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IL-7R-mediated signaling in T-cell acutelymphoblastic leukemia

Daniel Ribeiro 1, Alice Melão 1, João T. Barata*

Instituto de Medicina Molecular, Faculdade de Medicina, Unversidade de Lisboa, Av. Prof. Egas Moniz,1649-028 Lisboa, Portugal

a b s t r a c t

Interleukin-7 (IL-7), a cytokine produced in the bone marrow,thymus and other organs, is mandatory for normal human T-celldevelopment and peripheral homeostasis. Different studies,including phase I clinical trials, have indicated the potential ther-apeutic value of recombinant IL-7 in the context of anti-cancerimmunotherapy and as a booster of immune reconstitution.However, the two main pathways activated by IL-7, JAK/STAT5 andPI3K/Akt/mTOR, have both been implicated in cancer and there isconsiderable evidence that IL-7 and its receptor (IL-7R), formed byIL-7Ra (encoded by IL7R) and gc, may partake in T-cell acutelymphoblastic leukemia (T-ALL) development. In this context, themost compelling data comes from recent studies demonstratingthat around 10% of T-ALL patients display IL7R gain-of-functionmutations leading, in most cases, to disulfide bond-dependenthomodimerization of two mutant receptors and consequentconstitutive activation of downstream signaling, with ensuing celltransformation in vitro and tumorigenic ability in vivo. Here, wereview the data on the involvement of IL-7 and IL-7R in T-ALL,further discussing the peculiarities of IL-7R-mediated signaling inhuman leukemia T-cells that may be of therapeutic value, namelyregarding the potential use of PI3K and mTOR pharmacologicalinhibitors.

� 2012 Elsevier Ltd. All rights reserved.

* Corresponding author. Cancer Biology Unit, Instituto de Medicina Molecular, Faculdade de Medicina, Universidade deLisboa, Av. Prof. Egas Moniz, 1649-028 Lisboa, Portugal. Tel./fax: þ351217999524.

E-mail address: joao_barata@fm.ul.pt (J.T. Barata).1 These authors contributed equally to this article.

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2212-4926/$ – see front matter � 2012 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.jbior.2012.10.005

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Introduction

Although cancer is a genetic disease, there is accumulating evidence for the involvement ofmicroenvironmental cues in tumor progression. T-cell acute lymphoblastic leukemia (T-ALL), anaggressive subtype of the most frequent childhood cancer, is no exception. Interleukin-7 (IL-7) isproduced in the bone marrow, thymus and other organs, and it is absolutely required for normalhuman T-cell development and homeostasis of mature T-cells. Nevertheless, there is also considerableevidence, reviewed in the next sections, that IL-7 and its heterodimeric receptor (IL-7R), formed by IL-7Ra (encoded by IL7R and shared by the receptor for TSLP) and gc (which is shared by the receptors forIL-2, IL-4, IL-9, IL-15 and IL-21), may promote leukemia development. IL-7 stimulates viability and cellcycle progression, and triggers the activation of JAK/STAT5 and PI3K/Akt/mTOR pathways, in bothnormal and malignant T-cells. However, the mechanisms by which IL-7 promotes these effects differbetween normal and malignant T-cells, in ways that may have important therapeutic implications,namely regarding the differential functional consequences of pharmacological modulation of PI3K/Akt/mTOR signaling pathway. We will also discuss this below. Finally, we will briefly present the geneticevidence supporting the notion that aberrant activation of IL-7/IL-7R signaling is a frequent event in T-ALL. This notion stems from the fact that, in addition to the recently found gain-of-function mutationsin IL7R itself, there are alterations identified in T-ALL patients not only in the genes encoding thekinases that directly associate with the receptor, namely JAK1 and JAK3, but also in different elements ofPI3K/Akt/mTOR signaling pathway, particularly PI3K, PTEN and Akt.

