Ras-GTP Loading Research Articles

CalDAG-GEFIII controls Ca2+ entry via Rap1 and H-Ras, triggering distinct signaling pathways

Objective: Small GTPase Rap1 plays key functions in endothelium, including controlling Ca2+-dependent endothelial function (NO release). Our recent study suggests the Rap1A isoform restricts Ca2+ entry, to control eNOS activity. The activity of Rap1, a GDP to GTP switch, is controlled by Guanine Nucleotide Exchange Factors (GEFs). However, how Rap1 is activated in the context of Ca2+ signaling and regulation of eNOS activity is unknown. A Ca2+ and diacylglycerol (DAG)-activated CalDAG-GEFIII can activate Ha-Ras, R-Ras, and Rap1, but its functions in endothelial cells (ECs), and the signaling specificity of CalDAG-GEFIII through Rap1 and Ras pathways are not well understood. Our study aims to elucidate the role of CalDAG-GEFIII in regulating Ca2+ signaling in ECs. Approach and Results: To determine relative expression of Rap1 GEFs in ECs in different tissues, we reanalyzed the single-cell RNA sequencing (scRNA-Seq) data from eleven publicly available scRNA-seq datasets. We found RasGRP3 (encoding CalDAG-GEFIII) was highly expressed compared to other Rap1-GEFs in endothelial cells from different organs. To examine the involvement of CalDAG-GEFIII Rap1 and Ras activation in heart ECs, we determined the effect of on Vascular Endothelial Growth Factor (VEGF)-induced Rap1-GTP and Ras-GTP loading using pull-down assay. Knockdown of RasGRP3 blocked Rap1 and Ras activation downstream from VEGFR2, suggesting CalDAG-GEFIII acts as a GEF (activator) of both Rap1 and Ras in endothelial cells. To examine the involvement of CalDAG-GEFIII and H-Ras in Ca2+ signaling in ECs, we measured VEGF-induced intracellular Ca2+ using Fura2 dye, an intracellular Ca2+ indicator, in siRasGRP3, siH-Ras (most highly expressed Ras isoform) and siControl HUVECs. We found that Ca2+ release unchanged, but Ca2+ entry was increased in siRasGRP3 and siRas ECs, similarly to what we had found in siRap1A ECs. This suggests that Ras, like Rap1, limits ATP and TG-induced Ca2+ entry in HUVECs and that CalDAG-GEFIII may act upstream from both Rap1 and Ras. The elevated Ca2+ entry in siRap1A, siRasGRP3 ECs was partially normalized by exogenous expression of constitutively activated Rap1A or activation of Rap1 A by cAMP-dependent GEF, Epac. This indicates that Rap1A controls Ca2+ entry both acutely via a signaling mechanism, and at the transcript level. To determine the involvement of CalDAG-GEFIII in signaling by Rap1 and Ras, we determined the impact of siRNA knockdown of siRap1, siRas and siRasGRP3 on Ser1177 eNOS phosphorylation and Thr202/Tyr204 ERK phosphorylation in response to VEGF stimulation. Knockdown of RasGRP3 or Rap1 attenuated VEGF-induced Ser-1177 eNOS phosphorylation, while knockdown of RasGRP3 or Ras, but not Rap1, inhibited ERK phosphorylation in response to VEGF. Conclusions: Rap1 and Ras, activated downstream from CalDAG-GEFIII in response to VEGF activation, limit agonist-induced Ca2+ entry in endothelial cells. CalDAG-GEFIII promotes eNOS phosphorylation through Rap1B and promotes ERK phosphorylation via Ras, independent of Rap1. This suggests that CalDAG-GEFIII acts in two separate pathways, that are Rap1 and Ras dependent. NIH NHLBI: R01HL111582. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Contrasting Effects of Cancer-Associated Mutations in EphA3 and EphB2 Kinases.

Erythropoietin-producing hepatoma (Eph) receptors are a family of tyrosine kinases that can act as tumor promoters or tumor suppressors, depending on the receptor and cancer cell type. Cancer-associated somatic mutations have been identified in all Eph receptors, but in most cases, the functional effects of the mutations are unknown. In this study, we expressed and purified the kinase domains of wild-type (WT) EphA3 and EphB2 along with 16 cancer-associated mutants. We identified mutations that decrease EphA3 activity and both activating and inhibitory mutations in EphB2. To shed light on the mechanisms by which the mutations altered kinase activity, we measured the thermal stabilities of the enzymes and performed steady-state kinetic experiments. We also expressed the full-length receptors in HEK293T cells to determine the cellular effects. WT EphB2 promoted downstream ERK signaling, while a kinase-inactive mutant (S706F) was similar to the control cells. In contrast, WT EphA3 (but not loss-of-function mutants) inhibited ERK signaling. The reciprocal effects of EphB2 and EphA3 on ERK phosphorylation in HEK293T cells were also evident in Ras-GTP loading. Thus, consistent with the dual roles of Eph receptors as tumor promoters and tumor suppressors, somatic mutations have the potential to increase or decrease Eph function, resulting in changes in the downstream signaling transduction.

