliciclib,

cyc202), and a novel proteasome inhibitor. In the following section the infl uence of

the microenvironment on CLL survival is addressed. In chapter 4 a comparative study

of CLL cells in the peripheral blood and the lymph node compartments with respect

to mRNA and protein expression levels of various apoptosis regulatory molecules is

presented. In Chapter 5, in order to model the in vivo LN setting of CLL, the infl uence

of the CD40-signaling on the expression of apoptosis regulatory proteins is studied, in

relation to sensitivity to various current and novel chemotherapeutic drugs. In chapter

6 we describe an approach to overcome CD40L induced drug resistance in CLL using

cAbl kinase inhibitors. Finally, in chapter 7 an integrated discussion is presented and

future directions are suggested.


22


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chapter 33

Cross-talk among Bcl-2 family members in B-CLL: seliciclib acts via the Mcl-1/Noxa axis and gradual exhaustion of Bcl-2 protection

Delfi ne Y.H. Hallaert1,2, René Spijker1,2, Margot Jak1, Ingrid A.M. Derks2, Nuno L.

Alves2, Felix M. Wensveen2, Jan Paul de Boer3, Daphne de Jong3, Simon R Green4,

Marinus H.J. van Oers1 and Eric Eldering2

1 Dept. of Hematology, AMC, Amsterdam, The Netherlands2 Dept. of Experimental Immunology, AMC, Amsterdam, The Netherlands3 Dept. of Pathology, The Netherlands Cancer Institute/Antoni van Leeuwenhoek

Hospital, Amsterdam, The Netherlands4 Cyclacel Ltd., Dundee, UK

Cell Death and Differentiation, 2007; 14: 1958-1967

2


ABSTRACT

Seliciclib (R-roscovitine) is a cyclin-dependent kinase inhibitor in clinical development.

It triggers apoptosis by inhibiting de novo transcription of the short-lived Mcl-1 protein,

but it is unknown how this leads to Bax/Bak activation that is required for most forms

of cell death. Here, we studied the effects of seliciclib in B cell chronic lymphocytic

leukemia (B-CLL), a malignancy with aberrant expression of apoptosis regulators.

Although Seliciclib induced Mcl-1 degradation within 4 hrs, Bax/Bak activation

occurred between 16 and 20 hrs. During this period, no transcriptional changes in

apoptosis-related genes occurred. In untreated cells, pro-survival Mcl-1 was engaged

by the pro-apoptotic proteins Noxa and Bim. Upon drug treatment, Bim was quickly

released. The contribution of Noxa and Bim as a specifi c mediator of seliciclib-

induced apoptosis was demonstrated via RNAi. Signifi cantly, 16 hrs after seliciclib

treatment, there was accumulation of Bcl-2, Bim, and Bax in the ‘mitochondria-

rich’ insoluble fraction of the cell. This suggests that after Mcl-1 degradation, the

remaining apoptosis neutralising capacity of Bcl-2 is gradually overwhelmed, until

Bax forms large multimeric pores in the mitochondria. These data demonstrate in

primary leukemic cells hierarchical binding and cross-talk among Bcl-2 members,

and suggest that their functional interdependence can be exploited therapeutically.

32


33

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INTRODUCTION

B cell chronic lymphocytic leukemia (B-CLL) is a heterogeneous disease, typifi ed by

the accumulation of long-lived monoclonal B cells arrested at the G0/G1 phase of the

cell cycle1. B-CLL is considered a malignancy involving deregulated apoptosis. High

levels of pro-survival Bcl-2 and Mcl-1 proteins can be detected in B-CLL, which are

associated with aggressive disease and refractoriness to chemotherapy2;3. Contrasting

with these clinical observations, CLL cells succumb easily to ‘spontaneous’ apoptosis

during in vitro culture. So it remains unclear whether overexpression of these

anti-apoptosis proteins is strictly required to block imminent apoptosis, or affords

protection during stress or drug treatment. This aspect is particularly relevant in light

of the concept of ‘oncogene addiction’, which holds that each cancer type may strictly

depend on just a few particular oncogenes, like Bcl-24.

A pro-apoptotic subgroup within the Bcl-2 family consists of so called BH3-only proteins

and includes Bid, Bim, Noxa, Puma, Bad, Bmf, Bik and Hrk. By forming heterodimeric

complexes at the mitochondria, the interplay between BH3-only proteins and pro-

survival Bcl-2-like counterparts determines the activation of pro-apoptotic Bax-like

members and subsequent cell death5. Paradoxically, Noxa and Bmf are overexpressed

in circulating B-CLL cells6 although the functional signifi cance of these observations

remains unclear. Puma, a bona-fi de p53 target gene7;8, is rapidly induced in B-CLL

cells obtained from the majority of patients upon treatment with therapeutic drugs

such as fl udarabine and chlorambucil6. Still, it has been diffi cult to demonstrate an

improvement in overall survival upon treatment with these chemotherapeutic drugs

and disease relapse is inevitable in virtually all patients1. Up to 30% of B-CLL tumors

have a defect in the p53 pathway, which is often associated with resistance to therapy.

Therefore, there is an urgent need for novel drugs that work independently of p53.

Recently, the cyclin-dependent kinase (CDK) inhibitor seliciclib (CYC202,

R-roscovitine), has emerged as a potential drug for treatment of B cell malignancies

including B-CLL9. Another CDK inhibitor, fl avopiridol has recently been tested in CLL

with encouraging results10. Seliciclib induces apoptosis, most likely by inhibiting RNA

polymerase II dependent transcription and thereby resulting in loss of the short-lived

pro-survival Mcl-1 protein11. Yet, it remains unresolved how the decrease in Mcl-1

levels is coupled to apoptosis induction. Despite the notion that Bax and Bak are

indispensable for apoptosis12, the molecular cascade that mediates their activation is

presently a matter of debate. New developments have shed light on the interaction

between members of the Bcl-2 family. Specifi cally, the pivotal pro-survival Mcl-1

interacts, amongst others, with Noxa, Bim, Puma and Bak in model cell lines13;14.

These fi ndings may be relevant in connection with our observation of elevated


34expression of Noxa in B-CLL6, as well as in light of the above mentioned concept of

‘oncogene addiction’.

In the present study, we investigated the molecular mechanism underlying apoptosis

elicited by seliciclib in B-CLL, with emphasis on protein interactions between Bcl-2

family members. We show for the fi rst time that Mcl-1 is associated with Noxa in

primary leukemic cells and that Noxa is required to trigger the mitochondrial apoptosis

pathway upon seliciclib treatment. Furthermore, despite rapid proteasome-dependent

degradation of Mcl-1 (within 4 hrs), Bax/Bak activation and apoptosis are substantially

delayed (16-20 hrs). During this time Bim and Bax become associated with Bcl-2 and

these proteins eventually translocate to a mitochondria-rich insoluble fraction. Our

data suggest that in absence of Mcl-1, the remaining apoptosis neutralizing capacity

of Bcl-2 is gradually overwhelmed, resulting in Bax/Bak multimerisation and pore

formation in the mitochondria.

MATERIALS AND METHODS

B-CLL patients and isolation of leukemic B cells

Peripheral blood samples were obtained from B-CLL patients from the outpatient

clinic of the department of Hematology of the Academic Medical Center, Amsterdam;

the department of Internal Medicine, Meander Medical Center, Amersfoort and the

department of Internal Medicine, The Netherlands Cancer Institute, Amsterdam.

Informed consent was obtained according to the guidelines of the local Ethical

Review Board. Clinical characteristics of patients are presented in Table 1. This

study was conducted in accordance with the ethical standards in our institutional

medical committee on human experimentation, as well as in agreement with the

Helsinki Declaration of 1975, revised in 1983. Peripheral blood mononuclear cells

(PBMC), obtained via density-gradient centrifugation were frozen in 15% fetal calf

serum (FCS) containing 10% dimethyl sulphoxide (DMSO; Sigma Chemical Co., St

Louis, MO, USA) and stored in liquid nitrogen. Expression of CD5, CD19 and CD23

(all Becton Dickinson (BD) Biosciences, San Jose, CA, USA) on leukemic cells was

assessed by fl ow cytometry (FACScalibur, BD Biosciences) and CellQuest software

(BD Biosciences)6.


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35Cell lines, retroviral constructs and transduction

Cell lines were cultured in Iscove’s modifi ed Dulbecco’s medium (IMDM; Gibco Life

Technologies, Paisley, Scotland), supplemented with 10% (v/v) heat-inactivated

fetal calf serum (FCS) (ICN Biomedicals GmbH, Meckenhein, Germany), 5 mg/l

gentamycin and 5 mM L-glutamine (Gibco). The Burkitt lymphoma Ramos FSA clone

with enhanced response to CD95, the caspase-9-DN (C9DN) overexpressing Ramos

FSA cell line, Ramos 2G6 clone and the Bcl-2 overexpressing Ramos 2G6 cell line

have been described previously15;16. The B-CLL cell line Mec-117 was a kind gift of

Dr. Caligaris-Cappio (Milano, Italy). Mock and two Noxa siRNA sequences (N7 or

N8) were retrovirally transduced into the Ramos FSA, Mec-1 and J16 cell lines as

described previously18. Furthermore, siRNA targeting Bim (pSuper-Bim) was kindly

provided by Andreas Villunger (Biocenter, Innsbrück, Austria) and subcloned in the

EcoRI/Xho sites of pRetroSuper. After retroviral transduction of J16 cell line, the

B18 and B22 subclones were obtained after limiting dilution. The pcDNA3.1 vector

encoding Myc-hMcl-1 was obtained from Professor J. Borst (Netherlands Cancer

Institute). After digestion with BamH1 and Xba the product was inserted into the

BamH1 and SnaB1 blunt ended sites of the LZRS-IRES-GFP vector, resulting in

LZRS-Myc-hMcl-1-IRES-GFP.

Reagents

Seliciclib was obtained from Cyclacel Ltd (Dundee, UK). Fludarabine, staurosporine,

etoposide, carbonyl cyanide m-chlorophenylhydrazone (CCCP), Taxol and propidium

iodide (PI) were purchased from Sigma Chemical Co. Anti-human Fas10 (agonistic

antibody to the CD95 receptor) and anti-human IgM mAbs (CLB/MH15) were a kind

gift from Prof. Dr. L. Aarden (Sanquin, Amsterdam, The Netherlands). The pan-

caspase inhibitor zVAD-fmk was obtained from Alexis Biochemicals (Läuferlingen,

Switzerland). APC-labeled Annexin V was from IQ Products (Groningen, The

Netherlands) and MitoTracker Orange was from Molecular Probes (Leiden, The

Netherlands). Proteasome inhibitors MG132 and bortezomib, were obtained from

Calbiochem, (Amsterdam, The Netherlands) and Janssen-Cilag, (Tilburg, The

Netherlands), respectively.

Analysis of apoptosis

For cell stimulations, B-CLL cells (>90% CD19+CD5+ PBMC suspensions) were used

at a concentration of 1.7×106 cells/ml in 24-well plate, Ramos and Mec-1 cell lines

at a concentration of 5×105 cells/ml. Cells were harvested and washed in ice-cold

HEPES buffer (10 mM HEPES, 150 mM KCl, 1 mM MgCl2 and 1,3 mM CaCl

2, pH 7.4),


36and incubated with APC-labeled Annexin-V (BD Biosciences) for 20 minutes. Prior to

analyses, PI was added (fi nal concentration 5 µg/ml). Viable cells were defi ned by

Annexin V-/PI- staining. Alternatively, cells were incubated with 200 mM MitoTracker

for 30 mins at 37˚C. To assess activation of Bax and Bak, B-CLL cells (1×106) were

washed in PBS prior to fi xation in 4% formaldehyde for 5 min at room temperature.

Cells were washed in PBS and incubated in the presence of 1 µg/ml of anti-Bax (Ab-

6, clone 6A7, Calbiochem) or anti-Bak (Ab-1, Calbiochem), diluted in permeabilisation

buffer (0.5% BSA + 500 µg/ml digitonin in PBS) for 30 min at room temperature. Cells

were washed in PBA (PBS + 0.2% BSA + 0.02% NaN3) and incubated with goat

anti-mouse PE conjugated secondary antibody (Jackson ImmunoResearch Europe

Ltd., Suffolk, England), diluted 1/200 in permeabilization buffer for 30 min at room

temperature. Cells were washed with PBA, resuspended in PBS and analysed using

the FACS.

Western blotting and immuno-precipitation

Cell lysates for Western blotting were separated by 13% sodium dodecyl sulphate

polyacrylamide gel electrophoresis (SDS-PAGE) followed by western blotting as

described previously16. Blots were probed with the following antisera: monoclonal

anti-Mcl-1 (BD Pharmingen), monoclonal anti-Bax (BD Pharmingen), Bak monoclonal

antibody (mAb) (Upstate Biotechnology, Inc. Lake Placid, NY, USA), monoclonal anti-

Bim (Fig. 2 and Fig. 5A; Chemicon, Temecula, CA, USA), polyclonal anti-Bim (Fig.

3Aiii and Fig. 5B; Nventa, San Diego, CA, USA) monoclonal anti-Noxa from Imgenex

(San Diego, CA, USA), polyclonal anti-Bcl-2 (Alexis Biochemicals), polyclonal cleaved

caspase 3 (Asp175) antibody, polyclonal anti-p44/42-MAP Kinase (Cell signaling

Technology Inc., Danvers, MA, USA) antibody and antiserum to β-actin was from

Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

For immuno-precipitation (IP) of Mcl-1, 50×106 cells were washed twice in ice-

cold PBS (supplemented with 10 mM Na3VO

4) and lysed in 500 µL ice-cold Triton

X-100 lysis buffer (1% Triton-X100, 20 mM Tris-Cl (pH=7.4), 135 mM NaCl, 1.5 mM

MgCl2,10% Glycerol, 2 µg/ml Leupeptin, 1 mM PMSF and 2 µg/ml Trypsin inhibitor).

Whole cell lysates were precleared with normal rabbit serum precoupled to protein

A-sepharose and incubated for 3 hr with 5 µg polyclonal anti-Mcl-1 (BD Pharmingen)

precoupled to protein A-sepharose. Beads were pelleted, washed six times with lysis

buffer and boiled in SDS sample buffer, and further processed for Western blotting.

CHAPS lysates were used in certain experiments to avoid detergent-induced

conformational changes of Bax19, by lysing 50×106 cells (for IP) or 15×106 cells (for

Western Blot) for 30 min on ice in lysis buffer (1% Chaps, 20 mM Tris-Cl (pH=7.4),


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37135 mM NaCl, 1.5 mM MgCl

2,10% Glycerol, 1 mM EGTA, 2 mM Na

3VO

4, 10 mM

NaF, 2 µg/ml Leupeptin, 1 mM PMSF, 0.1 mM TLCK and 2 µg/ml Trypsin inhibitor).

After centrifugation (15 min; 13.000 × g; 4°C), the supernatant was used for IP or as

‘CHAPS soluble fraction’ on Western Blot. Pellets were solubilized in SDS sample

buffer and used in Western Blotting as the ‘CHAPS insoluble fraction’. Immuno-

precipitation of Bim (monoclonal anti-Bim (Chemicon)) was performed as described

above in 500 µL ice-cold CHAPS and lysates were precleared with normal rat serum

precoupled to protein G-sepharose.

Immunocytochemistry and confocal analysis

B-CLL cells were incubated with 200 nM MitoTracker for 24 hrs at 37˚C and treated

as described above (intracellular fl ow cytometry procedure). Cells (1 × 106) were

resuspended in 25 µl vectashield (Hardset with DAPI; Vector Laboratories, CA, USA)

and were spotted on cover slips. Images were independently captured by confocal

laser microscopy (model TCS 4D, Leica, Heidelberg, Germany) at the wavelengths

488 and 594 nm lines of the krypton/argon laser used for the excitation of FITC and

MitoTracker (Alexa594), respectively. Excitation of DAPI was by 594 nm.

Statistical analyses

The student’s t-test was used for analysis of differences between 2 groups. P-values

< 0.01 were considered statistically signifi cant.

RESULTS

Seliciclib induces apoptosis and rapid proteasome-dependent

degradation of Mcl-1

The effect of seliciclib on cell viability was investigated in 20 B-CLL samples. B-CLL

cells were >90% pure as determined by fl ow cytometry. Consistent with previous

reports20, in vitro culture of B-CLL cells was accompanied by moderate spontaneous

increase of apoptosis. Increasing doses (10, 25 and 50 µM) of seliciclib resulted in

dose-dependent increase of apoptosis (Fig. 1A). In comparison, cells treated with

high dose of fl udarabine (100 µM) showed a lower level of apoptosis at 24 hrs of

treatment (Fig. 1A), but this increased over time (data not shown). IgVH mutated

and unmutated B-CLL patients (n=8 vs. n=12) showed no difference in apoptosis

induction by seliciclib (data not shown).

Seliciclib was reported to result in rapid proteasomal breakdown of Mcl-1 protein11.


38

Mcl-1 is also described as a substrate for caspase-mediated degradation21. We

tested which of these pathways predominates in B-CLL. Seliciclib was applied in

combination with zVAD-fmk and the proteasome inhibitors MG132 and bortezomib.

As shown in Figure 1B, the degradation of Mcl-1 in B-CLL cells was at best partially

affected by zVAD-fmk (lane 2 vs. 3). In contrast, the proteasome inhibitors MG132

and bortezomib almost fully prevented Mcl-1 degradation (lane 2 vs. 4 and 5). Thus,

in B-CLL cells Mcl-1 is largely degraded already 4 hrs after addition of seliciclib,

predominantly via the proteasome.

In B-CLL, Mcl-1 associates with Noxa and Bim but not with Bak

Prior studies in model cell lines indicate that Mcl-1 selectively interacts with Bim,

Bak, Puma and Noxa, whereas Noxa has a strong preference for Mcl-1 and possibly

Bfl -113;14;22. In primary B-CLL cells the majority of Noxa protein was associated with

Mcl-1 (Fig. 2, compare IP with cleared lysate lanes). As also reported for normal tonsil

B cells23, Bim was clearly but not exclusively associated with Mcl-1. Cells were then

analysed after 2 hrs of Seliciclib treatment, i.e. before extensive degradation of Mcl-1

and zVAD-fmk or MG132 were added to block caspases or the proteasome. Rapid

dissociation of Bim from Mcl-1 could be observed, while Noxa was still largely bound

(Fig. 2, left panels).

Previous studies in HeLa cells indicated that Mcl-1 directly sequesters Noxa, Bim and

Bak, and upon UV treatment, Bak is displaced by Noxa24 . In B-CLL cells, Mcl-1 was

1 2 3 4 5 6 7 WB:

Mcl-1

β-actin

- + + + + - - Seliciclib (25 μM)

- - zVAD MG132 bort MG132 bort Inhibitors

48 kD —

37 kD —

B-CLL24

Figure 1. Apoptosis and Mcl-1 degradation in B-CLL upon seliciclib treatment.

(A) B-CLL cells (n=20) were treated with seliciclib (0, 10, 25 or 50µM) or fl udarabine (100 µM). Apoptosis

was determined via annexin-VAPC/PI staining at 0 and 24 hrs.

(B) B-CLL cells were untreated (lanes 1, 6 and 7) or treated with 25 µM seliciclib (lanes 2-5) for 4 hrs.

Where indicated, cells were pre-treated for 1 hr with 100 µM zVAD-fmk, 10 µM MG132 or 2 µM bortezomib.

Samples containing 30 µg of protein were analyzed by Western blotting for the presence of Mcl-1 (top panel)

and β-actin (bottom panel) as loading control. Experiments were repeated twice with similar results.

0 0 10 25 50 100

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39associated with Noxa and Bim, but we consistently could not detect association of

Mcl-1 with Bak, irrespective of seliciclib treatment, although Bak was present in CLL

lysates (Fig. 2, right panel). As expected, proteasome inhibition by MG132 resulted

in increased Mcl-1 protein, but also increased Noxa levels (Fig. 2, right panels). Bax

protein levels were unaffected by either seliciclib or MG132 treatment. In summary,

in primary B-CLL cells undergoing seliclib treatment Noxa remained bound to Mcl-1

while Bim dissociated.

Figure 2. In seliciclib treated B-CLL cells, Mcl-1 engages Noxa and Bim but not Bak.

B-CLL cells were treated with 25 μM seliciclib for 2 hrs. The pan-caspase inhibitors zVAD-fmk (100

μM) and proteasome inhibitor MG132 (10 μM) were added as indicated. Cell lysates were subjected to

immunoprecipitation (IP) assays using control Ab (CAb) or anti-Mcl-1 antibody. Bound (IP) and unbound

fractions (cleared lysate = 5% supernatant of lysate after IP) as well as total cell fractions (5% of total lysate)

were analysed by Western blotting to evaluate protein associations with Mcl-1. The blotting membrane was

rehybridized with anti-Bim, anti-Bak, anti-Bax and anti- -actin antibodies as indicated. In the case of Bim,

the data derive from a separate experiment. Experiments were repeated three times with similar results.

Noxa defi cient cells exhibit resistance to seliciclib-induced apoptosis

To test whether the association between Noxa and Mcl-1 was refl ected at a functional

level, the endogenous levels of Noxa and Mcl-1 were manipulated in model systems

via RNAi and overexpression. A signifi cant reduction of Noxa in Ramos FSA cells was

achieved with two different siRNA sequences (N7 and N8; Fig. 3Ai). Functionally, Noxa

RNAi conferred a signifi cant resistance to apoptosis induced by seliciclib compared

to the mock transduced cells (Fig. 3B), which correlated with the knock-down in Noxa

protein. These data were confi rmed in the Mec-1 cell line (Fig. 3C), originally derived

from a B-CLL patient, and the Jurkat T cell line18 (Fig. 3D). In contrast, upon several

other death stimuli like fl udarabine, staurosporine, CD95 and BCR stimulation,

no signifi cant effect of reduced Noxa levels was observed (Fig. 3B). Conversely,


40increased Mcl-1 levels (Fig. 3Aii) conferred resistance to seliciclib, but also to several

other apoptotic stimuli (Fig 3E). In agreement with the Mcl-1 association studies in

B-CLL presented in fi gure 2, knock-down of Bim also resulted in increased resistance

against seliciclib. As might be expected from its broader interaction range compared

to Noxa, Bim knock-down conferred resistance against diverse apoptotic stimuli,

among which taxol, in agreement with earlier studies25. In conclusion, these data

indicate that Noxa functions specifi cally in case of Mcl-1 degradation, such as after

seliciclib treatment.

Ai

Aii

Aiii

Figure 3. Noxa RNAi selectively affords resistance

to seliciclib, compared to Bim RNAi and Mcl-1

overexpression.

Noxa knock down was obtained using two different

Noxa RNAi constructs (N7 and N8) in Ramos and

Mec-1 cells. Ramos FSA was transduced with LZRS-

Mcl-1-IRES-GFP vector to obtain overexpression of

Mcl-1. Bim knock down in Jurkat T cells was achieved

with a Bim RNAi contstruct, yielding two independent

clones B18 and B22. Transduction with empty vectors

(M) served as control. Total cell lysates derived from

GFP+ cells were separated by SDS-PAGE.

(A) Noxa, exogenous Mcl-1 and Bim protein levels

were monitored by Western blotting in Ramos FSA,

Mec-1 and Jurkat T cells (J16). Mcl-1 was c-myc

tagged, which could be characterized with an anti-

c-myc antibody. -actin and a nonspecifi c band (*)

served as a loading control.

(B) Ramos FSA (M) (black), N7 (gray) and N8 (white)

were cultured 24 hrs in the absence or presence of

25 μM seliciclib, 100 μM fl udarabine (fl uda), 0.25 μM

staurosporine (stauro), 5 μg/ml α-CD95 or α-BCR

antibodies.

(C) Mec-1 (M) (black) and N8 (gray) were cultured 24

hrs in the absence or presence of 50 μM seliciclib,

0.25 μM staurosporine (stauro) and 5 μg/ml α-CD95.

Aiii

B C


D E41

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(D) J16 (M) (black), N7 (gray) and N8 (white) were cultured as described above with 25 μM seliciclib, 0.25

μM staurosporine (stauro) and 5 μg/ml α-CD95.

(E) Ramos FSA (M) (black) and Mcl-1 (gray) were cultured in the absence or presence of 25 μM seliciclib,

100 μM fl udarabine (fl uda), 0.25 μM staurosporine (stauro), 5 μg/ml α-CD95 or α-BCR antibodies.

(F) J16 (M) (black), J16-B18 (gray) and J16-B22 (white) were cultured 24 hrs in de absence or presence of

25 μM seliciclib, 5 μg/ml α-CD95, 3.1 μM etoposide, 25 μM CCCP, or 5 nM Taxol. Apoptosis was assessed

by fl ow cytometry analysis. Data represent mean ± SD from three or more independent experiments,

except in E; the CD95 and BCR triggering was done twice and no statistics were performed. P values

< 0.01 with Student’s T–test are indicated by an asterisk (*). In case of Bim knock-down (F) , only those

stimuli where both clones yielded P values <0.01 are marked.

Delay in Bax/Bak activation upon seliciclib treatment

Most forms of apoptosis ultimately require activation of Bax and/or Bak, which can

be monitored with conformation-specifi c antibodies26. Surprisingly, we observed that

at 4 hrs after seliciclib treatment, when Mcl-1 degradation was almost complete as

shown above, activation of Bax/Bak was still minimal (see Fig. 4,upper panel). At 12

hrs, Bax/Bak activation was still modest (middle panel). Only after 20 hrs substantial

conformational changes of Bax/Bak occurred. At all timepoints the mitochondrial

membrane potential as measured via MitoTracker staining mirrored the pattern of


Bax/Bak activation, and substantial damage occurred only at 20 hrs. These data

were obtained in various B-CLL samples tested (n=3) and demonstrate that there

was a substantial delay between Mcl-1 degradation and the actual onset of apoptosis.

During this period, there were no signifi cant changes in apoptosis-regulatory genes,

such as p53-responsive Puma, as detected by RT-MLPA6 (data not shown).

42

Figure 4. Seliciclib induced cell

death in B-CLL involves loss of the

mitochondrial membrane potential

and Bax/Bak conformational change.

B-CLL cells were incubated in the absence

(control, gray peak) or presence of 25 μM

seliciclib and monitored every 4 hrs up to

24 hrs. Only 4, 12 and 20 hr time points

are shown. Bax/Bak conformational

changes were determined by intracellular

fl ow cytometry using anti-Bax and anti-

Bak monoclonal antibodies (solid black

lines). For analysis of the mitochondrial

membrane potential (∆Ψm), cells were

incubated with MitoTracker Orange for

30 minutes.

Translocation of Bim, Bax and Bcl-2 to the mitochondria-rich

insoluble fraction subsequent to Mcl-1 degradation

In order to elucidate events occurring between Mcl-1 degradation and the onset of

apoptosis, we monitored candidate proteins to be potentially involved in this process.

This was done by co-immunoprecipitation of CHAPS-soluble lysates taken 0, 4

and 16 hrs after seliciclib treatment. The data shown in fi gure 2 suggested early

dissociation of Bim from Mcl-1. Since Bim can also bind Bcl-2 which is in general

highly expressed in B-CLL, we studied Bim associations over time. Bim was bound

to Bcl-2 at 0 hrs, and this association declined following prolonged seliciclib treatment

(Fig. 5A, left panel). These fi ndings were confi rmed in the reverse experiment by

immunoprecipitation of Bcl-2 (data not shown). Signifi cantly, at 16 hrs of treatment,

Bim, Bcl-2 and Bax were detected in the insoluble fraction of the cell (right panel).

Moreover, we could not detect association between Bim and Bax or Bak (the latter

is generally expressed at low levels in B-CLL cells) in viable nor in apoptotic cells.

