Oncogene (2013) 32, 2601–2613 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc

REVIEW JAK/STAT signaling in hematological malignancies

W Vainchenker1,2,3 and SN Constantinescu4,5

The (JAK)/signal transducer and activator of transcription (STAT) pathway is central to signaling by receptors, a superfamily of more than 30 transmembrane that recognize specific , and is critical in blood formation and immune response. Many of those receptors transmit anti-apoptotic, proliferative and differentiation signals, and their expression and functions are critical for the formation of blood lineages. Several cancers, including blood malignancies, have been associated with constitutive activation of members of the STAT family, which normally require JAK-mediated tyrosine phosphorylation for transcriptional activation. More recently, human myeloproliferative neoplasms were discovered to be associated with a unique acquired somatic in JAK2 (JAK2 V617F), rare exon 12 JAK2 , or mutations that constitutively activate wild-type JAK2. Prompted by these observations, many studies have explored the possibility that JAKs, cytokine receptors, or other components of the JAK/STAT pathway are mutated or upregulated in several hematological malignancies. This has been observed in certain pediatric acute lymphoblastic and adult T-cell lymphoblastic leukemias, and overexpression of JAK2 seems to be important in Hodgkin lymphoma. Here we discuss the nature and respective contribution of mutations dysregulating the JAK/STAT pathway in hematological malignancies and present examples in which such mutations drive the disease, contribute to the phenotype, or provide a survival and proliferative advantage. JAK inhibitors are making their way into the therapeutic arsenal (for example, in myelofibrosis), and we discuss the possibility that other hematological diseases might benefit from treatment with these inhibitors in combination with other agents.

Oncogene (2013) 32, 2601–2613; doi:10.1038/onc.2012.347; published online 6 August 2012 Keywords: JAK/STAT; hematological malignancies;

INTRODUCTION of rapamycin (mTOR) (Figure 1). Additionally, STAT proteins can be 10 The understanding of the mechanisms by which an extracellular activated by other kinases, such as SRC family kinases. signal can modify the biology of a cell by activating transcription Activation of STAT proteins, especially STAT5 and STAT3, is has been greatly advanced by investigating important in the pathogenesis of lymphoid and myeloid 11–14 signaling. Particularly, studies of transcriptional induction by the malignancies. Until recently, few genetic alterations had cytokines a, b and g via their cognate receptors have been found in this pathway, with the exception of mutations or facilitated defining the Janus kinase (JAK)/signal transducer and silencing of negative regulators and the presence of rare 15 activator of transcription (STAT) pathway.1,2 This pathway begins oncogenic JAK2 fusion proteins. The recent discovery of a by the binding of a cytokine to its cognate receptor, which basally recurrent activating somatic mutation in JAK2 in more than 50% of is completely inactive. Cytokine binding induces a conformational classical myeloproliferative neoplasms (MPNs) has emphasized the 16–19 change that either reorients a preformed dimer3–6 or induces role of JAKs in oncogenesis. Following this discovery, genetic receptor dimerization/oligomerization.7 As a consequence, JAKs, alterations in JAK2, JAK1 and JAK3 were discovered in hemato- 20–23 prebound to the receptors’ cytoplasmic region, become activated, logical malignancies as well as in other JAK regulators, such as leading to cross-phosphorylation and receptor tyrosine phos- cytokine receptors (myeloproliferative leukemia (MPL)/ phorylation, thus creating binding sites for cytoplasmic proteins (TPOR), cytokine receptor-like factor 2 24–27 28 such as the STAT proteins, which become substrates for JAKs.8 (CRLF2), -7 receptor a (IL-7 Ra)) and LNK. Phosphorylation leads to STAT homodimerization and trans- In this review, we focus on the role of the JAK/STAT pathway in location to and retention in the nucleus, where they act as hematological malignancies, particularly those with alterations in transcription factors (Figure 1). directly involved in this pathway and the role of JAKs as This pathway has a central role in the signaling of cytokines by potential therapeutic targets. regulating cell proliferation, survival, differentiation and immune response. Consequently, it has become clear that the JAK/STAT pathway also has an important role in oncogenesis.9,10 In addition THE JAK/STAT SIGNALING PATHWAY to STAT activation, JAK signaling activates other signaling The codes for four JAK (JAK1, JAK2, JAK3 and pathways such as mitogen-activated (MAPK), 2 [TYK2]) and seven STAT (STAT1, STAT2, STAT3, phosphatidylinositol-3’-kinase (PI3K) and AKT/mammalian target STAT4, STAT5A, STAT5B and STAT6) proteins, which mediate

1INSERM, UMR 1009, Hematopoı¨e`se Normale et Pathologique, Villejuif, France; 2Institut Gustave Roussy, UMR 1009, Villejuif, France; 3Universite´ Paris-Sud, UMR 1009, Villejuif, France; 4Ludwig Institute for Cancer Research, Brussels Branch, Brussels, Belgium and 5de Duve Institute, Universite´ Catholique de Louvain, Brussels, Belgium. Correspondence: Dr W Vainchenker, INSERM, UMR 1009, Hematopoı¨e`se normale et pathologique, Institut Gustave Roussy PR 1, 114, rue Edouart Vaillant, 94805 Villejuif Cedex, France or Dr SN Constantinescu, Universite´ Catholique de Louvain, de Duve Institute, Ludwig Institute for Cancer Research, Brussels Branch, Avenue Hippocrate 74, UCL 75–4, Brussels B1200, Belgium. E-mail: [email protected] or [email protected] Received 21 May 2012; revised 25 June 2012; accepted 26 June 2012; published online 6 August 2012 JAK/STAT signaling in hematological malignancies W Vainchenker and SN Constantinescu 2602