The Importance of IL-7 and IL-7R for Normal T-cell Development and Homeostasis

Interleukin-7 (IL-7) was originally cloned from B-cells, more than 30 years ago (Namen et al., 1988).Curiously, IL-7 is essential for normal B- and T-cell development in mice (Peschon et al., 1994), whereasin humans IL-7R receptor inactivating mutation were shown to lead to severe T-cell lymphopeniawithout affecting B-cell numbers (Noguchi et al., 1993; Puel et al., 1998). IL-7 is produced by stromalcells in the thymus and bone marrow, and also by vascular endothelial cells, intestinal epithelium,keratinocytes and follicular dendritic cells in different organs (Jiang et al., 2005). The IL-7 receptor (IL-7R) is a heterodimer comprised of the IL-7Ra (CD127) and the common gamma chain (gc; CD132),which is shared by the receptors for IL-2, -4, -9, -15 and -21. In humans, loss-of-function geneticalterations affecting either the a chain (Puel et al., 1998) or the gc (Noguchi, Yi, 1993) lead to thedevelopment of severe combined immunodeficiency (SCID) resulting from severely impaired thymo-poiesis and T-cell survival (Buckley, 2004; Puel et al., 1998). This syndrome, which is ultimately fatal,can only be corrected by bone marrow transplantation or gene therapy replacement of the defectivegene (Buckley, 2004), illustrating the non-redundant, critical role of IL-7 in T-cell development, whichhas also been demonstrated inmice (Boyman et al., 2008; Peschon et al., 1994; von Freeden-Jeffry et al.,1995).

T-cell development in the thymus is characterized by the sequential expression of CD4 and CD8 co-receptors in T-cell precursors (thymocytes), proceeding from CD8�CD4� double-negative (DN) intoCD8þCD4þ double-positive (DP) and finally, after negative and positive selection, CD8þ or CD4þsingle-positive (SP) thymocytes, that ultimately leave the thymus and move into the periphery asmature naïve T-cells. Inmice, IL-7was shown to be critical for the early thymocyte development, whereits absence results in a major developmental block at the DN stage. This is because IL-7 non-redun-dantly supports cell survival and proliferation of early DN thymocytes (CD44þCD25�, DN1 andCD44þCD25þ, DN2). At this stage IL-7 is also essential for T-cell receptor (TCR) g-chain gene rear-rangements (Ye et al., 2001). The rearrangement of other TCR genes can be promoted by, although notabsolutely dependent on, IL-7R-mediated signals (Chowdhury and Sen, 2003). At the DN3(CD44�CD25þ) stage, signals generated by a productively rearranged b-chain and pre-TCR (pTa) serveto maintain IL-7Ra expression, perhaps allowing responsiveness to limiting concentrations of IL-7 andsuccessful transition to the DN4 (CD44�CD25�) stage (Trigueros et al., 2003). Interestingly, thedownregulation of IL-7Ra appears to be required at the DN4 stage for further development to occurproperly, since forced IL-7Ra expression inhibits the transcription factors necessary for progression tothe DP stage, resulting in a blockade in T cell development (Balciunaite et al., 2005; Tussiwand et al.,

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2011). The expression of IL-7Ra is maintained at low to absent levels during most of the DP stage.However, after positive selection, thymocytes rapidly upregulate IL-7Ra. Re-expression of IL-7Ra in thethymus and the subsequent abundance of IL-7Ra on peripheral T-cells is not a passive process. It isapparently fine-tuned by positive selection signals in the thymus that have profound implications forT-cell fitness (Sinclair et al., 2011). This process will regulate IL-7 signaling during positive selection,which is required for CD8 T-cell lineage commitment (Park et al., 2010).

The essential role of IL-7 in the context of T-cell biology is not restricted to its functions duringthymocyte development. IL-7 has also a major impact on the homeostasis, differentiation and activityof mature T-cells both in mice (Azevedo et al., 2009; Lenz et al., 2004; Pellegrini et al., 2011; Prlic et al.,2002; Schluns et al., 2000; Seddon et al., 2003) and humans (Azevedo et al., 2009; Soares et al., 1998;Swainson et al., 2007). Notably, availability of IL-7 appears to be a limiting factor in T-cell expansion andit is critically conditioned by its utilization more than by regulation of its production (Guimond et al.,2009; Mazzucchelli and Durum, 2007). The IL-7Ra is expressed bymost resting T-cells as a result of theactivity of the transcription factors FOXO1 (Ouyang et al., 2009) and ETS1 (Grenningloh et al., 2011).However, IL-7Ra is transcriptionally downmodulated following IL-7-mediated signaling and/or T-cellactivation (Fry et al., 2003; Park et al., 2004). The implication is evident. Cells that have been exposedto, and benefited from, IL-7 lose their capacity to keep consuming the cytokine. Why does this happen?It is likely a smart strategy for maximizing the number of T-cells that gain access to this vital resource,inwhat has been proposed as the “altruistic model” (Mazzucchelli and Durum, 2007; Park et al., 2004).In fact, the mechanisms of IL-7-dependent downregulation of IL-7Ra are not restricted to, and canactually precede, the inhibition of IL7R transcription, since IL-7 induces rapid internalization of its ownreceptor in T-cells (Henriques et al., 2010). The prevalence of these mechanisms is suggestive of thebiological importance of diminishing the surface expression of IL-7Ra in activated T-cells. In otherwords, IL-7R downregulationmay have been naturally selected because of its ability to provide optimalimmunity in the context of a limited essential resource. Of course, this also illustrates the relevance offine-tuning the activity of IL-7-mediated signaling in peripheral T-cells. Accordingly, deregulation ofthe IL-7/IL-7R axis has been implicated in autoimmune diseases such as diabetes andmultiple sclerosis(Lee et al., 2012; Mazzucchelli et al., 2012), or chronic inflammatory diseases such as rheumatoidarthritis (Churchman and Ponchel, 2008).

