Cancer Letters 308 (2011) 1–13

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Cancer Letters

journal homepage: www.elsevier .com/locate /canlet

Mini-review

Notch-ing from T-cell to B-cell lymphoid malignancies

Leonardo Mirandola a,b,d, Paola Comi a, Everardo Cobos b,d, W. Martin Kast c,e,f,Maurizio Chiriva-Internati b,c,d,1, Raffaella Chiaramonte a,⇑,1

a Department of Medicine, Surgery and Dentistry, Università degli Studi di Milano, Milan, Italyb Division of Hematology & Oncology and Texas Tech University Health Sciences Center and the Southwest Cancer Treatment and Research Center, Lubbock, TX, USAc Kiromic, Inc., Lubbock, TX, USAd The Laura W. Bush Institute for Women’s Health and Center for Women’s Health, Texas Tech University Health Sciences Center, Amarillo, TX, USAe Departments of Molecular Microbiology & Immunology, Obstetrics & Gynecology and Urology, Norris Comprehensive Cancer Center,University of Southern California, Los Angeles, CA, USAf Cancer Research Center of Hawaii, University of Hawaii at Manoa, Honolulu, HI, USA

a r t i c l e i n f o a b s t r a c t

Article history:Received 21 January 2011Received in revised form 8 May 2011Accepted 12 May 2011

Keywords:NotchLymphoid malignanciesMultiple myelomaGamma-secretaseDrug resistance

0304-3835/$ - see front matter � 2011 Elsevier Ireldoi:10.1016/j.canlet.2011.05.009

⇑ Corresponding author. Address: Department of MDentistry, Università degli Studi di Milano, Via A.Milano, Italy. Tel.: +30 02 50323249; fax: +30 02 50

E-mail address: raffaella.chiaramonte@unimi.it (1 These authors contributed equally to the manusc

Notch receptors are transmembrane proteins critically determining cell fate and mainte-nance of progenitor cells in many developmental systems. Notch signaling is involved instem cell self-renewal and regulates the main functions of cell life at different levels ofdevelopment: cell proliferation, differentiation and apoptosis. By virtue of its involvementin the regulation of cell physiology, it is not surprising that a deregulation of the Notchpathway leads to the development of different tumors. In this review, we critically discussthe latest findings concerning Notch roles in hematologic oncology, with a special focus onT-cell acute lymphoblastic leukemia and B-cell malignancies. We also describe the molec-ular mediators of Notch-driven oncogenic effects and the current pharmacologicalapproaches targeting Notch signaling.

� 2011 Elsevier Ireland Ltd. All rights reserved.

1. Notch signaling in hematological malignancies

Notch receptors are a family of transmembrane proteinsexpressed by cells of different tissues. Regulating a delicatebalance of intracellular signals, Notch receptors criticallytune differentiation and proliferation processes [1–4]. Per-haps not surprisingly, mutations in Notch genes result indifferent diseased phenotypes, including cancer [2,5–7].The series of studies that lead to the discovery of Notchgenes started when genetics was born. The work by Mor-gan concerning Drosophila melanogaster ‘‘notched’’ wingphenotype [8] gave rise to further characterization of thegenetic locus associated with this developmental anomaly.In early 1980s, the study of Artavanis-Tsakonas et al.

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edicine, Surgery andDi Rudinì 8, I-20142,323254.

R. Chiaramonte).ript.

identified the Drosophila Notch gene on chromosome3C7 [9]. About 10 years later, the first evidence was giventhat Notch alterations can be associated with cancers whena translocation between chromosomes 7 and 9 was foundto be linked with T-cell acute lymphoblastic leukemia(T-ALL) [10]. Later, a number of point mutations, insertionsand deletions were reported to affect the Notch1 gene inT-ALL [11]. To date, Notch1 alterations also have been re-ported in other hematological cancers, such as multiplemyeloma (MM) [12,13], acute myeloid leukemia (AML)[14,15] and lymphoma [16,17].

2. Structure and activation mechanism

The Notch pathway plays different roles during celldevelopment and in different cell types. This is possible be-cause Notch signaling is modulated by four receptors,Notch1–4, which share sequence and structural homolo-gies. Neighboring cells send signals through five ligands,

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belonging to two different groups based on the homologywith the Drosophila genes Serrate or Delta. The first groupincludes Jagged1–2, the second group Delta-like -1, -3 and-4 (DLL-1, -3, -4).

Notch is synthesized as a single transmembrane poly-peptide. During translocation to the cell surface throughthe trans-Golgi system, post-translational modificationsoccur, such as glycosylation and cleavage by a furin-likeprotease which sheds the precursor in a functional hetero-dimeric receptor.

Fig. 1 displays the structure of mature Notch receptorsin mammals. The two intracellular and extracellular por-tions are stably associated through a non-covalent extra-cellular Ca++-dependent bond, which occurs at thehetero-dimerization (HD) domain. A proteolytic cleavagein the HD domain is triggered upon ligand binding, and ini-tiates Notch activation [14,18,19]. The extracellular sub-unit contains several epidermal growth factor-(EGF)related tandem repeats involved in the binding of Notch li-gands. Next, three copies of LIN12/Notch cysteine rich re-peats prevent receptor activation in the absence of aligand and its consequent translocation to the nucleuswhere it activates gene transcription.

The transmembrane region is followed by the RAM do-main, which is involved in the Notch-dependent transcrip-tional activity by interacting with the transcriptionrepressor, CSL (acronym for CBF1 in mammals, Su(H) inflies, and LAG-1 in Caenorhabditis elegans). Six ankyrin re-peats, folded into a helix-loop-helix structure, mediate fur-ther protein–protein interactions. The C-terminal domaincontains a PEST sequence (proline, glutamic acid, serineand threonine-rich region), which contains the signal se-quence for poly-ubiquitylation necessary to address theprotein to proteasomal degradation [20], and thereforecontrols the stability of activated Notch.