Figure 1. Cytokine receptors exist on the cell surface in an inactive state bound to JAKs via their cytosolic domains. The binding of a specific induces a conformational change in the preformed dimer, leading to tyrosine phosphorylation and cross-activation of JAKs, which phosphorylate intracellular receptor tyrosine residues (Y-P). In turn, the phosphorylated residues attract signaling adaptor proteins that recognize specific tyrosine phosphorylated sequences. Various adaptor proteins become substrates of JAKs, triggering signaling cascades. Cytokine receptors are linked to the STAT, Ras–MAPK, and phosphatidylinositol-3’-kinase (PI3K)–AKT pathways, which converge at the nucleus and regulate expression. Left, unliganded inactive cytokine receptor; middle, ligand-activated receptor, which is transient and induces anti-apoptotic, proliferative, and differentiation signals. Right, unliganded receptor that is constitutively active because of the attachment of JAK2 V617F, a constitutively active JAK, and is therefore recapitulating the cytokine-induced pathway, although in a persistent manner. Novel signaling molecules (X, Y and Z) become substrates of activated JAK, initiating novel interactions (X, Y and Z interactors) that change and/or induce novel epigenetic events.

signaling by more than 30 cytokine receptors that belong to the members signal mainly via JAK1, gp130 and other chains can also cytokine receptor superfamily.29 A principal feature of cytokine activate JAK2.29 receptors is the preassociation with JAK proteins, which promotes 30–33 traffic and stability and stringent cytokine dependency Lymphoid JAK/STAT signaling for signaling. Receptors interact with high affinity with cytokines, The JAK/STAT pathway also regulates formation of lymphoid cells. and 50% biologic activity can be obtained at 10% receptor o Cytokines that bind to receptors containing the common g chain, occupancy rates.34 such as IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21, are crucial for formation and function of T, natural killer and B cells.44 These receptors Myeloid JAK/STAT signaling activate JAK1 via cytokine-binding chains and JAK3 via the common g chain.45 Kinase-dead versions of JAK3, or an inhibitor Upon cytokine binding, JAK activation opens the path to that specifically blocks the catalytic activity of JAK3, but not of downstream signaling via particular STAT proteins, MAPK, and JAK1, still allow residual signaling via these receptors, suggesting PI3K/AKT pathways, in proportions that depend on each that a major role of JAK3 is that of a scaffold and partner of JAK1.46 receptor.35 The three type-I homodimeric proteins, erythro- These results suggest that highly specific JAK3 inhibitors may not poietin receptor (EPOR), MPL/TPOR, and granulocyte colony- prove as useful as once predicted in blocking autoimmune or stimulating factor receptor (G-CSFR) employ JAK2;36–38 however, oncogenic signaling via JAK3-associated cytokine receptors. Most MPL/TPOR can also activate TYK2, with low signaling output,33,37 receptor signaling via JAK1-JAK3 activate STAT3 and STAT5.47 and G-CSFR employs JAK1.39 EPOR activates mainly STAT5, whereas MPL/TPOR and G-CSFR strongly activate STAT3. MPL/ TPOR also induces STAT1 activation.40 MPL/TPOR and G-CSFR Termination of JAK/STAT signaling strongly activate the MAPK extracellular-regulated kinase 1/2 Physiologically, signaling by cytokine receptors is transient. The (ERK1/2) pathway, whereas EPOR activates this pathway to a lesser termination of the signal is due to (1) internalization and degree.38,41,42 Disrupting the physiological ratio between STAT5 degradation of the receptors by the lysosome and and STAT3 can lead to leukemia, as is seen with G-CSFR pathways, which require ubiquitination of intracellular domain truncations.43 In addition to the homodimeric receptors, GM-CSF lysines.33,48–51 JAK activation is required for degradation of and IL-3 utilize heterodimeric receptors that contain the common internalized receptors, as was demonstrated for TYK2 and b chain, which signals also via JAK2.29 Although IL-6 family interferon a, b, and o receptor 1;52 (2) phosphatase recruitment

