G12C Inhibitors Research Articles

Recent Developments, Challenges and Opportunities in Targeting the KRAS Pathway

Abstract: KRAS is a highly mutated gene among cancers, especially in the lung, pancreatic and colorectal cancers (CRC). Despite extensive efforts in the past three decades, KRAS remains undruggable. But lately, inhibitors selectively binding to cysteine mutant at 12th position (G12C) are in clinical trials as a single agent and in combination with other drugs. FDA approved LUMAKRAS (Sotorasib, AMG510) for metastatic NSCLC, while Adagrasib (MRTX849) is under clinical trials, and it is showing good safety and efficacy in pancreatic and GI tumors. Both these molecules demonstrated better response in NSCLC but have less efficacy in colorectal and other solid cancers. Among non-G12C KRAS mutant cancers, promising data are emerging from G12D inhibition. This review covers the status of KRAS G12C, non- G12C inhibitors, and discusses different nodal proteins in the RAS signaling pathway, mechanism of resistance in targeted therapy and combination approaches. Ongoing clinical trials with G12C inhibitors have promising results. However, a combination of KRAS inhibition with other inhibitors of signaling components in vertical axis (SHP2, SOS1, MEK, PI3K/AKT and EGFR inhibitors) might be beneficial for durable clinical response. Sotorasib in combination with MEK inhibitor showed promising results in preclinical xenograft and PDx models of KRAS mutated cancers. Although at a preclinical stage, other approaches like Tri complex inhibitor, KRAS (ON) inhibitors, and non-G12C inhibitors are of significant interest in KRAS drug discovery. Multiple targeted approaches are being explored based on tumor specific gene expression profiles considering dynamic nature of KRAS activity and heterogeneity of hard to treat tumors.

Prognostic Implication of KRAS G12C Mutation in a Real-World KRAS-Mutated Stage IV NSCLC Cohort Treated With Immunotherapy in The Netherlands

IntroductionWith the approval of G12C inhibitors as the second line of treatment for KRAS G12C-mutated NSCLC, and the expanding research regarding targeting KRAS, it is key to understand the prognostic implication of KRAS G12C in the current first line of treatment. We compared overall survival (OS) of patients with stage IV KRAS G12C-mutated NSCLC to those with a KRAS non-G12C mutation in a first-line setting of (chemo)immunotherapy. MethodsThis nationwide population-based study used real-world data from The Netherlands Cancer Registry. We selected patients with stage IV KRAS-mutated lung adenocarcinoma diagnosed in 2019 to 2020 who received first-line (chemo-)immunotherapy. Primary outcome was OS. ResultsFrom 28,120 registered patients with lung cancer, 1185 were selected with a KRAS mutation, of which 494 had a KRAS G12C mutation. Median OS was 15.5 months (95% confidence interval [CI]: 13.6–18.4) for KRAS G12C versus 14.0 months (95% CI:11.2–15.7) for KRAS non-G12C (p = 0.67). In multivariable analysis, KRAS subtype was not associated with OS (hazard ratio = 0.95, 95% CI: 0.82–1.10). For the subgroup with programmed death-ligand 1 at 0% to 49% who received chemoimmunotherapy, median OS was 13.3 months (95% CI: 10.5–15.2) for G12C and 9.8 months (95% CI: 8.6–11.3) for non-G12C (p = 0.48). For the subgroup with programmed death-ligand 1 more than or equal to 50% who received monoimmunotherapy, the median OS was 22.0 months (95% CI: 18.4–27.3) for G12C and 18.9 months (95% CI: 14.9–25.2) for non-G12C (p = 0.36). ConclusionsThere was no influence of KRAS subtype (G12C versus non-G12C) on OS in patients with KRAS-mutated stage IV NSCLC treated with first-line (chemo)immunotherapy.

Establishing the Role of Iridoids as Potential Kirsten Rat Sarcoma Viral Oncogene Homolog G12C Inhibitors Using Molecular Docking; Molecular Docking Simulation; Molecular Mechanics Poisson-Boltzmann Surface Area; Frontier Molecular Orbital Theory; Molecular Electrostatic Potential; and Absorption, Distribution, Metabolism, Excretion, and Toxicity Analysis.

The RAS gene family is one of the most frequently mutated oncogenes in human cancers. In KRAS, mutations of G12D and G12C are common. Here, 52 iridoids were selected and docked against 8AFB (KRAS G12C receptor) using Sotorasib as the standard. As per the docking interaction data, 6-O-trans-p-coumaroyl-8-O-acetylshanzhiside methyl ester (dock score: -9.9 kcal/mol), 6'-O-trans-para-coumaroyl geniposidic acid (dock score: -9.6 kcal/mol), 6-O-trans-cinnamoyl-secologanoside (dock score: -9.5 kcal/mol), Loganic acid 6'-O-beta-d-glucoside (dock score: -9.5 kcal/mol), 10-O-succinoylgeniposide (dock score: -9.4), Loganic acid (dock score: -9.4 kcal/mol), and Amphicoside (dock score: -9.2 kcal/mol) showed higher dock scores than standard Sotorasib (dock score: -9.1 kcal/mol). These common amino acid residues between iridoids and complexed ligands confirmed that all the iridoids perfectly docked within the receptor's active site. The 100 ns MD simulation data showed that RMSD, RMSF, radius of gyration, and SASA values were within range, with greater numbers of hydrogen bond donors and acceptors. MM/PBSA analysis showed maximum binding energy values of -7309 kJ/mol for 6-O-trans-p-coumaroyl-8-O-acetylshanzhiside methyl ester. FMO analysis showed that 6-O-trans-p-coumaroyl-8-O-acetylshanzhiside methyl ester was the most likely chemically reactive molecule. MEP analysis data highlighted the possible electrophilic and nucleophilic attack regions of the best-docked iridoids. Of all the best-docked iridoids, Loganic acid passed Lipinski, Pfizer, and GSK filters with a similar toxicity profile to Sotorasib. Thus, if we consider these iridoids to be KRAS G12C inhibitors, they will be a boon to mankind.