Abstract A025: Investigating the molecular mechanisms of regulation of the RAS guanine nucleotide exchange factor, SOS1 by Grb2 and 14-3-3

Abstract The MAPK/ERK signaling pathway is one of the most heavily mutated in human cancers. In normal cells, the stimulation of receptor tyrosine kinases (e.g. EGFR) on the plasma membrane signals the GTP-loading and activation of RAS through a mechanism catalyzed by the guanine nucleotide exchange factor (GEF) Son of Sevenless 1 (SOS1). RAS activation, in turn, initiates the RAF-MEK-ERK kinase cascade which triggers many downstream signals that can affect gene transcription, protein translation, cell proliferation, and feedback regulation. Several factors regulate SOS1 GEF activity, including two autoinhibitory domains within its own multi-domain architecture. First, the N-terminal domains of SOS1 occlude the allosteric RAS-GTP binding site in the RAS exchanger motif—thereby precluding SOS1 GEF activity—and this inhibition is relieved upon membrane recruitment and PIP2 phospholipid binding. Second, the C-terminal Proline-Rich Region (PRR) of SOS1 also occludes allosteric RAS-GTP binding by an unknown mechanism and is relieved when the growth factor receptor-bound protein 2 (Grb2) binds to motifs in the PRR. While the independent functions of these domains and interactions to inhibition and activation of GEF activity of SOS1 are known, the molecular mechanisms are poorly understood. An additional negative feedback loop involves the phosphorylation of the SOS1 PRR at two sites by the ribosomal protein S6 kinase 1 (RSK1), a substrate of downstream ERK. These modifications create 14-3-3 binding sites on SOS1, where 14-3-3 binding downregulates GEF activity, possibly by obstructing Grb2 association with the SOS1 PRR and membrane localization of SOS1. This work reconstitutes the SOS1:Grb2 and the SOS1:14-3-3 protein complexes in vitro to study the molecular mechanisms underlying SOS1 activity regulation. Citation Format: Orlando E. Martinez, Jawahar Sudhamsu. Investigating the molecular mechanisms of regulation of the RAS guanine nucleotide exchange factor, SOS1 by Grb2 and 14-3-3 [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A025.

Allosteric SHP2 Inhibitor RMC4550 Synergizes with Venetoclax in FLT3 and KIT Mutant Acute Myeloid Leukemia

Mutations in receptor tyrosine kinases (RTK) FLT3 and KIT occur frequently in Acute Myeloid Leukemia (AML) and are associated with high risk of relapse. FLT3 tyrosine kinase inhibitors (TKI) are clinically approved in AML, but resistance is common and involves emerging clones reliant on oncogenic signaling, particularly in the RAS/MAPK pathway. Patients who relapse on FLT3 TKIs have inauspicious prognoses and no specific therapeutic options, highlighting the unmet need for effective strategies to target oncogenic signaling and improve outcomes in relapsed/refractory (R/R) AML.The protein tyrosine phosphatase SHP2 (PTPN11) is a central node in RAS/MAPK activation downstream of various RTKs, including FLT3, acting as a scaffold for adaptor proteins that promote RAS-GTP loading. Novel allosteric inhibitors are being clinically investigated in cancers with signaling activating mutations.Here, we demonstrate that the allosteric SHP2 inhibitor RMC-4550 modulates expression of pro and anti-apoptotics in FLT3 and KIT mutant AML providing rationale for combinatorial targeting of SHP2 and BCL2 as a synergistic approach. We subsequently report the preclinical efficacy of RMC-4550 and the FDA-approved, BCL2 selective inhibitor, Venetoclax combination in both in vitro and in vivo AML models.We evaluated cell viability of multiple AML cell lines treated with RMC-4550. FLT3-ITD (Molm14, MV4-11) and KIT mutant (Kasumi1, SKNO1) lines were sensitive to SHP2 inhibition. RMC-4550 maintained its efficacy in FLT3-ITD Molm14 cells with secondary mutations in FLT3 tyrosine kinase domain (TKD) and in NRAS G12C. RMC-4550 biochemically represses pERK (Figure 1A) and transcriptionally downregulates mRNA expression of DUSP6 and anti-apoptotic BCL2 and MCL1.We functionally evaluated the mitochondrial outer membrane permeabilization (MOMP) in response to SHP2 inhibition using a dynamic iBH3 profiling assay. RMC-4550 increased the overall priming and the dependency on BCL2 in both Molm14 and MV4-11 cell lines (Figure 1A).To investigate the global transcriptomic changes induced by allosteric SHP2 inhibition, we performed total mRNA sequencing on Molm14, MV4-11 and SKNO1 cell lines. GSEA analysis revealed that RMC-4550 significantly upregulated expression of genes repressed by RAS activation, downregulated MYC targets, but also dysregulated genes mediating apoptosis. The most consistently upregulated pro-apoptotic gene was BMF (fold change: 4.39, FDR<0.001). BMF is a BH3-only protein found to be sequestered to motor filaments that, in response to cellular damage signals, is translocated in the cytoplasm and binds pro-survival Bcl2 proteins. The BMF transcript upregulation was confirmed by qPCR and western blot analysis showed a marked overexpression of the BMF protein level upon SHP2 inhibition, particularly in the cytoplasmic subcellular compartment (Figure 1B).We next treated Molm14, MV4-11, Kasumi and SKNO1 lines with incremental doses of RMC-4550 and Venetoclax in an 8x8 combination matrix to assess the synergy of the two compounds using cell viability and apoptosis readouts. The assay showed highly synergistic activity in both FLT3-ITD and KIT lines. Remarkably, we noted a potent synergy in Molm14 cells with concurrent mutation in NRAS G12C (Figure 1C).In a Molm14 cell line xenograft model, we demonstrated that the combination of RMC-4550 (30 mg/kg) and Venetoclax (100 mg/kg) administered orally 5 times a week for 28 days significantly decreased leukemia burden and improved survival (p<0.001) compared to control and single agents (Figure 1D).In a FLT3-ITD AML patient-derived xenograft (PDX) model, the combination of RMC-4550 and Venetoclax markedly decreased %hCD45 in both cardiac blood and spleen of NSGS mice compared to vehicle-treated control (Figure 1E).Supporting a potential therapeutic index for the combination, RMC-4550 and Venetoclax strongly inhibited colony formation in FLT3 AML primary samples compared to samples from healthy volunteers.Collectively, our data suggest that SHP2 inhibition increases the apoptotic dependency on BCL2 through up-regulation of the pro-apoptotic BMF, a mechanistic rationale to synergistically inhibit both targets. We provide preclinical evidence that co-targeting SHP2 and BCL2 is a potential effective therapeutic strategy in RTK-driven AML. [Display omitted] DisclosuresStahlhut: Revolutions Medicine: Current Employment, Current equity holder in publicly-traded company. Smith: Daiichi Sankyo: Consultancy; Amgen: Honoraria; AbbVie: Research Funding; Revolutions Medicine: Research Funding; FUJIFILM: Research Funding; Astellas Pharma: Consultancy, Research Funding.