The Bright Side of the Moon: IL-7 as an Anti-cancer Agent

The multiple roles of IL-7 extend beyond its impact on T-cell development and peripheralhomeostasis. IL-7 has been shown to potentiate anti-viral immune responses and anti-tumoralimmunity by different mechanisms (reviewed in (Fry and Mackall, 2002) and (Mackall et al., 2011)).In addition, IL-7 can have an extremely important role in boosting the immune system after chemo-therapy or bonemarrow transplantation (Mackall et al., 2011). This is particularly important since anti-cancer chemotherapeutic regimens frequently induce lymphopenia, and T-cell counts and repertoirediversity may remain affected for several years (Mackall et al., 1997; Mackall et al., 1994). Studies inmice have shown that treatment with IL-7 after administration of chemotherapeutic drugs or bonemarrow transplantation lead to expansion and recovery of the B- and T-cell compartments withincreased frequencies of naïve cells with normal function and BCR/TCR diversity (Komschlies et al.,1994; Morrissey et al., 1991). Furthermore, the reconstituted immune system also showed anti-tumor activity (Komschlies et al., 1994).

Overall, the evidence was strong suggesting that IL-7 administration could benefit cancer patients,not only by potentiating the immune response against the tumor, but also by preventing complicationsthat arise from therapy-induced immunodeficiency. This prompted the clinical testing of recombinanthuman IL-7 (rhIL-7) in cancer patients with refractory disease. At the time this manuscript is beingwritten there are, as far as we know, four registered cancer-related phase I and II clinical trials in theClinicalTrials.gov website that use rhIL-7 administration in their regimens (Table 1).

In the first study (NCT ID: NCT00062049) (Sportes et al., 2010; Sportes et al., 2008), rhIL-7 wasadministered to 16 lymphopenic subjects with refractory non-hematological malignancies. Treatmentwith rhIL-7 induced sustained peripheral CD4þ and CD8þ T-cell expansion, increased T-cell survivaland diversity of the TCR repertoire, independently of the age of the subject. Both CD4þ and CD8þ T-cell

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subsets were functional, and displayed a profile with increased proportion of naïve recent thymicemigrants and decreased proportion of regulatory T-cells. Importantly, no significant toxicities wereobserved. This suggests that administration of IL-7 could successfully promote immune reconstitutionafter chemotherapy-derived lymphopenia. Additionally, although this was a phase I trial, the study alsoevaluated potential anti-tumor effects of rhIL-7 administration (Sportes et al., 2010), demonstratingthat one of the patients had transient reduction in tumor mass size, whereas the remaining patientshad progressive disease.

In the second phase I trial, NCT ID: NCT00091338, twelve patients with metastatic melanoma weretreatedwithmelanoma antigen peptides together with different doses of rhIL-7. In accordancewith theprevious study, administration of exogenous IL-7 lead to increased circulating levels of functionalCD4þ and CD8þ T-cells, with a decrease in the proportion of regulatory T-cells. However, theadministration of rhIL-7 was not able to selectively promote the generation of immune precursorsagainst the peptides, when compared to the administration of the peptides alone (Rosenberg et al.,2006).

