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 ...

Primary myelofibrosis (PMF) is characterized by bone marrow fibrosis, myeloproliferation, extramedullary hematopoiesis, splenomegaly and leukemic progression. Moreover, the bone marrow and spleen of patients are full of atypical megakaryocytes that are postulated to contribute to fibrosis through the release of cytokines including TGF-β. Although the JAK inhibitor ruxolitinib provides symptomatic relief, it does not reduce the mutant allele burden or significantly reverse fibrosis. Here we show through pharmacologic and genetic studies that, apart from JAK2, Aurora kinase A (AURKA) is a novel therapeutic target in PMF. MLN8237, a selective AURKA inhibitor promoted polyploidization and differentiation of PMF megakaryocytes and displayed potent anti-fibrotic and anti-tumor activity in vivo. We also reveal that loss of one allele of AURKA is sufficient to ameliorate fibrosis and other PMF phenotypes in vivo. Our data suggest that megakaryocytes are drivers of fibrosis and that targeting them with AURKA inhibitors will provide therapeutic benefit in PMF.

Megakaryoblastic leukemia 1 (MAL) is a transcriptional coactivator of serum response factor (SRF). In acute megakaryoblastic leukemia, the MAL gene is translocated and fused with the gene encoding one twenty-two (OTT). Herein, we show that MAL expression increases during the late differentiation steps of neonate and adult human megakaryopoiesis and localized into the nucleus after Rho GTPase activation by adhesion on collagen I or convulxin. MAL knockdown in megakaryocyte progenitors reduced the percentage of cells forming filopodia, lamellipodia, and stress fibers after adhesion on the same substrates, and reduced proplatelet formation. MAL repression led to dysmorphic megakaryocytes with disorganized demarcation membranes and ␣ granules heterogeneously scattered in the cytoplasm. Gene expression profiling revealed a marked decrease in metalloproteinase 9 (MMP-9) and MYL9 expression after MAL inhibition. Luciferase assays in HEK293T cells and chromatin immunoprecipitation in primary megakaryocytes showed that the MAL/SRF complex directly regulates MYL9 and MMP9 in vitro. Megakaryocyte migration in response to stromal cell-derived factor 1, through Matrigel was considerably decreased after MAL knockdown, implicating MMP9 in migration. Finally, the use of a shRNA to decrease MYL9 expression showed that MYL9 was involved in proplatelet formation. MAL/SRF complex is thus involved in platelet formation and megakaryocyte migration by regulating MYL9 and MMP9. IntroductionSerum response factor (SRF) is a widely expressed transcription factor required for the expression of immediate early, musclespecific, and cytoskeletal genes. 1-4 SRF contains a MADS domain that mediates homodimerization and DNA binding, and that allows recruitment of transcriptional cofactors. SRF binds to a CArG box present in promoter/enhancer regions of SRF-regulated genes. 5 Depending on cell lines, different extracellular stimuli activate SRF through 2 main signaling pathways: the MAP-kinase pathway through members of the ternary complex factor (TCF) 6,7 and the small GTPases pathway through the Rho family 8 members regulating the myocardin-related transcription factors (MRTFs). The Rho-actin signaling pathway 9-12 stimulates SRF by 2 ubiquitous MRTFs, megakaryoblastic leukemia 1 (MAL; MKL1, MRTF-A, BSAC) and MAL16 (MKL2, MRTF-B).MAL was initially identified in acute megakaryoblastic leukemia (AMKL, M7) as a chromosome 22 encoded protein fused in 3Ј with RNA-binding motif protein 15 (RBM15; OTT) located on chromosome 1. [13][14][15] The translocation t(1,22)(p13;q13) leads to the in-frame fusion of the quasi-totality of OTT/RBM15 to the MAL gene. The OTT-MAL fusion protein is restricted to AMKL occurring de novo in infancy, 16,17 in children older than 1 year or, occasionally, in Down syndrome patients. 18,19 The subcellular localization of MAL is regulated through its association with globular actin by its RPEL motifs in the Nterminal region. The modification of the actin treadmilling by the Rho pathway results in the nuclear accumulation of ...