RAS and Other Molecular Targets in Pancreatic Cancer: The Next Wave Is Coming.

Since the discovery of oncogenes in the 1970s, cancer doctors and researchers alike have understood the promise of discovering drugs to block the dominantly acting function of mutated signaling proteins in cancer. This promise was delivered, first slowly, with early signals inhibiting HER2 and BCR-Abl in the 1990s and 2000s, and then quickly, with kinase inhibitors being approved hand over fist in non-small cell lung cancer, melanoma, and many other malignancies. The RAS proteins, however, remained recalcitrant to chemical inhibition for decades, despite being, by far, the most frequently mutated oncogenes in cancers of all types. Nowhere was this deficit more palpable than in pancreatic ductal adenocarcinoma (PDA), where > 90% of cases are driven by single nucleotide substitutions at a single codon of the KRAS gene. The ice began to crack in 2012 when Ostrem and colleagues (Nature 503(7477): 548-551, 2013) synthesized the first KRAS G12C inhibitors, which covalently bind to GDP-bound G12C-mutated KRAS and lock the oncoprotein in its inactive state. In the last decade, the scientific community has established a new foundation on this and other druggable pockets in mutant KRAS. Here we provide an up-to-date overview of drugs targeting KRAS and other molecular targets in pancreatic cancer.

Scribble mis-localization induces adaptive resistance to KRAS G12C inhibitors through feedback activation of MAPK signaling mediated by YAP-induced MRAS.

Tumor cells evade targeted drugs by rewiring their genetic and epigenetic networks. Here, we identified that inhibition of MAPK signaling rapidly induces an epithelial-to-mesenchymal transition program by promoting re-localization of an apical-basal polarity protein, Scribble, in oncogene-addicted lung cancer models. Mis-localization of Scribble suppressed Hippo-YAP signaling, leading to YAP nuclear translocation. Furthermore, we discovered that a RAS superfamily protein MRAS is a direct target of YAP. Treatment with KRAS G12C inhibitors induced MRAS expression, which formed a complex with SHOC2, precipitating feedback activation of MAPK signaling. Abrogation of YAP activation or MRAS induction enhanced the efficacy of KRAS G12C inhibitor treatment in vivo. These results highlight a role for protein localization in the induction of a non-genetic mechanism of resistance to targeted therapies in lung cancer. Furthermore, we demonstrate that induced MRAS expression is a key mechanism of adaptive resistance following KRAS G12C inhibitor treatment.

An allosteric pan-TEAD inhibitor blocks oncogenic YAP/TAZ signaling and overcomes KRAS G12C inhibitor resistance

The Hippo pathway is a key growth control pathway that is conserved across species. The downstream effectors of the Hippo pathway, YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif), are frequently activated in cancers to drive proliferation and survival. Based on the premise that sustained interactions between YAP/TAZ and TEADs (transcriptional enhanced associate domain) are central to their transcriptional activities, we discovered a potent small-molecule inhibitor (SMI), GNE-7883, that allosterically blocks the interactions between YAP/TAZ and all human TEAD paralogs through binding to the TEAD lipid pocket. GNE-7883 effectively reduces chromatin accessibility specifically at TEAD motifs, suppresses cell proliferation in a variety of cell line models and achieves strong antitumor efficacy in vivo. Furthermore, we uncovered that GNE-7883 effectively overcomes both intrinsic and acquired resistance to KRAS (Kirsten rat sarcoma viral oncogene homolog) G12C inhibitors in diverse preclinical models through the inhibition of YAP/TAZ activation. Taken together, this work demonstrates the activities of TEAD SMIs in YAP/TAZ-dependent cancers and highlights their potential broad applications in precision oncology and therapy resistance.

KRAS G12C-mutated pancreatic cancer: Clinical outcomes based on chemotherapeutic regimen.

4150 Background: Frontline treatment for advanced pancreatic ductal adenocarcinoma (PDAC) has been either 5-fluorouracil, oxaliplatin and irinotecan (FOLFIRINOX) or gemcitabine and nab-paclitaxel (GP) for the past decade. While the NAPOLI-3 trial, utilizing liposomal irinotecan, highlighted the superiority of a triplet regimen over GP, the question remains whether certain subgroups may derive particular benefit from GP. Pre-clinical data in lung cancer suggest that KRAS G12C may facilitate enhanced DNA adduct removal after platinum chemotherapy and confer resistance to this drug class. While multiple KRAS G12C inhibitors have shown early promise in PDAC, multi-agent chemotherapy remains the frontline standard and will likely remain an important therapeutic tool. This study aimed to investigate clinical outcomes after platinum and non-platinum-based chemotherapy in patients with advanced KRAS G12C-mutated PDAC. Methods: PDAC samples were tested using whole transcriptome sequencing (WTS; Illumina NovaSeq) and NextGen DNA sequencing (NextSeq, 592 Genes and NovaSEQ, WES) at Caris Life Sciences (Phoenix, AZ). Significance was determined by X2 and Fisher-Exact and p adjusted for multiple comparisons (q). Real-world overall survival (rwOS) was obtained from insurance claims data and calculated from first of treatment to last contact with comparison done by Kaplan-Meier test. Results: A total of 5,555 PDAC patients harboring KRAS pathogenic variants were identified, including 109 with a G12C mutation. For KRAS G12C mutants, median overall survival was 470 days for those who received GP (n=22) compared to 240 days for patients who received FOLFIRINOX (n=9) with a median difference of 230 days (HR 0.32, CI 0.12-0.82, p=0.013). In contrast, median overall survival was higher for patients harboring G12D (392 vs 297 days, p=0.003), G12V (500 vs 356 days, p=0.012) or G12R (469 vs 391 days, p=0.095) mutations who received FOLFIRINOX compared to GP. KRAS G12C mutated PDAC showed the highest rate of PDL1+ staining (27%) compared to other variants (G12R 13% and G12D 19%, q<0.01). TMB-H and MSI-H prevalence were highest in G12C compared to other variants (no statistical significance observed). Conclusions: In patients with advanced PDAC and a G12C mutation, median overall survival appears significantly longer in those treated with GP compared to FOLFIRINOX. The opposite trend was seen in patients with other KRAS variants including G12D, G12V, and G12R, consistent with the recently presented NAPOLI-3 trial. PDL1 staining was also highest in the KRAS G12C cohort. While this is the largest reported analysis of outcomes to frontline chemotherapy in KRAS G12C-mutated PDAC, the sample size is small and needs validation in additional datasets. Next steps include evaluating DNA mutational status and RNA expression of genes involved in DNA repair in G12C vs other KRAS variants to better understand the outcome data presented here.

