Genetic Basis and Molecular Pathophysiology of Classical Myeloproliferative Neoplasms

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MYELOPROLIFERATIVE NEOPLASMS Genetic basis and molecular pathophysiology of classical myeloproliferative neoplasms

William Vainchenker1-3 and Robert Kralovics4

1Unit´eMixte de Recherche (UMR) 1170, INSERM, Villejuif, France; 2Gustave Roussy, Villejuif, France; 3Universit´e Paris-Saclay, UMR 1170, Villejuif, France; and 4Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria

The genetic landscape of classical myelopro- vera, essential thrombocythemia (ET), and development of anemia or pancytopenia. liferative neoplasm (MPN) is in large part primary myelofibrosis (PMF) whereas CALR Both heterogeneity of classical MPNs and elucidated. The MPN-restricted driver muta- and MPL mutants are found in ET and PMF. prognosis are determined by a specific tions, including those in JAK2, calreticulin Other mutations in genes involved in epige- genomic landscape, that is, type of MPN (CALR), and myeloproliferative leukemia vi- netic regulation, splicing, and signaling co- driver mutations, association with other rus (MPL), abnormally activate the cytokine operate with the 3 MPN drivers and play a key mutations, and their order of acquisition. receptor/JAK2 pathway and their down- role in the PMF pathogenesis. Mutations in However, factors other than somatic mu- stream effectors, more particularly the STATs. epigenetic regulators TET2 and DNMT3A are tations play an important role in disease The most frequent mutation, JAK2V617F, involved in disease initiation and may pre- initiation as well as disease progression activates the 3 main myeloid cytokine recep- cede the acquisition of JAK2V617F. Other such as germ line predisposition, inflam- tors (erythropoietin receptor, granulocyte mutations in epigenetic regulators such as mation, and aging. Delineation of these colony-stimulating factor receptor, and MPL) EZH2 and ASXL1 also play a role in disease environmental factors will be important to whereas CALR or MPL mutants are re- initiation and disease progression. Muta- better understand the precise pathogene- stricted to MPL activation. This explains why tions in the splicing machinery are predom- sis of MPN. (Blood. 2017;129(6):667-679) JAK2V617F is associated with polycythemia inantly found in PMF and are implicated in the Introduction

The classical myeloproliferative neoplasms (MPNs), also called BCR- subtypes can be observed, as documented by the progression of ET and 2 ABL MPNs, are the most frequent diseases among the myeloprolif- PV to secondary myelofibrosis (MF). Furthermore, boundaries between erative disorders. MPNs are characterized by excessive production of these 3 disorders cannot be well established, especially between ET and terminally differentiated blood cells that are fully functional. Classical PMF. Thus, transitional entities may emerge describing disease states MPNs have been classified into 3 entities: polycythemia vera (PV), such as prefibrotic PMF (or early PMF) that displays an ET phenotype at essential thrombocythemia (ET), and primary myelofibrosis (PMF), diagnosis with typical bone marrow histology, but with a high probability which have frequent disease-related complications, such as venous and of progression to MF and worse prognosis than true ET.3 arterial thrombosis, hemorrhages, and transformation to acute myeloid Somatic mutations are responsible for clonal expansion of HSCs not leukemia (AML).1 All MPN entities arise from a single somatically only in MPNs, but also in most types of myeloid malignancies. High- mutated hematopoietic stem cell (HSC) that clonally expands and gives resolution genome analysis using microarray and next-generation rise to virtually all myeloid cells, and B and natural killer (NK) cells. sequencing (NGS) resulted in the discovery of several gene mutations The clonal expansion of the MPN HSC is accompanied by single or across all myeloid malignancies. Only relatively few of these gene multilineage hyperplasia. PV is characterized not only by an excess mutations are found associated with a single or limited number of of erythrocytes and predominant erythroid lineage involvement, but disease entities. Therefore, we classify these mutations into those that is also associated with a variable hyperplasia of the megakaryocytic/ are “restricted to MPN” and those that are “nonrestricted” and are found granulocytic lineages. ET is characterized by an increased platelet count also in other myeloid malignancies (Table 1). with a megakaryocytic hyperplasia, whereas PMF is a more heteroge- neous disorder both by its clinical and biological characteristics, defined by the presence of bone marrow fibrosis (excess of collagen fibers) and megakaryocytic hyperplasia. Myeloproliferation in PMF initially predominates in the bone marrow and later expands to extramedullary Mutations restricted to MPNs and their role in sites, such as the spleen. Although the clinical presentations of the 3 MPN the MPN pathogenesis entities are different in their typical forms, establishment of precise JAK2 gene mutations diagnosis at disease onset is often challenging. This is reflected by the 2 2016 revision of the World Health Organization (WHO) diagnostic Before 2005, the molecular pathogenesis of the BCR-ABL1 classical criteria for MPN.2 In many cases, a continuum between these disease MPNs was unknown. In 2005, a major breakthrough was the discovery

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668 VAINCHENKER and KRALOVICS BLOOD, 9 FEBRUARY 2017 x VOLUME 129, NUMBER 6

