REVIEW After 10 years of JAK2V617F: Disease biology and current management strategies in polycythaemia vera Jacob Grinfeld, Anna L Godfrey ⁎ Department of Haematology, Cambridge University Hospitals NHS Foundation Trust, Hills Rd, Cambridge CB2 0QQ, UK abstract article info Available online xxxx The JAK2V617F mutation accounts for the vast majority of patients with polycythaemia vera (PV) and around half of those with other Philadelphia-negative myeloproliferative neoplasms. Since its discovery in 2005, numerous insights have been gained into the pathways by which JAK2V617F causes myeloproliferation, including activation of JAK-STAT signalling but also through other canonical and non-canonical pathways. A variety of mechanisms explain how this one mutation can be associated with distinct clinical disorders, demonstrating how constitu- tional and acquired factors may interact in the presence of a single mutation to determine disease phenotype. Im- portant biological questions remain unanswered in PV, in particular how JAK2V617F affects stem cell function and what mechanisms drive myelofibrotic and leukaemic transformation. Whilst current management is largely centred on prevention of cardiovascular events, future therapies must aim to target the JAK2-mutant clone, to re- verse the underlying marrow pathology and to address the risk of transformation events. © 2016 Published by Elsevier Ltd. Keywords: Polycythaemia vera Erythrocytosis Myeloproliferative JAK2 1. Introduction Polycythaemia vera (PV), one of the Philadelphia-negative myelo- proliferative neoplasms (MPNs), is a clonal haematopoietic stem cell disorder characterised by an increased red cell mass, associated with proliferation of erythroid, granulocytic and megakaryocytic elements of the bone marrow [1]. In 2005 the identification of the JAK2V617F mu- tation elucidated a molecular basis for the disorder in over 95% of pa- tients [2–5], followed two years later by the identification of JAK2 exon 12 mutations in most remaining patients [6,7]. The vast majority of patients with PV therefore carry mutations in a single gene, and a wealth of studies has since illuminated the many molecular and cellular consequences of these mutations. Whilst JAK2 exon 12 mutations are specific to PV, the JAK2V617F mu- tation is also found in other myeloid disorders: 50 to 60% of those with essential thrombocythaemia (ET) or primary myelofibrosis (PMF) [2– 5], about half of patients with the MDS/MPN entity “refractory anaemia with ring sideroblasts and marked thrombocytosis” (RARS-T) [8], and lower frequencies in AML, other myeloproliferative and myelodysplastic disorders [9,10]. ET and PMF in particular share impor- tant clinical features with PV, including the presence of neutrophilia and/or thrombocytosis in some patients, hypercellularity of the bone marrow, frequent splenomegaly, and risks of thrombosis and haemorrhage [1]. In a minority of patients, PV, ET and PMF can all trans- form into acute myeloid leukaemia (AML) [11–13], those with ET may develop transformation to PV [14], and both ET and PV may transform into secondary myelofibrosis [11,13,14]. The identification of JAK2V617F offered some basis for the clinical similarities between these MPNs, but the molecular mechanisms behind the phenotypic dif- ferences between the JAK2V617F-positive disorders has remained the subject of intensive investigation. The identification of JAK2 mutations in PV led to the introduction of a rapid and non-invasive diagnostic test, with mutation testing now widely available and incorporated into national and international clini- cal guidelines [1,15–17]. Moreover the development of JAK2 inhibitors has led to a much-needed novel therapeutic avenue for patients with myelofibrosis [18,19], and most recently these drugs have also been shown to benefit certain patients with PV [20]. However, clinical man- agement of PV remains predominantly centred on the reduction of vas- cular complications and symptoms. In spite of significant advances that have been made in understanding the disease biology of PV, agents that can target the underlying neoplastic clone have remained elusive and the risk of disease transformation, with its almost inevitably poor out- come, remains an important and unmet clinical need. This review will first examine current understanding of the molecu- lar basis of PV, both in terms of the pathological effects of JAK2 muta- tions and the factors that may contribute to phenotypic differences between PV and other JAK2-mutated MPNs. Diagnostic criteria and their controversies will be considered. Key prognostic factors in PV will then be examined, followed by a discussion of current management strategies and unresolved clinical challenges. Blood Reviews xxx (2016) xxx–xxx ⁎ Corresponding author. E-mail addresses: [email protected] (J. Grinfeld), [email protected] (A.L. Godfrey). YBLRE-00462; No of Pages 18 http://dx.doi.org/10.1016/j.blre.2016.11.001 0268-960X/© 2016 Published by Elsevier Ltd. Contents lists available at ScienceDirect Blood Reviews journal homepage: www.elsevier.com/locate/blre Please cite this article as: Grinfeld J, Godfrey AL, After 10years of JAK2V617F: Disease biology and current management strategies in polycythaemia vera, Blood Rev (2016), http://dx.doi.org/10.1016/j.blre.2016.11.001
After 10 years of JAK2V617F: Disease biology and current management strategies in polycythaemia vera
Mar 08, 2023
Polycythaemia vera (PV), one of the Philadelphia-negative myeloproliferative neoplasms (MPNs), is a clonal haematopoietic stem cell disorder characterised by an increased red cell mass, associated with proliferation of erythroid, granulocytic and megakaryocytic elements of the bone marrow [1]. In 2005 the identification of the JAK2V617F mutation elucidated a molecular basis for the disorder in over 95% of patients [2–5], followed two years later by the identification of JAK2 exon 12 mutations in most remaining patients [6,7]. The vast majority of patients with PV therefore carry mutations in a single gene, and a wealth of studies has since illuminated the many molecular and cellular consequences of these mutations
Welcome message from author
The JAK2V617F mutation accounts for the vast majority of patients with polycythaemia vera (PV) and around half
of those with other Philadelphia-negative myeloproliferative neoplasms. Since its discovery in 2005, numerous
insights have been gained into the pathways by which JAK2V617F causes myeloproliferation, including activation
of JAK-STAT signalling but also through other canonical and non-canonical pathways. A variety of mechanisms
explain how this one mutation can be associated with distinct clinical disorders, demonstrating how constitutional and acquired factors may interact in the presence of a single mutation to determine disease phenotype. Important biological questions remain unanswered in PV, in particular how JAK2V617F affects stem cell function and
what mechanisms drive myelofibrotic and leukaemic transformation.