Upon ligand binding, a conformational change triggerstwo successive cleavages that release the Notch intracellu-lar portion (ICN), allowing it to translocate into the nu-cleus. Two proteases are responsible for the cleavages: anADAM/TACE metalloprotease acting outside the cell, andthe c-Secretase, which operates in the cytoplasm side[21]. Once in the nucleus, ICN displaces the transcriptionalrepressor complex CSL and recruits co-activators, such asPCAF, GCN5 [22], p300 [23] and MAML1 [24], thereby acti-vating the transcription of several downstream target

Fig. 1. Structure of mature Notch receptors in mammals. In the N-terminus extrrepeats (LIN12), which prevent activation in the absence of ligand, and the hetintracellular domain. This contains a RAM domain, followed by ankyrin repeats (AC-terminus contains a polyglutamine and a proline, glutamic acid, serine andproteasomal degradation.

genes. Among direct or indirect Notch effectors, severalare involved in proliferation, including c-myc, p21, p27,CycD1 [25–28], in cell migration, such as chemokine recep-tors CCR4, CCR8 and CXCR6 [29], CCR6 [30] and CCR7 [31],or are important cytokines or cytokine receptors, namelyIL-6 [32], IL-8 [33] VEGFR-2 [34] and IL7R [35].

3. The T-ALL paradigma

The Nobel Laureate, David Baltimore, was the first toshow that Notch1 may play a causative role in the develop-ment of T-ALL [36]. The first oncogenic form of Notch wasidentified as a partner in the t(7;9) (q34; q34.3) transloca-tion in a few rare cases of human T-ALL (>1%) [10]. The ret-rovirus-mediated expression of this truncated Notch1 genein bone marrow (BM) cells, induced leukemia upon trans-plantation into lethally irradiated syngeneic BALB/cByJmice with 50% frequency [36]. All tumors were composedof immature T-cells blocked at different maturation levels.

Besides the rare t(7;9) (q34; q34.3) translocation,Notch1 gene mutations are evident in nearly 60% ofT-ALL patients [37]. They are mainly located at the HD do-main (between exons 26 and 27), the extracellular juxta-membrane (JME) region (exon 28), or PEST domain (exon34). HD domain single amino acid substitutions and in-frame deletions/insertions weaken the association of thetwo subunits and favor ligand-independent Notch activa-tion [38]. JME mutations consist of tandem duplicationscausing expansions of the extracellular juxtamembrane re-gion [39]. Mutations in the PEST domain are small inser-tions or deletions which generate premature stop codonsand the loss of the C-terminal proteasome-targeting se-quence, causing an increased half-life [38]. Since the men-tioned pioneering reports indicate a causative role forNotch1 mutations in T-ALL [10,36,37], a number of studiesfocused on the analysis of Notch mutational status in thismalignancy. In T-ALL, all of the reported hyper-activatingmutations affected the Notch1 isoform, while Notch2, 3and 4 were not found to be altered [18,40–45]. For in-stance, a study [43] including 142 acute leukemias and192 tumors of different origin (lung, breast, colorectaland gastric carcinomas), revealed that Notch1 mutationswere selectively detected in T-ALL, but not in the othermalignancies. Oppositely, Notch2, 3 and 4 were not

acellular domain, the EGF-like repeats are followed by LIN12 cystein-richerodimerization domain (HD), which allows for the interaction with theNK). The RAM-ANK region is required for the control of transcription. Thethreonine rich region (PEST), which controls Notch ubiquitylation and

L. Mirandola et al. / Cancer Letters 308 (2011) 1–13 3

mutated in T-ALL [43]. Nonetheless evidences of theNotch3 ICN causative role in leukemogenesis have been re-ported in murine models [46]. The recent report by Masi-ero et al. showed that Notch3 promotes survival ofhuman T-ALL cells by up-regulating the mitogen-activatedprotein kinase phosphatase 1 (MKP-1) [47]. The clinicalsignificance of this result and the relative contribution ofNotch3 to T-ALL are still unknown. The absence of muta-tions affecting Notch3 in humans [48] suggests that themajor role in T-ALL progression is played by Notch1, evenif Notch3 could be indirectly altered by upstream path-ways. On these grounds, it is clear that the isoform 1 ofNotch receptors can be considered a specific target for T-ALL.

It is worth noting that all the described Notch1 muta-tions require c-Secretase cleavage to trigger intracellularsignals. The understanding of the different characteristicsdisplayed by T-ALL subgroups has been largely limited un-til systematic studies performed through gene expressionprofiling techniques [49]. Although T-ALL frequently arisesfrom the activation of oncogenes due to chromosomaltranslocations involving the TCR locus, five different onco-genes can be aberrantly expressed without chromosomallesions: HOX11, TAL1, LYL1, LMO1, and LMO2 [49].HOX11 activation was indicative of a favorable prognosis,while expression of TAL1, LYL1 and HOX11L2 was associ-ated with a poor response to treatment [49]. Notch1 recep-tor has been shown to affect and interact with suchoncogenes. Recently, the study by Riz et al. has demon-strated that HOX11 expression is strongly associated withactivating Notch1 mutations, and the two oncogenes co-regulate the T-ALL transcription program, by binding tothe Groucho-related TLE corepressors, increasing c-mycexpression and controlling CD1 and RAG genes transcrip-tion [50]. Tremblay et al. have elegantly shown that TAL1and LMO1 cooperate with Notch1 in T-ALL leukemogene-sis: [51] TAL1-LMO1 hyper-activation represents early on-set genetic lesions in differentiating thymocytes thattrigger leukemogenesis, but Notch1 activating mutationsare subsequently required to confer a selective advantageto TAL1-LMO1 mutated cells, bypassing the pre-TCR sig-naling requirement for survival [51].

4. Strategies to overcome Notch hyper-activation in T-ALL

The rationale of T-ALL treatment aimed to hamperNotch signaling stems from the vital role of Notch in thismalignancy and from the observation that Notch hyper-activating mutations occur with high frequency in thesetumors. As stated above, the Notch activation process is acomplex mechanism requiring two cleavages of the recep-tor. Therefore, theoretically any agent blocking at least oneof these two proteolytic steps could effectively impede orslow down the Notch signal. Historically, the first attempton this path relied on small molecules able to reversibly in-hibit the c-Secretase catalytic site. They are known withthe general name of c-Secretase inhibitors (GSIs). Initially,they were developed for the treatment of Alzheimer’s dis-ease, since c-Secretase is responsible for the accumulation

of the amyloid protein [52]. Studies on GSIs for the treat-ment of T-ALL initiated along with evaluations of the anti-cancer properties of these molecules in solid cancers. Forinstance, GSI-IX (EMD Chemicals, Inc.) efficacy againstmedulloblastoma was firstly reported in 2004 [53] and, 1year later, GSI-I (EMD Chemicals, Inc.) was shown to effec-tively induce apoptosis in Kaposi’s sarcoma [54]. At thesame time, the potential of GSI-XXI (EMD Chemicals, Inc.)to block the growth of T-ALL cells in vitro was described[11], and confirmed by further independent studies[55,56]. Unfortunately, accumulating evidences indicatethat GSIs are effective only in a limited sub-group of T-ALL, and the treatment response seems to be independentfrom the Notch1 mutational status [56–58]. This apparentdiscordance is due to the complex interactions betweenNotch and other intracellular signal mediators, amongwhich the most relevant are phosphatidylinositol 3-kinase(PI3K)/Akt [59,60] and c-Myc [61–63].