ECOG-ACRIN LUNG-MAP S1900E substudy: A phase II study of sotorasib in participants (Pts) with previously treated stage IV or recurrent KRAS G12C mutant non-squamous (Non-sq) non-small cell lung cancer (NSCLC).

TPS9143 Background: KRAS mutant NSCLC is further defined molecularly by specific mutation subtypes and co-mutations in tumor suppressor genes. Two targeted therapeutics, sotorasib and adagrasib, are currently approved for the treatment of previously treated advanced KRAS G12C mutant NSCLC. Sotorasib has also demonstrated superiority to docetaxel in the randomized phase III CodeBreaK 200 study ( de Langen et al Lancet. 2023 Feb 7 Epub). The impact of co-mutations in tumor suppressors, including TP53 and STK11, on the efficacy of first generation KRAS G12C inhibitors has been inconclusive. The S1900E substudy aims to prospectively understand, using rigorous biomarker testing/definitions, the impact of co-mutations on the clinical activity of sotorasib in pts with KRAS G12C mutant NSCLC. Methods: S1900E is a phase 2 open-label study of sotorasib in biomarker-defined cohorts for pts who have KRAS G12C stage IV/recurrent non-sq NSCLC, received at least one line of systemic treatment, and are naïve to KRAS G12C inhibitors. Pts are assigned to S1900E under the Lung-MAP screening protocol (NCT03851445) using FoundationOne F1CDx next generation sequencing assay on tissue (Foundation Medicine, Cambridge, MA). S1900E cohorts include: #1) Presence of TP53 co-mutation AND Wild Type STK11, KEAP1, NFE2L2, CUL3; #2) Presence of STK11 co-mutation AND Wild Type TP53, KEAP1, NFE2L2, CUL3; 3) All pts not eligible for Cohorts 1 and 2 (e.g. dual co-mutations in STK11 and TP53; co-mutations in KEAP1, NFE2L2, CUL3, or others; or lack of any co-mutations). Pts with treated, clinically controlled, and asymptomatic brain metastases are eligible and with amendment 3 (Mar 20 2022), eligibility expanded to pts with asymptomatic untreated brain metastases. Sotorasib is dosed 960 mg oral daily. The primary objective is to evaluate the RECIST 1.1 response rate (ORR) per cohort. For cohorts 1 and 3, the target sample size is 40 eligible pts based on a design with 92% exact power to rule out a 14% ORR at the 1-sided 5% level, if the true ORR were 34%; ≥10 responses would rule out a 14% ORR. Cohort 2 has a target sample size of 25 eligible pts with 93% exact power to rule out a 14% ORR at the 1-sided 5% level, if the true ORR were 40%; ≥7 responses would rule out a 14% ORR. Secondary endpoints include PFS, OS, duration of response, and toxicity. Circulating tumor DNA is being collected at day 1 of cycle 1, 2, and 3 and at progression. Enrollment started April 2 2021 with conservative accrual targets met, currently with 72% enrolled (84/116) as of Feb 14 2023, including 37, 16, and 31 pts, in cohorts 1, 2, and 3, respectively. Clinical trial information: NCT04625647 .

Clinical and genomic landscape of RAS pathway mutations in gynecologic cancers.

5611 Background: We aimed to describe RAS mutations in gynecologic cancers as they relate to clinicopathologic features, subtypes, co-mutations, and implications for therapy. Methods: Patients with gynecologic cancers who had next-generation sequencing at our institution between Feb 2010 and Aug 2022 were included. Clinical, histopathologic, and sequencing data were collected and analyzed. Overall survival was estimated using the Kaplan-Meier model. Results: Of the 3310 gynecologic cancer patients tested, 522 (15.8%) harbored a RAS mutation. For patients with mutation, median age was 56 (18-87) years, with cancer types including ovarian (43.5%), endometrial (43.4%), cervical (9.2%), and vulvovaginal (2.9%). Most patients (92.2%) had recurrence. The most common histology for each cancer included endometrioid in endometrial (60.8%), low grade serous in ovarian (34.4%), adenocarcinoma in cervical (60.4%), and melanoma in vulvovaginal (66.7%). Mucinous and clear cell differentiations were overrepresented (12.5% each). Median overall survival for all RAS-mutated patients was 69 months (95%CI 59-82), which was significantly better than in wildtype control of 56 months (95%CI 52-59, p = 0.022). Mutations observed were in KRAS (85.8%), NRAS (12.8%), and HRAS (3.3%). The most common mutant alleles for each were KRAS G12D (34.5%), KRAS G12V (31.7%), NRAS Q61R (34.3%), and HRAS G12S (17.6%). Of note, the incidence of KRAS G12C was 5.6% (0.8% overall). The most common co-mutations for RAS were PIK3CA (35.7%), PTEN (33.3%), TP53 (32.4%), and ARID1A (20.8%). This trend held for each mutation and cancer type. Of these, 64.7% were potentially actionable alterations, including 91.1% of PIK3CA, 83.7% of PTEN, and 50.0% of ARID1A. RAS pathway targeted therapies were administered to 62 patients (57 MEK, 5 BRAF, 5 KRAS G12C, 4 SHP2). The median overall survival for these patients was significantly improved (101 months, 95%CI 78-160) compared to RAS-mutated patients who did not receive a RAS pathway inhibitor (66 months, 95%CI 53-79, HR 0.7, p = 0.031). Of interest, novel KRAS G12C inhibitors showed durable benefit in 3 ovarian (high grade serous, low grade serous, clear cell) and 2 endometrial (mixed endometrioid, clear cell; mixed endometrioid, serous, mucinous) cancers with a clinical benefit rate of 100%, duration of objective response of 22 months, and ongoing responses at 5 and 16 months. Conclusions: Gynecologic cancers with RAS mutations have distinct histopathologic distribution and portend improved overall survival. The most commonly actionable co-mutations were PIK3CA, PTEN, and ARID1A, which may aid in combination therapy design. RAS pathway targeted therapy is associated with improved overall survival in this cohort and should be considered. [Table: see text]