Scaffold association factor B (SAFB) is required for expression of prenyltransferases and RAS membrane association

Inhibiting membrane association of RAS has long been considered a rational approach to anticancer therapy, which led to the development of farnesyltransferase inhibitors (FTIs). However, FTIs proved ineffective against KRAS-driven tumors. To reveal alternative therapeutic strategies, we carried out a genome-wide CRISPR-Cas9 screen designed to identify genes required for KRAS4B membrane association. We identified five enzymes in the prenylation pathway and SAFB, a nuclear protein with both DNA and RNA binding domains. Silencing SAFB led to marked mislocalization of all RAS isoforms as well as RAP1A but not RAB7A, a pattern that phenocopied silencing FNTA, the prenyltransferase α subunit shared by farnesyltransferase and geranylgeranyltransferase type I. We found that SAFB promoted RAS membrane association by controlling FNTA expression. SAFB knockdown decreased GTP loading of RAS, abrogated alternative prenylation, and sensitized RAS-mutant cells to growth inhibition by FTI. Our work establishes the prenylation pathway as paramount in KRAS membrane association, reveals a regulator of prenyltransferase expression, and suggests that reduction in FNTA expression may enhance the efficacy of FTIs.

Abstract 1889: Allosteric inhibition of SHP2 prevents adaptive resistance to MEK inhibition in neurofibromatosis type 1 associated malignant peripheral nerve sheath tumors

Abstract Introduction: Neurofibromatosis type 1 (NF1)-associated malignant peripheral nerve sheath tumors (MPNSTs) are malignant sarcomas occurring in approximately 15% of NF1 patients and always develop within benign precursor lesions (plexiform neurofibromas). The mortality caused by these tumors is high, approaching 100% in patients with unresectable, metastatic or recurrent disease. Targeted inhibition of the MAP kinase (MAPK) pathway has been attempted in clinical trials using MEK inhibitors (MEKi). While emerging data demonstrate effective tumor growth inhibition in benign plexiform neurofibromas, MEK inhibition blocks MPNST progression only to a limited extent. Combination strategies targeting both the MAPK and PI3K/AKT pathways (horizontal inhibition) have had limited clinical success due to excessive toxicities which precludes delivery of optimal therapeutic concentrations. Thus, novel approaches and complementary treatment strategies that are tolerable and prevent adaptive resistance mechanisms in NF1 MPNST are urgently needed. Methods: We have succeeded in establishing orthotopic in vivo models (patient-derived human xenografts-PDXs and genetically engineered mouse models- GEMMs) via implantation of tumor cells into the sciatic nerve, an approach that is critical, because it preserves the tumor microenvironment. Furthermore, we have optimized MPNST culture conditions, allowing us to establish robust primary cultures of MPNSTs while retaining key biological features of the original tumors. Summary of findings: Using this preclinical testing platform, we demonstrate that - 1. A feedback loop mediated by induction of receptor tyrosine kinase (RTK) signaling becomes hyperactivated upon MEK inhibition (MEKi). This subsequently activates the MAPK pathway to such an extent that MEKi alone is unable to completely block signaling to ERK, thereby maintaining tumor proliferation. 2. SHP2 is a protein tyrosine phosphatase (PTP) that functions as a positive signal transducer, acting between RTKs and RAS and thus, functions as physiological mediator for RAS activation. A treatment paradigm where MEKi is combined with SHP099, an allosteric SHP2 inhibitor to counteract the rebound increase in RTK mediated signaling to RAS-MEK-ERK results in effective attenuation of MEK-ERK signaling. In vivo studies demonstrate that the drug combination exhibits synergistic effects on tumor growth inhibition and significantly prolongs survival. 3. SHP099 disrupts a functional complex (SOS1/GRB2) which is essential to RAS-GTP loading and inhibits RAS-mediated downstream MAP kinase signaling in NF1 MPNST. Conclusions - Our results provide a mechanistic context for SHP2's precise role in the regulation of RAS-GTP and demonstrate that SHP2i can prevent adaptive resistance to MEKi. Given that allosteric SHP2 inhibitors are currently in clinical trials (NCT03114319), our studies can be rapidly translated into a clinical trial to evaluate a combination of SHP2i and MEKi as a novel treatment approach in NF1-MPNSTs. Citation Format: Sameer Farouk Sait, Kwan Tang, Rebecca Brown, Daochun Sun, Xunhua Xie, Benjamin Neel, Luis Parada. Allosteric inhibition of SHP2 prevents adaptive resistance to MEK inhibition in neurofibromatosis type 1 associated malignant peripheral nerve sheath tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1889.