C-myc positively regulates T-ALL cell growth, and itsexpression is directly controlled by nuclear Notch1 [11].In turns, c-myc has been reported to regulate Notch signalsby controlling the Notch-dependent activation of themammalian target of rapamycin (mTOR) [61]. Since mTORpromotes proliferation in response to growth factors [64]and c-myc activation is not directly affected by GSIs, it ispossible that a c-myc-driven, Notch-independent mecha-nism exists in GSIs-resistant T-ALL cells [61]. Accordingly,mutations stabilizing c-myc protein have been reportedto confer GSIs resistance to T-ALL cell lines [65]. TheFBW7 ubiquitin ligase-encoding gene is affected by inacti-vating mutations in resistant cells: this enzyme is requiredfor the proteasome-dependent degradation of both c-mycand Notch; not surprisingly, it has been found inactive inabout 30% of T-ALL cases [66]. More recently, the studyby O’Neil et al. showed that mutations in the Notch1 HDdomain are associated with FBW7 mutations, while muta-tions in Notch1 PEST domain and FBW7 are mutuallyexclusive [20], in accordance with their ability to controlproteasomal degradation of Notch. Because the target ofFBW7-mediated ubiquitylation of Notch1 is located inthe PEST domain, alterations of PEST produce a phenotypesimilar to that of FBW7 inactivation. However, the increaseof c-myc stability seems to be another major target ofFBW7, if we consider the more recent study by Dunwellet al. [67] which highlight a deregulation of another tumorsuppressor gene, the protein phosphatase 2 alpha (PP2A),with a similar effect in T-ALL patients. The promoter ofthe PP2A-encoding gene was reported to be inactive in69% of T-ALL patients, due to hyper-methylation mecha-nisms [67]. This finding is relevant in the context of GSIresistance, because PP2A-mediated dephosphorylation ofc-myc in Ser62 is required for c-myc proteasomal degrada-tion [63]. Therefore, not only Notch but at least two addi-tional factors, namely FBW7 and PP2A, control c-myclevels in T-ALL.

Concerning the PI3K/Akt pathway, it was reported to behyper-activated in GSI-resistant T-ALL cell lines, but not insensitive ones [60]. This is caused by the lack of the tumor-suppressor PTEN that in turn blocks PI3K-mediated activa-tion of Akt. Accordingly, the growth inhibitory effects ofGSI were reversed by forced AKT expression or by PTEN

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silencing. The clinical significance of this finding is evidentwhen considering that PTEN inactivating mutations repre-sent a rather frequent event in T-ALL patients (17%) [60].Nonetheless, the clinical relevance of PTEN loss in GSIresistance is still unclear. By analyzing primary human T-ALL samples obtained at diagnosis, a significant correlationbetween PTEN activation status and resistance to GSIs wasnot found [68].

In conclusion, it is evident that T-ALL treatments basedon Notch inhibition alone are likely to generate disappoint-ing or limited results, especially when translated from thebench to the bedside. Future successful approaches musttake into account the complex interactions that Notchreceptors establish in healthy or tumor cells. Accordingly,we foresee that the winning strategy will eventually con-sist of combined treatments to simultaneously block Notchactivity and Notch-associated pathways. Because deregula-tion of the latter appears to occur with different frequencyin different subjects, it is likely that the molecular charac-terization of each single patient will acquire increasing rel-evance to establish ‘‘personalized’’ therapies.

5. Prognostic significance of Notch status in T-ALL: anongoing debate

Because Notch1 is a key regulator of T-ALL blast biologyand it is frequently deregulated in patients, differentresearch studies have been undertaken to elucidate theclinical consequences of Notch1 mutational status,particularly concerning prognosis and treatment decisions[18,37,69,70]. Results and recommendations from theseevaluations are not univocal and larger clinical trials willbe needed to definitively answer the question whetherNotch1 gene analysis is a relevant prognostic factor forT-ALL patients. In 2006, van Grotel et al. studied the prog-nostic relevance of cytogenetic abnormalities in pediatricT-ALL patients through quantitative real-time PCR andfluorescence in situ hybridization [70]. Patients were strat-ified based on the mutational status of TAL1, HOX11/TLX1,HOX11L2/TLX3 and CALM-AF10 genes. Notch mutationswere identified in HOX11/TLX1 and TAL1-positive patientsubgroups, but no association of Notch1 mutation withthe outcome was found [70]. The first large study identify-ing Notch1 mutations as a positive prognostic factor inchildhood precursor T-ALL was reported by Breit et al.[37]. This consisted of a detailed and systematic analysisof Notch1 hot-spot mutations and their correlation withearly treatment response and long-term outcome in 157patients with T-ALL (median age at diagnosis 8.8 years) en-rolled in the ALL-Berling-Frankfurt-Munster 2000 Study.Results confirmed previous studies detecting Notch1mutations in 50% of pediatric T-ALL [11], specifically eitherin the HD (61.7%), PEST (15.9%) or in both domains (17%).Importantly, Notch1 activating mutations were associatedwith better prednisone response and reduced minimalresidual disease, regardless of sex, age, white blood cellcount and T-cell phenotype. Collectively, patients withNotch1 activating mutations were clusterized in a sub-group with better prognosis. The authors conclude thatNotch1 mutations are indicative of faster early treatment

response and favorable long-term outcome [11]. The re-cent work by Park et al. [69] supported these findingsand added elucidations on the role of FBW7 mutations.The study enrolled 55 T-ALL and 14 T-cell non-Hodgkinlymphoma (T-NHL) patients in the context of the JapanAssociation of Childhood Leukemia Study (JACLS). 14.6%of T-ALL and 21.4% of T-NHL patients had FBW7 mutations,while 30.9% of T-ALL and 42.9% of T-NHL displayed Notch1mutations. Only 5.4% of T-ALL and 1.4% of T-NHL hadmutations in both Notch1 and FBW7 genes. Results clearlyindicated that patients with Notch1 or FBW7 mutationshave an excellent long-term prognosis, with 95.5% 5-yearevent-free survival and 100% overall survival [69]. Theauthors highlight the relevance of assessing the mutationalstatus of Notch1 and FBW7 as a useful prognostic markerfor T-ALL.