In accordance with published fi ndings13;14, we could not detect association between

Bcl-2 and Noxa. The transition of Bim, Bax and Bcl-2 to the CHAPS-insoluble cell


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43fraction was confi rmed by Western blotting of 4 additional B-CLL samples taken after

0, 4 and 16 hrs of seliciclib treatment (Fig. 5B). Together, these data demonstrate that

Bim, Bcl-2 and Bax associations change during drug treatment, and this correlates

with transition to an insoluble state. The mitochondrial membrane protein porin/

VDAC2 was predominantly expressed in the ‘mitochondia rich’ insoluble fraction.

Presumably, after release from Mcl-1 and subsequently Bcl-2, Bim is responsible for

activation of Bax, although this is not detectable via direct interaction in the soluble

cell fraction.

As Bax is translocated to the membrane fraction during seliciclib-induced apoptosis,

we assessed its subcellular distribution using confocal microscopy. Figure 5C shows

that Bax is localized throughout the cytoplasm in untreated B-CLL cells. Following 16

hrs seliciclib treatment (+ pretreatment with zVAD), Bax showed a punctate distribution

and co-localization with the mitochondria, suggesting that Bax multimerizes at the

mitochondria.

A

B

Figure 5. Bim associates with Bcl-2 and Mcl-1

and shifts to the mitochondria-rich insoluble

fraction over time in B-CLL cells.

See color fi gures.

B-CLL cells were treated with 25 μM seliciclib for

0, 4 and 16 hrs.

(A) Cell lysates were subjected to immunopre-

cipitation (IP) assays using a Bim antibody. The

bound (IP) fraction (left panel) was analysed by

Western blotting to evaluate the association of

Bcl-2, Mcl-1, Bak and Bax with Bim over time. To-

tal cell fractions (5% of total lysate; middle panel)

and insoluble fractions (right panel) were moni-

tored by Western blotting. The blotting membrane

was hybridized with Bcl-2, Mcl-1, Bak, Bax, Bim,

-actin and Porin antibodies as indicated. (* Im-

munoglobulin heavy chain; ** Immunoglobulin

light chain)

(B) CHAPS- soluble and -insoluble fractions from

4 other B-CLL patients were analysed by Western

blotting to evaluate the localization of Bim, Bax

and Bcl-2 over time.

(C) Immunocytochemical staining for Bax of B-CLL

cells incubated in the absence or presence of 25

μM seliciclib for 24 hrs. MitoTracker Orange was

used for localization of the mitochondria.


C

Bcl-2 confers a temporary block to selicilib-induced apoptosis

To directly determine the role of Bcl-2 in the delayed onset of seliciclib-induced

apoptosis, we used a Ramos model cell line overexpressing Bcl-216. In parental

Ramos cells treated with seliciclib, Mcl-1 degradation (Fig. 6A; upper panel) occurred

within 4 hrs, similar to B-CLL cells (Fig. 1B). The decline in Mcl-1 levels occurred

independently of caspase activation, as demonstrated by overexpression of C9DN.

In contrast to B-CLL, Mcl-1 degradation was almost simultaneous with apoptosis

induction, shown by cleavage of effector caspase 3 (Fig. 6A; panel 3), and annexin-V

exposure (Fig. 6B). Overexpression of Bcl-2 in Ramos cell lines recapitulated

the delayed onset of apoptosis after seliciclib treatment observed in B-CLL cells.

Clearly, apoptosis was blocked after 4 hrs of treatment, whereas at 24 hrs virtually

all cells were apoptotic, despite the presence of Bcl-2 (Fig. 6B). In comparison, time

course analysis of Ramos cells with Noxa knock-down or Mcl-1 overexpression also

showed that the block in seliciclib induced apoptosis was not absolute. Here, the

fraction of apoptotic cells increased more gradually over time up to the last point

measured (48 hrs; Fig. 6C and 6D). The effect of seliciclib on Mcl-1 produced from

a recombinant vector may be less than on the endogenous transcript. However, the

recently described phosphorylation of Mcl-1 at Ser64 which enhances its apoptotic

activity is apparently also prevented by seliciclib27, therefore a complete block of

seliciclib-induced apoptosis of overexpressed Mcl-1 is not expected. Together, our

data indicate that manipulation of individual Bcl-2 family members is insuffi cient to

warrant full protection to seliciclib, which suggest they are subject to an intricate

functional interplay.

44


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Figure 6. Bcl-2 overexpression in Ramos cell line recapitulates delayed onset of apoptosis.

(A) Ramos 2G6 and Ramos FSA C9DN (DN9c) cells were treated with 25 μM seliciclib. At the indicated

time points samples were analyzed by Western blotting for the presence of Mcl-1 (top panel), Noxa (sec-

ond panel), activated caspase-3 (third panel) and -actin (bottom panel) as loading control.

(B) Ramos 2G6 and Bcl-2 cells (5×105/ml) were left untreated or treated with 50 μM seliciclib for 4 and 24

hrs.

(C) Ramos FSA and N8 cells (5×105/ml) were left untreated or treated with 25 μM seliciclib for 4, 24 and

48 hr.

(D) Ramos FSA and Mcl-1 cells (5×105/ml) were left untreated or treated with 25 μM seliciclib for 4, 24 and

48 hr. Viability in B, C and D was assessed by means of MitoTracker staining. Data represent mean ± SD

from three independent experiments.


DISCUSSION

In hematological cells, the primary apoptotic trigger ascribed to the novel cyclin-

dependent kinase inhibitor seliciclib is a rapid decrease in Mcl-1 levels independent

of p53 function9;11. This is of clinical importance in view of the poor prognosis of

B-CLL with functional defi ciency in the p53 pathway28 and the outgrowth of p53-

defi cient clones during treatment1. Furthermore, there is broad interest in the exact

mechanism of action of the various Bcl-2 family members, as it has become clear

that their protective function can be targeted also directly by pharmacological

compounds29. However, in the absence of a functional link to Bax and/or Bak

activation, drug-induced Mcl-1 decrease is in itself not suffi cient to explain cell death.

Our fi ndings emphasize two new aspects in the chain of events triggered in B-CLL

cells upon seliciclib treatment and Mcl-1 degradation. We found previously that the

BH3-only protein Noxa is expressed at high levels in B-CLL6, and here we establish

that the molecular effects of seliciclib rely on Noxa. Secondly, subsequent to Mcl-1

degradation, we observed a gradual deterioration of the protection afforded by Bcl-2,

which was most probably caused by liberated Bim.

Biochemical studies have shown that distinct BH3-only proteins have differential

binding affi nities to pro-survival Bcl-2 proteins13;14;30. Reportedly, Noxa has a strong

preference for binding to Mcl-1 which we confi rm here in primary B-CLL cells (Fig.

2). At present, the precise role of Noxa is not entirely understood, and our data point

to a new and specifi c role in drug-induced cell death. This was demonstrated by a

resistance to seliciclib in Noxa knock-down leukemic cell lines (Fig. 3). Difference in

Noxa expression did not affect apoptosis by other stimuli (fl udarabine, staurosporine,

CD95 and BCR). Together, these observations imply that the lethal effect of seliciclib

in B-CLL cells specifi cally involves Noxa function. In agreement with their broader

interaction range, Mcl-1 overexpression or Bim knock-down had an inhibitory effect

on multiple apoptotic stimuli.

Although Noxa levels are 5-10 times higher in B-CLL than in normal B cells6, this is

apparently in itself not a direct trigger for apoptosis, which fi ts well with the current

view of its function as an indirect apoptosis mediator13;14. The available evidence

indicates that Noxa acts by displacing pro-apoptotic Bcl-2 members from Mcl-124;30.

We have studied potential involvement of two pro-apoptotic candidates Bak and Bim

in seliciclib-mediated apoptosis in B-CLL. In agreement with studies in HeLa cells24,

we found that Noxa can liberate Bak from Mcl-1 in leukemic cell lines (data not shown).

However, we consistently could not detect Mcl-1 associated with Bak in primary B-CLL

cells. Instead, we observed that the level of Bim associated with Mcl-1 rapidly declined

upon seliciclib treatment, while Noxa remained bound (Fig. 2). This is consistent with

46


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47the differential binding affi nities among the BH3-only members reported by others13;22.

Once liberated from Mcl-1, Bim is reportedly capable of directly activating Bax and/

or Bak14;30. Direct association between Bax or Bak with purported direct BH3-only

activators such as Bid and Bim in model systems has been described31;32. Yet, direct

association of endogenous proteins under physiological conditions is diffi cult to

observe. We were also unable in various co-immunoprecipitation approaches to fi nd

Bim complexed with Bax or Bak in early or late apoptotic B-CLL cells. The aspect of

direct vs. indirect activation of Bax (or Bak) by BH3-only members such as Bim is

currently the subject of intense research and debate19;30;33. Although it is in fact still

unknown what actually causes transition of cytosolic Bax34, its activation is known to

result in multimerization and pore formation in mitochondria35. Therefore, we looked

for shifts from soluble to insoluble fraction upon apoptosis initiation, and could observe

transition of both Bim and Bax to the mitochondria-rich insoluble cell fraction (Fig.

5). Our fi ndings in primary B-CLL cells suggest that any direct interaction between

Bim and Bax is presumably short-lived, and/or induces rapid multimerization and

insertion of Bax in the mitochondrial membrane. At this point the interacting proteins

become insoluble and can no longer be detected by standard co-immunoprecipitation

approaches. This possible sequence of events upon seliciclib treatment of B-CLL

cells is schematically depicted in fi gure 7.

The proposed sequence of events from Mcl-1 degradation, Noxa-dependent Bim

displacement and liberation, and fi nally Bax activation is compatible with recent data

and interpretations concerning the hierarchical involvement of BH3-only members in

initiation of the mitochondrial apoptosis pathway13;30. Moreover, a functional linkage

between Noxa and Bim was very recently reported in cell lines36. During the period

between Mcl-1 degradation and eventual Bax/Bak activation in B-CLL cells, there

were no signifi cant changes in gene transcription of Bcl-2 family members (data not

shown). The available data suggest that after Mcl-1 degradation, Bcl-2 is temporarily

capable of withstanding the pro-apoptotic action of Bim and Bax, but is progressively

overwhelmed. In support of this, overexpression of Bcl-2 in a model system could

indeed delay but not prevent apoptosis induced by seliciclib treatment (Fig. 6). The

gradual undermining of Bcl-2 function could either be a direct effect of seliciclib by

an as yet unknown pathway, or indirectly result from shifts in the distribution of and

interactions between Bcl-2 family members.

The fi ndings reported here agree with and complement recent fi ndings concerning

the mechanism of action of the BH3-mimetic ABT-73737;38. This compound can

counteract the protective function of Bcl-2, Bcl-XL and Bcl-w and has potential as a

cancer therapeutic29. Cells overexpressing Mcl-1 are however resistant to ABT-737,

and strategies to target Mcl-1 levels enhance its effi cacy37;38. Based on our fi ndings


48with B-CLL cells reported here and elsewhere39, both direct targeting of Mcl-1 by drugs

such as seliciclib as well as indirect targeting by means of bortezomib to increase Noxa

levels, may be a promising combination with ABT-737 or similar compounds. Indeed,

it has been shown very recently that seliciclib treatment dramatically increased ABT-

737 lethality in human leukemia cell lines40. Overall, the functional cross-talk between

Bcl-2 family members suggest that the ‘addiction’ of leukemic cells to overexpression

of anti-apoptosis proteins such as Bcl-2 and Mcl-1 could be specifi cally targeted and

exploited in a therapeutic setting.

Figure 7. Model for the steps in apoptosis triggered by seliciclib in B-CLL cells.

In the initial phase (< 4hrs) Mcl-1 is degraded rapidly in a proteasome dependent manner. As Mcl-1 levels

fall, Bim is released while Noxa remains bound. Liberated Bim can be absorbed by Bcl-2 for a while, but

gradually there is a transition to a situation where Bcl-2 is no longer capable of binding excess Bim. What

is actually causing this transition is unknown. Both disrupting the balance between Bcl-2 family members

or unknown signaling events might eventually lead to Bcl-2 dysfunction. From that point on, Bim is able to

activate Bax, leading to multimerization and apoptosis. As indicated in the main text, whether Bim activates

Bax directly, indirectly or via transient interactions is currently the subject of controversy. Therefore, this

scheme only emphasizes that all three direct participants (Bcl-2, Bim and Bax) shift to an insoluble fraction

of the cell, which in the case of multimeric Bax is presumably mitochondrial.


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49

Of 24 patients the following characteristics were determined at time of inclusion in the study; sex (M=male,

F=female); age (in years); Rai stage: 0 (good prognosis), I-II (intermediate prognosis), and III-IV (poor

prognosis); IgVH mutation status (UM = unmutated IgV

H genes, M = mutated IgV

H genes) and treatment.

ND = not determined

Patient no. Sex Age (years) Rai (stage) lgVH

status Treatment

1 M 63 1 M ND

2 M 51 2 UM Yes

3 M 79 2 UM Yes

4 M 58 2 M No

5 M 81 0 M No

6 F 56 2 UM Yes

7 M 65 4 M No

8 M 64 ND UM Yes

9 F 81 0 M ND

10 M 75 0 M No

11 M 72 1 UM ND

12 M 56 2 UM Yes

13 F 49 1 M ND

14 M 71 1 M ND

15 M 73 2 M ND

16 M 70 2 UM ND

17 M 65 4 UM Yes

18 M 60 2 UM Yes

19 M 50 0 UM Yes

20 M 56 2 UM Yes

21 M 65 1 M Yes

22 M 65 ND M ND

23 F 71 0 M ND

24 F 75 3 M Yes

Table 1. Patient’s characteristics.


50 ACKNOWLEDGEMENT

We thank Bart de Goeij for technical assistance. In addition we thank the patients for

their blood donations. We also acknowledge Dr. Kramer, Dr. Wittebol and Dr. Baars

for including B-CLL patients in this study.


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54


chapter

3 -secretase inhibitor (GSI)-1 induces apoptosis in CLL cells via proteasome inhibition and Noxa upregulation

Delfi ne Y.H. Hallaert1,2, Heike Schmidlin3, Marinus H.J. van Oers1 and Eric

Eldering2

1 Dept of Hematology, AMC, Amsterdam, The Netherlands

2 Dept of Experimental Immunology, AMC, Amsterdam, The Netherlands

3 Dept of Cell Biology, AMC, Amsterdam, The Netherlands

Submitted for publication

γ


56 ABSTRACT

Deregulation of the Notch pathway has been suggested to contribute to the

pathogenesis of B cell chronic lymphocytic leukemia (CLL). γ-Secretase inhibitors

(GSIs) can block intracellular processing of all four different Notch receptors. We

found that GSI-1 (z-Leu-Leu-Nle-CHO) was a potent inducer of apoptosis in CLL,

but this appeared not to be due to inhibition of Notch signaling. Instead, effi cient

blocking of proteasomal activity by GSI-1 was observed, equivalent to established

proteasome inhibitors bortezomib and MG-132. In contrast with GSI-1, another

γ-secretase inhibitor GSI-9/DAPT neither affected proteasome activity nor induced

apoptosis in CLL. GSI-1-induced apoptosis was associated with a transcription-

independent accumulation of the BH3-only protein Noxa. The pivotal role of Noxa

in GSI-1 mediated apoptosis was demonstrated via RNAi in model systems. Our

data offer an explanation for certain confl icting notions on Notch signaling in B cell

malignancies, and suggest that GSI-1 or related compounds may hold promise for

therapeutic application in CLL.


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57INTRODUCTION

B cell chronic lymphocytic leukemia (CLL) is characterized by the relentless

accumulation of monoclonal B cells that coexpress CD5, CD19 and CD23. The

malignant B cells have a low proliferative activity, but prolonged cell survival. Although

over the last few years new (immuno)chemotherapy regimens have resulted in

strongly improved quality of remissions and progression free survival, at present

there still is no curative treatment1.

A prominent feature of CLL cells is the overexpression of the transmembrane

glycoprotein CD232. It was reported that Notch signaling is involved in the up-

regulation of the CD23a isoform in CLL cells, and that this may be linked with the

aberrant apoptosis regulation in CLL3;4. The Notch gene family consists of four

evolutionarily conserved transmembrane receptors that play a fundamental role in

cell fate decisions including cell proliferation, differentiation and apoptosis. Notch

signaling is initiated by receptor-ligand interaction resulting in two successive

proteolytic cleavages of the Notch receptor by TACE (TNF-α-converting enzyme) and

the γ-secretase/presenilin complex, which liberates the cytoplasmic domain of the

Notch receptor (Notch intracellular domain; NIC). The NIC enters the nucleus leading

to transcriptional activation of downstream target genes5. Recent reports suggest

that deregulated Notch signaling is associated with various malignancies, in which

Notch may function either as an oncogene or as a tumor suppressor6. Variable mRNA

expression levels of Notch1 and Notch2 (receptors); Delta-like 1 (ligand); Deltex

(a regulator molecule) and Hairy/Enhancer of Split-1 (Hes-1, the most well-known

target gene of Notch5) were found in CLL7. In general, studies into the role of Notch

signaling in B cell maligancies have led to disparate reports, with suggestions that

Notch activity induces apoptosis8, while others have indicated that in fact inhibition of

Notch signaling induces apoptosis9.

Various studies implicate the ubiquitin-proteasome pathway in the control of Notch

activity4;10;11, and proteasome inhibitors such as bortezomib induce apoptosis.

Following the successful application of proteasome inhibitors in multiple myeloma

(MM)12;13, this topic has attracted broad interest as novel treatment strategy for

cancer. Exposure to proteasome inhibitors results among others in induction of

the pro-apoptotic BH3-only protein Noxa, which was preceded in melanoma and

myeloma cells by enhanced transcription of Noxa mRNA14. We recently showed that

Bortezomib-mediated apoptosis in CLL also involves Noxa protein accumulation15.

In the present study we investigated the possible role of Notch signaling in CLL by

examining the effect of γ-secretase inhibitors. These chemical inhibitors can block

processing of all Notch receptors16;17. Unexpectedly, we observed that in CLL GSI-1


58is a potent inducer of apoptosis without discernable involvement of Notch signaling.

Instead, the compound effi ciently inhibits proteasomal activity, leading to accumulation

of the pro-apoptotic BH3-only protein Noxa, which has a pivotal role in GSI-1-induced

cell death.

MATERIALS & METHODS

Isolation of leukemic, normal B and normal T lymphocytes

Peripheral blood samples were obtained from CLL patients from the outpatient clinic

of the department of Hematology of the Academic Medical Center, Amsterdam;

the department of Internal Medicine, Meander Medical Center, Amersfoort and the

department of Internal Medicine, The Netherlands Cancer Institute, Amsterdam.

Informed consent was obtained according to the guidelines of the local Ethical

Review Board. Clinical characteristics of patients are presented in Table 1. This study

was conducted in accordance with the ethical standards in our institutional medical

committee on human experimentation, as well as in agreement with the Helsinki

Declaration of 1975, revised in 1983. Peripheral blood mononuclear cells (PBMC) from

CLL patients and healthy donors, obtained via density-gradient centrifugation were

frozen in 15% fetal calf serum (FCS) containing 10% dimethyl sulphoxide (DMSO;

Sigma Chemical Co., St Louis, MO, USA) and stored in liquid nitrogen before use or

directly used for experiments. Expression of CD3, CD5, CD19 (all antibodies from

Becton Dickinson (BD) Biosciences, San Jose, CA, USA) and CD23 (clone MHM6

from DAKO, Glostrup, Denmark) on leukemic cells were assessed by fl ow cytometry

(FACScalibur, BD Biosciences) and CellQuest software (BD Biosciences)15.

Cell lines

Cell lines were cultured in Iscove’s modifi ed Dulbecco’s medium (IMDM; Gibco Life

Technologies, Paisley, Scotland), supplemented with 10% (v/v) heat-inactivated fetal

FCS (ICN Biomedicals GmbH, Meckenhein, Germany), 100 U/ml penicillin, 100 µg/

ml streptomycin and 5 mM L-glutamine (Gibco). The RPMI myeloma cell line was

kindly provided by Dr. Spaargaren (Department of Pathology, AMC, Amsterdam, The

Netherlands). The CLL cell line Mec-1 was a kind gift of Dr. Caligaris-Cappio (Milano,

Italy).

Mock and Noxa shRNA sequence (N7, N818) were retrovirally transduced into Ramos

FSA (with enhanced sensitivity to CD95)19, Mec-1 and Jurkat (J16) cell lines. To

improve knockdown of Noxa, a limiting dilution of the Ramos FSA N8 cell population

was performed according to standard procedures. Resulting clones were selected


59for increased resistance to bortezomib and further characterized by Western blot to

assess reduced Noxa expression. Clone N8G10 was used for experiments in fi gure

7.

Reagents and antibodies

Roscovitine, fl udarabine and propidium iodide (PI) were purchased from Sigma

Chemical Co. (St. Louis, MO, USA). The pan-caspase inhibitor zVAD-fmk was

obtained from Alexis Biochemicals (Läuferlingen, Switzerland). APC-labeled Annexin

V was from IQ Products (Groningen, The Netherlands) and MitoTracker Orange was

from Molecular Probes (Leiden, The Netherlands). Proteasome inhibitors MG-132

and bortezomib, were obtained from Calbiochem, (Amsterdam, The Netherlands) and

Janssen-Cilag, (Tilburg, The Netherlands), respectively. -secretase inhibitors, GSI-

120 (Z-LLNle-CHO – Cat.nr. 565750) and GSI-921 (DAPT (Difl uorophenacetyl-L-alanyl)-

S-phenylglycine t-Butyl Ester) - Cat.nr. 565770) were purchased from Calbiochem.

Anti-human Fas10 (agonistic antibody to the CD95 receptor) was a kind gift from

Prof. Dr. L. Aarden (Sanquin, Amsterdam, The Netherlands). Monoclonal antibody

to CD123 (IL-3Rα) conjugated to PE was purchased from Becton Dickinson. Anti-

BDCA2-APC was obtained from Miltenyi Biotec (Bergisch Gladbach, Germany).

Analysis of apoptosis & Western Blot

For the analysis of apoptosis of CLL cells (>90% CD19+CD5+ PBMC suspensions)

and PBMCs from healthy donors, 5×106 cells/ml were incubated for indicated time

points with GSIs or other drugs. Apoptosis of CD19+ CLL and normal B cells and

CD3+ T cells was measured by Annexin-V22 or MitoTracker staining as described

before23.

Western blotting was performed as described previously24. Blots were probed with

monoclonal anti-Noxa from Imgenex (San Diego, CA, USA), monoclonal anti-Bim

Chemicon, Temecula, CA, USA), monoclonal anti-Mcl-1 (BD Biosciences) and

antiserum to β-actin from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Anti-ubiquitin was a kind gift from Prof. Rapoport (Department of Cell Biology,

Harvard Medical School, Boston, MA, USA). p53 functionality of CLL samples was

screened by the effect of radiation on the expression of p53 and p21 by Western Blot

analysis25.

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60Notch-dependent differentiation assay

Isolation of CD34+ cells from postnatal thymus tissue was described previously

by Dontje et al 26. The development of pDCs was assessed by coculturing 5 ×

104 CD34+CD1a- progenitor cells with 5 × 104 OP9 cells in MEMα (Invitrogen Life

Technologies, Carlsbad, CA, USA) with 20% FCS (Hyclone, Logan, UT,USA), 5 ng/

mL IL-7 and 5 ng/mL Flt3L. Differentiation assays for pDCs were analyzed after 1

week of coculture. Flow cytometric analyses were performed on an LSRII FACS

analyzer (Becton Dickinson).

Reverse transcription (RT) PCR

Total RNA was isolated using the Nucleospin RNA isolation kit (Macherey-nagel,

Düren, Germany). For RT-PCR (Reverse Transcriptase Polymerase Chain Reaction),

total RNA was extracted as described above, and cDNA was synthesized by means

of oligodeoxythymidine (oligo(dT)) and Superscript II RNase H-reverse trancriptase

(Invitrogen Life Technologies). Noxa, Notch1 and Notch2 transcripts were amplifi ed

by PCR as described before3;18. Products were electrophoresed on 1% agarose

gel. PCR for Hes-1 was performed on an iCycler PCR (Bio-Rad, Hercules, CA) as

described before26.

Proteasome activity assay

Cytoplasmic extracts (Assay buffer: 250 mM HEPES (pH7.5), 5 mM EDTA, 0.5%

NP-40 and 0.01% SDS) from freshly isolated PBMCs from CLL patients were used to

measure proteasome activity using a 20S proteasome activity assay kit (Chemicon,

part of Millipore; Billerica, USA) following the manufacturer’s instructions. The

assay is based on detection of the fl uorophore 7-amino-4-methylcoumarin (AMC)

after cleavage from the labeled substrate LVVY-AMC. The free AMC fl uorescence

was quantifi ed using a 380/460-nm fi lter set in a VICTOR2 D fl uorometer (Wallac-

PerkinElmer) PerkinElmer, Massachusetts, USA). Proteasomal activity was calculated

from the changes in fl uorescence over time and expressed per μg of protein in the

extract.


61RESULTS

GSI-1 is a potent, p53-independent inducer of apoptosis of primary

CLL cells

Notch expression has been characterized previously in CLL3;7, and we could

confi rm expression of Notch1 and Notch2 in CLL, while Notch3 and Notch4 were

not present in the samples tested (data not shown). γ-Secretase inhibitors (GSIs)

are pharmacological agents able to block Notch signaling16;17. GSI-1 (0.25 – 50 µM)

induced a signifi cant dose-dependent increase in apoptosis of CLL cells after 24

hours (Fig. 1A). Similar experiments were performed with GSI-9/DAPT, another class

of small molecule inhibitors of γ-secretase activity. DAPT, however, had no effect on

apoptosis in CLL (1.0 – 50 µM) (Fig. 1A). To determine the effect on normal B and T

lymphocytes, PBMCs from 3 healthy individuals were treated for 24 hrs with GSI-1 at

concentrations ranging from 0.5 to 50 µM. The cytotoxicity of GSI-1 towards normal

CD19+ B cells was comparable to that observed for CLL cells, while normal CD3+ T

cells were less sensitive to GSI-1 (Fig. 1B), and showed a maximum of approximately

50% apoptotic cells. Next, CLL samples of patients lacking functional p53 were

analyzed following incubation with GSI-1, in comparison with induction of apoptosis

by γ-irradiation (γ-IR). Both p53 defi cient (n=2) CLL samples showed prominent cell

death following treatment with 5 µM GSI-1, comparable to WT samples (see also

Fig. 1A), whereas they resisted radiation-induced apoptosis (Fig. 2). Taken together,

these results indicate that Notch1 and Notch2 are expressed in primary CLL cells and

treatment with GSI-1 but not DAPT results in rapid, p53-independent cell death.

Figure 1: Apoptosis of CLL cells, normal B and T lymphocytes upon GSI-1 treatment.

(A) Apoptosis response as analyzed by MitoTracker staining of CLL cells upon 24 hours incubation with

GSI-1 ( ; n=13; 0.25 - 50 µM) or DAPT ( ; n=3; 1 - 50 µM). Data represent mean ± SD.

(B) Apoptosis response as analyzed by MitoTracker staining of PBMCs from healthy donors (HD) (n=3).

CD19+ normal B cells ( ) and CD3+ normal T cells ( ) were treated with 1 – 50 µM GSI-1. Data represent

mean ± SD from 3 independent experiments.

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Treatment [μM]


62

Notch signaling does not contribute to CLL cell survival in vitro,

and GSI-1 cannot block Notch signaling

To test whether triggering via Notch infl uenced in vitro survival of CLL cells, freshly

isolated CLL cells were evaluated in two co-culture systems; murine fi broblast L cells27

or stromal OP-9 cells transfected with the Notch ligands Jag1 or DL126. CLL survival

was evaluated every day up to day 7. No signifi cant or reproducible difference in

survival compared to co-culture with untransfected control cells or in medium could

be observed (11 CLL samples tested at various cell densities, data not shown).

