KRASG12C Inhibitor Research Articles

A potent and selective pan-RAS inhibitor, ADT-1004, targeting complex KRAS mutations for pancreatic cancer.

e15085 Background: Pancreatic ductal adenocarcinoma (PDAC) is a challenging cancer with a 5-year survival rate of under 12%. More than 90% of PDAC cases involve KRAS mutations. KRASG12C inhibitors recently developed have limited use because only 3% of PDAC express this mutation, highlighting the urgent need for pan-RAS inhibitors to address the complex mutation landscape in PDAC while avoiding resistance mechanisms. We synthesized and evaluated a novel pan-RAS inhibitor, ADT-1004, in mouse tumor models transplanted with mouse PDAC cell lines or patient-derived xenografts. ADT-1004 is an orally bioavailable prodrug of ADT-007, a highly potent and selective pan-RAS inhibitor (doi.org/10.1101/2023.05.17.541233). Methods: Human and mouse PDAC cell lines were utilized to evaluate in vitro growth inhibitory potency and selectivity of ADT-007. For in vivo experiments, KPC-Luc (1 × 105) and 2838C3-Luc (1.5 × 105) PDAC cells harboring the KRASG12D mutation were injected into the pancreas of C57BL/6J mice. After one week, the mice were randomly divided into treatment groups (n = 7 per group) and received oral ADT-1004 at a dose of 40mg/kg body weight five times a week for 4 weeks. Tumor burden was assessed weekly by monitoring bioluminescence signals using the IVIS Xenogen imaging system. Four patient-derived xenografts from PDAC patients with KRASG12C, KRASG12D, KRASG12V, KRASG13Q mutations were subcutaneously implanted in NSG mice by a small incision in the right flank. A week later, when the tumors reached a size of 100mm3 mice were randomized (n = 7 per group) and treated orally with 40mg/kg of ADT-1004 five times a week for 6 weeks. Body weight and tumor size were measured twice weekly. Results: ADT-007 potently and selectively inhibited the growth of human and mouse PDAC cell lines. For example, the IC50 value of ADT-007 for KRASG12C MIA PaCA-2 PDAC cells was as low as 2 nM compared to 2500 nM for RASWT BxPC3 PDAC cells. Growth inhibition was dependent on activated RAS and associated with reduced GTP-RAS levels and MAPK/AKT signaling. ADT-1004 was well tolerated in mice at a dose up to 175 mg/kg bid orally with sustained plasma levels of ADT-007 far exceeding growth IC50 values. When administered orally at 40 mg/kg body weight, ADT-1004 displayed robust antitumor activity in mouse tumor models using patient or mouse derived PDAC cell lines with G12D, G12V, G12C, or G13Q KRAS mutations, causing significant inhibitory effects on ERK phosphorylation. Conclusions: ADT-1004 inhibited tumor growth of KRAS mutant PDA tumors by targeting activated RAS to disrupt downstream MAPK/AKT signaling. The results highlight the promise of ADT-1004 as a novel pan-RAS inhibitor and open new strategies for addressing the complex genetic landscape of PDAC and other RAS driven cancers.

Discovery of novel coumarin-based KRAS-G12C inhibitors from virtual screening and Rational structural optimization

KRAS-G12C inhibitors has been made significant progress in the treatment of KRAS-G12C mutant cancers, but their clinical application is limited due to the adaptive resistance, motivating development of novel structural inhibitors. Herein, series of coumarin derivatives as KRAS-G12C inhibitors were found through virtual screening and rational structural optimization. Especially, K45 exhibited strong antiproliferative potency on NCI-H23 and NCI-H358 cancer cells harboring KRAS-G12C with the IC50 values of 0.77 μM and 1.50 μM, which was 15 and 11 times as potent as positive drug ARS1620, respectively. Furthermore, K45 reduced the phosphorylation of KRAS downstream effectors ERK and AKT by reducing the active form of KRAS (KRAS GTP) in NCI-H23 cells. In addition, K45 induced cell apoptosis by increasing the expression of anti-apoptotic protein BAD and BAX in NCI-H23 cells. Docking studies displayed that the 3-naphthylmethoxy moiety of K45 extended into the cryptic pocket formed by the residues Gln99 and Val9, which enhanced the interaction with the KRAS-G12C protein. These results indicated that K45 was a potent KRAS-G12C inhibitor worthy of further study.

