Curr Hematol Malig Rep (2013) 8:299–306 DOI 10.1007/s11899-013-0184-z

MYELOPROLIFERATIVE DISORDERS (JJ KILADJIAN, SECTION EDITOR)

Genetic Basis of MPN: Beyond JAK2-V617F Nicole C. C. Them & Robert Kralovics

Published online: 5 November 2013 # Springer Science+Business Media New York 2013

Abstract The clonal blood disorders polycythemia vera, essential thrombocythemia and primary myelofibrosis belong to the BCR-ABL1-negative myeloproliferative neoplasms and are specified by increased production of terminally differentiated myeloid cells. Clonal evolution, disease initiation and progression are influenced by genetic alterations, often affecting cytokine signaling and gene expression. This review outlines somatic changes discovered in myeloproliferative neoplasms and how these genetic aberrations influence the pathogenesis of myeloproliferative neoplasms. Furthermore, genetic responses to drug treatments in myeloproliferative neoplasms are discussed. Keywords Myeloproliferative neoplasms . JAK2 . Clonal evolution . Oncogene . Tumor suppressor . Hematologic malignancy

Introduction Myeloproliferative neoplasms (MPNs) are characterized by excessive production of terminally differentiated myeloid cells. Polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) are the classical BCRABL1-negative MPNs which are defined by increased number

of erythrocytes, platelets and bone marrow fibrosis, respectively [1]. MPNs have a risk of developing thrombosis, bleeding and transforming to secondary acute myeloid leukemia (sAML). The reported risk of leukemic transformation is highest for PMF (around 20 %), median for PV (4.5 %) and lowest for ET (less than 1 %, for strictly WHO-classified ET cases) [2–4]. In health, a polyclonal stem cell pool contributes to hematopoiesis. Polyclonal hematopoiesis is changed to an abnormal monoclonal or oligoclonal hematopoiesis in myeloid malignancies, including MPN, and this change is caused by acquired mutations in hematopoietic stem cells (HSCs) [5]. Additional genetic lesions may direct clinical phenotype patterning of MPN and during the course of disease other mutations can promote disease progression as well as transformation to sAML. An important focus of the past and present MPN research is to reveal these MPN-associated genetic lesions. Somatic mutations in MPN pathogenesis are involved in a wide range of cellular pathways, predominantly cytokine receptor signaling and regulation of gene expression. This review will cover genetic changes found in MPN and outline their role in disease initiation, clonal evolution and disease progression. Moreover, we will discuss the genetic alterations to drug treatments seen in MPN.

JAK2 Mutation and its Role in MPN N. C. C. Them : R. Kralovics (*) CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Lazarettgasse 14, AKH BT25.3, 1090 Vienna, Austria e-mail: [email protected] N. C. C. Them e-mail: [email protected] R. Kralovics Department of Internal Medicine I, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria

The most prevalent mutation in MPN is the JAK2-V617F mutation, and it was discovered in 2005 [6–9]. Approximately 95 % of PV patients, 50-70 % of ET patients and 40-50 % of PMF patients possess this specific JAK2 mutation [10]. The JAK2-V617F mutation is located in exon 14 of the gene and abrogates the negative regulatory activity of the pseudokinase domain JH2 of the encoded JAK2 tyrosine kinase [11, 12]. Therefore, this mutation leads to a constitutive active JAK2 kinase signaling that is independent of cytokine stimuli, when

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homodimeric type 1 cytokine receptors are coexpressed [13]. Thus, this state is different from normal conditions where JAK2 is only activated after dimermerization or oligomerization of cytokine receptors that bound their respective ligands [14]. In PV patients JAK2 mutations in exon 12 can occur at low frequency [15]. Functional consequences of these mutations are thought to be equivalent to the V617F mutation. Aside from its important role in cytokine signaling, JAK2 was found to function as an epigenetic regulator through its nuclear localization where it can phosphorylate histone H3Y41 as well as the arginine methyltransferase PRMT5 [16, 17]. H3Y41 phosphorylation blocks the binding of heterochromatin protein 1α, thus, leading to gene expression changes [16]. Interestingly, if PRMT5 is phosphorylated by JAK2-V617F, it causes decreased PRMT5 methyltransferase activity and thereby reduces methylation of H2A and H4 [17]. In order to elucidate whether the JAK2-V617F mutation alone can induce MPN in vivo and to understand the role of JAK2 in the MPN pathogenesis, several mouse models were established, including bone marrow transplantation, transgenic and targeted knock-in models [18]. Although these murine models are resulting in slightly different MPN phenotypes, all were able to show that the JAK2 mutation is sufficient to cause MPN in mice.

