Philippe Depeille

Reduced VEGF production, angiogenesis, and vascular regrowth contribute to the antitumor properties of dual mTORC1/mTORC2 inhibitors

The mammalian target of rapamycin (mTOR) pathway is implicated widely in cancer pathophysiology. Dual inhibition of the mTOR kinase complexes mTORC1 and mTORC2 decreases tumor xenograft growth in vivo and VEGF secretion in vitro, but the relationship between these two effects are unclear. In this study, we examined the effects of mTORC1/2 dual inhibition on VEGF production, tumor angiogenesis, vascular regression, and vascular regrowth, and we compared the effects of dual inhibition to mTORC1 inhibition alone. ATP-competitive inhibitors OSI-027 and OXA-01 targeted both mTORC1 and mTORC2 signaling in vitro and in vivo, unlike rapamycin that only inhibited mTORC1 signaling. OXA-01 reduced VEGF production in tumors in a manner associated with decreased vessel sprouting but little vascular regression. In contrast, rapamycin exerted less effect on tumoral production of VEGF. Treatment with the selective VEGFR inhibitor OSI-930 reduced vessel sprouting and caused substantial vascular regression in tumors. However, following discontinuation of OSI-930 administration tumor regrowth could be slowed by OXA-01 treatment. Combining dual inhibitors of mTORC1 and mTORC2 with a VEGFR2 inhibitor decreased tumor growth more than either inhibitor alone. Together, these results indicate that dual inhibition of mTORC1/2 exerts antiangiogenic and antitumoral effects that are even more efficacious when combined with a VEGFR antagonist.

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 Ca2+-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 Ca2+ sensor STIM1, which controls the magnitude of Ca2+ 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 Ca2+-dependent Erk signaling as a critical proapoptotic pathway that mediates the negative selection of B cells.

Anthrax Lethal Toxin Inhibits Growth of and Vascular Endothelial Growth Factor Release from Endothelial Cells Expressing the Human Herpes Virus 8 Viral G Protein–Coupled Receptor

Purpose: In this study, we tested the hypothesis that inhibition of mitogen-activated protein kinase kinases (MKK) inhibits tumor growth by acting on angiogenic signaling and by extension may form the basis of an effective strategy for treatment of Kaposis sarcoma. Experimental Design: Murine endothelial cells expressing the human herpes virus 8 G protein–coupled receptor (vGPCR-SVEC) were treated with anthrax lethal toxin (LeTx). LeTx is a binary toxin ordinarily secreted by Bacillus anthracis and is composed of two proteins: protective antigen (the binding moiety) and lethal factor (the active moiety). Lethal factor is a protease that cleaves and inactivates MKKs. Results:In vitro, treatment of vGPCR-SVEC with LeTx inhibited MKK signaling, moderately inhibited cell proliferation, and blocked the ability of these cells to form colonies in soft agar. Treatment with LeTx also blocked the ability of these cells to release several angioproliferative cytokines, notably vascular endothelial growth factor (VEGF). In contrast, inhibition of mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 with U0126 caused a substantial inhibition of proliferation but only modestly inhibited VEGF release. In xenograft models, i.v. injection of LeTx caused reduced tumor growth characterized immunohistochemically by inhibition of MKK signaling, decreased rates of proliferation, and reduced levels of VEGF and VEGF receptor 2, with a corresponding decrease in vascular density. Conclusions: These data support a role for MKK signaling in tumor growth and vascularization and are consistent with the hypothesis that inhibition of MKK signaling by LeTx or a similar agent may be an effective strategy for the treatment of Kaposis sarcoma as well as other vascular tumors.

RasGRP1 opposes proliferative EGFR-SOS1-Ras signals and restricts intestinal epithelial cell growth

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.

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.

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.