Efficacy and safety of Kras inhibitors in pancreatic cancer: A systematic review of clinical trials.

e16297 Background: Pancreatic ductal adenocarcinoma (PDAC) is the most aggressive tumor and is one of the leading causes of cancer related deaths worldwide. Kirsten Rat Sarcoma (KRAS)mutation resulting in tumor cell proliferation and cancer progression is a frequently mutated oncogene in various cancers and is approximately seen in 90% of pancreatic ductal adenocarcinoma. Efforts are being done to target Kras mutation in the last 3–4 decades. Here in, we report the results of the systemic review of clinical trials done on chemotherapeutic agents targeting Kras mutated pancreatic cancer. Methods: We followed PRISMA guidelines to conduct this systematic review. A literature search was performed on PubMed, Clinicaltrials.gov, and Embase with mesh terms, “pancreatic neoplasm’’ and “kras” from the inception of data till 01/05/2023. We screened 4191 articles and included 3 clinical trials with published study results and 3 ongoing clinical trials. Results: In this systematic review, data on 2 chemotherapeutic agents Sotorasib (AMG510) and Adagrasib (MRTX849) which are Kras G12C inhibitors, are obtained from Code Break-100 and preliminary results of KRYSTAL-1 clinical trials, respectively. In these 2 clinical trials (N-48), all the patients had advanced pancreatic cancer with Kras G12C mutation and were previously treated with chemotherapy. 10 patients in Krystal-1 trial, who received adagrasib showed progression-free survival (PFS) of 6.6 months, disease control rate (DCR) of 100 % and partial response (PR) of 50%. 38 patients in the Code Break-100 trial, who received Sotorasib showed PFS of 4 months, DCR of 84.2% and PR of 21.1%. However, 50% of patients in the KRYSTAL-1 trial are still receiving treatment. Grade 3 /4 adverse events were seen in 21% and 16% of patients in the KRYSTAL-1 and Code Break-100 trial, respectively. Other Kras G12C inhibitors like D-1553, Sotorasib in combination with 2nd line chemotherapy are under investigation. Results of a clinical trial on anti KrasG12D siRNA done on locally advanced pancreatic cancer patients showed a median overall survival of 15.12 months with no dose limiting adverse events. A multinational randomized phase 2b clinical trial is ongoing for the evaluation of efficacy of siRNA based drug in combination with chemotherapy. Conclusions: Both Adagrasib and Sotorasib showed promising results in the pretreated Kras G12C pancreatic cancer patients with acceptable safety profile. Further exploration of these drugs and their combination with other chemotherapeutic agents is necessary. [Table: see text]

SPARK, studying pathways of resistance in KRAS-driven cancers: A remote plasma ctDNA participation study to identify mechanisms of resistance to KRAS inhibitors.

TPS3166 Background: KRAS mutations account for a significant proportion of oncogenic mutations in cancers of the lung, colon, pancreas, and other organs. Cancers with KRASG12C mutations are now targetable with direct KRASG12C inhibitors, and other allele-specific and pan-KRAS inhibitors are in clinical development. Recent work has shown that complex resistance mechanisms can arise after treatment with KRASG12C inhibitors, including polyclonal KRAS alterations, acquired bypass mechanisms, and acquired fusions. The SPARK protocol is a remote participation ctDNA collection study that will evaluate mechanisms of resistance to RAS inhibitors and combination therapies. Methods: SPARK (NCT05272423) is a non-interventional, single coordinating center, decentralized biospecimen collection study, with a planned accrual of up to 250 patients with a diagnosis of KRAS-mutant cancer who are experiencing disease progression on a specific KRAS inhibitor or combination of inhibitors. The study will enable a web-based remote consent (https://alcmi.net/research/spark/) and subjects will be sent blood collection kits with the necessary materials for local phlebotomy for the FoundationOne Liquid CDx (F1LCDx) Biopsy Assay. Plasma NGS results will be returned to the treating physician and the study team. Patients will initially be enrolled into two cohorts: Cohort 1A will be restricted to KRAS G12C mutant cancers who progressed after being treated with KRAS G12C inhibitors. Cohort 1B will enroll patients with KRAS mutant cancers whose tumors progressed on any KRAS inhibitor (other than a direct KRAS G12C inhibitor). Cohort 2 will enroll KRAS mutant patients that have progressed on KRAS inhibitors and who have already had a sequencing assay performed to determine the resistance mechanism to KRAS inhibitors. These patients will be invited to share their data and medical history with the study team. Plasma for F1LCDX analysis will be optional for Cohort 2. Mechanisms of acquired resistance will be summarized with descriptive statistics, and compared across drugs (e.g., sotorasib vs adagrasib), between tumor types (e.g., NSCLC vs CRC), and by regimen (e.g. monotherapy vs combination therapy) to determine if different resistance mutations arise in these settings. Progression-free survival (PFS), and overall survival (OS) with KRAS inhibitors will be analyzed depending on the resistance mechanism (e.g. on-target vs off-target). Kaplan-Meier method will be used estimate median survival, and log-rank test will be used to compare groups of interests. Cox-regression model will be used to estimate univariable and multivariable hazard ratios. Outcomes from subsequent treatment after the development of acquired resistance will also be explored with descriptive statistics. No interim analysis is planned. The study activated on November 1st 2022. Clinical trial information: NCT05272423 .

