JAK2 and MPL Mutations in Myeloproliferative Neoplasms: Discovery and Science

JAK2 and MPL mutations in myeloproliferative neoplasms: discovery and science

O Kilpivaara1 and RL Levine1,2

1Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA and 2Department of Medicine, Leukemia Service, Memorial Sloan Kettering Cancer Center, New York, NY, USA

Although it has long been known that the myeloproliferative Fialkow identified a single G6PD isoform in PV red blood cells, neoplasms (MPN) polycythemia vera (PV), essential thrombo- granulocytes, and platelets, consistent with clonal multilineage cythemia (ET) and primary myelofibrosis (PMF) are clonal expansion.7 Subsequent studies extended these findings to ET hematopoietic stem-cell disorders, for many years the genetic 8,9 basis for these disorders was elusive. A new era in MPN biology and PMF, providing evidence that these disorders arise in a began in 2005 with the discovery of a somatic point mutation in multipotent hematopoietic progenitor. At about the same time JAK2 tyrosine kinase (JAK2V617F), which was identified in a Jaroslav Prchal and Arthur Axelrad10 demonstrated that significant proportion of patients with PV, ET and PMF. Based erythroid colonies could be grown from PV bone marrow cells, on the hypothesis that JAK-STAT signaling is central to the but not from normal cells, in the absence of erythropoietin; pathogenesis of JAK2V617F-negative MPN, genomic studies have identified JAK2 exon 12 mutations in JAK2V617F-negative this has been termed endogenous erythroid colony formation, PV and activating mutations in MPL in patients with and can be demonstrated in some, but not all, patients with 10,11 JAK2V617F-negative ET and PMF. In this review, we will ET and PMF. In subsequent years, our understanding of the discuss the role of these mutant alleles in the pathogenesis molecular basis of PV, ET and PMF remained unchanged, with of PV, ET and PMF, the potential therapeutic implications of the notable exception of two papers in 1998; when Silva et al.12 these discoveries, and the implications of these discoveries for noted overexpression of BCL-XL in PV erythroid progenitors, and genomic studies of hematopoietic malignancies. 13 Leukemia (2008) 22, 1813–1817; doi:10.1038/leu.2008.229; Moliterno et al. noted downmodulation of thrombopoietin published online 28 August 2008 receptor expression on PV platelets. Keywords: myeloproliferative neoplasms; JAK2; MPL

