Endothelial JAK2V617F Expression Drives Inflammation and Cellular Senescence; New Evidence for the Roles of Endothelial Cells in MPN-Related Clotting Abnormalities?

Abstract

JAK2V617F is the primary pathogenic mutation in patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), a heterogeneous group of haematological malignancies characterised by increases in one or more myeloid lineages. The most common cause of morbidity and mortality in patients with MPNs are thrombohemorrhagic incidents, although the events leading to these clotting abnormalities remain unclear. It is now known that a subset of MPN patients express JAK2V617F in endothelial cells (ECs) which has been linked with a poorer prognosis and an increase in the incidence of thrombosis. Furthermore, using transgenic mouse models we have recently shown that expression of JAK2V617F in ECs plays a critical role in aberrant thrombosis in vivo (PNAS, 2014. 111(6): p. 2295-300). Here, we describe the transcriptomic and phenotypic profiles of JAK2V617F-positive ECs.Lentivirally transduced human umbilical vein ECs (HUVECs) showed a sustained increase in levels of activated JAK2, STAT3 and STAT1 in JAK2V617F-expressing cells compared to WT JAK2. In addition, JAK2V617F also caused a significant increase in expression of total STAT1 protein. Subsequent RNAseq analysis revealed an upregulation of several mRNAs related to inflammation and permeability in JAK2V617F-expressing cells. Expression of interferon-related genes, including serine protease inhibitor B2 (SERPINB2) and early growth response protein 1 (EGR1), and chemokine ligand 2 (CCL2) were also increased in JAK2V617F cells. SERPINB2 and EGR1 have roles in initiating cellular senescence and CCL2 was previously reported to induce permeability. These data, combined with increased expression of total STAT1, suggest that JAK2V617F expression in ECs induces an inflammatory response in this cell type. Moreover, in vitro permeability assays show increased permeability in HUVECs transduced with JAK2V617Fcompared to WT JAK2, which was significantly amplified following stimulation with thrombin.We then determined whether any functional differences existed between patient-derived normal and JAK2V617F-expressing ECs. For this we obtained blood from JAK2V617F-positive polycythaemia vera patients undergoing therapeutic phlebotomy and isolated blood outgrowth endothelial cells (BOECs) by repeated culture of PBMCs. Allelic burden of JAK2V617F, determined by quantitative allele-specific amplification (QuASA) was shown to vary between patient samples, (0% to 59.8% (n=9)) and increased expression of JAK2V617F appeared to correlate with decreased proliferation and significant changes in cellular morphology.In order to quantify these differences, ptychography, a novel label-free imaging technology was used to generate quantitative phase images of BOECs in time-lapse, to measure phenotypic changes in cell morphology, motility and proliferation. These results confirmed that JAK2V617F allelic burden was inversely related to BOEC proliferation. Cells which expressed only WT JAK2 or low levels of JAK2V617F exhibited exponential growth. However, an increase in JAK2V617F resulted in a decrease in BOEC proliferation. Indeed, cells which expressed high JAK2V617F levels (30-60%) failed to proliferate over the 48 hour imaging period. Furthermore, JAK2V617F-high BOECs were also shown to be larger in size, multi-nucleated and less motile compared to JAK2V617F-low. The increase in cell size and decreases in cell motility and proliferation suggest that increased JAK2V617F expression induces senescence in BOECs.These data describe features of ECs which are commonly associated with dysfunctional endothelium: sustained inflammation/permeability, limited cell growth and senescence. The critical role of ECs in maintaining hemostasis and the observed features of EC dysfunction in cells which express JAK2V617F, implicate mutation burden in ECs as a contributing factor towards the thrombotic abnormalities observed in patients with MPNs. DisclosuresNo relevant conflicts of interest to declare.