The two remaining clinical trials have no published data yet. The NCT ID: NCT00492440 study, wasa phase I trial to determine the optimal dose and biological activity of IL-7 in reconstituting the immunesystem and potentially promoting anti-tumor activity in patients with advanced-stage melanoma orkidney cancer. The trial has apparently been terminated. Themost recent study (NCT ID: NCT01368107)is a phase II clinical trialwhich is currently recruitingpatients. Theobjective is todetermine the impactofIL-7 on CD4þ T-cell lymphopenia and tumor progression in metastatic breast cancer.

Another phase I study (not registered in the ClinicalTrials.gov site) was performed using vaccinationwith autologous melanoma tumor cells engineered to express IL-7 (Moller et al., 1998). Three out of sixpatients developed anti-tumor reactive T-cells. However, although the vaccination was well tolerated,no major clinical responses were achieved.

The clinical trials already conducted provided good evidence for the potential of IL-7 in the contextof anti-cancer therapies, in the least as a booster of T-cell numbers and consequent improvement ofimmune reconstitution. However, it is important to take into account, in the design and interpretationof human studies, that IL-7 and its receptor may also have a “dark side” that relates to their capacity topromote the growth of tumor cells. For instance, in vitro evidence suggests that IL-7/IL-7R-mediatedsignaling may play a pro-tumorigenic role in some solid cancers, including breast, lung, melanoma,and colon cancer (Al-Rawi et al., 2003, 2004; Cosenza et al., 2002). Most strikingly, numerous studieshave gathered data suggesting the involvement of IL-7 and IL-7R in different lymphoid malignancies,which range from Hodkin’s lymphoma to chronic lymphocytic leukemia (Brown et al., 2003;Cattaruzza et al., 2009; Digel et al., 1991; Foss et al., 1994; Frishman et al., 1993; Long et al., 1995; Sassonet al., 2010). In the next sections, we will focus on T-cell acute lymphoblastic leukemia (T-ALL), forwhich the most convincing evidence has been gathered pinpointing the tumorigenic potential of theIL-7/IL-7R axis.

Table 1Cancer clinical trials using recombinant human IL-7.

Study Clinicaltrial.gov identifier(phase)

Status Startyear

Reference

Interleukin-7 in Treating PatientsWith Refractory Solid Tumors

NCT ID: NCT00062049(Phase I)

Completed 2003 (Sportes et al., 2010, 2008)

Interleukin-7 and Vaccine Therapyin Treating Patients With MetastaticMelanoma

NCT ID: NCT00091338(Phase I)

Completed 2004 (Rosenberg et al., 2006)

Interleukin-7 in Treating PatientsWith Metastatic Melanoma orLocally Advanced or MetastaticKidney Cancer

NCT ID: NCT00492440(Phase I)

Terminated 2007 –

Study Evaluating Impact of IL-7 onCD4 Lymphopenia, Risks of SevereHematological Toxicity and TumorProgression in Metastatic BreastCancer Patients

NCT ID: NCT01368107(Phase II)

Recruiting 2011 –

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The Dark Side of the Moon: IL-7 and IL-7R Involvement in T-ALL

T lymphocytes do not produce IL-7 and thus they depend exclusively of exogenous, microenvi-ronmental IL-7, which is produced by different cell types in several organs, most notably in the bonemarrow and the thymus (Alves et al., 2009; Goodwin et al., 1990; Hara et al., 2012; Mazzucchelli et al.,2009; Repass et al., 2009; Sakata et al., 1990; Tokoyoda et al., 2004; Wiles et al., 1992). T-ALL cells arethought to derive from thymic T-cell progenitors that colonize the bone marrow. It is therefore verylikely that T-cells undergoing malignant transformation encounter IL-7 in their microenvironment.This, in turn, implicates that IL-7 has the potential to modulate leukemogenesis. Several comple-mentary lines of evidence suggest that this may be the case.