A subset of flavaglines inhibits KRAS nanoclustering and activation.

The RAS oncogenes are frequently mutated in human cancers and among the three isoforms (KRAS, HRAS and NRAS), KRAS is the most frequently mutated oncogene. Here, we demonstrate that a subset of flavaglines, a class of natural anti-tumour drugs and chemical ligands of prohibitins, inhibit RAS GTP loading and oncogene activation in cells at nanomolar concentrations. Treatment with rocaglamide, the first discovered flavagline, inhibited the nanoclustering of KRAS, but not HRAS and NRAS, at specific phospholipid-enriched plasma membrane domains. We further demonstrate that plasma membrane-associated prohibitins directly interact with KRAS, phosphatidylserine and phosphatidic acid, and these interactions are disrupted by rocaglamide but not by the structurally related flavagline FL1. Depletion of prohibitin-1 phenocopied the rocaglamide-mediated effects on KRAS activation and stability. We also demonstrate that flavaglines inhibit the oncogenic growth of KRAS-mutated cells and that treatment with rocaglamide reduces non-small-cell lung carcinoma (NSCLC) tumour nodules in autochthonous KRAS-driven mouse models without severe side effects. Our data suggest that it will be promising to further develop flavagline derivatives as specific KRAS inhibitors for clinical applications.

Abstract B53: Differential response of distinct KRAS mutants to SHP2 inhibition

Abstract Introduction: RAS is mutated in 30% of tumors and is still considered “undruggable.” Among RAS genes KRAS is the most frequently mutated, mostly at residues G12, G13 and Q61, impairing its intrinsic and GAP-mediated GTP hydrolysis capacity (Prior et al., Cancer Res 2012). Nevertheless, specific mutations are differently distributed in human cancers (G12 89%, G13 9%, Q61 1%). In particular, G12D (36%) is prevalent over G12C (14%) and G12V (23%). Distinct oncogenic mutations differently affect RAS GTP hydrolysis capacity (Lu et al., Sci Rep 2016; Hunter et al., Mol Cancer Research 2015), with G12C preserving the highest GTPase activity and Q61 mutants being the most compromised. This implies that some KRAS mutants could be sensitive to upstream regulation, as suggested by the in vivo sensitivity of KRAS mutant tumors to SHP2 inhibitors (Mainardi et al. and Ruess et al., Nat Med 2018; Fedele et al., Cancer Discov 2018). In order to finely predict the response to the SHP2 inhibitor SHP099 (Chen et al., Nature 2016) in KRAS mutant patients, we used a panel of isogenic Rasless murine embryonic fibroblasts (MEFs) (Esposito et al., Semin Cancer Biol 2018) expressing human wild-type or mutant (G13D, G12C, G12D, G12V or Q61R) KRAS genes, and studied their response to SHP099, either alone or in combination with the MEK inhibitor AZD6244. Materials and Method: The panel of isogenic RAS-dependent MEFs was provided by the NCI RAS Initiative. In vitro, RAS-GTP loading was evaluated and dose-response curves generated based on cell viability assays. For xenograft experiments, 5x106 cells were subcutaneously injected in NSG mice. After tumor establishment (200mm3) mice were treated either with vehicle, SHP099 75mg/kg, AZD6244 25 mg/Kg or a combination of the last two. Results and Discussion: In vitro, we found that when cultured in 3% FCS, the most GTPase-proficient KRAS mutants (G13D and G12C) are more sensitive to SHP099 as compared to the most impaired (G12D and Q61R). Response to SHP099 was accompanied by a reduction in RAS-GTP loading. Surprisingly, in vivo results using xenograft models showed no correlation between residual GTPase activity of the mutants and sensitivity to SHP099. Nevertheless, synergy between SHP2 and MEK inhibitors treatment was more pronounced in the GTPase-proficent mutants, suggesting a possible correlation between residual intrinsic GTP hydrolysis capacity of the mutants and their resistance to MEK inhibitors through feedback activation of receptor tyrosine kinases (Sun et al., Cell Rep 2014). Conclusion: Our data confirm single-agent activity of SHP099 against proliferation driven by a subset of KRAS mutants, and suggest that mutants with higher residual GTPase capacity could better benefit from combined SHP2 and MEK inhibition. Citation Format: Sara Mainardi, Astrid Bosma, Antonio Mulero-Sánchez, René Bernards. Differential response of distinct KRAS mutants to SHP2 inhibition [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B53.