These studies raised initial enthusiasm in the scientificcommunity because of the possibility to differentiate thetreatment basing on a relatively small number of geneticanalyses, avoiding to expose patients with a favorable prog-nosis to high-dose and aggressive chemotherapy regimens.However, the complex interactions between Notch1 andother altered signaling pathways in T-ALL make the prog-nostic evaluation of this disease more complex than initiallyestimated. The recent investigation performed by Monsourand co-workers [18] evaluated Notch1 and FBW7 mutationsin adult T-ALL enrolled in the United Kingdom Acute Lym-phoblastic Leukemia XII (UKALLXII)/Eastern CooperativeOncology Group (ECOG) E2993 protocol. 88 T-ALL patientswere screened and their clinical outcome was studied: 60%of patients had Notch1 and 18% had FBW7 mutations, irre-spective of age or white blood cell count. Even if a trend to-wards poorer event-free survival was detected in patientswith wild-type Notch1 and FBW7, the authors reported nosignificant predictive value, concluding that the improve-ment in event-free survival for patients with Notch1 and/or FBW7 mutations is not high enough to justify a hypothet-ical reduction of therapy intensity [18].

6. Notch role in b-cell malignancies

The role of Notch in tumor development, progression,and drug resistance has been proven by increasing evi-dences, not only in T- but also in B-cell malignancies[71]. Notch deregulation has been detected in Hodgkin’slymphoma and large B-cell lymphoma [16,72], Burkitt’slymphoma [73], B-cell chronic lymphocytic leukemia[74], diffuse large B-cell lymphoma [75,76], primary effu-sion lymphomas associated with Kaposi’s sarcoma herpesvirus infection [77], and multiple myeloma (MM) [13,78].

6.1. B-cell precursor malignancies

Notch role in B-cell malignancies is still controversialand its outcome likely depends on the differentiation stagewhen the malignant transformation occurred. Several evi-dences indicate that Notch inhibits the maturation of earlyB-lymphoid progenitor cells. Inducible Notch1 deletion inadult mice drives to T cell development arrest and to thy-mus colonization by a B-lymphoid subpopulation [79].

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Moreover, BM reconstitution experiments show that BMcells constitutively transduced with active Notch1 origi-nate immature, double positive T cell progenitors in theBM, and are unable to originate mature B cells [80]. Finally,the B-lineage commitment factor Pax5 hampers Notch1expression [81]. Thus, Notch1 seems to facilitate the pro-gression of the common lymphoid progenitor along the Tcell lineage while blocking the B cell lineage. In accordance,we showed that Notch pathway is inactive in B-cell precur-sor acute lymphoblastic leukemia [14,82]. Morimura et al.[83] found that active Notch1 triggers cell cycle arrest inG0/G1 phase and apoptosis an avian immature B cells.

6.2. Malignancies of mature B cells

Notch1 transcript decreases during the development ofB cells in the BM, then low levels of Notch1 transcription issustained in peripheral B cells, where it contributes tomaintain the level of antibody-secreting B cells, possiblypromoting the class switching process [84]. Notch2 signal-ing is qualitatively distinct from that of Notch1 in B celldevelopment. Notch2 is a regulator of a B1–B2 differentia-tion pathway in the BM: it is essential for the specificationof the B1 lineage and the development of immature BCR+

B1 cells. In addition, differentiation of B cells in the mar-ginal zone requires Notch signaling, as well as BCR-specificsignals. Notch2 is also required for the differentiation offollicular B cells [84]. Accordingly, Notch signaling deregu-lation has been reported in the development of mature B-cell neoplasia, as we discuss below.

6.2.1. B-cell chronic lymphocytic leukemiaNotch2 plays an oncogenic role in B-cell chronic lym-

phocytic leukemia (B-CLL) through the induction of CD23[74]. B-CLL cells derive from naïve B cells. The pathogene-sis of the disease primarily involves defects that preventcell turnover by apoptosis, rather than alterations in cellcycle regulation. A well-known feature of B-CLL cells isthe high expression of CD23, which is closely related to cellsurvival and is transcriptionally regulated by Notch2,which is overexpressed in B-CLL. Enforced expression ofconstitutively active Notch2 rescues cells from apoptosisinduced by proteasome inhibitors [85], indicating thatNotch2 signaling could be responsible for the apoptosisfailure, characteristic of this disease.

6.2.2. Diffuse large B-cell lymphomasLee et al. [75] have identified gain-of-function muta-

tions in Notch2 gene at the HD and PEST domains in fivesamples of diffuse large B-cell lymphomas (DLBCL). Inaddition, Tohda and coworkers established a DLBCL cellline whose cell growth was suppressed by DAPT, GSI-Iand GSI-XII (Calbiochem, La Jolla, USA) [76].

6.2.3. Hodgkin’s lymphoma (HL)B-cell derived Hodgkin and Reed-Sternberg (HSR) cells

(which represent the clonal progeny of germinal B cells inmost cases of HL) are separable from non-Hodgkin cell lineson the basis of a Notch-associated gene expression signature[86]. HRS cells activate the Notch pathway in different ways.Jundt and colleagues analyzed Notch1 expression in

different B-cell lymphomas and reported a marked expres-sion of Notch1 in HSR cells [16]. Also, high levels of Notch2and Jagged2 are a common feature of HRS cell lines [86].An alternative mechanism triggers the inappropriateactivation of the Notch pathway in HL cell lines throughthe overexpression of an essential Notch co-activator, Mas-termind-like2 (MAML2). Therefore, it is not surprising thatthe inhibition of MAML2 reduces HL cell proliferation [86].