To address whether Notch signaling can be specifi cally blocked by GSI-1, we applied

an established cell culture system where lineage decision between T cells and

CD123+BDCA2+ plasmacytoid dendritic cells (pDCs) depends on Notch signals26.

The OP-9 cell line expressing human DL1 inhibits the development of pDCs from

CD34+CD1a- thymic progenitor cells, but as mentioned above it has no effect on

CLL survival. Addition of DAPT could overcome the inhibition of pDC development,

whereas GSI-1 could not (Fig. 3A). In fact, as can be seen from the FACS plots, GSI-

1 caused massive apoptosis at high concentration (5 µM). The effect of GSI-1 and

DAPT on the expression of the Notch specifi c target gene Hes-1 was evaluated by

quantitative RT-PCR (Fig. 3B). The mRNA levels were normalized to expression in

untreated CLL cells (relative value of 1) using β-actin as internal control. Expression of

Hes-1 shows large variation among untreated CLL cases and the median expression

of Hes-1 was lower than in normal B cells (0.1-17.5 % of the average of normal B

cells)7. However, incubation of CLL cells (n=2) with DAPT (1 or 10 µM) blocked the

basal Hes-1 expression, whereas GSI-1 (1 or 10 µM) showed no clear inhibitory

effect. Thus, triggering of Notch receptors on CLL did not affect their survival and

in contrast to DAPT, GSI-1 was unable to prevent Notch signaling in an established

system of lineage decision.

Figure 2: Apoptotic response to GSI-1 does

not require functional p53.

Apoptosis response of 3 CLL patients: CLL17

(black bar; p53 wt); CLL27 and CLL28 (gray bar

and white bar; p53 dysfunctional), to GSI-1 (5 µM)

and γ-IR (5Gy) assessed by MitoTracker staining

at 24 hours. (med = medium)


63

GSI-1-induced apoptosis is accompanied by Noxa protein accumulation

It was reported that in melanoma cells GSI-1 induced pro-apoptotic Bim and Noxa28.

Similarly, in CLL cells addition of GSI-1 resulted in a rapid and massive increase

in Noxa protein levels, surpassing the Noxa-inducing capacity of the proteasome

inhibitor bortezomib (Fig. 4A, upper panel). We recently showed that in CLL, Mcl-1

interacts with Noxa and Bim23, so we also evaluated protein expression of Mcl-1 and

Bim. No change in BimEL

and BimL protein levels occurred after 4 hours GSI-1 or

bortezomib exposure. After 24 hours, Bim protein levels declined (Fig. 4A, second

panel), which probably refl ects the transition of Bim to the insoluble fraction when

cells undergo apoptosis23. Mcl-1 levels increased after 4 hr treatment with 10 µM GSI-

1. The decrease in Mcl-1 protein in GSI-1 -and bortezomib treated cells at 24 hours

(Fig. 4A, third panel) is most probably the result of caspase-mediated degradation29,

as it could be inhibited by zVAD (data not shown).

To investigate whether Noxa upregulation was through increased transcription, we

performed RT-PCR of CLL cells incubated with GSI-1 or bortezomib. No induction of

Noxa mRNA was observed after 4 hours GSI-1 and bortezomib (Fig. 4B).

Figure 3: No effect of Notch ligation on CLL survival, and no effect of GSI-1 on Notch signaling.

(A) CD34+CD1a- cells were co-cultured with OP9-DL1 cells in the presence or absence of 10 µM DAPT, 1

or 5 µM GSI-1. Cultures were analyzed for the presence of CD123hiBDCA2+ pDCs after 1 week of culture.

The percentage of pDCs is indicated by FACS gates. The data are representative for 6 (DAPT) and 2

(GSI-1) experiments, respectively.

(B) RT-PCR analysis of Hes-1 was performed on two CLL samples (black and white bars) upon 4 hour

incubation with GSI-1 (1 or 10 µM) or DAPT (1 or 10 µM). Values were normalized to untreated CLL cells.

β-actin was used as internal control. The error bars represent SD of duplicate samples. Two CLL patients

are shown.

A B

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64After 24 hours of treatment with these agents, mRNA could hardly be extracted

because of the high proportion of dead cells (data not shown). In addition, Figure

4 B illustrates that in the myeloma cell line RPMI 8226, bortezomib does however

induce upregulation of Noxa mRNA at intermediate concentration, in agreement

with a previous report30. In conclusion, addition of GSI-1 to CLL cells causes a rapid

accumulation of Noxa protein levels, independent of transcriptional regulation.

A

B

GSI-1 inhibits proteasome activity and induces accumulation of

polyubiquitinated proteins

Given the aforementioned results, demonstrating Noxa protein accumulation upon

treatment with GSI-1 and bortezomib, we determined the effect of GSI-1 on the total

level of ubiquitinated proteins in HeLa cells and the proteasomal activity in primary CLL

samples. First, HeLa cells were incubated for 16 hours with GSI-1 or acknowledged

proteasome inhibitors bortezomib and MG-132. Figure 5A (upper panel) illustrates

that addition of GSI-1, bortezomib or MG-132 led to accumulation of polyubiquitinated

proteins. Concomitant with increased levels of ubiquitinated proteins, Noxa protein

levels accumulated over time (Fig. 5A; middle panel). Second, incubation of lysates

from freshly isolated CLL cells with GSI-1, bortezomib or MG-132 resulted in clear

inhibition of proteasome activity (Fig. 5B). In contrast, roscovitine and fl udarabine, both

chemotherapeutic agents capable of inducing apoptosis in CLL cells, or the gamma

secretase inhibitor DAPT had no measurable effect on proteasomal activity (Fig. 5B).

Figure 4: Effect of GSI-1 and

bortezomib on Noxa mRNA and

protein levels.

(A) CLL cells were treated for 4 and

24 hrs with GSI-1 (1 or 10 µM) or

bortezomib (B) (10 or 30 nM). Cell

lysates were probed for expression of

Noxa, Bim, Mcl1 and -actin protein

by Western Blot. Apoptosis of cells

was determinated by Annexin-VAPC/

PI staining and is indicated above the

lanes. Results are representative for 2

separate experiments.

(B) CLL cells or the myeloma cell

line RPMI 8226 were incubated for 4

hours with GSI-1 or bortezomib. RNA

was isolated and RT-PCR for Noxa

and GAPDH was performed. Results

are representative for 2 separate

experiments.


65In addition, proteasomal activity was measured in lysates obtained from CLL cells

after 4 and 24 hours incubation with various drugs. Again, GSI-1, bortezomib and

MG-132 showed blocking of the proteasome, whereas roscovitine did not (Fig. 5C).

Note that upon 24 hours after triggering with roscovitine proteasomal activity was

also undetectable, probably as a result of massive apoptosis, and subsequent block

in proteasomal activity31. Apoptosis responses under these various conditions are

represented in fi gure 5D. In conclusion, GSI-1 blocked proteasomal activity, leading

to accumulation of polyubiquitinated proteins, similar to the effects of well known

proteasome inhibitors such as MG-132 and bortezomib.

Figure 5: Effect of GSI-1 on ubiquitin / proteasome system.

(A) Accumulation of polyubiquitinated proteins after MG-132, bortezomib or GSI-1 treatment. HeLa cells

were treated with 10 µM MG-132, 10 nM bortezomib or 5 µM GSI-1 for16 hours. Lysates were probed for

expression of ubiquitinated proteins, Noxa and β-actin by Western blot.

(B) The enzymatic activity of the 20S proteasome was measured after adding bortezomib (20 nM), MG-132

(0.5 µM), GSI-1 (5 µM), DAPT (5 µM), roscovitine (25 µM) or fl udarabine (100 µM) to cytoplasmic extracts

from freshly isolated cells from 3 CLL patients. After 15 minutes incubation, the fl uorogenic proteasome

substrate LVVY-AMC was added. Results are expressed as change in AMC fl uorescence per minute per

μg protein.

(C) CLL cells were treated for 4 (black bar) and 24 (white bar) hrs with GSI-1 (1 or 5 µM), bortezomib (20

nM), MG-132 (0.5 µM) and roscovitine (25 µM). 20 S proteasomal activity was measured as described

above (4B). Data represent mean ± SD from four different CLL patients.

(D) Apoptosis response of CLL cells from the experiments described in C were measured by MitoTracker

staining after 4 (black bar) and 24 (white bar) hours.

A

C

B

D

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66Surface expression of CD23 is reduced upon proteasomal inhibition

Exposure of CLL cells to the proteasome inhibitors bortezomib and MG-132

induced apoptosis and decreased expression of CD23, which was proposed to

be a consequence of Notch inhibition4. To test the alternative possibility that CD23

down-regulation is a direct consequence of proteasomal inhibition, we incubated CLL

cells (n=3) with GSI-1, proteasome inhibitors bortezomib and MG-132, or the Notch

inhibitor DAPT. As can be seen in fi gure 6, GSI-1 led to a reduction of CD23 surface

expression similar to that seen with bortezomib or MG-132, while CD23 expression

remained essentially unchanged during 24 hours incubation with DAPT.

Figure 6: CD23 expression on CLL cells upon incubation with GSI-

1. CLL cells were cultured for 24 hours with 30 nM bortezomib (n=2),

0.5 μM MG-132 (n=2), 5 μM GSI-1 (n=4) and 5 μM DAPT (n=3). The

percentage CD23-positive cells was measured by FACS analysis: within

the viable population, CD5 and CD19 double positive cells were gated.

Data represent mean from ± SD.

Noxa knock-down results in decreased sensitivity to GSI-1

The previous experiments showed that Noxa protein accumulates upon GSI-1

treatment, but do not establish whether this is required to induce apoptosis. To

investigate a direct role for Noxa in GSI-1 mediated apoptosis, we studied various

cell lines transduced with retroviral vectors encoding two different Noxa shRNA

sequences (N7 and N8). Figure 7A illustrates signifi cant but not complete knock-down

of Noxa protein in the different cell lines. Incubation with GSI-1 (+/- pre-treatment

with zVAD-fmk) clearly raised Noxa protein levels, and this in fact still occurred in

the knock-down lines. Ramos FSA cells expressing two types of shRNA, exhibited a

decreased sensitivity to GSI-1-induced apoptosis compared to the mock transduced

cells, similar to results obtained with bortezomib (Fig. 7B).The effects of Noxa shRNA

on GSI-1-mediated cell death were reproducible in other cell lines, Mec-1 and Jurkat

(Fig. 7C&D). Consistent with previous results23, no effect of Noxa protein reduction was

observed on apoptosis triggered via other pathways, such as fl udarabine treatment

or triggering of the CD95 receptor (Fig. 7E). In all cases resistance to GSI-1-mediated


67apoptosis was not complete, most probably due to the fact that remaining levels of

Noxa protein in the knock-down lines were still augmented by GSI-1 (Fig. 7A). In

summary, these data demonstrate that decreased expression of Noxa has a direct

and specifi c effect on the susceptibility to apoptosis induced by GSI-1.

A

C

B

D E

Figure 7: Knockdown of Noxa with RNAi

specially prevents apoptosis induction

by GSI-1.

(A) Ramos FSA-M (=Mock, lane 1-4),

Ramos FSA-N7 (lane 5-8) and Ramos

FSA-N8G10 cells (lane 9-12) were treated

with the pan-caspase inhibitor zVAD-fmk

(100 µM) and GSI-1 (1µM) as indicated.

Noxa protein levels were monitored by

Western blotting. Noxa knockdown in Mec-

1 cells and Jurkat T (J16) cells has been

described previously 18;23. Equal protein

loading is shown by reprobing for β-actin.

(B) Mock ( ), N7 (▼) and N8.G10 ( )

Ramos FSA cells were incubated for

24 hours in the presence of indicated

concentrations of GSI-1 and bortezomib.

Viability was assessed by MitoTracker

staining and FACS analysis. Data

represent mean ± SD from 3 independent

experiments, and one experiment with N7

and bortezomib.

(C) Mock ( ) and N8 ( ) transduced Mec-

1 cells were incubated and analyzed as in

panel B. Data represent mean ± SD from

6 independent experiments for GSI-1 and

1 experiment for bortezomib treatment.

(D) Mock ( ) and N8 ( ) transduced J16

cells were incubated for 24 hours in the

presence of indicated concentrations of

GSI-1 and analyzed as in panel B. Data


experiments.

(E) Mock ( ) and N8.G10 ( ) transduced

Ramos FSA cells were incubated for

24 hours in the presence of 100 µM

fl udarabine (F) and 0.25 µM α-CD95, and

analyzed as in panel B. Medium = M. Data


experiments.

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68 DISCUSSION

The major fi ndings of our study are that GSI-1 is a potent inducer of apoptosis in CLL

and that this response does not involve Notch signaling. Rather, effi cient inhibition of

proteasomal activity and subsequent Noxa accumulation seems to play a key role.

Dysregulation of the Notch pathway has been suggested in CLL, mainly because of

the observation that overexpression of CD23 in CLL cells is regulated by Notch23.

In agreement with other reports3;7 we found that Notch1 and Notch2 receptors are

present in CLL cells. To explore a possible functional role of the Notch pathway in

CLL we performed two kinds of experiments. First we co-cultured primary CLL cells

with either L cells or OP9 cells expressing the Notch ligands DL1 and Jagged1.

This did not affect cell survival in CLL cells. To exclude that this observation is due

to already optimal autocrine or paracrine Notch stimulation in CLL we performed

a set of experiments aimed at blocking Notch signaling. To this end we used the

inhibitors DAPT21 and GSI-120, both assumed to specifi cally block γ-secretases.

Here we encountered a surprising dichotomy: whereas DAPT was unable to induce

apoptosis in CLL, it did inhibit pDC development in the presence of the Notch ligand

DL1, as described previously26. In contrast, this was not the case for GSI-1, which

was capable of potent induction of apoptosis in CLL. A similar dichotomy has been

decribed with respect to Notch2 dependent CD23 upregulation. Treatment of CLL

cells with proteasome inhibitors led to inhibition of nuclear Notch2 intracellular domain

(Notch2IC) activity and downregulation of CD234. However, CD23 down-regulation

could not be achieved with DAPT, as we have also observed. With respect to CD23

down-regulation we could confi rm that this is the result of proteasome- rather than

Notch inhibition (Fig. 6). CD23 is a known transcriptional target of NF-κB32, and

NFκB-p52 constitutively binds to the CD23 promotor region in murine cells33. Thus, a

possible explanation for the down-regulation of CD23 by proteasome inhibitors could

be their well-known negative effect on NF-κB signaling34. However, in experiments

using specifi c inhibitors of NF-κB signaling35 we observed no infl uence on the

expression of CD23 (data not shown).

One of the best characterized Notch targets is the HES family of transcription factors5.

The majority of the CLL patients display substantially lower HES-1 transcripts

compared to normal B cells7. In agreement, we could detect low HES-1 in two CLL

samples, which could further be decreased by DAPT but not signifi cantly by GSI-1

(Fig. 3B). Our data agree with previous studies showing Noxa upregulation and

apoptosis upon GSI in melanoma cell lines, melanoma xenografts and myeloma

cells9;14. To our knowledge, no data are available that would directly demonstrate

repression of Noxa by HES-1.


69In CLL, GSI-1-induced apoptosis was accompanied by rapid and extensive

accumulation of Noxa protein, due to blocking of proteasomal function, similar to

earlier fi ndings in CLL and other cell types that proteasome inhibitors like bortezomib

and MG-132 effectively block the proteasome and upregulate Noxa protein15. Effects

of GSI-1 on BH3-only proteins Noxa and Bim in melanoma cells14, were apparently

also regulated at transcriptional level14;36. The latter is clearly not the case in CLL,

since Noxa mRNA levels do not increase upon treatment with bortezomib or GSI-1. In

support of a direct role for Noxa accumulation, Noxa shRNA experiments demonstrated

a clear reduction in apoptosis mediated by GSI-1, whereas other apoptosis pathways

were unaffected.

The demonstration that GSI-1 acts as a proteasome inhibitor is perhaps not surprising

since the structure of GSI-1 (Z-LLNle-CHO) resembles that of other chymotrypsin

inhibitors and also that of MG-132 (z-LLL-CHO). Others have also remarked that the

aldehyde group on the GSI-1 peptide is probably able to covalently inhibit certain

serine protease20. Our data agree with that view and we propose that many, if not all,

of the apoptosis-inducing activities ascribed to GSI-1, and related compounds such

as GSI-12 (Z-IL-CHO)9 are probably due to proteasome rather than Notch inhibition.

An intriguing remaining question concerns the mechanism behind the rapid and

massive upregulation of Noxa protein by proteasome inhibition. Accumulation of

higher molecular weight species of Noxa, indicative of (poly)ubiquitination, were

never observed by us. Also, efforts to detect ubiquitination of Noxa in cells transiently

transfected with differentially tagged Noxa and ubiquitin, and treated with proteasome

inhibitors, were unsuccesfull (data not shown). A possible explanation for this might

be that Noxa accumulation occurs because its binding partner Mcl-1 is ubiquitinated

and degraded37;38, whereby Noxa dissociates and passively accumulates. Support

for such a chaperone-like role for Noxa in controlling the degradation of Mcl-1 has

indeed been obtained39.

In CLL, new therapies that-act independently of p53 are urgently required. We observed

that GSI-1 is effective in CLL cells from p53 dysfunctional patients, and a variety of

compounds related to GSI are already in the clinic for the treatment of Alzheimer’s

disease40. In that setting, side effects are limited41;42. Bortezomib has proven effi cacy

as single agent in multiple myeloma and some forms of non-Hodgkin’s lymphoma43-

45, although a number of toxicities are described46;47. For unknown reasons, clinical

trials with bortezomib in patients with fl udarabine-refractory CLL showed only limited

responses although biological activity was observed48. We propose that our studies

provide a basis to continued studies into alternative proteasome inihibitors such as

GSI-1 or related compounds as potential treatment for chemoresistant CLL.

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70 ACKNOWLEDGEMENTS

We thank René Spijker for technical assistance, and the patients for their blood

donations. We thank Bianca Blom for critical reading of the manuscript, and

acknowledge Dr. Kramer, Dr. Wittebol and Dr. Baars for including CLL patients in this

study.


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30. Gomez-Bougie P, Wuilleme-Toumi S,

Menoret E et al. Noxa up-regulation

and Mcl-1 cleavage are associated

to apoptosis induction by bortezomib

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31. Sun XM, Butterworth M, MacFarlane

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34. Nencioni A, Grunebach F, Patrone

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Proteasome inhibitors: antitumor

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C et al. Phase II clinical experience

with the novel proteasome inhibitor

bortezomib in patients with indolent

non-Hodgkin’s lymphoma and

mantle cell lymphoma. J.Clin.Oncol.

2005;23:676-684.

46. Lonial S, Waller EK, Richardson

PG et al. Risk factors and kinetics

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with bortezomib for relapsed,

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47. Richardson PG, Briemberg H,

Jagannath S et al. Frequency,

characteristics, and reversibility of

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of advanced multiple myeloma

with bortezomib. J.Clin.Oncol.

2006;24:3113-3120.

48. Faderl S, Rai K, Gribben J et al. Phase

II study of single-agent bortezomib

for the treatment of patients with

fl udarabine-refractory B-cell chronic

lymphocytic leukemia. Cancer

2006;107:916-924.

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et al. Differential regulation of noxa in

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ligand regulates access of MCL-1 to its

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5608.

38. Zhong Q, Gao W, Du F, Wang X. Mule/

ARF-BP1, a BH3-only E3 ubiquitin

ligase, catalyzes the polyubiquitination

of Mcl-1 and regulates apoptosis. Cell

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39. Czabotar PE, Lee EF, van Delft MF

et al. Structural insights into the

degradation of Mcl-1 induced by BH3

domains. Proc.Natl.Acad.Sci.U.S.A

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Hideshima T, Anderson KC.

Proteasome inhibition in the treatment

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74


chapter

4Differential Noxa/Mcl-1 balance in peripheral versus lymph node chronic lymphocytic leukemia cells correlates with survival capacity

Laura A. Smit1,4, Delfi ne Y.H. Hallaert2,3,4, René Spijker2,3, Bart de Goeij3, Annelieke

Jaspers2,3, Arnon P. Kater2, Marinus H.J. van Oers2, Carel J.M. van Noesel1, and

Eric Eldering2,3

1Dept. of Pathology, AMC, Amsterdam, the Netherlands

2Dept. of Hematology, AMC, Amsterdam, the Netherlands

3Dept. of Experimental Immunology, AMC, Amsterdam, The Netherlands

4These authors contributed equally to the manuscript

Blood, 2007; 109 (4): 1660-1668.


76 ABSTRACT

The gradual accumulation of chronic lymphocytic leukemia (B-CLL) cells is presumed

to derive from proliferation centers in lymph nodes and bone marrow. To what extent

these cells possess the purported anti-apoptotic phenotype of peripheral B-CLL cells

is unknown. Recently, we have described that in B-CLL samples from peripheral

blood, aberrant apoptosis gene expression was not limited to protective changes but

also included increased levels of pro-apoptotic BH3-only member Noxa. Here, we

compare apoptosis gene profi les from peripheral blood B-CLL (n=15) with lymph node

B-CLL (>90% CD5+/CD19+/CD23+ lymphocytes with Ki67+ centers; n=9). Apart from

expected differences in Survivin and Bcl-XL, a prominent distinction with peripheral

B-CLL cells was the decreased averaged level of Noxa in lymph nodes. Mcl-1 protein

expression showed a reverse trend. Noxa expression could also be reduced in vitro

by CD40 stimulation of peripheral blood B-CLL. Direct manipulation of Noxa protein

levels was achieved by proteasome inhibition in B-CLL and via RNAi in model cell

lines. In each instance, cell viability was directly linked with Noxa levels. These data

indicate that suppression of Noxa in the lymph node environment contributes to the

persistence of B-CLL at these sites and suggest that therapeutic targeting of Noxa

might be benefi cial.


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77INTRODUCTION

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by a progressive

accumulation of monoclonal CD5+ CD23+ mature B cells in the secondary lymphoid

tissues, bone marrow, and blood1. Previously, it was assumed that B-CLL is

associated with a defective regulation of programmed cell death (apoptosis), rather

than uncontrolled cell proliferation2. Indeed, high expression of the anti-apoptotic

proteins Bcl-2 and Mcl-1 has been associated with rapid disease progression and

a poor response to chemotherapy3;4. Paradoxically, investigation of virtually all

direct apoptosis regulators known at present revealed that, in addition to these anti-

apoptotic alterations, the pro-apoptotic proteins Noxa and Bmf are also abundantly

expressed in B-CLL5;6. How the elevated expression of these pro-apoptotic proteins

is associated with the reputed increased life span of the B-CLL cells is currently

unknown.

The vast majority of the circulating B-CLL cells is arrested in G0/G1 phase of the cell

cycle7, which has contributed to the view that B-CLL is an indolent disease. However,

isotopic labeling of leukemic cells in vivo revealed that a substantial fraction of the

B-CLL cells does proliferate8. It seems logical to assume that the generation of new

cells takes place in so called proliferation centers frequently found in lymph nodes

and bone marrow of B-CLL patients. This is supported by the numerous Ki67+ and

Survivin+ cells present in these structures1;9. The microenvironment not only plays an

essential role in the induction of proliferation but presumably also in the suppression

of apoptosis. In vitro experiments revealed that various cell types can support the

survival of B-CLL cells. Apart from follicular dendritic cells (FDC), bone marrow

stromal cells, IL-6 producing endothelial cells, VCAM-1 and SDF-producing nurse-

like cells, CD4+ T cells can also aid in providing a microenvironment where B-CLL

cells can survive and proliferate10-14. The importance of the microenvironment for

the survival of B-CLL cells is also shown by the fi nding that despite the relentless

accumulation of the B-CLL cells in vivo, culturing the leukemic cells in vitro results in

spontaneous apoptosis15;16. In vitro culture of B-CLL cells in the presence of CD40L

rescues the cells from spontaneous and drug-induced apoptosis, suggesting that

such co-stimulatory signals play a role in the survival of B-CLL cells in vivo and even

in the response to treatment9;17-19.

To date, B-CLL is an incurable disease. Although multi-agent treatment can result

in a profound peripheral lymphocyte depletion, the B-CLL cells in the bone marrow

and/or lymph nodes are less effectively targeted20. Persistence of B-CLL cells in

the bone marrow is associated with an increased risk of relapse21. Therefore, more

molecular data about the B-CLL cells in the lymphoid tissues and bone marrow are


78necessary, preferably coupled with assessment of effi cacy of therapeutics towards

B-CLL residing in those niches. We here initiated such an effort by comparing a large

panel of apoptosis regulators in circulating B-CLL cells and B-CLL cells residing in

lymph nodes. Although the expression of most apoptosis regulators was remarkably

comparable, a prominent difference was the expression of the BH3-only protein Noxa.

Furthermore, we demonstrate that CD40 engagement of peripheral B-CLL cells can

largely reproduce the altered apoptosis profi le found in lymph node B-CLL cells.

Finally, we show that in vitro manipulation of Noxa expression has a signifi cant and

direct effect on B-CLL cell survival. Together, these data provide a new link between

the anti-apoptotic microenvironment in the lymph nodes and suppression of Noxa,

which suggests that drugs that increase Noxa levels, such as proteasome inhibitors22-25,

may be of therapeutic benefi t in B-CLL.

MATERIAL EN METHODS

Patient material and cell lines

Patient material was obtained after routine diagnostic or follow-up procedures at

the departments of Hematology and Pathology of the Academic Medical Center

Amsterdam. All patients were diagnosed according to the WHO classifi cation system1.

Lymph node (LN) material diffusely infi ltrated by B-CLL cells was freshly frozen in

liquid nitrogen directly after surgical removal. Immuno-histochemical analysis (see

below) of these lymph nodes revealed that more than 90% of the tissue consisted

of tumor cells. Peripheral blood (PB) mononuclear cells (PBMC) of B-CLL patients

were obtained after Ficoll density centrifugation (Pharmacia Biotech, Roosendaal,

the Netherlands). PBMCs from B-CLL patients contained >75% CD5+, CD19+ cells

as assessed by fl ow cytometry and were stored in liquid nitrogen as cell suspensions

in 10% DMSO (Merck, Darmstadt, Germany) in heat-inactivated FCS (Invitrogen,

Breda, The Netherlands). Clone FSA of the Burkitt’s lymphoma cell line Ramos with

enhanced response to CD95 has been described previously24. Cell lines were cultured

in Iscove’s modifi ed Dulbecco’s medium (IMDM; Invitrogen), supplemented with 10%

(v/v) heat-inactivated FCS (ICN Biomedicals GmbH, Meckenheim, Germany), 100 U/

ml penicillin, 100 μg/ml streptomycin and 5 mM L-glutamine (Invitrogen).This study

was conducted in accordance with the ethical standards in our institutional medical

committee on human experimentation, as well as in agreement with the Helsinki

Declaration of 1975, revised in 1983.


79RNA isolation and reverse transcription-multiplex ligation-

dependent probe amplifi cation assay (RT-MLPA)

Total RNA was isolated using the Nucleospin RNA isolation kit (Macherey-nagel,

Düren, Germany). RT-MLPA procedure was performed as described previously5;25.

Briefl y, 100 ng total RNA was reverse transcribed using a gene-specifi c probe mix.