Transcript
After 10years of JAK2V617F: Disease biology and current management strategies in polycythaemia veraYBLRE-00462; No of Pages 18
Contents lists available at ScienceDirect
Blood Reviews
j ourna l homepage: www.e lsev ie r .com/ locate /b l re
REVIEW
After 10 years of JAK2V617F: Disease biology and current management strategies in polycythaemia vera
Jacob Grinfeld, Anna L Godfrey Department of Haematology, Cambridge University Hospitals NHS Foundation Trust, Hills Rd, Cambridge CB2 0QQ, UK
Corresponding author. E-mail addresses: [email protected] (J. Grinfeld), anna
(A.L. Godfrey).
http://dx.doi.org/10.1016/j.blre.2016.11.001 0268-960X/© 2016 Published by Elsevier Ltd.
Please cite this article as: Grinfeld J, God polycythaemia vera, Blood Rev (2016), http:
a b s t r a c t
a r t i c l e i n f o
Available online xxxx
The JAK2V617Fmutation accounts for the vastmajority of patients with polycythaemia vera (PV) and around half of those with other Philadelphia-negative myeloproliferative neoplasms. Since its discovery in 2005, numerous insights have been gained into the pathways bywhich JAK2V617F causesmyeloproliferation, including activation of JAK-STAT signalling but also through other canonical and non-canonical pathways. A variety of mechanisms explain how this one mutation can be associated with distinct clinical disorders, demonstrating how constitu- tional and acquired factorsmay interact in the presence of a singlemutation to determine disease phenotype. Im- portant biological questions remainunanswered in PV, in particular how JAK2V617F affects stem cell function and what mechanisms drive myelofibrotic and leukaemic transformation. Whilst current management is largely centred on prevention of cardiovascular events, future therapiesmust aim to target the JAK2-mutant clone, to re- verse the underlying marrow pathology and to address the risk of transformation events.
© 2016 Published by Elsevier Ltd.
Keywords: Polycythaemia vera Erythrocytosis Myeloproliferative JAK2
1. Introduction
Polycythaemia vera (PV), one of the Philadelphia-negative myelo- proliferative neoplasms (MPNs), is a clonal haematopoietic stem cell disorder characterised by an increased red cell mass, associated with proliferation of erythroid, granulocytic and megakaryocytic elements of the bonemarrow [1]. In 2005 the identification of the JAK2V617Fmu- tation elucidated a molecular basis for the disorder in over 95% of pa- tients [2–5], followed two years later by the identification of JAK2 exon 12 mutations in most remaining patients [6,7]. The vast majority of patients with PV therefore carry mutations in a single gene, and a wealth of studies has since illuminated themanymolecular and cellular consequences of these mutations.
Whilst JAK2 exon 12mutations are specific to PV, the JAK2V617Fmu- tation is also found in other myeloid disorders: 50 to 60% of those with essential thrombocythaemia (ET) or primary myelofibrosis (PMF) [2– 5], about half of patients with the MDS/MPN entity “refractory anaemia with ring sideroblasts and marked thrombocytosis” (RARS-T) [8], and lower frequencies in AML, other myeloproliferative and myelodysplastic disorders [9,10]. ET and PMF in particular share impor- tant clinical features with PV, including the presence of neutrophilia and/or thrombocytosis in some patients, hypercellularity of the bone marrow, frequent splenomegaly, and risks of thrombosis and
[email protected]
frey AL, After 10years of JA //dx.doi.org/10.1016/j.blre.20
haemorrhage [1]. In aminority of patients, PV, ET and PMF can all trans- form into acute myeloid leukaemia (AML) [11–13], those with ET may develop transformation to PV [14], and both ET and PV may transform into secondary myelofibrosis [11,13,14]. The identification of JAK2V617F offered some basis for the clinical similarities between these MPNs, but the molecular mechanisms behind the phenotypic dif- ferences between the JAK2V617F-positive disorders has remained the subject of intensive investigation.
The identification of JAK2mutations in PV led to the introduction of a rapid and non-invasive diagnostic test, with mutation testing now widely available and incorporated into national and international clini- cal guidelines [1,15–17]. Moreover the development of JAK2 inhibitors has led to a much-needed novel therapeutic avenue for patients with myelofibrosis [18,19], and most recently these drugs have also been shown to benefit certain patients with PV [20]. However, clinical man- agement of PV remains predominantly centred on the reduction of vas- cular complications and symptoms. In spite of significant advances that have beenmade in understanding the disease biology of PV, agents that can target the underlying neoplastic clone have remained elusive and the risk of disease transformation, with its almost inevitably poor out- come, remains an important and unmet clinical need.
This reviewwill first examine current understanding of themolecu- lar basis of PV, both in terms of the pathological effects of JAK2 muta- tions and the factors that may contribute to phenotypic differences between PV and other JAK2-mutated MPNs. Diagnostic criteria and their controversies will be considered. Key prognostic factors in PV will then be examined, followed by a discussion of currentmanagement strategies and unresolved clinical challenges.
K2V617F: Disease biology and current management strategies in 16.11.001
2. The molecular and cellular effects of JAK2 mutations
JAK2 is one of a family of cytoplasmic tyrosine kinases that mediates signal transduction from cell surface cytokine receptors, including the erythropoietin and thrombopoietin receptors [21]. Binding of ligand to receptor leads to JAK autophosphorylation, receptor transphosphorylation and creation of binding sites for signalling mole- cules such as STAT proteins. STATs are phosphorylated, translocate to the nucleus and affect target gene transcription. For example, binding of erythropoietin to its receptor (EpoR) causes phosphorylation of JAK2 and STAT5 [22], together with activation of other effectors includ- ing MAP kinase and phosphoinositide 3-kinase (PI3K)/Akt pathways [23].