Finally, a cell-contact dependent mechanism of Notchactivation has been reported involving the heterotypicinteraction between HRS cells and the infiltrating, Jag-ged1-expressing bystander cells in the lymph node. Jag-ged1 stimulation activates Notch signaling in vitro anddramatically induces proliferation and inhibition of apop-tosis [16]. The outcome of Notch signaling in HRS cells isthe repression of the B-cell differentiation program [72]and may explain the absence of the typical B-phenotypemarkers in malignant clones. At the molecular level, Notchhyper-activation was found to trigger NF-jB signal, whichpromotes HRS cell survival in synergism with Epstein-Barrvirus (EBV) [72].

6.2.4. KSHV primary effusion lymphomaAt least two B cell-infecting viruses, associated with

cancer development, affect Notch signaling: Kaposi’s sar-coma-associated herpes virus (KSHV) and Epstein-Barrvirus (EBV). KSHV and EBV infections are characteristic ofprimary effusion lymphomas (PEL). PEL cells display atranscriptional profile similar to that seen in plasma celltumors. KSHV latency-associated antigen, LANA, has multi-ple functions involving Notch signaling regulation. LANAbinds and antagonizes Sel10, an E3-ubiquitin ligase whichpromotes proteasomal degradation of the nuclear Notch.Therefore, LANA prolongs the half-life of activated Notchin KSHV infected cells [87]. In turn, ICN is involved in themaintenance of the malignant phenotype of KSHV-infectedPEL cells in vitro [87]. Accordingly, the onset of KSHV-in-fected PELs was significantly delayed in GSI-treated SCIDmice harboring PEL cell lines [77].

6.2.5. Burkitt’s lymphoma (BL)The second B-cell infecting virus which exploits the

Notch pathway machinery is EBV. It is found in several hu-man cancers, and displays B and epithelial cell tropism (re-viewed in [88]). It is present in almost 100% of BL inequatorial Africa, and acute infectious mononucleosis,nasopharyngeal carcinoma, HL, immunoblastic lymphoma,a subset of gastric carcinomas, rare T- and NK-cell lympho-mas, and leiomyosarcoma.

EBV is a highly efficient transforming virus in culture,able to convert more than 50% of all target cells (the restingB cell) into continuously proliferating, lymphoblastoid celllines (LCLs) within a few days. EBNA2 is necessary forimmortalization of B cells by EBV and functions by mim-icking Notch signaling thanks to its ability to interact withresponsive promoters through CSL (CBF1). Nonetheless,the interaction domain of EBNA2 appears to be differentfrom that of Notch1 and Notch2 [89]. Accordingly, Notchand EBNA2 exhibit several differences in the expressionof target genes [90]. The partially overlapping roles ofNotch and EBNA2 open the question if a treatment with

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GSI may be feasible as a therapy of BL, given that GSI blocksNotch activation but it is not expected to have any effect onEBNA2. Nonetheless, He and collaborators [73] reportedthat the Raji BL cell line is sensitive to GSI (Calbiochem,San Diego, CA) administration. The possible direct role ofNotch receptors in BL is still unclear and needs to be fur-ther elucidated. Specifically, Notch interaction with otherpathways, including BRC and c-myc, is unknown in BL.The role of c-myc in this context is controversial, since Rajicells carry the BL characteristic translocation t(8;14)(q24;q32) which places the Myc gene under the controlof IgH regulative region [17,73].

Other reports suggest a possible role of Notch in otherB-cell lymphomas on the basis of the expression of Notch1or MAML2 in mantle cell lymphoma, follicular center lym-phoma, MZ B-cell lymphoma, and B lymphoblastic leuke-mia [16,86]. By contrast, some authors reported a role ofNotch as a tumor suppressor gene in B-cell lymphomas.The NYC cell line, established from a clonal splenic murineB-cell tumor, represents a mature activated B1-like B-cellblast at the transition from the IgM-positive B cell to theantibody-secreting plasma cell stage. NYC can be inducedto undergo apoptosis by BCR cross linking. ActivatedNotch1 enhances BCR-driven apoptosis [91]. Finally, thegroup of Pear reported ICN mediated growth inhibitionand apoptosis in HL, MM and mixed lineage leukemia-translocated cell lines [92].

We suggest some possible mechanisms accounting forthe discrepancies reported so far, concerning Notch rolein B cell malignancies. Notch likely plays distinct roles inB-cell lymphomas, depending on the tumor differentiationstage. Notch signaling outcome may be dramatically influ-enced by other pathways, for instance the expression ofanti-apoptotic proteins. Finally, it is possible that Notch1and Notch2 isoforms play not redundant, complementaryroles.

6.3. Multiple myeloma: the importance of themicroenvironment

Recently, several studies focused on the role of Notchpathway in MM, a plasma cell malignancy characterizedby high mortality and aggressiveness. The pathologicalnature of MM lesions makes it a peculiar hematological tu-mor, strongly depending on the interaction with the micro-environment. Evidences so far available indicate a verycomplex picture, which involves Notch in the regulationof the interactions between MM plasma cells and the bonemarrow niche.

MM is the second most common hematologic malig-nancy, resulting from a clonal proliferation of plasma cellsin the bone marrow. This location is essential for malignantplasma cell interaction with bone marrow stromal cells.The latter drive MM cell growth and chemoresistanceand contribute to MM-associated bone disease.

Notch activation is tightly controlled during hematopoi-etic lineage differentiation. Under physiologic conditions,hematopoietic stem cells carry Notch receptors and receivesignals from Notch ligands expressed by BM stromal cells.Notch signaling plays a role in the regulation of stemcell renewal, survival and differentiation. This delicate

mechanism is exploited by malignant plasma cells whichestablish complex interactions with BM stromal cells lead-ing to MM cell proliferation, chemoresistance and bonedisease. Despite major advances in the treatment of MMin recent years, it still remains largely incurable and thefailure of the current therapeutic strategies is mainly dueto the MM cells’ ability to deregulate the complex BMmicroenvironment.

