Key Points• Enrichment of atypical MPL mutations in essential thrombocythemia.• MPLS204P and MPLY591N mutants are weak gain-offunction mutants.Mutations in signaling molecules of the cytokine receptor axis play a central role in myeloproliferative neoplasm (MPN) pathogenesis. Polycythemia vera is mainly related to JAK2 mutations, whereas a wider mutational spectrum is detected in essential thrombocythemia (ET) with mutations in JAK2, the thrombopoietin (TPO) receptor (MPL), and the calreticulin (CALR) genes. Here, we studied the mutational profile of 17 ET patients negative for JAK2V617F, MPLW515K/L, and CALR mutations, using whole-exome sequencing and next-generation sequencing (NGS) targeted on JAK2 and MPL. We found several signaling mutations including JAK2V617F at very low allele frequency, 1 homozygous SH2B3 mutation, 1 MPLS505N, 1 MPLW515R, and 2 MPLS204P mutations. In the remaining patients, 4 presented a clonal and 7 a polyclonal hematopoiesis, suggesting that certain triple-negative ETs are not MPNs. NGS on 26 additional triple-negative ETs detected only 1 MPLY591N mutation. Functional studies on MPLS204P and MPLY591N revealed that they are weak gain-of-function mutants increasing MPL signaling and conferring either TPO hypersensitivity or independence to expressing cells, but with a low efficiency. Further studies should be performed to precisely determine the frequency of MPLS204 and MPLY591 mutants in a bigger cohort of MPN. (Blood. 2016;127(3):333-342)

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Abexinostat is a pan histone deacetylase inhibitor (HDACi) that demonstrates efficacy in malignancy treatment. Like other HDACi, this drug induces a profound thrombocytopenia whose mechanism is only partially understood. We have analyzed its effect at doses reached in patient plasma on in vitro megakaryopoiesis derived from human CD34+ cells. When added at day 0 in culture, abexinostat inhibited CFU-MK growth, megakaryocyte (MK) proliferation and differentiation. These effects required only a short incubation period. Decreased proliferation was due to induction of apoptosis and was not related to a defect in TPO/MPL/JAK2/STAT signaling. When added later (day 8), the compound induced a dose-dependent decrease (up to 10-fold) in proplatelet (PPT) formation. Gene profiling from MK revealed a silencing in the expression of DNA repair genes with a marked RAD51 decrease at protein level. DNA double-strand breaks were increased as attested by elevated γH2AX phosphorylation level. Moreover, ATM was phosphorylated leading to p53 stabilization and increased BAX and p21 expression. The use of a p53 shRNA rescued apoptosis, and only partially the defect in PPT formation. These results suggest that HDACi induces a thrombocytopenia by a p53-dependent mechanism along MK differentiation and a p53-dependent and -independent mechanism for PPT formation.

Thrombocytopenia is a major side effect of a new class of anticancer agents that target histone deacetylase (HDAC). Their mechanism is poorly understood. Here, we show that HDAC6 inhibition and genetic knockdown lead to a strong decrease in human proplatelet formation (PPF). Unexpectedly, HDAC6 inhibition-induced tubulin hyperacetylation has no effect on PPF. The PPF decrease induced by HDAC6 inhibition is related to cortactin (CTTN) hyperacetylation associated with actin disorganization inducing important changes in the distribution of megakaryocyte (MK) organelles. CTTN silencing in human MKs phenocopies HDAC6 inactivation and knockdown leads to a strong PPF defect. This is rescued by forced expression of a deacetylated CTTN mimetic. Unexpectedly, unlike human-derived MKs, HDAC6 and CTTN are shown to be dispensable for mouse PPF in vitro and platelet production in vivo. Our results highlight an unexpected function of HDAC6–CTTN axis as a positive regulator of human but not mouse MK maturation.

Key Points• MPL P106L induces thrombocytosis due to an incomplete trafficking defect that allows very low cellsurface levels.• The P106L mutation uncouples MPL signaling from its THPO clearance functions.

Background Cytoskeletal rearrangements are essential for platelet release. The RHO small GTPase family, as regulators of the actin cytoskeleton, play an important role in proplatelet formation (PPF). In the neuronal system, CDC42 is involved in axon formation, a process that combines elongation and branching as for PPF. Objective To analyze the role of CDC42 and its effectors of the Wiskott-Aldrich syndrome protein (WASP) family in PPF. Methods Human megakaryocytes (MKs) were obtained from CD34 cells. Inhibition of CDC42 in MKs was performed with the chemical inhibitor CASIN or with an active or a dominant-negative form of CDC42. The knock-down of N-WASP was obtained with a small hairpin RNA strategy Results Herein, we show that CDC42 activity increased during MK differentiation. The use of the chemical inhibitor CASIN or of an active or a dominant-negative form of CDC42 demonstrated that CDC42 positively regulated PPF in vitro. We determined that N-WASP, but not WASP, regulated PPF. We found that N-WASP knockdown led to a marked decrease in PPF, owing to a defect in the demarcation membrane system (DMS). This was associated with RHOA activation, and a concomitant augmentation in the phosphorylation of mysosin light chain 2. Phosphorylation of N-WASP, creating a primed form of N-WASP, increased during MK differentiation. Phosphorylation inhibition by two Src family kinase inhibitors decreased PPF. Conclusions We conclude that N-WASP positively regulates DMS development and PPF, and that the Src family kinases in association with CDC42 regulate PPF through N-WASP.

The selenoprotein glutathione peroxidase 4 (GPX4), the only member of the glutathione peroxidase family able to directly reduce cell membrane–oxidized fatty acids and cholesterol, was recently identified as the central regulator of ferroptosis. GPX4 knockdown in mouse hematopoietic cells leads to hemolytic anemia and to increased spleen erythroid progenitor death. The role of GPX4 during human erythropoiesis is unknown. Using in vitro erythroid differentiation, we show here that GPX4-irreversible inhibition by 1S,3R-RSL3 (RSL3) and its short hairpin RNA–mediated knockdown strongly impaired enucleation in a ferroptosis-independent manner not restored by tocopherol or iron chelators. During enucleation, GPX4 localized with lipid rafts at the cleavage furrows between reticulocytes and pyrenocytes. Its inhibition impacted enucleation after nuclear condensation and polarization and was associated with a defect in lipid raft clustering (cholera toxin staining) and myosin-regulatory light-chain phosphorylation. Because selenoprotein translation and cholesterol synthesis share a common precursor, we investigated whether the enucleation defect could represent a compensatory mechanism favoring GPX4 synthesis at the expense of cholesterol, known to be abundant in lipid rafts. Lipidomics and filipin staining failed to show any quantitative difference in cholesterol content after RSL3 exposure. However, addition of cholesterol increased cholera toxin staining and myosin-regulatory light-chain phosphorylation, and improved enucleation despite GPX4 knockdown. In summary, we identified GPX4 as a new actor of human erythroid enucleation, independent of its function in ferroptosis control. We described its involvement in lipid raft organization required for contractile ring assembly and cytokinesis, leading in fine to nucleus extrusion.

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