ABCB1 attenuates brain exposure to the KRASG12C inhibitor opnurasib whereas binding to mouse carboxylesterase 1c influences its plasma exposure

Opnurasib (JDQ443) is a newly developed oral KRASG12C inhibitor, with a binding mechanism distinct from the registered KRASG12C inhibitors sotorasib and adagrasib. Phase I and II clinical trials for opnurasib in NSCLC are ongoing. We evaluated the pharmacokinetic roles of the ABCB1 (P-gp/MDR1) and ABCG2 (BCRP) efflux and OATP1 influx transporters, and of the metabolizing enzymes CYP3A and CES1 in plasma and tissue disposition of oral opnurasib, using genetically modified cell lines and mouse models. In vitro, opnurasib was potently transported by human (h)ABCB1 and slightly by mouse (m)Abcg2. In Abcb1a/b- and Abcb1a/b;Abcg2-deficient mice, a significant ∼100-fold increase in brain-to-plasma ratios was observed. Brain penetration was unchanged in Abcg2-/- mice. ABCB1 activity in the blood-brain barrier may therefore potentially limit the efficacy of opnurasib against brain metastases. The Abcb1a/b transporter activity could be almost completely reversed by co-administration of elacridar, a dual ABCB1/ABCG2 inhibitor, increasing the brain penetration without any behavioral or postural signs of acute CNS-related toxicity. No significant pharmacokinetic roles of the OATP1 transporters were observed. Transgenic human CYP3A4 did not substantially affect the plasma exposure of opnurasib, indicating that opnurasib is likely not a sensitive CYP3A4 substrate. Interestingly, Ces1-/- mice showed a 4-fold lower opnurasib plasma exposure compared to wild-type mice, whereas no strong effect was seen on the tissue distribution. Plasma Ces1c therefore likely binds opnurasib, increasing its retention in plasma. The obtained pharmacokinetic insights may be useful for further optimization of the clinical efficacy and safety of opnurasib, and might reveal potential drug-drug interaction risks.

Synergy of EGFR and AURKA Inhibitors in KRAS-mutated Non-small Cell Lung Cancers.

The introduction of specific KRAS G12C inhibitors to the clinical practice in lung cancer has opened up opportunities that did not exist before. However, G12C alterations are only a subtype of all KRAS mutations observed. Given the high expression of AURKA in KRASmut NSCLC, our study could point to a potential therapeutic option for this subgroup of patients.

D3S-001, a KRAS G12C Inhibitor with Rapid Target Engagement Kinetics, Overcomes Nucleotide Cycling, and Demonstrates Robust Preclinical and Clinical Activities.

First-generation KRAS G12C inhibitors, 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 guanosine diphosphate (GDP)- and guanosine triphosphate (GTP)-bound states, raising controversy about whether targeting the GDP-bound form can fully block this oncogenic driver. We herein report that D3S-001, a next-generation GDP-bound G12C inhibitor with faster target engagement (TE) kinetics, depletes cellular active KRAS G12C at nanomolar concentrations. In the presence of growth factors, such as epithelial growth factor and hepatocyte growth factor, the ability of sotorasib and adagrasib to inhibit KRAS was compromised whereas the TE kinetics of D3S-001 was nearly unaffected, a unique feature differentiating D3S-001 from other GDP-bound G12C inhibitors. Furthermore, the high covalent potency and cellular TE efficiency of D3S-001 contributed to robust antitumor activity preclinically and translated into promising clinical efficacy in an ongoing phase 1 trial (NCT05410145). Significance: The kinetic study presented in this work unveils, for the first time, that a GDP-bound conformation-selective KRAS G12C inhibitor can potentially deplete cellular active KRAS in the presence of growth factors and offers new insights into the critical features that drive preclinical and clinical efficacy for this class of drugs.