2.1. Biochemical effects of JAK2 mutations
In early studies, the V617Fmutationwas found to have activating ef- fects on JAK2 signalling activity, cytokine sensitivity and cytokine-de- pendent survival in cell lines [2–5]. This activation of JAK2 is thought to reflect the location of the mutation within the JH2 “pseudokinase” domain of the protein, which normally has auto-inhibitory effects on the catalytically active JH1 kinase domain [24–27]. Mechanismsmay in- clude the interference of the mutation with direct phosphorylation of JH1 by JH2 [28], and cooperation of V617F with other residues in JH2 to enhance JH1 kinase activity [29,30]. Interestingly, recent work on the JAK1 JH2 crystal structure has also suggested that JAK2V617F activa- tion requires a specific conformational change in the SH2-JH2 linker do- main [31], which is the area targeted by exon 12 mutations. Exon 12 mutations appear have similar activating effects to V617F in cell lines, including cytokine independence and constitutive activation of JAK2 and its downstream mediators in the absence of erythropoietin [6].
Several signalling changes appear to mediate the cellular effects of JAK2V617F. Increased phosphorylation of STAT5 has been demonstrated in numerous experimental systems, including cultured erythroid cells and bone marrow trephine biopsies from patients with MPNs, and in JAK2V617F knock-in mouse models [2,4,5,32–41]. The particular impor- tance of STAT5 activation in PV is illustrated by the observation that ex- pression of constitutively active STAT5 in erythroid progenitors can drive formation of the same erythropoietin-independent endogenous erythroid colonies that are typical of PV [42]. Moreover it has been shown more recently that Stat5 deletion in either a Jak2V617F knock- inmouse [43] or retroviral bonemarrow transplantationmodel [44] ab- rogates the marked erythrocytosis that is seen in the presence of Stat5.
Other signalling processes that appear to be activated by JAK2V617F include the PI3K/Akt and MAPK/ERK pathways, which have been stud- ied in many experimental systems [2,5,33,34,36,38,45]. There is evi- dence that the mutation has direct effects on apoptosis, both through the extrinsic pathway that acts through cell surface death receptors such as Fas [46], and through the intrinsic pathway, with increased levels of the anti-apoptotic protein Bcl-xL and impairment of its normal role in the DNA damage response [47–49]. Other mediators implicated in apoptotic dysregulation include ID1, Pim1/2 kinases, the BH3-only proteins Bad and Bim and the Bcl2-like protein Mcl-1, downstream of STAT3, STAT5 and ERK1/2 pathways [50–53]. Several studies have sug- gested that JAK2V617F may not only impair the normal cellular re- sponses to DNA damage but may also directly contribute to increased DNA damage and to levels of intracellular reactive oxygen species [39, 54–56].
Whilst activation of pathways such as STAT5 are considered canon- ical effects of JAK2V617F, several other “non-canonical” effects have been reported including modification of chromatin. JAK2 can enter the nucleus and phosphorylate histone H3 at Tyr41 (Y41), which prevents binding of the heterochromatin protein HP1α [57]. This allows JAK2 to have direct effects on the regulation of gene expression, a process which can occur in combination with, or independently of, STAT pro- teins [58]. JAK2V617F may also modulate chromatin by
Please cite this article as: Grinfeld J, Godfrey AL, After 10years of JA polycythaemia vera, Blood Rev (2016), http://dx.doi.org/10.1016/j.blre.20
phosphorylating the arginine methyltransferase PRMT5 and impairing its histone methylation activity, which may in turn promote growth and erythroid differentiation of haematopoietic cells [59].
There is evidence that JAK2V617Fmay not only act through intrinsic cellular pathways, but may have important effects through the micro- environment. For example, the mutation has been reported to increase levels of the cytokine TNFα, which is increased in the serumofMPN pa- tients and may paradoxically stimulate growth of JAK2-mutant cells [60]. An important role for the microenvironment was also suggested by two reports that macrophage depletion abrogates erythrocytosis in mouse models of JAK2V617F-driven polycythaemia [61,62].
2.2. Cellular effects of JAK2 mutations
Early studies of the cellular effects of JAK2V617F on haematopoiesis analysed cell populations at different stages of differentiation from pri- mary patient samples, with or without a period of ex vivo culture. These studies were mostly in keeping with an expansion advantage for JAK2V617F-mutant cells at later stages of myeloerythroid differentia- tion, particularly at low erythropoietin levels and in the presence of ho- mozygosity for the V617F mutation [63–67]. The effects of JAK2 mutations on haematopoietic cells have been elucidated further using mouse models. Importantly, early work demonstrated that expression of JAK2V617F in retroviral bone marrow transplantation models could cause a myeloproliferative phenotype [2,5], confirming the causal role of the mutation in human MPNs. These models had high, dysregulated levels of JAK2V617F expression, whilst knock-in models allow observa- tion of the phenotypes obtained when the gene is expressed at physio- logical levels, under the normal regulatory elements of the mouse Jak2 gene. These knock-in models have supported the conclusions from pa- tient studies, showing that JAK2V617F is associatedwith increased num- bers of lineage-restricted progenitors, including those of the megakaryocyte/erythroid and granulocyte/macrophage lineages [38– 41,68].
