KRAS Activation Research Articles

Abstract 657: Discovery of a novel brain penetrant SHP2 allosteric inhibitor with anti-tumor effects

Abstract KRAS is the most frequently dysregulated oncogene with high prevalence in non-small cell lung cancer (NSCLC), colorectal cancer (CRC), and pancreatic ductal adenocarcinoma (PDAC). Currently, FDA-approved sotorasib and adagrasib provide ground breaking through therapies for cancer patients harboring KRAS G12C mutation. However, there is still high unmet medical needs for inhibitors targeting broader KRAS-driven tumor, especially a substantial unmet medical need for agents that can penetrate BBB to control the brain metastases. SHP2, a key activator of KRAS located in the upstream of RAS pathway, facilitates the conversion of GDP-bound KRAS (off) state to GTP-bound KRAS (on) state serving as a key mechanism of KRAS activation. Therefore, SHP2 could be a promising therapeutic target to shut down the oncogenic RAS pathway as a broad combination partner with RAS/RAF/MEK/ERK pathway inhibitors. Here we report a highly potent and selective brain penetrant SHP2 inhibitor that is designed to target solid tumors metastazied to brain. KT-01766 showed excellent potency against SHP2 protein in various biochemical and cellular assays and showed the robust synergistic effect with RAS pathway inhibitors. KT-01766 as a mono therapy and in combination with KRAS G12C inhibitors demonstrated great anti-tumor efficacy with no apparent toxicity in mouse xenograft models harboring KRAS mutations. Furthermore, robust anti-tumor acitivty was confirmed in intracranial brain tumor model, supporting its potential to control brain-metastasized tumors. The present data suggest that KT-01766 is a potential candidate for combination therapy with agents targeting RAS pathway, as it is able to effectively suppress tumor growth both systemically and within the brain in KRAS mutated cancers. Citation Format: Miyeon Kim, Dongsu Kim, Yeejin Jeon, Kyeong Jin Yoon, Anna Jang, Mijung Lee, Dahye Jeon, Soyeon Jang, Jinhwan Kim, Eunji Kim, Jihoon Park, Victor Hong, Hayoun Jung, Sungpil Choi. Discovery of a novel brain penetrant SHP2 allosteric inhibitor with anti-tumor effects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 657.

Abstract 1596: Disrupting local immunosuppression by combined myCAF/myeloid targeting in pancreatic cancer

Abstract Immunotherapy has revolutionized clinical care for many patients, but some cancers, such as pancreatic ductal adenocarcinoma (PDAC) remain stubbornly resistant. Local immunosuppression (LIS) is one of the striking hallmarks of PDAC due to the combined effects of multiple different immunosuppressive cell types in the tumor microenvironment (TME). There is great need for an increased understanding of the cellular crosstalk within tumors to understand how stromal cells coordinate in their suppression of immune responses. Oncogenic KRAS activation in tumor cells promotes the invasion and proliferation of tumor-supporting stromal cells, while excluding cancer-targeted cytotoxic T cells. Prior attempts to reverse LIS in PDAC by targeting individual stromal cell populations have been unsuccessful, alluding to the contributions of multiple distinct cell types. Here we examine the interactions of cancer-associated fibroblasts (CAFs) and myeloid-derived suppressor cells (MDSCs) and how they coordinate to suppress immune responses in the PDAC TME. We interrogate PDAC stromal cell biology by using targeted therapies to perturb various cell populations both in vivo using genetically engineered mouse models, and ex vivo using PDAC tumor explants. Explants are short-term slice cultures that enable experimental study of intact tumor sections with a full complement of cell types. Importantly, PDAC explants maintain their histopathological architecture and cellular diversity over time. This medium-throughput platform allows for testing of multiple drugs and mechanistic hypotheses in the native PDAC TME. We show in preliminary data that Smoothened inhibition (SMOi) decreases the proliferation and activity of myCAFs, but provokes the expansion of CD11b-positive myeloid cells in vivo. Thus, we hypothesize that LIS in PDAC is maintained by a balance between myCAFs and myeloid cells, preventing effective T cell invasion. Single cell RNA-seq data comparing ctrl vs. SMOi-treated murine PDAC elucidates stromal subpopulations involved in the LIS phenotype and guides the identification of myeloid subtypes emerging after SMOi. Strikingly, we demonstrated that simultaneous SMOi and targeting myeloid cells via anti-Gr1 or CCR1 inhibition significantly elevates cytotoxic T cell numbers within the TME. We are currently investigating whether the activity of these T cells may be further potentiated through combination with immunomodulatory agents. By testing various treatment combination in the same TME, we will identify the best synergistic effects for future immunotherapy approaches in human PDAC. In summary, we are elucidating the complex mechanism behind LIS in PDAC by employing our novel explant culture system alongside in vivo studies. We aim to develop a translatable regimen to neutralize LIS, reactivating the cytotoxic T cells in the tumor periphery to invade, proliferate, and attack cancer cells. Citation Format: Marie C. Hasselluhn, Dafydd Thomas, Lukas Vlahos, Alvaro Curiel-Garcia, Amanda R. Decker-Farrell, Tanner C. Dalton, Stephen A. Sastra, Carmine F. Palermo, Andrea Califano, Kenneth P. Olive. Disrupting local immunosuppression by combined myCAF/myeloid targeting in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1596.