Precision medicine for pancreatic cancer: characterizing the clinicogenomic landscape and outcomes of KRAS G12C-mutated disease.

Mutated Kirsten rat sarcoma viral oncogene homolog (KRAS) is the most common oncogene alteration in pancreatic ductal adenocarcinoma, and KRAS glycine to cystine substitution at codon 12 (G12C) mutations (KRAS G12Cmut) are observed in 1%-2%. Several inhibitors of KRAS G12C have recently demonstrated promise in solid tumors, including pancreatic cancer. Little is known regarding clinical, genomics, and outcome data of this population. Patients with pancreatic cancer and KRAS G12Cmut were identified at Memorial Sloan Kettering Cancer Center and via the American Association of Cancer Research Project Genomics, Evidence, Neoplasia, Information, Exchange database. Clinical, treatment, genomic, and outcomes data were analyzed. A cohort of patients at Memorial Sloan Kettering Cancer Center with non-G12C KRAS pancreatic cancer was included for comparison. Among 3571 patients with pancreatic ductal adenocarcinoma, 39 (1.1%) with KRAS G12Cmut were identified. Median age was 67 years, and 56% were female. Median body mass index was 29.2 kg/m2, and 67% had a smoking history. Median overall survival was 13 months (95% CI: 9.4 months, not reached) for stage IV and 26 months (95% CI: 23 months, not reached) for stage I-III. Complete genomic data (via American Association of Cancer Research Project Genomics, Evidence, Neoplasia, Information, Exchange database) was available for 74 patients. Most common co-alterations included TP53 (73%), CDKN2A (33%), SMAD4 (28%), and ARID1A (21%). Compared with a large cohort (n = 2931) of non-G12C KRAS-mutated pancreatic ductal adenocarcinoma, ARID1A co-mutations were more frequent in KRAS G12Cmut (P < .05). Overall survival did not differ between KRAS G12Cmut and non-G12C KRAS pancreatic ductal adenocarcinoma. Germline pathogenic variants were identified in 17% of patients; 2 patients received KRAS G12C-directed therapy. Pancreatic cancer and KRAS G12Cmut may be associated with a distinct clinical phenotype. Genomic features are similar to non-G12C KRAS-mutated pancreatic cancer, although enrichment of ARID1A co-mutations was observed. Targeting of KRAS G12C in pancreatic cancer provides a precedent for broader KRAS targeting in pancreatic cancer.

KRAS G12C inhibitor combination therapies: current evidence and challenge.

Although KRAS G12C inhibitors have proven that KRAS is a "druggable" target of cancer, KRAS G12C inhibitor monotherapies have demonstrated limited clinical efficacy due to primary and acquired resistance mechanisms. Multiple combinations of KRAS G12C inhibitors with other targeted therapies, such as RTK, SHP2, and MEK inhibitors, have been investigated in clinical trials to overcome the resistance. They have demonstrated promising efficacy especially by combining KRAS G12C and EGFR inhibitors for KRAS G12C-mutated colorectal cancer. Many clinical trials of combinations of KRAS G12C inhibitors with other targeted therapies, such as SOS1, ERK, CDK4/6, and wild-type RAS, are ongoing. Furthermore, preclinical data have suggested additional promising KRAS G12C combinations with YAP/TAZ-TEAD inhibitors, FAK inhibitors, and farnesyltransferase inhibitors. The combinations of KRAS G12C inhibitors with immunotherapies and chemotherapies have also been investigated, and the preliminary results were reported. More recently, KRAS-targeted therapies not limited to KRAS G12C are being developed, potentially broadening the treatment landscape of KRAS-mutated cancers. Rationally combining KRAS inhibitors with other therapeutics is likely to play a significant role in future treatment for KRAS-mutated solid tumors.

Divarasib in the Evolving Landscape of KRAS G12C Inhibitors for NSCLC.