Oncogene (2013) 2601 – 2613 & 2013 Macmillan Publishers Limited JAK/STAT signaling in hematological malignancies W Vainchenker and SN Constantinescu 2603 to the receptor, thereby dephosphorylating JAKs;53 (3) recruitment Other oncogenic JAK2 fusion proteins have been described with of negative regulators that are transcriptionally induced by partner proteins such as breakpoint cluster region (BCR), single- cytokines, such as cytokine-induced SH2-containing protein 1 stranded DNA-binding protein 2, calmodulin-binding protein 3 and suppressors of cytokine signaling 1 to 7 (SOCS1–7) striatin and paired box 5 (PAX5) in MPNs and acute leukemia.71–73 proteins;43,54–58 and (4) interaction of STAT proteins with protein In pediatric ALL, PAX5, a master regulator of B-cell development, is inhibitors of STATs (PIAS) family members, which induce small rearranged in 2.5% of B-cell precursors, including the JAK2 fusion ubiquitin-related modifiers-(SUMO)–ylation of several transcription protein PAX5–JAK2, which has been implicated in tumorigen- factors and form signaling platforms localized to subnuclear esis.73 More recently, a fusion between SEC31A and JAK2 created regions.59 PIAS family members can repress transcriptional activity by a t(4;9)(q21;p24) was reported in B3% of patients with of transcription factors, such as the CCAAT/enhancer-binding Hodgkin lymphoma.74 SEC31A is involved in vesicular transport protein delta (C/EBPd) in an independent manner from the and has been shown to be present in another fusion with SUMOylation activity.60 anaplastic lymphoma in some forms of A substantial body of evidence has revealed that constitutively large B-cell lymphoma. The SEC31A–JAK2 leads to active cytokine receptors, obtained by in vitro mutagenesis, such constitutive JAK2 activity that can be blocked by JAK2 inhibitors. as those that are artificially dimerized by disulfide bonds in active Overall, in all cases, the expression of the fusion protein conformations, that is, EPOR R129C or MPL/TPOR S368C61,62 lead containing the JAK kinase domain is driven by the promoter of to permanent signaling. Thus, constitutively active STAT proteins the fusion partner, which induces oligomerization and activation in the nucleus overcome negative regulatory mechanisms, of the JAK kinase. eventually leading to oncogenesis.63 In addition, the JAK/STAT pathway may have an important role in the pathogenesis of Hodgkin lymphoma as the JAK2 is amplified in B30% of cases,74 supporting the notion that JAK overexpression can be oncogenic.75 In Hodgkin lymphoma, GENETIC ALTERATION OF JAKS IN HEMATOLOGICAL survival has been correlated with expression of a microRNA, MALIGNANCIES 76 miR-135a, which targets JAK2. Furthermore, loss-of-function JAK2 fusion proteins SOCS1 mutations and deletions in nonreceptor type protein Translocation ETS leukemia (TEL)–JAK2 was the first JAK2 fusion tyrosine phosphatase 2 (PTPN2) have been described, leading to protein to be discovered in a malignancy of a patient with T-cell activation of JAK/STAT signaling.77,78 childhood acute lymphocytic leukemia (ALL).15 TEL, also called ETS variant 6, has been found to be involved in other translocations associated with leukemias. The TEL–JAK2 protein results from a JAK mutations fusion between the oligomerization domain of TEL and the kinase More recently, JAK mutations have been found in hematological domain of JAK2 and exhibits constitutive tyrosine kinase activity malignancies. The classical non–BCR-ABL MPNs, polycythemia vera by promoting oligomerization of JAK2.15 The TEL–JAK2 fusion is (PV), essential thrombocythemia (ET), and primary MF (PMF), are associated with rare B-cell or T-cell leukemia as well as atypical chronic clonal disorders that arise from the hematopoietic stem chronic myeloid leukemia.64 The TEL–JAK2 fusion protein con- cells, clinically characterized by the production of an excess of stitutively activates STAT1 and STAT5 and the PI3K, RAS/ERK, p38 mature blood cells and biologically by a hypersensitivity or and NF-kB signaling pathways.65 In transplantation assays, TEL– independence to cytokines such as (EPO).79 Initially, JAK2 induces a fatal lymphomyeloproliferative disease, with JAK2 was an obvious candidate as a 9p loss of heterozygosity in a development that depends on STAT5.66 When human cord region that includes JAK2 had been described in 30% of cases of blood CD34 þ cells transduced with a vector encoding TEL–JAK2 PV.80 In 2005, an acquired mutation in exon 14 of JAK2, a guanine- are transplanted into immunodeficient mice, the mice develop to-thymidine substitution that results in a substitution of valine to myelofibrosis (MF), a myeloproliferative disorder.67 Additionally, phenylalanine at codon 617 (JAK2 V617F), was found in more than the pericentriolar material 1 (PCM1)–JAK2 fusion protein68 was 90% of patients with PV and 50% of those with ET and PMF.16–19 identified in several cases of chronic and acute leukemia69 and JAK2 V617F is a gain-of-function mutation located in the T-cell lymphoma.70 PCM1 encodes a centrosomal protein, the pseudokinase domain of JAK2 (JAK homology 2 [JH2]) (Figure 2), human autoantigen PCM1, predicted to contain a number of which negatively regulates the kinase domain (JH1), possibly via coiled-coil domains. ATP binding and autophosphorylation81 of sites known as

Figure 2. Domain structure of JAK2. JAK2 contains a tyrosine kinase domain (JAK homology 1 (JH1)), a pseudokinase domain (JH2), SH2-like domain, and a domain that resembles protein 4.1, ezrin, radixin and moesin (FERM). The latter domain is responsible for attachment to the cytosolic domains of cytokine receptors. The pseudokinase domain, JH2, functionally prevents the activation of the kinase domain, JH1 (curved arrow on the top). In contrast, mutations in the pseudokinase domain (red) lead to activation (arrow on the bottom) of JH1. The V617F mutation has been identified primarily in MPNs, whereas the R683 mutation (located in the hinge between the N- and C-lobes of JH2) has been uniquely identified in pediatric leukemia.

