doi:10.1182/blood-2008-05-155200Prepublished online December 4, 2008;   

 Wilkens, Brigitte Schlegelberger, Arnold Ganser and Christopher BaumYang, Jurgen Krauter, Nils von Neuhoff, Michael Heuser, Helmut Diedrich, Gudrun Gohring, Ludwig Zhixiong Li, Gernot Beutel, Mathias Rhein, Johann Meyer, Christian Koenecke, Thomas Neumann, Min High affinity neurotrophin receptors and ligands promote leukemogenesis

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High affinity neurotrophin receptors and ligands promote

leukemogenesis

Zhixiong Li1, Gernot Beutel2, Mathias Rhein1, Johann Meyer1, Christian Koenecke2, Thomas

Neumann1, Min Yang1, Jürgen Krauter2, Nils von Neuhoff3, Michael Heuser2, Helmut Diedrich2,

Gudrun Göhring3, Ludwig Wilkens3, Brigitte Schlegelberger3, Arnold Ganser2, Christopher Baum1,4

1Department of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany 2Department of Hematology, Hemostasis, Oncology, and Stem Cell Transplantation, Hannover Medical School, 30625 Hannover, Germany 3Institute of Cell and Molecular Pathology, Hannover Medical School, 30625 Hannover, Germany 4Division of Experimental Hematology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio 45229-3039, U.S.A.

ZL, GB, and MR contributed equally to this work.

Corresponding author:

Zhixiong Li, MD or Christopher Baum, MD Department of Experimental Hematology, OE6960 Hannover Medical School Carl-Neuberg-Straße 1 30625 Hannover Germany Phone: +49 511-532-5148 Fax: +49 511-532-5105 E-mail: li.zhixiong@mh-hannover.de Baum.christopher@mh-hannover.de Running title: leukemogenesis of TRK signaling

Category: neoplasia Part of the data was presented in an oral session at the 2007 Annual Meeting of the American Society

of Hematology in Atlanta.

Blood First Edition Paper, prepublished online December 4, 2008; DOI 10.1182/blood-2008-05-155200

Copyright © 2008 American Society of Hematology

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Abstract

Neurotrophins (NTs) and their receptors play a key role in neurogenesis and survival. The TRK

(tropomyosin-related kinase) receptor protein tyrosine kinases (TRKA, TRKB, TRKC) are high affinity

NT-receptors that are expressed in a variety of human tissues. Their role in normal and malignant

hematopoiesis is poorly understood. In a prospective study involving 94 adult patients (mean age 54.3

years), we demonstrate for the first time cell surface expression of the three TRKs and constitutive

activation in blasts from patients with de novo or secondary acute leukemia. At least one TRK was

expressed in 55% of the analyzed cases. We establish a clear correlation between the TRK

expression pattern and FAB classification. While only few point mutations were found in TRK

sequences by RT-PCR, we observed co-expression of BDNF (ligand for TRKB) in >50% of TRKB+

cases (16/30). Activation of TRKA or TRKB by NGF and BDNF, respectively, efficiently rescued

murine myeloid cells from irradiation-induced apoptosis. Co-expression of TRKB/BDNF or TRKA/NGF

in murine hematopoietic cells induced leukemia. Moreover, activation of TRKs was important for

survival of both human and murine leukemic cells. Our findings suggest that TRKs play an important

role in leukemogenesis and may serve as a new drug target.

Keyword: acute leukemia, TRKs, BDNF, autocrine loop, protein-tyrosine kinase

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Introduction

Current concepts of leukemogenesis postulate a collaboration of ‘class I’ mutations that result in for

example constitutively activated protein-tyrosine kinases (PTKs) with ‘class II’ mutations of

transcription factors such as AML/Runx or ETS proteins. In this scenario, class I mutations (such as

BCR/ABL and FLT3-ITD) promote proliferation but generally do not inhibit differentiation, while the

reverse is true for class II mutations.1 While the molecular analysis of patient samples has supported

this concept in human acute myeloid leukemia (AML), there still remain a substantial proportion of

patients in whom both types of mutations have not yet been demonstrated.

There is growing evidence for involvement of multiple PTK oncogenes, their immediate downstream

targets (e.g. phosphatidylinositol 3-kinase=PI3K), or of proteins regulating their function in

hematological malignancies.2-5 The fact that cytogenetic remissions can be achieved in the majority of

patients with chronic myeloid leukemia (CML) demonstrates a causal role of the BCR-ABL oncoprotein

in this disease.6 Analysis of activated PTK is also of clinical relevance.7,8 At least one third of AML

patients carry mutated FLT3 alleles and have unfavorable prognosis.8,9 It is thus important to identify

other PTKs that are activated in the remaining patients. Moreover, co-activation of receptor PTKs has

been suggested to be important for tumor development and to affect the tumor cell response to

targeted therapy.10 Oncogenic transformation by PTKs occurs in different ways,11 e.g. by genomic re-

arrangements, such as chromosomal translocations, gain-of function (GOF) mutations, PTK

overexpression or small deletions in receptor PTKs and cytoplasmic PTKs. Autocrine and/or paracrine

loops have been suggested as important mechanisms for aberrant kinase activation in human solid

tumors12 and leukemia,13 and may have therapeutic potential.14 However, few experimental studies

convincingly demonstrate the oncogenic potential of autocrine/paracrine circuits of PTKs in animal

models,12,15 and a prognostic role of autocrine loops in human leukemia has not been demonstrated.