Kristen Rat Sarcoma viral oncogene (KRAS) mutations are one of the most common oncogenic drivers found in 12-14% of non-small cell lung cancer (NSCLC) and 4% of colorectal cancer tumors. Although previously difficult to target, sotorasib and adagrasib are now approved for previously treated NSCLC patients with KRAS G12C mutations. In preclinical studies, divarasib was 5 to 20 times as potent and up to 50 times as selective as sotorasib and adagrasib. While sotorasib met its primary endpointin the phase III second line study against docetaxel, the progression-free survival (PFS) benefit was small and no overall survival (OS) benefit was observed. Adagrasib has demonstrated clinical benefitin the phase I/II KRYSTAL-1 study setting, however, 44.8% of patients reported grade 3 or higher toxicities. Divarasib has been studied in a phase I dose expansion cohort with promising efficacy [objective response (ORR) 53.4% and PFS 13.1 months]. Although most patients reported toxicities, the majority were low-grade and manageable with supportive care. Here we discuss these results in the context of the evolving KRAS G12C landscape.

A pivotal phase 2 single-arm study of glecirasib (JAB-21822) in patients with NSCLC harboring KRAS G12C mutation.

468214 Background: KRAS G12C mutation occurs in approximately 4% of non-small cell lung cancers (NSCLC) in China. Glecirasib (Jacobio, Beijing, PRC) is a potent and highly selective covalent oral inhibitor of KRAS G12C. Methods: This phase 2 single-arm study (NCT05009329) of glecirasib was conducted in China at 43 sites, enrolling participants with locally advanced or metastatic KRAS G12C mutated NSCLC. Subjects must have received prior platinum-based therapy and an immune checkpoint inhibitor (ICI) as well as appropriate targeted agents for any actionable mutations per local standards. Primary study objective is overall response rate (ORR) assessed by independent review committee (IRC). The secondary study objectives include duration of response (DOR), progression-free survival (PFS), overall survival (OS), disease control rate (DCR) and safety. Here we report topline results from this pivotal study. Results: A total of 119 patients with NSCLC harboring KRAS G12C mutation were enrolled and treated with single agent glecirasib at 800mg daily (median follow up 10.4 months). The median age was 62 years, 79% was male and 21% was female, and 80.7% had ECOG PS 1. The prior lines of therapy include 1-3, with 94.1% (112/119) patients having received prior platinum-based therapy and ICI. Confirmed ORR was 47.9% (56/117; 95% CI, 38.5%-57.3%) and confirmed DCR was 86.3% (101/117; 95% CI, 78.7%-92%) assessed by IRC. Median PFS per IRC was 8.2 months (95% CI, 5.5 – 13.1), and median OS was 13.6 months (95% CI, 10.9 – NE). Median DOR has not been reached (95% CI, 7.2 – NE ). Overall, treatment-related adverse events (TRAEs) of any grade were observed in 97.5% of subjects. Most common TRAEs [≥15%] were anemia (56.3%), blood bilirubin increased (48.7%), alanine aminotransferase increased (35.3%), aspartate aminotransferase increased (35.3%), hypertriglyceridemia (28.6%) and γ-Glutamyl transferase increased (15.1%). Grade 3 and 4 TRAEs were observed in 39.5%. No grade 5 TRAEs occurred. Most of patients can stay on the treatment and 5.0% patients discontinued the treatment due to TRAE. In contrast to other FDA approved KRAS G12C inhibitor, Gleciasib has 6.7% of nausea, 7.6% vomiting, 3.4% diarrhea in which only one grade 3 nausea is reported. Conclusion: The pivotal phase 2 study of Glecirasib has met the primary endpoints and demonstrates impressive ORR and PFS with promising clinical activity for patients with KRAS G12C mutated advanced NSCLC. Glecirasib is well tolerated, with an extremely low gastrointestinal toxicity profile, which may improve patient compliance with oral therapy. Clinical trial information: NCT05009329 .

The SOS1 Inhibitor MRTX0902 Blocks KRAS Activation and Demonstrates Antitumor Activity in Cancers Dependent on KRAS Nucleotide Loading.