The resulting cDNA was annealed overnight at 60°C to the MLPA probes. Annealed

oligonucleotides were covalently linked by Ligase-65 at 54°C (MRC, Amsterdam, The

Netherlands). Ligation products were amplifi ed by polymerase chain reaction (PCR;

33 cycles, 30 seconds at 95°C, 30 seconds at 60°C and 1 minute at 72°C) using

one unlabelled and one 6-carboxy-fl uorescein (FAM)-labeled primer (10 pM). PCR

products were run on an ABI 3100 capillary sequencer in the presence of 1pM ROX

500 size standard (Applied biosystems, Warrington, UK). Results were analyzed

using the programs Genescan analysis and Genotyper (Applied Biosystems).

Category tables containing the area for each assigned peak (scored in arbitrary

units) were compiled in Genotyper and exported for further analysis with Microsoft

Excel spreadsheet software. Data were normalised by setting the sum of all signals

at 100%, and expressing individual peaks relative to the 100% value.

Immunohistochemistry

Monoclonal antibodies specifi c for CD5 (clone 4C7 Lab vision, Neomarkers, Fremont,

CA), CD3 (clone SP7), CD23 (clone 1B12), Bcl-6 (clone PG-B67), were used on

formalin-fi xed paraffi n-embedded lymph node specimens. When necessary, antigen

retrieval was achieved using a TRIS-EDTA buffer pH 9.2. Antibody detection was

performed with the Powervision+ system (ImmunoVision Technologies, Daly City, CA)

which was succeeded, for the single antibody staining, by peroxidase visualization

with 3,3’-diaminobenzidine (DAB) (Sigma), 0.03% H2O

2 in Tris-HCl pH 7.6. Finally, the

sections were counterstained with haematoxylin, dehydrated and mounted in pertex.

For the CD3/Ki67 double stainings the Ki67 MIB-1 clone (Dako, Glostrup, Denmark)

was used, for the CD20/Ki67 double staining the Ki67 SP6 clone (Neomarkers),

and the L26 clone from Dako. After antibody detection with the Powervision+

system (ImmunoVision) the Liquid permanent red kit (Dako) was used, followed by

peroxidase visualization with DAB (Sigma). Finally the slides were counterstained

with haematoxylin and mounted in Vectamount.

Flow cytometry

Purifi ed B-CLL cells were incubated FITC- or PE-conjugated mAbs directed against

CD5 (Sanquin), CD19 (Sanquin) and CD3 (Becton and Dickinson, San Jose, CA) and

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80analyzed by fl ow cytometry with the CellQuest program on a FACS Calibur (Becton

and Dickinson).

In vitro CD40 stimulation

B-CLL samples were enriched to >95% purity from PBMCs via negative depletion as

described previously26. In brief, T cells, monocytes and granulocytes were depleted

using anti-CD3, anti-CD14 and anti-CD16 immunomagnetic beads on a magnetic

particle concentrator (Dynal A.S. Oslo, Norway). The B-CLL cells were stimulated for

three days in culture-treated 24-wells plates (Costar, Corning NY, USA). Each well

contained 5 x 106 B-CLL cells and 1.5 x 105 irradiated (30 Gy) CD40L-transfected or

untransfected fi broblast (NIH3T3).

Retroviral constructs and transduction

To knock-down Noxa, pRetro-super was used, which contains the polymerase III H1-

RNA promoter (pol3) for transcription of the siRNA probe and the phosphoglycerin

kinase (pgk)1 promoter driving GFP expression27. The siRNA sequences were: N7

5’GAAGGTGCATTCATGGTG3’ and N8 5’GTAATTATTGACACATTTC3’. The retroviral

plasmids were transfected into the helper virus amhotropic producer cell line Phoenix

with Fugen-6 (Roche Diagnostics, Almere, The Netherlands). For transduction,

Ramos-FSA cells were exposed overnight to viral supernatant (containing vector

GFP-only or one of the two Noxa RNAi-targeting sequences) on retronectin-coated

(Takara Shuzo, Otso, Japan) 24-well plates. GFP-positive cells were sorted using a

FACS-Aria (BD Biosciences) cell sorter to >90% purity for further experiments.

Analysis of apoptosis

PB B-CLL cells were stimulated at a concentration of 5x106 cells/ml with 20 nM

bortezomib (Janssen-Cilag, Tilburg, The Netherlands) for four hours. The cells were

washed twice with IMDM (when indicated) and incubated at given time points with

FITC-labeled Annexin-V (IQ products, Groningen, The Netherlands) for 20 minutes.

Prior to analyses, PI was added (fi nal concentration 5 µg/ml). Viable cells were

defi ned by Annexin V-/PI- staining. Ramos.FSA clones expressing either control-GFP

or Noxa-RNAi (N7 or N8) were stimulated at a concentration of 5x105 cells/ml with

30 nM bortezomib for 24 hours, harvested and incubated with 200 nM MitoTracker

Orange (Molecular Probes, Leiden, The Netherlands) for 30 minutes at 37°C,

washed and double-stained with APC-labeled Annexin-V (IQ products). Fludarabine,

staurosporine and propidium iodide (PI) were purchased from Sigma Chemical Co.

(St. Louis, MO, USA). Anti-human Fas10 (agonistic antibody to the CD95 receptor)

were a kind gift from Prof. Dr. L. Aarden (Sanquin, Amsterdam, The Netherlands).


81Western blotting

Western blotting was done as described previously5. Protein samples were separated

by 13% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)

followed by western blotting. Blots were probed with the following antisera: polyclonal

Mcl-1 (cat. no 554103, Pharmingen, BD Biosciences), monoclonal anti-Noxa (clone

114C307.1, Imgenex, San Diego, CA, USA), monoclonal anti-Bim (clone 14A8,

Chemicon, Temecula, CA, USA) and antiserum to -actin (clone I-19, Santa Cruz

Biotechnology, Inc., Santa Cruz, CA, USA). Protein bands were quantifi ed using high

resolution (1200 dpi) scanned images of exposed fi lms and AIDA image analyzer

software v3.5 (Raytest Gmbh; Straubenhardt, Germany). Exposed fi lms were only

considered when software indicated that bands were not overexposed. In each

sample, background corrected intensity of Mcl-1 or Noxa bands were normalized for

actin.

Statistical analyses

The Mann Whitney U test was used to analyze if differences in gene expression

between the PB and LN B-CLL were statistically signifi cant. P-values < 0.01 were

considered statistically signifi cant. Densitometric scans of western blots and MLPA

analyses of CD40-triggered CLL cells were analyzed with Student’s T-test. P-values

<0.05 were considered statistically signifi cant.

RESULTS

Patients characteristics and immunohistochemistry

Lymph nodes from 9 B-CLL patients and peripheral blood samples from 15 B-CLL

patients were included in the study. From 2 patients (B-CLL25 and B-CLL31) both

lymph node (LN) tissue and a peripheral blood (PB) sample was available (Table 1).

All B-CLL expressed CD5, CD23 and CD19/CD20. The B-CLL cells of the patients

with LN involvement expressed unmutated immunoglobulin heavy chain (IgVH)

genes. Of the peripheral blood B-CLL, 10 expressed mutated IgVH genes and 8

unmutated IgVH genes (Table 1). In the peripheral blood samples, at least 75% of

the leucocytes were lymphocytes. Due to low levels of CD5 expression, the standard

FACS gating yielded low percentage of CD5/CD19+ cells in some patients; Patient

30 had in addition low numbers of circulating cells and was fi rst diagnosed as small

lymphocytic leukemia (SLL). Immunohistochemistry demonstrated that >90% of the

LNs consisted of leukemic lymphocytes. Ki67+ cells were present in all LNs, either

diffusely or in proliferation centers. These cells were of B cell origin, as demonstrated

by double staining which showed that all Ki67+ cells were also CD20+. In contrast, the

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82scattered CD3+ T cells were generally negative for Ki67. Absence of clusters of Bcl-6+

or CD21+ (data not shown) cells excluded the presence of germinal center remnants

in these LNs (Fig.1).

Figure 1: Histology of lymph node infi ltrated by B-CLL cells. See color fi gures.

Ubiquitously present B-CLL cells were positive for CD23 and CD5. Scattered CD3+ T cells were present

throughout the LN. The absence of clusters of BCl-6+ cells excluded the presence of germinal center

remnants in the LNs. Ki67/CD20 and Ki67/CD3 double staining indicate that all cycling Ki67+ cells (pink)

were of CD20+ (brown) origin - see also inset -, while the CD3+ T cells were predominantly Ki67 negative.

Magnifi cation 40x.

Profi ling of apoptosis genes in peripheral blood and LN samples of B-CLL

The relative expression 34 known apoptosis regulators was investigated by RT-

MLPA5;25;28 in PB samples of 13 B-CLL patients, LN samples of 7 B-CLL patients (Fig.

2A) and paired PB and LN samples of 2 B-CLL patients (Fig. 2B and data not shown).

The relative expression of the majority of the investigated genes was remarkably

comparable between the PB and LN samples. We have described previously that,

compared to normal tonsillar B cell fractions, in PB B-CLL several anti- and pro-

apoptosis genes (e.g. Flip, Bcl-2, Noxa and Bmf) are aberrantly expressed5, and this

was also found in LN samples of B-CLL. Interestingly, 3 genes were differentially

expressed in PB B-CLL cells as compared to LN samples (Fig. 2C). In agreement

with previous reports, the IAP family member Survivin was not expressed in any of

the PB B-CLL samples whereas it was clearly expressed in LN B-CLL5;9 (P=0.0005).

Also, the anti-apoptotic Bcl-2-family member Bcl-XL was more abundantly expressed

in the LN samples (P=0.0003). The most striking difference in expression was

observed for the BH3-only member Noxa. As found previously, this apoptogenic gene

is abundantly expressed in PB B-CLL cells5, but its expression was clearly lower in LN


83B-CLL cells (averaged relative expression of 15.6 ± 9.8 in PB B-CLL versus 3.0±1.1

in LN B-CLL; P<0.0001 Fig. 2C). Of note, a difference in Noxa expression was also

observed between the paired PB- and LN samples of an individual patient (relative

expression 9.6 in the PB sample versus 3.4 in the LN sample) (Fig. 2B). Western

blot analyses confi rmed that the differences in Bcl-XL and Noxa mRNA expression

were also present at the protein level. The B-CLL LN samples generally expressed

lower levels of Noxa than the PB B-CLL samples, and the reverse was observed for

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Figure 2: Apoptosis gene expression profi le of B-CLL cells in peripheral blood and lymph nodes.

(A) Relative expression of 34 apoptosis regulators was investigated in 15 PB B-CLL (black bars) and 9

LN B-CLL (grey bars). Results of individual apoptosis regulatory genes are shown as expression relative

to the total signal in the sample, with standard deviation. Non-apoptosis genes included as housekeeping

genes are 2-microglobulin (B2M), Ferritin Light chain (FLT), -glucoronidase (GUS), and poly(A)-specifi c

ribonuclease (PARN).

(B) RT-MLPA data from PB and LN sample of B-CLL-25.

(C) The expression of Noxa, Survivin, Bcl-XL and Mcl-1 in individual patients are depicted as dots. Asterix (*)

indicates statistical signifi cance (P<0.001) of differences in gene expression between PB and LN B-CLL.


84

Figure 3: Comparison of Noxa, Mcl-1 and Bcl-XL protein in PB vs. LN B-CLL.

Protein lysates of 7 PB samples and 6 LN samples were subjected to Western blot analyses.

(A) Blots were stained with antibodies directed against Noxa, Mcl-1 or Bcl-XL, and reprobed with an

antibody against -actin as a loading control. In case of Bcl-XL, aspecifi c staining at the upper cutting edge

of the blot is visible and precluded analysis of the rightmost two samples.

(B) Densitometric scanning was performed, and averaged Noxa/actin and Mcl-1/actin ratios are plotted in

PB and LN samples. Unpaired T-test showed that Noxa ratios were statistically signifi cant (P=0.0026), and

Mcl-1 ratios showed a non-signifi cant trend.

A

B

Bcl-XL (Fig. 3). Comparison of the RT-MLPA data with the Western blot data revealed

a clear correlation between the levels of Noxa mRNA and Noxa protein. As reported

previously, no differences were observed in expression of these apoptosis genes

among IgVH-mutated versus unmutated cases5.

Since Noxa can selectively interact with the anti-apoptotic protein Mcl-1 and this may

infl uence the degradation of Mcl-129, we investigated the expression of this Bcl-2

family member in PB B-CLL and LN B-CLL. Although RT-MLPA showed no difference

in mRNA expression (Fig. 2C), Western blot analyses revealed that in most LN

B-CLL, where Noxa levels were low, Mcl-1 was slightly elevated. Furthermore, the

PB samples that expressed higher levels of Noxa showed a decreased expression

of Mcl-1 (Fig. 3). This is further illustrated by a paired PB/LN protein sample, where

in fact Noxa expression was almost equal, but in this case Mcl-1 protein levels were

clearly higher in the LN compartment. Densitometric scanning of Noxa and Mcl-1

protein levels also showed divergence between LN and PB, of which the differences

in Noxa levels were statistically signifi cant (Fig. 3B). In summary, the majority of the

apoptotic regulators are expressed equally in PB - and LN B-CLL, but a novel and

prominent distinction in Noxa level was found.


85Noxa expression is modulated by CD40 engagement in B-CLL cells

In the LNs the CD40+ B-CLL cells are in close contact with T cells that may express

CD40L2 (Fig. 1). To investigate the effect of this interaction on the expression of

the apoptotic regulators, PB B-CLL samples (n = 11) were co-cultured for 1-5

days with CD40L-transfected or untransfected 3T3 fi broblasts (Fig. 4). As reported

previously, CD40 stimulation resulted in increased expression of Bcl-XL, A1/Bfl -1,

Bid and Survivin9;17;18. Interestingly, in accordance with our fi ndings in the LN B-CLL

cells, CD40L-stimulated PB B-CLL cells also showed a diminished expression of

Noxa (Fig. 4A). The effects were observed after one day of CD40 stimulation and

RT-MLPA performed at day three and day fi ve showed that the expression of the

apoptosis regulators did not alter signifi cantly after that (Fig. 4B). It should be noted

that the levels of Noxa mRNA as compared to t=0 also decreased after culture on

the control 3T3 cells (p=0.019). The reason for this is not known, however a stronger

decline in Noxa levels was consistently observed after CD40 ligation (p=0.004), and

the difference between control and CD40-treated cells was statistically signifi cant

(p=0.016). These differences were further investigated at the protein level for three

patients (see Fig. 4C). Concordant with RT-MLPA analyses, Noxa levels decreased

after 3 days culture in presence of CD40L-expressing cells, and Bcl-XL levels

increased. Mcl-1 protein levels also clearly increased upon CD40-triggering, although

this was not observed via RT-MLPA. So, similar to fi ndings in LN samples (Fig. 3),

Mcl-1 levels were apparently under post-transcriptional control.

A prominent distinction between CD40-stimulated B-CLL and LN B-CLL was found

for expression of the apoptogenic BH3-only protein Bid. In contrast to LN B-CLL,

CD40L- stimulated B-CLL showed a strong and continuous induction of Bid (Fig. 4B).

Thus, the altered gene expression in LNs can be mimicked largely but not entirely by

in vitro CD40 engagement of B-CLL cells.

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86B

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Figure 4: CD40 stimulation of peripheral blood B-CLL results in an apoptosis gene expression

profi le similar to lymph node B-CLL.

(A) Apoptosis gene expression profi le was investigated by RT-MLPA in PB samples of 11 freshly isolated

B-CLL patients without culturing (black bars) and after three days of culturing on either 3T3 cells (grey bars)

or CD40L-transfected 3T3 cells (white bars). Data plus standard deviation are presented as in fi gure 2.

(B) The expression of Bcl-XL, Bfl -1/A1, Bid and Noxa are shown at day 1, 3 and 5 of culturing on 3T3

cells (white triangles) or CD40L-transfected 3T3 cells (black dots). Statistical analysis of day 0 vs. day 1

samples showed that in all cases the CD40L treated values were signifi cantly different (P<0.01). In case

of Noxa, there was also a small but signifi cant decrease for the 3T3 control cells (P=0.019), and a more

pronounced effect for CD40L treated cells (P=0.004; difference between 3T3 and CD40L-treated cells

P=0.0159, indicated by **).

(C) Western blot of t=0 samples in comparison of CD40L treated cells at day 3 for Noxa, Mcl-1 and Bcl-

XL showed that Noxa protein levels decrease while Mcl-1 and Bcl-X increase. For B-CLL sample 226 the

Mcl-1 levels at t=0 were in fact undetectable (see also Fig. 3).


87Bortezomib-induced Noxa upregulation causes apoptosis of PB

B-CLL cells

To establish a functional relationship between Noxa expression levels and apoptosis

sensitivity of B-CLL cells, we made use of recent fi ndings that proteasome inhibitors

rapidly and specifi cally upregulate Noxa22;23;30. To reduce a widespread impact of

proteasome inhibition on protein levels and transcription dependent processes31,

PB B-CLL cells were transiently exposed to bortezomib for 4 hours.The reversible

proteasome inhibitor was then either washed away or incubation was continued. As

expected, a pulse of bortezomib treatment already caused a rise in Noxa protein, and

this was suffi cient to impair survival of B-CLL cells (Fig. 5). Continuous exposure to

bortezomib resulted in a massive increase in Noxa levels that was accompanied by

almost 100% cell death at 48 hrs. Over the course of this experiment, Mcl-1 protein

levels fi rst increased (4 hr timepoint), most likely due to proteasome inhibition, and

then declined when cells went into apoptosis. This decline could be prevented by

blocking caspase activity with z-VAD (data not shown), and is thus in accord with

reports that Mcl-1 is a caspase substrate32;33. Next, we investigated whether the level

of Bim, another pro-apoptotic binding partner of Mcl-134, was also subject to change

upon bortezomib treatment, and might thereby trigger apoptosis. However, Bim levels

were unaffected, both as detected by RT-MLPA (data not shown), and by Western

blotting (Fig. 5A). Thus, pharmacological manipulation of the levels of Noxa protein in

B-CLL cells appeared to be directly related to viability in an in vitro setting.

Figure 5: Noxa upregulation via transient treatment with bortezomib impacts CLL survival.

Freshly isolated peripheral blood B-CLL cells were treated for 4 hrs with 20 nM of the proteasome inhibitor

bortezomib. Cells were then washed and cultured in fresh medium, or incubation was continued.

(A) At the indicated timepoints, cell lysates were prepared and probed for expression of Noxa, Mcl-1, Bim

and Actin protein by Western blot. Indicated below the lanes: untreated (M), bortezomib washed away

after 4 hrs (B+), and bortezomib without washing (B-). The decrease in Mcl-1 levels in bortezomib treated

cells at 24 and 48 hrs could be inhibited by the pan-caspase inhibitor z-VAD (data not shown). Due to

massive cell death after 48 hrs in the presence of bortezomib, these lysates did not yield suffi cient protein

for analysis.

(B) Apoptosis of cells was determined via AnnexinV staining. Spontaneous apoptosis in medium was

approximately 50%, which was increased by bortezomib treatment. Results are representative for 3

separate experiments.

A B

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88Noxa-defi cient cells exhibit resistance to bortezomib-induced cell death

Apoptosis regulatory genes as detected via RT-MLPA were not affected during the

short-term bortezomib treatment in the previous experiments (data not shown). Yet,

it can not be excluded that other genes and proteins besides Noxa that might impact

survival were affected by bortezomib. Therefore, to investigate a direct role for Noxa in

bortezomib-induced apoptosis, we employed a model system. Ramos B cells (clone

FSA)24 were transduced with distinct retroviral constructs encoding Noxa siRNAs (N7

or N8) or control-GFP. GFP-positive cells were sorted and Western blot analysis

revealed a suppression of Noxa-levels to approximately 50-75% compared to the

control-GFP (Fig. 6A). Both Ramos FSA cell lines expressing Noxa RNAi exhibited

a signifi cant resistance to bortezomib-induced apoptosis compared to the mock-

transduced cells (Fig. 6B). The partial resistance to proteasome inhibitor-mediated

apoptosis matched the partial knock-down of Noxa protein. Of note, also in Noxa

RNAi cells, bortezomib treatment caused a rapid increase in Noxa protein (data not

shown), thus explaining that apoptosis still occurred at higher concentration of the

drug. These data are in good agreement with effects of Noxa knock-down in other

celltypes (melanoma, mantle cell lymphoma and T cell leukemia)22;23;30. In addition,

we obtained similar fi ndings with another protease inhibitor (MG132; data not

shown). In contrast, no effect of Noxa protein reduction was observed on apoptosis

triggered via other pathways such as fl udarabine or staurosporin treatment, or

triggering of the CD95 receptor (Fig. 6C). In conclusion, these data demonstrate that

decreased expression of Noxa has a direct and specifi c impact on the susceptibility

to apoptosis induced by proteasome inhibitors. Conversely, the death-inducing effect

of proteasome inhibition observed in B-CLL cells may therefore rely predominantly

on shifts in Noxa expression.


89

DISCUSSION

There is increasing awareness that the B-CLL population in lymphoid proliferation

centers differs fundamentally from the well studied fraction in PB and that this

distinction may have clinical relevance8;35. Here, we present a fi rst direct comparison

of these two populations, focusing on the expression of 34 apoptosis regulatory

genes. Apart from expected differences in proliferation-related genes (Survivin

and Ki67) and anti-apoptotic Bcl-XL, we observed a prominent divergence in the

expression of pro-apoptotic Noxa. Previously we described that, compared to non-

malignant tonsil or peripheral B-cell fractions, B-CLL cells in the periphery display

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Figure 6: Noxa reduction via RNAi specifi cally

prevents apoptosis induction by proteasome

inhibitors.

Ramos Burkitt lymphoma cells were retrovirally

transduced with two RNAi constructs targeting

Noxa (N7 or N8), or GFP control.

(A) Western blot demonstrating reduced Noxa

expression in Ramos-N7 and -N8. Equal protein

loading is shown by reprobing for β-Actin.

(B) Mock, N7 and N8 transduced Ramos FSA

cells were cultured 24 hours in the presence of in-

dicated concentration of bortezomib. Viability was

assessed by AnnexinV/mitotracker staining and

FACS analysis. Data represent mean ± SD from

three independent experiments.

(C) Cells were incubated for 24 hrs in medium

containing 100 µM fl udarabine (fl uda), 0.25 µM

staurosporine (stauro), or 5 µg/ml α-CD95, and

analysed as in B.

A

B

C


90signifi cantly increased levels of this BH3-only member of the Bcl-2 family, in a p53-

independent manner5. The high levels of Noxa and another BH3-only member Bmf6,

contrasted with the purported anti-apoptotic phenotype of B-CLL cells2 but remained

functionally unexplained. Our new fi ndings show that the Noxa level is considerably

lower in LN CLL and that this is linked with survival capacity. Therefore, targeting

Noxa expression or function could be of clinical benefi t, also in p53 defi cient cases.

In vitro CD40 stimulation of PB B-CLL cells resulted in a clear reduction of Noxa

expression. Within the LN microenvironment, CD40 stimulation is most likely

delivered by CD40L+ T cells. Several groups have investigated the effects of in

vitro CD40 engagement in B-CLL cells9;14;17;18;36-39 but an effect on Noxa expression

was not yet reported. It is well known that CD40-stimulated B-CLL cells are more

resistant to spontaneous or drug-induced apoptosis. This is most probably due to the

induction the transcription factor nuclear factor κB (NFκB) and as a consequence, the

expression of various anti-apoptotic genes, such as Bcl-XL, cIAP2, A20 and Flip36.

Previously, Noxa was proposed to be a p53-response gene40, but in B-CLL cells,

Noxa is apparently not under control of p53, as illustrated by the clearly divergent

expression of Puma and Noxa upon p53 stimuli5;41. Later, various transcription factors

were proposed to regulate Noxa such as E2F1, p73 and hypoxia inducible factor

HIF-1α42-45. Therefore at present it is diffi cult to defi nitely assign a specifi c signaling

route that mediates Noxa expression. Very recently though, it was reported that HIF-

1α is overexpressed in peripheral B-CLL cells45, which may constitute a potential link

to the increased Noxa levels in B-CLL.

Although CD40 stimulation of PB B-CLL cells resulted in a similar apoptosis gene

expression profi le to LN B-CLL, several genes deviated from this profi le, most

prominently Bid, as reported previously17, but also A1/Bfl -1. This indicates that in the

LN, B-CLL cells also receive other stimuli than CD40. Indeed, apart from CD4+ T

cells expressing CD40L, other cell types can support survival of B-CLL cells. In vitro

culture with an FDC cell line or dendritic cells protects B-CLL cells from spontaneous

apoptosis14;46. FDC-mediated survival was reported to depend on the expression of

the Bcl-2 family member Mcl-114 and in vitro experiments revealed that Mcl-1 levels

decline in B-CLL cells undergoing apoptosis3;47. Interestingly, recent data indicate

that Mcl-1 is a preferred binding partner of Noxa34, and we have indeed observed

association of Mcl-1 with Noxa in primary B-CLL samples (D. Hallaert; manuscript

in preparation). Furthermore, in 293T cells, Noxa has been described to mediate

the degradation of Mcl-129. If this mechanism also holds true for B-CLL cells, it may

explain the increase in Mcl-1 protein we observed in LN B-CLL, which was not

accompanied by an increase in Mcl-1 mRNA. Accordingly, augmented Mcl-1 protein

levels are a consequence of the downregulation of Noxa in the LNs, rather than a


91environmental effect on Mcl-1 RNA expression. In addition, in vitro triggering of CD40

on B-CLL cells also infl uenced Mcl-1 levels in a post-transcriptional fashion (Fig. 4).

Thus, the differences in protein levels observed by us for Noxa, Mcl-1 and Bcl-XL

levels in the B-CLL LN environment, correspond with current models based on the

differential interaction potential of these Bcl-2 family members29;34, and support the

anti-apoptosis phenotype of B-CLL cells at this location compared to PB. In addition,

spontaneous apoptosis in vitro of B-CLL cells may be connected with the high levels

of Noxa which eventually saturate the short-lived Mcl-1 protein29;48.

Two separate experimental approaches supported a direct role for Noxa in survival

capacity of B-CLL cells. First, we used the recently discovered rapid and direct effect of

bortezomib on Noxa levels22;23;30 to demonstrate that short term bortezomib exposure

also quickly induced Noxa protein in B-CLL cells, with a corresponding decrease

in viability (Fig. 5). Although the levels of Bim did not change upon bortezomib

treatment, a role for Bim during the actual triggering phase of apoptosis cannot be

excluded. In model systems, Bim is capable of actively triggering Bax activation, while

Noxa functions in as ‘sensitiser’49;50. Secondly, a complementary experiment was

performed in a model system where only Noxa levels were modifi ed via RNAi. Here,

a clear inhibitory effect of Noxa reduction towards apoptosis mediated by proteasome

inhibition was observed, while other apoptosis pathways were unaffected (Fig. 6).

Taken together, our data support a model where the viability of the malignant B-CLL

clone within the LNs and possibly also the bone marrow corresponds with low levels

of Noxa and an upregulation of Bcl-XL and Mcl-1. In addition to these anti-apoptotic

gene expression alterations, the B-CLL cells also receive proliferative stimuli as

indicated by the Ki-67+ and Survivin+ cells. When the B-CLL cells enter the circulation

these stimuli are lost, Noxa is upregulated, and Bcl-XL, Mcl-1 and Survivin are

downregulated. As a result, the B-CLL cells may become prone to apoptosis, which

can however still be prevented by the continuous high expression of Bcl-2. To what

extent circulating B-CLL are actually undergoing apoptosis is diffi cult to detect directly.

Freshly isolated CLL cells are mostly non-apoptotic but undergo rapid ‘spontaneous’

apoptosis in vitro, and recent calculations point to appreciable in vivo death rates8.

