Hyperproliferation of myeloid and erythroid cells in myeloproliferative neoplasms (MPN) driven by the JAK2-V617F mutation is associated with altered metabolism. Given the central role of glutamine in anabolic and catabolic pathways, we examined the effects of pharmacologically inhibiting glutaminolysis, that is, the conversion of glutamine (Gln) to glutamate (Glu), using CB-839, a small molecular inhibitor of the enzyme glutaminase (GLS). We show that CB-839 strongly reduced the mitochondrial respiration rate of bone marrow cells from JAK2-V617F mutant (VF) mice, demonstrating a marked dependence of these cells on Gln-derived ATP production. Consistently, in vivo treatment with CB-839 normalized blood glucose levels, reduced splenomegaly and decreased erythrocytosis in VF mice. These effects were more pronounced when CB-839 was combined with the JAK1/2 inhibitor ruxolitinib or the glycolysis inhibitor 3PO, indicating possible synergies when cotargeting different metabolic and oncogenic pathways. Furthermore, we show that the inhibition of glutaminolysis with CB-839 preferentially lowered the proportion of JAK2-mutant hematopoietic stem cells (HSCs). The total number of HSCs was decreased by CB-839, primarily by reducing HSCs in the G1 phase of the cell cycle. CB-839 in combination with ruxolitinib also strongly reduced myelofibrosis at later stages of MPN. In line with the effects shown in mice, proliferation of CD34+ hematopoietic stem and progenitor cells from polycythemia vera patients was inhibited by CB-839 at nanomolar concentrations. These data suggest that inhibiting GLS alone or in combination with inhibitors of glycolysis or JAK2 inhibitors represents an attractive new therapeutic approach to MPN.

Arterial and venous thromboses are the most significant complications in patients with myeloproliferative neoplasms (MPNs), with the primary treatment goal being thrombotic risk reduction. In MPN with no history of thrombosis, primary prevention mainly involves the use of aspirin, and cytoreduction is added in high-risk patients. However, thrombotic complications can unveil an MPN in ∼20% of cases, necessitating the initiation of both antithrombotic therapy for the thrombosis and cytoreductive treatment for the MPN. The duration of anticoagulant therapy after an initial venous thromboembolic event is subject to discussion. Furthermore, the occurrence of a thrombotic complication in patients with a known diagnosis of MPN prompts a reconsideration of both antithrombotic and hematologic management. This review uses case-based discussions to explore the management of thrombotic complications in patients with MPN. It addresses the nature and duration of antithrombotic treatments, as well as the approach to cytoreduction. Special attention is given to the place of direct oral anticoagulants and to the management of patients with MPN with splanchnic vein thrombosis, which is disproportionately common in this group.

Background Acquired von Willebrand syndrome (AVWS) is frequent in patients with myeloproliferative neoplasms (MPNs). For von Willebrand factor (VWF) functional evaluation, ristocetin cofactor activity by aggregometry (VWF:RCo) is considered the gold standard but has limitations, and automated activity measurement has been developed such as the HemosIL VWF:RCo Werfen with particle agglutination (VWF:GPIbR). Objectives To evaluate the performance of VWF:GPIbR with HemosIL VWF:RCo Werfen (VWF:GPIbR) versus VWF:RCo in patients with thrombocytosis in the context of MPNs (T-MPNs) and in patients with secondary thrombocytosis (ST). Methods MPN patients with thrombocytosis >450 G/L (T-MPNs) were compared with patients with ST due to inflammation or iron deficiency. VWF activity (VWF:Act) was analyzed using VWF:RCo or VWF:GPIbR. VWF analysis was completed by analysis of VWF multimers and VWF collagen binding (CB) assay (VWF:CB). Results A total of 33 T-MPNs and 18 ST patients were included. Compared with aggregometry, evaluation of VWF:Act by VWF:GPIbR led to lower values in T-MPN patients, but also in ST patients. Interestingly, although the VWF:RCo/VWF:Ag ratio did not reveal differences between T-MPNs and ST patients, the VWF:GPIbR/VWF:Ag ratio analysis allowed us to suspect AVWS only in T-MPN patients. Using the distribution of VWF multimer analysis and VWF:CB, we here demonstrated that VWF:GPIbR allows AVWS diagnosis in nine T-MPNs as opposed to aggregometry. Conclusion Evaluation of VWF:Act using VWF:GPIbR has a greater sensitivity compared with aggregometry to detect AVWS in T-MPN patients. P.L., M.T., A.R., and M.F, contributed to designing and performing experiments, analysis and interpretation of data, and preparing the manuscript. A.R. and M.F. contributed to performing experiments, analysis and interpretation of data, and preparing the manuscript. E.R. contributed to providing patients' blood samples and preparing the manuscript. C.J. and A.G. contributed to supervising the study, designing experiments, analysis and interpretation of data, and preparing the manuscript. All authors discussed the results and commented on the manuscript. ^* These authors contributed equally to this work. Received: 23 November 2021 Accepted: 22 March 2022 Accepted Manuscript online: 23 March 2022 Article published online: 28 June 2022 © 2022. Thieme. All rights reserved. Georg Thieme Verlag KG Rüdigerstraße 14, 70469 Stuttgart, Germany

Myeloproliferative neoplasms (MPN) are clonal disorders of hematopoietic stem cells (HSC) that are most frequently caused by acquired somatic mutations in JAK2 . A number of conditional mouse models of JAK2- V617F-driven MPN have been generated that rely on Cre-LoxP mediated activation, resulting in polyclonal disease. To more closely mimic the monoclonal origin of human MPN, transplantations of single purified JAK2 -mutant HSCs or bone marrow (BM) at limiting dilutions into lethally irradiated recipient mice have been previously performed. However, irradiation is known to alter the BM microenvironment and also to induce transient aplasia accompanied by elevated cytokine levels that promotes the expansion of the mutant clone. To overcome these limitations, we examined whether JAK2- V617F-mutant HSCs are able to engraft and initiate MPN in non-conditioned recipients. We found that BM from two different MPN models, one expressing the human JAK2 -V617F, and another expressing the mouse Jak2 -V617F, efficiently engrafted and initiated MPN in non-irradiated immunocompromised Rag2 ^−/− recipients. MPN evolved even in transplantations at limiting dilutions, showing high competitiveness of single JAK2 -mutant HSCs. Thus, JAK2 -V617F mutant HSCs can outcompete resident non-mutated HSCs in the absence of elevated cytokine levels and without the need of emptying stem cell niches by irradiation. However, only BM from mice expressing the mouse Jak2 -V617F engrafted and initiated disease in non-conditioned C57BL/6 mice, while BM from mice expressing the human JAK2 -V617F was rejected, indicating that mouse Jak2 -V617F is ignored by the immune surveillance. These results provide a possible explanation why JAK2 -V617F is so frequently found in healthy individuals with clonal hematopoiesis.