STIM1, PKC-δ and RasGRP set a threshold for proapoptotic Erk signaling during B cell development.

Clonal deletion of autoreactive B cells is crucial for the prevention of autoimmunity, but the signaling mechanisms that regulate this checkpoint remain undefined. Here we characterize a previously unrecognized Ca(2+)-driven pathway for activation of the kinase Erk, which was proapoptotic and biochemically distinct from Erk activation induced by diacylglycerol (DAG). This pathway required protein kinase C-δ (PKC-δ) and the guanine nucleotide-exchange factor RasGRP and depended on the concentration of the Ca(2+) sensor STIM1, which controls the magnitude of Ca(2+) entry. Developmental regulation of these proteins was associated with selective activation of the pathway in B cells prone to negative selection. This checkpoint was impaired in PKC-δ-deficient mice, which developed B cell autoimmunity. Conversely, overexpression of STIM1 conferred a competitive disadvantage to developing B cells. Our findings establish Ca(2+)-dependent Erk signaling as a critical proapoptotic pathway that mediates the negative selection of B cells

RasGRP3 Mediates MAPK Pathway Activation in GNAQ Mutant Uveal Melanoma

Constitutive activation of Gαq signaling by mutations in GNAQ or GNA11 occurs in over 80% of uveal melanomas (UMs) and activates MAPK. Protein kinase C (PKC) has been implicated as a link, but the mechanistic details remained unclear. We identified PKC δ and ɛ as required and sufficient to activate MAPK in GNAQ mutant melanomas. MAPK activation depends on Ras and is caused by RasGRP3, which is significantly and selectively overexpressed in response to GNAQ/11 mutation in UM. RasGRP3 activation occurs via PKC δ- and ɛ-dependent phosphorylation and PKC-independent, DAG-mediated membrane recruitment, possibly explaining the limited effect of PKC inhibitors to durably suppress MAPK in UM. The findings nominate RasGRP3 as a therapeutic target for cancers driven by oncogenic GNAQ/11

Rôle de l'expression coordonnée d'enzymes de détoxification impliquant le glutathion dans la chimiorésistance du mélanome malin

Le mélanome malin (MM) est un cancer à grand potentiel métastatique dont l'incidence ne cesse d'augmenter. Dans les formes disséminées, le traitement standard est la chimiothérapie par des agents alkylants et des vinca-alcaloïdes, mais les taux de réponses n'excédent pas 20%, en raison d'une faible chimiosensibilité de ce type de tumeur. Plusieurs mécanismes de chimiorésistance ont été décrits parmi lesquels la détoxification par des protéines utilisant le glutathion, les glutathion-S-transférases (GSTs, conjugaison du glutathion) et les multidrug resistance proteins (MRPs, transporteurs membranaires). La GSTP1, la GSTM1 et la MRP1 sont respectivement exprimées dans 100%, 40% et 50% des tumeurs de MM mais leur rôle est mal connu. Afin d'étudier l'implication des GSTs et des MRPs dans la chimiorésistance du MM, deux modèles cellulaires ont été établis : un modèle de surexpression de la GSTM1 et un modèle d'inhibition de la GSTP1 par un ARN anti-sens inductible. La surexpression de la GSTM1 induit une résistance aux vinca-alcaloïdes (vincristine, vindésine, vinorelbine et vinflunine) et au chlorambucil. La GSTM1 et la MRP1 agissent en synergie dans la résistance des cellules à la vincristine et à la vindésine. Par contre la résistance à la vinorelbine et à la vinflunine induite par l'expression de la GSTM1 est indépendante des activités des GSTM1, MRP1 et MAP kinases. L'inhibition de la GSTP1, par l'expression d'un ARN anti-sens, a permis de mettre en évidence une action combinée de la GSTP1 et de la MRP1 dans la résistance des cellules à l'étoposide (inhibiteur de topoisomérase II). En conclusion l'implication, parfois coordonnée, des GSTs et des MRPs dans la chimiorésistance tumorale semble être spécifique d'une part de la combinaison anticancéreux/isoforme d'enzyme de détoxification (GST et/ou MRP) et d'autre part du type cellulaire ou du tissu concerné.MONTPELLIER-BU Pharmacie (341722105) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Increased baseline RASGRP1 signals enhance stem cell fitness during native hematopoiesis.