Discovery of JAK2V617F: four different stories

The year 2005 was a watershed in our understanding of the Introduction molecular pathogenesis of PV, ET, and PMF with the identification of the JAK2V617F allele in a significant proportion of The myeloproliferative neoplasms (MPN) were first described in patients with PV, ET and PMF.14–17 It is interesting to note that a landmark editorial in 1951 by William Dameshek (1900– 1 the different groups used disparate approaches to arrive at the 1969), an American hematologist. Dameshek recognized that same discovery, and these approaches are of value in consider- the different MPN are characterized by pan-myeloid prolifera- ing current and future efforts in cancer gene discovery. The tion, which he suggested might result from an ‘undiscovered group led by William Vainchenker began by developing a liquid stimulus’. In subsequent years, the disorders classified as MPN culture system to culture PV erythroid cells in the absence of have evolved to include chronic myeloid leukemia (CML), exogenous cytokines;18 this important technical advancement polycythemia vera (PV), essential thrombocythemia (ET), allowed them to use small molecule inhibitors to interrogate the primary myelofibrosis (PMF), chronic eosinophilic leukemia requirement for specific signaling pathways in PV erythroid cell (CEL), chronic myelomonocytic leukemia, and systemic masto- 2 proliferation. They first noted that small molecule inhibition of cytosis. In the past half century, a series of seminal cytogenetic JAK2 inhibited PV erythroid proliferation, and then subsequently and molecular biologic studies led to the identification of used siRNA to show that JAK2 expression was required for SPOTLIGHT disease-causing mutations in other MPNs, including BCR-ABL in 3 4 erythroid expansion in the absence of cytokines; this led them to CML, FIP1L1-PDGFRA in CEL, and PDGFRA/B translocations 15 5 sequence JAK2 in PV patient samples. This approach in chronic myelomonocytic leukemia, each of which has represents an important example of how functional genomics had diagnostic, biologic and therapeutic relevance for these can be used to evaluate the role of specific genes and pathways disorders. in oncogenic transformation, and as small molecule and siRNA In contrast, our understanding of the molecular pathogenesis libraries continue to improve in size and in ease of use, these of PV, ET, and PMF was largely limited to the work of Fialkow, approaches will facilitate investigation into the pathogenesis of Adamson, and Axelrad and other investigators in the 1970s. human malignancies on a much larger scale. Moreover, these Following up on Fialkow’s earlier work demonstrating that CML 6 approaches will complement the high-throughput genomic is a clonal hematopoietic stem-cell disorder, Adamson and studies being used to discover cancer-associated mutations, and allow dissection of which mutations represent ‘drivers’ in Correspondence: Dr RL Levine, Human Oncology and Pathogenesis oncogenesis and which are ‘passengers’ acquired by the Program, Leukemia Service, Department of Medicine, Memorial Sloan transformed clone during clonal selection but with no relevance Kettering Cancer Center, 1275 York Avenue, Box 20, New York, NY to tumorigenesis.19,20 10065, USA. E-mail: [email protected] Robert Kralovics and Radek Skoda began their efforts by Received 14 June 2008; revised 16 July 2008; accepted 28 July 2008; following up on the important discovery by Josef Prchal and published online 28 August 2008 Kralovics21 that loss of heterozygosity of chromosome 9p24 is a JAK2 and MPL mutations in myeloproliferative neoplasms O Kilpivaara and RL Levine 1814 relatively common event in PV. In this important study, Prchal Although these data do not allow us to elucidate how a single and coworkers showed that unlike most tumor suppressors, disease allele contributes to three distinct diseases, they provide where LOH is commonly the result of deletion of the remaining in vivo evidence of hematopoietic transformation by this wild-type copy of the gene, LOH at this locus in PV is the result oncogene, and provide a platform for testing JAK2-directed of acquired uniparental disomy (UPD), such that patients are left therapies in an in vivo setting.36 either with two paternal or two maternal copies of this region of the genome after mitotic recombination. Skoda and coworkers sequenced the genes in the minimal region of UPD to identify JAK2V617F: a breakthrough discovery, but the beginning of the JAK2V617F allele.16 As with the functional approach the journey undertaken by Vainchenker’s group, this approach also has important general relevance to the investigation of the genetic The discovery of the JAK2V617F allele was a landmark basis of cancer. Recent studies have shown that UPD, resulting discovery; however, it represents the start of what is an in homozygous gain-of-function alleles, is observed for other important journey into the genetic basis of PV, ET and PMF. oncogenic alleles, including FLT3 in acute myeloid leukemia,22 The first question, which has begun to be addressed, is the and MPL in PMF (RLL and DG Gilliland, unpublished molecular etiology of JAK2V617F-negative PV, ET and PMF. observation). The advent of high-resolution genomic platforms, Sensitive, allele-specific assays suggest that almost all patients most notably single nucleotide polymorphism arrays, will with PV are JAK2V617F-positive;37 however, a subset of patients facilitate the identification of regions of UPD,23,24 which can with PV (E5%) are JAK2V617F-negative. This led Scott, Green then be subjected