PLC-γ and PI3K Link Cytokines to ERK Activation in Hematopoietic Cells with Normal and Oncogenic Kras

Targeting signaling pathways upstream of oncogenic Ras may have therapeutic benefit in the treatment of leukemia. Target Upstream of Oncogenic Ras Members of the K-Ras family of small guanosine triphosphatases mediate signaling by cytokine and growth factor receptors to activate extracellular signal–regulated kinase (ERK), leading to cellular proliferation. Mutant K-Ras molecules, for example, K-RasG12D, accumulate in the active form and are associated with certain leukemias. Through flow cytometric analysis of phosphorylated proteins in mouse bone marrow cells, Diaz-Flores et al. showed that ERK activation downstream of K-RasG12D required cytokine receptor–dependent activation of phospholipase C–γ (PLC-γ) and phosphoinositide 3-kinase (PI3K) signaling. Treatment of mice with a clinically available PI3K inhibitor reduced ERK activation in cells expressing K-RasG12D, suggesting that molecules upstream of oncogenic Ras may provide therapeutic targets against some cancers. Oncogenic K-Ras proteins, such as K-RasG12D, accumulate in the active, guanosine triphosphate (GTP)–bound conformation and stimulate signaling through effector kinases. The presence of the K-RasG12D 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-RasG12D 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.

Abstract 2138: RasGRP3 mediates MAPK pathway activation in GNAQ mutant uveal melanoma

Uveal melanoma (UM) is the most common intraocular malignancy in adults and no effective treatment options are available for metastatic disease. Over 80% of UM show mutations in the Gαq family members GNAQ and GNA11. MAP-kinase pathway activation in part mediated by protein kinase C (PKC) has been shown as one critical contributing factor to GNAQ-mediated oncogenesis. However PKC inhibition alone does not completely suppress MAPK signaling. A more refined understanding of the signaling cascade linking MAPK signaling to mutant GNAQ or GNA11 is required to develop more effective strategies for targeted therapy. Among more than 10 different PKC isoforms, we identified both PKC δ and PKC e to be required and sufficient to activate MAPK pathway in GNAQ mutant melanomas by using siRNA mediated knock-down and co-transfection of GNAQ Q209L and specific PKC isoform cDNAs. Overexpression of GNAQ Q209L in 293FT cells increased Ras-GTP level and knock down of three Ras isoforms in GNAQ mutant uveal melanoma cell lines decreased MAPK signaling. Microarray analysis of 5 different GNAQ/11 mutant and 5 NRAS/BRAF-mutant melanoma cells revealed RasGRP3, a Ras-guanyl nucleotide exchange factor (RasGEF), ranks at the top of 487 differentially expressed genes between (cut off : p 2 or 100 fold) elevated RasGRP3 levels in GNAQ/11 mutant melanoma cells, while other RasGEFs were not significantly altered. Knock down of RasGRP3 decreased MAPK signaling and proliferation in GNAQ mutant melanoma cells, while it no effect on BRAF mutant cells. Mutating the PKC phosphorylation site of RasGRP3 (T133) partially attenuated RasGRP3-mediated MAPK signaling. Overexpression of PKC δ and PKC e increased RasGRP3 T133 phosphorylation but not PKC α and PKC ζ. While PKC inhibition completely abrogated RasGRP3 T133 phosphorylation, it did not fully suppress RasGRP3-mediated MAPK signaling, indicating that its activating effect only partly requires PKC. We found that RasGRP3 activation mediated by its diacylglycerol(DAG) binding C1 domain provides an independent conduit of activation, which is augmented by PKC-mediated phosphorylation at T133. DAG is a second messenger that is released by phospholipase C, a direct effector of activated GNAQ/11. Finally, we found that the markedly elevated RasGRP3 expression in GNAQ mutant melanoma cell lines is a direct consequence of the oncogenic signaling and is mediated by PKC δ and e. Hence, our data identify RasGRP3 as a critical signaling node linking oncogenic signaling downstream of GNAQ/11 to the Ras/Raf/MEK/ERK pathway through three mechanisms: phosphorylation by PKC δ and e, binding of the second messenger DAG, and by upregulation of its protein level as a consequence of oncogenic GNAQ-mediated activation of PKC δ and PKC e. The findings nominate RasGRP3 as a possible therapeutic target for cancers driven by oncogenic GNAQ/11. Citation Format: Xu Chen, Qiuxia Wu, Philippe Depeille, Jeroen P. Roose, Boris C. Bastian. RasGRP3 mediates MAPK pathway activation in GNAQ mutant uveal melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2138. doi:10.1158/1538-7445.AM2015-2138

Abstract LB-306: The Ras exchange factor RasGRP1 opposes proliferative EGFR-SOS1 Ras signals and restricts intestinal epithelial cell growth