The neurotrophins (NTs), which include nerve growth factor (NGF), brain-derived neurotrophic factor

(BDNF), NT-3, NT-4, and NT-6, play a major role in neuronal survival. NTs are unique in that they

utilize two different classes of receptors: the TRK (tropomyosin-related kinase) receptor protein

tyrosine kinases (TRKA, TRKB, TRKC) and the low affinity NGF receptor (LNGFR=p75NTR),16 a

member of the tumor necrosis factor cytokine receptor family. The biologically active receptors for

NGF, BDNF and NT-3 are TRKA, TRKB, and TRKC, respectively. NT-3 can bind to all of the TRK

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receptors, and NT-4 binds preferentially to TRKB. NT binding to TRK receptors leads to dimerization of

receptors and kinase activation. LNGFR may modulate the activity of this signaling complex but is not

required for its function.16 Of note, human embryonic stem cell survival has recently been shown to be

mediated through activation of TRK receptors by NTs.17 Members of the TRK family have been found

in several non-neural cell types,18,19 and may also play a crucial role in initiation, progression, and

metastasis of many tumors in humans, e.g. neuroblastoma, medullary thyroid carcinoma and breast

cancer.20-23 In addition, some data indicate relevance of TRK receptors as prognostic factors.20 For

example, TRKB is associated with bad prognosis in Wilms´ tumor.24 However, relatively little is known

about the mechanisms of oncogenesis mediated by altered TRK signaling.25,26

Many PTK oncogenes are derived from genes (e.g. Abl, FLT3, c-Kit and PDGFR-ß) normally involved

in the regulation of hematopoiesis or hematopoietic cell function.2 TRK receptors and their respective

ligands are also expressed at various stages of hematopoiesis.27,28 A role for neurotrophins in

hematopoiesis has yet to be confirmed using conditional knockouts. Nevertheless, recent data

suggested important functions of TRK signaling in hematopoiesis. TRKs promote proliferation and

survival of lymphocytes and monocytes/macrophages.29 TRKB expression is greatest in precursor

CD4-CD8- thymocytes and progressively declines throughout the T cell differentiation pathway.30

Importantly, there is increasing evidence for involvement of TRK receptors in leukemogenesis. A

cryptic translocation t(12;15) (p13;q25), which resulted in the chimeric transcript TEL-TrkC, was found

in an AML patient.25,31 Furthermore, a deleted form of TRKA (ΔTrkA), in which 75 amino acids are

lacking in the extracellular domain, was identified in another AML patient.32 In mouse models, we

found that ΔTrkA is a very potent oncogene that transforms cells mainly via PI3K and mTOR.33

Another study revealed the induction of TRKA and a contribution of NGF to survival signaling in

human cord blood cells transduced with retroviral vectors encoding the AML1-ETO oncogene.34 In

addition, we had evidence to suggest that a cytoplasmically deleted form of LNGFR may contribute to

leukemia in mice.35 Furthermore, we observed expression of LNGFR in patients with acute leukemia

(AL), preferentially in common ALL.36 Taken together, these data suggest a previously underestimated

role of NT signaling in leukemogenesis. However, with the exception of one report showing expression

of TRKA mRNA in primary leukemic cells in 44% of AML patients,37 the expression pattern of other

TRKs and NTs and their potential prognostic relevance have not been reported in primary leukemic

cells.

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Here, we screened AL patients for expression of TRKA, TRKB and TRKC. A distinct expression

pattern of these receptors in different leukemia subtypes as well as expression of NTs was observed.

Co-expression of TRKB and BDNF was associated with poor outcome and induced leukemia in a

murine model. Moreover, TRK signaling was important for maintenance of leukemic cells in vitro. This

study expands current concepts of leukemogenesis and encourages further evaluation of NT receptor

signaling as a drug target in leukemia therapy.

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Methods

TRK and NTs expression in leukemic blasts of AL patients

We studied tumor specimens from AL patients who had been diagnosed in the Hannover Medical

School from 2004 to 2006. Blood and/or bone marrow (BM) samples from AL patients were collected

at diagnosis after informed consent was obtained in accordance with the Declaration of Helsinki.

Mononuclear cells from all samples studied were immediately isolated by centrifugation over Ficoll

gradient and freshly used or stored at –1800C until further use. The following monoclonal antibodies

were used: anti-TRKA (clone H10, Biodesign), anti-TRKB (clone75133, R&D), anti-TRKC (clone

75219, R&D), anti-BDNF (Cat# GF35L, CALBIOCHEM), anti-NT-3 (clone 41512, R&D). A polyclonal

antibody against human NGF (cat# BAF256, R&D) was used for detection of NGF. Antibodies were

validated on cell lines that expressed retrovirally encoded TRK receptors and NTs. The study was

approved by the ethics committee of the Hannover Medical School. The majority of patients were

treated according to previously described protocols.38 For surviving patients, the median follow-up

period after diagnosis was 30 months. In all cases, cytomorphologic classification according to FAB

criteria was made on bone marrow and/or peripheral blood smears.

Retroviral vectors, vector production, retroviral transductions, in vivo tumorigenesis assays,

and tumor phenotyping

A retroviral vector encoding full-length human TRKA was kindly provided by Dr. Gary Reuther

(University of South Florida, Tampa, Florida).32 Plasmid SF91.IRES-EGFP.WPRE, which mediates

efficient transgene expression in hematopoietic cells, has been described.33 The retroviral vector is

referred to as SF91-IE. A PCR fragment containing the cDNA of human TRKB or BDNF was

generated and cloned into the NotI site before the IRES-EGFP cassette of SF91-IE. Resulting vectors

encoding TRKB or BDNF were named SF91-TRKB and SF91-BDNF, respectively. Cell free high-titer

supernatants containing the ecotropic envelope protein were generated as described.33 Retroviral

transductions, in vivo tumorigenesis assays, and tumor phenotyping were performed as previously

described (supplementary information).33

Radiation-induced apoptosis assay and leukemic cell growth assays

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2.5×105 32D cells were starved from IL-3 and serum for 3 hours, placed in 24-well plates, and

exposed to 5 Gy -irradiation. Immediately after irradiation, cells were supplemented with NGF, BDNF

(each 100 ng/mL), IL-3 (2 ng/mL), or no factor. Cell viability was analyzed using the Annexin-V assay.

