Key Points• JAK2V617F homozygosity drives a phenotypic switch between myeloproliferative neoplasms.• JAK2V617F homozygosity is insufficient to sustain clonal expansion.Genomic regions of acquired uniparental disomy (UPD) are common in malignancy and frequently harbor mutated oncogenes. Homozygosity for such gain-of-function mutations is thought to modulate tumor phenotype, but direct evidence has been elusive. Polycythemia vera (PV) and essential thrombocythemia (ET), 2 subtypes of myeloproliferative neoplasms, are associated with an identical acquired JAK2V617F mutation but the mechanisms responsible for distinct clinical phenotypes remain unclear. We provide direct genetic evidence and demonstrate that homozygosity for human JAK2V617F in knock-in mice results in a striking phenotypic switch from an ET-like to PV-like phenotype. The resultant erythrocytosis is driven by increased numbers of early erythroid progenitors and enhanced erythroblast proliferation, whereas reduced platelet numbers are associated with impaired platelet survival. JAK2V617F-homozygous mice developed a severe hematopoietic stem cell defect, suggesting that additional lesions are needed to sustain clonal expansion. Together, our results indicate that UPD for 9p plays a causal role in the PV phenotype in patients as a consequence of JAK2V617F homozygosity. The generation of a JAK2V617F allelic series of mice with a dose-dependent effect on hematopoiesis provides a powerful model for studying the consequences of mutant JAK2 homozygosity.
The principal morbidity and mortality in patients with essential thrombocythemia (ET) and polycythemia rubra vera (PV) stems from thrombotic events. Most patients with ET/PV harbor a JAK2V617F mutation, but its role in the thrombotic diathesis remains obscure. Platelet function studies in patients are difficult to interpret because of interindividual heterogeneity, reflecting variations in the proportion of platelets derived from the malignant clone, differences in the presence of additional mutations, and the effects of medical treatments. To circumvent these issues, we have studied a JAK2V617F knock-in mouse model of ET in which all megakaryocytes and platelets express JAK2V617F at a physiological level, equivalent to that present in human ET patients. We show that, in addition to increased differentiation, JAK2V617F-positive megakaryocytes display greater migratory ability and proplatelet formation. We demonstrate in a range of assays that platelet reactivity to agonists is enhanced, with a concomitant increase in platelet aggregation in vitro and a reduced duration of bleeding in vivo. These data suggest that JAK2V617F leads to intrinsic changes in both megakaryocyte and platelet biology beyond an increase in cell number. In support of this hypothesis, we identify multiple differentially expressed genes in JAK2V617F megakaryocytes that may underlie the observed biological differences.
A genome-wide scan in nearly 70,000 individuals showed that the common SNP rs8109288 in the first intron of the human TPM4 gene exerts an effect on the volume and count of platelets (Gieger et al. Nature 2011). We isolated a mouse line with an ENU-induced missense mutation in Tpm4. Mice carrying this mutation exhibited dose-dependent macrothrombocytopenia, while other blood cell counts were normal. Bone marrow transplant experiments demonstrated that the phenotype is intrinsic to hematopoietic cells. Notably, Tpm4 insufficiency did not affect the life span or in vitro function of mutant platelets, and there was no evidence of an increased propensity to bleeding. Megakaryocyte numbers in the bone marrow were increased, although maturation as measured by ploidy appeared normal. Mutant megakaryocytes displayed altered morphology indicating fragmentation, and markedly decreased proplatelet formation in vitro. Based on Gieger et al., we examined the functional requirement for TPM4 in human megakaryocytes. We found that the localisation of TPM4 in proplatelet-forming megakaryocytes was extremely similar to the localisation in their mouse counterparts, suggesting an identical role. Furthermore, knock down of TPM4by shRNA in human megakaryocytes did not affect maturation as measured by CD41 and CD42 expression, but significantly reduced the number of proplatelet-forming cells. The occasional megakaryocyte that did form proplatelets did not exhibit the typical “beads-on-a-string” phenotype. Typically, one large bulb at the end of a protrusion or a string with no clearly distinguishable beads was observed. We therefore performed a look-up in the BRIDGE consortium database, which enrolled 542 cases with inherited bleeding and platelet disorders of unknown aetiology in the NIHR BioResource for exome sequencing. Calling of variants revealed single nucleotide variants with consequences in TPM4which were absent from ~30,000 control haplotypes, in three BRIDGE cases. Two cases with a stop codon at residue 105 and R91H variant presented with macrothrombocytopenia and mild bleeding symptoms with platelet counts of 103 and 128 x10e9/L and volumes of 15.10 and 14.00 fl, respectively. The remaining case with variant D20N (not conserved, genomic evolutionary rate profiling score of 2.68 compared to 4.62 for the other variants) had a count in the normal range (232 x10e9/L) and a reduced platelet volume of 7.20 fl. Together, these results provide compelling evidence that Tropomyosin 4 is a crucial regulator of platelet production in mice and humans, being specifically required for the terminal stages of platelet formation. Our studies demonstrate that the common intronic variant exerts a subtle effect, whilst two extremely rare variants have a more robust effect on platelet formation leading to counts and volumes at the tails of the population distribution. The lack of concordance between mice and humans with regard to bleeding may be explained by strong modifiers at loci other than TPM4. Disclosures No relevant conflicts of interest to declare.
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