Abstract A21: Loss of Argonaute 2 leads to oncogene-induced senescence in mutant RAS-driven cancer

Abstract The RAS gene family is among the most commonly mutated genes within cancer, but little progress has been made in successfully targeting RAS mutations. Targeting binding partners of mutated RAS, however, presents a promising alternative therapeutic strategy. With the goal of uncovering novel interactors of RAS, we recently identified Argonaute 2 (AGO2) of the RNA-induced silencing complex (RISC) as a novel partner of the Switch II domain of KRAS. In order to assess the role of AGO2 in KRAS-G12D driven disease, we developed a mouse model of pancreatic cancer with conditional loss of AGO2. While AGO2 knockout did not prevent development of early precursor pancreatic intraepithelial (PanIN) lesions, loss of AGO2 prevented progression to late-stage PanINs, pancreactic ductal adenocarcinoma (PDAC), and metastatic disease. AGO2 null lesions displayed increased activation of the EGFR-RAS signaling axis during PanIN development. This signaling resulted in an increase in WT RAS-GTP activation, pEGFR-Y1068, and pERK levels leading to the development of oncogene-induced senescence in these PanIN lesions. Furthermore, we observed that EGFR-mediated phosphorylation of AGO2-Y393 disrupted the interaction between WT RAS and AGO2. This regulation by EGFR, however, was blocked in cells expressing mutant KRAS. These results suggested that the interaction of mutant RAS and AGO2 was vital to tumor development. To better assess the role of AGO2 loss in mutant RAS driven cancer, we performed AGO2 knockdown in multiple cell lines expressing mutations in either NRAS or HRAS isoforms. In each cell line, AGO2 directly interacted with KRAS, NRAS, and KRAS. In addition to suppressing growth in mutant RAS-driven cells (T24: HRAS-G12V, SK-MEL-2: NRAS-Q61H), loss of AGO2 produced marked increases in beta-galactosidase and p16 expression, as well as a decrease in cyclin D1, suggesting development of oncogene-induced senescence. Interestingly, upon AGO2 loss, cells displayed induction of pEGFR and pERK similar to what was observed in our pancreatic mouse model, and despite decreased expression of mutant RAS, WT RAS-GTP loading upon AGO2 loss was strongly induced. Together these results suggest a unique EGFR-AGO2-RAS signaling axis that requires AGO2-RAS interaction to prevent induction of oncogene-induced senescence in mutant RAS-driven cancers. Citation Format: Ronald F. Siebenaler, Sunita Shankar, Jean C. Tien, Vijaya L. Dommeti, Sylvia Zelenka-Wang, Jessica Waninger, Malay Mody, Seema Chugh, Chandan Kumar-Sinha, Arul M. Chinnaiyan. Loss of Argonaute 2 leads to oncogene-induced senescence in mutant RAS-driven cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A21.

BRAF inhibitors promote intermediate BRAF(V600E) conformations and binary interactions with activated RAS.

Oncogenic BRAF mutations initiate tumor formation by unleashing the autoinhibited kinase conformation and promoting RAS-decoupled proliferative RAF-MEK-ERK signaling. We have engineered luciferase-based biosensors to systematically track full-length BRAF conformations and interactions affected by tumorigenic kinase mutations and GTP loading of RAS. Binding of structurally diverse αC-helix-OUT BRAF inhibitors (BRAFi) showed differences in specificity and efficacy by shifting patient mutation-containing BRAF reporters from the definitive opened to more closed conformations. Unexpectedly, BRAFi engagement with the catalytic pocket of V600E-mutated BRAF stabilized an intermediate and inactive kinase conformation that enhanced binary RAS:RAF interactions, also independently of RAF dimerization in melanoma cells. We present evidence that the interference with RAS interactions and nanoclustering antagonizes the sequential formation of drug-induced RAS:RAF tetramers. This suggests a previously unappreciated allosteric effect of anticancer drug-driven intramolecular communication between the kinase and RAS-binding domains of mutated BRAF, which may further promote paradoxical kinase activation and drug resistance mechanisms.

Abstract 345: Novel RAS inhibitor, MCI-062, potently and selectively inhibits the growth of KRAS mutant pancreatic tumor cells by blocking GTP loading of RAS

Abstract Oncogenic mutations in RAS genes result in an intracellular elevation of active RAS and increased cellular propagation through downstream signaling pathways responsible for tumor cell growth and survival. Mutations in KRAS drive over 90% of pancreatic ductal adenocarcinomas (PDACs), presenting promising therapeutic potential for a RAS inhibitor. However, many have deemed RAS “undruggable” as a result of its relatively smooth structure and its high affinity for guanine nucleotide substrates. We have identified a novel series of indene derivatives that potently and selectively inhibits the growth of tumor cells with high levels of active RAS, while having minimal effects on tumor cells lacking constitutively active RAS, or cells derived from normal tissues. Here we report that our lead compound, MCI-062, potently and selectively inhibits the growth of KRAS mutant MIA PaCa-2 PDAC cells harboring oncogenic KRAS with an IC50 value of approximately 5 nM and greater than 300-fold selectivity over BxPC-3 PDAC cells that lack constitutively active RAS. MCI-062 also completely inhibits colony formation of a panel of PDAC cell lines with various KRAS mutations at low nanomolar concentrations. MCI-062 treatment of MIA PaCa-2 cells in basal and EGF-stimulated conditions depletes RAS-GTP levels in a dose-dependent manner. Consequently, MCI-062 treatment leads to inhibition of downstream MAPK and AKT signaling, cell cycle arrest, and induction of apoptosis in MIA PaCa-2 cells. Further investigation of the mechanism of action in cell-free systems demonstrated that this class of compounds inhibits GTP loading of RAS. MCI-062 inhibits binding of MANT-GTP to recombinant K-RAS in a dose-dependent manner. In a functional assay, MCI-062 reduces RAS-RAF-RBD binding when recombinant K-RAS is treated in a nucleotide-free state, but not when K-RAS is treated in a nucleotide-bound state. Testing in 3D spheroid models involving MIA PaCA-2 tumor cells revealed that MCI-062 potently inhibits growth of non-adherent cells. MCI-062 also has anti-tumor activity in a KRAS mutant CT26 mouse tumor xenograft model with no discernable toxicity. Western blotting of tumor lysates from mice treated with MCI-062 showed a reduction of active, GTP-bound RAS when compared to tumor lysates from control mice. These results provide in vitro and in vivo evidence that MCI-062 inhibits RAS-driven tumor cell growth by blocking GTP loading of RAS, supporting further evaluation of this novel class of RAS inhibitors for the treatment of pancreatic cancer, as well as other RAS-driven cancers. Funding provided by NCI grants R01CA131378, R01CA148817, R01CA197147, and R01CA155638. Citation Format: Tyler E. Mattox, Xi Chen, Jacob Valiyaveettil, Yulia Maxuitenko, Bing Zhu, Antonio Ward, Veronica Ramirez-Alcantara, Kristy Berry, Michael Boyd, Adam Keeton, Gary A. Piazza. Novel RAS inhibitor, MCI-062, potently and selectively inhibits the growth of KRAS mutant pancreatic tumor cells by blocking GTP loading of RAS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 345.