KRAS is the most frequently mutated oncogene in human cancer and facilitates uncontrolled growth through hyperactivation of the receptor tyrosine kinase (RTK)/mitogen-activated protein kinase (MAPK) pathway. The Son of Sevenless homolog 1 (SOS1) protein functions as a guanine nucleotide exchange factor (GEF) for the RAS subfamily of small GTPases and represents a druggable target in the pathway. Using a structure-based drug discovery approach, MRTX0902 was identified as a selective and potent SOS1 inhibitor that disrupts the KRAS:SOS1 protein-protein interaction to prevent SOS1-mediated nucleotide exchange on KRAS and translates into an anti-proliferative effect in cancer cell lines with genetic alterations of the KRAS-MAPK pathway. MRTX0902 augmented the antitumor activity of the KRAS G12C inhibitor adagrasib when dosed in combination in eight out of 12 KRAS G12C-mutant human non-small cell lung cancer and colorectal cancer xenograft models. Pharmacogenomic profiling in preclinical models identified cell cycle genes and the SOS2 homolog as genetic co-dependencies and implicated tumor suppressor genes (NF1 and PTEN) in resistance following combination treatment. Lastly, combined vertical inhibition of RTK/MAPK pathway signaling by MRTX0902 with inhibitors of EGFR or RAF/MEK led to greater downregulation of pathway signaling and improved antitumor responses in KRAS-MAPK pathway-mutant models. These studies demonstrate the potential clinical application of dual inhibition of SOS1 and KRAS G12C and additional SOS1 combination strategies that will aide in the understanding of SOS1 and RTK/MAPK biology in targeted cancer therapy.

CRISPR-Cas9 Screening Identifies KRAS-Induced COX2 as a Driver of Immunotherapy Resistance in Lung Cancer.

Oncogenic KRAS impairs antitumor immune responses. As effective strategies to combine KRAS inhibitors and immunotherapies have so far proven elusive, a better understanding of the mechanisms by which oncogenic KRAS drives immune evasion is needed to identify approaches that could sensitize KRAS-mutant lung cancer to immunotherapy. In vivo CRISPR-Cas9 screening in an immunogenic murine lung cancer model identified mechanisms by which oncogenic KRAS promotes immune evasion, most notably via upregulation of immunosuppressive COX2 in cancer cells. Oncogenic KRAS potently induced COX2 in both mouse and human lung cancer, which was suppressed using KRAS inhibitors. COX2 acted via prostaglandin E2 (PGE2) to promote resistance to immune checkpoint blockade (ICB) in lung adenocarcinoma. Targeting COX2/PGE2 remodeled the tumor microenvironment by inducing proinflammatory polarization of myeloid cells and influx of activated cytotoxic CD8+ T cells, which increased the efficacy of ICB. Restoration of COX2 expression contributed to tumor relapse after prolonged KRAS inhibition. These results provide the rationale for testing COX2/PGE2 pathway inhibitors in combination with KRASG12C inhibition or ICB in patients with KRAS-mutant lung cancer. Significance: COX2 signaling via prostaglandin E2 is a major mediator of immune evasion driven by oncogenic KRAS that promotes immunotherapy and KRAS-targeted therapy resistance, suggesting effective combination treatments for KRAS-mutant lung cancer.

Targeting KRASG12C in Non-Small-Cell Lung Cancer: Current Standards and Developments.