& 2013 Macmillan Publishers Limited Oncogene (2013) 2601 – 2613 JAK/STAT signaling in hematological malignancies W Vainchenker and SN Constantinescu 2604 negative regulatory sites (S523, Y570).82–84 In contrast to JAK2 JAK2 exon 12 mutations in PV fusion proteins, which signal autonomously, JAK2 V617F requires Activating JAK2 mutations are observed in more than 97% of activation of cytokine receptors for oncogenic signaling, especially patients with PV. Different somatic gain-of-function mutations in 85 dimeric receptors such as G-CSFR, EPOR and MPL/TPOR. exon 12 of JAK2 have been found in a fraction of patients with However, JAK2 V617F can also induce the canonical pathways of JAK2 V617F–negative PV.124 These genetic alterations are usually these receptors in the absence of cytokines (Figures 1 and 2). This complex but most frequently affect a single residue (K539). includes the activation of STATs, PI3K and MAPK/ERK pathways, Although not located in the pseudokinase domain, K539 mutants 17,19,86 respectively. The choice of activated STATs may largely may lead to the same change in JH2 domain structure as V617F. In depend of the receptor type, for example, essentially STAT5 for contrast to JAK2 V617F, exon 12 mutants are not associated with EPO-R and STAT3, STAT5 and STAT1 for MPL. When introduced in ET and PMF.125 The majority of patients with PV with JAK2 exon 12 cytokine-dependent cell lines, JAK2 V617F induced cytokine mutations exhibit clinical features of an idiopathic erythrocytosis, 17 independence and hypersensitivity, including to -like but these patients also progress to secondary MF.126 1.87 In different murine models, JAK2 V617F induced a myeloproliferative disorder, often phenocopying PV that progresses to MF, and an ET-like disorder.88–94 It has been suggested that both in humans and mice, the phenotype of the JAK MUTATIONS AND LEUKEMIA disease depends on the intensity of the signaling, which is related JAK2 to the number of JAK2 V617F copies.89 Supporting this hypothesis, A in the pseudokinase domain of JAK2 called JAK2DIREED most patients with PV have a homozygous mutation through a including R683 was initially discovered in a patients with Down mitotic recombination leading to the 9p loss of heterozygosity, syndrome (DS) and B-cell ALL (B-ALL).127 Expression of whereas the majority of patients with ET have a heterozygous JAK2DIREED in Ba/F3 cells induced constitutive activation of the mutation.95,96 The phenotype of the disease induced by JAK2 JAK/STAT pathway and growth factor–independent cell prolifera- V617F might also depend on the activation of specific tion.127 Subsequently, JAK2 mutations located in the downstream signals because PV, ET and PMF are associated with pseudokinase domain, especially at R683, but also in the kinase strong STAT5, STAT1 and STAT3 activation, respectively.86,97 In domain, were found in B20% of patients with DS and B-ALL.128,129 in vitro experiments, the EPO independence induced by JAK2 These JAK2 mutants are associated with CRLF2 rearrangements, V617F required both STAT5 and PI3K activation.98,99 leading to overexpression of this receptor.25 CRLF2, located on Two recent studies established that STAT5A/B activation is X, encodes a lymphoid cytokine receptor that crucial for development of JAK2 V617F–positive MPNs in mouse heterodimerizes with IL-7 Ra to form the receptor for thymic models100,101 using an inducible double STAT5A/B knockout stromal lymphopoietin. CRLF2 overexpression favors the strategy,102,103 suggesting that inhibitors of STAT5 could be transforming activity of JAK2 R683 mutants in Ba/F3 cells. useful in MPNs. Residual STAT5 activation after incomplete JAK2 Interestingly, the DS ALL with JAK mutations had similar gene inhibition might explain the apparent inability of JAK2 inhibitors expression signatures with BCR-ABL B-ALL, with frequent deletions to reduce the JAK2 V617F allele burden, although more data are in IKAROS family zinc finger 1 and cyclin-dependent kinase 2 required to resolve this issue.104–106 inhibitor A/B.130 This defines a subgroup of activated tyrosine There is also increasing evidence that other clonal mutations in kinase signature ALL that usually has a poor prognosis, which may epigenetic regulators such as TET2 and ASXL1 or in the splicing also include patients with genomic rearrangements in tyrosine machinery are present in a fraction of JAK2 V617F–positive MPNs kinases other than JAK and ABL, and therefore, may benefit from and other myeloid malignancies.107–109 This is especially true for new therapeutic approaches. PMF, in which such mutations are present in more than 50% of Importantly, JAK mutations and overexpression of CRLF2 have patients. Whether JAK2 V617F is the initiating event in these MPNs also been detected in other cases of pediatric B-ALL (without DS), is controversial and is presently under investigation, but JAK2 with a frequency between 4% and 7%.20,23,25 In nearly all cases, V617F seems to drive the phenotype of the disease. This was JAK mutations are associated with an overexpression of CRLF2. further confirmed in sideroblastic with thrombocytosis, a However, overexpression of CRLF2 is much more frequent than form of myelodysplastic syndromes (MDS), in which the JAK mutations and can be associated with other mutations such as sideroblastic anemia seems related to a splicing factor 3b IL-7 R.131 subunit 1 mutation110 and the thrombocytosis to JAK2 V617F.111 An activation mutation in JAK2 (T875N) has been described in a JAK2 V617F is not only a preactivated molecule but has also cell line derived from acute megakaryoblastic leukemia (AMKL), acquired additional properties: (1) resistance to SOCS3 negative CHRF-288–1, which has not yet been found in primary samples regulation;112 (2) tyrosine phosphorylation of protein arginine from patients with AMKL.132 methyltransferase 5,113 an arginine methyltransferase with different histone and nonhistone proteins substrates;114,115 (3) unconventional signaling to the chromatin by inducing histone JAK1 tyrosine phosphorylation;116 (4) decreased ability to promote MPL/ Somatic mutations in JAK1 have been found in 10 to 20% of cases TPOR cell surface expression, allowing megakaryocytes to escape of T-cell ALL (T-ALL)20,133,134 and less frequently in B-ALL. TPO-induced senescence;117,118 (5) preferred association with heat Mutations are located in the protein 4.1, ezrin, radixin and shock protein 90 (HSP90);119 and (6) specific requirement for the moesin (FERM) and the pseudokinase domain as the V658F pseudokinase domain F595 residue for constitutive signaling.120 mutant,20,133,134 equivalent to JAK2 V617F and shown previously Saturation mutagenesis at position V617 showed that Trp, Met, to be active.87 They also require signaling receptors such as IL-7R, Leu and Ile also induced constitutive activation of JAK2, with JAK2 IL-9 Ra, or IL-2 Rb for constitutive activation.135 Some JAK1 V617W being the strongest and Ile the weakest.121 Very rarely, mutations are associated with deletions in negative regulators patients with MF have been reported as carrying the JAK2 V617I such as PTPN2136 or CD45 (protein tyrosine phosphatase receptor mutation.122 For example, a JAK2 V617I germline mutation was C).137 As in B-ALL, the JAK/STAT pathway may have an important recently described in a family with hereditary autosomal dominant role in the pathogenesis of T-ALL as IL-7 R– and JAK3-activating thrombocytosis.123 Interestingly, this weaker mutation only mutations as well as loss-of-function mutations in SH2B3 (LNK) induced thrombocytosis, possibly fitting the model that low have been found in 15% of the cases.27,133 Rare (1%-2%) JAK1 JAK2 activation will induce ET, whereas stronger JAK2 activation mutations have also been described in will induce PV and PMF.123 (AML).21,22