MPN Associated Somatic Mutations Other Than JAK2 Mutations After the finding of JAK2 mutations, several other somatic mutations were discovered and implicated in the MPN pathogenesis. Affected pathways influence a variety of cellular functions and some examples are outlined in this section. JAK-STAT Pathway Hematopoiesis, under normal and stress conditions, is regulated mainly by hematopoietic cytokines, for instance granulocyte linage by granulocyte colony stimulating factor, erythroid linage by erythropoietin (EPO) and megakaryocytic linage as well as platelet production by thrombopoietin (TPO) [19–21]. Mutated genes found in MPN frequently target these cytokine signaling pathways, JAK2 being the most prominent. Myeloproliferative leukemia virus oncogene (MPL) encodes the receptor for TPO, which mediates signaling through the JAK-STAT pathway [22]. Several gain-of-function mutations in MPL were described in MPN and exon 10 MPL mutations are seen in up to 15 % of JAK2-V617F negative ET and PMF patients [23, 24]. The adaptor protein LNK (SH2B3) negatively regulates the TPO and EPO cytokine signaling by inhibiting JAK2 activation [25, 26]. Loss-of-function LNK

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mutations occur at low frequency and were initially found in JAK2- V617F negative MPN patients [27, 28]. Also suppressors of cytokine signaling (SOCSs) negatively regulate the JAK-STAT pathway [29]. In MPN patients mutations in SOCS genes are at low frequency, and, in addition, hypermethylation of CpG islands in these genes was described [30–32]. Another negative regulator of cytokine signaling is Casitas B-cell lymphoma (CBL), which acts as an adaptor protein and E3 ubiquitin ligase, targeting a diverse set of proteins for proteasomal degradation [33, 34]. Somatic CBL mutations have been found in diverse myeloid malignancies and within the group of MPNs, CBL is mostly mutated in PMF patients [35, 36]. In addition to the JAKSTAT pathway, cytokine signaling can be mediated by the MAPK signaling pathway [14]. Mutations affecting this pathway were also reported in MPN, including NRAS mutations as well as deletions of NF1 [37, 38]. Splicing Machinery Mutations of genes involved in RNA splicing, such as SF3B1, SRSF2 , U2AF1, have been identified in several myeloid malignancies [39•]. They largely occur in myelodysplastic syndrome (MDS) and around 9 % of MPN patients show mutations affecting the splicing machinery. Mutations in U2AF1 are thought to have a dominant-negative effect, causing abnormal splicing, apoptosis, cell cycle arrest and reduced reconstitution capacity of hematopoietic stem/ progenitor cells in vivo. Furthermore, it was speculated that the splicing defects are more related to changes in cell differentiation. This would be in accordance with the cytopenia seen in MDS patients, which is induced through ineffective hematopoiesis with elevated apoptosis. Transcription Factors Important elements of gene expression regulation are transcription factors, of which some were found to be deleted or mutated in MPN, suggesting that transcription factor networks might have a critical function in MPN pathogenesis [40••]. IKZF1, which encodes the transcription factor Ikaros, was shown to be the target of chromosome 7p deletions in MPNs and a late event in the clonal evolution from MPN to sAML [41]. Ikaros influences the development of T as well as B cells and an overexpressed dominant-negative form was reported to activate the JAK-STAT pathway [42, 43]. Deletions of chromosome 3p in MPN patients mapped to FOXP1, which is a transcription factor and speculated to be a tumor suppressor [40••, 44]. An additional common deleted region in MPN is located on chromosome 12p and was reported to map to ETV6 [40••]. This transcription factor is involved in a number of translocations in diverse hematologic malignancies, forming fusion genes with ABL1, RUNX1 and

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others [45, 46]. Another transcription factor, CUX1, is a target of chromosome 7q deletions in MPNs [40••]. CUX1 is implicated in cell cycle regulation, DNA damage response and hematopoiesis [47–49]. Moreover, the important transcription factor p53, which regulates as well cell cycle and DNA damage response, was found to be mutated in 1.6 % of MPN patients and was associated with leukemic transformation in post-MPN AML [40••]. Hematopoiesis is also regulated by the transcription factor RUNX1 which was found to be mutated in AML, post-MDS AML and post-MPN AML [50, 51•]. Recently mutations in nuclear factor erythroid 2 (NF-E2) and overexpression of this transcription factor were reported in MPN patients [52•, 53]. Mislocalization of NF-E2 was reported in PMF but not in ET patients, thus it was suggested that immunohistochemistry of NF-E2 could be used as a diagnostic tool to distinguish these two MPNs [54]. Moreover, increased expression of NF-E2 lead to an MPN phenotype in mice [55]. Epigenetic Regulators Gene expression is also influenced by epigenetic changes which encompass DNA as well as histone modifications. Various genes that have a role in epigenetic mechanisms were described to be mutated in myeloid malignancies [56]. Ten-eleven translocation 2 (TET2) mutations were associated with myeloid malignancies, with a mutational frequency of around 5 % in ET, 16 % in PV and 17 % in PMF [57]. TET enzymes catalyze the conversion of 5-methylcytosine to 5-hydroxymethylcytosine, which can subsequently lead to DNA demethylation [58]. TET2 mutations described in myeloid malignancies were shown to impair the enzymatic function of TET2 [59]. The enzymes isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) catalyze the conversion of isocitrate to α-ketogluterate, which in turn serves as a co-factor for TET2 [56]. Interestingly, not only somatic mutations in TET2 but also IDH1/2, were reported for MPN patients, which were at low frequency and more often found in post-MPN AML cases [60, 61]. IDH1/2 mutations were described to inhibit TET2 activity and lead to decreased DNA demethylation [62]. Moreover, it was reported that mutant IDH proteins act as neomorphic enzymes that catalyze the conversion of α-ketogluterate to 2-hydroxygluterate and show decreased enzymatic activity for isocitrate [63]. DNMT3A encodes a de novo methyltransferase that preferably adds a methyl group to unmethylated cytosine in CpG dinucleotides [64]. Somatic mutations in DNMT3A were reported in myeloid malignancies such as AML and in around 10 % of MPN patients [65, 66]. Mutations in that gene are speculated to cause a gain of function or a loss of function, thus the functional role of DNMT3A in myeloid malignancies is so far not clear. Additional sex combs like 1 (ASXL1) is a nuclear polycomb protein that can regulate transcription through diverse mechanisms such as histone modifications [67, 68]. Recently it was shown that loss of