Among the most common molecular alterations detected in non-small-cell lung cancer (NSCLC) are mutations in Kristen Rat Sarcoma viral oncogene homolog (KRAS). KRAS mutant NSCLC is a heterogenous group of diseases, different from other oncogene-driven tumors in terms of biology and response to therapies. Despite efforts to develop drugs aimed at inhibiting KRAS or its signaling pathways, KRAS had remained undruggable for decades. The discovery of a small pocket in the binding switch II region of KRASG12C has revolutionized the treatment of KRASG12C-mutated NSCLC patients. Sotorasib and adagrasib, direct KRASG12C inhibitors, have been approved by the US Food and Drug Administration (FDA) and other regulatory agencies for patients with previously treated KRASG12C-mutated NSCLC, and these advances have become practice changing. However, first-line treatment in KRASG12C-mutated NSCLC does not differ from NSCLC without actionable driver genomic alterations. Treatment with KRASG12C inhibitors is not curative and patients develop progressive disease, so understanding associated mechanisms of drug resistance is key. New KRASG12C inhibitors and several combination therapy strategies, including with immune checkpoint inhibitors, are being studied in clinical trials. The aim of this review is to explore the clinical impact of KRAS, and outline different treatment approaches, focusing on the novel treatment of KRASG12C-mutated NSCLC.

Management of sotorasib-related adverse events and hepatotoxicities following anti-PD-(L)1 therapy: Experience with sotorasib in two French anti-cancer centers and practical guidance proposal

IntroductionSotorasib is a first-in-class KRASG12C inhibitor that showed significant clinical activity in KRASG12C-mutated non-small cell lung cancer (NSCLC). The most frequent grade 3 or 4 sotorasib-related adverse events (AEs) were diarrhea (4–12 %) and hepatotoxicity (10.1–15.1 %). Data is lacking about the management of these AEs, especially in patients receiving sequential anti-PD-(L)1 and sotorasib therapy. Our aim was to report the management of grade ≥ 2 sotorasib-related AEs in real-world setting and to propose practical guidance for the management of grade ≥ 2 sotorasib-related AEs and more generally KRASG12C inhibitors-related AEs. Materials and methodsRecords from all consecutive patients who initiated sotorasib through expanded access program in two French anti-cancer centers from January 1st 2021 to April 1st 2023 were reviewed to identify and grade sotorasib-related AEs, according to NCI-CTCAE v5.0., and to collect AEs management data. Patients were included in the analysis if they presented a grade ≥ 2 sotorasib-related AE. ResultsFrom 57 patients identified, 21 met inclusion criteria including eighteen (86 %) who received sequential anti-PD-(L)1 and sotorasib therapy. Hepatotoxicity (76 %) and diarrhea (24 %) were the most common grade ≥ 2 sotorasib-related AEs. Among the 16 patients with a grade ≥ 2 hepatotoxicity, 12 (75 %) definitely discontinued sotorasib, among which 9 (56 %) after dose reductions and rechallenge, and five (32 %) received corticosteroids, allowing only one patient to resume sotorasib. Diarrhea and nausea were usually manageable and not associated with sotorasib discontinuation. We propose a step-by-step management practical guidance for sotorasib-related hepatotoxicity based on dose-reduction and careful monitoring. Liver biopsy is strongly encouraged for grade 3 and 4 hepatotoxicity to assess candidates for corticosteroids. DiscussionThe experience with sotorasib might help better prevent, screen and manage sotorasib-related and other KRASG12C inhibitors-related AEs, particularly hepatotoxicity.

Emerging Therapies in Kirsten Rat Sarcoma Virus (+) Non-Small-Cell Lung Cancer.

Kirsten rat sarcoma virus (KRAS) is the most frequently found oncogene in human cancers, including non-small-cell lung cancer (NSCLC). For many years, KRAS was considered "undruggable" due to its structure and difficult targeting. However, the discovery of the switch II region in the KRAS-G12C-mutated protein has changed the therapeutic landscape with the design and development of novel direct KRAS-G12C inhibitors. Sotorasib and adagrasib are FDA-approved targeted agents for pre-treated patients with KRAS-G12C-mutated NSCLC. Despite promising results, the efficacy of these novel inhibitors is limited by mechanisms of resistance. Ongoing studies are evaluating combination strategies for overcoming resistance. In this review, we summarize the biology of the KRAS protein and the characteristics of KRAS mutations. We then present current and emerging therapeutic approaches for targeting KRAS mutation subtypes intending to provide individualized treatment for lung cancer harboring this challenging driver mutation.