RAS nucleotide cycling underlies the SHP2 phosphatase dependence of mutant BRAF-, NF1- and RAS-driven cancers.

Oncogenic alterations in the RAS-RAF-MEK-ERK pathway drive the growth of a wide spectrum of cancers. While BRAF and MEK inhibitors are efficacious against BRAFV600E-driven cancers, effective targeted therapies are lacking for most cancers driven by other pathway alterations, including non-V600E oncogenic BRAF, RAS GTPase-activating protein (GAP) NF1 (neurofibromin 1) loss and oncogenic KRAS. Here, we show that targeting the PTPN11/SHP2 phosphatase with RMC-4550, a small molecule allosteric inhibitor, is effective in human cancer models bearing RAS-GTP dependent oncogenic BRAF (e.g. class 3 BRAF mutants), NF1 loss or nucleotide-cycling oncogenic RAS (e.g. KRASG12C). SHP2 inhibitor treatment decreases oncogenic RAS-RAF-MEK-ERK signaling and cancer growth by disrupting SOS1-mediated RAS-GTP loading. Our findings illuminate a critical function for SHP2 in promoting oncogenic RAS/MAPK pathway activation in cancers with RAS-GTP dependent oncogenic BRAF, NF1 loss and nucleotide-cycling oncogenic KRAS. SHP2 inhibition is a promising molecular therapeutic strategy for patients with cancers bearing these oncogenic drivers.

PO-019 PTPN11 is a therapeutic target in KRAS mutant lung cancer

IntroductionNon-Small-Cell Lung Cancer (NSCLC) accounts for approximately 85% of lung cancers, and mutations in the KRAS oncogene represent the most common driving event (Molina et al., Mayo Clin Proc 2008). So far, inhibition of the oncoprotein itself has proven difficult, and attempts to target downstream effectors have been hampered by the activation of compensatory resistance mechanisms (Bernards et al., Cell 2012). Notably, while MEK inhibition initially leads to ERK inactivation, P-ERK levels are quickly restored thanks to the upregulation of multiple growth factors receptors (Sun et al., Cell Reports 2014). Here we explored the possibility of targeting PTPN11 as a key mediator of receptor tyrosine kinase signalling in order to overcome intrinsic resistance to MEK inhibition.Material and methodsUsing a panel of 6 KRAS mutant NSCLC cell lines, we studied the biochemical and cell proliferation effect of combining the PTPN11 inhibitor SHP099 (Chen et al., Nature 2016) with the MEK inhibitor AZD6244. We confirmed our findings in CRISPR-based PTPN11 knockout clones of H2122 and H1944 cells. Importantly, we studied the effect of SHP099 in vivo in xenograft and PDX models, as well as in KRASLSLG12D p53fl/fl genetically engineered mouse model of NSCLC. To gain mechanistic insights into the effect of PTPN11 inhibition on mutant RAS activity, we used a panel of isogenic Rasless murine embryonic fibroblasts (Esposito et al., Semin Cancer Biol. 2018; NCI RAS Initiative) expressing human wild-type or mutant (G13D, G12C, G12D, G12V or Q61R) KRAS genes.Results and discussionsOur data show that depletion or inhibition of PTPN11 in KRAS mutant NSCLC cell lines invariably induces sensitivity to AZD6244. Surprisingly, in vivo models suggest that targeting PTPN11 is by itself sufficient to impair tumour growth, mainly due decreased RAS-GTP loading and induction of cellular senescence under growth factor-limiting conditions. Of note, our data suggest a correlation between the residual GTP hydrolysis capacity of the distinct KRAS mutants and their responsiveness to upstream regulators like PTPN11.ConclusionWhile in the past efforts have been focused on targeting KRAS downstream effectors, our data suggest that inhibiting upstream mediators like PTPN11 could be effective either alone or in combination with downstream inhibition. Our results suggest that targeting PTPN11 could be of clinical utility especially for tumours driven by KRAS mutants with high residual GTP hydrolysis capacity.