The effects of JAK2V617F on haematopoietic stem cells (HSCs) have beenmore controversial. Early studies used xenotransplantation exper- iments, inwhich CD34+ cells fromMPNpatientswere transplanted into NOD/SCIDmice.Whilst these cells could engraft in themice, the propor- tion of JAK2V617F-positive cells was frequently reduced in the reconstituted marrow compared to the original cells, arguing against an advantage for JAK2V617F-positive over wild-type HSCs [69,70]. In two knock-in models of murine Jak2V617F, an increase in the stem cell-enriched Lin−Sca-1+Kit+ (LSK) cell compartment was reported [38,68], whilst in another LSK numbers were unchanged [40]. With re- spect to HSC function, competitive transplantation experiments in one of these models suggested a selective advantage for JAK2V617F-mutant cells in primary transplantation, associatedwith an increase in HSC pro- liferation and decrease in apoptosis, but this advantage was not main- tained in secondary transplants [68]. Similarly, competitive transplantation experiments in a second model suggested normal early function but a late advantage in primary recipients [40,71], whilst subsequent data have suggested a lack of advantage in secondary recip- ients [72]. In the one knock-in model with a human JAK2V617F con- struct there was a quantitative and qualitative HSC defect, with reduced LSK cell numbers and evidence of increased DNA damage, re- duced cell cycling, and impaired function in competitive transplantation experiments, which was most marked in cells homozygous for JAK2V617F [39,73]. Further studies of this latter model suggested that the mutation may promote differentiation of HSCs at the expense of self-renewal [74].
Interestingly the concept of a JAK2V617F-induced HSC defect is con- sistent with other evidence from the human MPNs. For example, allele burdens for JAK2V617F are low in many patients [64] and may remain stable over years [75], and it was reported that JAK2V617F-positive cells did not expand in the recipient of an allogeneic stem cell transplant from a JAK2V617F-positive donor [76]. Conversely it was reported that a
K2V617F: Disease biology and current management strategies in 16.11.001
JAK2V617I mutation, found in a family with hereditary thrombocytosis in the absence of additional MPN-associated molecular abnormalities, was associated with increased numbers of phenotypic HSCs and an ad- vantage in xenotransplantation assays [77]. However this mutation also showed qualitative signalling differences to V617F andmay be associat- edwith a different HSC phenotype, andmoreover the constitutional na- ture of the V617I variant would obviate the requirement for a competitive advantage in human disease. The concept that oncogenes may not impart a stem cell advantage is also consistent with data from other malignancies. For example, the BCR-ABL fusion gene has been associated with reduced HSC self-renewal in vitro [78], in NOD/ SCID transplantation experiments [79] and in secondary transplants froma transgenicmousemodel [80], and oncogene-induced senescence is a recognised barrier to malignant progression in certain haematolog- ical and non-haematological neoplasms [81,82].
In summary, numerous studies have contributed to a body of knowl- edge regarding the biochemical and cellular consequences of JAK2 mu- tations. However, it has remained less clear how the various signalling perturbations contribute to the specific clinical features of MPNs, and which are the other factors that distinguish between the different JAK2-mutant MPN phenotypes. For example, STAT5 activation appears to play an important role in erythrocytosis, the defining feature of PV, since knockdown of Stat5 in JAK2V617F-driven models of erythrocytosis abrogates this aspect of the phenotype [43,44]. By con- trast Stat5 deletion did not prevent development of myelofibrosis in a Jak2V617F retroviral bone marrow transplantation model [44]. More- over studies of haematopoietic cells from MPN patients found that pat- terns of pSTAT3, pSTAT5, pERK1/2 and pAkt correlated better withMPN subtype rather than with the presence or burden of the JAK2V617Fmu- tation [83], highlighting a potential role for additional molecular mech- anisms in these signalling changes.
3. Factors determining phenotype in PV vs other JAK2-mutated MPNs
In 2005, analysis of ET patients in the PT1 trial demonstrated that in comparison to JAK2V617F-negative patients, JAK2V617F-positive pa- tients showed higher haemoglobin levels, neutrophil counts, more bone marrow erythropoiesis and granulopoiesis, lower platelet counts, mean corpuscular volume (MCV), serum erythropoietin and ferritin, and more frequent PV transformation [84]. Subsequent studies con- firmed these associations [14,85–88], suggesting that JAK2V617F-posi- tive ET has some characteristics of a mild form of PV, as well as a similar incidence of thrombosis [89]. These clinical similarities between PV and JAK2V617F-positive ET aremirrored by certain biological similar- ities (e.g. a low frequency of driver mutations additional to JAK2V617F [90]) and are in keeping with a model in which the two disorders form a phenotypic continuum [84]. However there are also important differences between PV and JAK2V617F-positive ET, which have prompted speculation and investigation of the factors that push an individual's phenotype towards one or other disorder. These factors in- clude the presence and size of clones homozygous for the JAK2V617F mutation; the presence of additional mutations and their subclonal hi- erarchy in relation to JAK2V617F; germline genetic factors; differences in gene expression; and other constitutional factors including age and gender. We discuss these factors in more detail below.
3.1. The role of JAK2V617F homozygosity in PV phenotype
A “homozygous” JAK2V617F sequence (N50%mutant) in granulocyte DNA was originally identified in 25–30% of those with PV, 9–20% with PMF, and 0–3% with ET [2–5]. Clinical studies next investigated how JAK2V617F allele burden, a measure of “gene dosage” for the mutation that ismost oftenmeasured in granulocyte DNA, correlatedwith certain clinical features. Initial studies divided PV patients into “heterozygous” (b50% mutant allele) and “homozygous” (N50% mutant allele) groups,
Please cite this article as: Grinfeld J, Godfrey AL, After 10years of JA polycythaemia vera, Blood Rev (2016), http://dx.doi.org/10.1016/j.blre.20
whilst subsequent studies analysed mutant allele burden as a continu- ous variable. Together these studies indicated that within PV, higher JAK2V617F allele burdens were associated with higher haemoglobin levels, higher white cell counts and lower platelet counts, together with other features suggesting a “more extreme” PV phenotype (lowerMCV, lower serum ferritin and erythropoietin, more splenomeg- aly, more pruritus and more need for cytoreductive therapy) [13,63,64, 91–95]. These data suggested that a higher JAK2V617F allele burden might promote a more “PV-like”, rather than “ET-like”, phenotype. Within ET, higher JAK2V617F allele burdens were associated with some of the same features as in PV (higher white cell counts and more splenomegaly), but in contrast to PV there was an association with higher platelet counts [63,64,88]. This may reflect a limitation of mea- suring allele burden in bulk granulocyte DNA: the assays did not dem- onstrate the contribution of JAK2V617F-heterozygous and homozygous cells to the overall mutant allele burden.
