JAK3, STAT1 and immunodeficiency
Mutations of the cytokine receptor common γ-chain account for a high proportion of cases of SCID (reviewed in Cavazzana-Calvo et al, 2005). In the mid-1990s, mutations in JAK3 that were associated with autosomal recessive SCID were discovered: affected individuals have absent T and NK cells with relatively normal numbers of poorly functional B cells (Macchi et al, 1995) and this affects around 10% of SCID cases (Cavazzana-Calvo et al, 2005). Most patients are compound heterozygotes, having inherited a distinct mutation from each parent and some individuals are homozygous for their mutations as a result of parental consanguinity (O'Shea et al, 2004a). The majority of described mutations have a major effect on protein expression and/or stability, or less commonly, result in mutated and non-functional Jak3 protein. Missense mutations and small in-frame deletions may permit protein expression but interfere with kinase activity, binding to the cytokine receptor γ-chain or intracellular trafficking (O'Shea et al, 2004a). As there are no mutational ‘hot spots’, diagnosis usually rests on sequencing the entire gene. Treatment of JAK3-deficient individuals has relied on stem cell transplantation but gene therapy approaches, akin to the treatment of γ-chain-deficient SCID, have been proposed (Cavazzana-Calvo et al, 2005).
Mutations in STAT pathways that cause immune deficiency states have also been described. Two kindred with the same heterozygous mutation in STAT1 (L706S) causing partial dominant STAT-1 deficiency (Dupuis et al, 2001) results in reduced responses to γ-interferon with a clinical picture of susceptibility to mycobacterial infections. Complete, autosomal recessive, STAT1 deficiency has also been reported (Dupuis et al, 2003) – this abolishes cellular responses to interferons and led to premature death from viral infection (Picard & Casanova, 2004).
JAK3 as a drug target
The highly specific immunodeficient phenotype seen in JAK3 deficiency has led to the proposition that, drugs that could selectively inhibit JAK3 may prove to be valuable in the management of organ transplantation and auto-immune disease. These could complement current immunosuppressants by having a novel mode of action. Due to the restricted tissue distribution of JAK3, an effective inhibitor has the potential to have much less toxicity compared with many immunosuppressants, such as ciclosporin (inhibitor of calcineurin), rapamycin (inhibitor of mTOR activation) and glucocorticoids (O'Shea et al, 2004b). The most advanced of the JAK3 inhibitors in drug development is known as CP-690,550 which has a 50% inhibitory concentration against JAK3 of 1 nmol/l (Changelian et al, 2003). Importantly, this compares with 20 nmol/l for JAK2 and 100 nmol/l for JAK1. CP-690,550 is orally bioavailable and has been shown to be effective in a number of allogeneic solid-organ transplant models and has entered clinical trials in renal transplant patients and rheumatoid arthritis (Changelian et al, 2003; O'Shea et al, 2005). The preclinical studies have not shown significant effects on haemoglobin (Hb) levels, platelet or neutrophil counts, suggesting that in vivo anti-JAK2 effects are minimal but data from the human trials is awaited.
JAKs in oncogenesis
Janus kinase signalling is activated in haematological malignancies by a number of mechanisms including the downregulation of negative regulators of JAK–STAT pathways, amplification of the JAK2 locus and the involvement of JAK2 in chromosomal translocations and, most recently, by the identification of an activating point mutation in JAK2. Human T lymphotropic virus type I (HTLV-1) infection, the pathogenic agent for the development of adult T-cell leukaemia/lymphoma, has been shown to result in the activation of JAK and STAT signalling pathways (Migone et al, 1995; Takemoto et al, 1997). Both JAK1 and JAK3 have been shown to be activated and this may be mediated, at least partly, by the HTLV-1 tax protein transactivating the promoters of a number of cytokines and their receptors, resulting in autocrine or paracrine signalling (Grassmann et al, 2005).