Oncogene (2013) 2601 – 2613 & 2013 Macmillan Publishers Limited JAK/STAT signaling in hematological malignancies W Vainchenker and SN Constantinescu 2605 JAK3 and TYK2 mutations in STAT1, STAT3 and STAT5 have yet been found in the JAK3 mutations have been detected in rare cases of adult T-cell other hematological malignancies. leukemia/lymphoma, cutaneous T-cell lymphoma,138 and more 133 recently in T-ALL. Interestingly, three gain-of-function (L156P, Cytokine receptors E183G, R172Q) mutations were found in the FERM homology 139 Gain-of-function mutations in cytokine receptors, which induce domain of JAK3 in adult T-cell leukemia/lymphoma. constitutive JAK activation, have been described in patients with Moreover, JAK3 mutations (JAK3 A572V, A573V, P132A) have 132,140,141 MPNs, ALL, and very rarely in patients with AML. In addition, been described in AMKL. These results suggest either activating mutations in tyrosine kinase receptors (RTKs) such as that a gc-coupled receptor expressed in the megakaryocyte FLT3 or KIT may activate STAT proteins independent of JAK lineage might be involved in supporting signaling by this JAK3 activation. mutant or that upon mutation, JAK3 acquires the ability to bind another receptor. Taken together, these data indicate that JAK mutants require MPL/TPOR and MPN cytokine receptor dimers/oligomers for constitutive signaling, and Several activating mutations of MPL/TPOR have been found with a it remains to be established whether mutant JAKs exert the same frequency of B5% and up to 15% in patients with ET and PMF.24 specificity as wild-type JAKs for cognate receptors. Most involve W515 in exon 10, resulting in its substitution to TYK2 mutations seem to be very rare in hematological , lysine, asparagine or alanine24,150–153 (Figure 3). Trypto- malignancies. Several nonsynonymous germline sequence var- phan-515 is located in an amphipathic motif (K/RWQFP) in the iants have been associated with AML but do not seem to be cytoplasmic region just after the transmembrane domain and consistent in affecting kinase activity.21 They seem to correspond prevents spontaneous activation of the receptor.154 These MPL to polymorphisms linked to a predisposition to leukemia, leading mutants induce a constitutive activation of JAK2 that is to either normal or decreased signaling function.21 A germline responsible for myeloproliferative disorders in animal models.24 TYK2 P1104A variation was previously linked to tumor tissue and In addition to JAK2 activation, MPL W515 mutants require tyrosine was predicted to exhibit impaired function.142 Similarly, it has 626 in the cytosolic domain to induce oncogenic signaling,153 been shown that the 46/1 haplotype related to the JAK2 locus which links the receptor to the STAT3 and MAPK-ERK1/2 increases the probability to develop JAK2 V617F MPN 3- to pathways. Altogether, this supports the contention that classical 5-fold.143–145 BCR-ABL–negative MPNs are related to an abnormal JAK2 activation. In addition, the MPL S505N mutation, initially described in familial thrombocytosis,155 was also found in rare 151,156 GENETIC ALTERATIONS IN DOWNSTREAM COMPONENTS OF sporadic MPNs (Figure 3). Furthermore, rare MPL mutations (three cases of MPL W515L and one case with a new mutation, THE JAK/STAT PATHWAY 132,157 STAT proteins T487A) have been described in primary or secondary AMKL. Although constitutive STAT activation is very frequent in hematological malignancies, very few mutations in STAT genes IL-7R and CRLF2 in ALL have been yet described. Mutations in the STAT6 DNA-binding In B- and T-ALL, somatic gain-of-function IL-7R exon 6 mutations domain have been found in primary mediastinal B-cell lymphoma have been found with a frequency of B9% in patients with (36%), which may lead to a loss-of-function.146 However, the T-ALL.27,158 In most cases, these mutations introduce an unpaired oncogenic properties of these mutants remain to be determined. cysteine in the extracellular juxtamembrane–transmembrane A possible hypothesis may be related to a cross-talk between region and promote dimerization of the receptor, inducing STAT1 and STAT6.147 Thus, these loss-of-function mutations may constitutive JAK1 activation. In B-ALL, these mutations are inhibit negative regulation mediated by STAT1. Like for some solid associated with an overexpression of CRLF2 (see above) forming tumors,148 recurrent activating mutations of STAT3 have been a functional, spontaneously activated receptor for thymic stromal found in large granular lymphocytic leukemia.149 No recurrent lymphopoietin.158 Interestingly, mutations in CRLF2 have also

Figure 3. The MPL/TPOR contains a unique RWQFP cytosolic juxtamembrane domain motif. W515 in the motif has been found to be mutated in 5% to 10% of patients with essential ET and primary myelofibrosis (PMF) that do not harbor JAK2 V617F. These W515 mutations lead to cytokine-independent activation of the receptor, which then signals constitutively via JAK2. Activating TPOR transmembrane S505N mutations have been identified in familial ET. These receptors form active dimers in the absence of the TPO ligand and signal constitutively via JAK2. In rare human megakaryoblastic leukemia cell lines, an extracellular juxtamembrane T487A-activating mutation has been identified.