to high-throughput DNA resequencing for and their coworkers to hypothesize that JAK-STAT signaling SPOTLIGHT somatic mutations.25–27 would be activated by novel mutations in this subset of PV, and The remaining two groups used candidate gene approaches to led to the identification of somatic mutations in exon 12 of JAK2 discover the JAK2V617F allele. Anthony Green and coworkers in JAK2V617F-negative PV.31 To date at least eight different used candidate resequencing of genes relevant to signaling somatic missense, deletion and insertion mutations in JAK2 exon pathways important in oncogenic transformation to identify the 12 have been identified in JAK2V617F-negative PV-involving JAK2V617F allele in PV, ET and PMF.14 The group led by Gary residues 538–543.38–41 In vitro studies demonstrated that these Gilliland resequenced conserved domains of all tyrosine kinases alleles transform hematopoietic cells to cytokine-independent on a large set of MPN patients who provided samples using an growth, and expression of JAK2 Exon 12 alleles in vivo causes Internet-based collection protocol.17 This ‘tyrosine kinome’ polycythemia and leukocytosis, as is observed for JAK2V617F. resequencing strategy has been used to identify oncogenic Perhaps the most interesting aspect of the discovery of JAK2 alleles in a spectrum of human malignancies, including color- exon 12 mutations is the absence of the phenotypic pleiotropy ectal cancer,25 lung cancer,26 and more recently in acute associated with the canonical JAK2V617F allele. Studies have myeloid leukemia.27 These candidate gene resequencing studies found these alleles in PV and not in ET/PMF, and the majority of have become easier to perform as standard resequencing patients with JAK2 exon 12 mutations exhibit a specific clinical decreases in cost and as next-generation sequencing technolo- phenotype, with polycythemia, variable leukocytosis, but not gies become feasible for larger resequencing efforts; however, thrombocytosis. This suggests that different mutations in the these approaches will likely lead to the identification of ‘driver’ same gene have different effects on signaling which result in and ‘passenger’ alleles, and as discussed above functional distinct clinical phenotypes. From a clinical perspective, the studies will be of paramount importance to elucidate those identification of JAK2 exon 12 mutations provides a diagnostic mutations that actually contribute to oncogenic transformation. test for JAK2V617F-negative patients who present with erythro- Moreover, it will be important to assess whether candidate cytosis, and the presence of JAK2 exon 12 mutations should be mutations are acquired or inherited through analysis of matched taken as evidence of a clonal MPN in this context. normal tissue, as was done to show JAK2V617F was somatic; As opposed to PV, where JAK2V617F and JAK2 exon 12 therefore analysis of tumor and matched normal DNA is mutations account for virtually all patients, as many as half of ET absolutely required for future high-throughput genomic studies. and PMF are JAK2V617F-negative. No group has identified The mutation at codon 617 of JAK2 is always the same, a mutations in JAK2 in JAK2V617F-negative ET and PMF. Given guanine-to-thymidine transversion, which changes valine to the likelihood that JAK-STAT signaling would be involved in phenylalanine. Given that substitution of tryptophan, methio- JAK2V617F-negative ET and PMF, Pikman et al.42 asked nine, isoleucine and leucine for valine at codon 617 constitu- whether mutations in the type I cytokine receptors known to tively activates JAK2,28 it is surprising that no other alleles have interact with JAK2 could be identified in these patients. This led been identified at this position. Moreover, there are additional to the identification of mutations of the thrombopoietin receptor activating mutations in other domains of JAK2, including the (MPL) at codon 515. Unlike valine 617, substitution of leucine, T875N kinase domain mutation,29 the IREED deletion,30 and lysine or alanine for tryptophan 515 have all been reported JAK2 exon 12 mutations.31 Why then do virtually all patients at codon 515, and these mutations are all transforming acquire the same mutation at codon 617? It is hoped that future in vitro.42–44 These mutations occur in a subset of patients genetic and functional studies will elucidate why JAK2V617F with JAK2V617F-negative ET and PMF, including 8.5% of predominates in PV, ET and PMF. JAK2V617F-negative ET patients,45 and approximately 10% of A spectrum of in vitro and in vivo studies have been JAK2V617F-negative PMF patients.42–44 Three patients have also performed, which validate JAK2V617F as an oncogene; how- recently been described with somatic MPLS505N mutations,45 ever, the most important studies performed to date are in vivo an allele which had previously been associated with inherited assays in which JAK2V617F is expressed in the murine bone thrombocytosis.46 MPLW515-positive ET patients have higher marrow transplantation assay.32–35 In this context, expression of platelet counts and lower hemoglobin levels than JAK2V617F- Jak2V617F, but not wild-type Jak2, in recipient mice results in a positive ET patients,45,47 and MPLW515-positive PMF patients short latency, fully penetrant MPN notable for marked poly- present with more severe anemia.48 Moreover, endogenous cythemia, strain-specific leukocytosis, extramedullary hemato- megakaryocyte colonies, but not endogenous erythroid colony, poiesis and interestingly, an absence of thrombocytosis. can be grown from MPLW515-positive patient cells. These data