Abstract LB077: CSF-1R inhibition with Pimicotinib (ABSK021) enhanced anti-tumor efficacy of KRASG12C inhibitors in preclinical non-small cell lung cancer mouse models

Abstract Background: KRAS mutations are highly prevalent oncogenic drivers in human cancers and KRASG12C is the most frequent mutations found in KRAS-mutant non-small lung cancer (NSCLC). It is reported that tumor associated macrophages (TAMs) are enriched in KRAS-driven transgenic lung cancer models. Depletion of macrophage results in a significant decrease in tumor burden and increased survival. Sotorasib (AMG510), the first KRASG12C inhibitor approved by FDA, has been found to modulate tumor immunity, impacting the recruitment of macrophages. CSF-1R signaling is critical for macrophage proliferation and survival. CSF-1R inhibition depletes TAMs and reprograms tumor microenvironment (TME), resulting in enhancement of anti-tumor immunity. Therefore, the combination of KRASG12C and CSF-1R inhibitors may synergize to enhance anti-tumor efficacy. Pimicotinib (ABSK021) is a potential best-in-class small molecule inhibitor of CSF-1R in clinical development of multiple indications. Here, we conducted in vivo experiments to explore the combined effects of KRASG12C and CSF-1R inhibitors. Methods: In vivo efficacy studies were performed to evaluate the combined treatment of KRASG12C inhibitors (AMG510 and ABSK071, a next-generation inhibitor targeting KRASG12C with better inhibitory activity) and CSF-1R inhibitor (ABSK021) in NCI-H2122 PBMC humanized mice and LLC syngeneic mice. RNA sequencing was employed to investigate the mechanism of action underlying the synergistic anti-tumor effect. Immunohistochemistry (IHC) was utilized to examine the changes of immune cell populations in TME. Results: The combinatory regimen of ABSK021 and KRASG12C inhibitors showed improved anti-tumor effects than each single agent alone in both mouse NSCLC models. Bioinformatics and follow-up analysis revealed a reduction in immune suppressive TAMs in tumors treated with ABSK021 alone or in combination with KRASG12C inhibitors. Furthermore, modulation of TME related to anti-tumor immunity, such as activated CD4 T, CD8 T and NK cells, was increased, and pro-tumor immunity, such as MDSC, regulatory T cells, was decreased in combination group. IHC results confirmed that ABSK021 addition led to macrophage depletion and more CD8 T cell infiltration in TME. Conclusions: For the first time, we demonstrate that the combined inhibition of KRASG12C and CSF-1R leads to superior therapeutic efficacy in preclinical NSCLC mouse models. This results suggests a potential novel therapeutic regimen that could yield improved clinical benefit to patients. Citation Format: Nannan Zhang, Bin Shen, Shenyan Liu, Cheng Dai, Haiyan Ying, Fushen Guo, Zhixuan Zhu, Wan Shi, Min Tu, Jie Wang, Jie Zhang, Manqi Liu, Qi Zhang, Zhui Chen. CSF-1R inhibition with Pimicotinib (ABSK021) enhanced anti-tumor efficacy of KRASG12C inhibitors in preclinical non-small cell lung cancer mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB077.

Abstract ND07: BBO-8520, a first-in-class, direct inhibitor of KRASG12C (ON), locks GTP-bound KRASG12C in the state 1 conformation resulting in rapid and complete blockade of effector binding