Together, this suggests that apoptotic B-CLL cells are rapidly cleared from circulation

in vivo. It is generally assumed that in the LNs and bone marrow the B-CLL cells are

relatively protected against therapeutic drugs20. The circulating B-CLL cells that are

already prone to apoptosis are more easily targeted, but the residual B-CLL cells in

the LN/bone marrow will eventually lead to a relapse. Currently, there is much interest

in application of novel, p53-independent drugs to treat B-CLL51-53. Our data provide

new insight into the regulation of the apoptotic behavior of B-CLL cells, and also

afford new clues for therapeutic intervention by targeting Noxa expression.

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ACKNOWLEDGEMENTWe are grateful to the patients for donating samples and the clinicians involved for

their collaboration. This study was initiated after suggestions from Professor Steven

Pals (Dept of Pathology of the AMC) that investigation into survival and apoptosis of

B-CLL cells should include lymph nodes. The authors would like to thank JBG Mulder

and AR Musler for immunohistochemical stainings.

92


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LN indicates lymph node; PB, peripheral blood; ND, not done.

*Lymphocytes were investigated in the lymph node samples by immunohistochemistry and in the peripheral

blood samples by FACS analysis.

†IgVH mutations were positive if 2% of the IgV

H gene was mutated.

‡These samples were used only for Western blot analyses.

§These samples displayed low CD5 staining; therefore, the combined CD5/CD19 gate yielded low

values.

Patient Location Age,

year

Rai

stage

Lymph,*

%

CD5+CD19+,

%

CD19+,

%

CD3+,

%

IgVH

mutations †

B-CLL24 LN 52 >90 ND ND ND -

B-CLL27 LN 64 1 >90 76 76 10 ND

B-CLL28 LN 80 4 >90 64 93 8 -

B-CLL30 LN 46 3 >90 37# 72 26 -

B-CLL33 LN 76 2 >90 ND ND ND -

B-CLL35 LN 67 2 >90 ND ND ND -

B-CLL36 LN 59 4 >90 93 93 5 -

B-CLL37 PB 62 1 ND ND ND ND +

B-CLL38 PB 62 0 98 97 97 2 +

B-CLL39 PB 72 3 ND 94 98 3 +

B-CLL40 PB 70 3 93 48# 98 1 -

B-CLL41 PB ND 4 94 99 99 1 -

B-CLL42 PB 48 0 69 76 76 17 -

B-CLL43 PB 54 2 91 96 96 4 -

B-CLL44 PB 55 2 86 91 91 0 +

B-CLL45 PB 63 4 82 98 98 2 +

B-CLL46 PB ND ND 82 ND ND ND ND

B-CLL47 PB 54 2 83 ND ND 7 -

B-CLL48 PB 59 2 75 89 89 4 +

B-CLL50 PB 69 0 ND 99 99 3 +

B-CLL167‡ PB 70 0 62 84 86 11 +

B-CLL183‡ PB 78 0 47 94 95 4 +

B-CLL226‡ PB 64 1 70 98 98 1 -

B-CLL261‡ PB 77 4 32 92 92 4 +

B-CLL25 PB 68 2 ND ND 78 13 -

LN 2 >90 82 83 20 -

B-CLL31 PB 72 2 ND 81 81 8 -

LN 2 >90 72 81 12 -

Table 1. Patient and B-CLL sample characteristics.


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98


chapter

5

11

Persistent Mcl-1/Bim protein signature after CD40 ligation in Chronic Lymphocytic Leukemia is associated with specific drug sensitivity

Delfi ne Y.H. Hallaert1,2, Annelieke Jaspers1, Arnon P. Kater1, Marinus H.J. van Oers1,

Eric Eldering2


2Dept. of Experimental Immunology, AMC, Amsterdam, the Netherlands

Manuscript in preparation


100ABSTRACT

Relapse in CLL might originate from a niche where CLL cells proliferate and are

protected from chemotherapeutic drugs. It is therefore important to understand how

CLL cells from lymph nodes (LN) differ from the cells which have moved into the

circulation. To mimic this in vivo LN setting we used CLL cells stimulated via CD40.

The aim of the present study was to monitor expression of apoptosis regulatory genes,

in relation to sensitivity for various types of drugs (fl udarabine, bortezomib, GSI-1 and

roscovitine) during and following a CD40-ligand stimulus. CD40 ligation resulted in

enhanced NF-κB activity and increased expression of target genes Bcl-XL and Bfl -

1. Furthermore, Mcl-1 and BimEL

protein, but not RNA levels, were increased and

decreased respectively. Four days after cessation of CD40L stimulation there was a

dichotomy: NF-κB activity, Bcl-XL and Bfl -1 gene expression gradually declined, but

Mcl-1 and BimEL

protein changes persisted. This was accompanied by reversal of

resistance to drugs, except for roscovitine, a cyclin dependent kinase (CDK) inhibitor,

where the apoptosis-inducing capacity strongly depends on Mcl-1 and Bim. These

data illustrate the long-lasting, non-transcriptional effects of CD40 signals on Mcl-1

and Bim in CLL.


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INTRODUCTION

Chronic lymphocytic leukemia (CLL) remains an incurable disease and although

multi-agent treatment can result in peripheral lymphocyte depletion, the CLL cells in

the protected microenvironmental niches (lymph node (LN), bone marrow (BM) and

spleen) are less effectively targeted. Therefore, it is important to assess the effi cacy

of therapeutics directed towards CLL cells residing in those niches1.

CLL cells in the peripheral blood (PB) are arrested in the G0/G

1 phase of the cell

cycle, and replication occurs in proliferation centers (pseudofollicles). Recently it was

shown that the disease process is more dynamic than previously considered and is

characterized by proliferating as well as dying cells2. Selected microenvironmental

signals delivered by accessory cells, such as follicular dendritic cells (FDCs), BM

stromal cells, IL-6-producing endothelial cells, SDF-producing nurse-like cells, or

CD40 ligand (CD40L/CD154) expressing CD4+ T cells, have been shown to increase

the apoptotic threshold in vitro3-7. CD40L is expressed on activated T cells and plays

an important role in B cell activation, proliferation, isotype switching, normal germinal

centre formation8;9 and normal specifi c antibody responses10;11. In B cells, the response

to CD40 is complex and it is well established that CD40 can support cell survival

through upregulation of the expression of genes encoding antiapoptotic proteins such

as Bcl-XL and Bfl -112-15. The roles of the phosphoinositide 3-kinase (PI3K), mitogen-

activated protein kinase (MAPK), and nuclear factor-kappa B (NF-κB) pathways in

mediating CD40 stimulation are well described16. NF-κB signaling is divided into

two pathways: the canonical pathway and the alternative pathway. Activation of

the canonical pathway proceeds through the degradation of phosporylated IκBα

and subsequent nuclear translocation of active heterodimers (composed of p50,

p65, and/or c-Rel). Activation of the alternative NF-κB pathway results in nuclear

translocation of p52 along with RelB. Translocation of active heterodimers to the

nucleus infl uences the expression of various genes17;18. Studies suggest that CLL

cells may have constitutively activated canonical NF-κB activity, which enhances cell

survival19;20.

The Bcl-2 family of proteins plays an important role in the response to chemotherapeutic

drugs and includes anti-apoptotic and pro-apoptotic members. The Bcl-2 family

members Bax and Bak directly trigger cytochrome c release from the mitochondria.

In turn, this is under the control of the anti-apoptotic Bcl-2 family members (Bcl-2,

Bfl -1, Mcl-1 and Bcl-XL). This balance can be tipped by the pro-apoptotic BH3-only

subgroup of the family21;22.

In a recent comparative ex vivo study of apoptosis regulatory genes and proteins in

neoplastic B cells derived from CLL LN proliferation centers and from PB, we observed

101


specifi c changes in mRNA expression: Bcl-XL and Bfl -1 were upregulated and Noxa

was downregulated. Furthermore, although Mcl-1 mRNA levels were unaltered,

protein expression was upregulated in LN samples14. Studies on Bcl-2 family members

in CLL cells stimulated in vitro with CD40L showed upregulation in mRNA expression

of Bcl-XL, Bfl -1, Bid, and survivin and downregulation of Noxa12-15. Again, Mcl-1 was

clearly increased upon CD40 triggering13;14, on the protein level, but not at the RNA

level. Thus, in vitro CD40L stimulated CLL and ex vivo LN CLL cells displayed a highly

analogous anti-apoptotic phenotype compared to PB CLL. Therefore, in vitro CD40L

stimulation of CLL cells seems to be a useful model to mimic the in vivo LN setting.

Mapping this transit from LN to PB at the molecular level in relation to drug sensitivity

could provide new insights into the effi cacy of chemotherapeutic regimens. We

observed a distinctive trend where reversible transcriptional changes are contrasted

with long-lasting post-transcriptional modulation of Mcl-1 and Bim proteins. Together,

these changes dictate short- and long-term effects with respect to drug sensitivity.


CLL cells

PB from CLL patients was obtained in the setting of routine diagnostic or follow-

up procedures at the department of Hematology of the Academic Medical Center

Amsterdam. Patients had to give informed consent and the study was approved by the

AMC Ethical Review Board. This study was conducted in accordance with the ethical

standards in our institute and in agreement with the Helsinki Declaration of 1975,

revised in 1983. Peripheral blood (PB) mononuclear cells (PBMCs) obtained after Ficoll

density gradient centrifugation (Pharmacia Biotech, Roosendaal, The Netherlands)

were frozen in Iscove’s Modifi ed Dulbecco’s Medium (IMDM) supplemented with

L-Glutamine, 25 mM HEPES (Biowhittaker, Europe) containing, 2mM L-Glutamine

(Invitrogen), 50 mg Gentamycine (Invitrogen) 3.57 x 10-4% (v/v) -mercapto ethanol

(Merck, Darmstadt, Germanyand), 10 % dimethyl sulphoxide (DMSO; Sigma Chemical

Co., St. Louis, MO, USA) and 15% fetal calf serum, (FCS; ICN Biomedicals GnbH,

Mecken heim, Germany), and stored in liquid nitrogen. Expression of CD5 and CD19

(both Bekton Dickinson (BD) Biosciences, San Jose, CA, USA) on leukemic cells

(>90% purity) was assessed by fl ow cytometry (FACScalibur, BD Biosciences) and

analysed with CellQuest software (BD Biosciences).

102


RNA isolation and RT-MLPA

Total RNA was isolated using the GenElute Mammalian Total RNA Miniprep Kit (Sigma

Aldrich). Reverse transcription–multiplex ligation-dependent probe amplifi cation

assay (RT-MLPA) procedure was performed as described previously23;24.

Reagents

The proteasome inhibitor bortezomib was obtained from Janssen-Cilag (Tilburg the

Netherlands). GSI-125 (gamma-secretase inhibitor-1; Z-LLNle-CHO – Cat.nr. 565750)

and cycloheximide were obtained from Calbiochem (Amsterdam, the Netherlands).

Roscovitine and fl udarabine were purchased from Sigma Chemical Co. (St. Louis,

MO, USA).

Analysis of apoptosis, Western blot and antibodies

For apoptosis induction, 5×106 CLL/ml cells were incubated with 100μM fl udarabine

(48 hrs), 30nM bortezomib, 25 μM roscovitine or 5 μM GSI-1 (24 hrs), and stained

with 200 nM MitoTracker Orange (Molecular Probes, Leiden, The Netherlands) for

30 minutes at 37°C and analysed by FACS. Western blotting was performed as

described previously23. Cells were lysed in Laemmli Sample Buffer, and samples

(10-30 μg protein) were separated by 13% sodium dodecyl sulfate polyacrylamide

gel electrophoresis (10% gels for Erk). Blots were probed with the following

antibodies: polyclonal anti-Mcl-1 (catalog no. 554103; Pharmingen, BD Biosciences),

monoclonal anti-Noxa (clone 114C307.1; Imgenex, San Diego, CA), monoclonal anti-

Bim (clone 14A8; Chemicon, Temecula, CA), antiserum to -actin (clone I-19; Santa

Cruz Biotechnology, Santa Cruz, CA), polyclonal anti-Bcl-XL (catalog no. 620211,

BD Biosciences), polyclonal anti-Bcl-2 (catalog no. 210-701-C100, Kordia, Leiden

the Netherlands) polyclonal anti-phospho-Erk (catalog no. #9102 Cell Signaling),

polyclonal anti-Erk (catalog no. #9101 Cell Signaling), monoclonal anti-phospho-IκBα

(clone 5A5, Cell Signaling), polyclonal anti-IκBα (catalog no. #9242, Cell Signaling),

polyclonal anti-p100/p52 (catalog no. #4882, Cell Signaling), polyclonal anti-phospho-

GSK-3β (sc-11757, Santa Cruz Biotechnology) and polyclonal anti-GSK-3β (clone

1H8, Santa Cruz Biotechnology).

In vitro CD40 stimulation

CLL cells were stimulated by co-culture with fi broblasts (NIH3T3) that had been

stably transfected with a plasmid encoding human CD40L (3T40L). Fibroblasts

were irradiated (30Gy) and plated in culture-treated 6-wells plates (6x105 cells/well).

CLL cells were thawed and 5x106 cells per well were added (day-3) to the adhered

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fi broblasts in 3 ml IMDM containing 10% FCS and incubated at 37oC. After 3 days

(day 0) CLL cells were gently removed and transferred to new 6-well plates and

incubated for 4 days (day 1-4) in 3 ml IMDM containing 10% FCS. There were no

residual fi broblasts in the transferred CLL cell culture.

Proteasome activity assay

Cytoplasmic extracts (Assay buffer: 250 mM HEPES (pH7.5), 5 mM EDTA, 0.5%

NP-40 and 0.01% SDS) from freshly isolated PBMCs from CLL patients (CD40L

stimulation and time point of lyses is indicated in legend) were used to measure

proteasome activity using a 20S proteasome activity assay kit (Chemicon, part of

Millipore; Billerica, USA) following the manufacturer’s instructions. The assay is based

on detection of the fl uorophore 7-amino-4-methylcoumarin (AMC) after cleavage

from the labeled substrate LVVY-AMC. The free AMC fl uorescence was quantifi ed

using a 380/460-nm fi lter set in a VICTOR2 D fl uorometer (Wallac-PerkinElmer,

Massachusetts, USA). Proteasomal activity was calculated from the changes in

fl uorescence over time and expressed per μg of protein in the extract.

Statistical analysis

The Mann Whitney U test was used to analyze weather differences in proteasomal

activity between control 3T3 and 3T40L stimulated CLL cells at day 0 and day 4

(4 days after cessation of CD40 stimulation) were statistically signifi cant. P values

below 0.05 were considered statistically signifi cant.

RESULTS

The anti-apoptotic expression profi le resets after termination of

CD40 stimulation

CLL cells were co-cultured with non transfected 3T3 cells (control), or with 3T3 cells

stably transfected with a huCD40L-encoding plasmid (3T40L). We evaluated mRNA

expression profi les of different apoptosis genes by means of RT-MLPA14;24 from cells

of CLL patients during, and at different time points after, CD40 triggering. Cells were

stimulated for three days (day -3 to day 0), followed by four days culture in the absence

of CD40 signals (day 1-4). As reported previously, CD40L stimulation resulted in

increased mRNA expression of Bcl-XL, Bfl -1, Bid and Survivin and decreased mRNA

expression of Noxa (Fig. 1; day 0)12-14;26. Expression of Bcl-XL, Bfl -1 and Bid mRNA

declined gradually to pre-stimulation levels after cessation of CD40 stimulation. Noxa

downregulation, however, was not only observed in CLL cells cultured on 3T40L cells

104


Figure 1. Apoptosis gene expression profi le of CLL cells after termination of CD40 stimulation.

Changes in the mRNA expression of Bcl-XL, Bfl -1, Mcl-1, Noxa, Bim, Bid, survivin and GUS were

investigated by RT-MLPA. CLL cells were co-cultured for 3 days on control 3T3 cells (dashed lines) or

CD40L-transfected 3T3 cells (solid lines). After detachment and washing, cells were incubated in medium

for 4 days without stimulation. mRNA expression is shown at day -3 (prior to stimulation), 0 (3 days co-

culture), 1, 2, 3 and 4 (days after termination of stimulation). Data represent mean ± SD from 5 independent

experiments.

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105


A

but also on 3T3 cells and the mRNA levels remained low under both conditions. Mcl-1

and Bim mRNA expression was not altered upon CD40, similar to the housekeeping

gene GUS which showed stable expression throughout the experimental period. In

conclusion, the altered gene expression by in vitro CD40 engagement of CLL cells

was in agreement with previous results12-14;26. Cessation of CD40 stimulation resulted

in a gradual decline to baseline expression.

CLL cells selectively regain chemosensitivity after termination of

CD40 stimulation

Next, sensitivity to different chemotherapeutic drugs was tested, in relation to

106

Figure 2. Apoptosis response after termination of CD40 stimulation in CLL.

(A) CLL cells were co-cultured for 3 days on 3T3 cells (dashed lines) or CD40L-transfected 3T3 cells

(solid lines). After detachment and washing, cells were directly or after 1-4 days of additional CD40L free

culture, incubated with bortezomib (30 nM) or GSI-1 (5 µM) for 24 hours and with fl udarabine (100 µM) for

48 hours. Apoptosis response was analyzed by MitoTracker staining. Data represent mean ± SD from 6

independent experiments.

(B) Apoptosis response upon 24 hours incubation with roscovitine (25 µM) of CLL cells cultured as

described in A. Data represent mean ± SD from 4 independent experiments.

(C) CLL cells were co-cultured for 3 days on 3T3 cells (black bars) or CD40L-transfected 3T3 cells (white

bars). After detachment and washing, cells were incubated with fl udarabine (F) (100 µM) for 48 hours or

roscovitine (R) (25 µM), bortezomib (B) (30 nM) and GSI-1 (G) (5 µM) for 24 hours. Also the following

combinations of drugs were used: bortezomib (30 nM) + roscovitine (25 µM) (B30/R25) or GSI-1 (5 µM) +

roscovitine (25 µM) (G5/R25). M=medium. Apoptosis response was analyzed by MitoTracker staining. %

Apoptosis is shown at day 0 (+/- 3 days CD40 stimulation). Data represent mean ± SD from 6 independent

experiments.


the culture regimen described above. Figure 2 shows that at day 0 CLL cells had

become resistant to all drugs tested: the purine analog fl udarabine, the proteasome

inhibitors bortezomib and GSI-1 (gamma-secretase inhibitor-1), and the CDK inhibitor

roscovitine. Also the combination of roscovitine with bortezomib, GSI-1 (Fig. 2C)

or fl udarabine (data not shown), did not induce apoptosis at day 0. After cessation

of CD40 stimulation CLL cells became gradually sensitive again to fl udarabine,

bortezomib and GSI-1, but not to roscovitine (Fig. 2A&B; day0-4). This surprising

dichotomy was further investigated.

Prolonged changes in Mcl-1 and BimEL protein expression after

termination of CD40 triggering in CLL

Previous research showed that roscovitine induced apoptosis via the Mcl-1/Noxa

axis. Rapid (4 hours) Mcl-1 degradation is the initial event in roscovitine-induced

apoptosis27;28 and the BH3-only proteins Noxa and Bim are involved as specifi c

mediators in this process27.

Changes in Mcl-1, Bcl-XL and Noxa were determined in CLL cells treated for 24

hours with roscovitine or bortezomib at two different time points; after three days of

CD40L stimulation (day 0) and four days after termination of CD40L stimulation (day

4). In accord with previous data12-14;27;28, Mcl-1 and Bcl-XL protein expression was

upregulated after CD40L stimulation at day 0 compared to unstimulated (3T3) CLL

cells (Fig. 3A). At day 4, the levels of Bcl-XL were declined compared to day 0, whereas

Mcl-1 protein expression was still upregulated. Also, Noxa protein expression was

higher after CD40L stimulation at day 0 and 4, in contrast with mRNA levels observed

in fi gure 1. Finally, CD40 stimulation increases proteasomal degradation of BimEL

.

Figure 3B illustrated the decrease in BimEL

protein, which persisted up to day 4.

Concerning drug responses in this setup, roscovitine was functional, as measured

by Mcl-1 degradation, at day 4 but not at day 0. Nevertheless, at both time points

107

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B C


A

B

108

Figure 3. Protein expression of Mcl-1, Bcl-XL, Noxa and Bim after termination of CD40 stimulation

in CLL.

(A) CLL cells were co-cultured with control 3T3 or CD40L-transfected 3T3 cells for 3 days (day 0). After

detachment and washing, cells were directly (day 0) or after 4 days (day 4) of CD40L free culture, incubated

with the indicated drugs (24 hours). Lysates were probed for Mcl-1, Noxa and Bcl-XL as indicated and actin

as loading control.

(B) CLL cells were co-cultured as in A. Lysates were probed for Bim as indicated and actin as loading

control.


roscovitine did not induce apoptosis. Furthermore, as expected bortezomib induced

accumulation of Noxa and Mcl-114;29, at both time points (Fig 3A).

In conclusion, Mcl-1 and Bcl-XL protein expression were increased after CD40

triggering. After termination of CD40 triggering, Bcl-XL expression gradually decreased,

however, Mcl-1 and Noxa remained high. Also the Erk-mediated BimEL

degradation

continued after cessation of the CD40 signal.

CD40 signaling results in NF-κB, in GSK-3β and long-lasting ERK

activation

Various signaling pathways are known to affect levels of Bcl-2 family members. First,

we examined the canonical and alternative NF-κB activity in three CLL samples

stimulated with CD40L. Figure 4A illustrates the phosphorylation of IκBα and the

processing of p100 to p52 at day 0, demonstrating activation of the canonical

and alternative NF-κB pathway upon CD40 triggering. At day 4, activation of the

canonical pathway was decreased to baseline expression in CLL 4&5 and reduced

approximately 50% in CLL 6. The alternative pathway was decreased in CLL 4&6, but

still increased in CLL 5.

Next, it is known that Erk signaling mediates the proteasomal degradation of BimEL

in model systems30;31. Figure 4B shows that Erk is phosphorylated upon CD40

stimulation and this was maintained and even increased at day 4.

Third, Mcl-1 is a known target for phosphorylation and subsequent proteasomal

degradation. Cytokine withdrawal in murine cell lines causes decreased PI3K-Akt/

PKB signaling to activate GSK-3β which in turn phosporylates Mcl-1, thus marking

it for proteasomal degradation32. In CLL, CD40 ligation induced both total and

phosphorylated (inactive) forms of GSK-3β (Fig. 4C).

Taken together, these data demonstrated activation of various CD40L-induced survival

pathways in CLL. After cessation of the CD40 signal, NF-κB in general returns to

baseline, as also indicated by mRNA data (indicated by Bcl-XL and Bfl -1 expression).

Erk remained signifi cantly activated, while GSK-3β appeared persistently blocked.

Proteasomal activity does not account for dysregulated protein

expression after CD40 stimulation in CLL

Next, we tested whether the altered protein profi le (of Mcl-1 and BimEL

) observed

after CD40 stimulation was a consequence of altered proteasomal activity. In fi gure

5 proteasomal activity was measured in CLL cell lysates obtained after 3 days

incubation of the CLL cells with CD40L (day 0; n=13) and four days after cessation

of CD40L stimulation (day 4; n=4). Proteasomal activity was signifi cantly increased

after CD40 stimulation at day 0. This persisted four days after termination of CD40

stimulation.

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109


B

A

C

Figure 4. Activity of NF-κB, Erk and GSK-3β upon CD40 engagement in CLL.

(A) CLL cells were co-cultured with control 3T3 or CD40L-transfected 3T3 cells for 3 days (day0). After

detachment and washing (day 0), cells were incubated for 4 days in medium (day 4). Lysates were probed

for IκBα, p-IκBα and p100/52 as indicated. Shown are representative examples of 3 CLL patients.

(B) CLL cells were co-cultured with control 3T3 or CD40L-transfected 3T3 cells for 3 days (day0). Lysates

were probed for Erk and p-Erk as indicated and actin as loading control. Shown are representative

examples of 3 CLL patients.

(C) CLL cells were co-cultured with control 3T3 or CD40L-transfected 3T3 cells for 3 days (day 0). Lysates

were probed for GSK-3β and p-GSK-3β as indicated. Shown are representative examples of 2 CLL

patients.

110

Figure 5. Proteasomal activity in CLL cells upon

CD40 stimulation.

CLL cells were co-cultured with control 3T3 or 3T40L

cells for 3 days. After detachment and washing (day 0).

CLL cells were incubated for 4 days in medium (day

4). At day 0 (n=13) and 4 (n=4), CLL cells were lysed,

the fl uorogenic proteasome substrate LVVY-AMC was

added and the enzymatic activity of the 20S proteasome

was measured as the amount of AMC fl uorescence per

minute (counts/min). Results are expressed as change

in AMC fl uorescence per minute per μg protein.

P<0.01 with the Mann Whitney U test are indicated by

an asterisk (*).

NS = not signifi cant.


A

BFigure 6. Mcl-1 turnover upon CD40

stimulation.

(A) CLL cells were co-cultured with 3T40L cells for

3 days. After detachment and washing, cells were

incubated with 5 µg/ml cycloheximide. Cells were

harvested at indicated time points (0, 30, 60, 120,

240 and 360 minutes). Lysates were probed for

Mcl-1 and actin as loading control.

(B) The experiment described in A, was quantifi ed

with AIDA evaluation software. Results are

presented as the percentage of Mcl-1 expression

at indicated time points after addition of

cycloheximide (5 µg/ml).

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CD40 ligation does not infl uence Mcl-1 and Bim protein turnover

in CLL

Finally, we examined whether the half-life of Mcl-1 changed upon CD40 ligation.

Therefore, the stability of Mcl-1 was assessed following inhibition of protein synthesis

by treatment with cycloheximide. Previous reports in cell lines showed that Mcl-1

protein was rapidly lost with a half-life of around 30 minutes33;34. In CLL, after CD40

stimulation, the half-life of Mcl-1 was similar (Fig. 6). These results suggest that CD40

ligation does not considerably infl uence the half-life of Mcl-1 in CLL cells.

DISCUSSION

The aim of this study was to mimic the transition of CLL cells as they egress from LN

to PB, and relate this to drug sensitivity. The major fi nding of the present work was

that CD40 stimulation has long-lasting effects on protein expression of Mcl-1 and Bim,

and this was paralleled by prolonged resistance to the CDK inhibitor roscovitine.

In CLL, CD40 ligation affects various signaling pathways with subsequent transcriptional

consequences16;20;35. We observed that both the canonical and alternative NF-κB

pathways were stimulated, and as a result, the NF-κB target genes Bcl-XL and Bfl -


1, were upregulated. This expression signature was accompanied by broad drug

resistance. Termination of CD40 signals resulted in diminished NF-κB activity,

subsequent Bcl-XL and Bfl -1 downregulation and pre-sensitization to proteasome

inhibitors and fl udarabine. Importantly, the resistance to the CDK inhibitor roscovitine

persisted. Recent studies by us, and others, have shown that Noxa, Bim and Mcl-1

are crucial mediators in roscovitine-induced apoptosis27. Therefore, the persistent

increased Mcl-1/Bim protein ratio observed after cessation of CD40 triggering might

mediate the resistance to roscovitine. Remarkably, although roscovitine did initiate

Mcl-1 degradation at day 4, this obviously did not trigger apoptosis. We speculate that

the Mcl-1 reservoir still dominates and thus continuously neutralizes pro-apoptotic

proteins, such as Bim and Noxa. In previous studies we demonstrated the role of Mcl-

1, Bim and Noxa in roscovitine-induced cell death27. It would be interesting; however,

to test the specifi c role of these proteins in the resistance to roscovitine through gene

knockdown or overexpression experiments in CD40 stimulated CLL cells. Additionally,

there might be unknown mechanisms of action of roscovitine, other than the described

fast effect on Mcl-127. In this context, it would be interesting to test the effects of the BH3-

mimetic ABT-73736;37 or similar compounds, in combination with roscovitine, as it has

been shown that roscovitine increased ABT-737 toxicity in human leukemia cell lines38.