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ASXL1 leads to decreased histone H3K27 trimethylation and that ASXL1 interacts with the polycomb repressive complex 2 (PRC2) [69]. ASXL1 mutations occur in MPN patients and are more frequent in PMF as well as secondary myelofibrosis (sMF) than in PVand ET [70]. Interestingly, also PRC2 members were found to be mutated in myeloid malignancies [71•, 72•]. The PRC2 complex modifies histones to cause transcriptional repression and is implicated in the regulation of development, differentiation as well as cell proliferation [73]. The core PRC2 complex consists of SUZ12, EED, RBAP48 and the methyltransferase EZH2 (or EZH1) [74].

Clonal Evolution During Aging and MPN After the discovery of JAK2-V617F mutations in MPN, two hypothesis emerged, either that the JAK2 mutation is the main causative aberration in MPN or that clonal hematopoiesis is established by other mutations and subsequent acquisition of the JAK2 mutation causes the MPN phenotype [75]. Several studies could show that MPN patients exhibited a clonal disease in which the JAK2-V617F mutation was only present in a subset of clonal cells, supporting the second hypothesis [76, 77]. However, overall it is suggested that the somatic mutations in MPN are stochastically acquired and do not necessarily follow a certain order [75]. This genetic heterogeneity leads to clonal diversities among patients and also coexisting clonal evolutions within patients. Interestingly, sAML can arise from the same clone that caused the MPN phenotype or an independent one (Fig. 1) [37, 78]. Therefore, an important MPN research focus recently was to identify mutations in addition to JAK2 that would account for the disease initiation, progression and leukemic transformation (examples of described mutations were outlined in the previous section). Clonal Hematopoiesis with Genetic Aberrations in the Normal Aged Population A changed HSC compartment with a bias for the myeloid linage and an elevated risk of myeloid malignancies are attributes of the aging hematopoietic system [79, 80]. It was postulated that a clonal expansion of HSCs causes the myeloid linage bias. Recent work contributed to the understanding of clonal hematopoiesis in healthy individuals [81••, 82••, 83••, 84••]. One of these studies reported in a cohort of twins an accumulation of somatic structural variants with age, which might account for the age-related decrease in blood cell clonality [81••]. Interestingly, chromosome 5q and 20q deletions were detected in healthy individuals, both being aberrations associated with myeloid malignancies [40••, 81••]. Two additional studies examined chromosomal aberrations in the general population by utilizing a large

302 Fig. 1 Leukemic transformation after chronic myeloproliferative neoplasms (MPNs). Diverse genetic alterations can lead to an MPN phenotype, such as the most common mutation JAK2-V617F. MPN clones can acquire additional genetic changes, for instance TP53 mutations, which could lead to secondary acute myeloid leukemia (sAML). However, leukemic progression can also be caused by an independent clone that gained mutations, for example in FLT3 or NPM1. The therapy prescribed at the chronic MPN phase might restrict clonal expansion but could also select for drug resistant clones. It could be that genetic changes leading to drug resistance are the same that are important for evolution to sAML (TP53 mutations might be potential candidates)

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JAK2

JAK2

TP53 FLT3/NPM1

JAK2

JAK2 de novo

JAK2

JAK2

TP53 JAK2

number of single nucleotide polymorphism (SNP) array data that was generated for diverse genome-wide association studies [82••, 83••]. Both could show that the frequency of somatic chromosomal anomalies increases with age. Moreover, the acquired chromosomal aberrations seem non-random and correlated with genomic changes observed in myeloid malignancies, including acquired uniparental disomies (aUPDs) of chromosome 9 and 14; trisomy of chromosome 8 as well as deletions on chromosome 4q, 13q and 20q [40••, 82••, 83••]. Individuals with somatic chromosomal anomalies were shown to have a 10 fold higher risk of developing hematological cancer as compared to individuals without detectable aberrations [83••]. Another study postulated that somatic mutations, which occur in the aging hematopoietic system, would account for the clonal hematopoiesis seen in the elderly population [84••]. Indeed, they found somatic TET2 mutations in 5.6 % of elderly individuals that showed clonal hematopoiesis, but had no hematological malignancy. As outlined in the previous section, TET2 mutations are recurrently seen in myeloid malignancies. Overall these recent findings reveal genetic aberrations in the aging hematopoietic system that associate with clonal hematopoiesis and are often seen in hematological malignancies. This leads to the suggestion that aberrations occurring in the aging hematopoietic system account for the clonal expansion and might constitute an early event in the development of hematological malignancies.

