A mechanistic study on thrombus formation in JAK2 V617F and CALR mutated chronic myeloproliferative neoplasia (CMN)

JAK2-V617F and CALR mutations are the most common genetic aberrations in classic Philadelphia-Chromosome negative chronic myeloproliferative neoplasia (CMN). A major cause of morbidity and mortality in patients carrying these mutations is a marked prothrombotic state leading to venous and arterial thrombosis. Based on a large body of evidence, in recent years, granulocytes and monocytes were identified as key players in induction of venous thrombosis.
Previously, our group found that JAK2-V617F aberrantly activates ß1 and ß2 integrins (VLA4 and LFA1) on leukocytes in CMN and identified some of the critical inside-out signaling molecules involved (e.g. small GTPase Rap1, CALDAG-GEF1). Interestingly, inhibition of VLA4 and LFA1 using neutralizing antibodies suppressed JAK2-V617F induced thrombus formation in-vivo (inferior vena cava stenosis model).
Based on these studies, we aim to elucidate the precise underlying molecular mechanisms that trigger and sustain the process of venous thrombosis in CALR- and JAK2-V617F-mutated CMN. Our comprehensive analysis will include characterisation of integrin-mediated granulocyte adhesion and of key signaling molecules driving integrin activation in granulocytes. Our experimental approach will employ various suitable cell lines, JAK2-V617F knock-in and CALR mutated mouse models, primary leukocytes derived from patients and a thrombosis in-vivo model (inferior vena cava stenosis) which is well established in our lab. Molecules involved will be targeted using neutralising antibodies and selective small molecule inhibitors. Further, we will employ 2-photon microscopy in saphenous vein thrombosis model to intravitally investigate a potential difference in rolling, crawling, adhesion and aggregation (thrombus formation) of JAK2-V617F positive and CALR mutated granulocytes, respectively. Further, these investigations will also focus on the involvement of neutrophil extracellular traps (NETs), including a potential activation of peptidylarginine deiminase 4 (PAD4) by mutated CALR. In an in vitro study, we previously showed that in JAK2-V617F positive leukocytes, BTK and the small GTPase RhoA were constitutively activated. Thus, we hypothesize that signaling molecules upstream and downstream of BTK are activated in CALR mutated leukocytes and may represent an integration point for development of thrombosis. This part of the project may allow to explore BTK as a potential therapeutic drug target in CMN. Finally, based in previous results showing differential activation of the small GTPase Rap1 in granulocytes isolated from CALR mutated patients, we aim to dissect the molecular mechanisms involved in differential Rap1 activation in CALR and JAK2V617F mutated granulocytes.
Identification of the precise molecular pathways involved in the pro-thrombotic state of JAK2-V617F positive and CALR mutated patients may ultimately provide novel targets for prophylaxis and therapy of venous thrombosis in CMN.