It is known for long that IL-7 transgenic mice display accelerated mortality due to T- and B-lymphoma development (Abraham et al., 2005; Osborne et al., 2010; Rich et al., 1993). Moreover, AKR/J mice, which naturally overexpress IL-7Ra, tend to spontaneously develop thymic T-cell lymphomas.Overexpression of IL-7Ra in thymocytes from AKR/J mice associates with increased survival andselective advantage in competitive transplantation assays (Laouar et al., 2004). Notably, IL-7Ra istranscriptionally upregulated by NOTCH1, one of the most commonly mutated genes in T-ALL (Wenget al., 2004), and appears to be involved in Notch-mediated leukemia cell maintenance (Gonzalez-Garcia et al., 2009; Li and von Boehmer, 2011). These studies suggest that deregulation of the IL-7/IL-7R signaling axis can be oncogenic or, at least, partake in leukemia progression. In contrast,there is evidence from mouse studies with Il7r -/- mice that restraining IL-7R-mediated signaling canaccelerate lymphomagenesis in the context of p53 deficiency (Kibe et al., 2012), which suggest thatthe leukemogenic potential of the IL-7/IL-7R axis may depend on particular cellular scenarios.Nonetheless, in agreement with the notion that IL-7R-mediated signal transduction can promoteleukemia development, malignant cells collected from human T-ALL patients at diagnosis frequentlyexpress the IL-7 receptor (Digel et al., 1991; Eder et al., 1990; Scupoli et al., 2007; Silva et al., 2011a;Touw et al., 1990) and the anti-apoptotic effect of IL-7 in vitro on T-ALL cells (Barata et al., 2001; Digelet al., 1991; Karawajew et al., 2000) appears to correlate with their IL-7Ra surface levels (Karawajewet al., 2000). Moreover, IL-7 clearly promotes T-ALL cell proliferation in vitro, as demonstrated bynumerous early independent studies (Barata et al., 2001; Barata et al., 2004b; Digel et al., 1991; Ederet al., 1990; Makrynikola et al., 1991; Masuda et al., 1990). Notably, we have shown that the prolif-erative effect of IL-7 occurs in a majority of T-ALL cases (more than 70%), independently of their stageof maturation block (Barata et al., 2004b). These observations extend to in vitro systems that mimicthe thymic and bone marrow microenvironments. Scupoli et al showed that thymic epithelial cellsand bone marrow stromal cells cultured in vitro promote T-ALL cell viability via IL-7 production(Scupoli et al., 2007, 2003), and our studies using chimeric fetal thymus organ cultures providedevidence for the importance of IL-7 produced in the thymus for T-ALL cell proliferation (Barata et al.,2006). Most importantly, IL-7 can accelerate T-cell leukemia development in vivo. We have recentlyshown that human T-ALL cells initiate leukemia more slowly when engrafted to immunocompro-mised Rag2-/- Il2rg-/- mice lacking IL-7 as compared to control Rag2-/- Il2rg-/- mice. IL-7 deficiencydecreases the expansion of leukemia cells in the bone marrow, diminishes the efficiency of infiltrationinto other organs and delays leukemia-associated death of transplanted mice (Silva et al., 2011b).Importantly, clinical measurements of IL-7 plasma levels and IL-7 receptor (IL-7R) expression in T-ALLpatients versus healthy controls strongly suggest that IL-7 effectively stimulates human leukemia cells(Silva et al., 2011b).

While the studies summarized above were generated from in vitro analyses, mouse models orcorrelative data collected from patient samples, their biological relevance for human T-cell leukemiahas very recently been validated by the work of different groups providing direct evidence for theinvolvement of IL-7R signaling in T-ALL. We and others have found that around 10% of T-ALL patientsdisplay gain-of-function mutations in the IL7R gene (encoding IL-7Ra), which lead to constitutiveactivation of the receptor promoting cell transformation in vitro (Shochat et al., 2011; Zenatti et al.,2011) and tumor formation in vivo (Zenatti et al., 2011). The mutations are heterozygous, somatic andlocated in exon 6. Most of them create an unpaired cysteine in the extracellular juxtamembrane–transmembrane portion of IL-7Ra, leading to disulfide bond-dependent receptor homodimerization(Zenatti et al., 2011) and constitutive signaling in a manner that does not require the ligand, the other

