Oncogenic mutations in RAS genes, like KRAS^G12D or NRAS^G12D, 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 KRAS^G12D or NRAS^G12D, 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.

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.

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.

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.

Oncogenic K-Ras proteins, such as K-Ras(G12D), accumulate in the active, guanosine triphosphate (GTP)-bound conformation and stimulate signaling through effector kinases. The presence of the K-Ras(G12D) oncoprotein at a similar abundance to that of endogenous wild-type K-Ras results in only minimal phosphorylation and activation of the canonical Raf-mitogen-activated or extracellular signal-regulated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) signaling cascades in primary hematopoietic cells, and these pathways remain dependent on growth factors for efficient activation. We showed that phospholipase C-γ (PLC-γ), PI3K, and their generated second messengers link activated cytokine receptors to Ras and ERK signaling in differentiated bone marrow cells and in a cell population enriched for leukemia stem cells. Cells expressing endogenous oncogenic K-Ras(G12D) remained dependent on the second messenger diacylglycerol for the efficient activation of Ras-ERK signaling. These data raise the unexpected possibility of therapeutically targeting proteins that function upstream of oncogenic Ras in cancer.

The character of EGFR signals can influence cell fate but mechanistic insights into intestinal EGFR-Ras signalling are limited. Here we show that two distinct Ras nucleotide exchange factors, RasGRP1 and SOS1, lie downstream of EGFR but act in functional opposition. RasGRP1 is expressed in intestinal crypts where it restricts epithelial growth. High RasGRP1 expression in colorectal cancer (CRC) patient samples correlates with a better clinical outcome. Biochemically, we find that RasGRP1 creates a negative feedback loop that limits proliferative EGFR-SOS1-Ras signals in CRC cells. Genetic Rasgrp1 depletion from mice with either an activating mutation in KRas or with aberrant Wnt signalling due to a mutation in Apc resulted in both cases in exacerbated Ras-ERK signalling and cell proliferation. The unexpected opposing cell biological effects of EGFR-RasGRP1 and EGFR-SOS1 signals in the same cell shed light on the intricacy of EGFR-Ras signalling in normal epithelium and carcinoma.

Colorectal cancer (CRC) is the third most frequent neoplastic disorder and is a main cause of tumor-related mortality as many patients progress to stage IV metastatic CRC. Standard care consists of combination chemotherapy (FOLFIRI or FOLFOX). Patients with WT KRAS typing are eligible to receive anti-EGFR therapy combined with chemotherapy. Unfortunately, predicting efficacy of CRC anti-EGFR therapy has remained challenging. Here we uncover that the EGFR-pathway component RasGRP1 acts as CRC tumor suppressor in the context of aberrant Wnt signaling. We find that RasGRP1 suppresses EGF-driven proliferation of colonic epithelial organoids. Having established that RasGRP1 dosage levels impacts biology, we focused on CRC patients next. Mining five different data platforms, we establish that RasGRP1 expression levels decrease with CRC progression and predict poor clinical outcome of patients. Lastly, deletion of one or two Rasgrp1 alleles makes CRC spheroids more susceptible to EGFR inhibition. Retrospective analysis of the CALGB80203 clinical trial shows that addition of anti-EGFR therapy to chemotherapy significantly improves outcome for CRC patients when tumors express low RasGRP1 suppressor levels. In sum, RasGRP1 is a unique biomarker positioned in the EGFR pathway and of potential relevance to anti-EGFR therapy for CRC patients.

Colorectal cancer (CRC) is the third most frequent neoplastic disorder and is a main cause of tumor-related mortality as many patients progress to stage IV metastatic CRC. Standard care consists of combination chemotherapy (FOLFIRI or FOLFOX). Patients with WT KRAS typing are eligible to receive anti-EGFR therapy combined with chemotherapy. Unfortunately, predicting efficacy of CRC anti-EGFR therapy has remained challenging. Here we uncover that the EGFR-pathway component RasGRP1 acts as CRC tumor suppressor in the context of aberrant Wnt signaling. We find that RasGRP1 suppresses EGF-driven proliferation of colonic epithelial organoids. Having established that RasGRP1 dosage levels impacts biology, we focused on CRC patients next. Mining five different data platforms, we establish that RasGRP1 expression levels decrease with CRC progression and predict poor clinical outcome of patients. Lastly, deletion of one or two Rasgrp1 alleles makes CRC spheroids more susceptible to EGFR inhibition. Retrospective analysis of the CALGB80203 clinical trial shows that addition of anti-EGFR therapy to chemotherapy significantly improves outcome for CRC patients when tumors express low RasGRP1 suppressor levels. In sum, RasGRP1 is a unique biomarker positioned in the EGFR pathway and of potential relevance to anti-EGFR therapy for CRC patients.

Modulation of Wnt signaling has untapped potential in regenerative medicine due to its essential functions in stem cell homeostasis. However, Wnt lipidation and Wnt-Frizzled (Fzd) cross-reactivity have hindered translational Wnt applications. Here, we designed and engineered water-soluble, Fzd subtype-specific "next-generation surrogate" (NGS) Wnts that hetero-dimerize Fzd and Lrp6. NGS Wnt supports long-term expansion of multiple different types of organoids, including kidney, colon, hepatocyte, ovarian, and breast. NGS Wnts are superior to Wnt3a conditioned media in organoid expansion and single-cell organoid outgrowth. Administration of Fzd subtype-specific NGS Wnt in vivo reveals that adult intestinal crypt proliferation can be promoted by agonism of Fzd5 and/or Fzd8 receptors, while a broad spectrum of Fzd receptors can induce liver zonation. Thus, NGS Wnts offer a unified organoid expansion protocol and a laboratory "tool kit" for dissecting the functions of Fzd subtypes in stem cell biology.