Leukemia JAK2 and MPL mutations in myeloproliferative neoplasms O Kilpivaara and RL Levine 1815 suggest that there are differences in signaling between genetic variation is important to the phenotypic diversity of JAK2V617F and MPLW515 mutations, which are of clinical these diseases and sets the stage for future efforts aimed at relevance. The murine data supports this notion, as expression elucidating the role of germ line variation in phenotype, of MPLW515L causes a phenotype which is distinct from response to specific therapies and MPN-associated JAK2V617F in the murine BMT model, with thrombocytosis, complications. leukocytosis and myelofibrosis, but not polycythemia, in recipient mice. The most important aspect of the discovery of JAK2 exon 12 Predisposition alleles 54 and MPL mutations in JAK2V617F-negative MPN is the In families with a predisposition to the development of MPN, 55,56 demonstration that activation of JAK2 signaling is seen in both JAK2V617F is acquired as a somatic allele. This suggests JAK2V617F-positive and JAK2V617F-negative disease, either that there are MPN predisposition alleles, which provide a through mutations in JAK2 itself or in cytokine receptors. The specific selective advantage to the development of mutations in question remains, though as to whether patients with JAK2/MPL- the JAK2-signaling pathway. We would predict these alleles are negative MPN have evidence of JAK2 activation and depen- relevant to signaling in this pathway, given that kindreds have dence on this pathway for proliferation. A spectrum of in vitro been described with JAK2V617F-positive and JAK2 exon 12 39 and in vivo assays can be used to assess this possibility; positive MPN, and there are patients with JAK2V617F- 43 however, the most convincing data will emerge from clinical positive, MPLW515L-positive disease, suggesting that there trials of JAK2 inhibitors that will allow us to know whether is extremely strong selection for JAK2-activating alleles in this patients with JAK2/MPL-negative MPN are sensitive to JAK2 context. Moreover, a recent study noted an increased risk of 57 inhibitors at similar doses to JAK2/MPL-positive patients. MPN for first-degree relatives of MPN patients, suggesting there are common MPN susceptibility alleles which might be relevant to presumed sporadic MPN. Dilemma of one mutation and three phenotypes Additional somatic mutations The second unanswered question, which is perhaps the most Cytogenetic abnormalities have been observed in patients with important from a pathogenetic standpoint, is how does a single MPN, most notably including deletion in chromosome 20q.58 In disease allele contribute to the pathogenesis of PV, ET and PMF, addition, patients have been observed where all hematopoietic three distinct clinical disorders. A number of hypotheses have cells had deletions of 20q loss and only a subset were positive been posited to explain the phenotypic pleiotropy of these for JAK2V617F.59 These data suggest there are ‘pre-JAK2 alleles’ disorders. which arise before the acquisition of the JAK2V617F allele in some patients. The most intriguing data comes from patients with a JAK2V617F-positive MPN transform to a JAK2V617F- Gene dosage 60,61 negative acute leukemia, suggesting there is a ‘pre-JAK2’- As discussed above, homozygosity for JAK2V617F is the result of transformed hematopoietic progenitor. The identity of these UPD at chromosome 9p24, and the initial studies noted that alleles remains a mystery. homozygosity for JAK2V617F was much more common in PV than in ET.14–17 The gene dosage hypothesis is also supported by 49 clonogenic colony data, as Scott et al. showed that homo- JAK2/MPL mutations and signaling zygous JAK2V617F mutant erythroid colonies are observed in most patients with PV but are rare in ET. Therefore we can The relevance of specific-signaling pathways in JAK2V617F- surmise that 9p24 UPD and JAK2V617F homozygosity is mediated hematopoietic transformation has not been fully common, and early, in the development of PV, but not ET, uncovered. Many groups have focused on the STAT family of suggesting that a high level of JAK2 signaling favors an erythroid transcription factors, including STAT5A and STAT5B,62,63 based phenotype and a low JAK2 state favors a megakaryocyte on previous studies showing that STAT5A and STAT5B are phenotype. In vivo studies are consistent with this hypothesis, necessary for TEL-JAK2-mediated myeloproliferation.64 Addi- as overexpression of Jak2V617F in the hematopoietic compart- tional studies are needed to ascertain the relevance of specific ment results in polycythemia and leukocytosis without STAT transcription factors, the MAP kinase signaling pathway, 32–35 SPOTLIGHT associated thrombocytosis, and transgenic expression PI3 kinase and other signaling pathways activated by of JAK2V617F at a much lower level results in thrombo- JAK2V617F to MPN pathogenesis. Moreover, it remains to be 50–52 cytosis. It is likely there are differences in constitutive seen whether there are differences in activation of signaling by signaling in cells heterozygous and homozygous for JAK2V617F JAK2V617F, JAK2 exon 12 mutations and by MPL mutations, that are relevant to MPN phenotype. Future studies are needed and whether these differences are of clinical and/or pathogenic to elucidate the mechanism by which JAK2V617F gene dosage relevance. affects MPN pathogenesis.