Abstract 1842: Global proteomics reveals integrin signaling as a driver of infiltration in glioblastoma

Abstract Background: Glioblastoma (GBM), a notably aggressive brain cancer, presents significant treatment challenges due to its propensity for deep, diffuse infiltration into healthy brain tissue. This trait enables GBM to resist and survive following surgery and conventional adjuvant therapies. Emerging evidence suggests that signaling between GBM stem cells (GSCs) may be instrumental in this invasive behavior. Our study aimed to unravel the intricacies of these intercellular communication networks within glioblastoma by employing comprehensive proteomic profiling across a diverse array of patient-derived GSC lines. Methods: We conducted our study using a sex-stratified cohort comprising 17 patient-derived GSC lines. Proteomic profiling encompassed analyses of both GSC lysates and conditioned media under various oxygen conditions (normoxia at 20% O2, hypoxia at 1% and 0.2% O2). Additionally, we incorporated the glioma cerebral organoid (GLICO) system, facilitating co-cultures of GSCs with human cerebral organoids. For in-depth proteomic analysis, we utilized advanced TMT-based liquid chromatography-tandem mass spectrometry (LC-MS/MS; Q-Exactive, Thermo Fisher). Results: Our findings corroborate that GSCs adhere to the tripartite model of proliferation, invasion, and hypoxic response. A salient discovery was the consistent enrichment of integrin signaling pathways in GSCs under hypoxic conditions. Specifically, we observed a significant association between upregulation in integrin signaling (BIOCARTA_INTEGRIN_PATHWAY) and elevated KRAS activity (SWEET_KRAS_TARGETS_UP: r=0.43, p<0.01), as well as hypoxic responses (WINTER_HYPOXIA_UP: r=0.76, p<0.0001) in GSCs. Notably, in the GLICO model, both integrin signaling and KRAS pathways were markedly upregulated in the presence of GSCs compared to cerebral organoids alone (p<0.05). Conclusions: This research highlights the pivotal role of integrin signaling in mediating the invasive attributes of glioblastoma. The data underscore the importance of further exploring key modulators within the integrin signaling pathway, presenting a promising avenue for the discovery of novel therapeutic targets in glioblastoma treatment. Citation Format: Lennart J. van Winden, Rifat Shahriar Sajid, Okty Abbasi Borhani, Evelyn Rose Kamski-Hennekam, Nakita Gopal, Alberto Leon, Phedias Diamandis. Global proteomics reveals integrin signaling as a driver of infiltration in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1842.

Abstract 6503: D3S-001, a second-generation GDP-bound KRAS G12C inhibitor, overcomes nucleotide cycling and demonstrates robust preclinical and clinical activities

Abstract First-generation KRAS G12C inhibitors [G12Ci(s)], such as sotorasib and adagrasib, are limited by the depth and duration of clinical responses. One potential explanation for their modest clinical activity is the dynamic “cycling” of KRAS between its GDP- and GTP-bound states, raising controversy whether targeting the GDP-bound form can fully block this oncogenic driver. We herein report D3S-001, a second-generation GDP-bound G12Ci with faster target engagement (TE) kinetics, depletes cellular active KRAS G12C at sub-nanomolar concentrations, overcomes nucleotide cycling, and demonstrates durable anti-tumor activities preclinically and in a phase 1 clinical trial (NCT05410145). Covalent efficiency and TE kinetics were measured by SPR, LC-MS and ELISA. Anti-tumor efficacy was assessed in cell line-derived xenograft (CDX) and patient-derived xenograft or organoid (PDX or PDO) models. Activity in brain metastatic tumors and tumors with acquired resistance to sotorasib was investigated. Preclinically, D3S-001 exhibited exceptionally high covalent efficiency with a kinact/KI of 1.43 × 106 M−1s−1, the highest among all reported G12Ci(s) to date and a 20- to 70-fold improvement over adagrasib and sotorasib, respectively. In whole cells, D3S-001 depleted GTP-bound active KRAS G12C with an inactivation t1/2 of 5.8 minutes, allowing >95% TE (5x t1/2) in 30 minutes which is within the time required for epithelial growth factor (EGF) to induce KRAS GDP to GTP transition. By contrast, sotorasib and adagrasib required 3.7 and 2.8 hours, respectively, to reach 95% TE. In the presence of EGF, the ability of sotorasib and adagrasib to block KRAS was compromised by over 55%, whereas TE kinetics of D3S-001 was nearly unaffected, a unique feature differentiating D3S-001 from earlier GDP-bound G12Ci(s). The covalent efficiency of D3S-001 correlated with robust anti-tumor activity at sub-nanomolar concentrations and durable tumor regressions in CDX and PDX/PDO models of different cancer types. In a sotorasib-resistant PDX model, D3S-001 exhibited robust efficacy of tumor regression, while sotorasib and adagrasib showed no effect. Treatment with D3S-001 also resulted in sustained intracranial tumor remission in brain metastasis models. In the first-in-human trial of D3S-001, durable RECIST responses in patients were observed across all dose cohorts from 50 mg to 900 mg QD. Durable intracranial activity was also observed. Response rate and duration are reported in a companion clinical trial abstract submitted to this AACR meeting. In conclusion, D3S-001 is a second-generation GDP-bound KRAS G12Ci that overcomes GDP-to-GTP cycling. Its high covalent efficiency and rapid TE kinetics correlated with robust anti-tumor activity preclinically and translated into promising clinical activity with a high response rate and durable responses in a phase 1 trial. Citation Format: Jing Zhang, Sun Min Lim, Mi Ra Yu, Cheng Chen, Jia Wang, Wenqian Wang, Haopeng Rui, Jingtao Lu, Shun Lu, Tony Mok, Zhi Jian Chen, Byoung Chul Cho. D3S-001, a second-generation GDP-bound KRAS G12C inhibitor, overcomes nucleotide cycling and demonstrates robust preclinical and clinical activities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6503.