Cells staining negative for both Annexin-V and propidium iodide were counted as viable cells. To

analyze clonal growth, 2x102 or 2x103 murine cells were plated per dish in M3234 media (StemCell

Technologies, Vancouver, Canada) in the presence of signal transduction inhibitors. The assays were

plated as duplicates or quadruplicates, and colonies were counted on day 6. Mononuclear cells from

patients were cultured in RPMI 1640 supplemented with 10% fetal calf serum (FCS) and exposed to

inhibitors and/or idarubicin.39 Inhibitors K252a, AG879 (Calbiochem, Merck, Bad Soden, Germany)

and anti-human BDNF antibody (Promega, Mannheim, Germany) were used.

Western blot analysis

For signal transduction analysis, leukemic cell extracts were prepared following established

protocols.33 Cell lysates were used as indicated in results. Antibodies were purchased from Santa

Cruz Biotechnology (Santa Cruz, California, U.S.A.).

RNA extraction and reverse transcriptase– polymerase chain reaction (RT-PCR), small

interfering RNA (siRNA)-mediated knockdown, BDNF-ELISA, and statistical analysis

Please refer to supplementary information.

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Results

Frequent expression of TRK receptors in leukemic blasts of AL patients

We analyzed expression of TRKs in AL patients. 94 adult patients (42 female, 52 male) with a mean

age of 54.3 years and diagnosis of primary or secondary AML (87%), ALL (12%), or AUL (1%) were

enrolled after informed consent. The patients´ clinical characteristics are summarized in Table 1.

Expression of TRKA, TRKB and TRKC was detected by flow cytometry using monoclonal antibodies. If

>20% of leukemic blasts expressed at least one of the TRK receptors, cases were considered TRK+

(Figure 1A, B, S1).40 Thus, 55% of the analyzed cases expressed at least one TRK receptor, without

statistically meaningful differences in expression rates between AML (43/82) and ALL (8/11). We

observed expression of TRKA on AML blasts, which is in agreement with a previous study that

demonstrated expression of TRKA on the RNA level.37

For the first time, we found expression of TRKB and TRKC in human leukemia. Interestingly, while

TRKB can be expressed alone in blasts, TRKA or TRKC expression always occurred concomitantly

with TRKB. About 18% of leukemia cases co-expressed all TRK receptors. Co-expression of two or

more TRK receptors (i.e. TRKA+TRKB, TRKB+TRKC and TRKA+TRKB+TRKC) was observed in

AML, while ALL blasts exclusively expressed only TRKB. Although TRK mRNA was found in

hematopoietic cells from healthy volunteers,27,37 we did not detect TRK receptors on the surface of

normal mononuclear cells by flow cytometry (data not shown), which is in agreement with previous

studies.41

Correlation of TRK expression and French-American-British (FAB) leukemia classification

Next, we analyzed the relationship between patient age, FAB subtype, white blood cell (WBC) counts,

ECOG status, cytogenetics, FLT3 mutation and TRK expression. In agreement with recent

publications,8 we found internal tandem duplications of the juxtamembrane region of the FLT3 receptor

(FLT3-ITD) in 25% (17/67) of AML patients. There was no significant correlation of patient age, WBC

counts, ECOG status, FLT3-ITD or cytogenetics with TRK expression (Table 1). However, in contrast

to a previous study,37 we established a clear correlation of TRK expression pattern and the FAB

classification (Table 1). In particular, TRKA was expressed in 21 of 34 myelo-monocytic/monocytic

leukemias (AML M4 and M5) (62%) whereas only 5 of 48 non-myelo-monocytic/monocytic leukemias

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(10%) were positive (p<0.001). The same observation was made for TRKB and TRKC (71% vs. 38%,

p<0.005 and 47% vs. 8%, p<0.001, respectively). Co-expression of at least two of the three TRK

receptors was observed in 62% (21/34) of patients with AML M4 and M5, but in only 13% (6/48) of

other subtypes (p<0.001). The latter cases were often secondary AML after a myelodysplastic

syndrome (MDS). Thus, our data show that TRK expression in AML is closely linked to monocytic

differentiation (AML M4 and M5), unless the AL developed on the basis of MDS. Corroboratively, we

found expression of TRKA in >98% of THP1 cells (a commonly used human monoblastic leukemia cell

line) by flow cytometry (data not shown).

Expression of NTs in leukemia and constitutive activation of TRK in leukemic cells

To assess potential mutations and deletions, we sequenced the 2nd Ig-like domain, transmembrane

domain and whole intracellular domain (focusing on the kinase domain) of TRKA, TRKB and TRKC

receptors following RT-PCR of RNA isolated from leukemic cells. Mutations, small deletions and

duplications in these regions have been reported as potential mechanisms for constitutive activation of

TRKs.32,42 RT-PCR and direct sequencing of PCR fragments were successfully performed in 98% and

97% of patients, respectively. Unexpectedly, only four different point mutations (TRKB: T573I, Y707N,

and V684I; TRKC: Y800H) were found in four patients by RT-PCR. We did not observe deletions or

duplications in TRKs. As oncogenic TRKA was originally cloned in a patient with colon carcinoma as a

TPM3/TRK transcript caused by translocation within chromosome 1,43 we also searched for the

TPM3/TRK translocation in our patients, yet without success (n=32).