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The majority of patients with BCR-ABL1-negative myeloproliferative neoplasms (MPN) harbor mutations in JAK2 or MPL, which lead to constitutive activation of the JAK/STAT, PI3K, and ERK signaling pathways. JAK inhibitors by themselves are inadequate in producing selective clonal suppression in MPN and are associated with hematopoietic toxicities. MK-2206 is a potent allosteric AKT inhibitor that was well tolerated, including no evidence of myelosuppression, in a phase I study of solid tumors. Herein, we show that inhibition of PI3K/AKT signaling by MK-2206 affected the growth of both JAK2V617F or MPLW515L-expressing cells via reduced phosphorylation of AKT and inhibition of its downstream signaling molecules. Moreover, we demonstrate that MK-2206 synergizes with Ruxolitinib in suppressing the growth of JAK2V617F mutant SET2 cells. Importantly MK-2206 suppressed colony formation from hematopoietic progenitor cells in patients with primary myelofibrosis (PMF) and alleviated hepatosplenomegaly and reduced megakaryocyte burden in the bone marrows, livers and spleens of mice with MPLW515L-induced MPN. Together, these findings establish AKT as a rational therapeutic target in the MPNs.

The molecular mechanisms that regulate megakaryocyte (MK) ploidization are poorly understood. Using MK differentiation from primary human CD34 ؉ cells, we observed that p19 INK4D expression was increased both at the mRNA and protein levels during ploidization. p19 INK4D knockdown led to a moderate increase (31.7% ؎ 5%) in the mean ploidy of MKs suggesting a role of p19 INK4D in the endomitotic arrest. This increase in ploidy was associated with a decrease in the more mature MK population (CD41 high CD42 high ) at day 9 of culture, which was related to a delay in differentiation. Inversely, p19 INK4D Until now, the best-studied model of polyploidization concerns megakaryocytes (MKs) in which after several replicative rounds, the process of classic mitosis is replaced by an endomitotic process. MK endomitosis is very similar to mitosis, but MKs skip anaphase B, telophase, and cytokinesis giving rise to a polyploid cell with a single polylobulated nucleus. 1,2 Although polyploidization is a part of the MK differentiation program, terminal differentiation that is associated with an increased platelet protein synthesis and the development of specific organelles requires an arrest of the cell cycle. Both the mitotic and endomitotic processes require an amplification of the DNA content. However, while mitosis leads to an increase in cell number, polyploidization results in an increase in the cell size and protein mass. In the case of MK, the platelet precursor, polyploidization increases platelet production.DNA replication is a regulated mechanism that depends on a well-organized network of the cyclin-dependent kinases (CDKs) and cyclin-dependent inhibitors (CDKIs) of the cell cycle. Progression through G1 to S phase of cell cycle in mammalian cells requires the activity of G1-cyclins (D and E) associated with their catalytic subunits (CDK4 and CDK6). The mitogen-stimulated cyclin D and CDK4/6 complexes phosphorylate the retinoblastoma protein (Rb) allowing its dissociation from E2Fs transcription factors. Activated E2Fs then regulate the expression of genes necessary for DNA synthesis during the S phase of cell cycle. Cells exit cell cycle and accumulate in a quiescent G 0 /G 1 state either in the absence of mitogenic stimuli when the cyclin D-dependent activity is lost or after CDK4/6 inhibition. CDKs are regulated by inhibitory phosphorylations mediated by the WEE1 and MYT1 kinases 3 or by induction of specific inhibitors from the INK4 and CIP/KIP families. Till now, 4 members of the INK4 family (p19 INK4D , p18 INK4C , p16 INK4A , and p15 INK4B ) are described to specifically block the activity of CDK4/6 either by forming inactive ternary INK4-CDK4/6-cyclin D or binary INK4-CDK4/6 complexes. 4 The members of the CIP/KIP family (p21 CIP1/WAF1 , p27 KIP1 , and p57 KIP2 ) inhibit cyclin A-or cyclin E-associated CDK2 activity, but stabilize cyclin D-associated CDK4/6 activity. 5,6 In MKs, a high level of 2 CIP/KIP family members, p21 CIP1 and p27 KIP1 ,7,8 and 1 member of the INK4 family, p16 INK4A , 7 was detected...