JAK2 de novo

Genetic Changes that Associate with Disease Progression MPNs can progress to sAML, which has a diagnosis criterion of at least 20 % blasts in the bone marrow and/or peripheral blood [1]. After leukemic transformation, patients have poor prognosis with an adverse outcome within a few months [51•]. Although the genetic basis of disease progression in MPN is still poorly understood, several genetic alterations could be linked to leukemic progression over the past years. IDH1/2 mutations were reported at low frequency in MPN cases (1.9 % PV; 0.8 % ET; 4.2 % PMF) as opposed to post-MPN AML patients (21.6 %), indicating an association with leukemic transformation [60, 61]. Moreover, it was reported that mutations in IDH1/2 predict reduced survival in PMF patients [85]. Several studies described in AML, post-MPN AML and post-MDS AML mutations in RUNX1, encoding an important transcription factor in hematopoiesis [50, 51•]. PostMPN AML patients were shown to exhibit significantly more chromosomal aberrations than MPN patients [40••]. Several chromosomal anomalies could be linked to post-MPN AML, such as aUPD of chromosome 22q, gains of chromosome 1q and 3q, and deletions of chromosome 5q, 6p, 7p and 7q. Interestingly, the minimal amplified region of chromosome 1q contains MDM4, which encodes an inhibitor of the tumor suppressor p53 [86•]. In accordance, also TP53 mutations were described to be associated with post-MPN AML and as an independent prognostic factor for poor survival in sAML [51•, 86•]. P53-related aberrations, either gain of chromosome 1q or mutation in TP53, were reported for 45.5 % of post-

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MPN AML patients [86•]. These findings together suggest an important role of the p53 pathway in leukemic transformation of MPNs.

Genetic Adaptation to Drug Therapies in MPN An ongoing question is whether the therapy administered at the chronic MPN phase has an effect on disease progression and evolution to sAML (Fig. 1). Radioactive phosphorus (P32) and the alklylating agent chlorambucil have an established leukemogenic potential [87, 88, 89•]. In addition, another alkylating agent, pipobroman, was recently shown to increase the risk of leukemic transformation in PV patients [89•, 90]. Whether the therapy with the ribonucleotide reductase inhibitor hydroxyurea (HU) or the alkylating agent busulfan (BU) increases the progression to sAML is controversial. For instance, a retrospective study described an elevated risk of evolving second hematological malignancies in BU-treated ET patients, whereas HU was not associated with an increased risk [91]. In contrast, a recent study reported no association of leukemic transformation with the treatment of BU and HU in PV patients [89•]. However, the final results of the French Polycythemia Study Group showed that for HU-treated PV patients the risk of evolution to sAML was higher than previously described, even though the natural disease progression of PV should be kept in mind [90]. It is not well understood what influence the chronic MPN treatment has on the acquisition of genetic aberrations associated with leukemic transformation (Fig. 1). It is possible that genetic changes that cause drug resistance at the same time lead to leukemic transformation in MPN. A potential candidate might be p53 [92]. P32, alkylating agents and HU cause DNA damage and p53 has an important role in the DNA damage response [93]. Moreover, the p53 pathway is important in the evolution of MPN to sAML. Interestingly, HU-treated patients that progressed to AML or MDS were shown to have a high proportion of chromosome 17p deletions [94]. Together these data suggest that DNA damage inducing agents administered during the chronic MPN phase would increase the risk of acquiring TP53 mutations or chromosome 17p deletions, thereby escaping drug induced cell death and promote evolution to sAML. One of the emerging therapies for MPNs is interferon alpha (IFNα). Remarkably, IFNα treatment leads to hematological and molecular responses in the majority of PVand ET patients [95]. Moreover, IFNα is not leukemogenic and is able to induce complete molecular remission in a subset of patients [96, 97]. Still the administration of IFNα in MPNs was limited due to its toxicity and early treatment discontinuations [95]. The use of polyethylene glycol (peg)-IFNα decreased toxicity and increased plasma half-life in hepatitis patients [98]. In

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accordance, PV patients treated with peg-IFNα-2a showed limited toxicity [96]. Which genetic aberrations might determine drug sensitivity or resistance is not well understood. In patients that possess JAK2 and TET2 mutations, it was described that the treatment with peg-IFNα-2a is able to reduce JAK2-V617F clones but not the TET2 mutant one [99]. It was proposed that the persistent TET 2 mutation would lead to a clonal hematopoiesis without an MPN phenotype. Moreover, a recent study showed that peg-IFNα-2a treated patients with JAK2 and TET2 mutations possessed at the beginning of the therapy a higher JAK2-V617F mutant allele burden and a less significant reduction of this burden during treatment in contrast to patients with JAK2 but without TET2 mutations [100•]. The same study described that peg-IFNα-2a treated patients without a complete molecular remission were prone to have more mutations beyond JAK2.

Conclusion Over the past years a number of genetic changes were identified that are involved in the MPN pathogenesis. The variety of these aberrations suggests that several cellular pathways have an impact on the clonal evolution in MPN. These affected pathways include cytokine signaling and regulation of gene expression by changes in the splicing machinery, transcription factors or epigenetic regulators. Interestingly, MPN associated aberrations were found in the aging hematopoietic system, which might explain clonal expansion and indicate an early event in hematological malignancies. Genetic changes that cause disease progression and leukemic transformation are a current research focus. In addition to the natural risk of MPN to progress to sAML, the treatment given at the chronic MPN phase might influence the disease progression. In order to understand the impact of the therapy on MPN pathogenesis and leukemic transformation, it will be important to assess clonal evolution in respect to the applied therapy. Highthroughput sequencing will still enhance the genetic complexity and knowledge of MPN. Depending on their somatic mutation profile, MPN patients might be classified into groups to aid clinical management and treatment decisions. Acknowledgments This study was supported by the Austrian Science Fund (P23257-B12) and the MPN Research Foundation. Compliance with Ethics Guidelines Conflict of Interest Nicole C.C. Them and Robert Kralovics declare that they have no conflict of interest. Human and Animal Rights and Informed Consent This article does not contain any studies with human or animal subjects performed by any of the authors.