Oncogenic mutations in RAS genes, like KRASG12D or NRASG12D, trap Ras in the active state and cause myeloproliferative disorder and T cell leukemia (T-ALL) when induced in the bone marrow via Mx1CRE. The RAS exchange factor RASGRP1 is frequently overexpressed in T-ALL patients. In T-ALL cell lines overexpression of RASGRP1 increases flux through the RASGTP/RasGDP cycle. Here we expanded RASGRP1 expression surveys in pediatric T-ALL and generated a RoLoRiG mouse model crossed to Mx1CRE to determine the consequences of induced RASGRP1 overexpression in primary hematopoietic cells. RASGRP1-overexpressing, GFP-positive cells outcompeted wild type cells and dominated the peripheral blood compartment over time. RASGRP1 overexpression bestows gain-of-function colony formation properties to bone marrow progenitors in medium containing limited growth factors. RASGRP1 overexpression enhances baseline mTOR-S6 signaling in the bone marrow, but not in vitro cytokine-induced signals. In agreement with these mechanistic findings, hRASGRP1-ires-EGFP enhances fitness of stem- and progenitor- cells, but only in the context of native hematopoiesis. RASGRP1 overexpression is distinct from KRASG12D or NRASG12D, does not cause acute leukemia on its own, and leukemia virus insertion frequencies predict that RASGRP1 overexpression can effectively cooperate with lesions in many other genes to cause acute T-ALL

Human iPS Cell-Derived Neurons Uncover the Impact of Increased Ras Signaling in Costello Syndrome

Increasing evidence implicates abnormal Ras signaling as a major contributor in neurodevelopmental disorders, yet how such signaling causes cortical pathogenesis is unknown. We examined the consequences of aberrant Ras signaling in the developing mouse brain and uncovered several critical phenotypes, including increased production of cortical neurons and morphological deficits. To determine whether these phenotypes are recapitulated in humans, we generated induced pluripotent stem (iPS) cell lines from patients with Costello syndrome (CS), a developmental disorder caused by abnormal Ras signaling and characterized by neurodevelopmental abnormalities, such as cognitive impairment and autism. Directed differentiation toward a neuroectodermal fate revealed an extended progenitor phase and subsequent increased production of cortical neurons. Morphological analysis of mature neurons revealed significantly altered neurite length and soma size in CS patients. This study demonstrates the synergy between mouse and human models and validates the use of iPS cells as a platform to study the underlying cellular pathologies resulting from signaling deficits.Significance statementIncreasing evidence implicates Ras signaling dysfunction as a major contributor in psychiatric and neurodevelopmental disorders, such as cognitive impairment and autism, but the underlying cortical cellular pathogenesis remains unclear. This study is the first to reveal human neuronal pathogenesis resulting from abnormal Ras signaling and provides insights into how these phenotypic abnormalities likely contribute to neurodevelopmental disorders. We also demonstrate the synergy between mouse and human models, thereby validating the use of iPS cells as a platform to study underlying cellular pathologies resulting from signaling deficits. Recapitulating human cellular pathologies in vitro facilitates the future high throughput screening of potential therapeutic agents that may reverse phenotypic and behavioral deficits

Human iPS Cell-Derived Neurons Uncover the Impact of Increased Ras Signaling in Costello Syndrome

Increasing evidence implicates abnormal Ras signaling as a major contributor in neurodevelopmental disorders, yet how such signaling causes cortical pathogenesis is unknown. We examined the consequences of aberrant Ras signaling in the developing mouse brain and uncovered several critical phenotypes, including increased production of cortical neurons and morphological deficits. To determine whether these phenotypes are recapitulated in humans, we generated induced pluripotent stem (iPS) cell lines from patients with Costello syndrome (CS), a developmental disorder caused by abnormal Ras signaling and characterized by neurodevelopmental abnormalities, such as cognitive impairment and autism. Directed differentiation toward a neuroectodermal fate revealed an extended progenitor phase and subsequent increased production of cortical neurons. Morphological analysis of mature neurons revealed significantly altered neurite length and soma size in CS patients. This study demonstrates the synergy between mouse and human models and validates the use of iPS cells as a platform to study the underlying cellular pathologies resulting from signaling deficits. SIGNIFICANCE STATEMENT Increasing evidence implicates Ras signaling dysfunction as a major contributor in psychiatric and neurodevelopmental disorders, such as cognitive impairment and autism, but the underlying cortical cellular pathogenesis remains unclear. This study is the first to reveal human neuronal pathogenesis resulting from abnormal Ras signaling and provides insights into how these phenotypic abnormalities likely contribute to neurodevelopmental disorders. We also demonstrate the synergy between mouse and human models, thereby validating the use of iPS cells as a platform to study underlying cellular pathologies resulting from signaling deficits. Recapitulating human cellular pathologies in vitro facilitates the future high throughput screening of potential therapeutic agents that may reverse phenotypic and behavioral deficits