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IL-7R subunit (gc), or JAK3 (which is normally associated with gc), and is dependent on JAK1 (whichis normally associated with IL-7Ra) (Fig. 1) (Zenatti et al., 2011). The remaining IL7R mutationsintroduce either a tryptophan residue or an SxxxG related motif within the transmembrane region,both of which have been implicated in facilitating homo- or heterodimer formation (Ridder et al.,2005; Russ and Engelman, 2000). However, the exact mechanism by which these mutations oper-ate and the evaluation of their in vivo leukemogenic potential remain to be determined. Nonetheless,these results revealing the presence of IL7R gain-of-function mutations in T-ALL, which have beenconfirmed by subsequent studies (Bains et al., 2012; Porcu et al., 2012; Zhang et al., 2012), indicatethat targeting IL-7R-mediated downstream signaling might be a potentially useful therapeuticapproach for the treatment of IL7R-mutated patients. We have generated proof-of-concept data inthis direction by showing that different clinical-stage JAK small molecule inhibitors are able to killIL7R-mutant cells (Zenatti et al., 2011). Similar to IL-7 stimulation (Barata et al., 2004a; Barata et al.,2004c; Batista et al., 2011; Silva et al., 2011a), IL7R mutations trigger not only JAK/STAT5 but alsoPI3K/Akt/mTOR signaling pathway (Shochat et al., 2011; Zenatti et al., 2011) in T-ALL cells. Thus,targeting the latter pathway might be therapeutically beneficial not only to prevent the effects ofmicroenvironmental IL-7 stimulation of T-ALL cells but also in the context of cell-autonomousaberrations involving the IL-7R. In this context, it is important to emphasize the singularities of IL-7/IL-7R-mediated signaling in T-ALL cells versus normal T-cells, and how subtle differences maypossibly open important therapeutic windows.

Fig. 1. Physiological (IL-7-dependent) versus mutant IL-7R-mediated (constitutive) signaling in leukemia. Physiological, IL-7-dependent, signaling relies on heterodimerization of the two IL-7R subunits (IL-7Ra/CD127 and gc/CD132), which is required foractivation of both JAK1 and JAK3 and consequent downstream signaling. It fluctuates between ON and OFF states according to ligandavailability. In contrast, IL7R cysteine-introducing mutations promote disulfide bond formation between two IL-7Ra/CD127 mutantchains and receptor homodimerization. This leads to the close proximity and consequent activation of two JAK1 molecules asso-ciated with each IL-7Ra cytoplasmic tail. The corollary is that IL-7 is no longer required for triggering signaling, which is perma-nently turned ON.

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IL-7/IL-7R-Mediated Signaling in T-ALL versus Normal T-cells: Subtle Differences – MajorConsequences?

IL-7 binding to its receptor initiates signaling by promoting IL-7Ra and gc heterodimerization andassociated conformational changes (McElroy et al., 2012) that allow for JAK1 and JAK3 trans-phosphorylation and consequent phosphorylation of tyrosine residues in the cytoplasmic tail of IL-7Ra (of which the conserved Y449 residue appears to be critical for downstream signaling), therebycreating docking sites for effector molecules, most notably PI3K and STAT5 (reviewed in (Kittipatarinand Khaled, 2007; Mazzucchelli and Durum, 2007)).

STAT5 is phosphorylated by JAKs following recruitment to the IL-7Ra, consequently dimerizing andtranslocating to the nucleus where it induces transcription of several genes involved in cell survival andproliferation. The JAK/STAT pathway is critical for IL-7-dependent homeostasis of naïve CD4 and CD8 T-cells, as demonstrated by the fact that overexpression of SOCS1 (a negative regulator of JAK-STATsignaling) results in reduction of the number of naïve cells in the peripheral T-cell pool(Ramanathan et al., 2006). During thymic development, there is evidence that STAT5 is involved in IL-7-mediated immature thymocyte differentiation rather than mediating the survival/proliferativeeffects of the cytokine, which are PI3K-dependent (Pallard et al., 1999). The role of JAK/STAT5 pathwayin the development of human T-ALL is not clear, although there is evidence from mouse studies thatSTAT5 is required for IL-7-dependent T-cell lymphomagenesis (Abraham et al., 2005). However, thedetails and possible specificities of JAK/STAT pathway modulation by IL-7 stimulation in T-ALL cells ascompared to their normal counterparts remain to be dissected.