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References Papers of particular interest, published recently, have been highlighted as: • Of importance •• Of major importance 1. Vardiman JW, Thiele J, Arber DA, et al. The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes. Blood. 2009;114:937–51. 2. Mesa RA, Li CY, Ketterling RP, et al. Leukemic transformation in myelofibrosis with myeloid metaplasia: a single-institution experience with 91 cases. Blood. 2005;105:973–7. 3. Crisa E, Venturino E, Passera R, et al. A retrospective study on 226 polycythemia vera patients: impact of median hematocrit value on clinical outcomes and survival improvement with anti-thrombotic prophylaxis and non-alkylating drugs. Ann Hematol. 2010;89:691–9. 4. Barbui T, Thiele J, Passamonti F, et al. Survival and disease progression in essential thrombocythemia are significantly influenced by accurate morphologic diagnosis: an international study. J Clin Oncol. 2011;29:3179–84. 5. Tefferi A, Vainchenker W. Myeloproliferative neoplasms: molecular pathophysiology, essential clinical understanding, and treatment strategies. J Clin Oncol. 2011;29:573–82. 6. Baxter EJ, Scott LM, Campbell PJ, et al. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet. 2005;365:1054–61. 7. James C, Ugo V, Le Couedic JP, et al. A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature. 2005;434:1144–8. 8. Kralovics R, Passamonti F, Buser AS, et al. A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med. 2005;352:1779–90. 9. Levine RL, Wadleigh M, Cools J, et al. Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell. 2005;7:387–97. 10. Plo I, Vainchenker W. Molecular and genetic bases of myeloproliferative disorders: questions and perspectives. Clin Lymphoma Myeloma. 2009;9 Suppl 3:S329–39. 11. Saharinen P, Silvennoinen O. The pseudokinase domain is required for suppression of basal activity of Jak2 and Jak3 tyrosine kinases and for cytokine-inducible activation of signal transduction. J Biol Chem. 2002;277:47954–63. 12. Lindauer K, Loerting T, Liedl KR, Kroemer RT. Prediction of the structure of human Janus kinase 2 (JAK2) comprising the two carboxy-terminal domains reveals a mechanism for autoregulation. Protein Eng. 2001;14:27–37. 13. Lu X, Levine R, Tong W, et al. Expression of a homodimeric type I cytokine receptor is required for JAK2V617F-mediated transformation. Proc Natl Acad Sci U S A. 2005;102:18962–7. 14. Baker SJ, Rane SG, Reddy EP. Hematopoietic cytokine receptor signaling. Oncogene. 2007;26:6724–37. 15. Scott LM, Tong W, Levine RL, et al. JAK2 exon 12 mutations in polycythemia vera and idiopathic erythrocytosis. N Engl J Med. 2007;356:459–68. 16. Dawson MA, Bannister AJ, Gottgens B, et al. JAK2 phosphorylates histone H3Y41 and excludes HP1alpha from chromatin. Nature. 2009;461:819–22. 17. Liu F, Zhao X, Perna F, et al. JAK2V617F-mediated phosphorylation of PRMT5 downregulates its methyltransferase activity and promotes myeloproliferation. Cancer Cell. 2011;19:283–94.

Curr Hematol Malig Rep (2013) 8:299–306 18. Li J, Kent DG, Chen E, Green AR. Mouse models of myeloproliferative neoplasms: JAK of all grades. Dis Model Mech. 2011;4:311–7. 19. Nicola NA, Metcalf D, Matsumoto M, Johnson GR. Purification of a factor inducing differentiation in murine myelomonocytic leukemia cells. Identification as granulocyte colony-stimulating factor. J Biol Chem. 1983;258:9017–23. 20. Jelkmann W. Regulation of erythropoietin production. J Physiol. 2011;589:1251–8. 21. Kaushansky K, Lok S, Holly RD, et al. Promotion of megakaryocyte progenitor expansion and differentiation by the cMpl ligand thrombopoietin. Nature. 1994;369:568–71. 22. Fishley B, Alexander WS. Thrombopoietin signalling in physiology and disease. Growth Factors. 2004;22:151–5. 23. Pardanani AD, Levine RL, Lasho T, et al. MPL515 mutations in myeloproliferative and other myeloid disorders: a study of 1182 patients. Blood. 2006;108:3472–6. 24. Pietra D, Brisci A, Rumi E, et al. Deep sequencing reveals double mutations in cis of MPL exon 10 in myeloproliferative neoplasms. Haematologica. 2011;96:607–11. 25. Tong W, Zhang J, Lodish HF. Lnk inhibits erythropoiesis and Epodependent JAK2 activation and downstream signaling pathways. Blood. 2005;105:4604–12. 26. Tong W, Lodish HF. Lnk inhibits Tpo-mpl signaling and Tpomediated megakaryocytopoiesis. J Exp Med. 2004;200:569–80. 27. Oh ST, Simonds EF, Jones C, et al. Novel mutations in the inhibitory adaptor protein LNK drive JAK-STAT signaling in patients with myeloproliferative neoplasms. Blood. 2010;116:988–92. 28. Lasho TL, Pardanani A, Tefferi A. LNK mutations in JAK2 mutationnegative erythrocytosis. N Engl J Med. 2010;363:1189–90. 29. Krebs DL, Hilton DJ. SOCS: physiological suppressors of cytokine signaling. J Cell Sci. 2000;113(Pt 16):2813–9. 30. Jost E, do ON, Dahl E, et al. Epigenetic alterations complement mutation of JAK2 tyrosine kinase in patients with BCR/ABLnegative myeloproliferative disorders. Leukemia. 2007;21:505–10. 31. Teofili L, Martini M, Cenci T, et al. Epigenetic alteration of SOCS family members is a possible pathogenetic mechanism in JAK2 wild type myeloproliferative diseases. Int J Cancer. 2008;123: 1586–92. 32. Suessmuth Y, Elliott J, Percy MJ, et al. A new polycythaemia veraassociated SOCS3 SH2 mutant (SOCS3F136L) cannot regulate erythropoietin responses. Br J Haematol. 2009;147:450–8. 33. Schmidt MH, Dikic I. The Cbl interactome and its functions. Nat Rev Mol Cell Biol. 2005;6:907–18. 34. Saur SJ, Sangkhae V, Geddis AE, et al. Ubiquitination and degradation of the thrombopoietin receptor c-Mpl. Blood. 2010;115:1254–63. 35. Grand FH, Hidalgo-Curtis CE, Ernst T, et al. Frequent CBL mutations associated with 11q acquired uniparental disomy in myeloproliferative neoplasms. Blood. 2009;113:6182–92. 36. Dunbar AJ, Gondek LP, O'Keefe CL, et al. 250K single nucleotide polymorphism array karyotyping identifies acquired uniparental disomy and homozygous mutations, including novel missense substitutions of c-Cbl, in myeloid malignancies. Cancer Res. 2008;68:10349–57. 37. Beer PA, Delhommeau F, LeCouedic JP, et al. Two routes to leukemic transformation after a JAK2 mutation-positive myeloproliferative neoplasm. Blood. 2010;115:2891–900. 38. Stegelmann F, Bullinger L, Griesshammer M, et al. High-resolution single-nucleotide polymorphism array-profiling in myeloproliferative neoplasms identifies novel genomic aberrations. Haematologica. 2010;95:666–9. 39. • Yoshida K, Sanada M, Shiraishi Y, et al. Frequent pathway mutations of splicing machinery in myelodysplasia. Nature. 2011;478:64–9. This study reported mutations of genes involved in RNA splicing in diverse myeloid malignancies.