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The character of EGFR signals can influence cell fate. Anti-EGFR treatment has been successful as cancer therapy but not in colorectal cancer (CRC) where activating somatic KRAS mutations (KRASMUT) are prevalent. While relevant to CRC, intestinal EGFR signaling is poorly understood, particularly in the context of KRASMUT. Here we show that two distinct Ras nucleotide exchange factors, RasGRP1 and SOS1, lie downstream of the EGFR but act in functional opposition to one another in the context of KRASMUT. RasGRP1 creates a negative feedback loop that limits EGFR-SOS1-Ras signals and restricts CRC growth. Genetic Rasgrp1 depletion from KRasMUT mice exacerbates intestinal epithelial cell growth and dysplasia, leading to a highly serrated colonic epithelium. Furthermore, low RasGRP1 expression in CRC patient specimens correlates with poor clinical outcome. The unexpected inhibitory role of EGFR-RasGRP1 signals in KRASMUT gastrointestinal malignancies reveals an intricacy of EGFR signaling that should be considered for future molecular therapeutics. Citation Format: Philippe Depeille, Linda M. Henricks, Robert van de Ven, Ed Lemmens, Mary Matli, Kevin M. Haigis, David Donner, Robert Warren, Jeroen P. Roose. The Ras exchange factor RasGRP1 opposes proliferative EGFR-SOS1 Ras signals and restricts intestinal epithelial cell growth. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-306. doi:10.1158/1538-7445.AM2014-LB-306

Abstract B1: Dual mTORC1/mTORC2 inhibition limits tumor growth, VEGF production and vascular regrowth

Inhibitors of VEGF signaling target tumor angiogenesis and inhibit tumor growth; however, the effects of antiangiogenic therapy are often transient. Cessation of treatment or dose interruption can induce rapid regrowth of tumor vessels and tumor relapse. Preclinical studies have demonstrated that upon withdrawal of VEGFR inhibitors, tumor vessels can rapidly repopulate the tumor on the tracks of the empty basement membrane sleeves (1,2). This rapid vessel regrowth is mediated, at least in part, by the interaction of VEGF produced by the tumor and VEGF receptors expressed on endothelial cells. Neutralization of the VEGF ligand combined with VEGFR inhibition may provide more complete VEGF signaling blockade, however the combination of bevacizumb, a monoclonal antibody to VEGF ligand, and sunitinib, a mulitkinase/pan-VEGFR inhibitor, has resulted in significant clinical toxicity (3). As an alternative approach, we reasoned that inhibition of VEGF production would decelerate the regrowth of tumor vessels following VEGFR inhibition. We sought to reduce VEGF production in the tumor by targeting the mechanism for VEGF translation using a selective inhibitor of mTOR. The mTOR kinase is a critical signaling hub which regulates multiple cellular networks including cap-dependent translation. The translation of many cellular growth factors, including VEGF isoforms, is regulated by the 4E-BP1 complex, which is in turn regulated by mTOR. ATP-competitive, selective inhibitors of mTORC1/mTORC2, OXA-01 and OSI-027 (ASP4786) can attenuate 4E-BP1 phosphorylation more completely than rapamycin, an allosteric inhibitor of mTORC1. In RIP-TAg mouse pancreatic tumors, OXA-01 dramatically reduced VEGF production whereas rapamycin, was less effective. Interestingly, OXA-01-mediated reduction in VEGF was associated with decreased vessel growth and normalization of the vascular architecture, but not vascular regression. In contrast, treatment with a selective inhibitor of VEGF receptors, OSI-930, resulted in substantial vascular regression but no decrease in tumor VEGF levels. Upon discontinuation of OSI-930 tumor vessels rapidly regrew, and this regrowth was diminished by OXA-01 treatment but not with rapamycin. Another key mTORC2 activity is regulation of the Akt signaling cascade, a central mediator of cellular survival. Inhibition of mTORC1/mTORC2 with OXA-01 induced tumor cell apoptosis in vivo, and this was enhanced when combined with the VEGFR inhibitor, OSI-930. In order to explore the clinical feasibility of this total VEGF blockade approach, we tested the combination of OSI-027, an mTORC1/mTORC2 inhibitor in clinical trials, and sunitinib, an approved multikinase/VEGFR2 inhibitor, in human tumor xenografts. The combination decreased the growth of human tumor xenografts to a greater degree than either single agent. Together these data demonstrate one mechanism for co-targeting angiogenic ligand production as well as inhibition of VEGFR signaling to provide maximal blockade of VEGF signaling and to limit vessel regrowth after cessation of the VEGFR inhibitor.