& 2013 Macmillan Publishers Limited Oncogene (2013) 2601 – 2613 JAK/STAT signaling in hematological malignancies W Vainchenker and SN Constantinescu 2606 been described (F232C) that result in the addition of a cysteine in observed in some malignancies, allowing for resistance to the anti- the juxtamembranous domain of the cytokine receptor.159 proliferative effect of .172

G-CSFR and AML LNK LNK, also called SH2B3, is a member of the SH2B family, which Activating mutations of G-CSFR (colony-stimulating factor 3 173 receptor (CSF3R)) are rare in AML but have been described in a contains two other members and has an important role in 160 hematopoiesis by negatively regulating JAK2 activation through few cases and, furthermore, may be secondary mutations 174,175 160 its SH2 domain. In murine models, the knockout of LNK occurring during disease progression. The mutation reported in 176 these cases (Thr617Asn) leads to JAK2 activation and can be found leads to myeloproliferation associated with MF. In humans, rare 161 mutations in LNK have been found in patients with ET and PMF in familial neutrophilia. Acquired mutations of G-CSFR have 28 been described in children with severe congenital neutropenia without other mutations in signaling molecules. LNK mutations undergoing granulopoiesis-stimulating factor treatment.162 These lead to loss of regulatory functions and consequently to increased JAK2/STAT signaling. LNK mutations have also been described mutations lead to a truncation in the G-CSFR cytoplasmic domain, 177 impairing its ligand-induced internalization and leading to during disease progression to AML. In some cases, LNK mutations were associated with JAK2 V617F, suggesting, as in prolonged STAT5 activation and decreased SOCS3 binding that 178 can precede the development of MDS/leukemia from a few the murine models, cooperation between these mutants. More recently, mutations in LNK have been found in chronic months to several years. During disease progression, other 133 mutations of G-CSFR located in the extracellular domain can myelomonocytic leukemia, MPN/MDS, and in a case of T-ALL. appear and may lead to cytokine independence.163 SOCS1, SOCS2 and SOCS3 SOCS proteins are also important negative regulators of JAK 179 MOLECULAR ALTERATION IN RTKs AND THE JAK/STAT signaling through a classic feedback loop. In addition, SOCS1 PATHWAY may be involved in the regulation of NF-kB signaling and function.180 Mutations in SOCS1 have been found in different Acquired fusion proteins or gain-of-function mutations in several pathologies, particularly in Hodgkin lymphoma,77 in which they RTKs such as FLT3, KIT, platelet-derived , are associated with high levels of phospho-STAT5 and in primary fibroblast growth factor receptor, and anaplastic lymphoma mediastinal B-cell lymphoma (10% of the cases).181 Mutations in receptor tyrosine kinase have been described in different SOCS are infrequent in MPNs,182 possibly owing to the inability of hematological malignancies including leukemia, mastocytosis SOCS3 to downregulate JAK2 V617F or exon 12 JAK2 mutant and MPNs,164 which are associated with strong activation of signaling.112,182 Hypermethylation of CpG islands in SOCS1 and STAT proteins such as STAT1, STAT3 and STAT5.165–167 There is SOCS3 associated with decreased expression has been described also convincing evidence that STAT activation, especially that of in several hematological malignancies, notably in lymphoid STAT5, is essential in the transformation mediated by most of malignancies183 and JAK2 V617F-positive and -negative MPNs.184,185 these mutants, especially for FLT3 ITD and KIT D816V.168,169 The However, SOCS proteins can be overexpressed in T-cell wild-type receptor either weakly or does not activate the STAT lymphoma.172 proteins, and thus, mutations induce an important switch in initiating STAT activation. The mechanism of this activation remains unclear, but there is evidence that the location of the PTPN1 and PTPN2 activating receptor in the endoplasmic reticulum and signaling PTPN1 and PTPN2, also called protein tyrosine phosphatase 1B from the cytoplasmic compartment, along with resistance to and T-cell protein tyrosine phosphatase, respectively, are capable ubiquitin-dependent degradation, may greatly contribute to of dephosphorylating JAKs. Deletion of the entire PTPN2 locus has activation.168,170 There is also evidence that the kinase domain been demonstrated in T-ALL (6% of the case), usually associated of the receptor can directly phosphorylate different STAT proteins with activating mutations of JAK1.136 A similar inactivation can be and that indirect phosphorylation can occur through SRC family observed in cell lines derived from Hodgkin lymphoma but are kinases or the JAKs. Interestingly, it has been shown that in the rare in primary cells from patients.78 mast cell line HMC-1.1 (KIT V560G) and 1.2 (KIT V560G and D816V), JAK2 was found to be constitutively activated and TG101348, a Protein tyrosine phosphatase receptor C (CD45) JAK2 inhibitor, inhibited JAK2 phosphorylation as well as STAT3 CD45 is a transmembrane phosphatase, which regulates JAK2, and STAT5.171 Thus, the role of JAK in STAT activation depends on particularly in response to IL-3 and EPO.186,187 Recently, loss-of- both the type of RTK mutation and on the cell type. function mutations in CD45 were found in T-ALL primary cells and cell lines.137 To this end, knockdown of CD45 in T-ALL increased JAK/STAT signaling and the signaling of JAK1 and IL-7Ra JAK/STAT NEGATIVE REGULATORS mutants.137 As mentioned previously, the JAK/STAT pathway is negatively regulated by several types of proteins acting on the degradation SHP1 (PTPN6) of JAKs and STAT proteins (SOCS, PIAS), on JAKs or receptor SHP1 mutations have not been described in hematopoietic phosphorylation (LNK, phosphatase), or on the degradation of the malignancies. However, hypermethylation of its promoter has cytokine receptors (Casitas B-cell lymphoma (CBL)). Genes coding been described in multiple myeloma and lymphoma primary cells for most of these proteins can be considered tumor suppressor or cell lines associated with hyperphosphorylation of STAT3.188,189 genes, and their inactivation (mutations, silencing) potentiates JAK/STAT signaling when mutations of JAK are present. In certain cases, they may be the main event in dysregulating the pathway. SHP2 (PTPN11) However, some of these molecules may also have the opposite Gain-of-function mutations in SHP2 are associated with hemato- effect. For example, some phosphatases, such as SHP2, are directly logical malignancies. Germline mutations are present in 50% of involved in positive signaling and are associated with gain-of- cases of Noonan syndrome, a syndrome characterized by multiple function mutations, which affect the RAS pathway more than the malformations including heart disease. This PTPN11 germline JAK/STAT pathway. Furthermore, SOCS overexpression can be mutations as the somatic mutations may lead to juvenile