Table 1. Somatic mutations in MPN Gene function and symbol Location Type of mutations Protein function Frequency Consequences Reference Signaling MPN driver JAK2 9p24 JAK2V617F Tyrosine kinase associated with 95% PV, 50%-60% PMF and ET Increased RBC, platelet, and 4-7 cytokine receptors granulocyte production JAK2 exon 12 3% PV Increased RBC 17 MPL 1p34 MPL515L/K/A/R TPOR 2%-3% ET Increased platelet production 19, 22, 23 MPLS505N Other missense 3-5% PMF 25 mutations CALR 19p13 Indel exon 9 Mutant: activator of MPL 20%-25% ET, 25%-30% PMF Increased platelet production 28, 29 Other signaling LNK 12q24 Missense (loss of Negative regulator of JAK2 1% ET, 2% PMF Synergy with JAK2V617F- 35 function) deletion Disease progression CBL 11q23;3 Missense (loss of Cytokine receptor internalization 4% PMF Disease progression 110 function) (progression to AML) NRAS 1p13.2 Missense (activation) ERK/MAPK signaling Rare PMF Progression to leukemia (5%- 65 10% in secondary AML) NF1 17q11 Missense deletion ERK/MAPK signaling Rare PMF Progression to leukemia (5%- 66 10% in secondary AML) FLT3 13q12 FLT3-ITD Cytokine receptor (FLT3-L) MPN (,3%) Progression to leukemia (10%- 100 15% in secondary AML) DNA methylation TET2 4q24 Missense, nonsense a-Ketoglutarate–dependent 10%-20% MPN (ET, PV, and Initiation, 70 deletion dioxygenase PMF) Oxidation of 5mC into 5hmC and Mutations on 2 alleles active 5mC demethylation associated with progression DNMT3A 2p23 Missense, hotspot DNA methylase, de novo 5%-10% MPN (ET, PV, and Initiation 74 methylation PMF) IDH1 2q33.3 Missense, hotspot Neomorphic enzyme, generation 1%-3% PMF Initiation, Disease progression 111 of 2-hydroxyglutarate blocking a-ketoglutarate–dependent enzymes IDH2 15q26.1 Missense, hotspot Neomorphic enzyme, generation 1%-3% PMF Initiation, Disease progression 111 of 2-hydroxyglutarate blocking a-ketoglutarate–dependent enzymes Histone modifications EZH2 7q35-36 Missense, indel H3K27 methyltransferase, loss 5%-10% PMF Initiation 82, 83 of function Disease progression ASXL1 20q11 Nonsense/ indel Chromatin-binding protein 25% PMF Initiation 69 associated with PRC1 and 2 1%-3% ET/PV Rapid progression Transcription factors TP53 17p13.1 Missense/ indel Transcription factor regulating ,5% (20% of sAML) Progression to leukemia 98 cell cycle, DNA repair and (mutations on both alleles) apoptosis complex karyotype CUX1 7q22 Deletion 7p Transcription factor regulating ,3% Progression to leukemia 112 TP53 and ATM IKZF1 7p12.2 Deletion 7p, indel Master transcription factor in ,3% Progression to leukemia 112 lymphopoiesis ETV6 12p13 Missense/ indel Transcription factor of the ETs ,3% Progression to leukemia 112 family RUNX1 21q22.3 Nonsense/ missense/ Master transcription factor in ,3% (10% of sAML) Progression to leukemia 112 indel hematopoiesis RNA splicing SRSF2 17q25.1 Missense, hotspot Serine/arginine-rich pre- RNA ,2% ET 10%-15% PMF Initiation? Progression 94 splicing factor (association with IDH mutations) SF3B1 2q33.1 Missense RNA-splicing factor 3b subunit ,3% ET Phenotypic change (anemia) 113 1, part of U2 U2AF1 21q22.3 Missense U2 small nuclear RNA-splicing 10%-15% PMF Phenotypic change (anemia) 94 factor

ATM, ataxia-telangiectasia mutated; RBC, red blood cell; sAML, secondary AML. From www.bloodjournal.org by guest on March 16, 2017. For personal use only.

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Figure 1. Role of cytokine receptors in the oncogenic properties of JAK2V617F and CALR mutants. (A) JAK2V617F activates signaling through the 3 main homodimeric receptors EPOR, MPL, and G-CSFR, which are involved in erythrocytosis, thrombocytosis, and neutrophilia, respectively. (B) The CALR mutants mainly activate MPL and at a low level the G-CSFR but not the EPOR, explaining the thrombocytosis associated with these mutants. Professional illustration by Som- ersault18:24.

of a G to T somatic mutation at nucleotide 1849, in exon 14 of JAK2, (SH2) and the pseudokinase domains, a region between amino acids resulting in the substitution of valine to phenylalanine at codon 536 and 547, around Lys 539. The most frequent mutations being the 617 (JAK2V617F) in the pseudokinase domain.4-7 This mutation can N542-E543del (23%), E543-D544del (11%), and F537-K539delinsL be found in around 70% of MPNs: 95% of PV and 50% to 60% of ET and K539L (10%), respectively. The VAF in granulocytes can be low and PMF. The JAK2V617F mutation often undergoes a transition and their detection may require speciﬁctechniques. from heterozygosity to homozygosity due to occurrence of mitotic recombination resulting in copy-neutral loss of heterozygosity along a Thrombopoietin receptor gene (MPL) mutations variable size region on the short arm of chromosome 9 (9pLOH).5,8 The JAK2V617F mutation arises in a multipotent hematopoietic progenitor, Two main types of myeloproliferative leukemia virus (MPL;the is present in all myeloid lineages, and can be also detected in lymphoid thrombopoietin [TPO] receptor [TPOR]) mutations located in exon cells, mainly B and NK cells and more rarely and later in disease in 10 have been associated with MPNs. The most frequent are mutations T cells. It is absent in nonhematopoietic cells although ithasbeenfound on the tryptophan W515 located at the boundary of the transmembrane in endothelial cells of the spleen and of splanchnic veins of patients with and the cytosolic domains of MPL, the most prominent mutations MF and/or splanchnic thrombosis as the Budd-Chiari syndrome.9 The being MPLW515L and K.19 However, several other substitutions have variant allele frequency (VAF) in granulocytes is highly variable, with a been described such as W515R, W515A, and W515G.20 Mutations continuum between the threshold of detection (around 1%) to 100%. are usually heterozygous, but can be homozygous during disease The VAF is usually low in ET (around 25%), is higher in PV (frequently progression.21 Mutations on MPLW515 are restricted to ET (around over 50%), and is close to 100% in post-PV or post-ET MF. In PMF, the 3%)andPMF(around5%)andcanbealsofoundinRARS-T.22-24 The VAF is variable, but frequently high.10 The JAK2V617F is mainly other mutation, MPLS505N, is even rarer and is located in the trans- restricted to classical MPNs with the exception of refractory anemia membrane domain, stabilizing receptors in active dimeric orienta- with ring sideroblasts and thrombocytosis (RARS-T).11 It can rarely be tions. Originally, this mutation was described in hereditary form foundinsomeothermalignant hemopathies.12 However, JAK2V617F of thrombocytosis as a germ line mutation.25 Later on, acquired has been detected at very low level (lower than 1%) in the normal MPLS505N somatic mutations were described in ET in ,1% of the population, including in a neonate.13-15 It is 1 of the most frequent cases.22,23 This underscores the similarities in mechanisms of mutations found in the clonal hematopoiesis associated with aging thrombocytosis between hereditary thrombocytosis and ET. More (clonal hematopoiesis of indeterminate potential).16 recently, a number of “noncanonical” mutations of MPL were found in JAK2 exon 12 mutations have been also found in MPNs and are triple-negative ET (ET negative for JAK2V617F, calreticulin [CALR] present in the majority of JAK2V617F2 PV.17 They are not usually mutations, and MPLW515L/K / MPLS505N). These mutations are rare associated with ET and PMF, but JAK2 exon 12 mutation can progress and lead to amino acid changes either in the extracellular (S204 or to secondary MF.18 Several mutations have been described, most of E230) or in the intracellular (Y591) domains.26,27 These noncanonical them being in-frame small insertions and deletions. Exon 12 JAK2 mutations are more often acquired and lead to a true MPN. In addition, mutations are all located in the linker between the Src homology 2 germ line noncanonical mutations of MPL can also be detected, From www.bloodjournal.org by guest on March 16, 2017. For personal use only.