Whilst these early patient studies showed a correlation between JAK2V617F allele burden and PV phenotype, mouse models have since been used to establish the causal nature of this relationship. One trans- genicmousemodelwas consistentwith the concept that a phenotype of ET or PV could result from lower and higher levels of JAK2V617F expres- sion levels, respectively, suggesting a role for gene dosage in determin- ing MPN phenotype [96]. However the same switch from ET to PV was not seen with increasing gene dosage in other transgenic models [97, 98], and all transgenicmodels are complicated by variable transgene ac- tivation between cells.
More recently the development of knock-in mouse models has allowed study of JAK2V617F when expressed at physiological levels under its normal regulatory elements [38–41,68]. Four knock-inmodels with a heterozygous murine mutant Jak2 allele developed a phenotype consistentwith PVwith erythrocytosis, leucocytosis, splenomegaly, var- iable thrombocytosis and increased megakaryocyte-erythroid progeni- tors [38,40,41,68]. One of these studies reported generation of a homozygous-mutant mouse, which showed more pronounced leucocytosis, thrombocytosis and splenomegaly but reduced or un- changed haemoglobin levels compared to heterozygousmice [38]; sim- ilar phenotypes were observed in a hemizygous mouse with deletion of the wild-type Jak2 allele [99]. By contrast in a fifth model with a human JAK2V617F allele, mice heterozygous for themutation showed amoder- ate thrombocytosis with minimal erythrocytosis, normal spleen size and plasma erythropoietin levels, whilst homozygosity was associated with marked erythrocytosis and reduced platelet counts, significant splenomegaly and suppressed plasma erythropoietin levels. These fea- tures closely parallel those of human ET and PV, respectively, and pro- vided direct genetic evidence that homozygosity for human JAK2V617F can be associatedwith a switch from ET-like disease to a PV-like pheno- type [73]. The reasons behind the phenotypic variation between the dif- ferent knock-in models remain unclear however, and may reflect differences in technical aspects of the targeting strategies or inherent differences in the mutant human and mouse proteins.
Further analysis of the precise role of JAK2V617F homozygosity in human PV has utilised genotyping of individual erythroid colonies grown fromMPN patient samples. This approach circumvents the limi- tations of allele burden studies in bulk granulocyte DNA, by investigat- ing the presence and frequency of homozygosity at the level of single precursors. An early study of BFU-E colonies grown in high erythropoi- etin conditions found that while virtually all PV and ET patients pro- duced both wild-type and mutant colonies, JAK2V617F-homozygous colonies were detectable in almost all of the PV patients, but in none of the cohort with ET [100]. Subsequent studies largely supported these observations [63,64], but raised the possibility that JAK2V617F-ho- mozygous erythroid colonies could be found in a small number of pa- tients with ET [63,64,101], whilst PV patients lacking JAK2V617F- homozygous colonies were also described [63,64]. A large study of PV and ET patients then analysed the genotypes of erythroid colonies grown in low erythropoietin conditions, aiming to select for even the
K2V617F: Disease biology and current management strategies in 16.11.001
smallest homozygous-mutant clones. This study confirmed that JAK2V617F-homozygous precursors could be identified from approxi- mately 80% of PV patients [102]. A higher proportion…
Contents lists available at ScienceDirect
Blood Reviews
j ourna l homepage: www.e lsev ie r .com/ locate /b l re
REVIEW
After 10 years of JAK2V617F: Disease biology and current management strategies in polycythaemia vera
Jacob Grinfeld, Anna L Godfrey Department of Haematology, Cambridge University Hospitals NHS Foundation Trust, Hills Rd, Cambridge CB2 0QQ, UK
Corresponding author. E-mail addresses: [email protected] (J. Grinfeld), anna
(A.L. Godfrey).
http://dx.doi.org/10.1016/j.blre.2016.11.001 0268-960X/© 2016 Published by Elsevier Ltd.
Please cite this article as: Grinfeld J, God polycythaemia vera, Blood Rev (2016), http:
a b s t r a c t
a r t i c l e i n f o
Available online xxxx
The JAK2V617Fmutation accounts for the vastmajority of patients with polycythaemia vera (PV) and around half of those with other Philadelphia-negative myeloproliferative neoplasms. Since its discovery in 2005, numerous insights have been gained into the pathways bywhich JAK2V617F causesmyeloproliferation, including activation of JAK-STAT signalling but also through other canonical and non-canonical pathways. A variety of mechanisms explain how this one mutation can be associated with distinct clinical disorders, demonstrating how constitu- tional and acquired factorsmay interact in the presence of a singlemutation to determine disease phenotype. Im- portant biological questions remainunanswered in PV, in particular how JAK2V617F affects stem cell function and what mechanisms drive myelofibrotic and leukaemic transformation. Whilst current management is largely centred on prevention of cardiovascular events, future therapiesmust aim to target the JAK2-mutant clone, to re- verse the underlying marrow pathology and to address the risk of transformation events.
© 2016 Published by Elsevier Ltd.
Keywords: Polycythaemia vera Erythrocytosis Myeloproliferative JAK2
1. Introduction
Polycythaemia vera (PV), one of the Philadelphia-negative myelo- proliferative neoplasms (MPNs), is a clonal haematopoietic stem cell disorder characterised by an increased red cell mass, associated with proliferation of erythroid, granulocytic and megakaryocytic elements of the bonemarrow [1]. In 2005 the identification of the JAK2V617Fmu- tation elucidated a molecular basis for the disorder in over 95% of pa- tients [2–5], followed two years later by the identification of JAK2 exon 12 mutations in most remaining patients [6,7]. The vast majority of patients with PV therefore carry mutations in a single gene, and a wealth of studies has since illuminated themanymolecular and cellular consequences of these mutations.