Mutations influence the conformational dynamics of the GDP/KRAS complex

Mutations near allosteric sites can have a significant impact on the function of KRAS. Three specific mutations, K104Q, G12D/K104Q, and G12D/G75A, which are located near allosteric positions, were selected to investigate the molecular mechanisms behind mutation-induced influences on the activity of KRAS. Gaussian accelerated molecular dynamics (GaMD) simulations followed by the principal component analysis (PCA) were performed to improve the sampling of conformational states. The results revealed that these mutations significantly alter the structural flexibility, correlated motions, and dynamic behavior of the switch regions that are essential for KRAS binding to effectors or regulators. Furthermore, the mutations have a significant impact on the hydrogen bonding interactions between GDP and the switch regions, as well as on the electrostatic interactions of magnesium ions (Mg2+) with these regions. Our results verified that these mutations strongly influence the binding of KRAS to its effectors or regulators and allosterically regulate the activity. We believe that this work can provide valuable theoretical insights into a deeper understanding of KRAS function. Communicated by Ramaswamy H. Sarma

Biophysical and Biochemical Characterization of Structurally Diverse Small Molecule Hits for KRAS Inhibition.

We describe six compounds as early hits for the development of direct inhibitors of KRAS, an important anticancer drug target. We show that these compounds bind to KRAS with affinities in the low micromolar range and exert different effects on its interactions with binding partners. Some of the compounds exhibit selective binding to the activated form of KRAS and inhibit signal transduction through both the MAPK or the phosphatidylinositide 3-kinase PI3K-protein kinase B (AKT) pathway in cells expressing mutant KRAS. Most inhibit intrinsic and/or SOS-mediated KRAS activation while others inhibit RAS-effector interaction. We propose these compounds as starting points for the development of non-covalent allosteric KRAS inhibitors.

Haploinsufficiency of Adenomatous Polyposis Coli Coupled with Kirsten Rat Sarcoma Viral Oncogene Homologue Activation and P53 Loss Provokes High-Grade Glioblastoma Formation in Mice.

Glioblastoma multiforme (GBM) is the most common and deadly type of brain tumor originating from glial cells. Despite decades of clinical trials and research, there has been limited success in improving survival rates. However, molecular pathology studies have provided a detailed understanding of the genetic alterations associated with the formation and progression of glioblastoma-such as Kirsten rat sarcoma viral oncogene homolog (KRAS) signaling activation (5%), P53 mutations (25%), and adenomatous polyposis coli (APC) alterations (2%)-laying the groundwork for further investigation into the biological and biochemical basis of this malignancy. These analyses have been crucial in revealing the sequential appearance of specific genetic lesions at distinct histopathological stages during the development of GBM. To further explore the pathogenesis and progression of glioblastoma, here, we developed the glial-fibrillary-acidic-protein (GFAP)-Cre-driven mouse model and demonstrated that activated KRAS and p53 deficiencies play distinct and cooperative roles in initiating glioma tumorigenesis. Additionally, the combination of APC haploinsufficiency with mutant Kras activation and p53 deletion resulted in the rapid progression of GBM, characterized by perivascular inflammation, large necrotic areas, and multinucleated giant cells. Consequently, our GBM models have proven to be invaluable resources for identifying early disease biomarkers in glioblastoma, as they closely mimic the human disease. The insights gained from these models may pave the way for potential advancements in the diagnosis and treatment of this challenging brain tumor.

A novel MTORC2-AKT-ROS axis triggers mitofission and mitophagy-associated execution of colorectal cancer cells upon drug-induced activation of mutant KRAS

ABSTRACT RAS is one of the most commonly mutated oncogenes associated with multiple cancer hallmarks. Notably, RAS activation induces intracellular reactive oxygen species (ROS) generation, which we previously demonstrated as a trigger for autophagy-associated execution of mutant KRAS-expressing cancer cells. Here we report that drug (merodantoin; C1)-induced activation of mutant KRAS promotes phospho-AKT S473-dependent ROS-mediated S616 phosphorylation and mitochondrial localization of DNM1L/DRP1 (dynamin 1 like) and cleavage of the fusion-associated protein OPA1 (OPA1 mitochondrial dynamin like GTPase). Interestingly, accumulation of the outer mitochondrial membrane protein VDAC1 (voltage dependent anion channel 1) is observed in mutant KRAS-expressing cells upon exposure to C1. Conversely, silencing VDAC1 abolishes C1-induced mitophagy, and gene knockdown of either KRAS, AKT or DNM1L rescues ROS-dependent VDAC1 accumulation and stability, thus suggesting an axis of mutant active KRAS-phospho-AKT S473-ROS-DNM1L-VDAC1 in mitochondrial morphology change and cancer cell execution. Importantly, we identified MTOR (mechanistic target of rapamycin kinsase) complex 2 (MTORC2) as the upstream mediator of AKT phosphorylation at S473 in our model. Pharmacological or genetic inhibition of MTORC2 abrogated C1-induced phosphorylation of AKT S473, ROS generation and mitophagy induction, as well as rescued tumor colony forming ability and migratory capacity. Finally, increase in thermal stability of KRAS, AKT and DNM1L were observed upon exposure to C1 only in mutant KRAS-expressing cells. Taken together, our work has unraveled a novel mechanism of selective targeting of mutant KRAS-expressing cancers via MTORC2-mediated AKT activation and ROS-dependent mitofission, which could have potential therapeutic implications given the relative lack of direct RAS-targeting strategies in cancer. Abbreviations: ACTB/ß-actin: actin beta; AKT: AKT serine/threonine kinase; C1/merodantoin: 1,3-dibutyl-2-thiooxo-imidazoldine-4,5-dione; CAT: catalase; CETSA: cellular thermal shift assay; CHX: cycloheximide; DKO: double knockout; DNM1L/DRP1: dynamin 1 like; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; H2O2: hydrogen peroxide; HSPA1A/HSP70-1: heat shock protein family A (Hsp70) member 1A; HSP90AA1/HSP90: heat shock protein 90 alpha family class A member 1; KRAS: KRAS proto-oncogene, GTPase; MAP1LC3B/LC3B, microtubule associated protein 1 light chain 3 beta; LC3B-I: unlipidated form of LC3B; LC3B-II: phosphatidylethanolamine-conjugated form of LC3B; MAPKAP1/SIN1: MAPK associated protein 1; MAPK1/ERK2: mitogen-activated protein kinase 1; MAPK3/ERK1: mitogen-activated protein kinase 3; MFI: mean fluorescence intensity; MiNA: Mitochondrial Network Analysis; MTOR: mechanistic target of rapamycin kinase; MTORC1: mechanistic target of rapamycin kinase complex 1; MTORC2: mechanistic target of rapamycin kinase complex 2; O2.−: superoxide; OMA1: OMA1 zinc metallopeptidase; OPA1: OPA1 mitochondrial dynamin like GTPase; RICTOR: RPTOR independent companion of MTOR complex 2; ROS: reactive oxygen species; RPTOR/raptor: regulatory associated protein of MTOR complex 1; SOD1: superoxide dismutase 1; SOD2: superoxide dismutase 2; SQSTM1/p62: sequestosome 1; VDAC1: voltage dependent anion channel 1; VDAC2: voltage dependent anion channel 2.