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Figure 2. The CALR mutants bind and activate MPL. (A) WT CALR binds to MPL in the ER by interaction of its lectin domain with MPL N-glycosylation, controls the quality of the proteins, and then comes away from the receptor, which traffics to the cell surface mainly by the conven- tional route to the cell surface. A completely glycosylated MPL (mature form) is expressed at the cell surface where it binds TPO to be activated. CALR mutants bind to MPL in the ER by the same interaction as the WT but the interaction is reinforced by the new C terminus. (B) The mutant CALR remains bound to MPL and probably traffics to the membrane both conventionally and un- conventionally (autophagosomes and pre-Golgi) to the membrane. This leads to the presence of an immature MPL form on which the mutant CALR is bound. Activation of MPL is dependent on the new C terminus and also takes place on the membrane (Stefan Constantinescu, W.V., unpublished results). In cell lines, this leads to activation of mainly the STAT pathway whereas the extracellular signal-regulated kinase (ERK) is slightly activated and the PI3K pathway even less. In cell lines, MPL that binds mutant CALR can only be slightly activated by TPO. Professional illustration by Somersault18:24.

strongly suggesting that these triple-negative ET cases are in fact CALR mutations are usually heterozygous although a few cases of hereditary thrombocytosis. homozygous mutations have been observed, more particularly for type 2 mutations.28,29 Interestingly, homozygous CALR mutations are Mutations in the calreticulin gene: CALR associated with a defect in myeloperoxidase accumulation in primary granules of granulocytes, and thus, can be suspected in an ET when a At the end of 2013, frameshift mutations in the CALR gene were “myeloperoxidase deficiency” is detected by using a flow cytometer– discovered in the majority of JAK22 and MPL2 ET and PMF (50%- based hematology analyzer.33 60% ET and 75% PMF).28,29 There are .50 mutations now described, In contrast to JAK2V617F, the VAF for mutant CALR in ET is high, but all are located in exon 9 inducing a 11(2112) frameshift. At the on average around 40%.10 In rare cases of ET, the VAF can be lower, time of writing, only the mutations leading to this 11frameshiftare but almost never below 15%. This implies that CALR mutants might known to be pathogenic. The other nonpathogenic mutations are give a stronger clonal advantage when compared with JAK2V617F. usually germ line variants of CALR.Specific shifting of the reading The molecular diagnosis is rendered easier by this high VAF and can be frame leads to a new C terminus devoid of the KDEL motif, important performed by numerous techniques and routinely by sizing fluores- for the protein retention in the endoplasmic reticulum (ER). The cently labeled polymerase chain reaction products of exon 9 of CALR.28 mutated CALR protein contains a common new amino acid sequence AfewCALR polymorphisms have been detected not only as single- that bears positive charges. In contrast to wild-type (WT) CALR, the nucleotide polymorphisms, but also in phase deletions of exon 9.34 negative charges required for calcium binding are lost at different There is presently no evidence that these variants have any role in extents, depending on the mutant. The 2 most frequent mutations pathogenesis. correspond to a 52-bp deletion (p.L367fs*46), also called type 1 and a 5-bp insertion (p.K385fs*47), also called type 2. They represent nearly LNK mutations the 2 extremes of modifications observed in exon 9: the del52 having lost most of the WT exon 9 sequence and calcium-binding sites, and In 2010, somatic mutations in exon 2 of LNK (SH2B3), an adaptor ins5 being closer to the WT sequence having kept around 50% of protein which regulates JAK2 activation, were detected in 2 patients negative charges. According to these structural changes, the other (PMF and ET).35 These mutations had a functional impact on mutations have been classified as type 1–like and type 2–like using proliferation and were considered as the driver of the disease. Recent algorithms based on the preservation of an a helix close to WT in studies have questioned the role of LNK as a true driver of the type 2–like mutation.30 There are great differences in the frequency disease.36 LNK mutations have been found in many different types between type 1 and type 2 mutations in ET and PMF: in ET, type 1 and of hemopathies, more particularly in erythrocytosis.37 Some of type 2 mutations are closely distributed (55% vs 35%), whereas in these mutations can be germ line, including the initially described PMF, type 1 are largely predominant (75% vs 15%).31 Thepresenceof LNKE208Q; some others are secondary somatic mutations associated CALR mutants in exceptional cases of PV has been reported, but their with JAK2V617F or CALR mutations, more particularly during disease role in the pathogenesis of the PV remains unclear.32 progression.38 Thus, rather than primary drivers, LNK mutations appear From www.bloodjournal.org by guest on March 16, 2017. For personal use only.