The PI3K/Akt/mTOR pathway is anothermajor signalingmodule involved in thymocyte developmentand IL-7-mediated effects. Phosphatidylinositol 3-kinase (PI3K) catalyzes the production of phosphati-dylinositol 3,4,5 trisphosphate (PIP3), therebyactivatinganumberofdownstreamtargets, includingmostprominently Akt/PKB and its downstream targetmTOR. In vitro studieswith human thymocytes indicatethat activationofPI3K/Aktpathway is required for IL-7-inducedproliferationand/or survival of immatureCD34þ thymocytes (Pallard et al.,1999). Interestingly, IL-7was shown to induce rapid phosphorylation ofAkt/PKB in CD34þ thymocytes, which was not detected in more differentiated CD34- thymocytes(Johnson et al., 2008). In humanmature peripheral T-cells IL-7 induces PI3K/Akt pathway activation onlyafter at least one hour of stimulation. This late, but sustained, induction of PI3K/Akt signaling activity isnecessary for T-cell proliferation (Lali et al., 2004) and, as judged by studies in mouse peripheral T-cells,STAT5 transcriptional activitymight be required for Akt activation (Wofford et al., 2008). This contrasts towhat happens in T-ALL cells, which generally respond to IL-7 in a manner that resembles that of CD34þthymocytes in the sense that PI3K/Akt/mTOR pathway is rapidly activated even in CD34- T-ALL cases((Barata et al., 2004a; Barataet al., 2004c; Batista et al., 2011; Silva et al., 2011a) andourunpublisheddata).This ability to promote early, STAT5-independent activation of PI3K/Akt/mTORpathwaymight reflect theexistence of an immature precursor program that remains active in T-ALL cells irrespectively of theirdevelopmental stage. Whether this is indeed the case requires investigation.

IL-7 prevents T-cell apoptosis and induces cell cycle entry, thereby promoting in vitro proliferation ofboth normal and malignant T-cells (e.g. (Barata et al., 2001; Barata et al., 2004c; Fox et al., 2005; Khaledet al., 2002; Lali et al., 2004; Pellegrini et al., 2004; Rathmell et al., 2001; Sereti et al., 2009; Swainsonet al.,2007; vonFreeden-Jeffryet al.,1997)–however, themechanismsbywhich IL-7does sodiffer in each case.In healthy T-cells PI3K/Akt/mTOR activation is indispensable for T-cell cell cycle progression (Lali et al.,2004; Swainson et al., 2007), but does not regulate Bcl-2 expression or cell survival (Fox et al., 2005;Rathmell et al., 2001). In sharp contrast, PI3K/Akt/mTOR activation in IL-7-stimulated T-ALL cells isfundamental not only for p27kip1 downregulation and cell cycle progression but also for Bcl-2 upregu-lation and increased cell viability (Barata et al., 2001; Barata et al., 2004c). Although the exact reasons forthese functional differences warrant investigation, they have clear therapeutic implications. PI3K ormTORsmallmolecule inhibitorsareessentiallycytostatic for IL-7-exposedhealthyT-cells (Swainsonetal.,2007) and do not significantly affect the viability ofmature T-cells (Fox et al., 2005; Rathmell et al., 2001)or thymocytes (our unpublished data). In contrast, the same class of drugs is clearly cytotoxic to IL-7-stimulated T-ALL cells, blocking cell cycle progression and promoting apoptosis (Barata et al., 2001;Barata et al., 2004c). Most patients at diagnosis present with leukemia cells that are IL-7-responsivein vitro (Barata et al., 2004b) and that are likely benefiting from IL-7 exposure in vivo (Silva et al.,

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2011b). Therefore, if integrated into combinatorial therapeutic protocols these drugs have thepotential tocontribute to the selective elimination of the malignant cells without having a major impact on theirnormal counterparts. Moreover, the therapeutic potential of PI3K/Akt/mTOR signaling inhibitors mayextend beyond the context of IL-7R-mediated signaling, since it is known that the vast majority of T-ALLdisplayconstitutive activationof the pathway (Silva et al., 2008). In this regard it isworthmentioning thatpre-clinical in vitro studies with several clinical-stage PI3K/Akt/mTOR pharmacological inhibitors haveshownpromising results (Chiarini et al., 2010; Evangelisti et al., 2011; Grimaldi et al., 2012; Simioni et al.,2012).

Information on other pathways that have been reported to be activated by IL-7 in lymphocytes canbe found elsewhere (Barata et al., 2005; Jiang et al., 2005; Khaled and Durum, 2002, 2003).

Genetic Evidence for the Relevance of IL-7R-Mediated Signaling in T-ALL

In the previous section, we have summarized the main signaling pathways elicited by IL-7 stimu-lation of normal and malignant T-cells. This allows us to better contextualize the genetic evidence onthe relevance of cell-autonomous deregulation of IL-7R-mediated signaling networks in T-ALL biology,which extends beyond the IL7R mutations we already described.