Therefore, we investigated other major mechanisms by which TRK expression could contribute to

transformation or differentiation. Autocrine and/or paracrine loop has been suggested as an important

mechanism of PTK activation in human cancers.12,13 Elevated expression of TRKB and/or BDNF has

also been reported to occur frequently in multiple myeloma (24 and 12 out of 25 cases studied,

respectively), promoting myeloma cell survival.44 Thus, we next investigated expression of NTs in

blasts from patients with AL. We chose flow cytometry to detect intracellular expression of NGF, BDNF

or NT-3. Co-expression of BDNF (primary ligand for TRKB) was observed in over half of TRKB+ cases

(53.3%, 16/30) (Figure 1C, S1). Moreover, we found expression of NGF or NT-3 in 2 patients

expressing TRKs (Figure S2). Importantly, we observed constitutive phosphorylation of TRKs in all

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analyzed primary leukemia samples (n=13, Figure 1D), suggesting a role of TRK signaling in leukemic

transformation.

Co-expression of TRKB and BDNF is associated with poor survival

The good response rate on days 15 and 21 after induction therapy was not significantly different

according to TRK expression (62% vs. 81% for TRK– and TRK+, respectively, p=0.16). However, we

found that expression of TRKs and BDNF was associated with bad prognosis (Table 2). Patients

whose blasts express TRKA had a shorter median survival compared with patients whose blasts do

not (312 vs. 547 days, hazard ratio 1.44, p=0.23). Interestingly, in agreement with other reports,45 it

seems that expression of LNGFR is associated with better prognosis (Figure S3). These differences

may reach statistical significance if investigated in larger cohorts.

The survival difference was even more pronounced for patients whose blasts co-express TRKB and

BDNF (Table 2). These patients had significantly shorter overall survival when compared with patients

whose blasts express neither TRKB nor BDNF (8% vs. 30% at 3 years, respectively, p<0.05) (Figure

2). However, event free survival displayed no statistical difference. Importantly, there was no

significant difference regarding incidence of FLT3-ITD in both groups of patients. While this finding is

reminiscent of recent data demonstrating association of the TRKB/BDNF autocrine survival pathway

with poor outcome in human solid tumors,24 we are not aware of a previous report demonstrating a

prognostic relevance of an autocrine or paracrine loop in AL.

Activation of TRK signaling protects myeloid cells from apoptosis and supports proliferation

On cell types other than neuronal cells including mast cells, keratinocytes, monocytes and B cells,

TRK signals potentially stimulate cell proliferation through anti-apoptotic effects.18,46 To examine the

role of TRKs in the regulation of apoptosis in myeloid cells, we used 32D cells transduced with

retroviral vectors expressing TRKA or TRKB.33 We tested whether stimulation of 32D/TRKA cells by

NGF could rescue cells from irradiation-induced apoptosis. 32D/TRKA cells were highly sensitive to

irradiation, with less than 10% viable cells 26 hours after irradiation. Upon NGF exposure, up to 88% of

32D/TRKA cells escaped apoptosis (Figure 3A). Similar data were obtained with 32D cells expressing

TRKB after stimulation with BDNF (Figure 3A and 3B). For yet unknown reasons, activation of TRKB

was not as potent as TRKA in protecting the cells from apoptosis at a later time point (54h). Exposure

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to NGF or BDNF rescued less than 8% of non-transduced 32D cells from apoptosis (Figure 3A).

Importantly, treatment of 32D TRKB cells with K252a (a widely used TRK inhibitor17,47,48) significantly

counteracted the anti-apoptotic effect of BDNF activation (Figure 3C), suggesting that kinase activity

of TRKs is absolutely required for their anti-apoptotic function. Moreover, activation of TRKA by NGF

or TRKB by BDNF also supported proliferation of 32D cells in liquid culture (>1 month) and

methycellulose in the absence of IL-3 (data not shown).

Co-expression of TRKA/NGF and TRKB/BDNF efficiently transforms hematopoietic cells and

induces leukemia in mouse models

To address the in vivo leukemogenic potential of autocrine activation of TRKA and TRKB, we first

used a model based on 32D cells as previously described.33 Retroviral vector-mediated expression of

NGF in 32D/TRKA cells caused growth-factor independence in vitro. Even without prior selection for

growth-factor independence in vitro, retroviral vector-mediated co-expression of TRKA and NGF or

TRKB and BDNF in 32D cells elicited a fatal AML in all syngeneic C3H/Hej recipients (n=4/4 for

TRKA/NGF, n=2/2 for TRKB/BDNF). In contrast, only 4 out of 26 animals transplanted with cells

expressing TRKA alone developed leukemia, and none of the animals transplanted with cells

expressing TRKB or NGF or BDNF alone (n=6) developed donor-cell derived leukemia. Interestingly,

in agreement with the less potent anti-apoptotic effect of TRKB observed in 32D cells, transformation

induced by autocrine activation of TRKB required a longer latency in comparison with TRKA/NGF (13

vs. 3.5 weeks). Importantly, while only 14% of cells were positive for TRKB/BDNF on the day of

transplantation (Figure 4A), the majority of leukemic cells recovered from animals with overt disease

co-expressed both vectors (Figure 4B), strongly suggesting selection for leukemic transformation by

autocrine activation. The developing leukemias led to elevated WBC counts (Figure 4C),

splenomegaly, and hepatomegaly. Cytology and histopathology revealed extensive infiltration of blasts

in the BM, spleen, and liver (Figure 4D-H), and in some cases also in the lung, and kidney (data not

shown). As in the patient samples, constitutive activation of TRKB was observed, suggesting a crucial

contribution of TRK signaling to leukemogenesis (Figure 4I).

We next assessed the ability of autocrine activation of TRKB to transform primary murine

hematopoietic cells. Hematopoietic stem/progenitor cells enriched lineage negative (Lin-) BM cells

were transduced with retroviral vectors expressing TRKB (SF91-TRKB), BDNF (SF91-BDNF), or both.