Activation of MAP Kinase Pathway by Polyisoprenylated Cysteinyl Amide Inhibitors Causes Apoptosis and Disrupts Breast Cancer Cell Invasion.

Prognoses for TNBC remain poor due to its aggressive nature and the lack of therapies that target its "drivers". RASA1, a RAS-GAP or GTPase-activating protein whose activity inhibits RAS signaling, is downregulated in up to 77% of TNBC cases. As such, RAS proteins become hyperactive and similar in effect to mutant hyperactive RAS proteins with impaired GTPase activities. PCAIs are a novel class of agents designed to target and disrupt the activities of KRAS and other G-proteins that are hyperactive in various cancers. This study shows the anticancer mechanisms of the PCAIs in two breast cancer cell lines, MDA-MB-468 and MDA-MB-231. PCAIs (NSL-YHJ-2-27) treatment increased BRAF phosphorylation, whereas CRAF phosphorylation significantly decreased in both cell lines. Moreover, the PCAIs also stimulated the phosphorylation of MEK, ERK, and p90RSK by 116, 340, and 240% in MDA-MB-468 cells, respectively. However, in MDA-MB-231 cells, a significant increase of 105% was observed only in p90RSK phosphorylation. Opposing effects were observed for AKT phosphorylation, whereby an increase was detected in MDA-MB-468 cells and a decrease in MDA-MB-231 cells. The PCAIs also induced apoptosis, as observed in the increased pro-apoptotic protein BAK1, by 51%, after treatment. The proportion of live cells in PCAIs-treated spheroids decreased by 42 and 34% in MDA-MB-468 and MDA-MB-231 cells, respectively, which further explains the PCAIs-induced apoptosis. The movement of the cells through the Matrigel was also inhibited by 74% after PCAIs exposure, which could have been due to the depleted levels of F-actin and vinculin punctate, resulting in the shrinkage of the cells by 76%, thereby impeding cell movement. These results show promise for PCAIs as potential therapies for TNBC as they significantly inhibit the hallmark processes and pathways that promote cell proliferation, migration, and invasion, which result in poor prognoses for breast cancer patients.

Genome-Wide Analysis of DNA Methylation in Pseudomyxoma Peritonei Originated from Appendiceal Neoplasms

Introduction: Pseudomyxoma peritonei (PMP) is a disease characterized by progressive accumulation of intraperitoneal mucinous ascites produced by neoplasms in the abdominal cavity. Since the prognosis of patients with PMP remains unsatisfactory, the development of effective therapeutic drug(s) is a matter of pressing concern. Genetic analyses of PMP have clarified the frequent activation of GNAS and/or KRAS. However, the involvement of global epigenetic alterations in PMPs has not been reported. Methods: To clarify the genetic background of the 15 PMP tumors, we performed genetic analysis using AmpliSeq Cancer HotSpot Panel v2. We further investigated global DNA methylation in the 15 tumors and eight noncancerous colonic epithelial tissues using MethylationEPIC array BeadChip (Infinium 850k) containing a total of 865,918 probes. Results: This is the first report of comprehensive DNA methylation profiles of PMPs in the world. We clarified that the 15 PMPs could be classified into at least two epigenotypes, unique methylation epigenotype (UME) and normal-like methylation epigenotype (NLME), and that genes associated with neuronal development and synaptic signaling may be involved in the development of PMPs. In addition, we identified a set of hypermethylation marker genes such as HOXD1 and TSPYL5 in the 15 PMPs. Conclusions: These findings may help the understanding of the molecular mechanism(s) of PMP and contribute to the development of therapeutic strategies for this life-threatening disease.