Efficacy and Imaging-Enabled Pharmacodynamic Profiling of KRAS G12C Inhibitors in Xenograft and Genetically Engineered Mouse Models of Cancer.

KRAS is one of the most commonly mutated oncogenes in lung, colorectal, and pancreatic cancers. Recent clinical trials directly targeting KRAS G12C presented encouraging results for a large population of non-small cell lung cancer (NSCLC), but resistance to treatment is a concern. Continued exploration of new inhibitors and preclinical models is needed to address resistance mechanisms and improve duration of patient responses. To further enable the development of KRAS G12C inhibitors, we present a preclinical framework involving translational, non-invasive imaging modalities (CT and PET) and histopathology in a conventional xenograft model and a novel KRAS G12C knock-in mouse model of NSCLC. We utilized an in-house developed KRAS G12C inhibitor (Compound A) as a tool to demonstrate the value of this framework in studying in vivo pharmacokinetic/pharmacodynamic (PK/PD) relationship and anti-tumor efficacy. We characterized the Kras G12C-driven genetically engineered mouse model (GEMM) and identify tumor growth and signaling differences compared to its Kras G12D-driven counterpart. We also find that Compound A has comparable efficacy to sotorasib in the Kras G12C-driven lung tumors arising in the GEMM, but like observations in the clinic, some tumors inevitably progress on treatment. These findings establish a foundation for evaluating future KRAS G12C inhibitors that is not limited to xenograft studies and can be applied in a translationally relevant mouse model that mirrors human disease progression and resistance.

An integrated model for predicting KRAS dependency.

The clinical approvals of KRAS G12C inhibitors have been a revolutionary advance in precision oncology, but response rates are often modest. To improve patient selection, we developed an integrated model to predict KRAS dependency. By integrating molecular profiles of a large panel of cell lines from the DEMETER2 dataset, we built a binary classifier to predict a tumor's KRAS dependency. Monte Carlo cross validation via ElasticNet within the training set was used to compare model performance and to tune parameters α and λ. The final model was then applied to the validation set. We validated the model with genetic depletion assays and an external dataset of lung cancer cells treated with a G12C inhibitor. We then applied the model to several Cancer Genome Atlas (TCGA) datasets. The final "K20" model contains 20 features, including expression of 19 genes and KRAS mutation status. In the validation cohort, K20 had an AUC of 0.94 and accurately predicted KRAS dependency in both mutant and KRAS wild-type cell lines following genetic depletion. It was also highly predictive across an external dataset of lung cancer lines treated with KRAS G12C inhibition. When applied to TCGA datasets, specific subpopulations such as the invasive subtype in colorectal cancer and copy number high pancreatic adenocarcinoma were predicted to have higher KRAS dependency. The K20 model has simple yet robust predictive capabilities that may provide a useful tool to select patients with KRAS mutant tumors that are most likely to respond to direct KRAS inhibitors.

Abstract B025: The RAF/MEK clamp avutometinib (VS-6766) enhances antitumor efficacy of KRAS G12C and G12D inhibitors through vertical inhibition of RAS, RAF and MEK. </title>

Abstract KRAS is the most frequently mutated RAS family member. Within non-small cell lung cancer (NSCLC), G12C is the most prevalent KRAS mutation (~13%) and the KRAS G12C inhibitors (G12Ci) sotorasib and adagrasib are now FDA approved. However, it has been shown that simultaneous targeting of multiple nodes in the MAPK pathway may be optimal for deeper and more durable responses. Furthermore, acquired mutations in the MAPK pathway occur clinically upon progression on sotorasib or adagrasib, altogether supporting the need for clinical combinations with G12Ci. Based on the clinical success of G12Ci, several KRAS G12D inhibitors (G12Di) are now being developed as G12D is the most prevalent KRAS mutation across human cancers including pancreatic (~28%) and colorectal (~11%) cancers. Avutometinib (VS-6766; avuto) is a RAF/MEK clamp that potently inhibits MEK kinase activity and induces a dominant negative RAF-MEK complex preventing phosphorylation of MEK by ARAF, BRAF and CRAF. This unique mechanism allows avuto to block MEK signaling without the compensatory re-activation of MEK that appears to limit the efficacy of MEK-only inhibitors. Here, we tested whether addition of avuto to G12Ci or G12Di could improve MAPK pathway blockade and anti-tumor efficacy. Avuto was synergistic with sotorasib and adagrasib in reducing viability of a panel of KRAS G12C cancer cell lines in 3D culture, and the combination of avuto + G12Ci showed improved depth and duration of MAPK pathway inhibition, stronger inhibition of cell cycle/proliferation markers and stronger activation of pro-apoptotic markers relative to G12Ci alone. In mouse models, avuto enhanced efficacy of G12Ci in KRAS G12C NSCLC xenograft models. Furthermore, avuto potentiated G12Ci efficacy in a KRAS(+/G12C);p53null NSCLC GEMM model with avuto + G12Ci combination inducing complete response in 25% of the mice. To evaluate efficacy of avuto against KRAS mutations that have been observed clinically upon acquired resistance to G12Ci, proliferation of Ba/F3 cells expressing these mutations was assessed. While avuto potently inhibited (IC50 < 15 nM) proliferation of cells bearing KRAS G12C/R68S, G12C/H95D or G12C/Y96C mutations, adagrasib showed diminished potency (IC50 ≥ 85 nM) against all 3 of these mutations and sotorasib showed diminished potency (IC50 ≥ 150 nM) against G12C/R68S and G12C/Y96C. Accordingly, avuto strongly inhibited tumor growth of an in vivo model expressing KRAS G12C/Y96D, while sotorasib was ineffective. Similarly, avuto was found to be synergistic with the G12Di MRTX1133 in reducing viability of a panel of KRAS G12D cancer cell lines, and avuto enhanced anti-tumor efficacy of MRTX1133 in KRAS G12D colorectal (CR3300) and pancreatic (PA1252) cancer PDX models. These results support ongoing clinical studies of avutometinib in combination with sotorasib (NCT05074810) and adagrasib (NCT05375994) for patients with KRAS G12C NSCLC. Additionally, these preclinical data support combination of avutometinib with a G12D inhibitor for patients with KRAS G12D colorectal or pancreatic cancers. Citation Format: Silvia Coma, Monica Musteanu, Cristina Caffarra, Alessia Mira, Enrico Patrucco, Julien Dilly, Andrew J. Aguirre, Mariano Barbacid, Chiara Ambrogio, Jonathan A. Pachter. The RAF/MEK clamp avutometinib (VS-6766) enhances antitumor efficacy of KRAS G12C and G12D inhibitors through vertical inhibition of RAS, RAF and MEK. </title> [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 B025.