Mcl-114 and Bim protein levels in CD40 triggered CLL are under posttranscriptional

control. Both proteins are known targets for phosphorylation and proteasomal

degradation, but via different upstream routes. Activated Erk can mediate BimEL

degradation30;39;40, whereas termination of PKB signaling has been linked with GSK-

3β-mediated Mcl-1 phosphorylation and subsequent degradation32. Since both

phosphorylated Erk and reduced BimEL

protein levels persist after termination of

CD40 signaling (Fig. 3B), it appears likely that this pathway is indeed continuously

active in the CLL cells. The control of Mcl-1 protein levels seems less clear. In the

two CLL samples tested, CD40 stimulation induced phosphorylation of GSK-3 ,

thereby inactivating it. In growth factor withdrawal-mediated apoptosis, this depends

on decreased PKB activity and lead to decreased Mcl-1 turnover32. PKB activity

was however unaffected upon CD40 stimulation in CLL and the PI3 kinase inhibitor

LY294002 did not induce apoptosis (Hallaert, unpublished observation). More

surprisingly, although protein levels were persistently increased, the half-life of Mcl-1

was still quite short (<45 minutes). This value is close to the reported basal half-life

of approximately 30 minutes in other cells33;34. It would seem therefore, that CD40

triggering does not result in a drastically increased half-life of Mcl-1, although direct

comparison with un-triggered CLL cells was not feasible due to very low Mcl-1 levels

in those cells. In view of these observations, another possible control mechanism

may be involved. Other studies suggested translational regulation of Mcl-1 through

112


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113the initiation factor eIF41;42. It is known that phosphorylation eIF2α can repress

translation of Mcl-1 resulting in downregulation of Mcl-1 protein41. Whether Mcl-1

protein expression after CD40 triggering in CLL is also regulated via eIF2α or other

posttranscriptional mechanisms remains to be determined.

Our earlier work has shown that Noxa is highly expressed in circulating CLL levels23,

but is low in LN14;23This can be mimicked to a certain extent by in vitro CD40 signals,

but some inconsistencies concerning Noxa protein levels have come to light in the

present studies. First, prolonged in vitro culture of CLL on 3T40L cells but surprisingly

also 3T3 cells caused persistent lowering of Noxa mRNA as detected by MLPA. In

various CLL samples however, a rise of Noxa protein was observed upon prolonged

CD40 stimulation. Since the regulation of Noxa at transcriptional level and especially

protein level is uncertain43-46, we can only speculate about the underlying mechanism.

First, the rapid suppressive effect of CD40 triggering as well as the slower effect of

prolonged co-culture with 3T3 cells on Noxa mRNA indicates that in vivo circulating

CLL cells lack signals that might suppress this high basal transcriptional rate. In support

of this, the basal Noxa mRNA levels are low in LN niches14. This difference is unlikely

to be due to CD40 signals, since we observed that prolonged in vitro CD40 triggering

can in fact cause a gradual increase in Noxa protein. The underlying mechanism of

this puzzling discrepancy between Noxa mRNA and protein remains unexplained. It

could on one hand be caused by unknown modes of post-transcriptional regulation of

Noxa, or also result from the various forms of regulation of its binding partner Mcl-1.

If the latter were true, the observed changes in Noxa are not an intrinsic property, but

would result merely from passive accumulation as Mcl-1 protein levels change. More

research is required to discriminate among these different options.

Taken together, prolonged CD40 triggering induced not only reversible changes

in transcription patterns, but also seems to have long-lasting effects on post-

transcriptional regulation of Mcl-1 and Bim. Using this model system to mimic the in

vivo LN setting could help to predict responses to new drugs.


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18. Karin M. Nuclear factor-kappaB in

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20. Cuni S, Perez-Aciego P, Perez-Chacon

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21. Chipuk JE, Green DR. How do BCL-2

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2005;65:5399-5407.

29. Perez-Galan P, Roue G, Villamor

N et al. The proteasome inhibitor

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generation of ROS and Noxa activation

independent of p53 status. Blood

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30. Ley R, Balmanno K, Hadfi eld K,

Weston C, Cook SJ. Activation of the

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only protein, Bim. J.Biol.Chem.

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31. Luciano F, Jacquel A, Colosetti P et al.

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on serine 69 promotes its degradation

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32. Maurer U, Charvet C, Wagman AS,

Dejardin E, Green DR. Glycogen

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116


chapter

6c-Abl Kinase Inhibitors Overcome CD40Mediated Drug Resistance in CLL; Implications for Therapeutic Targeting of Chemoresistant Niches

Delfi ne Y.H. Hallaert1,3, Annelieke Jaspers1, Carel J.M. van Noesel2, Marinus H.J.

van Oers1, Arnon P. Kater1, Eric Eldering3


2Dept. of Pathology, AMC, Amsterdam, the Netherlands

3Dept. of Experimental Immunology, AMC, Amsterdam, the Netherlands

Blood: accepted for publication


ABSTRACT

In lymph node (LN) proliferation centers in chronic lymphocytic leukemia (CLL), the

environment protects from apoptotic and cytotoxic triggers. Here, we aimed to defi ne

the molecular basis for the increased drug resistance and searched for novel strategies

to circumvent it. The situation in CLL LN could be mimicked by prolonged in vitro

CD40 stimulation, which resulted in upregulation of anti-apoptotic Bcl-xL, A1/Bfl -1

and Mcl-1 proteins, and afforded resistance to various classes of drugs (fl udarabine,

bortezomib, roscovitine). CD40 stimulation also caused ERK-dependent reduction of

BimEL

protein, but ERK inhibition did not prevent drug resistance. Drugs combined with

sublethal doses of the BH3-mimetic ABT-737 displayed partial and variable effects

per individual CD40-stimulated CLL. The anti-apoptotic profi le of CD40-triggered CLL

resembled BCR-Abl-dependent changes seen in CML, which prompted application

of c-Abl inhibitors imatinib or dasatinib. Both compounds, but especially dasatinib,

prevented the entire anti-apoptotic CD40 program in CLL cells, and restored drug

sensitivity. These effects also occurred in CLL samples with dysfunctional p53.

Importantly, ex vivo CLL LN samples also displayed strong ERK activation together

with high Bcl-xL and Mcl-1 but low BimEL

levels. These data indicate that CLL cells in

chemoresistant niches may be sensitive to therapeutic strategies that include c-Abl

inhibitors.

118


INTRODUCTION

Chronic lymphocytic leukemia (CLL) is a CD5+ B-cell malignancy that is still considered

incurable, although novel treatment combinations of monoclonal antibodies and

chemotherapy1 seem promising. Many patients eventually develop drug resistance

through several pathways including mutation of the p53 tumor suppressor gene, or

involving the gene encoding the ataxia telangectasia mutated (ATM), which is a kinase

required for p53 function. Such genetic lesions are uncommon in CLL at diagnosis,

but increase in frequency as the disease progresses2. Since the cytoreductive

activity of most current chemotherapeutic agents requires functional p53, loss of p53

is associated with drug resistance and poor prognosis3. Because of these aspects,

different agents with p53-independent modes of action are clearly needed.

CLL has been considered a smoldering disease characterized by long-lived tumor

cells arrested in the G0/G

1-phase of the cell cycle and possessing intrinsic apoptosis

defects4. This concept was largely based on analyses of peripheral blood derived

CLL cells. A study of in vivo cellular kinetics however suggested that CLL is a dynamic

disease with substantial proliferation rates as well as increased death rates compared

to normal B-cells5. Prior to this, it was already known that proliferation and especially

increased survival of the malignant B-cells may not result primarily from intrinsic

defects, but appear to depend largely on interactions with microenvironmental

bystander cells. Interactions between CLL cells and follicular dendritic cells, bone

marrow stromal cells, IL-6-producing endothelial cells, SDF-producing nurselike cells,

or CD40L expressing CD4+ T cells cells6-9 have been shown to increase the apoptotic

threshold in vitro. In a recent comparative survey of apoptosis regulatory genes and

proteins in neoplastic B-cells derived from CLL lymph node (LN) proliferation centers

and from peripheral blood10, we observed specifi c changes including increased

expression of anti-apoptotic proteins such as Mcl-1, Bcl-xL and A1/Bfl -1 in LN cells.

Extended cell survival of tumor cells within the LN microenvironment may create an

intracellular milieu permissive for genetic instability and for the accumulation of gene

mutations that favors disease progression. Furthermore, these microenvironmental

interactions may provide a safe haven from cytotoxic anticancer drugs, thus serving

as a tumor reservoir from which relapse occurs (reviewed by Pedersen and Reed11).

This concept is supported by the observation that prolonged CD40 activation, which

to a large extent recapitulates the anti-apoptotic expression profi le of LN derived

CLL cells, renders CLL cells resistant to current chemotherapeutics12;13. The currently

widely applied drug fl udarabine relies on an intact p53 response which induces

expression of the Bcl-2 member Puma, thereby triggering apoptosis14-16. Alternative,

p53-independent drugs such as the proteasome inhibitor bortezomib or the cyclin

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dependent kinase inhibitor roscovitine engage other pro-apoptotic Bcl-2 members

such as Noxa and Bim. Especially Bim is a potent pro-apoptosis member of the BH3-

only subgroup of the Bcl-2 family, engaged by a variety of apoptotic triggers17-20. A

potential means of suppressing the lethal capacity of Bim involves the pro-survival

kinase ERK. In model systems activation of ERK leads to phosphorylation and

subsequent proteasomal degradation of the BimEL

splice variant21;22.

In the present study we used in vitro CD40 stimulation as a model for chemoresistant

LN CLL, and searched for means to circumvent it. CD40 stimulation of CLL cells

strongly induced Bcl-xL, Mcl-1 and A1/Bfl -1 proteins, resulting in a broad drug

resistance. Various aspects of this anti-apoptotic program also occur in chronic

myeloid leukemia (CML), a disease for which current treatment includes kinase

inhibitors that were developed to target BCR-Abl signaling23. Therefore, we next

applied the c-Abl inhibitors imatinib (Gleevec, STI-571) or dasatinib (Sprycel, BMS-

354825) in conjunction with CD40. Both drugs caused a profound reversal of the

protective CD40 effects, and restored drug sensitivity. Probing of LN CLL samples

demonstrated that in these protective niches similar pro-survival signaling pathways

are active as upon CD40 triggering in vitro. Collectively, these data suggest that

CLL cells residing in LN might be therapeutically targeted by drug combinations that

include c-Abl inhibitors.


Patient material

Patient material was obtained after routine diagnostic or follow-up procedures at

the departments of Hematology and Pathology of the Academic Medical Center

Amsterdam. This study was conducted after informed consent and approved by the

AMC medical committee on human experimentation, in agreement with the Helsinki

Declaration of 1975, revised in 1983. Lymph node (LN) material, diffusely infi ltrated

by CLL cells, was freshly frozen in liquid nitrogen directly after surgical removal.

Immunohistochemical analysis of these lymph nodes revealed that greater than 90%

of the tissue consisted of tumor cells10. Peripheral blood (PB) mononuclear cells

(PBMCs) of patients with CLL, obtained after Ficoll density gradient centrifugation

(Pharmacia Biotech, Roosendaal, The Netherlands) were frozen in Iscove’s Modifi ed

Dulbecco’s Medium (IMDM) supplemented with L-Glutamine, 25 mM HEPES

(Biowhittaker, Europe) containing, 2mM L-Glutamin (Invitrogen), 50 mg Gentamycin

(Invitrogen) 3.57 x 10-4% (v/v) -mercapto ethanol (Merck, Darmstadt, Germany), 10

120


% dimethyl sulphoxide (DMSO; Sigma Chemical Co., St. Louis, MO, USA) and 15%

fetal calf serum, (FCS; ICN Biomedicals GnbH, Meckenheim, Germany), and stored

in liquid nitrogen. Expression of CD5 and CD19 (antibodies obtained from Bekton

Dickinson (BD) Biosciences, San Jose, CA, USA) on leukemic cells was assessed by

fl ow cytometry (FACScalibur, BD Biosciences) and analysed with CellQuest software

(BD Biosciences).

RNA isolation and RT-MLPA

Total RNA was isolated using the GenElute Mammalian Total RNA Miniprep Kit (Sigma

Aldrich). Reverse transcription–multiplex ligation-dependent probe amplifi cation

assay (RT-MLPA) procedure was performed as described previously16;24.

Reagents

The proteasome inhibitor bortezomib was obtained from Janssen-Cilag (Tilburg

the Netherlands). The -secretase inhibitor GSI-1, the Erk inhibitor PD-98059, the

NF-κB inhibitor BAY-11-7082 and the proteasome inhibitor MG132 were obtained

from Calbiochem (Amsterdam, the Netherlands). Roscovitine and fl udarabine

(F-Ara-A) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). ABT-

737 was obtained under MTA from Abbott (courtesy Dr S Rosenberg, Abbott Park,

Illinois, USA). The kinase inhibitors, imatinib and dasatinib were from Novartis

(Basel, Switzerland) and Bristol-Myers Squibb (New York, NY, USA) respectively.

Analysis of apoptosis, Western Blot and antibodies

For apoptosis induction, cells at a density of 1.5.106/ml in culture medium were

treated with 100μM fl udarabine (48 hrs), 30nM bortezomib, 25 μM roscovitine or 5

μM GSI1 (24 hrs), and stained with 200 nM MitoTracker Orange (Molecular Probes,

Leiden, The Netherlands) for 30 minutes at 37°C and analysed by FACS. Western

blotting was performed as described previously16. Cells were lysed in Laemmli

Sample Buffer, and samples (10-30 μg protein) were separated by 13% sodium

dodecyl sulfate polyacrylamide gel electrophoresis (10% gels for ERK). To screen for

p53 functionality, cells were irradiated (5Gy) and after O/N incubation tested for the

expression of p53 and p21 by western blot analysis as described before25. Blots were

probed with polyclonal anti-Mcl-1 (catalog no. 554103; Pharmingen, BD Biosciences),

monoclonal anti-Noxa (clone 114C307.1; Imgenex, San Diego, CA), monoclonal anti-

Bim (clone 14A8; Chemicon, Temecula, CA), antiserum to -actin (clone I-19; Santa

Cruz Biotechnology, Santa Cruz, CA), polyclonal anti-Bcl-xL (catalog no. 620211,

BD Biosciences), polyclonal anti-Bcl-2 (catalog no 210-701-C100, Kordia, Leiden

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the Netherlands) polyclonal anti-phospho-Erk (catalog no. #9102 Cell Signaling),

polyclonal anti-Erk (catalog no. #9101 Cell Signaling), polyclonal antibodies against

A1/Bfl -1 and Bid were a kind gift of Prof. Dr. J. Borst (The Netherlands Cancer

Institute, Amsterdam, The Netherlands).

In vitro CD40 stimulation and cell lines

BCR-Abl positive K562 cells and NIH3T3 fi broblasts were cultured in IMDM as

described above for CLL cells. CD40-ligand (CD40L, CD154) was expressed on

NIH3T3 fi broblasts, stably transfected with a plasmid encoding human CD40L.

Fibroblasts were irradiated (30Gy) and plated in culture-treated 6-wells plates (6x 105

cells/well). CLL cells were thawed and 5 x 106 cells per well were added to the adhered

fi broblasts in 3 ml IMDM containing 10% FCS and incubated for 48 hrs at 37oC. To

test the effect of c-Abl kinase inhibitors, imatinib and dasatinib, and the effect of Erk-

inhibitor PD-58059, CLL cells were pre-treated with 80 μM imatinib or dasatinib, or

50 μM PD-58059 for 30 minutes. After pre-incubation CLL cells were stimulated for

48 hrs at 37oC with CD40L with or without 30 μM imatinib or dasatinib or 50 μM PD-

58059. In the case of dasatinib, also other regimens and concentrations were used,

where CLL cells were fi rst co-cultured for 48 hrs with CD40L-expressing or control

3T3 fi broblasts, detached and washed and subsequently incubated in medium for an

additional 48 hrs in the presence of varying dasatinib concentrations (30nM-30μM),

followed by testing sensitivity to cytotoxic drugs, as described above.

RESULTS

Prolonged CD40 stimulation of CLL cells results in broad drug

resistance, which is independent of ERK- mediated decrease in

BimEL levels

In vitro stimulation via CD40 renders CLL cells resistant to fl udarabine and induces

expression of various anti-apoptotic proteins such as Bcl-xL and A1/Bfl -1 via de

novo transcription12;13;26. In addition to previously described transcriptional effects

of prolonged CD40 triggering, several novel effects on protein levels of various

apoptosis regulators were observed. In particular, the BimEL

splice variant decreased

while Mcl-1 levels increased (see Fig. 1A).

Since it is known that ERK signaling can affect BimEL

protein levels in model

systems21;27 this aspect was investigated further. Over the course of several days of

CD40 stimulation, a signifi cant reduction in BimEL

protein levels occurred, although

Bim mRNA levels remained constant10 (see also Fig. 4 below). In Figure 1B, the larger

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two Bim species represent BimEL

and most presumably a splice variant Bimα128,

which became visible in certain samples upon prolonged migration in SDS-PAGE.

Under the experimental conditions applied a short-lived phosphorylated form of Bim

(p-Bim) is probably also present21, but in our hands this form of Bim could not be

observed in primary samples either with the antibody used here, nor with commercial

antibodies specifi cally generated against p-Bim. The activation status of ERK upon

CD40 triggering was increased, and addition of the specifi c ERK inhibitor PD-98059

during CD40 stimulation prevented the reduction of BimEL

(Fig. 1C). Addition of the

proteaseome inhibitor MG132 after CD40 stimulation demonstrated that BimEL

levels

Figure 1. Anti-apoptotic changes in CLL cells upon CD40 stimulation include ERK- mediated

decrease in BimEL

levels.

(A) Changes in expression of apoptosis regulators upon 48 hrs CD40 triggering were monitored by Western

blot for the indicated proteins. Results are from a representative CLL sample from >10 patients studied.

Equal protein loading was confi rmed by staining for actin as loading control.

(B) Time course of BimEL

decrease monitored in 2 CLL samples. Shown are samples taken on consecutive

days of co-culture in absence or presence of CD40 stimulation. In sample 11-166 a decrease in BimEL

can

be observed on day 2, and in sample 12 on day 1. Position of BimEL

is indicated by a triangle, the faster

migrating species is probably the Bimα1 splice variant.

(C) Effects of ERK inhibition on BimEL

levels and phosphorylated ERK. CLL cells were stimulated with CD40

in the presence of ERK inhibitor as indicated, and lysates were probed for Bim protein, phosphorylated

and total ERK levels.

were controlled via increased protein turnover, confi rming previous reports21;22;29 (data

not shown).

Next, CLL cells triggered via CD40 in the absence or presence of ERK inhibition were

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investigated for sensitivity to drugs that are in current clinical use or in preclinical

development. As can be seen in fi gure 2. prolonged CD40 stimulation rendered

the cells resistant to fl udarabine, as observed before12;13,the proteasome inhibitor

bortezomib and the cyclin-dependent kinase inhibitor roscovitine. In addition, the

-secretase inhibitor GSI-1 was included, which is considered to be an inhibitor of

Notch signaling30. We have recently observed that GSI-1 is in fact an inhibitor of the

proteasome and a potent inducer of apoptosis in CLL (Hallaert et al., submitted).

CD40 triggering also rendered CLL cells resistant to GSI-1. For multiple CLL isolates

tested, addition of ERK inhibitors did not alleviate the broad drug resistance afforded

Figure 2. Broad drug resistance of CLL cells upon CD40 stimulation is not prevented by ERK

inhibition.

CLL cells were co-cultured with control 3T3 (control) or CD40L-expressing cells for 48 hrs, in the presence

of ERK inhibitor PD-98059 as indicated. After detachment and washing, cells were incubated with the

indicated drugs as described in detail in Methods, and analyzed for apoptosis by MitoTracker staining

after 24 hrs (roscovitine, bortezomib and GSI-1) or 48 hrs (fl udarabine). Cells cultured on 3T3 cells (black

bars) are sensitive to all drugs, but CD40 stimulation (white bars) confers broad drug resistance and this

is maintained when ERK is inhibited (grey bars). The data shown for untreated samples (medium) were

measured at 24hr. Apoptosis levels of medium samples at 48hrs were comparable.

via prolonged CD40 stimulation (Fig. 2). Together these data indicate that although

CD40 signaling activates ERK and thereby causes a decline in BimEL

levels, this is

not the cause for the observed broad drug resistance.

c-Abl inhibors prevent the anti-apoptotic protein profi le of CD40-

treated CLL cells

Another aspect of prolonged CD40 triggering of CLL cells was an increase in Mcl-1

protein (see Fig. 1A) which was, similar to the changes in Bim, independent from

increased transcription (see also below). Mcl-1 has recently been recognized as

promising target for drugs31, and has been implicated in anti-apoptotic signaling via

BCR-Abl in chronic myeloid leukemia32-34. Furthermore, other anti-apoptotic changes

124


in our in vitro CD40-CLL system, such as increased Bcl-xL and decreased Bim, have

also been implicated in BCR-Abl signaling32;35-37. Lastly, it was recently reported that

c-Abl protein expression correlates positively with tumor burden and disease stage

in CLL38. Therefore, we next tested the c-Abl inhibitor STI-571/gleevec/imatinib as a

potential suppressor of CD40-mediated pro-survival effects in CLL cells. In fi gure 3 it

can be seen that imatinib caused a clear reversal of almost all effects of CD40 stimu-

lation regarding Bcl-xL, Mcl-1, A1/Bfl -1 and BimEL

levels (left panel). This was also

observed for the second generation Abl inhibitor sprycel/dasatinib (middle panel).

A B

Figure 3. Anti-apoptotic gene and protein profi le of CLL

induced by CD40 stimulation is reversed by kinase

inhibitors imatinib and dasatinib.

(A) CLL cells were co-cultured with control 3T3 or CD40L-

expressing cells for 48 hrs, in the presence of PD-98059,

imatinib or dasatinib as indicated. Lysates were probed for

Bim, Mcl-1, Bcl-xL, A1/Bfl -1 and Bcl-2 as indicated and actin

as loading control. Shown are representative examples of 2

CLL samples with WT p53 function (left and middle panel),

and 1 CLL with p53 dysfunction (right panel; note different

order of samples). The upregulation of Mcl-1, Bcl-xL and

A1/Bfl -1 is not affected by ERK inhibition, but prevented by

imatinib or dasatinib, irrespective of p53 functionality.

(B) RNA was collected from CLL cells stimulated for 48

hrs with CD40 and inhibitors as indicated, and assayed for

expression of 34 apoptosis genes by MLPA. Shown are

averaged relative expression levels ± SD (in percentage

of total normalized signal) of selected genes in samples

from p53 WT (n=4) and p53 dysfunctional (n=3) CLL cells.

The CD40-mediated positive effects on transcription of

A1/Bfl -1 and Bcl-xL are reversed by Abl kinase inhibitors.

Examples of genes that are not signifi cantly affected

at the transciptional level are Mcl-1, Bim and GUS

( -Glucuronidase, a housekeeping gene).

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Rela

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This compound has a higher specifi c activity towards c-Abl, but is also less specifi c

for Abl kinase and targets other kinases such as Btk, Lyn and Tec23;39. The effects

of imatinib and dasatinib with respect to reversing the CD40 effects on pro-survival

parameters were also observed in CLL cells with a dysfunction in the p53 pathway

(Fig. 3, right panel).

The various kinase inhibitors were also monitored for their effects on transcription

using a multiplex assay able to quantify expression of 34 apoptosis regulatory genes.

As described previously, prolonged in vitro CD40 stimulation of CLL cells induces

transcription of Bcl-xL and A1/Bfl -1, as well as a reduction in Noxa10;13. For the ERK

inhibitor PD-98059 no effects on transcription of these genes were found. In contrast,

the c-Abl inhibitors prevented upregulation of Bcl-xL and A1/Bfl -1 transcripts, while for

example Mcl-1 and Bim transcripts were hardly affected by these drugs (Fig. 3B black

bars), although they did display changes at the protein level (Fig. 3A). The effects of

the Abl kinase inhibitors on Bcl-xL and A1/Bfl -1 were similar to those observed when

CLL cells were exposed to NF-κB inhibitor BAY-11-7082 during stimulation via CD40

(Supplementary Fig. 1). The inhibitory effects of especially dasatinib on Bcl-xL and

A1/Bfl -1 transcription were also detected in cells with a dysfunctional p53 response

(Fig. 3B white bars). In these cases, the effects of imatinib on CD40-induced gene

transcription were limited, suggesting that perhaps the suppressive effects of imatinib

may require p53 function. The complete dataset for all genes interrogated by the MLPA

probe set is represented in supplementary fi gure 2. Together these data demonstrate

that imatinib/dasatinib have a clear impact on signaling pathways leading to gene

transcription such as NF-κB, and also on mechanisms controlling protein turnover of

Mcl-1 and Bim.

Contribution to drug resistance of pro-survival proteins probed

by ABT-737

Anti-apoptotic Bcl-2 family members can be counteracted by BH3 mimetics such

as ABT-737, a widely studied compound in preclinical development40. ABT-737 is

very effective against Bcl-2 and Bcl-xL, but does not bind to Mcl-1 or A1/Bfl -131;41.

As reported before42, CLL cells are quite sensitive to ABT-737, but upon stimulation

with CD40 this is reduced approximately 100-fold (Fig.4 A&B). We tested whether

sublethal doses of ABT-737 could synergize with other drugs in this setting. There

was a slight increase in apoptosis of CD40-stimulated cells when 0.1μM ABT-737

was combined with various other drugs. This was further restored to levels observed

in medium or control cultures with 3T3 cells by using 1 μM ABT-737. In fi gure 4C

the averaged data from 4 CLL patients are shown. Individual sample responses to

126


ABT-737 showed divergent patterns, with some patient’s cells displaying full reversal

of drug sensitivity at 1.0 μm ABT-737 for all drugs tested, while others displayed

different patterns depending on the drug tested (Supplemental Fig. 3). This seemed

consistent with the clear patient to patient variation in the degree of upregulation of

Mcl-1 and A1/Bfl -1 (Fig. 3A). These results indicate that the contribution of Bcl-2 and

Bcl-xL to the observed drug resistance in this in vitro model is substantial, but could

generally be counteracted by ABT-737. The effi ciency and effective dose with which

ABT-737 acts differs per patient sample and this probably correlates with the degree

of increase in Mcl-1, and possibly A1/Bfl -1, obtained with CD40 stimulation.

c-Abl kinase inhibitors prevent drug resistance of CD40-treated

CLL cells

In a similar fasion as above for ABT-737, the effect of c-Abl kinase inhibitors on the

drug resistance afforded by CD40 triggering was measured. The apoptosis-inducing

effects of the Abl inhibitors themselves on control samples co-cultured with 3T3 cells

and CD40L-expressing cells were minimal (Fig. 5A, medium samples). Only at high

Figure 4. Contribution of Mcl-1 to drug resistance probed by ABT-737.

(A) CLL cells were treated immediately after thawing with the indicated concentrations of ABT-737 or the

inactive enantiomer. After 24 hrs, apoptosis was measured by MitoTracker staining.

(B) CLL were cultured for 2 days in medium, with 3T3 control cells or with 3T40L cells before treatment with

ABT-737 as above. Data in A and B represent averages ± SD from 3 different CLL samples.

(C) Sublethal doses of ABT-737 after CD40 stimulation as determined in B) (0.1 and 1.0μM) were combined

with various other drugs as indicated to test synergy in reversal of drug resistance. Data are averages ± SD

from 5 (0.1 μM) or 4 (1μM) patient samples, tested in 3 independent experiments.