Somatic JAK1 gain-of-function mutations are relatively prevalent in adult T-ALL (18% of the cases)and can also be found in childhood T-ALL (2% of the cases) (Flex et al., 2008; Zhang et al., 2012). Themutations affect conserved residues within the FERM, SH2, pseudokinase, and kinase domains of JAK1.Importantly, the mutants induce constitutive activation of STATs, Akt and ERK proteins, and confergrowth factor independence to Ba/F3 cells, indicative of their transforming ability. Clinically, JAK1mutations are associated with relapse and decreased overall survival (Flex et al., 2008). Themechanismof action of these mutations is not completely understood, particularly given their heterogeneousdomain location, although some data indicate that they might play a role in inducing conformationalchanges which promote the catalytic activation of the protein (Radtke et al., 2005; Zhou et al., 2001).

JAK3 gain-of-functionmutations have been reported in differentmalignancies, such as breast cancer(Jeong et al., 2008), adult T-cell leukemia/lymphoma (Elliott et al., 2011) and megakaryoblasticleukemia (Walters et al., 2006). They have been recently described also in T-ALL (Bains et al., 2012;Kalender Atak et al., 2012; Zhang et al., 2012). Similar to JAK1, the JAK3 mutations occur within theFERM, pseudokinase and kinase domains and are transforming in vitro. However, their mechanism ofaction still needs to be elucidated.

In addition to these alterations in IL-7R-associated JAKs, there is evidence that cell-autonomouslesions in downstream mediators of IL-7R signaling are frequent in T-ALL cells, most notably withinPI3K/Akt pathway. Hyperactivation of this pathway is very frequent in T-ALL and contributes toleukemia cell maintenance (Silva et al., 2008). The major negative regulator of PI3K/Akt pathway is thelipid phosphatase PTEN (phosphatase and tensin deleted on chromosome 10), which removes the 3-phosphate from PIP3, dephosphorylating PIP3 into PIP2 and thereby down-modulating Akt andmTOR signaling (Fruman and Bismuth, 2009). PTENmutations aremostly clustered in exon 7 (Gutierrezet al., 2009; Jotta et al., 2010; Maser et al., 2007; Silva et al., 2008), which encodes the C2 domain of theprotein, and are usually nonsense or frame-shift, resulting in a truncated, non-functional protein(Gutierrez et al., 2009). They occur in 5–30% of the T-ALL samples and have been associated withdecreased overall survival and risk of relapse (Jotta et al., 2010). PTEN gene deletion is less frequent,occurs in up to 9% of primary T-ALL and has also been associated with poor prognosis (Gutierrez et al.,2009; Remke et al., 2009). PIK3CA (p110a) gain-of-function mutations in T-ALL are uncommon (5% ofthe cases) and affect mostly exons 9 and 20. PI3KR1 (p85a) inactivating mutations have also beenreported in 5% of T-ALL patients. AKTmutations are even less frequent (around 2% of T-ALLs) (Gutierrezet al., 2009). The frequencies of mutations for each gene are relatively low. However, if analyzedtogether as part of a common pathway, genetic alterations in the PTEN-PI3K-AKT pathway can accountup to 48% of the T-ALL cases (Gutierrez et al., 2009). Evidently, this pathway is involved in manyprocesses in the cell that do not relate to IL-7R-mediated signaling. Nonetheless, one should take intoaccount that PI3K/Akt/mTOR is one of the major IL-7R downstream signaling effectors, with a clearimpact on T-ALL cell cycle progression and cell viability. Therefore, it seems logical to include thegenetic alterations summarized above that affect PI3K/Akt/mTOR signaling as evidence, together with

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the mutations affecting JAK1, JAK3 and IL7R itself, for the high prevalence of mutations affecting the IL-7/IL-7R signaling axis in T-ALL. Overall, the studies summarized in this review hint on the majorimportance of IL-7/IL-7R-mediated signaling deregulation for human T-cell leukemogenesis.

Conflict of Interest Statement

The authors have no conflict of interest to declare.The research work in JTB’s lab related to the present reviewwas supported by grants from Fundação

para a Ciência e a Tecnologia, FCT, Portugal. The funding agency did not have any influence on theplanning, execution or writing of the manuscript.

Acknowledgments

The research work in JTB’s lab related to the present reviewwas supported by grants from Fundaçãopara a Ciência e a Tecnologia, FCT, Portugal (PTDC/SAU-OBD/104816/2008, PTDC/SAU-ONC/122428/2010 and PIC/IC/83023/2007). DR and AM have FCT PhD fellowships. We apologize to the authorswhose work, although of relevance to the field, may have not been included in this review.

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