Notch activation can occur through different ways (de-picted in Fig. 2) and displays different outcomes, depend-ing on the cell type (Table 1). MM cells canautonomously activate Notch signaling through homotypicinteractions, since they simultaneously express Notch-1, -2and -3 receptors and their ligands. Among these, Jagged-2plays a central role as it is over-expressed in MM patients.Jagged-2 overexpression can be driven by epigenetic dere-gulations including promoter hypomethylation [12], andconstitutive core promoter acetylation, due to reduced lev-els of the SMRT co-repressor [93]. A deregulation can occurat the post-transcriptional level, involving the over-expres-sion of Jagged-2-specific ubiquitin-ligase Skeleotrophin[94]. Notch ligands are also abundantly expressed by stro-mal cells and macrophages [95] which, therefore, can acti-vate Notch in MM cells through heterotypic interactions.Houde et al. [12] confirmed the relevance of Jagged-2deregulation in patients. Jagged-2 overexpression is anearly event, and is positively correlated with disease stage.

Notch signaling activation in MM cells affects both tu-mor cells and the surrounding environment. As previouslydiscussed, Notch signaling can be activated in MM cells byneighboring tumor cells or by BM stroma cells, osteoclastsand macrophages [95].

The consequences of Notch activation on MM cells areapoptosis inhibition [13,96] and decreased sensitivity tochemotherapeutics [13].

Notch signaling withdrawal, mediated by GSI-XII(Calbiochem) and GSI-15 (RH02015SC, Maybridge, AcrosOrganics, Belgium), increased the level of apoptotic cells inMM cell lines [13,78]. Consistently, Notch pathway up-regulation exerted a protective effect from drug-inducedapoptosis, when MM cell lines were co-cultured with Jag-ged1-expressing stromal cells, or after administration of sol-uble Jagged1 [97]. The protective effects of Notch may be dueto its ability to stop the cell cycle in the G0/G1 phase, prevent-ing apoptosis. After GSI-XII-mediated Notch withdrawal,Noxa, an important pro-apoptotic protein, was up regulated,and a reduced AKT signaling pathway was observed [78].

The role of Notch in the regulation of MM cell cycle pro-gression is far less clear. Both a proliferative effect and ananti-proliferative effect have been reported. According toSchwarzer and colleagues, GSI-XV (Calbiochem) reducesproliferation after a 48 h treatment [96]. This data wereconfirmed by the group of Gabrilovic by the use of GSI-XII (Calbiochem) [78]. Nonetheless, the same group previ-ously observed that MM cell lines co-cultured on a layer ofJagged1-expressing cells were blocked in the G0/G1 phase[13]. Consistently with this data, the forced expression ofICN by an inducible retroviral vector reduced the numberof cells, beginning from 48 h, and up to 8 days [92].

By contrast, the work of Jundt et al. [97] shows a differ-ent picture. The Jagged1-expressing stromal cell line, HtTA

Fig. 2. Roles of Notch receptors and ligands in the vicious circle established by MM cells and the bone marrow microenvironment interactions. Notchsignaling is activated by Jagged-1,2 ligands expressed by MM cells and bone marrow stroma cells (BMSCs). ICN triggers proliferative and anti-apoptoticsignals in malignant cells. MM cell-expressed Jagged-1,2 ligands also prompt Notch signaling in BMSCs and osteoclasts (OCLs). Upon Notch stimulation,BMSC secretes MM growth factors, such as IL-6, VEGF and IGF; further, it is possible that Notch receptor also control the expression of IL-8, MMPs and SDF1by BMSCs, contributing to tumor burden. Particularly, SDF1 activates the chemokine receptor CXCR4 in MM cells, promoting their proliferation andrecruitment to the bone marrow, while MMPs contribute to bone lesions and MM cell growth. MM-driven Notch activation in OCL stimulates boneresorption mechanisms. Although the main factor controlling CCR6 expression in OCLs is BMSC-derived RANK ligand (RANK-L), the possibly exists thatNotch1 activation increases CCR6 levels, that in turn, mediates OCL recruitment to osteolysis sites and OCL activation. More details are in the text.

Table 1Mechanisms and most relevant effects of Notch receptor activation in the three main cell subtypes residing in MM-induced bone marrow lesions.

Target cell Mechanism Outcome Refs.

MM plasmacell

Homotypic activation of Notch-1,-2 and -3 by aberrantlyexpressed Jagged-2

Proliferation, resistance to apoptosis andchemotherapy

[12,13,92–96,98]

Growth arrestActivation induced by BM stromal cells Secretion of IL-6, VEGF, IGF

Expression of CXCR4

Osteoclast Notch activation by MM cell-expressed Jagged-2 Increased osteoclastogenesis and boneresorption activity

[30,95,96,103]

Increased RANKL secretion Hyper-expression of Jagged-1 and Notch-2NF-jB and NFATc1 activationCCR6 expression

BM stromalcell

Notch activation by Jagged-2 expressed on MM cells Increased secretion of IL-6, VEGF and IGF [12,95]

L. Mirandola et al. / Cancer Letters 308 (2011) 1–13 7

forces MM cells to enter the S phase during the first 24 h ofco-culture. This evidence excludes that GSIs biological out-come can be simply due to an unspecific side effect, and isin accordance with Notch inhibition experiments in bothmurine xenografts, reporting an anti-tumor activity of GSIs[78]. In-depth studies are necessary to explain the incon-sistency of the reported results. Nonetheless, it is possibleto observe that during gain-of-function experiments, thesurvey time of cell proliferation measurement was differ-ent and could be compatible with a biphasic effect. Withinthe first 24 h, as reported by Jundt et al. [97], Notch activa-tion might induce an initial proliferation increase, followedby the reinforcement of anti-proliferative signals which

determine cell growth arrest [13]. One of the most relevantNotch transcriptional targets is c-Myc [62], the well-known transcription factor that could be responsible ofthe initial G0/G1 phase entry; but, on the other side, Notchactivation triggers p21 up-regulation in MM cells consis-tently with the observed cell cycle stop [13]. A thoroughgene expression analysis in MM cells could elucidate ifboth of these and other antithetic signals co-exist in thefirst hours following Notch modulation and if they areresponsible for the observed dual effects.