Downregulation of negative regulators. Hypermethylation of normally unmethylated CpG islands in gene promoter elements can lead to downregulation of transcription and hence protein levels, and has been widely described in cancer biology with respect to tumour suppressor genes (Baylin & Ohm, 2006). SHP1 hypermethylation has been described to occur at high frequencies in a number of haemopoietic tumours including myeloma, anaplastic lymphoma, mantle cell and follicular lymphomas, and acute leukaemias (Oka et al, 2002; Chim et al, 2004a,b; Johan et al, 2005a). In myeloma cells, treatment with the demethylating agent 5-azacytidine led to re-expression of SHP1 and a downregulation in levels of phosphorylated STAT3 (Chim et al, 2004a). It has been suggested that hypermethylation of SHP1 may be mediated by STAT3, resulting in a signalling amplification loop (Zhang et al, 2005). Hypermethylation of the SOCS3 promoter has been reported in a variety of epithelial tumours, including lung and hepatocellular carcinomas, but not so far in haematological malignancies (He et al, 2003; Niwa et al, 2005). Hypermethylation of CpG islands in the SOCS1 gene has also been described (Yoshikawa et al, 2001) and lie within what is now thought to be exon 2 of the gene and not in a promoter region (Melzner & Moller, 2003). This correlates with reduced expression of SOCS1 mRNA, which increases after treatment with demethylating agents and has been described in acute leukaemias, myelodysplastic syndrome (MDS) and myeloma amongst others (Galm et al, 2003; Watanabe et al, 2004; Brakensiek et al, 2005).
Recently, mutations in SOCS1 have been described in lymphomas. In primary mediastinal B-cell lymphomas (PMBL), there is frequent gain of genetic material at 9p24 which includes the JAK2 locus (Bentz et al, 2001) and gene expression profiling has shown increased JAK2 mRNA levels (Savage et al, 2003). Melzner et al (2005) found that JAK2 protein was not overexpressed in the MedB1 PMBL line, which has trisomy 9, but that the protein half-life was increased due to delayed degradation. They showed that these cells harbour biallelic SOCS1 mutations that abolish SOCS box activity and also found deletion mutations in nine of 20 primary PMBL tumours which result in truncated or aberrant transcripts (Melzner et al, 2006). This group has also described similar deletions in Hodgkin lymphoma cell lines and in micro-dissected Hodgkin cell samples from eight of 19 primary cases (Weniger et al, 2006). In these cases, there was a high correlation with the presence of phosphorylated STAT5 by immunohistochemistry, suggesting JAK activation in the tumour.
In the course of evaluating the mechanisms for constitutive STAT3 activation in anaplastic lymphomas, Zhang et al (2002) found that levels of PIAS3, which is an inhibitor of STAT3, were absent in the majority of anaplastic large cell lymphoma (ALCL) cell lines. The mechanism for this was not identified. Ueda et al (2003) showed that the mRNA expression of PIASy was significantly reduced in AC133+ samples from patients with advanced MDS [refractory anaemia (RA) with excess blasts, MDS-acute myeloid leukaemia (AML)] compared with samples from patients with RA.
Fusion proteins. Three distinct fusion proteins involving JAK2 have been described –TEL/Ets translocation variant (ETV)6–JAK2, BCR–JAK2 and pericentriolar material (PCM1)–JAK2. These are relatively rare and involve different lineages and clinical phenotypes.
The TEL–JAK2, as a consequence of t(9;12)(p24;p13), has been identified in single cases of pre-B acute lymphoblastic leukaemia (ALL), atypical CML in transformation and T-ALL (Lacronique et al, 1997; Peeters et al, 1997). The fusion results in a constitutively active kinase due to the oligomerisation motifs in TEL bringing together JAK2 kinase domains. In addition, two of the breakpoints result in loss or disruption of the inhibitory JH2 domain, possibly contributing to activation (Lacronique et al, 1997). Murine bone marrow transplantation experiments with cells infected with TEL–JAK2 retroviruses generated a mixed T-lymphoid/myeloproliferative disorder (MPD) – transformation was dependent on JAK2 kinase activity and on the TEL oligomerisation domain (Schwaller et al, 1998). Although TEL–JAK2 expression results in the activation of a number of downstream signalling pathways including phosphoinositide-3 kinase (PI3K) and MAPK, STAT5 is required for transformation – STAT5ab−/− mice fail to develop lympho-myeloproliferative disease in response to TEL–JAK2 (Schwaller et al, 2000).
BCR–JAK2, associated with t(9;22)(p24;q11.2), has been described in a single case of Philadelphia-negative CML which was resistant to imatinib therapy (Griesinger et al, 2005). Recently, several groups have identified a recurrent translocation t(8;9)(p22;p24) that fuses the great majority of the human autoantigen PCM1 gene to the C terminal two-thirds of JAK2 (Bousquet et al, 2005; Murati et al, 2005; Reiter et al, 2005; Adelaide et al, 2006). This includes the JH2 and JH1 domains and is predicted to result in a constitutively active kinase due to multiple coiled-coil oligomerisation motifs in PCM1. Of 15 patients so far described, 10 had atypical MPDs and the others had AML, pre-B ALL and T lymphoblastic lymphoma.