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levels and upon prolonged exposure did imatinib and dasatinib induce signifi cant

apoptosis in CLL cells, in contrast to e.g. K562 cells which are very sensitive due

to their dependence on the BCR-Abl fusion protein for survival (Supplemental Fig. 4).

Remarkably however, imatinib and especially dasatinib prevented the resistance

towards various drugs normally observed upon CD40 treatment of CLL cells. This

appeared true for CLL samples with mutated as well as unmutated IgVH gene

sequences (both n=2). The sensitising effect of these inhibitors was also seen in CLL

cells with a dysfunctional p53 pathway (Fig. 5B). Especially the cytotoxic effect of

128

A C

B D

Figure 5. Drug resistance of CD40-stimulated CLL cells is reversed by c-Abl kinase inhibitors.

(A) CLL samples (n=4) were cultured on 3T3 (control) or CD40L-expressing cells in the presence of the

indicated inhibitors for 48 hrs, and after detachment and washing cultured for 24 hrs in medium or with the

cytotoxic drugs. Average results for apoptosis measured via MitoTracker staining are shown.

(B) The same as in A) for an experiment with p53 dysfunctional cells. Data are representative for 3 similar

experiments performed; the variation among samples in particular for background apoptosis in the absence

of external stimuli precluded averaging.

(C) A similar experiment as in A) was performed with decreasing concentrations of dasatinib as indicated.

Drug susceptibility was assessed by incubation with 5 μM GSI-1 for 24 hrs. Results represent averages of

4 experiments or 2 where indicated. At 3 nM there was no effect of dasatinib detectable (not shown).

(D) Sequential CD40 stimulation followed by incubation with c-Abl kinase inhibitors. CLL cells were co-

cultured with 3T3 cells expressing CD40L for 48 hrs, detached and washed before addition of dasatinib

(300 nM) for an additional 48 hrs, and were then tested for drug susceptibility. Results represent average

data of 3 experiments.


proteasome inhibitors (bortezomib and GSI-1) was potentiated by treatment of CLL

cells with c-Abl inhibitors during CD40 exposure. In general, the effects of dasatinib

were stronger than those of imatinib at the concentrations used (30 μM), as was

also observed for the effects on protein levels (see Fig. 3). Since dasatinib has a

higher specifi c activity towards its target kinases than imatinib23;43 we also tested its

effects over a lower range of concentrations. The capacity of dasatinib to modulate

the drug sensitivity of CD40-treated CLL cells could also be observed at substantially

lower concentrations (30nM-3μM). This is demonstrated in fi gure 5C for the results

obtained with GSI-1, which was in general the strongest inducer of apoptosis in CLL

cells among the drugs tested.

The results thusfar were obtained with simultaneous administration of CD40 signals

and kinase inhibitors. To better refl ect the actual situation of LN CLL cells already

exposed to a protective environment, isolated PB CLL cells were fi rst stimulated

via CD40 for 48 hrs, followed by separate addition of dasatinib (0.3μM) and drug-

sensitivity tests. Also in this set-up, a reversal of resistance towards various drugs

(fl udarabine, bortezomib, roscovitine) could be observed (Fig. 5D). Thus, dasatinib

has a clear capacity to interfere with the protective effects afforded by prolonged

CD40 stimulation.

Similar apoptosis protein signature in ex vivo LN samples as

upon in vitro CD40 triggering

To relate the effects of in vitro CD40 stimulation with the in vivo situation, samples

from CLL lymph nodes were lysed directly in SDS-containing sample buffer and

probed for the presence of proteins involved in apoptosis regulation. As observed

before, a clear increase of Bcl-xL protein was present in LN samples compared to

peripheral blood (PB) samples10. This was also found for Mcl-110 and A1/Bfl -1 (data

not shown). Regarding the expression levels of other signature proteins involved

in CD40-mediated anti-apoptosis pathways, a strong increase in both total and

phosphorylated ERK was found, concomitant with decreased levels of BimEL

(Fig. 6).

These fi ndings indicate that in CLL lymph nodes similar survival pathways are

operational as those that can be induced in peripheral blood CLL cells by prolonged

in vitro CD40 stimulation.

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DISCUSSION

Previous reports have described effects of inhibitors of BCR-Abl kinase on single

anti-apoptosis proteins (predominantly Mcl-1, Bcl-xL or Bim) in CML or model cell

lines35-37. This study provides an overview on the effects of c-Abl inhibitors on all Bcl-2

members in the context of CD40 signaling in CLL cells. The rationale for the present

study was two-fold. First, there is the growing concept that CLL is a dynamic disease,

with proliferation centers in LN and possibly also BM. These protective niches, where

cells are prone to be more drug resistant, are presumably the source of relapsing

clones. Second, the potential of novel drugs such as kinase inhibitors, to target pro-

survival signaling pathways to which malignant cells have become addicted.

We have observed that our in vitro CLL culture model setting provides strong and

probably supra-physiological CD40 signals, with longlasting protective effects

which continue after detachment of CLL cells from CD40L cells (data not shown).

Nevertheless, comparison between LN samples and PB CLL cells stimulated in vitro

via CD40 indicated the presence of a comparable pro-survival signature as implied

by ERK activation and BimEL

levels. Previously, we have shown that in LN samples

also increased levels of Bcl-xL and Mcl-1 are detectable10. Together, the available

data indicate that the pro-survival signature triggered via CD40 stimulation in vitro is

also found in CLL lymph nodes, and imply that our experimental data hold promise

for extrapolation towards a therapeutic setting.

With respect to post-transcriptional effects of CD40 stimulation on CLL cells, both Bim

and Mcl-1 proteins are known targets for phosphorylation and subsequent increased

proteasomal degradation. Cytokine withdrawal in murine cell lines causes decreased

PI3K-Akt/PKB signaling to activate GSK3 which in turn phosporylates Mcl-1, thus

marking it for proteasomal degradation44. In the case of CLL cells, our data indicate

that upon CD40 stimulation PKB phosphorylation was undetectable, the PI3 kinase

130 Figure 6. Anti-apoptotic protein

signature in CLL lymph nodes.

Protein lysates obtained from

peripheral blood (PB, n= 5) and

lymph node (LN, n=6) were probed

for phosphorylated-ERK, total

ERK, Bim and actin as indicated.

The expression of these proteins in

ex vivo LN was similar to changes

observed upon in vitro stimulation

of PB CLL cells with CD40.


inhibitor LY294002 did not trigger apoptosis, and the rate of Mcl-1 protein turnover was

not changed (unpublished observations). Since Mcl-1 transcription in CLL cells was

also not affected by CD40, this suggests that the increase in Mcl-1 protein is possibly

controlled at the level of translation by a non-PKB dependent mechanism. Recent

evidence from other experimental systems indeed points at translational repression

of Mcl-1 via eIF initiation factors as yet another point of regulation45;46. If this system

is operational under our experimental conditions and whether it may be connected

with the other recently described pathway implicating antigen receptor/PI3-K/PKB

signaling in affecting Mcl-1 levels47 remains to be determined. In contrast to the

situation in AML cells41, in primary CLL cells the ERK pathway seems not responsible

for increased Mcl-1 protein, as the ERK inhibitor PD-98059 did not block its increase,

and did not affect drug susceptibility (Fig. 2&3). Whether or not increased Mcl-1 plays

an important role in vivo in survival of CLL in lymph nodes seems an important issue

with respect to therapeutic application of ABT-737. Our data and those of others31;41

indicate that variations in Mcl-1 and possibly also A1/Bfl -1 levels will determine the

effective dose of ABT-737 both as a single agent and in drug combinations. Of note,

the combination of ABT-737 with roscovitine, which should counteract Bcl-2, Bcl-xL as

well as Mcl-131, was not effective in all patients (Supplemental Fig. 4). This suggests

that either roscovitine is unable to reduce Mcl-1 in this setting or that perhaps in these

samples A1/Bfl -1 is a dominant factor. Our observations on increased BimEL

turnover

are in accord with an established pathway of ERK-mediated phosphorylation and

proteasomal degradation21;22;29. To our knowledge, this is the fi rst example of this

pathway operating in primary tumor cells upon CD40 stimulation, and in CLL LN

samples.

In our experience, neither imatinib nor dasatinib are effi cient inducers of apoptosis as

single agents, in contrast to their effects on K562 cells, which depend for survival on

the BCR-Abl fusion oncogene (Supplemental Fig. 3). In a recent study, considerable

variation in apoptosis susceptibility in untreated and dasatinib-treated peripheral

blood samples was found using 5 μM dasatinib, and the response was correlated

with IgVH mutation and ZAP70 status48. This and other studies performed to date

agree that, in CLL cells from peripheral blood, dasatinib has a strong synergistic

effect in combination with p53-pathway dependent and -independent agents48-50.

Transcriptional effects of imatinib and dasatinib on Bcl-xL and A1/Bfl -1 were similar

to those of inhibitors of NF-κB. We noted that the effects of the c-Abl inhibitors on

reversing ERK phosphorylation status, and the corresponding changes in BimEL

levels varied among patients, without apparent correlation with prognostic factors

such as mutation or p53 status (data not shown and Fig. 3). These signaling

pathways are affected/reversed by imatinib and dasatinib although the actual

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131


target(s) remains unknown. Recent analyses of the spectrum of kinase targets of

these compounds points to various candidates involved in T- and/or B-cell activation

such as Src kinases including Lck and Fyn, Btk and Tec kinase23;39. The spectrum of

non-Abl kinases targeted by dasatinib is in fact quite broad (>20 kinases), and an

immunosuppressive effect was predicted23, and recently confi rmed for T cells51. Our

preliminary analyses do not show a similar inhibitory effect of dasatinib on in vitro

B-cell proliferation however (A. Jaspers, unpublished observation). From the kinases

targeted by dasatinib no obvious candidate(s) for exclusive participation in the CD40

pathway is apparent, although the Ser/Thr kinase p38α and upstream MAP kinases

appear likely as participants. A clue for the participation of Btk or Tec kinases comes

from a recent report that their expression level is regulated via NF-κB in a positive

feedback-loop. This loop can be interrupted by proteasome inhibitors52, which fi ts

with our observation that the combination of bortezomib or GSI-1 with dasatinib has

the strongest effect on apoptosis of CD40-stimulated CLL cells (Fig. 5A&B).

Obviously, c-Abl kinase itself may very well be involved, and there is evidence

that levels of c-Abl protein expression correlate positively with tumor burden and

disease stage in CLL53. Another study reported that c-Abl becomes active upon CD40

triggering and then induces p7354. This signaling route is predicted to bypass p53

and may therefore be therapeutically relevant. Both these studies used imatinib and/

or introduction of recombinant c-Abl, so they cannot provide defi nitive evidence of

endogenous c-Abl kinase activity in CLL. The majority of studies on activity have

been done with the BCR-Abl-positive cell line K562 or primary CML samples where

expression levels of the oncogenic fusion protein are augmented. Our preliminary

efforts to detect active endogenous c-Abl either in un-stimulated, CD40-triggered or

LN CLL cells by western blotting with commercial antibodies were inconsistent.

At present, two independent mechanisms are attributed to the development of

chemoresistance in CLL. The fi rst is a shift in the balance between pro- and anti-

apoptotic regulators, and both Mcl-155 and Bfl -1/A156 have been associated with

resistance to chemotherapy. Signifi cantly, these hallmarks are very similar to the

CD40-activated CLL phenotype we use as a model. The second mechanism is based

on acquired mutations resulting in a dysfunctional p53 response3. A recent phase

II evaluation of dasatinib as single agent in relapsed and refractory CLL showed

limited effects, but in good correlation with our data a reduction of lymph node size

was observed in a major fraction of patients57. Our data indicate that c-Abl inhibitors,

notably dasatinib, overcome the protective profi le within the micro-environment

resulting in susceptibility to p53 pathway dependent drugs (fl udarabine) as well as

to p53-independent agents (roscovitine, bortezomib, ABT-737). Thus, from a clinical

perspective it may be more effective to apply combination strategies of dasatinib

132


with other drugs. Our data provide a rationale to combine dasatinib both with purine-

analogues but also with drug regimens that do not exclusively rely on p53 function

for effi cacy.

ACKNOWLEDGEMENTS

We thank Dr. M. Kramer, Dr. S. Wittebol, (department of Internal Medicine, Meander

Medical Centre, Amersfoort) and Dr. J. Baars (department of Internal Medicine, The

Netherlands Cancer Institute, Amsterdam) for including CLL patients in this study, and

René van Lier for his insightful comments and critical reading of the manuscript.

This work was supported by the Dutch Cancer Foundation (DCF) grant nr. UVA2004-

3039. A.P.K. is supported by a ‘Veni’ grant from ZonMw (The Netherlands Organization

for Health Research and Development).

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134

Patient Rai

stage

WBC

10E9/L

Lymphocytes

(%)CD5+/

CD19+2

CD3+

(%)

IgVH

3

mut.

status

p53

status4

CD38

(CD20+)

Chromosomal

abnomalities5

CLL01 l 113.0 90.0 88.3 4.4 + functional 81 none

CLL07 II 93.3 86.9 96.8 2.4 + functional 87 11q-

CLL08 0 58.5 91.0 92.7 4.3 + ND 24 none

CLL11 II 49.3 88.8 91.4 5.5 - functional 92 13q-

CLL12 I 72.2 86.3 93.0 5.0 + functional 33 none

CLL16 II 80.1 73.7 92.0 6.0 + ND 39 13q-

CLL17 II 102.0 92.1 93.3 4.1 - ND 47 none

CLL18 0 48.0 89.2 90.5 7.7 + ND 29 ND

CLL20 II 61.4 89.0 95.0 4.0 + functional 20 ND

CLL21 IV 60.6 89.8 86.7 3.1 - ND 24 ND

CLL22 I 60.1 94.4 81.7 6.5 - dysfunc-

tional

44 17p-

CLL23 IV 137.0 94.8 83.5 3.8 + dysfunc-

tional

57 17p-;13q-

CLL25 I 79.0 93.3 84.7 3.4 - dysfunc-

tional

39 17p-

CLL29 I 117.0 95.8 95.0 1.5 + ND 9.5 ND

CLL31 II 68.8 94.7 90.8 4.7 + ND 1.9 13q-

CLL32 0 73.8 86.9 85.2 7.2 + ND 2.4 ND

CLL40 IV 232.0 96.1 98.5 1.2 - ND 97 ND

1 Patient data for the lymph node samples used in this study can be found in ref 10.2 Percentage of cells positive for CD5 and CD19 surface expression.3 Mutated IgV

H gene (+) denotes >2% mutations compared to germline sequence.

4 P53 functional status was measured via radiation-induced RNA expression of p53 target genes Puma

and Bax, or by p53 and p21 protein upregulation via western blot, as described in refs 16 and 25. Patient

25 had a so-called type A dysfunction. (ND = not determined)5 As determined by FISH. Probes for 11q22.3 (ATM), 13q14 (D13S319) and 17p13 (TP53) were obtained

from Abbott-Vysis. Samples with >10% aberrant signals were considered abnormal.

Table 1. Patient characteristics (peripheral blood samples)1.


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3457.


SUPPLEMENTAL FIGURE LEGENDS

Figure S1. NF-кB inhibitor BAY-11-7082 blocks CD40-mediated Bcl-xL and Bfl -1 upregulation.

CLL cells were stimulated with or without CD40L for 48 hrs in the presence of ERK inhibitor PD-98059 or

of BAY-11-7082, an irreversible inhibitor of IκB-α phosphorylation (1 and 5 μM). Cells were lysed in SDS-

containing buffer and analyzed by western blot. Ch

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Figure S2. RT-MLPA analysis of p53 WT and p53 dysfunctional CLL cells, stimulated via CD40 in

absence or presence of kinase inhibitors.

Cells were stimulated for 48 hrs as in fi gure 3b, and apoptotic gene expression profi le was analyzed by

RT-MLPA. Results were normalized and expressed relative to total signal. Housekeeping genes included

in this assay were, GUS, B2M, FLT and PARN. (Note that probes for IAP2 are non functional58). Graphs

represent averaged data ± SD of n=4 p53 functional (A) and n=3 dysfunctional (B) samples.

A

B


Figure S3. Apoptosis sensitivity of individual CLL samples after co-culture with 3T3 or 3T40L cells,

followed by treatment with various drugs in conjunction with ABT-737.

CLL samples were cultured as described in legend to fi gure 4, followed by incubation with the indicated

drugs in the presence of 1μM ABT-737. Apoptosis was determined after 24 hrs by Mito Tracker staining.

140

Figure S4. Peripheral blood-derived and CD40-stimulated CLL cells are insensitive to imatinib or

dasatinib.

(A,B) Peripheral blood-derived CLL cells (n=3) were stimulated with increasing concentration of imatinib

or dasatinib for 24, 48 or 72 hrs.

(C,D) CLL cells (n=4) were cultured for 48 hrs in medium or together with 3T3, or 3T40L cells, followed

by stimulation with different concentrations imatinib or dasatinib for 48 hrs. The BCR-Abl positive cell

line K562 was used as a positive control for imatinib and dasatinib-induced apoptosis. Apoptosis was

determined by MitoTracker staining.

A

C

B

D


141


142


chapter

7Concluding remarks


144


One focus of the studies described in this thesis is the mechanism of apoptosis

induction by novel drugs that act independently of the p53 response pathway. In

chapter 2 we discussed the insights obtained from the studies on these drugs in

relation to the current debate regarding the interactions and functional mechanism of

the Bcl-2 family members. Our conclusions are summarized in a model (chapter 2)1.

A second major aspect of this thesis concerned the functional consequences of CD40

triggering and the identifi cation of abnormalities in apoptosis pathways in CLL cells,

in relation to drug sensitivity (chapter 4-5-6). In this fi nal chapter the relevance of our

fi ndings and the implications with respect to future treatment strategies for CLL are

discussed.

CD40L expressed on the surface of fi broblast or epithelial cell lines has been widely

used to enhance the in vitro survival of CLL cells2-9. We have used this system to study

the effects of CD40 ligation on drug sensitivity. The basic premise underlying these

studies is that CD40-stimulated peripheral blood (PB) CLL cells are a representative

model for CLL cells residing in lymph node (LN) proliferation centers, the site where

they receive pro-survival signals from the microenvironment. Thus, a key question is

whether the model is indeed appropriate and relevant. To our opinion the answer to

this question should be affi rmative because of the following arguments.

(1) Histochemical studies have shown that in vivo CLL cells may be exposed to

CD40L. T lymphocytes have been found interspersed between CLL cells in LN and

bone marrow (BM) proliferation centers10;11 and CD40L-positive T lymphocytes have

been detected in LN pseudofollicles12. However, the functional activity of these T cells

remains to be proven. It is known that ex vivo CD40 stimulation provides signifi cant

survival signals to normal tonsil-derived B lymphocytes13;14. Similar experiments with

LN-derived CLL cells have to our knowledge not been published, but it appears likely

that these would yield comparable results.

(2) We and others have shown remarkable similarities between in vitro CD40L

stimulated PB CLL cells and in vivo LN CLL cells regarding changes in expression

pattern of apoptosis regulating proteins. Table 1 summarizes the expression of

important members of the Bcl-2 family (Bcl-XL, Mcl-1, Bid, Bim

EL and Noxa), the

activated form of the MAP kinase ERK (p-ERK), XIAP and survivin. Both in CD40-

triggered PB CLL cells and in LN CLL cells expression of Bcl-XL, Mcl-1, p-ERK, XIAP,

survivin and Bid are increased, whereas Noxa and BimEL

are decreased.

(3) In CD40 stimulated PB CLL cells and in LN CLL cells the expression pattern

of these proteins is comparable not only qualitatively but also quantitatively. In our

experience, the observed changes compared to PB CLL are of the same order of

magnitude. As a corollary, this notion provides an argument against the possibility that

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the in vitro CD40 system provides unphysiological stimuli. Of course, some caution

must be exerted in directly extrapolating the data to the in vivo situation because it

has to be assumed that in vivo CLL cells will receive a variety of signals.

Protein LN CD40L Reference

Bcl-XL

Up Up [3, 4, 7, 15]

Mcl-1 up Up [3, 4, 7, 15]

Bid up* Up* [2, 7]

BimEL

Down Down Chapter 5 and 6

Noxa Down Down [15]

p-ERK Up Up Chapter 5 and 6

XIAP Up* Up [4, 5]

Survivin Up Up [16]

Protein expression of indicated proteins was evaluated in LN CLL cells and in PB CLL cells after

in vitro CD40 triggering. Changes as compared to non-stimulated PB CLL cells are indicated by

up- or down-regulation.

* (Hallaert, unpublished observations)

These observations have 2 important implications.

The proliferation centers in LN, BM and spleen might be considered as 1.

‘sanctuary sites’ where CLL cells are protected from the effects of cytotoxic

drugs. Thus, these sites probably are the source of the relentless relapses

that characterize the clinical course of CLL, explaining why thus far curative

treatment is lacking. Relapses occur even with recently introduced, very potent

drug combinations (like FCR) which result in minimal residual disease (MRD)

negativity in the PB and BM in a high percentage of patients17. Whereas in

the later phases of the disease (acquired) p53 dysfunction is an important

cause of therapy resistance, at diagnosis only 10-15% of patients have a 17p

deletion18;19. In contrast, the microenvironmentally induced chemo-resistance

is of great importance in all phases of CLL.

Novel therapies should be tested on CD40L stimulated CLL cells. 2. To

date, the potential effi cacy of novel cytotoxic agents is usually assessed

on PB CLL cells. This disregards the potential protective signals from the

microenvironment. As a consequence, various compounds induce signifi cant

apoptosis in vitro, but the responses in vivo remain disappointing.

146

Table 1. Protein expression in LN CLL and in vitro CD40L stimulated PB CLL cells is similar.


It can be concluded that the ideal treatment of CLL should at the same time be able to

overcome resistance in the sanctuary sites and induce apoptosis independent of p53

function. A promising drug combination in this perspective is the c-Abl kinase inhibitor

with proteasome inhibitors (chapter 6). In clinical trials in CLL however, dasatinib and

bortezomib administered as single agent yielded disappointing effects, underscoring

the need for combination regimens. To explore this, in the near future a clinical trial

with dasatinib plus fl udarabine will be started in fl udarabine refractory CLL.

In the more distant future, the CD40 system might not only be useful for new drug

testing in general but may also enable pre-treatment assessment of the potential

responsiveness to (combinations of) drugs in individual patients, opening possibilities

to attain the important goal of individualized, “tailor made” therapy in CLL.

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147


8. Biagi E, Yvon E, Dotti G et al.

Bystander transfer of functional human

CD40 ligand from gene-modifi ed

fi broblasts to B-chronic lymphocytic

leukemia cells. Hum.Gene Ther.

2003;14:545-559.

9. Fluckiger AC, Rossi JF, Bussel A et al.

Responsiveness of chronic lymphocytic

leukemia B cells activated via surface

Igs or CD40 to B-cell tropic factors.

Blood 1992;80:3173-3181.

10. Ghia P, Strola G, Granziero L et al.

Chronic lymphocytic leukemia B

cells are endowed with the capacity

to attract CD4+, CD40L+ T cells by

producing CCL22. Eur.J.Immunol.

2002;32:1403-1413.

11. Schmid C, Isaacson PG. Proliferation

centres in B-cell malignant

lymphoma, lymphocytic (B-CLL):

an immunophenotypic study.

Histopathology 1994;24:445-451.

12. Carbone A, Gloghini A, Gruss

HJ, Pinto A. CD40 ligand is

constitutively expressed in a subset

of T cell lymphomas and on the

microenvironmental reactive T cells of

follicular lymphomas and Hodgkin’s

disease. Am.J.Pathol. 1995;147:912-

922.

13. Hennino A, Berard M, Krammer

PH, Defrance T. FLICE-inhibitory

protein is a key regulator of germinal

center B cell apoptosis. J.Exp.Med.

2001;193:447-458.

14. Lagresle C, Mondiere P, Bella C,

Krammer PH, Defrance T. Concurrent

engagement of CD40 and the

antigen receptor protects naive and

memory human B cells from APO-1/

Fas-mediated apoptosis. J.Exp.Med.

1996;183:1377-1388.

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1999;106:995-1004.

5. Cuni S, Perez-Aciego P, Perez-Chacon

G et al. A sustained activation of PI3K/

NF-kappaB pathway is critical for

the survival of chronic lymphocytic

leukemia B cells. Leukemia

2004;18:1391-1400.





2002;100:1795-1801.





Br.J.Haematol. 2007;138:721-732.

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15. Smit LA, Hallaert DY, Spijker R et

al. Differential Noxa/Mcl-1 balance

in peripheral versus lymph node

chronic lymphocitic leukemia cells

correlates with survival capacity.

Blood 2007; 109: 1660-1668






2001;97:2777-2783.

17. Tam CS, O’Brien S, Wierda W et al.

Long term results of the fl udarabine,

cyclophosphamide & rituximab regimen

as initial therapy of chronic lymphocytic

leukemia. Blood 2008;112:975-980

18. Dohner H, Fischer K, Bentz M et al.

p53 gene deletion predicts for poor

survival and non-response to therapy

with purine analogs in chronic B-cell

leukemias. Blood 1995;85:1580-1589.






3157.

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150


chapter

Summary

8


152


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153The work described in this thesis can be divided into two parts. The fi rst part focuses

on the mechanism of apoptosis induction by novel drugs that act independently of

the p53 response pathway (chapter 2-3). The second part focuses on the functional

consequences of CD40 stimulation and the understanding of aberrant apoptosis

pathways in CLL. These studies were designed to test drug sensitivity and to fi nd

future treatment strategies for CLL (chapter 4-5-6).

Chapter 1 introduces the current understanding in therapy, apoptosis and the

microenvironment in CLL and provides an outline for the studies presented.

In chapter 2 we investigated the mechanism underlying apoptosis elicited by the

cyclin dependent kinase inhibitor, roscovitine, in CLL. Biochemical analysis showed

that the BH3-only protein Noxa was associated with the Bcl-2 homologue Mcl-1 in

CLL cells. Furthermore, this apoptosis route required the BH3-only member Bim.

Defi ciency of these Bcl-2 family proteins, assessed by RNA interference techniques,

protected CLL cells from roscovitine induced apoptosis. We concluded that CLL

survival depends on the balance between these pro- and anti-apoptotic Bcl-2 family

members,and that roscovitine activates a selective apoptosis pathway, described as

the Mcl-1/Noxa axis.

In chapter 3 we focused on the effect of a chemical compound, γ-secretase inhibitor

(GSI-1), that is used in the treatment of Alzheimer’s disease. We found that GSI-1

was a potent inducer of apoptosis in CLL through effi cient blocking of proteasomal

activity. Furthermore, inhibition of proteasomal activity triggered the accumulation of

the pro-apoptotic molecule Noxa. The role of Noxa was also demonstrated via RNA

interference experiments. We suggest that GSI-1 or related compounds may hold

promise for therapeutic applications in CLL.

In chapter 4 we compared apoptosis gene profi les from peripheral blood with

lymph node material of CLL patients. A prominent difference between CLL cells in

the peripheral blood and the lymph node was the decreased expression of the pro-

apoptotic protein Noxa and the increased expression of anti-apoptotic Mcl-1 and Bcl-

XL in the lymph node. This differential expression pattern was also observed by in

vitro CD40 stimulation of peripheral blood CLL cells. Direct manipulation of Noxa

protein levels was achieved by proteasome inhibition (increase in Noxa expression,

see chapter 3) in CLL cells and RNA interference techniques (decrease in Noxa

expression) in model cell lines. In both experiments, cell viability was directly linked

with the levels of Noxa. We concluded that the suppression of Noxa in the lymph


154node microenvironment contributes to the resistance of the CLL cells at these sites.