Abstract A104: Targeting RABL3 to disrupt KRAS trafficking and activity in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal cancers with limited treatment options. Nearly all PDAC harbors a KRAS mutation, and oncogenic KRAS signaling is a key driver. Localization of the KRAS protein to the plasma membrane is absolutely required for downstream tumorigenic activities, making trafficking of KRAS to the plasma membrane a compelling target for treatment. In a familial pancreatic cancer syndrome with a striking five cases of PDAC, we recently discovered a rare, inherited gain-of-function mutation in the gene RABL3 that impacts the kinetics of KRAS intracellular trafficking. RABL3 was found to indirectly interact with KRAS, and the familial RABL3 mutation accelerates trafficking of KRAS to the plasma membrane. Here, we present pre-clinical experiments evaluating the therapeutic potential of targeting RABL3 to disrupt KRAS trafficking and activity. To achieve inducible knockdown of RABL3, we have engineered KPCY cells (tumor cells derived from KrasLSL-G12D/+; Trp53LSL-R172H/+; Pdx1-Cre; Rosa26YFP/YFP mice) to express a doxycycline-inducible shRNA targeting RABL3. In vitro, RABL3 knockdown results in robust suppression of KPCY cell proliferation. Moreover, the knockdown of RABL3 abrogated tumor growth of KPCY cells in immune-competent subcutaneous and orthotopic pancreatic cancer mouse models. Single-cell transcriptome profiling of tumors revealed immune infiltration of T cells, B cells, macrophages, and dendritic cells following RABL3 knockdown, suggesting that RABL3 knockdown creates vulnerabilities seen with suppression of KRAS activity. Validating these findings in a human context, we find a significantly elevated level of RABL3 expression across multiple human PDAC cell lines, consistent with a driver role in PDAC and a critical role in regulating KRAS trafficking. Similarly, analysis of human datasets revealed elevated RABL3 transcript levels in PDAC TCGA specimens compared to matched normal tissue as well as in PDAC patient-derived organoids compared to the GTEx cohort of normal pancreas tissues. Furthermore, there is a trend for worse patient survival with higher RABL3 expression levels. Therefore, to evaluate the potential of RABL3 knockdown to improve the potency, efficacy, and toxicity when combined with other PDAC therapeutic targets, we are currently testing the impacts of RABL3 knockdown on the therapeutic profile of newly available small-molecular KRAS inhibitors. Our findings provide pre-clinical support for targeting RABL3 to disrupt KRAS intracellular trafficking and inhibit KRAS activity for the treatment of PDAC. Citation Format: Woo-Jeong Jeong, Katherine J. Aney, Ethan Chen, Annan Yang, Giselle A. Uribe, Andrew Aguirre, Sahar Nissim. Targeting RABL3 to disrupt KRAS trafficking and activity in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A104.

Abstract C082: Decoding the KRAS-dependent proteome through the ribosomal lens

Abstract Our study addresses the gap in understanding the KRAS-specific translation landscape and functional proteome in cancer. We demonstrate that mutant KRAS drives distinct mechanisms of translational control to fuel cancer growth. By leveraging ribosome footprinting and advanced sequencing techniques, we investigate the impact of sotorasib, a targeted inhibitor of G12C mutant KRAS activity, on translation. Notably, KRAS (G12C) inhibition profoundly alters the translation of ribosomal proteins and translation elongation factors. Additionally, we reveal that KRAS inhibition resulted in ribosome stalling on a specific subset of mRNAs. Acute treatment with sotorasib hampers the translation of crucial oncogenic proteins, including MYC, as well as proteins involved in metabolism. These translational changes are facilitated by specific RNA motifs and RNA binding proteins that respond to KRAS activity. In essence, our study delineates the KRAS (G12C)-specific regulation of translation and elucidates the mechanism underlying KRAS (G12C)-mediated control over the cancer proteome. Citation Format: Kamini Singh, Trang Uyen Nguyen, Eric Nels Pederson, Vinay Kumar, Xiaoling Yun, Zhengqing Ouyang. Decoding the KRAS-dependent proteome through the ribosomal lens [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C082.

Abstract B100: Netrin-1/Neogenin-1 interaction modulates pancreatic innervation to promote tumorigenesis and accelerates cancer progression