Abstract B018: The development of macrocyclic peptide Ras inhibitors

Abstract While there has been tremendous progress developing small molecule covalent K-Ras G12C inhibitors that bind Ras-off (GDP) and inhibit nucleotide exchange, there remains a need for additional K-Ras inhibitors with different Ras isoform/mutant selectivity profiles and Ras-on (GTP) mechanisms of inhibition. Of particular interest are compounds with a pan-K-Ras profile that inhibit multiple oncogenic mutants and wild type K-Ras, but achieve a therapeutic window by sparing H and N-Ras. In addition, addressing cancers driven by K-Ras oncogenic mutants beyond G12C, K-Ras amplification, or upstream dysregulation where K-Ras is maintained in the GTP-bound state will require “Ras-on” compounds that prevent effector binding. We were interested in the potential of peptide macrocycles that bind the Switch II pocket of K-Ras, in a different conformation than the small molecules, to inhibit Ras-on by allosterically blocking Raf and other effector binding. KRpep-2d is a 19-residue disulfide bridged peptide macrocycle discovered by phage display against K-Ras G12D bound to GDP. Due to its high molecular weight (2561 Da), polarity and metabolic instability, KRpep2d is inactive in in vitro cell assays. And while biochemical experiments have shown that it binds the Ras-off GDP-bound conformation of K-Ras with high affinity to inhibit nucleotide exchange, it has low affinity for K-Ras GTP and does not allosterically inhibit Raf binding. Using parallel macrocyclic peptide chemistry, we made 180 analogs of KRpep-2d to explore truncation of the exocyclic residues and replacement of the labile disulfide bond. UNP-6752, the best compound from this exploratory library, had K-Ras affinities similar to KRpep2d with a 50-fold K-Ras GDP/GTP preference, but was only 11 residues and cyclized with a non-reducible linker. Critically this compound lacked the eight arginine residues of KrPep-2d, which both preclude passive-cell permeability and induce mast cell degranulation. A second iteration of 350 compounds designed to explore single or multiple amino acid substitutions in the core of UNP-6752 led to compound UNP-7572 with sub-micromolar affinity for K-Ras GTP and an IC50 in a K-Ras-Raf TR-FRET assay of ~800 nM. Working from the UNP-7572 we are tuning selectivity for K-Ras and our progress further improving allosteric Ras-Raf inhibition, cell permeability and activity will be presented. Citation Format: Kristopher Josephson, Anthony Silvestri, Jennifer L. Fribourgh, Walter M. Bray, Brian David, Cameron C. Hanna, Sai Kumar Chakka, Alex Hyatt, Monique Porcella, Dio Dieppa-Matos, Cameron R. Pye, Joshua Schwochert. The development of macrocyclic peptide Ras inhibitors [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 B018.

Abstract B007: Proximal RTK signaling regulates tumor initiating cell survival and therapeutic responsiveness in EGFR- and KRAS-mutated lung adenocarcinoma