The hypothesis is that proliferating CLL cells in the lymph node are protected from

apoptosis through the modulation of pro- and anti-apoptotic molecules by CD40

signaling. Once CLL cells leave their lymph node ‘cradle’, the nurturing CD40 signal

is lost and a strong increase in Noxa levels occurs, which corresponds with the high

apoptosis rate of peripheral blood-derived CLL in vitro. We propose that Noxa is a

promising therapeutic target to treat CLL.

In chapter 5 we gained more insight in how CLL cells in the lymph node differ from

the CLL cells which have moved into the circulation. We mimicked this transit in vitro

from the lymph node to the peripheral blood by temporary CD40 receptor triggering

of CLL cells (CD40 model) and we monitored the expression of apoptosis regulatory

genes in relation to sensitivity to various types of drugs during and following CD40

triggering. We showed that CD40 induced changes in the apoptosis gene expression

profi le and that the observed broad drug resistance was reversible after cessation

of CD40 stimulation. In contrast, however, Bim and Mcl-1 protein levels remained

unchanged and also roscovitine resistance was sustained. We conclude that this

model system to mimic the in vivo lymph node setting could help to predict responses

of CLL to new drugs.

Chapter 6 aimed to defi ne the molecular basis for the increased drug resistance

observed after CD40 stimulation in CLL and searched for novel strategies to

circumvent this resistance. As shown in chapters 4 and 5, CD40 triggering resulted

in an anti-apoptotic RNA and protein profi le and resulted in resistance to various

chemotherapeutic drugs. The anti-apoptotic profi le of CD40-stimulated CLL cells

resembles BCR-Abl-dependent changes seen in Chronic Myeloid Leukemia (CML).

This abnormality is characterized by a translocation between one chromosome 9 and

one chromosome 22, resulting in the BCR/Abl fusion-gene. This prompted us to use

c-Abl inhibitors imatinib and dasatinib, which prevented the entire anti-apoptotic profi le

of CD40 triggered CLL, and restored drug sensitivity. These effects also occurred

in CLL samples with a dysfunctional p53. We concluded that treatment settings,

combining c-Abl inhibitors such as imatinib or dasatinib with other chemotherapeutic

drugs may be promising for the treatment of CLL.


chapter

9Nederlandse samenvatting


156


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157ACHTERGROND

Apoptose

Apoptose of geprogrammeerde celdood is een strict gereguleerd proces dat het

organisme beschermt door niet gewenste cellen dood te laten gaan. Verstoring

van apoptose speelt een belangrijke rol bij de maligne transformatie van cellen

en draagt bij tot resistentie tegen chemotherapie. Apoptose geschiedt door middel

van activatie van een specifi eke groep enzymen (caspases). Deze enzymen zetten

de cel aan tot de degradatie van, voor de cel belangrijke, eiwitten, wat uiteindelijk

resulteert in celdood. Er zijn twee belangrijke apoptotische signaleringspaden die

caspases kunnen activeren (zie hoofdstuk 1, Fig.1). Als eerste de intrinsieke route

of mitochondriale apoptoseroute. Hier vindt signalering plaats via de mitochondriën,

de energiecentrale van de cel. Ten tweede is er de extrinsieke route, waarbij

signalering vanaf het celoppervlak plaats vindt. Regulatie van caspase activatie wordt

gecontroleerd door twee families van eiwitten: de Bcl-2- en inhibitor-of-apoptosis

(IAP) familie. De Bcl-2 familie bestaat uit eiwitten met een pro- (stimuleren apoptose)

of anti-apoptotische (remmen apoptose af) functie. Het evenwicht tussen deze twee

groepen eiwitten bepalen uiteindelijk de gevoeligheid van de cel voor apoptotische

signalen. IAP eiwitten hebben een anti-apoptotische werking doordat ze kunnen

binden aan caspases en daarmee hun activiteit remmen.

In verschillende soorten kanker worden afwijkingen gevonden in genen die apoptose

reguleren. Een voorbeeld is het tumor suppressor gen p53 dat bij celschade

de celgroei zal stopzetten (waarbij het DNA hersteld kan worden) of apoptose

zal induceren (indien de schade te erg is). Hierdoor wordt het ontstaan van een

tumor verhinderd. Echter, bij kanker is p53 vaak gemuteerd wat kan leiden tot een

verminderde gevoeligheid voor apoptose en chemoresistentie.

Chronisch Lymfatische Leukemie

Chronisch Lymfatische Leukemie (CLL) is een bij ouderen veel voorkomende

bloedkanker (leukemie), of preciezer: kanker van witte bloedcellen (lymfocyten). Er

zijn drie types lymfocyten en in CLL is het de kwaadaardige B lymfocyt die de ziekte

veroorzaakt. In het bloed kunnen enorme aantallen kwaadaardige B lymfocyten

voorkomen. Eerst werd er verondersteld dat de toename van de B lymfocyten eerder

het gevolg is van defecten in apoptose regulatie waardoor deze cellen niet verwijderd

kunnen worden. Tegenwoordig is deze opvatting hervat en staat het vrijwel vast

dat er ook een langzame celgroei (deling) is die voornamelijk in de lymfeklieren

en het beenmerg, de zogenaamde micro-omgeving, plaatsvindt. Met de huidige


158chemotherapie is het mogelijk de prognose van de ziekte tijdelijk te verbeteren door

de cellen in het bloed te laten verdwijnen, maar volledige genezing is niet mogelijk.

Er is nog onvoldoende inzicht in de onderliggende oorzaak van de kwaadaardige

groei van de B lymfocyten in lymfeklieren en de resistentie tegen chemotherapeutica.

Daarom is het noodzakelijk onderzoek te doen naar de samenhang tussen deze

aspecten en nieuwe behandelingsmogelijkheden.

DOELSTELLING VAN DIT PROEFSCHRIFT

De studies beschreven in dit proefschrift hebben als doelstelling een beter inzicht te

verwerven in:

Het werkingsmechanisme van nieuwe chemotherapeutica die onafhankelijk 1.

van het p53 pad werken. Hierbij wordt dieper ingegaan op het effect van

chemotherapeutica op de apoptose signalering in CLL.

Het mechanisme van de verstoorde apoptose en de overleving van CLL cellen 2.

die zich in zowel het perifeer bloed als in de lymfeklier bevinden. Aan de hand

van deze bevindingen wordt gezocht naar optimale therapeutische combinaties

om resistentie in CLL tegen te gaan.

In hoofdstukken 2 en 3 werd het apoptose pad onderzocht van twee potentiële

chemotherapeutica: een cyclin-dependent kinase (CDK) inhibitor (remt de celgroei)

roscovitine en een inhibitor van het proteasoom (het eiwit complex in de cel dat

zorgt voor de afbraak van overbodige en beschadigde eiwitten). De invloed van

de micro-omgeving (in de micro-omgeving, zoals de lymfeklier en het beenmerg,

vindt er interactie plaats tussen de tumorcel en niet tumorale cellen) werd in de

volgende hoofdstukken bestudeerd. In hoofdstuk 4 werd onderzocht wat het verschil

is in de expressie van apoptose regulerende eiwitten in CLL cellen in het perifeer

bloed versus de lymfeklier en wat de functionele gevolgen van die verschillen zijn. In

hoofdstuk 5 en 6 werd verder ingegaan op de functionele gevolgen met betrekking

tot het overwinnen van resistentie tegen chemotherapeutica. Hiervoor werd gebruik

gemaakt van een in vitro (buiten het lichaam) cel systeem (model) dat de micro-

omgeving gedeeltelijk nabootst.


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159DE RESULTATEN VAN HET PROMOTIE ONDERZOEK

Hoofdstuk 2

In dit hoofdstuk werd de apoptose route van een nieuw apoptose inducerend

chemotherapeuticum, roscovitine, bestudeerd op moleculair vlak in CLL. We vonden

dat in CLL cellen het pro-apoptotisch BH3-only eiwit Noxa gebonden is met het Bcl-2

homologe anti-apoptotisch eiwit Mcl-1. Tevens toonden we dat bij deze apoptose

route ook het pro-apoptotisch BH3-only eiwit Bim betrokken is. Het belang van deze

eiwitten in roscovitine geïnduceerde celdood werd bevestigd met behulp van RNA

interferentie (remmende) technieken. Er kon geconcludeerd worden dat er in CLL

een functionele interactie is tussen pro- en anti-apoptotische eiwitten en dat apoptose

geïnduceerd door roscovitine een selectieve route volgt, genaamd de Mcl-1/Noxa

route.

Hoofdstuk 3

In dit hoofdstuk werd aangetoond dat γ-secretase remmers, een geneesmiddel voor

de behandeling van Alzheimer, ook effectief is in het induceren van apoptose in CLL.

Wij ontdekten dat het toedienen van dergelijk geneesmiddel aan CLL cellen leidt tot

een signifi cante remming van het proteasoom. Het blokkeren van het proteasoom

heeft als gevolg dat eiwitten zich ophopen in de cel, waarbij de accumulatie van Noxa

het meest prominent was. De sleutelrol van Noxa kon worden aangetoond met RNA

interferentie technieken. Deze bevindingen werpen licht op nieuwe geneesmiddelen

voor de behandeling van CLL, maar ook voor andere types kanker. Doordat er

een groot aantal γ-secretase remmers reeds beschikbaar zijn in de kliniek voor de

behandeling van de ziekte van Alzheimer, zal er veel sneller een antwoord zijn voor

de toepassing ervan in kanker.

Hoofdstuk 4

Eerder was er al aangetoond dat de regulatie van celdood van CLL cellen verstoord is.

Er werd een uitgebreid onderzoek gedaan naar een scala van apoptose regulerende

eiwitten. Het resultaat was echter paradoxaal, want naast het waargenomen anti-

apoptotisch profi el was ook de expressie van de pro-apoptotische ‘BH3-only’

eiwitten Noxa en Bmf fors gestegen in CLL B lymfocyten t.o.v. B lymfocyten van

een gezonde donor. Echter, deze analyses zijn gedaan op CLL B lymfocyten uit het

perifeer bloed, die bij kweek in het laboratorium (in vitro) spontaan in apoptose gaan.

Deze spontane apoptose kon voorkomen worden door condities te simuleren zoals

deze voorkomen in lymfeklieren. Dit geschiedde middels het kweken van CLL cellen

in de aanwezigheid van ‘hulp’ cellen (fi broblasten) die de CD40 receptor op CLL


160cellen stimuleren (CD40 model). Deze vaststelling leidde ertoe dat er (in hoofdstuk

4) in klierweefsel van CLL patienten (ex vivo) werd gekeken naar het expressie

profi el van apoptose regulerende moleculen om zo onderliggende stoornissen

in apoptose genen in deze compartimenten (perifeer bloed en lymfeklier) in kaart

te brengen. We konden zien dat, net als in het in vitro CD40 model, er ook in de

lymfeklier een anti-apoptotisch profi el aanwezig is: de anti-apoptotische eiwiten

(Mcl-1, Bcl-XL en A1) waren hoog en het pro-apoptotische eiwit Noxa was laag.

Onze hypothese is dat op de plek waar groei van CLL cellen plaatsvindt door

omgevingsfactoren, waarschijnlijk in de lymfeklier, de dodelijke apoptose

genen onderdrukt worden waardoor er bij die cellen resistentie optreedt voor

chemotherapie. Wanneer de cellen in het perifeer bloed terecht komen valt die

bescherming weg, waardoor ze gevoelig worden voor apoptose en chemotherapie.

Hoofdstuk 5

Om meer inzicht te verwerven in onze hierboven opgestelde hypothese werd er

in hoofdstuk 5 onderzoek gedaan naar veranderingen in het apoptotische profi el

en de consequenties voor chemotherapie gevoeligheid tussen CLL cellen in de

lymfeklier en cellen die de lymfeklier micro-omgeving hebben verlaten om in de

periferie verder te circuleren. Deze situatie werd nagebootst in vitro door CLL cellen

gedurende een bepaalde periode in kweek te brengen bij ‘hulp’ cellen (het hierboven

beschreven CD40 model). Onze bevindingen toonden aan dat het gewijzigd apoptose

genexpressie profi el dat wordt waargenomen na CD40 stimulatie in het CD40 model

systeem normaliseert na het wegnemen van deze stimulus. Onverwachts werd echter

gevonden dat het pro-apoptotisch eiwit Bim en het anti-apoptotisch eiwit Mcl-1 nog

een lange tijd ontregeld waren en dit had consequenties voor de gevoeligheid voor

sommige chemotherapeutica. CLL cellen werden opnieuw gevoelig voor proteasoom

remmers en voor fl udarabine (tegenwoordig eerstelijns therapie in CLL), maar niet

voor roscovitine. Verondersteld werd dat Bim en Mcl-1 cruciaal zijn voor apoptose

geïnduceerd door roscovitine. Er kon geconcludeerd worden dat de bepaling van het

apoptotisch profi el en de gevoeligheid voor chemotherapeutica in het CD40 in vitro

model waardevolle informatie zou kunnen geven voor de keuze van een geschikte

therapie in CLL.

Hoofdstuk 6

In hoodstuk 6 werd er gezocht naar therapeutische strategieën om resistentie in

CLL te omzeilen. Ook hier werd het CD40 model gebruikt om CLL cellen te

stimuleren. Hier kon opnieuw bevestigd worden dat CLL cellen gestimuleerd via de

CD40 receptor een zelfde anti-apoptotisch eiwit profi el vertonen als in de lymfeklier


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161(ex vivo) en dat deze cellen ongevoelig zijn voor alle geteste chemotherapeutica. In

Chronische Myeloide Leukemie (CML) wordt hetzelfde anti-apoptotische eiwit profi el

waargenomen. Bij CML is er sprake van een translocatie tussen het 9e en het 22e

chromosoom, waardoor er het BCR/Abl fusie-gen ontstaat. Dit is een versmelting

tussen het Abl-(Abelson mouse leukemia) gen afkomstig van chromosoom 9 en BCR-

(breakpoint cluster region) gen van chromosoom 22. Veelbelovend in de behandeling

van CML zijn de Abl-inhibitors imatinib en dasatinib. Beide middelen veroorzaakten

een normalisatie van het anti-apoptotisch eiwit profi el, met als gevolg dat de CLL

cellen opnieuw gevoelig werden voor alle geteste chemotherapeutica. Dit was ook

het geval voor cellen van CLL patienten waarvan het p53 eiwit niet functioneel was.

Er kon zo geconcludeerd worden dat combinaties van medicijnen cruciaal zijn in

de behandeling van resistentie in CLL. De combinatie van Abl-inhibitors imatinib of

dasatinib met andere chemotherapeutica lijken een veelbelovende benadering voor

de behandeling van CLL.


162


Dankwoord


164


Het onderzoek beschreven in dit proefschrift begon 4 jaar geleden ... in een enorme

fi le op de A2 richting Amsterdam. Eenmaal aangekomen op mijn sollicitatiegesprek

was alle fi le-ellende snel vergeten en was ik ten volle overtuigd van dit project en

mijn nieuwe omgeving! Ik was klaar voor de overzet naar het noorden! Het einde van

een periode van hard werken is nu nabij en ik moet eerlijk toegeven dat het niet altijd

mee zat. Dit AIO-schap zorgde voor een bijzondere confrontatie met mezelf. Nu kan

ik oprecht zeggen dat ik erg trots en verheugd ben en dit is te danken aan heel veel

mensen om me heen!

Allereerst de begeleiders, die dit promotieonderzoek hebben gestart en opgevolgd.

Eric, hoewel onze communicatie niet altijd vlekkenloos verliep, heb ik veel van je

bijgeleerd. Je altijd kritische blik op het plannen, uitvoeren en uitwerken van proeven,

hebben mij zeer zeker wetenschappelijk gevormd. Je geduld en toewijding in het tot

stand brengen van kwalitatieve publicaties waren onontbeerlijk voor het succesvol

afronden van het promotietraject.

Rien, ik bewonder hoe je steeds met ethousiasme het onderzoek benadert. Je goede

begeleiding, maar ook je betrokkenheid vormden een belangrijke steun. Ik kon altijd

bij je terecht, hartelijk dank daarvoor.

René van Lier, ook voor jouw inzet in dit werk ben ik erg dankbaar. Elke donderdag,

tijdens de werkbespreking, stond je klaar voor onze CLL groep om de data kritisch te

analyseren en bij te sturen. Je verbaasde me telkens weer met je kennis en snelheid

van denken.

Eric, Rien en René, bedankt dat ik bij jullie kan promoveren!

En dan de onmisbare hulp van Annelieke en René Spijker.

Annelieke, liefste paranimf, ik bewonder je gedrevenheid. Ik wil je bedanken voor je

essentiële bijdragen aan dit proefschrift. Niet alleen onze samenwerking in het CD40

werk, maar ook onze congresbezoekjes en andere spontane gelegenheden waren

altijd geweldig. Ik vergeet nooit onze ‘shopping-acts’, hoe kan ik dat vergeten, mijn

hele huis ligt vol met die zooi! En hopelijk komen er nog veel leuke momenten in de

toekomst.

René, volgens mij ben jij degene aan wie ik het meest vragen heb gesteld tijdens mijn

hele AIO periode. Je bent daarom mijn ‘AMC-Carlo’. Bedankt voor je altijd luisterend

oor en waardevolle discussies over wetenschap. Ook al je inzet in de langdurige

zoektocht naar Bim wil ik hier nog eens in het licht zetten. Zonder jouw IP-kunsten

was hoofdstuk 2 niet wat het nu is. Ik wens je veel succes en geluk toe met je nieuwe

baan.

Dan

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Overige leden van de CLL-apoptose groep,

Arnon, je altijd enthousiaste samenwerking heb ik enorm gewaardeerd. Je bent niet

alleen een goede arts en onderzoeker, maar ook een gezellige collega. Ik ben je

erg dankbaar voor je inzet in dit proefschrift. Rogier, we zijn ongeveer tergelijkertijd

begonnen en we eindigen ongeveer samen in oktober in de Lutherse kerk. Wat goed

zeg! Ik wil je bedanken voor de leuke tijd samen. Zowel in het lab als ernaast. Felix,

gezellige altijd vrolijke collega, bedankt voor de leuke tijd en veel success met je

Noxa beestjes (Ik stem alleen niet volledig in met je keuze van ‘decoratie’ in onze

oude AIO kamer). Margot en Jacqueline, jullie kwamen er pas het laatste jaar van

mijn AIO-schap bij, bedankt voor de gezellige samenwerking. Dieuwertje, bedankt

voor het opwerken van alle patiëntenmateriaal. Dankzij jou kunnen wij meer proeven

doen! Ook de hulp van Ingrid was onmisbaar. Bedankt voor de vele keren dat ik

je mocht storen met weer eens ‘een vraagje’. Bovendien wil ik mijn student Bart

bedanken voor het nuttige ‘bartezomib’ werk, alsook de nieuwe studenten Chantal en

Iris. Jullie motivatie en inzet voor het CLL onderzoek vind ik geweldig!

Oude en nieuwe kamergenootjes,

Henrike, liefste paranimf, rechter-kant-in-de-kamer van me, een speciaal woord van

dank voor jou. We zijn echte maatjes geworden! Ik wil je van harte bedanken voor de

waardevolle tijd samen. Onze hilarische ‘hupslesjes’ gaven ons telkens weer nieuwe

energie (zelfs de psychological warfare met die twee keno’s daar). To be continued!

Daphne, ‘panter-verzorgster’, ook voor jou een speciaal woord van dank. Ik heb

genoten van onze gezellige tijd samen zowel binnen als buiten het AMC. Bedankt

voor alle ‘catsitting’ en dat ‘cadeautje’ dat je nooit terug mag geven is misschien wel

het proberen waard?

Niet te vergeten zijn de oude kamergenootjes. Ester van Leeuwen, bedankt om mij

bij de hand te nemen in het eerste jaar van mijn AIO-schap. Je vertrek naar San

Diego heeft me niet koud gelaten. Nuno (Nuni), I admire your qualities as a scientist.

Thank you for all the helpful commentaries and discussions on my work. Ook bedankt

aan alle andere kamergenootjes, Godelieve, Elsa, Kirsten, Marjolein, Madeleine en

Alek.

Uiteraard wil ik alle stafl eden, Kris, Jörg en René Lutter en verder ook Martijn en

Robert bedanken voor jullie waardevol technisch en inhoudelijk advies op mijn werk.

Ook alle andere collega’s van het Laboratorium voor Experimentele Immunologie

en het Laboratorium Speciële Hematologie wil ik bedanken voor hun input en

collegialiteit. Bovendien wil ik Carel en Laura van de afdeling pathologie bedanken

voor de bijdrage aan dit proefschrift, met name voor het tot stand komen van hoofdstuk

166


4 en de immunohistochemische kleuringen van klierweefsel.

I would like to give a special word of gratitude to Fiona and Fabrizio. I feel lucky for

both meeting you at that interesting apoptosis congress in Rolduc. Fiona, my running

mate, in this last two years of my PhD, you were very helpful to me. Not only on the

level of research but also personal. Thank you so much and I am honoured to be

invited to your wedding in Oxford. It will be amazing! Fabrizio, special friend of mine,

thank you for our interesting discussions, both research and non-research, thank you

for reading parts of this work and for helping me dealing with ‘Kinsky’!

En als laatst de onschatbare waarde van mijn familie.

Liefste mammie en pappie, bedankt voor al wat je mij gegeven hebt. Het resultaat

is er! Jullie kleinste ‘achterkomertje’ waar jullie zo trots op zijn volgt haar tao tot ze

haar doel bereikt en ze is er ontzettend gelukkig om! Mama, ik bewonder je altijd

aanwezige kracht. Papa, ik bewonder je stille bron van intelligentie. Papa, bedankt

voor je ch’an-lesjes.

Dimitri, wonder broer, het bewijs dat we geboren zijn voor wetenschap en geneeskunde

is er. Onze urenlange gesprekken over het ontstaan van het heelal, over ‘oneindig’

(al werd ik er soms helemaal gek van), over biologie, fysiologie, anatomie, en nog zo

veel meer, blijven me eeuwig bij. Bedankt voor al je inzet in mijn vorming. Bedankt

grote broer!

Lientje, van paarden-freaks tot schoonzussen tot soulmates! For ever!

Sophie, mijn grote zus, Thierry, Magalie en Florence, bedankt voor jullie altijd

aanwezige interesse en steun in mijn vak.

Joachim, broere (‘Chipje’, ‘John’), Isabel, Manou en Elias, ook jullie zijn er altijd voor

mij met even grote interesse en waardering. Bedankt!

Ook de belangstelling van mijn schoonfamilie voor mijn werk stel ik ten zeerste op prijs.

Liefste schoonpa en schoonmama, bedankt voor alle mooie ontspannende tijden met

jullie die zeer zeker hebben bijgedragen tot wat ik nu ben. Yves Jr., Charlotte, Céleste

en klein wondertje; Catherine en Mikaël; Caroline en Michaël, bedankt voor jullie

interesse en steun. Met name Caroline, hartelijk dank voor je inzet bij het layouten

van dit werk.

Tot slot, Guy, liefde van mijn leven, hoe zeer ik jou wil bedanken voor alles wat je voor

me doet is eigenlijk onbeschrijfl ijk. Zonder jou had ik het nooit gered! Je bent mijn

wederhelft en ik laat je nooit meer los! Ti amo!!!

Dan

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168


Curriculum vitae


170


De auteur van dit proefschrift, Delfi ne Hallaert, werd op 9 december 1977 geboren

te Brugge, België. In 1996 behaalde ze haar middelbaar diploma aan het Sint-

Jozefsinstituut te Brugge. In 1998 startte zij de studie Biomedische Laboratorium

Technologie aan de Katholieke Hoge School, Brugge, België. Tijdens deze studie liep

zij stage bij de afdeling Klinische Chemie aan het Sint-Jan ziekenhuis te Brugge (Prof.

Blaton). In 2001 behaalde zij haar diploma met onderscheiding. Aansluitend studeerde

zij Biomedische Wetenschappen aan de Vrije Universiteit Brussel, te Brussel, België.

Tijdens deze studie liep zij stage bij de afdeling Immunologie aan de Vrije Universiteit

Brussel, België (Prof.dr. Thielemans). In 2004 behaalde zij haar diploma met grote

onderscheiding. In dit zelfde jaar begon zij als assistent in opleiding op de afdeling

Hematologie (hoofd Prof.dr. M.H.J. van Oers) en Experimentele Immunologie (hoofd

Prof.dr. R.A.W. van Lier), aan het Academisch Medisch Centrum, Universiteit van

Amsterdam, te Amsterdam, om onder leiding van Prof.dr. van Oers en dr. Eldering

aan het in dit proefschrift beschreven onderzoek te werken. In september 2008 start

zij met een verkort programma van de opleiding Geneeskunde aan de Universiteit

van Nijmegen.

Cu

rricu

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vita

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173LIST OF PUBLICATIONS

1. Smit L, Hallaert DYH, Spijker R, de Goeij B, Jaspers A, Kater AP, van Oers MHJ,

van Noesel CJ, Eldering E. Differential Noxa/Mcl-1 balance in peripheral versus

lymph node chronic lymphocytic leukemia cells correlates with survival capacity.

Blood, 2007; 109 (4): 1660-1668.

2. Hallaert DYH, Spijker, Jak M, Derks IAM, Alves NL, Wensveen FM, de Boer

JP, de Jong D, Green SR, van Oers MHJ and Eldering E. Cross-talk among Bcl-2

family members in B-CLL: seliciclib acts via the Mcl-1/Noxa axis and gradual

exhaustion of Bcl-2 protection.

Cell Death and Differentiation, 2007; 14: 1958-1967.

3. Hallaert DYH, Jaspers A, van Noesel CJ, van Oers MHJ, Kater AP, Eldering E. c-Abl

Kinase Inhibitors Overcome CD40-Mediated Drug Resistance in CLL; Implications for

Therapeutic Targeting of Chemoresistant Niches. Blood (accepted for publication).

Lis

t of p

ub

licatio

ns


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Co

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ure

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COLOR FIGURES

Chapter 1. Figure 1. The two main pathways leading to apoptosis. The extrinsic pathway is

triggered by ligation of cell surface receptors, such as Fas, resulting in activation of caspase-8.

The intrinsic pathway is activated by cytotoxic stimuli, such as DNA damage, which leads to

the release of apoptosis promoting factors from the mitochondria. In the cytosol, cytochrome c

results in the activation of caspase-9. This pathway is regulated by the Bcl-2 family of proteins.

Activated caspase-8 and -9 in turn activate effector caspase-3, -6 and -7. Cross-talk between

the pathways occurs through Bid, which is cleaved by caspase-8 and then can activate the

intrinsic pathway.


Chapter 2. Figure 5C. Bim and Bax association with Bcl-2 shifts to the mitochondria-rich

insoluble fraction over time in B-CLL cells.

Immunocytochemical staining for Bax of B-CLL cells incubated in the absence or pres-

ence of 25 μM seliciclib for 24 hrs. MitoTracker Orange was used for localization of the

mitochondria.

chapter 4. Figure 1: Histology of lymph node infi ltrated by B-CLL cells.

Ubiquitously present B-CLL cells were positive for CD23 and CD5. Scattered CD3+ T cells were

present throughout the LN. The absence of clusters of BCl-6+ cells excluded the presence of

germinal center remnants in the LNs. Ki67/CD20 and Ki67/CD3 double staining indicate that all

cycling Ki67+ cells (pink) were of CD20+ (brown) origin - see also inset -, while the CD3+ T cells

were predominantly Ki67 negative. Magnifi cation 40x.

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Crosstalk among Bcl-2 family members in B-CLL: seliciclib acts via the Mcl-1/Noxa axis and gradual...

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