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Three of 6 animals co-expressing TRKB and BDNF developed lymphoblastic leukemia within 14

weeks after transplantation (Figure S4). The other 3 remained healthy for another 5 months, but had

no or less than 5% transgene expression. All animals (n=6) transduced with TRKB or BDNF alone

showed normal hematopoiesis. Similarly, we observed development of myeloid leukemia in one out of

3 animals transplanted with Lin- cells co-expressing TRKA and NGF (data not shown). Thus, in

agreement with our clinical observation, activation of TRKs induced both myeloid and lymphoblastic

leukemia in our mouse models. Moreover, our data (patients and mouse) support the view that

LNGFR is not absolutely required for TRK-mediated responses.49

Autocrine loop TRKB/BDNF is a survival factor for murine leukemic cells

Leukemic cells from mouse #681 (Figure 4) grew factor-independently. Importantly, supernatant

collected from #681 cells supported growth of 32D cells expressing TRKB without cytokine

supplementation. BDNF was measured to be >300pg/ml by ELISA (in comparison, concentration of

BDNF in serum of human healthy control was 27pg/ml44) and no membrane-bound BDNF was found

by FACS analysis (data not shown). This strongly suggests existence of a TRKB/BDNF autocrine loop

in #681 cells.

We examined the ability of a neutralizing antibody to BDNF, or the TRK inhibitor K252a, to interfere

with the autocrine activation loop. In the absence of treatment, autophosphorylation of TRKB was

detected in leukemic cells isolated from mouse #681, suggesting that the autocrine loop is

constitutively active. Both anti-BDNF neutralizing antibody and K252a dramatically blocked

phosphorylation of TRKB (Figure 4I). If leukemia development is mediated through activation of TRKs

by NT, then pharmacological inhibition of TRK signaling or blocking the action of NT should reduce

leukemia cell survival. To test this hypothesis, survival of leukemic cells isolated from mouse #681

was measured by colony formation in the presence or absence of BDNF-neutralizing antibody and

K252a.17 Anti-human BDNF antibody showed a dose-dependent effect on leukemic cell survival

(Figure 4J). K252a induced up to 50% growth inhibition of leukemic cells (data not shown). In contrast,

addition of either the anti-BDNF neutralizing antibody (Figure 4J) or K252a did not alter the survival of

control cells. Further, we generated 2 lentiviral vectors expressing siRNAs targeted against different

regions on the TRKB mRNA sequence.50 Compared with control vector transduced cells, siRNA

expression reduced colony formation up to 13 fold, and induced cell death in the absence of IL-3 (data

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not shown). Moreover, leukemic cells from mice co-expressing TRKA/NGF were very sensitive to

treatment of K252a and AG879, a TRKA inhibitor (Figure S5). Collectively, these data reveal that an

autocrine loop involving TRK receptors is a major survival factor for leukemic cells in the murine

model.

TRK signaling is important for survival of human AML cells

We evaluated apoptosis in cultured leukemic cells obtained from 4 patients with AML. Cells were

cultured in RPMI 1640 (10% FCS) and exposed to the TRK inhibitor K252a (100nM – 400nM) for

approximately 18 hours. We found that K252a induced apoptosis over basal levels in all cases and led

to up to 65% reduction of living cells by FACS analysis (Figure 5A, 5B), while K252a had minimal

effect on leukemic cells from patients T17 and T56 not expressing TRKs (Figure 5A, 5B). Moreover,

we also observed dephosphorylation of TRK proteins after treatment (data not shown), suggesting that

the apoptotic effect of K252a was mainly due to inhibition of TRK signaling. In patient T72 (co-

expressing TRKB/BDNF only), a moderate reduction of living cells was observed upon anti-BDNF

antibody treatment alone or in the combination with K252a (Figure S6). This indicates that survival

activity of a portion of leukemic cells is indeed due to an autocrine loop of TRKB/BDNF. However, no

or less apoptotic effect of anti-BDNF antibody was observed in cells from patients T0 (negative for

BDNF) and T10 (expressing TRKs and BDNF) (Figure S6). In the latter case, it is possible that the

potential effect of the anti-BDNF antibody was compensated by signaling driven from TRKA and/or

TRKC. K252a also enhanced idarubicin-induced apoptosis in leukemic cells from patients T72 and T0

(data not shown). However, cells from patients with ALL (T14 and T20) were resistant or less sensitive

to K252a treatment compared with AML patients (data not shown), suggesting different sensitivities to

TRKs signal transduction inhibition among patients. Generally, the growth inhibition effects of K252a

and anti-BDNF antibody we observed in human leukemic cells (Figure 5, S6) is less strong than in

murine leukemic cells (Figure 4), reflecting the more complex leukemogenesis in humans. However,

our findings collectively suggest that TRK signaling is involved in survival of human blasts, particularly

in AML.

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Discussion

NTs and their receptors regulate proliferation, differentiation, and death of normal and neoplastic

neuronal cells and also have been implicated in the formation of various human cancers.16,24 Although

expression of TRKs has been shown in different stages of hematopoiesis, their functional role has

remained largely unclear. By RT-PCR, expression of TRKs and NTs in adult human BM cells was

much lower than in fetal BM cells, suggesting down-regulation in adult hematopoiesis.27 Consistently,

we and others did not observe surface expression of TRKs in normal mononuclear cells.41 In contrast,

expression of TRKA has been shown in different leukemia cell lines.41 Multiple myeloma cells

expressed TRKB, and responded to BDNF by activating MAPK and PI3K/Akt signaling cascades.44 So

far only one group analyzed expression of TRK in human AML, detecting TRKA mRNA in leukemic

cells of less than half of the patients tested.37 However, expression on the protein level and a potential

correlation with leukemia subtypes or prognosis were not assessed. Using stringent criteria, our study

provides direct evidence for frequent and high expression of TRK receptors in human AL. As the TRK

expression pattern did not only depend on the leukemia subtype but was also correlated with

prognosis, we hypothesize that this receptor system may play a pathogenetic role in human leukemia.