Curr Hematol Malig Rep (2013) 8:299–306 40. •• Klampfl T, Harutyunyan A, Berg T, et al. Genome integrity of myeloproliferative neoplasms in chronic phase and during disease progression. Blood. 2011;118:167–76. Investigation of chromosomal aberrations in MPN that identified commonly affected chromosomal regions and lesions that associated with disease progression. 41. Jager R, Gisslinger H, Passamonti F, et al. Deletions of the transcription factor Ikaros in myeloproliferative neoplasms. Leukemia. 2010;24:1290–8. 42. Georgopoulos K. Haematopoietic cell-fate decisions, chromatin regulation and ikaros. Nat Rev Immunol. 2002;2:162–74. 43. Kano G, Morimoto A, Takanashi M, et al. Ikaros dominant negative isoform (Ik6) induces IL-3-independent survival of murine pro-B lymphocytes by activating JAK-STAT and up-regulating Bcl-xl levels. Leuk Lymphoma. 2008;49:965–73. 44. Banham AH, Beasley N, Campo E, et al. The FOXP1 winged helix transcription factor is a novel candidate tumor suppressor gene on chromosome 3p. Cancer Res. 2001;61:8820–9. 45. Odero MD, Carlson K, Calasanz MJ, et al. Identification of new translocations involving ETV6 in hematologic malignancies by fluorescence in situ hybridization and spectral karyotyping. Gene Chromosome Cancer. 2001;31:134–42. 46. Bohlander SK. ETV6: a versatile player in leukemogenesis. Semin Cancer Biol. 2005;15:162–74. 47. Truscott M, Harada R, Vadnais C, et al. p110 CUX1 cooperates with E2F transcription factors in the transcriptional activation of cell cycle-regulated genes. Mol Cell Biol. 2008;28: 3127–38. 48. Vadnais C, Davoudi S, Afshin M, et al. CUX1 transcription factor is required for optimal ATM/ATR-mediated responses to DNA damage. Nucleic Acids Res. 2012;40:4483–95. 49. Cadieux C, Fournier S, Peterson AC, et al. Transgenic mice expressing the p75 CCAAT-displacement protein/Cut homeobox isoform develop a myeloproliferative disease-like myeloid leukemia. Cancer Res. 2006;66:9492–501. 50. Harada H, Harada Y, Niimi H, et al. High incidence of somatic mutations in the AML1/RUNX1 gene in myelodysplastic syndrome and low blast percentage myeloid leukemia with myelodysplasia. Blood. 2004;103:2316–24. 51. • Milosevic JD, Puda A, Malcovati L, et al. Clinical significance of genetic aberrations in secondary acute myeloid leukemia. Am J Hematol. 2012;87:1010–6. This study compared genetic aberrations occurring in secondary and de novo AML and identified mutant TP53 as an independent adverse prognostic factor for overall survival in secondary AML. 52. • Jutzi JS, Bogeska R, Nikoloski G, et al. MPN patients harbor recurrent truncating mutations in transcription factor NF-E2. J Exp Med. 2013;210:1003–19. This study reported mutations in nuclear factor erythroid 2 in MPN patients. 53. Wang W, Schwemmers S, Hexner EO, Pahl HL. AML1 is overexpressed in patients with myeloproliferative neoplasms and mediates JAK2V617F-independent overexpression of NF-E2. Blood. 2010;116:254–66. 54. Aumann K, Frey AV, May AM, et al. Subcellular mislocalization of the transcription factor NF-E2 in erythroid cells discriminates prefibrotic primary myelofibrosis from essential thrombocythemia. Blood. 2013;122:93–9. 55. Kaufmann KB, Grunder A, Hadlich T, et al. A novel murine model of myeloproliferative disorders generated by overexpression of the transcription factor NF-E2. J Exp Med. 2012;209:35–50. 56. Shih AH, Abdel-Wahab O, Patel JP, Levine RL. The role of mutations in epigenetic regulators in myeloid malignancies. Nat Rev Cancer. 2012;12:599–612. 57. Tefferi A, Pardanani A, Lim KH, et al. TET2 mutations and their clinical correlates in polycythemia vera, essential thrombocythemia and myelofibrosis. Leukemia. 2009;23:905–11.