Targeted therapy for MPNs Germ line modifiers Pardanani et al.53 asked whether single nucleotide polymor- We are all hopeful that inhibitors of JAK2 will be safe and phisms in JAK2, MPL, EPOR and GCSFR influence phenotype. effective, and specific inhibitors of JAK2 kinase activity have Analysis of 32 single nucleotide polymorphisms in 179 patients entered the clinic in phase I/II trials in PMF and post-PV/ET with MPN allowed them to determine that specific single myelofibrosis.65 It is important to consider there may be nucleotide polymorphisms were associated with PV or with ET. significant toxicities associated with JAK2 inhibition, making it Although these data are from a single cohort, they provide likely that JAK2 inhibitors will have to demonstrate safety in evidence that host genetic variation is relevant to MPN patients with PMF or post-PV/ET-PMF before being tested in PV phenotype. Nonetheless, these data provide evidence that host and ET. Colony assays suggest that JAK2 inhibitors will be of

Leukemia JAK2 and MPL mutations in myeloproliferative neoplasms O Kilpivaara and RL Levine 1816 value for patients with JAK2 exon 12 or MPL mutations,41 and leukemia with t(5;12) chromosomal translocation. Cell 1994; 77: JAK2 inhibitor treatment in patients with JAK2/MPL-negative 307–316. MPN would allow a direct test of the hypothesis that JAK-STAT 6 Fialkow P, Gartler SM, Yoshida A. Clonal origin of chronic myelocytic signaling is central to the pathogenesis of PV, ET and PMF. leukemia in man. Proc Natl Acad Sci USA 1967; 58: 1468–1471. 7 Adamson JW, Fialkow PJ, Murphy S, Prchal JF, Steinmann L. Although there is considerable enthusiasm for JAK2 inhibitor Polycythemia vera: stem-cell and probable clonal origin of the development in these disorders, we must keep in mind a number disease. N Engl J Med 1976; 295: 913–916. of important concerns about these agents as they enter the 8 Tsukamoto N, Morita K, Maehara T, Okamoto K, Sakai H, clinical arena. First, resistance is likely to emerge in some Karasawa M et al. Clonality in chronic myeloproliferative disorders patients treated with JAK2 inhibitors, which should be studied defined by X-chromosome linked probes: demonstration of using in vitro systems. Moreover, the data showing that heterogeneity in lineage involvement. British Journal of Haematology 1994; 86: 253–258. JAK2V617F-negative acute myeloid leukemia occurs frequently 60,61 9 El Kassar N, Hetet G, Li Y, Briere J, Grandchamp B. Clonal analysis in patients with a JAK2V617F-positive MPN, raises the of haemopoietic cells in essential thrombocythaemia. Br J Haem troubling possibility that JAK2 inhibitor therapy might increase 1995; 90: 131–137. the risk of leukemic transformation. As we move forward into 10 Prchal JF, Axelrad AA. Letter: Bone-marrow responses in poly- the clinic with these agents, it will be important to ask clinically cythemia vera. N Engl J Med 1974; 290: 1382. and biologically relevant questions, and to ascertain the 11 Lutton JD, Levere RD. Endogenous erythroid colony formation by peripheral blood mononuclear cells from patients with myelo- potential limitations of these agents to derive the most benefit fibrosis and polycythemia vera. Acta Haematol 1979; 62: 94–99. for patients with the different MPN. 12 Silva M, Richard C, Benito A, Sanz C, Olalla I, Fernandez-Luna JL.

SPOTLIGHT Expression of Bcl-x in erythroid precursors from patients with polycythemia vera. N Engl J Med 1998; 338: 564–571. Future directions 13 Moliterno AR, Hankins WD, Spivak JL. Impaired expression of the thrombopoietin receptor by platelets from patients with poly- Although the recent discovery of JAK2V617F and MPL muta- cythemia vera. N Engl J Med 1998; 338: 572–580. tions have provided important insight into the biology of PV, ET 14 Baxter EJ, Scott LM, Campbell PJ, East C, Fourouclas N, Swanton S and PMF, there is much more to be done to unravel the mystery et al. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet 2005; 365: 1054–1061. of these MPN. The identity of the causative alleles in JAK2/MPL- 15 James C, Ugo V, Le Couedic JP, Staerk J, Delhommeau F, Lacout C negative MPN are a mystery, and the biologic differences et al. A unique clonal JAK2 mutation leading to constitutive signal- between JAK2V617F, JAK2 exon 12 alleles and MPL alleles are ling causes polycythaemia vera. Nature 2005; 434: 1144–1148. not known. Moreover, the basis of the phenotypic pleiotropy in 16 Kralovics R, Passamonti F, Buser AS, Teo SS, Tiedt R, Passweg JR JAK2V617F-positive MPN, and the role of additional inherited et al. A gain-of-function mutation of JAK2 in myeloproliferative and acquired disease alleles remains to be delineated. We can disorders. N Engl J Med 2005; 352: 1779–1790. 17 Levine RL, Wadleigh M, Cools J, Ebert BL, Wernig G, Huntly BJ truly say that the post-JAK2V617F era is an exciting era in MPN et al. Activating mutation in the tyrosine kinase JAK2 in biology and therapy, and the best is yet to come! polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell 2005; 7: 387–397. 18 Ugo V, Marzac C, Teyssandier I, Larbret F, Lecluse Y, Debili N Acknowledgements et al. 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