The JAK2 V617F mutation and MPDs
The MPDs, originally grouped together by Dameshek (1951), are characterised by increased numbers of differentiated blood cells and are believed to arise in a multipotential haemopoietic progenitor. They include polycythaemia vera (PV), ET, primary idiopathic myelofibrosis (IMF) (the classic MPDs), as well as chronic eosinophilic leukaemia/hypereosinophilic syndrome (CEL/HES), systemic mastocytosis and CML. ET and PV share several features: a hypercellular marrow with overproduction and predominance of one lineage; hypersensitivity to cytokines such as EPO; presence of extramedullary haemopoiesis; progression in a significant proportion of cases to myelofibrosis and a relatively low propensity for evolution to acute leukaemia in the absence of the use of leukaemogenic cytoreductive treatment. The role of the BCR-ABL tyrosine kinase in CML pathophysiology has now been established for some years and more recently it has become apparent that aberrant tyrosine kinase activity is also associated with other MPDs (De Keersmaecker & Cools, 2006). A number of fusion partners have been described for the fibroblast growth factor receptor 1 (FGFR1) tyrosine kinase that typically result in a ‘8p11’ myeloproliferative syndrome associated with eosinophilia, lymphoblastic lymphoma and a propensity to transform to AML (Macdonald et al, 2002), although the BCR-FGFR1 fusion is associated with a CML-like phenotype (Demiroglu et al, 2001; Roumiantsev et al, 2004). Systemic mast cell disorders are frequently found to have activating point mutations in the SCF receptor, KIT (D816V) (Lennartsson et al, 2005). An interstitial deletion on chromosome 4q12 results in a fusion of the FIP1L1 gene to the PDGFRA gene and is causally implicated in the pathogenesis of HES/CEL and is also associated with mast cell disorders with associated eosinophilia (Cools et al, 2003; Tefferi & Pardanani, 2004). Translocations involving the PDGFRB locus at 5q33 have been described in a number of patients with atypical MPDs (Reilly, 2003). Diseases involving ABL, PDGFRA and PDGFRB kinases are all responsive to the inhibitor imatinib mesylate (Hannah, 2005).
Until just over a year ago, no abnormalities in TK signalling had been identified in the classic MPDs: PV, ET and IMF. Then, in a relatively short space of time, five publications detailed the identification of a somatic mutation in JAK2 at high frequency in these diseases (Baxter et al, 2005; James et al, 2005; Kralovics et al, 2005; Levine et al, 2005a; Zhao et al, 2005). This G–T mutation results in phenylalanine being substituted for valine at position 617 (V617F) in the pseudokinase/JH2 domain and results in a protein with increased kinase activity and hyper-responsiveness to cytokine signalling. Within the last 12 months, there has been a remarkable effort to characterise the biological and clinical correlates of this mutation with a large number of articles published.
The JAK2 V617F mutation was identified using a variety of approaches. The group of Vainchenker found that a kinase inhibitor of JAK2 or knockdown of JAK2 expression using small interfering RNA technology could inhibit the formation of eythropoietin-independent erythroid colonies that are a hallmark of PV (James et al, 2005). This led to sequencing of JAK2 and the detection of the mutation. Work from Kralovics et al had previously identified loss of heterozygosity (LOH) of a region on chromosome 9p in PV and identified a 6·2-Mbp region common to all 51 patients screened. As this region contained JAK2, with its known role in erythropoiesis, this was screened further for mutations (Kralovics et al, 2005). The three other groups targeted JAK2 as part of a general sequencing screen of tyrosine kinases and phosphatases in MPDs (Baxter et al, 2005; Levine et al, 2005a; Zhao et al, 2005).
A number of groups have now reported on the frequency of JAK2 V617F in MPDs (Tables III–V). The prevalence of the mutation in PV is very high and ranges in various series from 65% to 100% with an average of 82% in nearly 1000 reported cases. The differences in reported rates are likely to be due at least three reasons: first, the stringency of the criteria used to diagnose PV; secondly, the sensitivity of the method used to detect mutations and finally, the source of DNA. The majority of studies have used peripheral blood neutrophils as they are thought to be derived from the same clonal progenitor that is transformed in PV (reviewed in Tefferi & Gilliland, 2005; Tefferi & Spivak, 2005; Vainchenker & Constantinescu, 2005; De Keersmaecker & Cools, 2006; Nelson & Steensma, 2006). Direct sequencing techniques are likely to have a lower sensitivity than techniques that employ PCR amplification of the mutant allele (Campbell et al, 2006a).
