RAS GTPases mediate a wide variety of cellular functions, including cell proliferation, survival, and differentiation. Recent studies have revealed that germline mutations and mosaicism for classical RAS mutations, including those in HRAS, KRAS, and NRAS, cause a wide spectrum of genetic disorders. These include Noonan syndrome and related disorders (RAS/mitogen-activated protein kinase [RAS/MAPK] pathway syndromes, or RASopathies), nevus sebaceous, and Schimmelpenning syndrome. In the present study, we identified a total of nine missense, nonsynonymous mutations in RIT1, encoding a member of the RAS subfamily, in 17 of 180 individuals (9%) with Noonan syndrome or a related condition but with no detectable mutations in known Noonan-related genes. Clinical manifestations in the RIT1-mutation-positive individuals are consistent with those of Noonan syndrome, which is characterized by distinctive facial features, short stature, and congenital heart defects. Seventy percent of mutation-positive individuals presented with hypertrophic cardiomyopathy; this frequency is high relative to the overall 20% incidence in individuals with Noonan syndrome. Luciferase assays in NIH 3T3 cells showed that five RIT1 alterations identified in children with Noonan syndrome enhanced ELK1 transactivation. The introduction of mRNAs of mutant RIT1 into 1-cell-stage zebrafish embryos was found to result in a significant increase of embryos with craniofacial abnormalities, incomplete looping, a hypoplastic chamber in the heart, and an elongated yolk sac. These results demonstrate that gain-of-function mutations in RIT1 cause Noonan syndrome and show a similar biological effect to mutations in other RASopathy-related genes.

Heartbeat is required for normal development of the heart, and perturbation of intracardiac flow leads to morphological defects resembling congenital heart diseases. These observations implicate intracardiac haemodynamics in cardiogenesis, but the signalling cascades connecting physical forces, gene expression and morphogenesis are largely unknown. Here we use a zebrafish model to show that the microRNA, miR-21, is crucial for regulation of heart valve formation. Expression of miR-21 is rapidly switched on and off by blood flow. Vasoconstriction and increasing shear stress induce ectopic expression of miR-21 in the head vasculature and heart. Flow-dependent expression of mir-21 governs valvulogenesis by regulating the expression of the same targets as mouse/human miR-21 (sprouty, pdcd4, ptenb) and induces cell proliferation in the valve-forming endocardium at constrictions in the heart tube where shear stress is highest. We conclude that miR-21 is a central component of a flow-controlled mechanotransduction system in a physicogenetic regulatory loop.

Cardiogenesis proceeds with concomitant changes in hemodynamics to accommodate the circulatory demands of developing organs and tissues. In adults, circulatory adaptation is critical for the homeostatic regulation of blood circulation. In these hemodynamics-dependent processes of morphogenesis and adaptation, a mechanotransduction pathway, which converts mechanical stimuli into biological outputs, plays an essential role, although its molecular nature is largely unknown. Here, we report that expression of zebrafish miR-143 is dependent on heartbeat. Knocking-down miR-143 results in de-repression of retinoic acid signaling, and produces abnormalities in the outflow tracts and ventricles. Our data uncover a novel epigenetic link between heartbeat and cardiac development, with miR-143 as an essential component of the mechanotransduction cascade.

No abstract

Enhancer of zeste homolog (EZH) 1 and its close homolog EZH2 are component of polycomb repressive complex 2 (PRC2), and play a partially redundant and crucial role for the maintenance of transcriptional repression by tri-methylating histone H3 lysine 27 (H3K27). Hyper tri-methylation of H3K27 have been associated with lymphoma and myeloma progression, suggesting PRC2 is a therapeutic target for hematological malignancies. We have developed a novel EZH1 and EZH2 dual inhibitor valemetostat (DS-3201b), which simultaneously inhibited the enzymatic activity of EZH1 and EZH2 in nano-molar concentration. Valemetostat demonstrated anti-proliferative activities against the Activated B-cell-like (ABC) and Germinal Center B-cell-like (GCB) subtypes of Diffuse Large B-cell Lymphoma (DLBCL) cells. Furthermore, valemetostat induced apoptosis in DLBCL cell lines, regardless of subtype. We revealed that the pleiotropic effects of valemetostat on the expression levels of B-cell receptor signaling molecules by western blotting analysis. In particular, valemetostat suppressed the expression level of BCL6 protein, a key oncogene in B cell lymphoma. Transcriptome analysis of 16 DLBCL cell lines using RNA sequencing suggested that tumor suppressor genes, DNA damage response related genes and cell cycle related genes were affected by valemetostat treatment. In particular, valemetostat down regulated c-myc signaling in valemetostat-sensitive cells. Valemetostat also demonstrated synergistic anti-tumor activity with standard of care therapy against a DLBCL cell line KARPAS-422 xenografted model. In conclusion, our results suggested that valemetostat has therapeutic activity in DLBCL cells by inhibiting B-cell receptor signaling and c-myc signaling pathway. A phase 1 clinical study of valemetostat mono-therapy is now ongoing in patients with non-Hodgkin lymphoma including DLBCL (Clinical trial information: NCT02732275). Disclosures Hama: Daiichi Sankyo Co., Ltd.: Employment. Banjo:Daiichi Sankyo Co., Ltd.: Employment. Honma:Daiichi Sankyo Co., Ltd.: Employment. Takata:Daiichi Sankyo Co., Ltd.: Employment. Nosaka:Daiichi Sankyo Co., Ltd.: Employment. Shiroishi:Daiichi Sankyo Co., Ltd.: Employment. Watanabe:Daiichi Sankyo Co., Ltd.: Employment. Yamamoto:Daiichi Sankyo RD Novare Co., Ltd.: Employment. Hirata:Daiichi Sankyo RD Novare Co., Ltd.: Employment. Nakano:Daiichi Sankyo RD Novare Co., Ltd.: Employment. Inaki:Daiichi Sankyo Co., Ltd.: Employment. Goto:Daiichi Sankyo RD Novare Co., Ltd.: Employment. Totoki:Daiichi Sankyo RD Novare Co., Ltd.: Employment. Kataoka:Daiichi Sankyo RD Novare Co., Ltd.: Employment. Lim:Daiichi Sankyo RD Novare Co., Ltd.: Employment. Wada:Daiichi Sankyo RD Novare Co., Ltd.: Employment. Kumazawa:Daiichi Sankyo RD Novare Co., Ltd.: Employment. Tsutsumi:Daiichi Sankyo Co., Ltd.: Employment.