Abstract Background: Nerves are a major component of the tumor microenvironment, with active crosstalk between cancer cells and nerves that may contribute to cancer progression. Netrin1 (Ntn1) is an axon guidance molecule that is important during early neural development. Recent studies suggest that Ntn1 may also play a role in tumorigenesis but its role in pancreatic cancer is not well understood. We hypothesized that Ntn1 plays a pro-tumorigenic role in pancreatic ductal adenocarcinoma (PDAC) progression and metastasis in part through modulation of nerves in the microenvironment. Methods: Expression of Ntn1 and its receptor Neogenin1(Neo1) were investigated in mouse models of spontaneous PDAC (LSL-Kras+/G12D/Pdx1-Cre; KC, LSL-Kras+/G12D/LSL-Trp53+/R172H/Pdx1-Cre; KPC). The influence of Ntn1 on pancreatic cancer progression and innervation was evaluated by KC mice with or without conditional knockout of Ntn1 (LSL-Kras+/G12D/Pdx1-Cre/Netrin1fl/fl; KCN). Murine PDAC cell lines and organoids were used to examine the functional role of Ntn1 and Neo1 through genetic and pharmacological modulations. The capacity of these cell lines to generate metastasis and the effect on neurite outgrowth was studied in a co-culture system with Dorsal Root Ganglia (DRG). Results: Expression of Ntn1 was not observed in normal acinar cells or ductal cells, but was found in both PanIN lesions and PDAC. Neo1 expression was similar to Ntn1 and there was a positive correlation between the expression of Ntn1 and Neo1. A lower rate of PanIN progression was observed in KCN mice compared to KC mice. In the metastatic model, overexpression of Ntn1 increased tumor burden and decreased survival while injections of a neutralizing Ntn1 antibody or genetic knockdown of Neo1 improved both endpoints. Ntn1 and Neo1 were upregulated in pancreatic organoids upon KRAS activation. Recombinant Ntn1 (rNTN1) promoted organoid forming capacity and upregulate Sox2 and Sox9 gene expression, which was inhibited by genetic knockdown of Neo1 or an anti-Neo1 antibody, supporting a direct autocrine role in modulating stemness. Nerve fibers were significantly increased at the site of PDAC liver metastasis, with Ntn1 showing the highest expression level among neurotrophins, as it was significantly increased in metastatic PDAC cells. Consistent with this observation, we observed less innervation in KCN mice, which correlated with reduced PanIN progression. Co-culture of DRGs with PDAC cells that overexpressed Ntn1 resulted in much greater axonal elongation consistent with a role of Ntn1 in promoting innervation. Conclusion: The Netrin1/Neogenin1 interaction is an important regulator of PDAC progression, acting both directly on PDAC cells to promote stemness, and indirectly via modulating tumor innervation. Blockade of Ntn1/Neo1 interaction reduces the capacity of PDAC to metastasize to the liver and improves survival. Overall, targeting the Ntn1/Neo1 interaction may represent a potential new therapeutic approach for the treatment of pancreatic cancer. Citation Format: Yosuke Ochiai, Sunagawa Masaki, Ermanno Malagola, Hiroki Kobayashi, Feijing Wu, Ruth A. White, Leah B. Zamechek, Timothy C. Wang. Netrin-1/Neogenin-1 interaction modulates pancreatic innervation to promote tumorigenesis and accelerates cancer progression [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B100.

Revealing the mechanism of action of a first-in-class covalent inhibitor of KRASG12C (ON) and other functional properties of oncogenic KRAS by 31P NMR

Individual oncogenic KRAS mutants confer distinct differences in biochemical properties and signaling for reasons that are not well understood. KRAS activity is closely coupled to protein dynamics and is regulated through two interconverting conformations: state 1 (inactive, effector binding deficient), and state 2 (active, effector binding enabled). Here we use 31P NMR to delineate the differences in state 1 and state 2 populations present in wild-type (WT) and common KRAS oncogenic mutants (G12C, G12D, G12V, G13D, and Q61L) bound to its natural substrate GTP or a commonly used nonhydrolyzable analogue GppNHp. Our results show that GppNHp-bound proteins exhibit significant state 1 population, whereas GTP-bound KRAS is primarily (90% or more) in the state 2 conformation. This observation suggests that the predominance of state 1 shown here and in other studies is related to GppNHp and is most likely nonexistent in cells. We characterize the impact of this differential conformational equilibrium of oncogenic KRAS on RAF1 kinase effector RBD (RAS Binding Domain) binding and intrinsic hydrolysis. Through a KRAS G12C drug discovery, we have identified a novel small molecule inhibitor, BBO-8956, which is effective against both GDP and GTP-bound KRAS G12C. We show that binding of this inhibitor significantly perturbs the state 1 - state 2 equilibrium and induces an inactive state 1 conformation in GTP-bound KRAS G12C. In the presence of BBO-8956, RAF1 RBD is unable to induce a signaling competent state 2 conformation within the ternary complex, demonstrating the mechanism of action (MOA) for this novel, active-conformation inhibitor.

Abstract B081: SOAT1 as a targetable KRAS dependency in PDAC

Abstract Metabolic rewiring is an emerging hallmark of neoplasia. In pancreatic ductal adenocarcinoma (PDAC), KRAS alters myriad metabolic programs to promote cellular transformation. Among them, the cholesterol synthesis pathway has emerged as a vulnerability of cancer cells due to the importance of cholesterol in membrane fluidity and the production of isoprenoid intermediates that enable KRAS membrane localization and activation in pancreatic cancer. We recently reported that the non-essential gene SOAT1 (Sterol-O-Acyl transferase 1), encoding an enzyme that esterifies free cholesterol into an insoluble form for storage and transport, is markedly upregulated in metastatic PDAC cells. SOAT1 promotes the hyperactivation of the mevalonate synthesis pathway due to loss of negative feedback by free cholesterol, thereby leading to increased production of isoprenoids such as farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), which are required for RAS and RHO membrane localization and activity, respectively. Genetic ablation of Soat1 reduced RAS and RHO membrane localization and inhibited cellular proliferation and mitochondrial function in vitro, and these effects were largely rescued by exogenous FPP and GGPP. Therefore, our study nominated SOAT1 as a new candidate gene for therapeutic targeting. Based on SuFEx Click Chemistry (ASCC) technology, we have designed and synthesized a panel of drug-like candidates. The first series of Soat1 probes selectively impair the proliferation of PDAC cells that express SOAT1, reduce SOAT1 protein content in cells, and covalently bind to SOAT1. In mice, the most active SOAT1 probes were tolerated in a 28-day daily dosing study up to 120 mg/kg. Marked tumor growth inhibition was noted in mice bearing PDAC xenografts and GEMM mice treated as monotherapy with 15 mg/kg/d of the most active SOAT1 probe, and tissue analysis demonstrated tumor necrosis, decreased proliferation, and increased apoptosis. Mechanistically, SOAT1 loss or inhibition targets multiple important components of cellular transformation in PDAC including KRAS, RHO, and mitochondrial biology, and our work suggests that it is an attractive target to pursue for further drug development in this cancer. Citation Format: Wenjun Lan, Shoujun Sun, Jeremy Nigri, Josh Homer, Young Park, Astrid Deschenes, Paolo Cifani, Justin Cross, Victoria Gaeth, Sunny Kim, Shawn Ting, Rachel Rubino, John Moses, David Tuveson. SOAT1 as a targetable KRAS dependency in PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B081.