The JAK2 V617F mutation has been detected in progenitors and myeloid cells including cells with HSC, common myeloid progenitor, megakaryocyte–erythroid progenitor phenotypes as well as colony-forming cells and more mature progeny, such as neutrophils and platelets (Baxter et al, 2005; Jamieson et al, 2006; Kiladjian et al, 2006a). So far, mutant JAK2 V617F has not been reported in T or B lymphocytes (James et al, 2005; Lasho et al, 2005). The mutation is somatic and has not been detected in any normal individuals or patients with secondary erythrocytosis. An initial report suggesting that JAK2 V617F could be detected in buccal samples from a small proportion of PV patients is likely to be due to contamination of such specimens with granulocyte DNA (Kralovics et al, 2005; Levine et al, 2005a). In just over a quarter of cases, only the mutant JAK2 V617F allele is found and this has been shown to be due to LOH at the JAK2 locus on chromosome 9p (Kralovics et al, 2005). LOH in this case is not due to gene deletion but to both copies being from a single parental origin due to mitotic recombination (acquired uniparental disomy) (Kralovics et al, 2002). This results in two mutant copies increasing the JAK2 V617F dosage. This feature is unusual for a gain-of-function mutation and LOH is normally associated with the loss of a tumour suppressor gene (Payne & Kemp, 2005).
The JAK2 V617F mutation is also found in IMF and ET with a prevalence of around 50%. Homozygous mutations are uncommon in ET compared with PV. The mutation has been screened for in a number of other haematological malignancies – it is found in some cases of atypical MPD (Jones et al, 2005; Steensma et al, 2005), in a subset of patients with MDS perhaps in association with 5q− (Ingram et al, 2006) and rarely in AML unless it is secondary to a previous MPD (Jelinek et al, 2005; Levine et al, 2005b; Frohling et al, 2006; Lee et al, 2006; Steensma et al, 2006). No cases have been described in lymphoid malignancies (Levine et al, 2005b) although a distinct mutation, JAK2 L611S, was discovered in one case of pre-B-ALL during a screen for JAK2 mutations using denaturing high-performance liquid chromatography (Kratz et al, 2006). The JAK2 V617F mutation is also found at similar frequencies to that seen in sporadic disease in familial MPDs. Germline transmission of JAK2 V617F has not been detected but the presence of this acquired mutation in these familial cases suggests that this can precipitate disease on the background of an underlying genetic predisposition (Cario et al, 2005; Bellanne-Chantelot et al, 2006).
Biological effects of JAK2 V617F. As discussed above, the JH2 domain exerts an inhibitory effect on JAK activity and the V617F mutation is predicted to disrupt this inhibition (Lindauer et al, 2001). Ectopic expression of JAK2 V617F in either epithelial or haemopoietic cell lines results in autophosphorylation of mutant JAK2 [which is not seen with wild-type (WT) JAK2] and activation of downstream signalling (James et al, 2005; Levine et al, 2005b). BAF3 or FDCP cell lines expressing the EPOR and engineered to stably express JAK2 V617F showed a degree of factor independence as well as marked hypersensitivity to EPO. James et al (2005) showed that these responses were inhibited by the presence of WT JAK2, perhaps due to competition for limiting numbers of cytokine receptor subunits. Work from the group of Lodish has shown that co-expression of JAK2 V617F with a homodimeric type 1 cytokine receptor (EPOR, TPOR or G-CSFR) facilitates the transformation of cells to growth-factor independence, suggesting that the mutant JAK2 requires a receptor scaffold so as to be active (likely due to the need for proximity required for transphosphorylation and activation) (Lu et al, 2005). This contrasts with the effects of the TEL–JAK2 fusion which can readily transform cells on its own, presumably because of the strong homo-dimerisation effects of the TEL moiety (Lacronique et al, 1997). Ectopic expression of JAK2 V617F can also sensitise cells to the effects of IGF1, a common feature of PV progenitors (Staerk et al, 2005).
