Identification of regulators of polyploidization presents therapeutic targets for treatment of AMKL.

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. Our study implicates five networks of kinases that regulate the switch to polyploidy. Moreover, we find that dimethylfasudil (diMF, H-1152P) selectively increased polyploidization, mature cell-surface marker expression, and apoptosis of malignant megakaryocytes. An integrated target identification approach employing proteomic and shRNA screening revealed that a major target of diMF is Aurora kinase A (AURKA). We further find that MLN8237 (Alisertib), a selective inhibitor of AURKA, induced polyploidization and expression of mature megakaryocyte markers in acute megakaryocytic leukemia (AMKL) blasts and displayed potent anti-AMKL activity in vivo. Our findings provide a rationale to support clinical trials of MLN8237 and other inducers of polyploidization and differentiation in AMKL.

Chemical and Olfactory Analysis of the Volatile Fraction of Ocimum gratissimum Concrete from Madagascar.

The chemical composition of the volatile fraction of Ocimum gratissimum concrete (romba) from Madagascar has been determined for the first time by GC/MS and GC-FID. A methyl cinnamate chemotype has been determined for this material, along with a set of compounds typical in essential oils and extracts from plants of the Ocimum genus. Variability was mostly observed on terpenes and terpenoids components. GC-O-MS was also used for a sensory evaluation of this material performed by a master perfumer. The chemical composition of this O. gratissimum extract was then compared with literature data to assess subtle differences between chemotypes of the same species and other species of the same genus within natural variability. A mapping illustrates the occurrence of the cinnamate chemotype in Eastern Africa, India and now Madagascar, while other origins generally present eugenol, thymol, camphor, or linalool chemotypes.

Dysmegakaryopoiesis of FPD/AML pedigrees with constitutional RUNX1 mutations is linked to myosin II deregulated expression.

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.

Down-regulation of the RUNX1-target gene NR4A3 contributes to hematopoiesis deregulation in familial platelet disorder/acute myelogenous leukemia.

RUNX1 encodes a DNA-binding α subunit of the core-binding factor, a heterodimeric transcription factor. RUNX1 is a master regulatory gene in hematopoiesis and its disruption is one of the most common aberrations in acute leukemia. Inactivating or dominant-negative mutations in the RUNX1 gene have been also identified in pedigrees of familial platelet disorders with a variable propensity to develop acute myeloid leukemia (FPD/AML). We performed analysis of hematopoiesis from 2 FPD/AML pedigrees with 2 distinct RUNX1 germline mutations, that is, the R139X in a pedigree without AML and the R174Q mutation in a pedigree with AML. Both mutations induced a marked increase in the clonogenic potential of immature CD34(+)CD38(-) progenitors, with some self-renewal capacities observed only for R174Q mutation. This increased proliferation correlated with reduction in the expression of NR4A3, a gene previously implicated in leukemia development. We demonstrated that NR4A3 was a direct target of RUNX1 and that restoration of NR4A3 expression partially reduced the clonogenic potential of patient progenitors. We propose that the down-regulation of NR4A3 in RUNX1-mutated hematopoietic progenitors leads to an increase in the pool of cells susceptible to be hit by secondary leukemic genetic events.

Spirocyclic Compounds in Fragrance Chemistry: Synthesis and Olfactory Properties.

In this Review, both the scientific literature and patents have been analysed to gather, examine and classify synthetic strategies developed to access spirocyclic molecules having olfactory properties of interest for the perfume industry. New structures have been reported at a steady state since 1961 with a particularly intense activity in the 80's. In the diversity of olfactory properties observed, spirocyclic molecules were found to be quite frequently woody, as well as fruity and ambery, among other tonalities. More recent data reflect the evolution of modern synthetic organic chemistry towards bioinspiration, enantioselective reactions, green and sustainable chemistry and biotechnology.

MAL/SRF complex is involved in platelet formation and megakaryocyte migration by regulating MYL9 (MLC2) and MMP9.

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 alpha 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.

JAK inhibitors for the treatment of myeloproliferative neoplasms and other disorders.

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.

Downregulation of GATA1 drives impaired hematopoiesis in primary myelofibrosis.