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Table 2. Mouse models Mouse models Phenotype Reference JAK2V617F Retrovirus C57Bl/6 PV progressing to MF 114-116 BALB/c Severe lethal PV to MF 115, 116 Transgenic Vav huJAK2V617F ET/PV progressing to MF 117 H2K muJak2V617F ET/PV progressing to MF 118 Inducible JAK2min promoter huJAK2V617F Vav Cre ET 57 Mx Cre PV with thrombocytosis 57 Pf4Cre (MK specific) No phenotype 119 Mild ET and a PV after transplantation 120 Tie2Cre (HSC and endothelial cell) ET with neutrophilia progressing to osteopetrosis 119 with bleeding diathesis Knock-in Constitutive muJak2V617F PV with thrombocytosis progressing to MF 121 Germ line induction of muJAK2V617F PV progressing to MF 55 E2Acre Germ line induction of huJAK2V617F (cDNA) Stella Cre Heterozygous Mild ET 122 Homozygous PV 122 muJAK2V617F Mx Cre Heterozygous ET/PV progressing to MF 123 Homozygous Severe PV progressing to MF 123 EpoR Cre (erythroid specific) Heterozygous Mild erythrocytosis 124 Homozygous Erythrocytosis 124 muJAK2V617F heterozygous Vav Cre ET progressing to PV progressing to MF 56 Scl Cre PV progressing to MF 125 huJAK2V617F (cDNA) heterozygous Mild ET progressing in PV (10% cases) 58 Mx Cre CALR Retrovirus CALRdel52 Mild ET, ET progressing to MF 50,51 CALRins5 Mild ET 51 Transgenic H-2Kb promoter ET 126 Knock-in Constitutive muCALRdel 61 ET 60 Inducible humanized CALRdel52 Caroline Marty, Isabelle Plo, Harini Nivarthi, R.K., Vav Cre ET (short-term follow-up) W.V., unpublished results SCL CreER ET (short-term follow-up

cDNA, complementary DNA.

more as predisposition mutations when germ line or secondary noncovalent attachment of JAKs to cytokine receptors depends on mutations, increasing the pathogenicity of JAK2V617F and CALR the FERM domain. The JAK family kinases can be considered as when acquired.36 the catalytic part of the hematopoietic cytokine receptor family as they are constitutively bound to the receptors intracellularly. In addition, the association of JAKs with receptors is important for their pro- per trafﬁcking to the cell surface. Homodimeric receptors such as MPN-restricted mutations activate JAK2 erythropoietin (EPO) receptor (EPOR), MPL, and granulocyte colony- kinase–dependent cytokine receptor stimulating factor receptor (G-CSFR) use JAK2, whereas heteromeric pathways receptors use JAK1 and JAK2/tyrosine kinase 2 (TYK2) or JAK3. Cytokine binding induces changes in the conformation of receptors, JAK2 is a member of the JAK family characterized by 2 kinase which activate the JAKs bound to the receptor by trans-phosphorylation. domains: 1 catalytically active at the C terminus and 1 catalytically The activated JAKs will phosphorylate the receptors, which are inactive (or very weak) pseudokinase that prevents self-activation of the subsequently used as a docking site for other signaling molecules, kinase domain.39 At the N terminus, JAKs possess a four-point-one more particularly the STATs. In the canonical pathway, STATs are ezrin radixin moesin (FERM)-like domain and a SH2-like domain. The phosphorylated by the JAKs, which induce their homodimerization From www.bloodjournal.org by guest on March 16, 2017. For personal use only.

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Figure 3. MKs play a central role in MPN pathogenesis. The MPL/JAK2 pathway is activated by the 3 MPN-restricted mutations (JAK2V617F, CALR mutants, and MPL mutants) placing MK hyperplasia and eventually dysplasia as a central determinant in MPN. MKs are mainly involved in platelet production, and also play an important role in the hematopoietic niche by regulating HSCs, remodeling the marrow by secretion of TGF-b1and other cytokines (platelet-derived growth factor [PDGF], vascular endothelial growth factor [VEGF]) which ultimately lead to MF, and also inducing a neo-osteogenesis by inducing an osteoblastic differentiation through TGF-b1 and inhibiting osteoclast differentiation through osteoprotegerin. Furthermore, MKs secrete numerous inflammatory cytokines such as IL1a. The mechanisms of local activation of TGF-b1 are poorly known. OPG, osteoprotegerin; RANK, receptor activator of NF-kB; RANKL, RANK ligand; TGF, transforming growth factor; TSP, thrombospondin. Professional illustration by Somer- sault18:24.

or heterodimerization and subsequent trafficking to the nucleus domain of MPL, which prevents spontaneous activation of MPL.47 All where they regulate transcription of their target genes (Figure 1). substitutions of W515, except W515C and W515P, will lead to the The pseudokinase domain of JAK2 has 2 roles: 1 is to inhibit the kinase activation of MPL in the absence of TPO and will activate JAK2. The domain and the other is to promote cytokine-dependent activation. S505N mutation induces a stable active dimer. However, both types of V617F activates JAK2 by a mechanism, which is not completely mutations activate constitutive signaling through induction of pro- understood, but both structural and functional data have established ductive dimerization of the transmembrane helix of MPL.20,25 that the first conformational change induced by V617F involves CALR in contrast is not a signaling molecule, but an ER chaperone the helix C of the pseudokinase domain.40,41 Mutations in exon 12 involved in the quality control of N-glycosylated proteins and in are located upstream of the pseudokinase domain, and the mecha- calcium storage. The initial publication on CALR mutations has shown nism of activation appears different than that for JAK2V617F.42 that CALRdel52 was able to activate STAT5 and this was impeded by JAK2V617F or JAK2 exon 12 mutations, when expressed in JAK2 inhibition.28 Recent studies have established that CALR mutants interleukin-3 (IL-3)-dependent cell lines, induce cytokine hypersen- can activate MPL and subsequently JAK248-52 (Figure 2). Mutant sitivity or cytokine independence. The presence of homodimeric CALR requires both its mutant C terminus and MPL for oncogenic receptors such as EPOR greatly facilitates this biological effect.43 transformation.49,50,53 WTCALRandmutantCALRbindtothe The presence of a cytokine receptor is absolutely required to induce N-glycosylated residue of the extracellular domain of MPL in the ER, signaling by JAK2V617F at low expression levels. JAK2V617F and the lectin-binding activity of CALR mutants as well as the new tail induces constitutive activation of STATs, and of the phosphatidy- are required for MPL activation49 (Figure 2). This binding is reinforced linositol 3-kinase (PI3K) and MAPK pathways. This cytokine by the new C terminus and its positive charges, which lead to activation hypersensitivity or independence induced by JAK2V617F in cell of the receptor.50,53 It is unknown at which cellular compartment MPL lines mimics the EPO hypersensitivity or independence previously is activated, but there is evidence that CALR mutants behave as described in PV erythroid progenitors.4,44 In addition to this acti- an abnormal chaperone and traffics with MPL to the cell surface.47 In vation of the canonical pathway of cytokine receptors, 2 reports have this case, MPL activation can occur anywhere from the ER to the described that JAK2V617F acts on chromatin by phosphorylating cell surface. Moreover, CALR mutants are secreted and may induce H3Y41, decreasing the binding of heterochromatin protein 1a and cytokine secretion by monocytes.54 CALR mutants also appear to PRMT5, impairing its ability to methylate histone substrates.45,46 It is slightly activate the G-CSFR, but not the other cytokine receptors.49 unknown whether these effects on chromatin are really important in Whether this activation of G-CSFR plays a role in the pathogenesis of the pathogenesis of MPNs. mutant CALR1 MPN is unknown (Figure 1). Activation of MPL by TPO induces the activation of JAK2 and Overall, the main MPN-associated mutations, JAK2V617F, JAK2 TYK2, which are constitutively bound to the receptor, but only exon 12 mutants, MPLW515L/K, and CALR mutants, activate the the activation of JAK2 is indispensable for signaling and induction cytokine/receptor/JAK2 pathways and their downstream signaling, of proliferation by MPL. MPLW515 is located in the amphipathic JAK2V617F, through the 3 homodimeric receptors (EPOR, MPL, From www.bloodjournal.org by guest on March 16, 2017. For personal use only.