Expression of JAK2 V617F in murine HSC in a transplantation model has been reported. James et al (2005) showed that this resulted in erythrocytosis without a major effect on granulocyte or platelet counts. Wernig et al (2006) have recently shown that JAK2 V617F expression produces an increased haematocrit and splenomegaly in recipient mice with a variable degree of leucocytosis and marrow fibrosis depending on the strain of mice used. Although bone marrow examination showed megakaryocyte hyperplasia, no increase in platelet counts was seen. The phenotype of these mice contrasts markedly with those transplanted with other MPD-associated activated TKs, such as BCR-ABL or TEL-PDGFRA where a MPD without elevated haematocrit develops (Wernig et al, 2006). These results suggest that the presence of the JAK2 V617F may be sufficient to induce PV but the disease phenotype may be affected by other, so far unknown, genetic modifiers. Lacout et al (2006) found that JAK2 V617F expression in transplanted C57BL/6J mice resulted in polycythaemia accompanied by neutrophilia. This was followed by marrow fibrosis with resulting anaemia in a manner akin to the ‘spent phase’ and fibrosis seen in the natural history of some cases of PV. In one group of secondarily transplanted animals, where there was a relatively low level of JAK2 V617F expression, a brief transient period of thrombocytosis was seen. It has been suggested that this is a situation analogous to that seen in clinical ET where JAK2 V617F homozygosity is rare and may provide some of the explanation for this phenotype. However, the animal data so far are limited and will require further study, preferably using an approach where the mutant JAK2 is ‘knocked-in’ to the endogenous JAK2 locus.
JAK2 V617F in ET. The frequency of JAK2 V617F is significantly lower in ET and semiquantitative analysis shows that JAK2 V617F levels may be low, suggesting the presence of both mutant clonal and residual normal haemopoietic cells (Antonioli et al, 2005; Campbell et al, 2005; Wolanskyj et al, 2005). These studies were mainly carried out with granulocyte DNA but the results are unlikely to be due to selective involvement of the megakaryocyte lineage as one report shows good concordance between platelet and granulocyte levels of JAK2 mutant (Campbell et al, 2005) and in another, V617F JAK2 was discovered in the platelets of only two of 24 patients negative by granulocyte PCR (Kiladjian et al, 2006a).
In ET, using techniques to detect X-chromosome inactivation patterns (XCIP), a significant proportion of cases have been shown to have polyclonal haemopoiesis (Harrison et al, 1999). Polyclonal cases of ET have been shown to have mutant JAK2 V617F at a similar prevalence to monoclonal cases (Antonioli et al, 2005; Kiladjian et al, 2006a; Levine et al, 2006). This has been interpreted as a failure of relatively insensitive clonality assays in picking up low-level clones that can be detected by JAK2 mutation analysis. It is perhaps surprising that the mutant clone does not come to dominate considering its potential proliferative advantage and this finding is distinct from other MPDs, such as BCR-ABL-driven CML, where the mutant cells comprise the majority of cells. Further, it is not easy to reconcile the presence of a minor JAK2 mutant population, perhaps detectable only by PCR-based techniques, with the presence of clinical disease, i.e. how does a clone that is only, say, at the 15% level cause a platelet count over 1000 × 109/l. To add to the complexity of JAK mutations in ET, some monoclonal cases of ET (by XCIP analysis) have only low levels of mutation suggesting that the JAK2 V617F is a secondary change in a subpopulation (Levine et al, 2006) and a significant proportion of monoclonal cases of ET have WT JAK2 (Antonioli et al, 2005; Levine et al, 2006). The lack of significant thrombocytosis in some murine transplantation models as well as these clinico-pathological findings indicate a more complex role for JAK2 V617F in ET which needs further clarification.
JAK2 V617F in IMF. There are variable reports of the clinical correlates of JAK2 V617F in myelofibrosis with a link to higher white cell count but not to rate of leukaemic transformation (Mesa et al, 2006a,b) and also to an older age at presentation as well as an association with pruritus and thrombosis (Tefferi et al, 2005a). Campbell et al (2006b) have reported a reduced red cell transfusion requirement and worse survival in mutant-positive patients. The reduced red cell requirements have been interpreted as being consistent with erythroid stimulation by the mutant JAK2 but no data are given in this study as to how cases of myelofibrosis secondary to PV were excluded (e.g. red cell mass estimation). The use of JAK2 mutant status as a tool to assist in the choice of therapies such as stem cell transplantation is not appropriate at present and will require additional data from other investigators.