Abstract C003: RAMP 205: A phase 1b/2a study of gemcitabine, nab-paclitaxel, avutometinib, and defactinib in untreated metastatic pancreatic ductal adenocarcinoma

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) accounts for ~90% of pancreatic cancer. Surgery is the only curative treatment. Unfortunately, most patients are diagnosed with advanced disease and are not candidates for resection. Combination chemotherapy approaches have provided incremental improvements to PDAC outcomes. However, impact on survival for patients with inoperable disease remains modest, with a 2-year survival rate of less than 7%. Activating mutations in the KRAS oncogene are a key initiating event in ~80-90% of PDAC. Permanent activation of KRAS drives tumor cell proliferation and survival, tumor microenvironment modulation, and metabolic alterations in PDAC. Patients with KRAS mutant PDAC have worse outcomes compared to those with KRAS wild-type PDAC. In addition, hyperactivation of focal adhesion kinase (FAK) has been correlated with high stromal density, resulting in therapeutic resistance and poor survival. Avutometinib is a first-in-class oral RAF/MEK clamp that potently inhibits MEK kinase activity while also blocking the compensatory reactivation of MEK by upstream RAF. Defactinib is a selective inhibitor of focal adhesion kinase (FAK), which has been shown to mediate resistance to multiple anticancer agents. Preclinical data have shown that the combination of avutometinib + FAK inhibition (FAKi) + chemotherapy (gemcitabine + paclitaxel as a surrogate for nab-paclitaxel) induced tumor regression in a autochthonous KRAS/TP53 mutant PDAC mouse model. Prior clinical experience of defactinib + pembrolizumab + chemotherapy was safe, showed responses, decreased stromal density, and increased CD8+ T cell infiltration in patients with PDAC (NCT02546531). Methods: RAMP 205 is a multi-center, open-label, single arm Phase 1b/2a study of gemcitabine and nab-paclitaxel in combination with avutometinib and defactinib in patients with previously untreated metastatic PDAC (NCT05669482). The study is being conducted in 2 parts: Parts A (dose evaluation) and Part B (dose expansion). The starting dose is 2.4 mg avutometinib orally (PO) twice weekly + 200 mg defactinib PO twice per day, in combination with standard once weekly gemcitabine/nab-paclitaxel (QW IV). All study drugs are dosed 3 weeks on, 1 week off. Following determination of the Recommended Phase 2 Dose (RP2D) in Part A, Part B will evaluate efficacy of the RP2D via objective response rate (primary endpoint). Mandatory tumor biopsies and blood samples will be collected for correlative studies, including circulating tumor DNA, molecular profiling and pharmacodynamic markers. Eligible patients must have histologic or cytologic evidence of metastatic PDAC and measurable disease according to RECIST v1.1. Patients with prior or concomitant treatment for metastatic PDAC or prior treatment with RAS/MAPK or FAK inhibitors are not eligible. The study will enroll ~35 patients with 6-12 patients in Part A, and 23 patients in Part B. Citation Format: Kian-Haut Lim, Manuel Hidalgo, Mark H. O'Hara, Kristen R. Spencer, Ignacio Garrido-Laguna, David G. DeNardo, Vijeta Bhambhani, Gloria Patrick, Yaofeng Cheng, Silvia Coma, Jonathan A. Pachter, Louis J. Denis. RAMP 205: A phase 1b/2a study of gemcitabine, nab-paclitaxel, avutometinib, and defactinib in untreated metastatic pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C003.

Abstract B078: A complex of NF1 and SPRED2 non-canonically behave as tumor promoters in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy with poor prognosis due to late detection and lack of effective therapeutic options. >90% of PDAC tumors are driven by activating mutations in the KRAS oncogene, making it an appealing subject for further investigation into understanding PDAC biology and offering a potential genetic vulnerability to target in patients. Our laboratory has previously reported a mutant-KRAS-proximal complex at the plasma membrane that consists of tumor-suppressor Neurofibromatosis type 1 (NF1), Sprouty Related EVH1 Domain Containing 2 (SPRED2), and Ribosomal S6 Kinase 1 (RSK1). This complex mechanistically suppresses WT KRAS signaling via RSK1, a suppression which is then relieved upon mutant KRAS inhibition, ultimately promoting adaptive resistance for PDAC cells under these conditions. We find that both knockout of either NF1 or SPRED2 in PDAC organoids induces a substantial growth defect in vivo. Our preliminary data suggests that NF1 and SPRED2 support KRAS activity in mouse pancreatic cancer in vivo. Moreover, both SPRED2 and NF1 KO show decreased proliferation, invasion, and attachment on collagen family members in vitro. Given NF1's well-established role as a tumor-suppressor, our observations of decreased tumorigenic properties upon NF1 deletion are unexpected. We hypothesize that NF1 and SPRED2 promote tumorigenesis via recruitment of RSK1 to the plasma membrane, inducing the phosphorylation of putative substrates to stimulate a pro-migratory cellular behavior. Our work suggests the existence of a unique RSK1 program localized at the plasma membrane by mutant KRAS, providing a new facet of oncogenesis to investigate for fundamental understanding and potential therapeutic opportunities. Citation Format: Sun Kim, Hsiu-Chi Ting, Astrid Deschenes, Jonathan Kastan, Youngkyu Park, David Tuveson. A complex of NF1 and SPRED2 non-canonically behave as tumor promoters in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B078.