Abstract Lung cancer is the leading cause of cancer-related death worldwide; adenocarcinoma is the most common subtype of lung cancer. Oncogenic driver mutations in the RTK/RAS pathway occur in 75-90% of LUAD. Oncogene-targeted therapies substantially improve outcomes in LUAD; however, recalcitrant tumors invariably emerge necessitating novel therapeutic approaches that either delay therapeutic resistance or treat resistant cancers to enhance patient outcomes. Resistance to oncogene-targeted therapies in LUAD is commonly due to RTK pathway reactivation. Resistance to osimertinib in EGFR-mutated tumors, KRASG12C inhibitors (sotorasib, adagrasib) in KRASG12C-mutated tumors, or MEK inhibitors (trametinib) in KRAS (non-G12C)-mutated tumors often develops via activation of multiple RTKs. The multiplicity of RTKs that can be activated to drive resistance suggests that individual RTK inhibitors will be ineffective alone, while broad inhibition of RTK signaling can enhance the efficacy of and delay resistance to targeted therapies in LUAD. Tumor initiating cells (TICs) are a functional subset of LUAD cells that exhibit self-renewal and are recalcitrant to oncogene-targeted therapies; it has been hypothesized that therapy-resistant TICs are the sanctuary population within the bulk tumor responsible for therapeutic resistance. Targeted ablation of TICs has the potential to enhance the efficacy of and delay resistance to targeted therapies in LUAD. We found that proximal RTK inhibition (SOS1-I and/or SOS2 KO) enhanced the killing effect of oncogene-targeted therapies under 3D culture conditions. SOS1 inhibition and/or SOS2 KO further delayed resistance to oncogenic targeted therapies in both EGFR- and KRASG12C-mutated cell lines. Since TICs are proposed as driving therapeutic resistance, these data suggested that SOS1/2 may regulate TIC survival. TICs can be functionally defined by their ability to proliferate in 3D culture from a single cell. Using this functional definition, we found that 72-h treatment with the G12C-Is adagrasib or sotorasib enriched for functional TICs 2-3 fold in multiple KRASG12C-mutated cell lines, suggesting that TICs could act as a sanctuary population of G12C-I resistant cells. We further isolated cells based on ALDH activity and found isolated ALDHhigh TICs are markedly recalcitrant to short-term G12C-I treatment. In both cases, we found that SOS1-I and/or SOS2 KO inhibited the recalcitrance of TICs to G12C inhibitors to synergistically inhibit TIC survival. These data suggest that SOS1 inhibition and/or SOS2 KO impedes the development of resistance to oncogenic targeted therapy by targeting TIC outgrowth. Citation Format: Brianna Daley, Rob Kortum. Proximal RTK signaling regulates tumor initiating cell survival and therapeutic responsiveness in EGFR- and KRAS-mutated lung adenocarcinoma [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 B007.

Abstract B030: Using the ResCu system for preclinical testing of KRAS G12C inhibitors

Abstract Introduction: Recent colorectal cancer trials with KRAS G12C inhibitors have faced resistance challenges, and a preclinical model is needed to test resistance inhibition strategies. Experimental Methods: We have developed a novel culturing system, ResCu, that allows for long-term culture of cells without the need for passaging, enabling us to determine treatment resistance that can develop over time in a physiologically relevant system that conserves the resistance pathways found in patients. Using this system, we evolved resistance to a KRAS G12C inhibitor, sotorasib, after only two weeks of culture. The evolved cells were characterized for cross-resistance and cross-sensitivity by exposing these cells to a panel of drugs for seven days. Cell viability was assessed by luminescence. Based on the drug screen results, the sotorasib evolved cells were further evolved to additional drug classes to identify novel therapeutic combinations. Whole population and single-cell transcriptomic analysis were used to identify resistance mechanisms and synthetic lethality. Results: Using the ResCu system, we evolved resistance to sotorasib, a KRAS G12C inhibitor, and other drugs in combination with sotorasib. We identified a synergistic combination of sotorasib and a secondary inhibitor that initially suppressed resistance in the sotorasib evolved cells. We identified potential mechanisms underlying this synergy using transcriptomic analysis to enable future mechanistic characterization. Subsequently, the ResCu system drove resistance evolution to the sotarasib synergistic combination. We identified splicing alterations associated with this resistance. Conclusion: We have developed a novel resistance culturing system, ResCu, that allows for preclinical testing of KRAS G12C inhibitors and determines the most promising compounds for inhibiting specific resistance pathways. This system allows for long-term culturing of cells without passaging, thereby maintaining the various phenotypes of the original populations that are commonly lost in traditional passaging cultures. Citation Format: Victor M. Ruiz, Lindsay Hill, Manasa Gadde, Christopher Bulow, Maurizio Morri, Nicholas Goldner. Using the ResCu system for preclinical testing of KRAS G12C inhibitors [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 B030.

Abstract B012: Effects of adagrasib on oncogenic signaling, immune cell regulation and biomarkers of response in preliminary clinical analyses

Abstract The recent approval of KRAS G12C inhibitors has provided novel treatment options for lung, colorectal and other cancer patients harboring this mutation and has changed the landscape of research for these cancers. Current priorities in the field include characterizing the mechanism of action of KRAS G12C inhibitors and the mechanisms underlying resistance to KRAS inhibition. Utilizing access to pre and post-treatment patient samples from KRYSTAL-1, preliminary gene expression analyses from baseline and post-adagrasib (Cycle 1, Day 8) treated patient samples demonstrated multiple oncogenic pathway gene signatures, including MYC, mTOR, cell cycle, EMT and inflammation-related signatures, were significantly regulated by adagrasib. KRAS/MAPK pathway and cell cycle-related genes including ETV4, CCND1, DUSP6, TOP1, and CENPA, as well as the Singh KRAS dependency signature were significantly downregulated in Cycle1, Day 8 compared to baseline tumor biopsies. Genes implicated in inflammation and the immune response were significantly upregulated in Cycle 1, Day 8 compared to baseline tumor biopsies. In agreement with the gene expression data as well as findings from syngeneic mouse models, preliminary immunohistochemical analysis on matched baseline, post-adagrasib treated (Cycle 1, Day 8), and end of treatment FFPE patient samples revealed marked changes in tumor cell mechanistic biomarkers and immune cell types following adagrasib treatment. These observations included decreased tumor Ki67 and increased tumor PD-L1, as well as marked alterations in tumor immune cell composition, including increased CD8+ T cells and decreased MDSCs following adagrasib treatment in several patients. Finally, flow cytometry and TCRb sequencing data from adagrasib and pembrolizumab-treated patient blood samples demonstrated an increase in several activated CD8+ T cell populations and the emergence of new T cell clones in a subset of patients after combination treatment. Together, these data suggest robust evaluation of patient biopsies pre and post adagrasib treatment may better predict outcomes that can be achieved in patients harboring KRASG12C mutant cancers and can guide early clinical development strategies, including support for combining adagrasib with immune checkpoint inhibitors. Citation Format: Jill Hallin, Laura Hover, Julio Fernandez-Benet, Adam Pavlicek, Kenna Anderes, Pasi A. Jänne, Gregory J. Riely, Alexander I. Spira, Jun Zhang, Peter Olson, James G. Christensen. Effects of adagrasib on oncogenic signaling, immune cell regulation and biomarkers of response in preliminary clinical analyses [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 B012.