Other clinico-pathological correlates of JAK2 V617F. The presence of JAK2 mutation in PV, ET and IMF has been reported to correlate with other biomarkers such as polycythaemia rubra vera type 1 (PRV1) expression and endogenous erythroid colony formation (Goerttler et al, 2005; Tefferi et al, 2005a,b). The association of PRV1 expression with JAK2 V617F in ET and IMF has since been questioned (Antonioli et al, 2005; Bellosillo et al, 2006; Vannucchi et al, 2006a). In patients with PV, homozygous JAK2 V617F is associated with an increased Hb at diagnosis and increased rate of fibrotic change, but not with thrombosis or bleeding risk or with duration of disease (Tefferi et al, 2006). In ET, a large series from the UK showed that mutation is associated with features that resemble PV, including increased Hb and neutrophils, risk of transformation to PV, low ferritin and EPO and increased risk of venous thromboses (Campbell et al, 2005). Similar results have been found by other groups (Antonioli et al, 2005) and have led to suggestions that patients with JAK2 V617F-positive ET may have a forme fruste of PV with the level of erythrocytosis influenced by genetic or acquired modifiers (Campbell et al, 2005). The presence of JAK2 V617F is also associated with lower platelet counts and a requirement for lower doses of hydroxycarbamide but not anagrelide (Campbell et al, 2005). V617F-positive patients treated with hydroxycarbamide had significantly lower rates of arterial thrombosis compared with those treated with anagrelide, an effect not seen in the JAK2-WT group.
Quantification of mutant JAK2 V617F following treatment with either imatinib or α-interferon has been reported by two groups. Jones et al (2006) reported that significant molecular responses to imatinib were uncommon; however, clinical response rates in the reported cases were also low and in the two cases with complete haematological response showed a several fold reduction in mutant level. Kiladjian et al (2006b) showed that patients treated with interferon, who had a much higher rate of clinical response, showed modest but significant decreases in mutant JAK2 level although with reduction below detection limits in only one case. These results suggest that in some cases of PV, the mutant cells are more sensitive to antiproliferative agents albeit with a modest differential compared with residual non-mutant cells.
Diagnostic implications of JAK2 V617F. The very high frequency of JAK2 V617F in PV has implications for the diagnosis of this disease. Incorporating a mutation screen early into the algorithm for investigating a case of suspected polycythaemia could help to streamline diagnosis although clearly, the presence of a JAK2 mutation alone does not distinguish PV from IMF or ET. James et al (2006) have shown that, in the investigation of 88 patients with erythrocytosis, according to World Health Organization criteria, the JAK2 V617F mutation was found in 57 of 61 cases diagnosed as PV and 0 of 11 as idiopathic erythrocytosis. This contrasted with 43 of 45 and eight of 21, respectively, on PVSG criteria. They, and others (Tefferi & Pardanani, 2006) have suggested that mutation analysis can be used to help screen individuals and that this may reduce the need for further investigations, such as red cell mass and bone marrow biopsy. However, the utility of these types of approach remains to be demonstrated in a prospective clinical study. In ET, where differentiating a primary proliferative condition from a reactive one is notoriously difficult, the use of JAK2 mutation analysis may assist in identifying patients with a stem cell disorder. JAK2 analysis has broader applicability as clonality assays are restricted to females and are also not evaluable in older women due to the phenomenon of age-related skewing. Of course, it has to be remembered that patients without a JAK2 mutation can still have a primary MPD.
Implications for therapy. The identification of JAK mutations in MPDs, and the role that aberrant JAK signalling plays in other haematological malignancies, has raised the exciting prospect of developing specific inhibitors for clinical use and significant efforts to screen for such compounds are under way. In the rare patients with malignant disease associated with strongly activating JAK2 translocations, such as those with PCM1-JAK2, such inhibitors, by analogy with imatinib in CML, may have significant clinical activity. In the management of chronic MPDs, there are a few outstanding questions that will need careful evaluation. First, whether it is possible to obtain an inhibitor that has preferential activity against mutant rather than WT JAK2 and which does not produce significant haematological toxicity. Secondly, as the management of many patients with PV with conventional therapies, including venesection, aspirin and hydroxycarbamide, has a reasonable outcome at relatively modest costs, a cost–benefit analysis of potentially expensive long-term targeted therapy will be needed. Thirdly, in ET and IMF, the role of JAK2 V617F as the driving force in disease pathophysiology is not as yet clear and the potential impact of JAK2-directed therapy is uncertain. With the huge effort being expended in investigating JAK signalling, we can be certain that the next few years will bring further developments in this fast-evolving field.