The mechanisms underlying aberrant expression of TRKs and BDNF in leukemic cells are unknown.

By real-time RT-PCR, we observed upregulation of TRKA and BDNF but not TRKB and TRKC in

analyzed samples (n=4, data not shown), suggesting that posttranscriptional events may contribute

the expression of TRKB and TRKC.

One characteristic finding in the present study is that TRK expression is associated with

myelomonocytic and monocytic leukemia (Figure 1 and Table 1). It is possible that the expression of

different members of the TRK family simply reflects the cell of origin, without a significant role in tumor

biology. However, although TRKA can be found in B lymphocytes,18 T lymphocytes,29 and

monocytes/macrophages,46,51 we only observed TRKA expression in AML patients by flow cytometry.

Therefore, it is more likely that differential expression of TRK receptors and activation of their

respective signal transduction pathways directly affects the biologic behavior of the cells, which leads

to differentiation, survival, and/or proliferation. For example, tumors with functional TRKB may be

particularly aggressive because TRKB provides a growth advantage and may protect them from

chemotherapy.24 In the present study, expression of TRKs was associated with shorter survival,

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particularly for patients co-expressing TRKB and BDNF. Our data thus support the concept that TRKB

may play an important role in human tumorigenesis.24,52 In neuroblastoma, TRKB expression has been

demonstrated preferentially in aggressive, MYCN-amplified tumors, and is associated with a poor

prognosis. Activation of the BDNF/TRKB signaling pathway in neuroblastoma cells expressing TRKB

increases cell survival,53 and may protect neuroblastoma cells from chemotherapy and thereby

contribute to a more chemoresistant phenotype.54 In a recent functional genomic screen for genes that

suppress anoikis, TRKB was identified as an oncoprotein associated with metastatic capacity.21

Overexpression of TRKB rendered nonmalignant epithelial cells anoikis (apoptosis)-resistant and

highly tumorigenic. Consistent with the model that suppression of anoikis facilitates metastasis, TRKB-

expressing cells formed highly invasive and metastatic tumors in nude mice, with very short

latencies.21 TRKB kinase activity is required and also sufficient for anoikis suppression, tumor

formation, and experimental metastasis.52 Consistently, we found that TRKB kinase activity was

absolutely required for its anti-apoptotic effect (Figure 3D), potentially a major mediator for

transformation induced by TRK signaling. We also demonstrated that targeting TRKs by TRK inhibitor

or siRNA efficiently inhibited growth of leukemic cells in vitro (Figure 4 and 5). These results suggest

that targeting the enzymatic activity of TRKB or its downstream effectors might be beneficial in therapy

of both solid tumors and leukemia.

Autocrine circuits of tyrosine kinases such as FLT313 or KDR14 are potentially involved in human

leukemia. Multiple myeloma cells were found to express TRKB, with potential survival signaling

induced by autocrine BDNF.44 In all of these cases, a prognostic role remains to be determined.

Additionally, there are so far no animal studies which directly confirm their tumorigenic potential,

although blocking autocrine loop function/activity has been shown to be important for treatment of

leukemia in vitro and in animal models.14 Here, we found a high incidence of expression of

TRKB/BDNF and its association with poor prognosis in our patient cohort (Figure 2). Our mouse

models showed direct evidence for the leukemogenic potential of autocrine activation of TRKA/NGF

and TRKB/BDNF (Figure 4). Consistent with recent publications showing <2% of point mutations in

>90 tyrosine kinases (including TRKs) in >300 AML patients,55,56 we found very few point mutations in

TRK receptors, indirectly supporting the hypothesis that autocrine activation represents a major

mechanism for transformation by TRKs, and probably also for many other receptor PTKs. However,

reflecting the finding that NGF and BDNF are also expressed by stromal cells in bone marrow, and the

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recent finding that TRK receptors can also be activated in the absence of NTs,47,57 we do not rule out

the possibility of other mechanisms for activation of TRKs in leukemogenesis, e.g. paracrine or

intracrine mechanism. In fact, we also observed constitutive activation of TRKs in patients expressing

TRKs only (T20, T42 in Figure 1). Interestingly, autocrine activation of TRK has also been reported in

inflammatory cells including monocytes and has been suggested to be an important factor in the

establishment of autoimmune diseases.46 Moreover, recent data demonstrate that NGF is an autocrine

factor essential for the survival of macrophages infected with HIV. These cells take advantage of their

autocrine NGF, survive for a very long period of time and continuously produce virus particles. Thus,

exploring the effects of inhibiting autocrine TRK signaling may open new therapeutic strategies for

both leukemia and latent HIV infection.51

Development of AML is believed to require the co-operation of class I and class II mutations.1 Our

finding that over half of the patients investigated showed expression/mutations of TRKs reduces the

proportion of patients in whom so far no activation of PTKs (class I) has been identified. Therapies

targeting receptor PTKs have provided remarkable responses in both hematologic cancers and solid

tumors, but their clinical efficacy has been limited in many cases, and few, if any, patients are cured.