Abstract 3993: Efficacy of SHP2 phosphatase inhibition in cancers with nucleotide-cycling oncogenic RAS, NF1 loss and RAS-GTP-dependent oncogenic BRAF

Abstract Oncogenic alterations in the RAS-RAF-MEK-ERK pathway, including mutant forms of KRAS, BRAF, and loss of the tumor suppressor and RAS GTPase-activating protein (GAP) NF1, drive the growth of a wide spectrum of cancers. While BRAF and MEK inhibitors are efficacious against BRAFV600E-driven cancers, effective targeted therapies are lacking for most cancers driven by other pathway alterations, including oncogenic KRAS, NF1 loss, and non-V600E oncogenic BRAF. Here, we show that targeting the PTPN11/SHP2 phosphatase with a novel small molecule allosteric inhibitor is effective in human cancer models bearing nucleotide-cycling oncogenic RAS (e.g. KRASG12C), NF1 loss, or RAS-GTP dependent oncogenic BRAF (e.g. class 3 BRAF mutants). SHP2 inhibitor treatment decreases oncogenic RAS-RAF-MEK-ERK signaling and cancer growth by disrupting SOS1-mediated RAS-GTP loading. Our findings illuminate a critical function for SHP2 in promoting oncogenic RAS activation and downstream signaling in cancers with nucleotide-cycling oncogenic RAS, RAS-GTP dependent oncogenic BRAF, and NF1 loss. SHP2 inhibition is a promising molecular therapeutic strategy for patients with cancers bearing these oncogenic drivers. Citation Format: Trever G. Bivona, Robert Nichols, Franziska Haderk, Carlos Stahlhut,Christopher Schulze, Golzar Hemmati, David Wildes. Efficacy of SHP2 phosphatase inhibition in cancers with nucleotide-cycling oncogenic RAS, NF1 loss and RAS-GTP-dependent oncogenic BRAF [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3993.

Targeting prohibitins with chemical ligands inhibits KRAS-mediated lung tumours.

This corrects the article DOI: 10.1038/onc.2017.93.

Abstract 1007: SHP2 inhibition restores sensitivity to ALK inhibition in resistant ALK-rearranged non-small cell lung cancer (NSCLC)

Abstract Despite development of highly potent and selective inhibitors (e.g., ceritinib, alectinib, lorlatinib) targeting anaplastic lymphoma kinase (ALK), resistance invariably develops and limits the efficacy of these inhibitors in the clinic. The major classes of resistance are on-target genetic alterations (e.g., secondary ALK kinase domain mutations) and activation of alternative or bypass signaling pathways. While most patients are responsive to sequential treatment with two or more ALK inhibitors, ALK-independent resistance eventually emerges and leads to failure of further ALK-directed monotherapy. We used a synthetic lethal pooled shRNA screen to discover loss-of-function events that could sensitize resistant patient-derived cell lines to ALK inhibition. In addition to identifying known bypass targets such as FGFR, EGFR and SRC, we also identified PTPN11 (which encodes SHP2, a non-receptor protein tyrosine phosphatase that modulates signaling downstream of growth factor receptors) as a common hit shared by cell lines exhibiting different mechanisms of bypass activation. In parallel with the shRNA screen, we also performed a high throughput combination compound screen in the same patient-derived models, and identified activation of the same bypass signaling pathways. We showed that the highly potent and selective small-molecule SHP2 inhibitor SHP099 could sensitize resistant cell lines to ALK inhibition. In biochemical studies, co-targeting of ALK and SHP2 overcame resistance mediated by ALK-independent bypass mechanisms by decreasing RAS-GTP loading potential of cells and inhibiting phospho-ERK rebound. These results suggest that dual ALK and SHP2 inhibition may represent a new therapeutic strategy for ALK-positive patients, whose lung cancers have evolved ALK-independent mechanisms of resistance, including activation of bypass signaling pathways. Citation Format: Leila Dardaei, Hui Qin Wang, Paul Fordjour, Manrose Singh, Grainne Kerr, Satoshi Yoda, Jinsheng Liang, Yichen Cao, Yan Chen, Justin F. Gainor, Luc Friboulet, Ibiayi Dagogo-Jack, David T. Myers, Emma Labrot, David Ruddy, Melissa Parks, Dana Lee, Richard H. DiCecca, Susan Moody, Huaixiang Hao, Morvarid Mohseni, Matthew LaMarche, Juliet Williams, Keith Hoffmaster, Giordano Caponigro, Cyril H. Benes, Alice T. Shaw, Aaron N. Hata, Fang Li, Jeffrey A. Engelman. SHP2 inhibition restores sensitivity to ALK inhibition in resistant ALK-rearranged non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1007. doi:10.1158/1538-7445.AM2017-1007

Targeting prohibitins with chemical ligands inhibits KRAS-mediated lung tumours.

KRAS is one of the most frequently mutated oncogenes in human non-small cell lung cancers (NSCLCs). RAS proteins trigger multiple effector signalling pathways including the highly conserved RAF-MAPK pathway. CRAF, a direct RAS effector protein, is required for KRAS-mediated tumourigenesis. Thus, the molecular mechanisms driving the activation of CRAF are intensively studied. Prohibitin 1 (PHB1) is an evolutionarily conserved adaptor protein and interaction of CRAF with PHB1 at the plasma membrane is essential for CRAF activation. Here, we demonstrate that PHB1 is highly expressed in NSCLC patients and correlates with poor survival. Targeting of PHB1 with two chemical ligands (rocaglamide and fluorizoline) inhibits epidermal growth factor (EGF)/RAS-induced CRAF activation. Consistently, treatment with rocaglamide inhibited proliferation, migration and anchorage-independent growth of KRAS-mutated lung carcinoma cell lines. Surprisingly, rocaglamide treatment inhibited Ras-GTP loading in KRAS-mutated cells as well as in EGF-stimulated cells. Rocaglamide treatment further prevented the oncogenic growth of KRAS-driven lung cancer allografts and xenografts in mouse models. Our results suggest rocaglamide as a RAS inhibitor and that targeting plasma membrane-associated PHB1 with chemical ligands would be a viable therapeutic strategy to combat KRAS-mediated NSCLCs.

