Summary The mechanism by which cells decide to skip mitosis to become polyploid is largely undefined. Here we used a high-content image-based screen to identify small-molecule probes that induce polyploidization of megakaryocytic leukemia cells and serve as perturbagens to help understand this process. We found that dimethylfasudil (diMF, H-1152P) selectively increased polyploidization, mature cell-surface marker expression, and apoptosis of malignant megakaryocytes. A broadly applicable, highly integrated target identification approach employing proteomic and shRNA screening revealed that a major target of diMF is Aurora A kinase (AURKA), which has not been studied extensively in megakaryocytes. Moreover, we discovered that MLN8237 (Alisertib), a selective inhibitor of AURKA, induced polyploidization and expression of mature megakaryocyte markers in AMKL blasts and displayed potent anti-AMKL activity in vivo. This research provides the rationale to support clinical trials of MLN8237 and other inducers of polyploidization in AMKL. Finally, we have identified five networks of kinases that regulate the switch to polyploidy.

JAK inhibitors have been developed following the discovery of the JAK2V617F in 2005 as the driver mutation of the majority of non- BCR-ABL1 myeloproliferative neoplasms (MPNs). Subsequently, the search for JAK2 inhibitors continued with the discovery that the other driver mutations ( CALR and MPL) also exhibited persistent JAK2 activation. Several type I ATP-competitive JAK inhibitors with different specificities were assessed in clinical trials and exhibited minimal hematologic toxicity. Interestingly, these JAK inhibitors display potent anti-inflammatory activity. Thus, JAK inhibitors targeting preferentially JAK1 and JAK3 have been developed to treat inflammation, autoimmune diseases, and graft-versus-host disease. Ten years after the beginning of clinical trials, only two drugs have been approved by the US Food and Drug Administration: one JAK2/JAK1 inhibitor (ruxolitinib) in intermediate-2 and high-risk myelofibrosis and hydroxyurea-resistant or -intolerant polycythemia vera and one JAK1/JAK3 inhibitor (tofacitinib) in methotrexate-resistant rheumatoid arthritis. The non-approved compounds exhibited many off-target effects leading to neurological and gastrointestinal toxicities, as seen in clinical trials for MPNs. Ruxolitinib is a well-tolerated drug with mostly anti-inflammatory properties. Despite a weak effect on the cause of the disease itself in MPNs, it improves the clinical state of patients and increases survival in myelofibrosis. This limited effect is related to the fact that ruxolitinib, like the other type I JAK2 inhibitors, inhibits equally mutated and wild-type JAK2 (JAK2WT) and also the JAK2 oncogenic activation. Thus, other approaches need to be developed and could be based on either (1) the development of new inhibitors specifically targeting JAK2V617F or (2) the combination of the actual JAK2 inhibitors with other therapies, in particular with molecules targeting pathways downstream of JAK2 activation or the stability of JAK2 molecule. In contrast, the strong anti-inflammatory effects of the JAK inhibitors appear as a very promising therapeutic approach for many inflammatory and auto-immune diseases.

FPD/AML is a familial platelet disorder characterized by platelet defects, predisposition to acute myelogenous leukemia (AML) and germ-line heterozygous RUNX1 alterations. Here we studied the in vitro megakaryopoiesis of 3 FPD/AML pedigrees. A 60% to 80% decrease in the output of megakaryocytes (MKs) from CD34 ؉ was observed. MK ploidy level was low and mature MKs displayed a major defect in proplatelet formation. To explain these defects, we focused on myosin II expression as RUNX1 has been shown to regulate MYL9 and MYH10 in an inverse way. In FPD/AML MKs, expression of MYL9 and MYH9 was decreased, whereas MYH10 expression was increased and the MYH10 protein was still present in the cytoplasm of mature MKs. Myosin II activity inhibition by blebbistatin rescued the ploidy defect of FPD/AML MKs. Finally, we demonstrate that MYH9 is a direct target of RUNX1 by chromatin immunoprecipitation and luciferase assays and we identified new RUNX1 binding sites in the MYL9 promoter region.Together, these results demonstrate that the defects in megakaryopoiesis observed in FPD/AML are, in part, related to a deregulation of myosin IIA and IIB expression leading to both a defect in ploidization and proplatelet formation. IntroductionFamilial platelet disorder with predisposition to acute myeloid leukemia (FPD/AML, OMIM 601399) is an autosomal dominant disorder characterized by dysmegakaryopoiesis, qualitative and quantitative platelet defects, and a propensity to develop myelodysplastic syndromes (MDSs) and/or AML. Several types of heterozygous germ-line mutations or deletions in RUNX1, including missense, frameshift, and nonsense mutations or large intragenic deletion or single nucleotide deletion in the Runt domain have been identified in FPD/AML. The progression to AML is often linked to the somatic alteration of the second RUNX1 allele, 1 supporting the fact that RUNX1 acts as a tumor suppressor gene. RUNX1 (also known as AML1, PEBP2aB, or CBFA2) is 1 of the 3 DNA-binding ␣ subunits of the hematopoietic transcription complex called core binding factor (CBF). RUNX1 contains both a runt homology domain (RHD), which mediates DNA binding and heterodimerization with the core binding factor ␤ (CBF␤) subunit to stabilize the interaction of the complex with DNA and to protect CBF from proteolytic degradation. The C-terminal domain of RUNX1 is responsible for transcriptional activation. RUNX1 can act as a repressor or an activator depending on the cellular context. It regulates positively different hematopoietic genes encoding cytokines and their receptors, such as IL-3, 2 GM-CSF, and M-CSF or negatively the CD4 gene contributing thus to impaired T-cell development. 3 Somatic alterations in RUNX1 are frequently found in AML, MDS, and chronic myelomonocytic leukemia (CMML).In different mouse models, RUNX1 was shown to be essential for establishing definitive hematopoiesis. 4 It is required for the generation of hematopoietic stem cells (HSCs) from the aorta, but not later on. Targeted deletion of RUNX1 in adult HSCs led to their ...