Whilst JAK2 exon 12mutations are specific to PV, the JAK2V617Fmu- tation is also found in other myeloid disorders: 50 to 60% of those with essential thrombocythaemia (ET) or primary myelofibrosis (PMF) [2– 5], about half of patients with the MDS/MPN entity “refractory anaemia with ring sideroblasts and marked thrombocytosis” (RARS-T) [8], and lower frequencies in AML, other myeloproliferative and myelodysplastic disorders [9,10]. ET and PMF in particular share impor- tant clinical features with PV, including the presence of neutrophilia and/or thrombocytosis in some patients, hypercellularity of the bone marrow, frequent splenomegaly, and risks of thrombosis and
[email protected]
frey AL, After 10years of JA //dx.doi.org/10.1016/j.blre.20
haemorrhage [1]. In aminority of patients, PV, ET and PMF can all trans- form into acute myeloid leukaemia (AML) [11–13], those with ET may develop transformation to PV [14], and both ET and PV may transform into secondary myelofibrosis [11,13,14]. The identification of JAK2V617F offered some basis for the clinical similarities between these MPNs, but the molecular mechanisms behind the phenotypic dif- ferences between the JAK2V617F-positive disorders has remained the subject of intensive investigation.
The identification of JAK2mutations in PV led to the introduction of a rapid and non-invasive diagnostic test, with mutation testing now widely available and incorporated into national and international clini- cal guidelines [1,15–17]. Moreover the development of JAK2 inhibitors has led to a much-needed novel therapeutic avenue for patients with myelofibrosis [18,19], and most recently these drugs have also been shown to benefit certain patients with PV [20]. However, clinical man- agement of PV remains predominantly centred on the reduction of vas- cular complications and symptoms. In spite of significant advances that have beenmade in understanding the disease biology of PV, agents that can target the underlying neoplastic clone have remained elusive and the risk of disease transformation, with its almost inevitably poor out- come, remains an important and unmet clinical need.
This reviewwill first examine current understanding of themolecu- lar basis of PV, both in terms of the pathological effects of JAK2 muta- tions and the factors that may contribute to phenotypic differences between PV and other JAK2-mutated MPNs. Diagnostic criteria and their controversies will be considered. Key prognostic factors in PV will then be examined, followed by a discussion of currentmanagement strategies and unresolved clinical challenges.
K2V617F: Disease biology and current management strategies in 16.11.001
2. The molecular and cellular effects of JAK2 mutations
JAK2 is one of a family of cytoplasmic tyrosine kinases that mediates signal transduction from cell surface cytokine receptors, including the erythropoietin and thrombopoietin receptors [21]. Binding of ligand to receptor leads to JAK autophosphorylation, receptor transphosphorylation and creation of binding sites for signalling mole- cules such as STAT proteins. STATs are phosphorylated, translocate to the nucleus and affect target gene transcription. For example, binding of erythropoietin to its receptor (EpoR) causes phosphorylation of JAK2 and STAT5 [22], together with activation of other effectors includ- ing MAP kinase and phosphoinositide 3-kinase (PI3K)/Akt pathways [23].
2.1. Biochemical effects of JAK2 mutations
In early studies, the V617Fmutationwas found to have activating ef- fects on JAK2 signalling activity, cytokine sensitivity and cytokine-de- pendent survival in cell lines [2–5]. This activation of JAK2 is thought to reflect the location of the mutation within the JH2 “pseudokinase” domain of the protein, which normally has auto-inhibitory effects on the catalytically active JH1 kinase domain [24–27]. Mechanismsmay in- clude the interference of the mutation with direct phosphorylation of JH1 by JH2 [28], and cooperation of V617F with other residues in JH2 to enhance JH1 kinase activity [29,30]. Interestingly, recent work on the JAK1 JH2 crystal structure has also suggested that JAK2V617F activa- tion requires a specific conformational change in the SH2-JH2 linker do- main [31], which is the area targeted by exon 12 mutations. Exon 12 mutations appear have similar activating effects to V617F in cell lines, including cytokine independence and constitutive activation of JAK2 and its downstream mediators in the absence of erythropoietin [6].
Several signalling changes appear to mediate the cellular effects of JAK2V617F. Increased phosphorylation of STAT5 has been demonstrated in numerous experimental systems, including cultured erythroid cells and bone marrow trephine biopsies from patients with MPNs, and in JAK2V617F knock-in mouse models [2,4,5,32–41]. The particular impor- tance of STAT5 activation in PV is illustrated by the observation that ex- pression of constitutively active STAT5 in erythroid progenitors can drive formation of the same erythropoietin-independent endogenous erythroid colonies that are typical of PV [42]. Moreover it has been shown more recently that Stat5 deletion in either a Jak2V617F knock- inmouse [43] or retroviral bonemarrow transplantationmodel [44] ab- rogates the marked erythrocytosis that is seen in the presence of Stat5.
Other signalling processes that appear to be activated by JAK2V617F include the PI3K/Akt and MAPK/ERK pathways, which have been stud- ied in many experimental systems [2,5,33,34,36,38,45]. There is evi- dence that the mutation has direct effects on apoptosis, both through the extrinsic pathway that acts through cell surface death receptors such as Fas [46], and through the intrinsic pathway, with increased levels of the anti-apoptotic protein Bcl-xL and impairment of its normal role in the DNA damage response [47–49]. Other mediators implicated in apoptotic dysregulation include ID1, Pim1/2 kinases, the BH3-only proteins Bad and Bim and the Bcl2-like protein Mcl-1, downstream of STAT3, STAT5 and ERK1/2 pathways [50–53]. Several studies have sug- gested that JAK2V617F may not only impair the normal cellular re- sponses to DNA damage but may also directly contribute to increased DNA damage and to levels of intracellular reactive oxygen species [39, 54–56].
Whilst activation of pathways such as STAT5 are considered canon- ical effects of JAK2V617F, several other “non-canonical” effects have been reported including modification of chromatin. JAK2 can enter the nucleus and phosphorylate histone H3 at Tyr41 (Y41), which prevents binding of the heterochromatin protein HP1α [57]. This allows JAK2 to have direct effects on the regulation of gene expression, a process which can occur in combination with, or independently of, STAT pro- teins [58]. JAK2V617F may also modulate chromatin by
Please cite this article as: Grinfeld J, Godfrey AL, After 10years of JA polycythaemia vera, Blood Rev (2016), http://dx.doi.org/10.1016/j.blre.20
phosphorylating the arginine methyltransferase PRMT5 and impairing its histone methylation activity, which may in turn promote growth and erythroid differentiation of haematopoietic cells [59].