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Figure 4. Genes involved in epigenetic regulation and leukemic transformation. The mechanisms by which the genes involved in the epigenetic regulation described in Table 1 lead to modifications in gene regulation are detailed. Some genes involved in leukemic transformation (N-Ras pathway and transcription factors such as p53, RUNX1) are also described. MEK, MAPK/ERK-kinase; RAF, rapidly accelerated fibrosarcoma; SOS, Son of Sevenless; TF, transcription factor. Professional illustration by Somersault18:24.

G-CSFR) and CALR mutants through mainly MPL (Figure 1). This metabolism including decreased expression in hepcidin and increased may explain also why these different mutations are usually mutually expression in transferrin receptor-1 and erythroferrone that favor iron exclusive. However, rarely, 2 of these mutations can be detected in availability for erythroblasts. the same patient, but usually they are present in different clones or For MPLW515K/L/A, only retroviral models have been described. subclones. Although these models are far from ideal because they lead to the expression of MPLW515 mutant in all types of hematopoietic cells, an ET-like disorder rapidly progressing to MF is usually observed, a MPN-restricted mutations drive myeloproliferative disorders 19 and give rise to different diseases in murine models phenotype close to the mice expressing a high level of TPO. For CALR mutants, retroviral models have been used to induce All 3 MPN mutation types have been expressed in mice to investigate a myeloproliferative disorder reminiscent of an ET-like pheno- whether they are drivers of MPNs and to model the disease (Table 2). type, which in the case of CALRdel52 may progress to MF. In this JAK2V617F has been expressed in mouse hematopoiesis by several mouse model, the presence of MPL is indispensable for disease approaches: retroviral transduction of HSC followed by bone marrow development.50,51 A recent transgenic model for CALRdel52 also led transplantation, transgenic mice, and constitutive or inducible knock-in to an ET-like phenotype that was ameliorated by ruxolitinib, similar (KI) mice. Whatever the model, expression of JAK2V617F induces results being obtained in del61 or del52 KI mice60 (Caroline Marty, a myeloproliferative phenotype with certain differences. The most Isabelle Plo, Harini Nivarthi, W.V., R.K., unpublished results). frequent phenotype has been a PV-like disorder progressing to MF.55,56 In conclusion, the 3 MPN oncogenes are true drivers of the disease A correlation between the level of expression and the phenotype phenotype with JAK2 exon 12 giving only an erythrocytosis pheno- has been found: low expression being associated with an ET-like type, JAK2V617F giving rise to ET, PV, and MF, whereas CALR mutant phenotype and a higher expression (30% of the WT or more) with a PV- and MPLW515L/K/A are associated with ET and MF, resembling the like phenotype.57 Further phenotypic variability may be related to phenotype observed in patients. The current data sustain the hypothesis the use of human vs murine JAK2V617F. The heterozygous KI of the that the phenotype of MPNs is mostly related to the types of receptors human JAK2V61F displays a very mild ET phenotype and the ho- that are activated. However, in all models, MPL activation plays a mozygous KI induces an erythrocytosis.58 Thus, in mouse models, central role by its role on HSCs and on megakaryocytes (MKs).19,51,61 JAK2V617F is the driver of the myeloproliferative disorder and the MK hyperplasia plays a central role in the pathogenesis of MPNs not level of expression in great part determines the phenotype of the MPN, a only by increasing platelet production, but also by serving as a niche situation close to the human setting. Recently, JAK2 exon 12 transgenic for HSCs, by remodeling bone marrow leading to MF and also by mice have been obtained that developed isolated erythrocytosis.59 inducing neo-osteogenesis (Figure 3). However, in none of the models The erythroid phenotype was even more marked than in JAK2V617F did mice develop a PMF, whereas they did develop secondary MF transgenic mice. This was attributed to important changes in iron (post-ET or post-PV); no leukemic transformation is observed during From www.bloodjournal.org by guest on March 16, 2017. For personal use only.