Abstract B021: Pan-RAS IMM-1-104 activity in humanized 3D tumor models is independent of specific amino acid substitution

Abstract Introduction: Novel dual-MEK inhibitor IMM-1-104 is under clinical investigation for use in RAS-addicted solid tumors. Approved KRAS G12C inhibitors are available but cover a limited subset of patients. For example, the KRAS G12C substitution occurs in only 1-3% of pancreatic cancers. We assessed response to IMM-1-104 across RAS mutant preclinical models to determine whether a preference was observed for mutation position, or for specific amino acid substitutions. Experimental Procedures: Response to IMM-1-104 was measured in a humanized 3D tumor growth assay across 133 tumor models. Sixty-nine of these models have a reported RAS mutation, and all models are being mutationally profiled by whole exome sequencing, with the majority (~75%) completed to date. The RAS-mutant panel spans 11 tissue types and includes a subset of 30 confirmed KRAS G12 mutated cell lines drawn from three major indications: 12 pancreatic, 11 lung, and 7 colorectal cancer models. Based on the 3D assay, cell lines were classified into sensitive, intermediate, and resistant to IMM-1-104. The distribution of responses was then assessed across mutation position and amino acid substitutions. Summary of New Data: In the full dataset across 69 RAS-mutant models, at least one model displayed response to IMM-1-104 (sensitive or intermediate) for each observed mutation in K/N/HRAS. That is, no particular mutation position or amino acid substitution was exclusively found to be resistant. Association of response with amino acid identity was further evaluated in a subset of lines for the most frequently altered residue in KRAS, G12. A distribution of responses was observed for each amino acid substitution. The G12 substitutions having at least four cell lines each total across the three indications included G12C (8 lines), D (5 lines), R (4 lines), and V (11 lines). Across cell lines for each of these substitutions, multiple response categories were observed. In each case, half or more lines fell into the intermediate category with the rest falling into one or both of the other response categories. For example, out of the 8 KRAS G12C lines, 6 showed intermediate response, 1 showed resistance, and 1 showed sensitivity. Examining these distributions together, no significant statistical relationship was seen between the amino acid substitution and response categories by Fisher’s exact test. Conclusions: Across 69 RAS-mutated cell lines, each mutation position or amino acid substitution was associated with sensitive or intermediate response in at least one line. Looking at the most commonly mutated position in KRAS, across 30 KRAS G12 mutated cell lines from three cancer indications, no significant preference was observed with respect to response to IMM-1-104 for a particular amino acid at G12. These observations suggest potential relevance of IMM-1-104 to a broad RAS-driven patient population. Citation Format: Sarah Kolitz, Praveen Nair, Mai Johnson, Jason Funt, Peter J. King, Kevin D. Fowler, Anna Travesa, Frank Wang, John Brothers II, Amy Axel, Scott Barrett, Benjamin J. Zeskind, Brett Hall. Pan-RAS IMM-1-104 activity in humanized 3D tumor models is independent of specific amino acid substitution [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 B021.

Abstract IA20: The impact of RAS inhibition on tumor immune evasion

Abstract The approval of two KRAS G12C mutant-specific inhibitory drugs, sotorasib and adagrasib, has markedly improved the treatment of lung cancer patients harbouring KRAS G12C oncogenic mutations. However, development of acquired drug resistance is very common after initial responses, and impact on overall survival is small. Additional therapies will therefore need to be combined with KRASG12C inhibitors if long-term cures are to be achieved. Due to the immunosuppressive nature of the signaling network controlled by oncogenic KRAS, targeted KRAS G12C inhibition can indirectly affect anti-tumour immunity. This has served as a rationale for combination with immune checkpoint blockade (ICB), which we have shown leads to additional therapeutic benefit in immunogenic/hot, but not immune evasive/cold, pre-clinical lung tumour models. However, in clinical trials in KRAS G12C mutant lung cancer, combination of KRAS G12C inhibitors with PD-(L)1 blockade has yet to show improved outcome, either due to toxicities or lack of efficacy, possibly linked to prior progression on immunotherapy. Study of the response of KRAS G12C mutant lung tumours to KRAS G12C inhibitory drugs using spatial and multi-omic technologies has highlighted the role of particular populations of immune cells in the tumor microenvironment in impeding immune attack on the tumor. In immune evasive lung cancer, following KRAS G12C inhibitor treatment a community of cells is identified within the tumor containing dendritic cells, CD8+ T cells and dying tumor cells, which appears to function as an antigen presentation hub. This community is restrained by T reg cells, whose coordinate targeting markedly improves the impact of KRAS G12C inhibitors. In an immunogenic/hot mouse model of KRAS G12C mutant lung cancer, tertiary lymphoid structures are seen along with tumor-binding antibodies, which target endogenous retrovirus (ERV) envelope glycoproteins on the tumor cells. ERV-targeting B cell responses are amplified by ICB in both humans and mice, and by targeted KRAS G12C inhibition in the murine model. ERV-reactive antibodies exert anti-tumour activity that extend survival in the murine model and ERV expression predicts the outcome of ICB in human lung adenocarcinoma. Rational, mechanism-based exploitation of the impact of KRAS inhibition on tumor immune evasion provides opportunities for successful combination therapy of RAS mutant cancers. Citation Format: Julian Downward. The impact of RAS inhibition on tumor immune evasion [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 IA20.