305 58. Tahiliani M, Koh KP, Shen Y, et al. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science. 2009;324:930–5. 59. Ko M, Huang Y, Jankowska AM, et al. Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2. Nature. 2010;468:839–43. 60. Tefferi A, Lasho TL, Abdel-Wahab O, et al. IDH1 and IDH2 mutation studies in 1473 patients with chronic-, fibrotic- or blastphase essential thrombocythemia, polycythemia vera or myelofibrosis. Leukemia. 2010;24:1302–9. 61. Pardanani A, Lasho TL, Finke CM, et al. IDH1 and IDH2 mutation analysis in chronic- and blast-phase myeloproliferative neoplasms. Leukemia. 2010;24:1146–51. 62. Figueroa ME, Abdel-Wahab O, Lu C, et al. Leukemic IDH1 and IDH2 mutations result in a hypermethylation phenotype, disrupt TET2 function, and impair hematopoietic differentiation. Cancer Cell. 2010;18:553–67. 63. Ward PS, Patel J, Wise DR, et al. The common feature of leukemiaassociated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate. Cancer Cell. 2010;17:225–34. 64. Yokochi T, Robertson KD. Preferential methylation of unmethylated DNA by Mammalian de novo DNA methyltransferase Dnmt3a. J Biol Chem. 2002;277:11735–45. 65. Ley TJ, Ding L, Walter MJ, et al. DNMT3A mutations in acute myeloid leukemia. N Engl J Med. 2010;363:2424–33. 66. Stegelmann F, Bullinger L, Schlenk RF, et al. DNMT3A mutations in myeloproliferative neoplasms. Leukemia. 2011;25:1217–9. 67. Cho YS, Kim EJ, Park UH, et al. Additional sex comb-like 1 (ASXL1), in cooperation with SRC-1, acts as a ligand-dependent coactivator for retinoic acid receptor. J Biol Chem. 2006;281: 17588–98. 68. Kim K, Choi J, Heo K, et al. Isolation and characterization of a novel H1.2 complex that acts as a repressor of p53-mediated transcription. J Biol Chem. 2008;283:9113–26. 69. Abdel-Wahab O, Adli M, LaFave LM, et al. ASXL1 mutations promote myeloid transformation through loss of PRC2-mediated gene repression. Cancer Cell. 2012;22:180–93. 70. Stein BL, Williams DM, O'Keefe C, et al. Disruption of the ASXL1 gene is frequent in primary, post-essential thrombocytosis and postpolycythemia vera myelofibrosis, but not essential thrombocytosis or polycythemia vera: analysis of molecular genetics and clinical phenotypes. Haematologica. 2011;96:1462–9. 71. • Score J, Hidalgo-Curtis C, Jones AV, et al. Inactivation of polycomb repressive complex 2 components in myeloproliferative and myelodysplastic/myeloproliferative neoplasms. Blood. 2012;119: 1208–13. Inactivating mutations in polycomb repressive complex 2 members were reported in MPN and MDS/MPN patients. 72. • Puda A, Milosevic JD, Berg T, et al. Frequent deletions of JARID2 in leukemic transformation of chronic myeloid malignancies. Am J Hematol. 2012;87:245–50. This study reported frequent deletions of JARID2 and suggested that mutations in polycomb repressive complex 2 components are important for leukemic transformation. 73. Sauvageau M, Sauvageau G. Polycomb group proteins: multifaceted regulators of somatic stem cells and cancer. Cell Stem Cell. 2010;7:299–313. 74. Margueron R, Reinberg D. The Polycomb complex PRC2 and its mark in life. Nature. 2011;469:343–9. 75. Kralovics R. Genetic complexity of myeloproliferative neoplasms. Leukemia. 2008;22:1841–8. 76. Kralovics R, Teo SS, Li S, et al. Acquisition of the V617F mutation of JAK2 is a late genetic event in a subset of patients with myeloproliferative disorders. Blood. 2006;108:1377–80. 77. Nussenzveig RH, Swierczek SI, Jelinek J, et al. Polycythemia vera is not initiated by JAK2V617F mutation. Exp Hematol. 2007;35: 32–8.