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Table 3. Co-occurrence of mutations PV JAK2V617F ET JAK2V617F ET CALRmut ET MPLm PMF JAK2V617F PMF CALRmut PMF MPLm

% (No. of patients)

TET2 8.33 (4) 5.26 (3) 0 (0) 0 (0) 28.95 (11) 2.63 (1) 2.63 (1) DNMT3A 6.25 (3) 7.02 (4) 0 (0) 3.51 (2) 7.89 (3) 0 (0) 0 (0) ASXL1 0 (0) 3.51 (2) 0 (0) 1.75 (1) 15.79 (6) 7.89 (3) 0 (0) IDH1/IDH2 2.08 (1) 1.75 (1) 0 (0) 0 (0) 2.63 (1) 0 (0) 0 (0) EZH2 2.08 (1) 0 (0) 1.75 (1) 0 (0) 2.63 (1) 2.63 (1) 0 (0) SF3B1 4.17 (2) 0 (0) 0 (0) 0 (0) 2.63 (1) 0 (0) 0 (0) U2AF1 0 (0) 0 (0) 0 (0) 0 (0) 7.89 (3) 0 (0) 0 (0)

Results are extracted from Nangalia et al29 because it is one of the rare studies in which all types of MPNs are available. Overall, 143 patients: PV, 43; ET, 57; and PMF, 38. Percentages are expressed for each MPN, irrespective of the MPN drive mutation. Thus, percentages are calculated on a very low number of patients due to the heterogeneity of the disorder, explaining differences with other studies, more particularly for the mutations in splicing genes. disease progression. The different STATs may play an important role extremely rare in CALR PMF in comparison with JAK2V617F. Thus, in disease phenotype: STAT5 being required for the PV development, PMF appears not to be a pure MPN, but a mixed myeloproliferative/ STAT3 inhibiting the thrombocytosis, and STAT1 increasing the myelodysplastic syndrome. The presence of mutations in genes other thrombocytosis.62,63 than the 3 MPN-restricted driver genes increases the myelodysplastic features of the disease and the severity of the disease, explaining the continuity between MPN, MPN/MDS, and MDS (Figure 5). It is beyond the scope of this review to cover all the recurrent gene mutations in detail. Therefore, we will focus on TET2 and DNMT3A,as “Nonrestricted” acquired mutations and their they may represent the most important modifiers of the MPN-restricted role in the MPN pathogenesis oncogenic mutations, on EZH2 whose role has been recently studied in detail, and on addition of sex combs like 1 (ASXL1)andSRSF2,which The 3 main driver mutations do not explain the entire heterogeneity of 2 are all associated with a poor prognosis in MF. the classical BCR-ABL MPNs. The development of NGS, as well as fi other whole-genome analysis techniques, allowed the identi cation of TET2 and DNMT3A mutations and their role in disease initiation several acquired mutations in MPNs (Table 1). However, none of these mutations is restricted to MPNs andtheyareevenmorefrequentin The TET and DNMT3A protein families play a central role in the myelodysplastic syndromes (MDSs) and AML. This underscores the regulation of DNA methylation at cytosine guanine dinucleotide (CpG) continuum between the different myeloid malignancies, and most sequences (Figure 4): the DNMT3 protein family is involved in the of these mutations are involved in phenotypic changes and disease methylation of cytosines (5mC), whereas the TET protein family in progression in MPNs. active demethylation through oxidation of 5mC into 5hmC.68 TET2 Mutations identified in myeloid malignancies target DNA methyl- mutations have been the first mutations, along with ASXL1,tobe 1 ation regulators (TET2, DNMT3A, IDH1/2), histone modifiers identified in JAK2V617F MPNs.69,70 All TET2 mutations are loss-of- (Polycomb repressor complex 1 and 2 members, IDH1/2), transcription function point mutations or deletions, usually on 1 allele, but more factors (TP53, CUX1, IKZF1, FOXP1, ETV6, RUNX1), proteins rarely on both somatic alleles. TET2 mutations have been identified involved in signaling (NF1, NRAS, KRAS, LNK, CBL, FLT3), and initially as mutations preceding JAK2V617F and are responsible for splicing factors (SF3B1, SRSF2, U2AF1, ZRSR2)(Figure4).64-66 As clonal dominance.70 Their frequency ranges from 10% to 20% and they the majority of the mutations are loss of function and act as dominant- are found in all MPN subtypes. For comparison, TET2 mutations negative or via haploinsufficiency or complete homozygous loss, it are present in 50% to 60% of chronic myelomonocytic leukemia seems that most of the mutated genes are myeloid tumor suppressors. In (CMML).71 TET2 mutations can be also secondary mutations after MPNs, targeted gene panel sequencing studies identified patients with JAK2V617F or CALR mutations and induce an expansion of double- multiple gene mutations. Although recurrent single-gene mutations mutated stem cells and progenitors.72 It has been suggested that when were identified, individual gene mutations had too low frequency to TET2 mutates first, the subsequent occurrence of JAK2V617F induces allow convincing evidence of association with disease progression. an ET phenotype, in contrast to the situation in which JAK2V617F However, the number of detected mutations (as an indirect measure of mutates first and the phenotype is more likely PV. Secondary TET2 genetic complexity or progression of clonal evolution) allowed the mutations can also be associated with progression, more particularly identification of high-risk patients.67 when the mutation is homozygous.73 However, in ET and PV, in the majority of cases, only 1 mutation in DNMT3A mutations are less frequent than TET2 mutations in a MPN driver gene is found. In contrast, in the majority of PMF, more MPNs (5%-10%) and in the majority of the cases precede JAK2V617F somatic mutations are found (3 mutations or more). Some differences or MPL mutations.74 As in other malignant hemopathies, the R882H are observed in the coexpression of mutations along the type of MPNs. is the most prevalent DNMT3A mutation. Both TET2 and DNMT3A An example obtained by whole-exome sequencing is illustrated mutations increase the self-renewal capacities of HSCs in both humans in Table 3. However, in several studies using targeted gene panel and mice.75,76 The precise mechanism is not completely understood, sequencing studies, mutations in spliceosome genes appear very rare in but TET2 and DNMT3A control the expression of genes involved in PV, in comparison with ET. This may be due to the continuum between HSC properties as well as differentiation. It has been recently suggested ET and RARS-T or prefibrosis/PMF.Similarly,inPMF,thereare that TET2-deficient HSCs are more prone to differentiation than normal no major differences with respect to the association of JAK2V617F, HSCs, with the derepression of transcription factors such as Klf1.68 and CALR or MPL mutants with the different mutations in epige- TET2 and DNMT3A are the 2 most frequently mutated genes netic regulators. On the other hand, spliceosome genes mutations are associated with clonal hematopoiesis during aging.77-79 On the other From www.bloodjournal.org by guest on March 16, 2017. For personal use only.