Oncogene (2013) 2601 – 2613 & 2013 Macmillan Publishers Limited JAK/STAT signaling in hematological malignancies W Vainchenker and SN Constantinescu 2607 myelomonocytic leukemia (35% of the case). In addition, somatic A common signaling event in many hematological malignancies PTPN11 mutation may lead to other pediatric malignancies such is represented by constitutive activation of STAT3 and STAT5, as B-ALL, AML and MDS, but very rarely in adults.190 There is which is translated by the persistent presence of phosphorylated evidence that dysregulation of the N-RAS-ERK pathway is critical STAT proteins on chromatin.11–14 With the exception of genetic in transformation by mutated SHP2, but dysregulation of the alterations in cytokine receptors or in JAKs, the kinase responsible JAK2/STAT pathway seems also to be involved in this process. for phosphorylating STAT proteins remains controversial. In chronic myeloid leukemia, STAT5 activation also participates in the development of MPNs.100 In addition, resistance to CBL may be mediated by an increased expression of STAT5.201 Recent The CBL proteins are multifunctional adapter proteins with evidence has suggested that the development of MPNs is ubiquitin activity and major regulatory roles in RTK negative independent of JAK2 and that activation of STAT5 is directly regulation. However, CBL proteins may have numerous targets mediated by BCR-ABL.202 other than RTK including JAK2, STAT5, and cytokine receptors In AML, it has been suggested that SRC kinases promote STAT such as MPL/TPOR,191 and therefore, loss-of-function in CBL may phosphorylation and survival.203 Genes targeted by constitutively lead to increased JAK/STAT signaling. CBL is mutated in a variety active STAT proteins are not always the same as those targeted by of myeloid malignancies,192,193 with the highest frequency in cytokine-induced STAT protein204 (Figure 4). For example, chronic myelomonocytic leukemia and juvenile myelomonocytic constitutively active STAT5B, but not cytokine-activated STAT5, leukemia. CBL mutations have also been found in a low induces the lipoma preferred partner gene, which is the host gene percentage of patients with PMF (6%).194 for miR-28, a microRNA that targets the 3’-untranslated region of MPL in MPNs. This miR is pathologically expressed in platelets from 30% of patients with MPN.205 ACTIVATION OF THE JAK/STAT PATHWAY BY CYTOKINES AND SIGNALING BY CONSTITUTIVELY ACTIVE STAT PROTEINS Autocrine or paracrine cytokine stimulation has a fundamental TARGETING THE JAK/STAT PATHWAY role in promoting solid tumor proliferation and in angiogenesis. As described above, STAT proteins are constitutively activated in This is less common in hematological malignancies, with the the majority of hematological malignancies. However, only in a exception of some lymphoid malignancies, the best example minority of them could it be demonstrated that JAK/STAT being multiple myeloma. The interaction of myeloma cells with signaling is directly altered, such as in BCR-ABL–negative MPN, a bone marrow induces an increased secretion of cytokines from subset of T- and B-ALL, very rare AML, Hodgkin lymphoma, B-cell both cell types.195–197 Among them, IL-6 induces the activation of mediastinal lymphoma, and multiple myeloma. The discovery of JAK2 and STAT3, critical for proliferation and survival. However, JAK2 mutants linked to MPNs prompted the development of JAK2 this STAT3 phosphorylation may also depend on mechanisms inhibitors as targeted therapy for MPNs. Several JAK2 ATP other than JAK2 activation. Several JAK2 inhibitors have been competitive type I kinase inhibitors are being tested in clinical investigated for their effect on the growth and survival of multiple trials for primary and secondary MF in patients positive or myeloma cell lines and primary plasma cells, and all inhibited negative for JAK2 mutations.206–208 has recently been cell proliferation and increased apoptosis.198–200 In addition, approved by the Food and Drug Administration for treatment of ruxolitinib, a potent JAK2 inhibitor, enhanced the anti-tumor intermediate- or high-risk MF, and the Committee for Medicinal activity of chemotherapeutic agents in xenografts, offering Products for Human Use of the European Medicines Agency promising preclinical results.198 recently recommended the approval of ruxolitinib for the

Figure 4. Constitutive STAT3 and STAT5 activation in hematological malignancies. STAT proteins are phosphorylated on tyrosine residues by cytokine-activated JAKs but also by c-SRC, constitutively activated or overexpressed JAKs, fusion proteins involving kinases (ABL, platelet- derived growth factor (PDGFR)), mutated cytokine receptors (MPL/TPOR, G-CSFR, or mutated tyrosine kinase receptors (KIT, FLT3), and by receptor tyrosine kinases such as insulin-like growth factor 1 receptor (IGF1R) in certain transformed cells. Physiological targets of cytokine- activated JAK/STAT pathway signaling are persistently activated in transformed cells, along with novel targets that require a prolonged period of phosphorylated STAT proteins in the nucleus and/or association with other transcription factors.