There is evidence that JAK2V617Fmay not only act through intrinsic cellular pathways, but may have important effects through the micro- environment. For example, the mutation has been reported to increase levels of the cytokine TNFα, which is increased in the serumofMPN pa- tients and may paradoxically stimulate growth of JAK2-mutant cells [60]. An important role for the microenvironment was also suggested by two reports that macrophage depletion abrogates erythrocytosis in mouse models of JAK2V617F-driven polycythaemia [61,62].
2.2. Cellular effects of JAK2 mutations
Early studies of the cellular effects of JAK2V617F on haematopoiesis analysed cell populations at different stages of differentiation from pri- mary patient samples, with or without a period of ex vivo culture. These studies were mostly in keeping with an expansion advantage for JAK2V617F-mutant cells at later stages of myeloerythroid differentia- tion, particularly at low erythropoietin levels and in the presence of ho- mozygosity for the V617F mutation [63–67]. The effects of JAK2 mutations on haematopoietic cells have been elucidated further using mouse models. Importantly, early work demonstrated that expression of JAK2V617F in retroviral bone marrow transplantation models could cause a myeloproliferative phenotype [2,5], confirming the causal role of the mutation in human MPNs. These models had high, dysregulated levels of JAK2V617F expression, whilst knock-in models allow observa- tion of the phenotypes obtained when the gene is expressed at physio- logical levels, under the normal regulatory elements of the mouse Jak2 gene. These knock-in models have supported the conclusions from pa- tient studies, showing that JAK2V617F is associatedwith increased num- bers of lineage-restricted progenitors, including those of the megakaryocyte/erythroid and granulocyte/macrophage lineages [38– 41,68].
The effects of JAK2V617F on haematopoietic stem cells (HSCs) have beenmore controversial. Early studies used xenotransplantation exper- iments, inwhich CD34+ cells fromMPNpatientswere transplanted into NOD/SCIDmice.Whilst these cells could engraft in themice, the propor- tion of JAK2V617F-positive cells was frequently reduced in the reconstituted marrow compared to the original cells, arguing against an advantage for JAK2V617F-positive over wild-type HSCs [69,70]. In two knock-in models of murine Jak2V617F, an increase in the stem cell-enriched Lin−Sca-1+Kit+ (LSK) cell compartment was reported [38,68], whilst in another LSK numbers were unchanged [40]. With re- spect to HSC function, competitive transplantation experiments in one of these models suggested a selective advantage for JAK2V617F-mutant cells in primary transplantation, associatedwith an increase in HSC pro- liferation and decrease in apoptosis, but this advantage was not main- tained in secondary transplants [68]. Similarly, competitive transplantation experiments in a second model suggested normal early function but a late advantage in primary recipients [40,71], whilst subsequent data have suggested a lack of advantage in secondary recip- ients [72]. In the one knock-in model with a human JAK2V617F con- struct there was a quantitative and qualitative HSC defect, with reduced LSK cell numbers and evidence of increased DNA damage, re- duced cell cycling, and impaired function in competitive transplantation experiments, which was most marked in cells homozygous for JAK2V617F [39,73]. Further studies of this latter model suggested that the mutation may promote differentiation of HSCs at the expense of self-renewal [74].
Interestingly the concept of a JAK2V617F-induced HSC defect is con- sistent with other evidence from the human MPNs. For example, allele burdens for JAK2V617F are low in many patients [64] and may remain stable over years [75], and it was reported that JAK2V617F-positive cells did not expand in the recipient of an allogeneic stem cell transplant from a JAK2V617F-positive donor [76]. Conversely it was reported that a
K2V617F: Disease biology and current management strategies in 16.11.001
JAK2V617I mutation, found in a family with hereditary thrombocytosis in the absence of additional MPN-associated molecular abnormalities, was associated with increased numbers of phenotypic HSCs and an ad- vantage in xenotransplantation assays [77]. However this mutation also showed qualitative signalling differences to V617F andmay be associat- edwith a different HSC phenotype, andmoreover the constitutional na- ture of the V617I variant would obviate the requirement for a competitive advantage in human disease. The concept that oncogenes may not impart a stem cell advantage is also consistent with data from other malignancies. For example, the BCR-ABL fusion gene has been associated with reduced HSC self-renewal in vitro [78], in NOD/ SCID transplantation experiments [79] and in secondary transplants froma transgenicmousemodel [80], and oncogene-induced senescence is a recognised barrier to malignant progression in certain haematolog- ical and non-haematological neoplasms [81,82].
In summary, numerous studies have contributed to a body of knowl- edge regarding the biochemical and cellular consequences of JAK2 mu- tations. However, it has remained less clear how the various signalling perturbations contribute to the specific clinical features of MPNs, and which are the other factors that distinguish between the different JAK2-mutant MPN phenotypes. For example, STAT5 activation appears to play an important role in erythrocytosis, the defining feature of PV, since knockdown of Stat5 in JAK2V617F-driven models of erythrocytosis abrogates this aspect of the phenotype [43,44]. By con- trast Stat5 deletion did not prevent development of myelofibrosis in a Jak2V617F retroviral bone marrow transplantation model [44]. More- over studies of haematopoietic cells from MPN patients found that pat- terns of pSTAT3, pSTAT5, pERK1/2 and pAkt correlated better withMPN subtype rather than with the presence or burden of the JAK2V617Fmu- tation [83], highlighting a potential role for additional molecular mech- anisms in these signalling changes.