Recently, Stommel and colleagues have identified obstacles to meaningful response to single-agent

therapies targeting receptor PTKs utilizing a glioblastoma model.10 They found three or more activated

receptor PTKs in each tumor, and up to 10 activated receptor PTKs in some cases. Combinations of

PTK inhibitors, but not single agents, inhibited PI3K signaling and related sequelae. Consistently,

single-agent targeted therapies for AML patients with FLT3-ITD or c-Kit mutations showed only

moderate efficacy in some cases.8 In the present study, we found a significant proportion of AML

patients with both FLT3-ITD and TRK expression. We speculate that assessment of TRK expression

and signaling in these patients, and combination of targeted therapies or multifunctional kinase

inhibitors directed at activated receptor PTKs may improve outcome.58

In summary, we demonstrate that primary AL cells frequently express TRK receptors and BDNF on

the protein level. We found a significant correlation with TRK expression and morphologic subtypes as

established in the FAB classification, and poor outcome in patients with co-expression of TRKB and

BDNF. The leukemogenic activity of this autocrine loop was confirmed in mouse models. Moreover,

activation of TRKs was an important survival factor for leukemic cells from both patients and mice.

Collectively, our findings suggest that TRKs play an important role in leukemogenesis with autocrine

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stimulation as a potential mechanism for oncogenic activity. Thus, TRKs and their downstream

signaling partners might serve as novel therapeutic targets in AL. To improve classification and

treatment of hematological malignancies, we would recommend the prospective assessment of NTs,

their receptors and downstream pathways in larger cohorts and additional malignancies of

hematopoietic origin.

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Acknowledgments

This study was supported by the Deutsche Krebshilfe (grant: 10-2090-Li I) and by the Deutsche

Forschungsgemeinschaft (DFG, excellence cluster REBIRTH). CB was also supported by the National

Cancer Institute (R01-CA107492-01A2). MR is a student of the MD/PhD program at Hannover Medical

School (MHH), and received support from the Deutsche José Carreras Leukämie-Stiftung (grant:

DJCLS F05/10). CK was supported by DFG (KO 3582/1-1). We are very grateful to Stefan Bartels and

Ludwig Hoy for help with statistical analysis, Axel Schambach for providing vector backbones, Michael

Morgan for providing reagents and critical reading of this manuscript, Peter Horn and Martin Sauer for

providing cells, Vanessa Prox, Christine Garen, Rene Kirstein, Ellen Neumann, Elke Stürmer, Elvira

Lux, and Cindy Elfers for technical assistance, Rolf Baumann, Hans Grundtke, Jörg Frühauf, Anne

Koop, and Bernd Polivka (all MHH) for irradiation of animals and cells. We also thank Dr. D. Martin-

Zanca for providing cDNA of TPM3/TRK.

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Authorship

Z.L., J.M., A.G., and C.B. designed the study. Z.L. and C.B. interpreted the data and wrote the

manuscript. Z.L., M.R., T.N., and M.Y. performed immunophenotyping, mutation analysis, colony

assays, apoptosis assays and animals studies. G.B., C.K., M.H., H.D., and Z.L. collected patients’

data and samples. M.R. and J.M. contributed Western blot analysis. N.N., G.G., L.W., J.K., and B.S.

performed cytogentic and molecular genetic analysis and provided samples. G.B. performed statistical

analysis.

Conflict of interest: The authors declare that no conflict of interest exists.

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Tables

Table 1 patient characteristics according to TRK expression

Characteristic Total TRK-

(% of

total=Column 2 )

TRK+

(% of total=Column 2 )

P

(between TRK+ and

TRK-)

Patients, n 94 42 (45%) 52 (55%)

Age, y

Mean 54.3 52.9 55.5 0.41

Range 16 - 79 20 – 77 16 – 79

Sex, n 0.17

Male 52 27 25

Female 42 15 27

Leukemia 0.358

AML, n 82 39 (48%) 43 (52%)

ALL, n 11 3 (27%) 8 (73%)

AUL, n 1 0 1

Diagnosis (AML), n 0.057

De novo 58 32 (55%) 26 (45%)

Post-MDS/secondary 24 7 (29%) 17 (71%)

FAB subtype AML 0.003

M0 3 3 0

M1 25 16 9

M2 17 10 7

M3 1 1 0

M4 21 9 12

M5 13 0 13

M6 1 0 1

M7 1 0 1

% Blasts

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Bone marrow 73 76 70 0.280

Peripheral blood 44 49 39 0.173

WBC counts, x109/L

Mean 24.4 61.0 0.113

(cutoff: 20.000) 59

Range 0.8 - 159.0 0.6 - 454.0

ECOG status 0.764

0 22 10 12

1 52 25 27

2 13 7 6

3 1 0 1

Cytogenetics 0.481

Normal karyotype 39 17 (44%) 22 (56%)

t(8;21) (q22;q22) 1 1 0

inv(16) (p13q12) 1 1 0

t(15:17)(q22;q11-21) 1 1 0

t(11q23) 2 1 1

Complex karyotype* 21 11 10

Other aberrations 24 8 16

FLT3-ITD-, n (%) 50 26 (52%) 24 (48%) 0.363

FLT3-ITD+, n (%) 17 6 (35%) 11 (65%)

missing 17 (18%)

*Complex karyotype was defined as 3 or more cytogenetic abnormalities in the absence of t(8;21),

inv(16), t(15;17), or t(11q23).60

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Table 2 Univariate Cox regression analysis for TRKs and BDNF

median survival (days)

Hazard Ratio (95% CI) p-value receptor

positive

receptor

negative

TRKA 1.44 (0.80–2.62) 0.23 312 547

TRKB 1.17 (0.66–2.07) 0.60 359 547

TRKC 1.32 (0.70–2.50) 0.40 359 535

BDNF 1.38 (0.68–2.82) 0.37 225 480

TRKB / BDNF 2.26 (1.07–4.80) 0.03 212 547

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Figure legends

Figure 1. Expression of TRK receptors and NTs in patients with AL. (A) Showing the gate for the

blast population. Left panel: Patient T10 (AML M5), middle panel T14 (T-cell ALL), right panel: Patient

T78 (AML M1). (B) Top row showing expression of all three TRK receptors in the blasts of patient T10.