Another possibility is that the complexity of Notch ef-fect in MM could be ascribed to the MM-cell ability to pro-duce membrane and soluble molecules necessary for the

8 L. Mirandola et al. / Cancer Letters 308 (2011) 1–13

observed cross-talking with other neighboring MM cellsand stroma cells. Several evidences indicate the existenceof Notch-mediated interactions between malignant plas-ma-cells and microenvironment. Cell lines and malignantplasma cells from MM patients overexpressing Jagged2trigger the secretion of IL-6, vascular endothelial growthfactor (VEGF), and insulin-like growth factor (IGF); theseare important mediators of MM cell survival and prolifera-tion [12]. At the same time, Notch signaling plays a role inthe communication from MM cells to stromal cells. Jagged-2-overexpressing MM cells activate Notch signaling in pre-osteoclasts and osteoclasts (OCLs) inducing differentiation,activation and secretion of pro-inflammatory mediators in-volved in the increase of osteolytic bone lesions [13,96].Schwarzer and colleagues provided the first evidence thatNotch signaling is involved in specific tumor–stroma inter-actions between MM cells and primary OCLs through di-rect cell–cell contact. In their work, the authors displayedthat the OPM-2 MM cell line is able to increase OCL num-bers and activity through the activation of Notch signalingin these cells [96]. A mechanism was proposed for explain-ing the Notch role in osteoclastogenesis involving RANKsignaling [95]. RANK is a transmembrane heterotrimericreceptor of RANKL expressed on hematopoietic OCL pro-genitors and mature OCLs. The RANK-RANKL interactionpromotes OCL differentiation by activating several intra-cellular pathways, including nuclear factors of activatingT cells (NFAT) and NF-kB. Stimulation with RANKL inducedJagged-1 and Notch-2 expression during OCL differentia-tion and, in turn, Notch2 activity enhanced osteoclasto-genesis by cooperating with NF-kB to activate NFATc1transcription factors, essential for OCL maturation.

Notably, recent evidence indicates Notch signaling as akey element in creating the complex system constituted bysoluble and membrane bound factors relevant in MMmaintenance and progression. Indeed several of these fac-tors have been found under Notch signaling control, albeitin a different cellular context: (i) CXCR4 is under Notchcontrol in bone marrow-derived dendritic and endothelialcells [98,99]; this chemokine receptor has a role in MMmalignancy by promoting tumor cell recruitment in BM[100], due to chemotactic attraction of the ligand chemo-kine SDF-1 secreted by BM stromal cells, and transendo-thelial migration, stimulated by cell adhesion to theendothelium [101]. Also, SDF-1 increases proliferationand drug resistance of both MM cell lines and primaryMM cells [102]. (ii) CCR6 expression is upregulated byNotch1 in human Langerhans cells [28]. MIP3-alpha/CCR6axis contributes to MM malignancy favoring bone lesiondevelopment in MM patients. As a matter of fact, signifi-cantly higher MIP3-alpha levels were detected in MM pa-tients with bone involvement versus those withoutosteolytic lesions. The mechanism involves Notch-medi-ated MM cell ability to promote MIP3-alpha secretionand CCR6 expression by BM osteoprogenitor cell; in turnCCR6 signaling enhances differentiation of OCL [103]: (iii)IL-8 is a potent activator of osteoclastic differentiationand bone resorption [104] and acts as a growth and che-motactic factor for MM cell lines and patients’ plasma cells[105]; Notch1 positively influences its expression in boneinflammatory disease [33]. (iv) IL-6 is reported by Houde

and colleagues to be secreted by stromal cells followingNotch signaling activation induced by Jagged2 over-expressing MM cells [12]. A paracrine (by the stromal cells)production of IL-6 has also been reported. Once secreted,IL-6 plays an essential role in promoting the proliferationof malignant plasma cells in MM and IL-6 levels directlycorrelate with tumor burden, bone destruction and drugresistance [106]. It is clear that MM cells regulate IL-6 pro-duction largely in a cell contact–dependent manner [107].(v) Matrix metalloproteinases (MMPs) are implicated in tu-mor-stroma interactions, tumor invasion and metastasis.BM stromal cells of MM patients are frequently character-ized by production of high levels of MMP-1and MMP-2[108]. The same MM cell produces MMP-9 and MMP-2,which trigger degradation of collagen IV, the major constit-uent of the basement membrane [109]. The role of MMPsin MM was demonstrated by means of 5T2 MM mousemodel, which develops a disease similar to human MM.Metalloproteinase inhibitors reduced angiogenesis andprovided protection from the development of osteolyticbone disease [110], therefore accounting for tumor burdendecrease. In two different contexts, pancreatic cancer [111]and in inflammatory settings such as osteoarthritic disease[32], Notch was reported to play a role in the regulation ofMMPs.

7. Opportunities from the notch pathways for mm

Studies exploring the feasibility of a Notch-targetingtherapy are promising, although scarce. Encouragingin vitro works have assessed the effect of Notch withdrawalon MM tumor growth and osteoclastogenic activity.Gamma-Secretase inhibitors have been used, namelyDAPT, GSI-XII (EMD Chemicals, Inc) and the recently syn-thesized GSI-15 (RH02015SC, Maybridge, Acros Organics,Belgium).

Jundt and colleagues [97] reported that a 24 h treat-ment of MM cell lines with DAPT resulted in a 40–75% de-crease in proliferation rate. Both GSI-XII and GSI-15 wereused on MM cell lines, inducing a reduction of tumor cellproliferation, at least partially due to apoptosis increase[13,78,96]. Up to now, the underlining mechanism wasnot completely clarified. Notch-induced resistance toapoptosis was attributed to growth inhibition associatedwith up-regulation of p21WAF/Cip [13]. More recently,the same authors displayed that the cytotoxic effects in-duced by GSI-XII- triggered Notch signaling withdrawalwas mediated by Hes-1 and up-regulation of theproapoptotic protein, Noxa [78]. This group associatedthe Notch-dependent apoptosis resistance to chemoresis-tance, another important effect relevant for the possibleuse of Notch-targeting therapy. They observed that forcedretroviral expression of Notch-1, but not Notch-2,preserved tumor cells from melphalan- and mitoxan-trone-induced apoptosis [13].