Primary myelofibrosis (PMF) is a clonal hematologic malignancy characterized by BM fibrosis, extramedullary hematopoiesis, circulating CD34+ cells, splenomegaly, and a propensity to evolve to acute myeloid leukemia. Moreover, the spleen and BM of patients harbor atypical, clustered megakaryocytes, which contribute to the disease by secreting profibrotic cytokines. Here, we have revealed that megakaryocytes in PMF show impaired maturation that is associated with reduced GATA1 protein. In investigating the cause of GATA1 downregulation, our gene-expression study revealed the presence of the RPS14-deficient gene signature, which is associated with defective ribosomal protein function and linked to the erythroid lineage in 5q deletion myelodysplastic syndrome. Surprisingly, reduced GATA1 expression and impaired differentiation were limited to megakaryocytes, consistent with a proproliferative effect of a GATA1 deficiency on this lineage. Importantly, expression of GATA1 effectively rescued maturation of PMF megakaryocytes. Together, these results suggest that ribosomal deficiency contributes to impaired megakaryopoiesis in myeloproliferative neoplasms.

Targeting megakaryocytic-induced fibrosis in myeloproliferative neoplasms by AURKA inhibition.

Primary myelofibrosis (PMF) is characterized by bone marrow fibrosis, myeloproliferation, extramedullary hematopoiesis, splenomegaly and leukemic progression. Moreover, the bone marrow and spleens of individuals with PMF contain large numbers of atypical megakaryocytes that are postulated to contribute to fibrosis through the release of cytokines, including transforming growth factor (TGF)-ß. Although the Janus kinase inhibitor ruxolitinib provides symptomatic relief, it does not reduce the mutant allele burden or substantially reverse fibrosis. Here we show through pharmacologic and genetic studies that aurora kinase A (AURKA) represents a new therapeutic target in PMF. Treatment with MLN8237, a selective AURKA inhibitor, promoted polyploidization and differentiation of megakaryocytes with PMF-associated mutations and had potent antifibrotic and antitumor activity in vivo in mouse models of PMF. Moreover, heterozygous deletion of Aurka was sufficient to ameliorate fibrosis and other PMF features in vivo. Our data suggest that megakaryocytes drive fibrosis in PMF and that targeting them with AURKA inhibitors has the potential to provide therapeutic benefit.

p19 INK4d controls hematopoietic stem cells in a cell-autonomous manner during genotoxic stress and through the microenvironment during aging.

Hematopoietic stem cells (HSCs) are characterized by the capacity for self-renewal and the ability to reconstitute the entire hematopoietic compartment. Thrombopoietin maintains adult HSCs in a quiescent state through the induction of cell cycle inhibitors p57(Kip2) and p19(INK4d). Using the p19(INK4d-/-) mouse model, we investigated the role of p19(INK4d) in basal and stress-induced hematopoiesis. We demonstrate that p19(INK4d) is involved in the regulation of HSC quiescence by inhibition of the G0/G1 cell cycle transition. Under genotoxic stress conditions, the absence of p19(INK4d) in HSCs leads to accelerated cell cycle exit, accumulation of DNA double-strand breaks, and apoptosis when cells progress to the S/G2-M stages of the cell cycle. Moreover, p19(INK4d) controls the HSC microenvironment through negative regulation of megakaryopoiesis. Deletion of p19(INK4d) results in megakaryocyte hyperproliferation and increased transforming growth factor ß1 secretion. This leads to fibrosis in the bone marrow and spleen, followed by loss of HSCs during aging.

P19INK4D links endomitotic arrest and megakaryocyte maturation and is regulated by AML-1.

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) overexpression in CD34(+) cells resulted in a decrease in mean ploidy level associated with an increase in CD41 and CD42 expression in each ploidy class. Confirming these in vitro results, bone marrow MKs from p19(INK4D) KO mice exhibited an increase in mean ploidy level from 18.7N (+/- 0.58N) to 52.7N (+/- 12.3N). Chromatin immunoprecipitation assays performed in human MKs revealed that AML-1 binds in vivo the p19(INK4D) promoter. Moreover, AML-1 inhibition led to the p19(INK4D) down-regulation in human MKs. These results may explain the molecular link at the transcriptional level between the arrest of endomitosis and the acceleration of MK differentiation.