Middle row: patient T14 expressed only TRKB. Bottom row: patient T78 was negative for TRK receptors.

(C) BDNF was also expressed in patients T10 (left panel) and T14 (middle panel), but not in patient T78

(right panel). The isotype controls are shown as insets. (D) Constitutive phosphorylation of p145TRK in

primary leukemic cells. Total cell lysates were blotted and probed with an anti-pTRK (E-6) antibody

detecting phosphorylated forms of all three TRK receptors. The blot was stripped and reprobed with the

anti-TRKB (794) antibody. Please see flow cytometry diagrams in Figures S1 and S3 showing

expression of TRKs and BDNF in the blasts of the patients not shown here.

Figure 2. Kaplan-Meier estimates of overall survival for patients with AML according to TRKs and

BDNF status. Co-expression of TRKB and BDNF was associated with statistically significant poor

outcome. The log-rank test was used to compare differences between survival curves.

Figure 3. Ligand-dependent resistance to radiation-induced apoptosis. (A) 32D cells expressing

TRKA, TRKB, and 32D wild-type cells. Cells were starved for 3 hours and exposed to 5 Gy irradiation.

Cells that were Annexin-V and propidium iodide (PI)-negative were counted as viable cells. Viability was

calculated as the percentage of these cells over the total cell population. (B) BDNF prevented apoptosis

of 32D cells expressing TRKB almost as efficiently as IL-3. Cells were analyzed by flow cytometry 26h

after irradiation. Combined Annexin-V and PI staining was used to distinguish early apoptotic (Annexin-

V+/PI-) and later apoptotic cells (Annexin-V+/PI+). (C) K252a dramatically inhibited anti-apoptotic effects

of BDNF-mediated activation of TRKB, while only slight inhibition was observed if cells were cultured in

the presence of IL-3 alone. Results presented are the mean ± SD of at least 2 independent experiments.

NGF/BDNF concentration was 100 ng/mL, murine IL-3 2ng/ml.

Figure 4. Development of leukemia in mouse #681 transplanted with TRKB and BDNF modified

hematopoietic cells (32D). FACS analysis showing co-expression of TRKB and BDNF before

transplantation (A) and in myeloid blasts recovered from the animal (B). (C) Peripheral blood smear

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showing marked leukocytosis consisting predominantly of immature myeloid cells. (D) Cytospin of BM

showing myeloblasts with an abundant cytoplasm (x 1000). (E, F) Diffuse myeloblastic infiltration in

spleen and complete effacement of its normal structure (x 100, x 1000). (G, H) Extensive infiltration of

leukemic cells in liver, primarily in portal areas, but also diffusely in the sinusoid (x 100, x 1000). (I)

Constitutive activation of TRKB in leukemic cells and blocking of the TRKB/BDNF autocrine loop by anti-

BDNF neutralizing antibody and K252a. Leukemic cells were treated with either 20µg/ml anti-BDNF

antibody or 100nM K252a for 12h. Total cell lysates (300µg) from untreated and treated cells were

immunoprecipitated with the anti-TRK (C-14), separated on SDS-PAGE, blotted and probed with an anti-

pTRK (E-6) antibody (top and middle panels with short and long exposition, respectively). The blot was

stripped and reprobed with the anti-TRKB (H181) antibody. Arrows point to the phosphorylated forms of

TRK. Note different expression pattern of human TRKB in human cells (only 145KDa form in Figure 1)

and rodent cells (both 145KDa and 120KDA forms) as previously described.52 (J) Anti-human BDNF

antibody (10µg/ml and 20µg/ml) inhibited growth of leukemic cells in colony-forming assays. Results are

presented as the average percentage of colonies formed in the presence of antibody (100% value

derived from untreated control). Results presented are the mean ± SD of 2 independent experiments.

Figure 5. K252a induces apoptosis of primary AML cells and. Cell viability was analyzed using the

Annexin-V assay. Results are presented as the percentage of living cells in the presence of K252a

(100% value derived from untreated control). (A) Cells were from patient T72 expressing TRKB and

BDNF, both patients T10 and T29 expressing TRKs (TRKA, TRKB and TRKC) and BDNF, and T0 (only

TRKs). Patients T17 and T56 were negative for TRKs and NTs. Results presented for T10 (K 200nM)

and T29 (K 400nM) are the mean ± SD of 2 independent experiments. Note that treatment schedule was

not completely applied to all patients tested due to limited number of cells. The concentrations of K252a

that we used have been well documented for TRK inhibition in the literature.17,47,48 K=K252a. (B) Flow

cytometry diagram of apoptosis of blasts from patient T17 and T29, cultured with K252a for 18h before

analysis.

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A

B

Li et al., 2008Figure 3

CmIL-3

100 102 104100

102

10414%

12%72%

BDNF

100 102 104100

102

10424%

14%59%

w/o cytokines

100 102 104100

102

10470%

21%7%

Annexin

IP

TRKA

0 10 20 30 40 50 600

25

50

75

100no factormIL-3hNGF

Time [hours]

TRKB

0 10 20 30 40 50 600

25

50

75

100no factormIL-3BDNF

Time [hours]

32Dwt

0 10 20 30 40 50 600

25

50

75

100no factormIL-3hNGFBDNF

Time [hours]

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A

Li et al., 2008Figure 4

B

14% 14%

38%

100 102 104100

102

104

3%

70%4%

100 102 104100

102

104

FN

DB

TRKB

C D

E F

G H

I J

α− pTRKB

α− TRKB

lm/gμ02 bA

Mn001 a252K

detaertnU

For personal use only. by guest on June 8, 2013. bloodjournal.hematologylibrary.orgFrom

For personal use only. by guest on June 8, 2013. bloodjournal.hematologylibrary.orgFrom