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Figure 5. Presence of myeloproliferative and myelodysplastic features in MPN. (A-C) The 3 MPN-restricted drivers lead to a myeloproliferative phenotype. All of the additional mutations in genes involved in epigenetics and splicing modify the differentiation and give a myelodysplastic phenotype even if some of them such as TET2, DNMT3A, and EZH2 clearly play an important role in initiation. Moreover, there are additional mutations involved in splicing that induce myelodysplastic features leading to MF, cytopenia, and eventually progression to leukemia. Professional illustration by Somersault18: 24.

hand, there is no clear evidence that these mutations induce gene gives a poor prognosis.84 Three recent studies have shown that Ezh2 instability. However, by inducing a clonal hematopoiesis and thus loss in a murine MPN model expressing JAK2V617F dramatically increasing replication of mutated stem cells, they may indirectly induce modifies the MPN phenotype. JAK2V617F/Ezh22/2 double mutant secondary mutations. They may also favor leukemogenesis by progres- mice had significantly reduced survival due to rapid acceleration to sively modifying the equilibrium between self-renewal and differen- lethal MF.85-87 Furthermore, strong cooperation between Ezh2 loss tiation. DNMT3A mutation may predispose to leukemic progression and JAK2V617F on disease initiation by increasing the fitness of in MPNs. In mouse models, Tet2 loss increased the competitive JAK2V617F stem cells was reported. At the cellular level, Ezh2 loss reconstitution capacities of Jak2V617F-expressing HSCs and also increased megakaryopoiesis at the expense of erythropoiesis. The 2 2 worsened the MPN phenotype with an increase in extramedullary phenotype of the JAK2V617F/Ezh2 / double mutant mice was hematopoiesis without leukemic transformation.80,81 related to changes in the expressionofPRC2targetgenesandmore Overall, these 2 types of mutations increase the self-renewal of particularly of the Lin28b/let7/Hmga2 axis. The overexpression of JAK2V617F HSCs and play an important role in disease initiation. Hmga2, a gene that was previously found deregulated in human PMF, However, they can also induce disease progression when they occur as appears to play an important role in this phenotype.86,87 secondary mutations, but their role in MF and leukemic transformation remains elusive. ASXL1 SRSF2 Loss of EZH2 function promotes MF development and and mutations are associated poor prognosis with a poor prognosis

EZH2 is 1 of the 2 histone methyltransferases of the PRC2 complex and ASXL1 encodes for an epigenetic regulator, which binds to chromatin. is involved in the repressive H3K27 trimethylation (H3K27m3) Interestingly, it has been shown that ASXL1 recruits the PRC2 com- (Figure 4). Loss-of-function mutations and cytogenetic lesions in EZH2 plex to speciﬁc loci through a direct interaction between ASXL1 and and other PRC2 members (SUZ12, JARID2, EED) have been described EZH288 (Figure4).However,theroleofASXL1iswiderthanEZH2 in MPNs and across all myeloid malignancies.82,83 EZH2 is mutated in because it can be also involved in the PRC1 complex by its associa- 5% to 10% of PMF, more particularly associated with JAK2V617F and tion with the deubiquitinating enzyme (DUB) BRCA-1–associated From www.bloodjournal.org by guest on March 16, 2017. For personal use only.

676 VAINCHENKER and KRALOVICS BLOOD, 9 FEBRUARY 2017 x VOLUME 129, NUMBER 6 protein (BAP1), a critical tumor suppressor in solid tumor.89 In addition, role of therapy in the induction or on the selection of TP53-mutated ASXL1 also binds to nuclear hormone receptors such as RARa and clones is presently unknown. TP53 as well as DNMT3A mutations are estrogen receptors. The ASXL family plays an important role in apparently more frequent in post-PV or -ET AML than on post-MF development as shown in Drosophila by regulating Hox genes. In leukemia.100 Other secondary leukemia are mainly associated with humans, de novo or germ line ASXL1 mutations are responsible for mutations in ASXL1, SRSF2, IDH1/2, CBL,andLNK. Late events a developmental syndrome called the Bohring-Opitz syndrome.90 include RUNX1 mutations (30% of the cases), FLT3–internal tandem Somatic mutations in ASXL1 were first described by Birnbaum’s duplication (15% of the cases), N-RAS, NF1, and deletion in IKZF1 and group, first in MDS and CMML and then in MPN.69,91 They are the CUX1100,101 (Figure 4). The complex mutational profile of patients in second mutations by their frequency in epigenetic regulators after the leukemic stage indicates that all patients at this stage are genetically TET2 in MPNs. However, they are essentially associated with PMF unique with complex clonal hierarchies. Treatment is challenging and with a frequency around 25%. The ASXL1 mutations are associated relapse almost always occurs. with an aggravation in the prognosis, whatever the International Prognostic Scoring System (iPSS) classification. Mutations are loss of function and are associated with a higher frequency of AML transformation. These genetic alterations are either focal deletion or nonsense mutation or insertion/deletion leading to frameshift, the Important factors for initiation most common variant being the c.1934dupG;p.Gly646TrpfsX12 Aging variant. In mice, Asxl1 deletion leads to profound cytopenia with a dysplasia associated with a profound defect in HSC self-renewal There is increasing evidence that JAK2V617F is relatively frequent in 13 properties that are corrected by Tet2 deletion.92 the aging healthy population and is presently estimated to be ,0.5%. SRSF2 mutations are also associated with MPNs having a poor This indicates that JAK2V617F alone is sufficient to engender clonal prognosis.93 Mutations in genes encoding spliceosome proteins were hematopoiesis and suggests it has a disease-initiating role in MPN. detected in different hematopoietic malignancies in 2011, but more However, JAK2V617F being a frequent mutation, it has a low particularly in MDS.94 Spliceosome gene mutations in MPNs are penetrance to give a MPN, suggesting that other factors must be essentially restricted to ET and MF. The most frequent mutations associated to JAK2V617F to give rise to a disease. Aging is generally include SRSF2, SF3B1,andU2AF1 and are associated with anemia and associated with a deregulation of HSCs, which lose their function and thrombocytopenia.95 SRSF2 encodes for a protein, which is a member become myeloid-biased and less quiescent as a consequence of intrinsic of the serine/arginine-rich splicing factor family that binds to and environmental changes. Overall, this suggests that JAK2V617F exonic splicing enhancer (ESE) sequences in the pre–messenger HSCs have higher competitive properties in this context, explaining RNA (mRNA). There is increasing evidence that SRSF2 mutations, why MPNs are age-related diseases. essentially on the proline 95 are not loss of functions, but recognize preferentially the CCNG ESE motifs whereas the WT sequence Inflammation recognizes both the CCNG and GGNG ESE motifs. This alters the splicing of numerous pre-mRNA and leads to numerous functionally All MPNs, particularly PMF, are associated with an important in- relevant misspliced events. In consequence, mice with a heterozygous flammatory response as demonstrated by the presence of high Srsf2P95H mutant develop a cytopenia by myelodysplasia whereas a plasma levels of inflammatory cytokines as well as constitutional homozygous Srsf2 knockout when induced inadults leads tocytopenia symptoms alleviated by anti-inflammatory therapies such corticoid 102 due to a decrease in hematopoietic progenitors associated with and ruxolitinib. These inflammatory cytokines are synthetized by increased apoptosis mimicking a bone marrow aplasia.96 Particularly, the mutated and nonmutated hematopoietic cells as well as by non- 103 it has been shown that the Srsf2 P95H mutation in mice can lead to a hematopoietic cells such as mesenchymal stromal cells. More missplicing and nonsense-mediated decay of Ezh2.97 Knowing that particularly, it has been demonstrated that the JAK2V617F progenitors 1 SRSF2 mutations are enriched in leukemic transformation of MPNs secrete IL1b, which induces the apopotic death of nestin cells 104 (19% of the cases), this further establishes that EZH2 and the PRC2 providing a favorable environment for JAK2V617F HSCs. More- complex are important factors in the development of MF and over, it has been shown that tumor necrosis factor a (TNFa) gives a 105 progression toward leukemia. competitive advantage to JAK2V617F HSCs. IL33 may have similar properties.106 The inflammatory cytokines might also be involved in extramedullar hematopoiesis by favoring colonization of JAK2V617F HSCs and progenitors in the spleen. The p53 pathway and the development of secondary AML Predisposition