Cereblon-based Bifunctional Degrader of SOS1, BTX-6654, Targets Multiple KRAS Mutations and Inhibits Tumor Growth.

Mutations within the oncogene KRAS drive an estimated 25% of all cancers. Only allele-specific KRAS G12C inhibitors are currently available and are associated with the emergence of acquired resistance, partly due to upstream pathway reactivation. Given its upstream role in the activation of KRAS, son of sevenless homolog 1 (SOS1), has emerged as an attractive therapeutic target. Agents that target SOS1 for degradation could represent a potential pan-KRAS modality that may be capable of circumventing certain acquired resistance mechanisms. Here, we report the development of two SOS1 cereblon-based bifunctional degraders, BTX-6654 and BTX-7312, cereblon-based bifunctional SOS1 degraders. Both compounds exhibited potent target-dependent and -specific SOS1 degradation. BTX-6654 and BTX-7312 reduced downstream signaling markers, pERK and pS6, and displayed antiproliferative activity in cells harboring various KRAS mutations. In two KRAS G12C xenograft models, BTX-6654 degraded SOS1 in a dose-dependent manner correlating with tumor growth inhibition, additionally exhibiting synergy with KRAS and MEK inhibitors. Altogether, BTX-6654 provided preclinical proof of concept for single-agent and combination use of bifunctional SOS1 degraders in KRAS-driven cancers.

Ganoderma microsporum immunomodulatory protein combined with KRASG12C inhibitor impedes intracellular AKT/ERK network to suppress lung cancer cells with KRAS mutation

KRAS mutations are tightly associated with lung cancer progression. Despite the unprecedented clinical success of KRASG12C inhibitors, recurrent mechanisms of resistance and other KRAS mutations require further therapeutic approaches. GMI, a protein from the medicinal mushroom Ganoderma microsporum, possesses antitumor activity; whereas, the biological function of GMI on regulating KRAS mutant lung cancer cells remains unknown. Herein, RNA-sequencing and bioinformatics showed that GMI may regulate KRAS-modulated MAPK and PI3K-AKT pathways in A549 (KRASG12S) cells. Further experiments demonstrated that GMI inhibited KRAS activation and suppressed ERK1/2 and AKT signaling in A549 cells. Intriguingly, GMI inhibited AKT signaling but increased phosphorylation of ERK in H358 (KRASG12C) cells. GMI significantly suppressed tumor growth in LLC1 cells-allograft and H358 cells-xenograft mice. GMI showed a synergistic effect with KRASG12C inhibitors in inhibiting cell growth, KRAS activation and KRAS-mediated downstream signaling, leading to apoptosis in H358 cells. Combination of GMI and KRASG12C inhibitor, AMG 510, resulted in more durable inhibition of tumor growth and KRAS activity in H358 cells-xenograft mice. This study highlights the potential of GMI, a dietary fungal protein, as a viable therapeutic avenue for KRAS-mutant lung cancer in combination with KRASG12C inhibitors.

CDKN2A-p16 Deletion and Activated KRASG12D Drive Barrett’s-Like Gland Hyperplasia-Metaplasia and Synergize in the Development of Dysplasia Precancer Lesions

Background & AimsBarrett’s esophagus (BE) is the precursor of esophageal dysplasia and adenocarcinoma (EAC). CDKN2A-p16 deletions were reported in 34-74% of BE patients who progressed to dysplasia and EAC, suggesting that p16 loss may drive neoplastic progression. KRAS activation frequently occurs in EAC and pre-cancer lesions. LGR5+ stem cells in the squamocolumnar-junction (SCJ) of mouse stomach, contribute as BE progenitors. We aimed to determine the functional effects of p16 loss and KRAS activation in Barrett’s-like metaplasia and dysplasia development. MethodsWe established mouse models with conditional knockout of CDKN2A-p16 (p16KO) and/or activated KRASG12D expression in interleukin-1b transgenic mice targeting SCJ LGR5+ cells and characterized histologic alterations (mucous-gland hyperplasia/metaplasia, inflammation, and dysplasia) in mouse SCJ. Gene expression was determined by microarray, RNAseq, and immunohistochemistry of SCJ tissues and cultured 3D organoids. Resultsp16KO mice exhibited increased mucous-gland hyperplasia/metaplasia vs. controls (p=.0051). Combined p16KO+KRASG12D resulted in more frequent dysplasia and higher dysplasia scores with 82% of p16KO+KRASG12D mice developing high-grade dysplasia (p=0.0036). SCJ transcriptome analysis showed several activated pathways in p16KO vs. control mice (apoptosis, TNF-alpha/NFkB, proteasome degradation, p53 signaling, MAPK, KRAS, and G1-to-S transition). Conclusionsp16 deletion in LGR5+ cell precursors triggers increased SCJ mucous-gland hyperplasia/metaplasia. KRASG12D synergizes with p16 deletion resulting in higher grades of SCJ glandular dysplasia, mimicking Barrett’s high-grade dysplasia. These genetically modified mouse models establish a functional role of p16 and activated KRAS in the progression of Barrett’s like lesions to dysplasia in mice, representing an in vivo model of esophageal pre-cancer. Derived 3D organoid models further provide in vitro modeling opportunities of esophageal pre-cancer stages.