& 2013 Macmillan Publishers Limited Oncogene (2013) 2601 – 2613 JAK/STAT signaling in hematological malignancies W Vainchenker and SN Constantinescu 2608 treatment of MF-related or symptoms. Because progenitors, that is, erythroid in PV or megakaryocytic in ET/PMF, none of the present compounds discriminate between wild-type are addicted to certain pathways in contrast to normal progeni- and mutated JAK2, as a consequence of efficacy and their tors. Along these lines, inhibitors of STAT5, STAT3, MAP-kinase, mechanism of action, JAK2 inhibitors may induce significant levels PI3K, AKT and mTOR exist, and at least in cell lines and primary of anemia and thrombocytopenia, which is considered a toxic cells, mTOR inhibitors have proven useful.220 effect and can lead to treatment interruption. Additionally, it is It is possible that the combination of JAK2 inhibitors with unknown whether these inhibitors can target heterodimers such inhibitors of downstream effectors including BCL2/BCL-XL (ABT- as JAK1/JAK2 and JAK2/TYK2, which may participate in G-CSF or 737) might be more effective.221 Furthermore, a promising TPO signaling. approach may be represented by the combination with Treatment of patients with MF with ruxolitinib has a modest epigenetic therapy as several major epigenetic regulators have impact in reducing allele burden and decreasing marrow fibrosis. been found to be mutated in MPNs;222 JAK2 may induce Strikingly, and particularly in ruxolitinib treatment, two important epigenetic regulation;116 and constitutive STAT5 signaling might positive effects of JAK2 inhibitors are substantial and rapid have different target genes than the cytokine-induced STAT5 reduction of spleen size and decrease in constitutional symp- protein.205 JAK inhibitors and such combined therapeutic toms.105,106,209–212 These effects are exerted both by JAK1/JAK2 strategies might be extended in the future to subsets of ALL inhibitors such as ruxolitinib and by other JAK2-specific inhibitors, with dysregulation of the JAK/STAT signaling and eventually to as the SAR302503 (TG101348). For constitutional symptoms, therapy-refractory Hodgkin lymphoma. inhibitors might prove beneficial in preventing the cytokine Certainly, it may be important to directly target STAT5 and storm present in MF.209 In contrast, the molecular basis for the STAT3, as their activation is involved in most hematological spleen-reducing effects of JAK2 inhibitors remains unexplained. malignancies. Recently, it was shown that a molecule already Clearly, the understanding of the players involved in these JAK2 developed, pimozide, was capable of inhibiting STAT5 activation effects will be important for future therapeutic improvement. in BCR-ABL cell lines.223 Furthermore, the recent specific The development of mutant specific JAK2 inhibitors remains involvement of STAT1 in ET and not in PV86 suggests that STAT1 difficult because of the lack of a high-resolution crystal structure of inhibitors might limit thrombocytosis in certain situations. On the both the entire JAK2 and the prevailing JAK2 V617F mutant. other hand, inhibition of these pathways is likely to exert Recent structure and function studies suggest that targeting of secondary effects that might limit their use. the pseudokinase domain helix C-F595 and surrounding residues might uncouple inhibition of JAK2 V617F constitutive signaling from cytokine-stimulated signaling.120 Similarly, inhibition of CONCLUSION signaling by MPL/TPOR W515 or S505N mutants would require Numerous genetic alterations involving the JAK/STAT pathway, detailed structures of such mutated receptors that activate wild- including JAK1, JAK2 and JAK3, have been discovered in type JAK2. A molecule specifically blocking cytosolic tyrosine Y626 hematological malignancies these last years. As in MPNs, they of MPL/TPOR may be able to prevent the MF-inducing effects of 153 can drive the disease. JAK inhibitors have been developed with MPL mutants, but such phosphotyrosine blocking molecules some clinical results, but they were moderate in comparison to the are very difficult to obtain. A more global inhibitor of MPL/TPOR expectancies. A future task will be to develop new molecules or signaling may also be useful in certain cases of MPNs that progress therapeutic strategies targeting more specifically the mutated to MF because of excessive MPL/TPOR signaling. However, forms of JAKs; a major challenge will be the development of inhibition of MPL/TPOR signaling in hematopoietic stem cells molecules directly targeting the STAT proteins. might lead to a reduction of hematopoietic stem cell quiescence and reservoir.213–215 Whether the conformation of receptor-JAK complexes differ when the receptor or JAK are mutated remains to CONFLICT OF INTEREST be established, but if this were the case, small molecules or There is potential conflict of interest. Both WV and SNC have participated in ad-hoc that would disrupt JAK2 V617F binding to cytokine Scientific Advisory Boards at Novartis related to JAK inhibitors. receptors would prove useful (Figure 2). Apart from directly targeting JAK2 and MPL/TPOR, recently it was shown that preventing the association of JAK2 mutants with ACKNOWLEDGEMENTS HSP90 leads to JAK2 degradation via ubiquitination.119 Inhibitors of histone deacetylase 6, which is involved in deacetylation of Financial support for medical editorial and graphic design assistance was provided by Novartis Pharmaceuticals. We thank Matthew Hoelzle PhD, Daniel Hutta, PhD and HSP90, are attractive because acetylated HSP90 can no longer Isabelle Plo PhD for their assistance with this manuscript. associate with JAK2. Givinostat, a pan-histone deacetylase inhibitor, induced a hematological response in most patients with PV and some patients with MF in a recent phase II clinical 216 trial. 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