306 78. Green A, Beer P. Somatic mutations of IDH1 and IDH2 in the leukemic transformation of myeloproliferative neoplasms. N Engl J Med. 2010;362:369–70. 79. Beerman I, Maloney WJ, Weissmann IL, Rossi DJ. Stem cells and the aging hematopoietic system. Curr Opin Immunol. 2010;22:500–6. 80. Beerman I, Bhattacharya D, Zandi S, et al. Functionally distinct hematopoietic stem cells modulate hematopoietic lineage potential during aging by a mechanism of clonal expansion. Proc Natl Acad Sci U S A. 2010;107:5465–70. 81. •• Forsberg LA, Rasi C, Razzaghian HR, et al. Age-related somatic structural changes in the nuclear genome of human blood cells. Am J Hum Genet. 2012;90:217–28. This study found in a cohort of twins an accumulation of somatic structural variants with age and somatic changes that are seen in myeloid malignancies. 82. •• Jacobs KB, Yeager M, Zhou W, et al. Detectable clonal mosaicism and its relationship to aging and cancer. Nat Genet. 2012;44:651–8. As in reference 83, this study reported that the frequency of somatic chromosomal changes increases with age and that these lesions correlated with genomic aberrations described for myeloid malignancies. 83. •• Laurie CC, Laurie CA, Rice K, et al. Detectable clonal mosaicism from birth to old age and its relationship to cancer. Nat Genet. 2012;44:642–50. As in reference 82, this study reported that the frequency of somatic chromosomal changes increases with age and that these lesions correlated with genomic aberrations described for myeloid malignancies. 84. •• Busque L, Patel JP, Figueroa ME, et al. Recurrent somatic TET2 mutations in normal elderly individuals with clonal hematopoiesis. Nat Genet. 2012;44:1179–81. This study found somatic mutations in TET2 in elderly individuals with clonal hematopoiesis. 85. Tefferi A, Jimma T, Sulai NH, et al. IDH mutations in primary myelofibrosis predict leukemic transformation and shortened survival: clinical evidence for leukemogenic collaboration with JAK2V617F. Leukemia. 2012;26:475–80. 86. • Harutyunyan A, Klampfl T, Cazzola M, Kralovics R. p53 lesions in leukemic transformation. N Engl J Med. 2011;364:488–90. P53related aberrations were associated with post-MPN AML, which suggests that the p53 pathway is important for leukemic transformation. 87. Berk PD, Goldberg JD, Silverstein MN, et al. Increased incidence of acute leukemia in polycythemia vera associated with chlorambucil therapy. N Engl J Med. 1981;304:441–7. 88. Bjorkholm M, Derolf AR, Hultcrantz M, et al. Treatment-related risk factors for transformation to acute myeloid leukemia and myelodysplastic syndromes in myeloproliferative neoplasms. J Clin Oncol. 2011;29:2410–5.

Curr Hematol Malig Rep (2013) 8:299–306 89. • Tefferi A, Rumi E, Finazzi G, et al.: Survival and prognosis among 1545 patients with contemporary polycythemia vera: an international study. Leukemia 2013. This study encompassed seven centers and investigated survival and leukemic transformation in polycythemia vera patients. 90. Kiladjian JJ, Chevret S, Dosquet C, et al. Treatment of polycythemia vera with hydroxyurea and pipobroman: final results of a randomized trial initiated in 1980. J Clin Oncol. 2011;29:3907–13. 91. Radaelli F, Onida F, Rossi FG, et al. Second malignancies in essential thrombocythemia (ET): a retrospective analysis of 331 patients with long-term follow-up from a single institution. Hematology. 2008;13:195–202. 92. Spivak JL, Hasselbalch H. Hydroxycarbamide: a user's guide for chronic myeloproliferative disorders. Expert Rev Anticancer Ther. 2011;11:403–14. 93. Reinhardt HC, Schumacher B. The p53 network: cellular and systemic DNA damage responses in aging and cancer. Trends Genet. 2012;28:128–36. 94. Sterkers Y, Preudhomme C, Lai JL, et al. Acute myeloid leukemia a n d m ye l o d y sp l a s t i c s yn d ro m e s f o l l o w i n g es s e n t i a l thrombocythemia treated with hydroxyurea: high proportion of cases with 17p deletion. Blood. 1998;91:616–22. 95. Kiladjian JJ, Chomienne C, Fenaux P. Interferon-alpha therapy in bcr-abl-negative myeloproliferative neoplasms. Leukemia. 2008;22:1990–8. 96. Kiladjian JJ, Cassinat B, Chevret S, et al. Pegylated interferon-alfa2a induces complete hematologic and molecular responses with low toxicity in polycythemia vera. Blood. 2008;112:3065–72. 97. Quintas-Cardama A, Kantarjian H, Manshouri T, et al. Pegylated interferon alfa-2a yields high rates of hematologic and molecular response in patients with advanced essential thrombocythemia and polycythemia vera. J Clin Oncol. 2009;27:5418–24. 98. Kozlowski A, Charles SA, Harris JM. Development of pegylated interferons for the treatment of chronic hepatitis C. BioDrugs. 2001;15:419–29. 99. Kiladjian JJ, Masse A, Cassinat B, et al. Clonal analysis of erythroid progenitors suggests that pegylated interferon alpha-2a treatment targets JAK2V617F clones without affecting TET2 mutant cells. Leukemia. 2010;24:1519–23. 100. • Quintas-Cardama A, Abdel-Wahab O, Manshouri T, et al.: Molecular analysis of patients with polycythemia vera or essential thrombocythemia receiving pegylated interferon alpha-2a. Blood 2013. This follow-up study of a phase II trial with pegylated interferon alpha-2a focused on the molecular analysis of the interferon treated MPN patients and suggested that genetic aberrations might influence drug response.