Various mutations and cytogenetic lesions targeting the TP53 tumor- Several susceptibility alleles that correspond to common polymor- suppressor function are found in around 40% to 50% of secondary phisms in genes such as JAK2, particularly the JAK2 46/1 haplotype, leukemia of MPNs. This usually includes missense mutations or TERT, MECOM, SH2B3, CHEK2, PINT,andGFI1B,mayweakly(by deletions in the TP53 gene or ampliﬁcation of chromosome 1q targeting twofold to sixfold) increase the development of the disease.13,107 The MDM4, a TP53 transcriptional inhibitor.98 Heterozygous TP53- mechanism is presently unknown but these predisposition variants mutated clones can be found early in MPN, but evolution to leukemia involve genes that play a role either in DNA damage response (CHEK2, is associated with its clonal dominance and transition from heterozy- TERT, JAK2 46/1 haplotype) or in the JAK2/STAT pathway. Recently, gosity to homozygosity for the mutation.98 This mode of transformation predisposition factors were discovered in the true MPN families with an mimics therapy-related leukemia and is quite different from sporadic autosomal pattern of inheritance but with an incomplete penetrance, leukemia.99 Presently, the only mouse model which leads to leukemia is suggesting that they are strong predisposing factors. Although RBBP6 the overexpression of JAK2V617F in a Tp53 knockout model.65 The mutations were found in MF families, a copy-number variation of From www.bloodjournal.org by guest on March 16, 2017. For personal use only.

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6 genes with 2 genes involved (ATG2B and GSKIP)wasidentiﬁed in studies. Future work on the pathogenesis of MPNs will be more focused MPN families with a particularly aggressive phenotype.108,109 on the interaction between extrinsic factors and acquired genetic abnormalities in disease initiation and progression.

Conclusions

The mutational landscape in classical MPN is much more complex Acknowledgments than initially thought. However, abnormal activation of JAK2 is the common feature of disease, causing mutations in the 3 genes The authors are indebted to Isabelle Plo, Caroline Marty, and Stefan driving the MPNs. The genetic cause remains unknown in around Constantinescu for their help to improve the manuscript. 15% of chronic thrombocytosis classified as ET and in ,10% of This work was supported by grants from Ligue Nationale Contre “ ” PMF. The rare triple-negative PMF appears more likely to be MDSs le Cancer ( Equipe labellisee´ 2016 ), from INSERM, and from with secondary fibrosis. In the majority of ET and PV, only Laboratory of Excellence Globule Rouge-Excellence. W.V. was funded “ ’ ” mutations in JAK2, CALR,andMPL can be found, suggesting that by the program Investissements d avenir . R.K. received funding from the deregulation in the cytokine receptor/JAK2 pathway is suffi- the Austrian Science Fund (SFB F4702 and P29018-B30). cient to induce a clonal myeloproliferative disorder reinforcing the concept that JAK2 is the main therapeutic target. The mechanism of JAK2 activation may define 2 main types of MPNs: (1) those driven by JAK2V61F, leading to ET, PV, and PMF and associated with Authorship proliferation and activation of the 3 main myeloid lineages (erythroid, granulocytic, and MK/platelet lineages) and (2) those Contribution: W.V. and R.K. wrote the manuscript. driven by CALR and MPL mutants which are associated with ET Conflict-of-interest disclosure: The authors declare no competing and PMF and target essentially the MK/platelet lineage. financial interests. PMF represents a different disorder, which may be genetically ORCID profiles: R.K., 0000-0002-6997-8359. closer to MDS, and its development may require the occurrence of Correspondence: William Vainchenker, INSERM UMR 1170, several drivers preceding the JAK2V617F mutations and the presence Institut Gustave Roussy, 39, Rue Camille Desmoulins, Villejuif, of inflammation in the bone marrow microenvironment. The clinical 94805 France; e-mail: [email protected]; and Robert Kralovics, Re- heterogeneity seems to be mainly related to the combination of search Center for Molecular Medicine of the Austrian Academy of mutations and perhaps their order of acquisition. The precise role of Sciences, Lazarettgasse 14, AKH BT 25.3, A-1090 Vienna, Austria; each mutation and their impact on MPN phenotype will require further e-mail: [email protected].

References

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2017 129: 667-679 doi:10.1182/blood-2016-10-695940 originally published online December 27, 2016

Genetic basis and molecular pathophysiology of classical myeloproliferative neoplasms

William Vainchenker and Robert Kralovics

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