EGFR inhibition evokes innate drug resistance in lung cancer cells by preventing Akt activity and thus inactivating Ets-1 function

Nonsmall cell lung cancer (NSCLC) is the leading cause of cancer death worldwide. About 14% of NSCLCs harbor mutations in epidermal growth factor receptor (EGFR). Despite remarkable progress in treatment with tyrosine kinase inhibitors (TKIs), only 5% of patients achieve tumor reduction >90%. The limited primary responses are attributed partly to drug resistance inherent in the tumor cells before therapy begins. Recent reports showed that activation of receptor tyrosine kinases (RTKs) is an important determinant of this innate drug resistance. In contrast, we demonstrate that EGFR inhibition promotes innate drug resistance despite blockade of RTK activity in NSCLC cells. EGFR TKIs decrease both the mitogen-activated protein kinase (MAPK) and Akt protein kinase pathways for a short time, after which the Ras/MAPK pathway becomes reactivated. Akt inhibition selectively blocks the transcriptional activation of Ets-1, which inhibits its target gene, dual specificity phosphatase 6 (DUSP6), a negative regulator specific for ERK1/2. As a result, ERK1/2 is activated. Furthermore, elevated c-Src stimulates Ras GTP-loading and activates Raf and MEK kinases. These observations suggest that not only ERK1/2 but also Akt activity is essential to maintain Ets-1 in an active state. Therefore, despite high levels of ERK1/2, Ets-1 target genes including DUSP6 and cyclins D1, D3, and E2 remain suppressed by Akt inhibition. Reduction of DUSP6 in combination with elevated c-Src renews activation of the Ras/MAPK pathway, which enhances cell survival by accelerating Bim protein turnover. Thus, EGFR TKIs evoke innate drug resistance by preventing Akt activity and inactivating Ets-1 function in NSCLC cells.

Abstract 5486: Cancer therapeutics and the βGBP cytokine. Activation of death pathways prior to and after PI3K inhibition, and efficacy in vivo.

Abstract Phosphatidylinositol-3-OH kinase (PI3K) signaling is crucial to the viability of cancers and represents a selective target for therapeutic intervention. Our strategy against cancer is based on the use of a newly identified cytokine (βGBP), a natural inhibitor of both class IA and class IB PI3K that in cancer cells, but not in normal cells, is a potent activator of cell death: a novel therapeutic concept which carries no chemotherapeutic disadvantages and that can be tested and utilised in clinical trials. Cell death by Hu-r-βGBP follows two modes: 1) Apoptosis consequent to downregulation of signaling downstream of PI3K where inhibition of PI3K activity by Hu-r-βGBP has two major outcomes: suppression of Ras-GTP loading leading to block of ERK activation, and negation of akt gene expression leading to loss of Akt, conditions that either by blocking the ability of cancer cells to proliferate or by impairing their ability to survive erase oncogenicity in many cancer cells including those from prostate, HER2-driven and triple negative breast cancer. 2) Apoptosis consequent to induction of protein-protein interactions from βGBP receptor to PI3K where conformational changes conveyed by allosteric modulation from the βGBP receptor to p110 lead to changes in PIP2 spatial distribution at the plasma membrane, loss of PIP2-actin linkages, increased macromolecular mobility within the plane of the plasma membrane, clustering of death receptors and devastating death in Kras-mutant cancer cells, therapeutically reproducible in vivo. Citation Format: Livio Mallucci, Valerie Wells. Cancer therapeutics and the βGBP cytokine. Activation of death pathways prior to and after PI3K inhibition, and efficacy in vivo. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5486. doi:10.1158/1538-7445.AM2013-5486

N terminus of ASPP2 binds to Ras and enhances Ras/Raf/MEK/ERK activation to promote oncogene-induced senescence

The ASPP2 (also known as 53BP2L) tumor suppressor is a proapoptotic member of a family of p53 binding proteins that functions in part by enhancing p53-dependent apoptosis via its C-terminal p53-binding domain. Mounting evidence also suggests that ASPP2 harbors important nonapoptotic p53-independent functions. Structural studies identify a small G protein Ras-association domain in the ASPP2 N terminus. Because Ras-induced senescence is a barrier to tumor formation in normal cells, we investigated whether ASPP2 could bind Ras and stimulate the protein kinase Raf/MEK/ERK signaling cascade. We now show that ASPP2 binds to Ras-GTP at the plasma membrane and stimulates Ras-induced signaling and pERK1/2 levels via promoting Ras-GTP loading, B-Raf/C-Raf dimerization, and C-Raf phosphorylation. These functions require the ASPP2 N terminus because BBP (also known as 53BP2S), an alternatively spliced ASPP2 isoform lacking the N terminus, was defective in binding Ras-GTP and stimulating Raf/MEK/ERK signaling. Decreased ASPP2 levels attenuated H-RasV12-induced senescence in normal human fibroblasts and neonatal human epidermal keratinocytes. Together, our results reveal a mechanism for ASPP2 tumor suppressor function via direct interaction with Ras-GTP to stimulate Ras-induced senescence in nontransformed human cells.