Although the paucity of in vivo data about GSI effect onMM, results on two different MM animal models are con-cordant. Xenograft and SCID-hu mice demonstrated a sub-stantial antitumor effect of GSI-XII (Calbiochem) and theability to increase sensitivity to chemotherapeutic drugs,

L. Mirandola et al. / Cancer Letters 308 (2011) 1–13 9

such as doxorubicin and melphalan [78]. These datarepresent the first indication that Notch is a promisingtherapeutic target in MM although different pictures indi-cate a different role of Notch in MM in respect to T-ALL.Definitely, clarifications on the complex Notch cross-talk-ing with other onco-proteins are necessary to predict thebiological effect of Notch withdrawal, the effectiveness ofNotch inhibition therapies and possibly to plan combinedtreatments directed to the key deregulated elements in dif-ferent patient subsets.

8. Perspectives of pharmacological interventionstargeting the Notch pathway in hematopoietic tumors

The large amount of data about the molecular and bio-logical effects of GSIs in in vitro models of T-ALL is rela-tively recent [11,60,62,65,112]; thus, studies undertakenin human subjects are still limited and need furtheranalyses.

At present, two phase I clinical trials are being evalu-ated for the rationale of the use of GSIs in clinical practice:one is related to the use of Merck’s MK0752 treatment ofbreast cancer (identifier number NCT00106145); the otheris focused on Notch inhibition in advanced T-ALL and lym-phoma (identifier number NCT00100152). Results are notencouraging. Despite the mentioned efficacy in vitro, GSIdisplayed significant gastrointestinal toxicity and no clini-cal response in relapsed and refractory T-ALL [113]. Gas-trointestinal side-effects have been predicted on the basisof studies in animal models, where chronic administrationof GSIs boosted the formation of secretory goblet cells inthe intestine [114–116]. Nonetheless, the absence of a clin-ical response was quite unexpected and an explanationwas provided by later research, showing that primaryresistance to Notch1 inhibition is a common feature in hu-man T-ALL cell lines [60,65,115]. To overcome such clinicalissues, Real and colleagues proposed a combined therapywith GSI and glucocorticoids, displaying promising resultsas they were able to improve glucocorticoids responsethrough GSI administration, and contemporary to mitigateGSI cytotoxicity in the gut through glucocorticoids [42]. Sofar, our knowledge of GSI resistant mechanisms and inci-dence in humans has been limited to cell lines. Recent ad-vances indicate that resistance to c-secretase inhibition inprimary T-ALL may not be as common as previously fore-seen. The works by Medyouf et al. [68] and Armstronget al. [117] demonstrated that in the majority of T-ALLcases, leukemic cells obtained from patients at diagnosiswere still dependent on Notch signaling for leukemia initi-ating activity, and were sensitive to GSI treatment. Despitethe fact that c-secretase complex is the most studied phar-macological target for the treatment of T-ALL, interestingalternatives are available. As stated, gastrointestinal toxic-ity is the main factor limiting the dose and time of GSItreatment in patients [115]. This is due to the inability ofGSIs to distinguish Notch 1 from Notch 2 [118]. The latteris critical for the differentiation and survival of intestinalepithelium cells. Furthermore, at first glance, Notch2seems to give a wider contribution to the development ofB cells [84]. Therefore, potential toxic effects of GSIs could

additionally rise from hampering B cell differentiation.Recently, antibodies selectively blocking Notch1 or Notch2have been developed [119,120]. While pan-Notch inhibi-tion results in severe globet cell metaplasia, selectiveanti-Notch1 antibodies effectively block tumor cell growthin vivo, without any detectable gastrointestinal side effects[119]. Soluble Dll ligands have been shown to effectivelyblock Notch signaling in animal models of lung cancer[121,122]. Anti-Dll4 antibodies reduced cancer stem cellfrequency and tumor-associated angiogenesis, acting onNotch signaling in human breast cancer xenograft murinemodels [123]. Since the majority of Notch hyper-activatingmutations detected in T-ALL patients make the receptorless dependent from ligand binding [11], the possibility ex-ists that blocking Notch ligands may result ineffectively insuch tumors. Direct inhibition of Notch transcriptionalactivity in the nucleus has been recently obtained by Moel-lering et al. [124] through the hydrocarbon-stapled peptideSAHM1 which is able to hamper the assembly of the activeCBF-1 transcriptional complex in human T-ALL cell lines,reducing Notch signaling and leukemic initiation potentialboth in vitro and in vivo. Given its high specificity of actionand low toxicity in animal models, this innovative ap-proach is expected to prove useful for the future ofNotch-based therapies of T-ALL.

Investigation on Notch role in MM is very recent com-pared to the research in acute leukemia. It is thereforenot surprising that no data are available concerning Notchinhibition in MM patients. Nonetheless, in vitro and in vivostudies in animal models indicate that targeting the c-secretase complex may represent the basis of a potenttherapy of MM. Indeed, in this hematological malignancy,blocking Notch signaling not only results in reduced tumorcell growth and increased apoptosis, but it also abolishesthe vicious cycle due to the cross-talk between MM cellsand the bone marrow microenvironment [78,96]. Further,we hold that antibodies blocking the Notch ligands mayrepresent a potentially effective alternative to GSI in MMsince Jagged-2 over-expression by MM and BM-residentcells are major causes of Notch signaling abnormalities inthis pathology and contribute to bone osteolytic disease[12,93,94,124], while no mechanism of Notch receptor li-gand-independent activation has been reported to date inMM.

In conclusion, the reported results indicate that a moredetailed understanding of Notch-driven oncogenic andphysiologic cellular mechanisms is needed to overcomethe clinical issues raised by Notch inhibitors in vivo. Partic-ularly in the short run, it is likely that effective Notch-targeted therapies will be based on GSI-glucocorticoidcombinations to reduce side-effects and increase the ther-apeutic window at the same time. Alternative inhibitors,acting either on Notch/ligand interaction or Notch tran-scriptional activity, are more specific and less toxic, butwill need more time and further evaluations before theirclinical use becomes a reality.

Conflict of interest

The authors declare no conflict of interest.

10 L. Mirandola et al. / Cancer Letters 308 (2011) 1–13

Acknowledgements

R.C. is supported by Ministry of Education, University andResearch (PRIN) and by the Milan University Program forresearch grants (PUR), P.C. from the Ministry of Health(Research on specific issues – Ordinary project-IRCCS). Wethank Teri Fields for her assistance in editing this manuscript.WMK holds the Walter A. Richter Cancer Research Chair.

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