3. Factors determining phenotype in PV vs other JAK2-mutated MPNs
In 2005, analysis of ET patients in the PT1 trial demonstrated that in comparison to JAK2V617F-negative patients, JAK2V617F-positive pa- tients showed higher haemoglobin levels, neutrophil counts, more bone marrow erythropoiesis and granulopoiesis, lower platelet counts, mean corpuscular volume (MCV), serum erythropoietin and ferritin, and more frequent PV transformation [84]. Subsequent studies con- firmed these associations [14,85–88], suggesting that JAK2V617F-posi- tive ET has some characteristics of a mild form of PV, as well as a similar incidence of thrombosis [89]. These clinical similarities between PV and JAK2V617F-positive ET aremirrored by certain biological similar- ities (e.g. a low frequency of driver mutations additional to JAK2V617F [90]) and are in keeping with a model in which the two disorders form a phenotypic continuum [84]. However there are also important differences between PV and JAK2V617F-positive ET, which have prompted speculation and investigation of the factors that push an individual's phenotype towards one or other disorder. These factors in- clude the presence and size of clones homozygous for the JAK2V617F mutation; the presence of additional mutations and their subclonal hi- erarchy in relation to JAK2V617F; germline genetic factors; differences in gene expression; and other constitutional factors including age and gender. We discuss these factors in more detail below.
3.1. The role of JAK2V617F homozygosity in PV phenotype
A “homozygous” JAK2V617F sequence (N50%mutant) in granulocyte DNA was originally identified in 25–30% of those with PV, 9–20% with PMF, and 0–3% with ET [2–5]. Clinical studies next investigated how JAK2V617F allele burden, a measure of “gene dosage” for the mutation that ismost oftenmeasured in granulocyte DNA, correlatedwith certain clinical features. Initial studies divided PV patients into “heterozygous” (b50% mutant allele) and “homozygous” (N50% mutant allele) groups,
Please cite this article as: Grinfeld J, Godfrey AL, After 10years of JA polycythaemia vera, Blood Rev (2016), http://dx.doi.org/10.1016/j.blre.20
whilst subsequent studies analysed mutant allele burden as a continu- ous variable. Together these studies indicated that within PV, higher JAK2V617F allele burdens were associated with higher haemoglobin levels, higher white cell counts and lower platelet counts, together with other features suggesting a “more extreme” PV phenotype (lowerMCV, lower serum ferritin and erythropoietin, more splenomeg- aly, more pruritus and more need for cytoreductive therapy) [13,63,64, 91–95]. These data suggested that a higher JAK2V617F allele burden might promote a more “PV-like”, rather than “ET-like”, phenotype. Within ET, higher JAK2V617F allele burdens were associated with some of the same features as in PV (higher white cell counts and more splenomegaly), but in contrast to PV there was an association with higher platelet counts [63,64,88]. This may reflect a limitation of mea- suring allele burden in bulk granulocyte DNA: the assays did not dem- onstrate the contribution of JAK2V617F-heterozygous and homozygous cells to the overall mutant allele burden.
Whilst these early patient studies showed a correlation between JAK2V617F allele burden and PV phenotype, mouse models have since been used to establish the causal nature of this relationship. One trans- genicmousemodelwas consistentwith the concept that a phenotype of ET or PV could result from lower and higher levels of JAK2V617F expres- sion levels, respectively, suggesting a role for gene dosage in determin- ing MPN phenotype [96]. However the same switch from ET to PV was not seen with increasing gene dosage in other transgenic models [97, 98], and all transgenicmodels are complicated by variable transgene ac- tivation between cells.
More recently the development of knock-in mouse models has allowed study of JAK2V617F when expressed at physiological levels under its normal regulatory elements [38–41,68]. Four knock-inmodels with a heterozygous murine mutant Jak2 allele developed a phenotype consistentwith PVwith erythrocytosis, leucocytosis, splenomegaly, var- iable thrombocytosis and increased megakaryocyte-erythroid progeni- tors [38,40,41,68]. One of these studies reported generation of a homozygous-mutant mouse, which showed more pronounced leucocytosis, thrombocytosis and splenomegaly but reduced or un- changed haemoglobin levels compared to heterozygousmice [38]; sim- ilar phenotypes were observed in a hemizygous mouse with deletion of the wild-type Jak2 allele [99]. By contrast in a fifth model with a human JAK2V617F allele, mice heterozygous for themutation showed amoder- ate thrombocytosis with minimal erythrocytosis, normal spleen size and plasma erythropoietin levels, whilst homozygosity was associated with marked erythrocytosis and reduced platelet counts, significant splenomegaly and suppressed plasma erythropoietin levels. These fea- tures closely parallel those of human ET and PV, respectively, and pro- vided direct genetic evidence that homozygosity for human JAK2V617F can be associatedwith a switch from ET-like disease to a PV-like pheno- type [73]. The reasons behind the phenotypic variation between the dif- ferent knock-in models remain unclear however, and may reflect differences in technical aspects of the targeting strategies or inherent differences in the mutant human and mouse proteins.
Further analysis of the precise role of JAK2V617F homozygosity in human PV has utilised genotyping of individual erythroid colonies grown fromMPN patient samples. This approach circumvents the limi- tations of allele burden studies in bulk granulocyte DNA, by investigat- ing the presence and frequency of homozygosity at the level of single precursors. An early study of BFU-E colonies grown in high erythropoi- etin conditions found that while virtually all PV and ET patients pro- duced both wild-type and mutant colonies, JAK2V617F-homozygous colonies were detectable in almost all of the PV patients, but in none of the cohort with ET [100]. Subsequent studies largely supported these observations [63,64], but raised the possibility that JAK2V617F-ho- mozygous erythroid colonies could be found in a small number of pa- tients with ET [63,64,101], whilst PV patients lacking JAK2V617F- homozygous colonies were also described [63,64]. A large study of PV and ET patients then analysed the genotypes of erythroid colonies grown in low erythropoietin conditions, aiming to select for even the
K2V617F: Disease biology and current management strategies in 16.11.001
smallest homozygous-mutant clones. This study confirmed that JAK2V617F-homozygous precursors could be identified from approxi- mately 80% of PV patients [102]. A higher proportion…
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