Abstract The glycine to cysteine mutation on codon 12 of KRAS (KRASG12C) is found in ~15% of non-small cell lung cancers and in a low percentage of colorectal and pancreatic adenocarcinomas. This activating mutation pushes the balance of cellular KRAS towards its active, GTP-bound (ON) state that signals downstream and drives cellular proliferation. Recently approved inhibitors of KRASG12C that bind and sequester the oncogenic protein in its inactive GDP-bound (OFF) state, have demonstrated clinical efficacy; however, median duration of response has been &amp;lt; 9 months. Rapid tumor cell adaptation to KRASG12C (OFF)-only inhibitors like sotorasib and adagrasib has been attributed to reactivation of MAPK signaling through increased RTK flux and KRASG12C gene amplification that result in increased KRASG12C (ON) presence and activity. To block rapid KRASG12C (ON)-driven adaptation to KRASG12C (OFF) inhibitors, we have developed a first-in-class, direct, small molecule covalent inhibitor of both KRASG12C (ON) and (OFF) states. BBO-8520 binds in the switch II pocket and covalently modifies both the (ON) and (OFF) forms of KRASG12C independently of any other partner proteins. Mass spectrometry analysis of KRASG12C covalent engagement by BBO-8520 shows complete modification of both KRASG12C states within 15 minutes, while sotorasib and adagrasib, only modify the (OFF) state. 31P NMR studies demonstrate that BBO-8520 inhibits KRASG12C (ON) by locking the GTP-bound protein in state 1, a conformation where it is unable to bind effectors. The ability to directly bind KRASG12C (ON) leads to potent (~30 nM) inhibition in an effector (Raf1) binding assay where inactive (OFF) inhibitors demonstrate no measurable potency. BBO-8520 displays highly significant binding to KRASG12C in a global cysteine proteome analysis and is 100x more selective for KRASG12C than for WT KRAS and other mutant isoforms, with no measurable activity against N- or H-RAS. Cellular signaling and viability assays show that BBO-8520 has sub-nanomolar potency against KRASG12C mutant cell lines. In effector binding assays, BBO-8520 rapidly and completely blocks the RAS-RAF1 interaction, clearly differentiating it from inactive (OFF) inhibitors that require longer times to allow for cycling to the inactive (OFF) state. Additionally, long-term clonogenic assays that detect the emergence of resistance in the presence of inhibitors show that BBO-8520 is at least 30x more potent than sotorasib and adagrasib at preventing outgrowth. Importantly, the presence of growth factors (e.g., EGF) that push KRASG12C into its (ON) state, and significantly lower the potency of inactive (OFF) inhibitors (&amp;gt;20x), have only minor effects on BBO-8520’s potency. Drug-like pharmacokinetic properties allow BBO-8520 to achieve strong dose- and time-dependent pharmacodynamic effects (&amp;gt;80% inhibition of pERK) following a single, oral dose in KRASG12C mutant tumor bearing mice. In vivo target engagement and pERK inhibition in the MIAPaCa-2 and H358 KRASG12C mutant tumor models resulted in durable tumor regressions at 10 mg/kg. Similarly, daily dosing of 10 mg/kg of BBO-8520 in the KrasG12C-p53 driven GEMM model for 6 weeks resulted in better than 50% lung tumor volume regression. BBO-8520’s potent activity against KRASG12C (ON) presents, for the first time, the potential opportunity to directly target all mutant KRASG12C in cells, including the active form of KRASG12C, enabling efficacy following complete or near-complete target inhibition. Citation Format: Anna E. Maciag, James Stice, Bin Wang, Alok Sharma, Albert Chan, Ken Lin, Devansh Singh, Marcin Dyba, Yue Yang, Saman Setoodeh, Brian P. Smith, Dana Rabara, Zuhui Zhang, Erik K. Larsen, Dom Esposito, John Paul Denson, Michela Ranieri, Mary Meynardie, Sadaf Mehdizadeh, Patrick Alexander, Maria Abreu Blanco, David Turner, Rui Xu, Felice C. Lightstone, Kwok Kin Wong, Dhirendra Simanshu, Keshi Wang, Andrew G. Stephen, Kerstin Sinkevicius, Dwight V. Nissley, Eli Wallace, Frank McCormick, Pedro J. Beltran. BBO-8520, a first-in-class, direct inhibitor of KRASG12C (ON), locks GTP-bound KRASG12C in the state 1 conformation resulting in rapid and complete blockade of effector binding [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr ND07.

Abstract LB220: Farnesyltransferase inhibitors show synergistic anticancer effects in combination with novel KRAS G12C inhibitors

Abstract Background. Farnesyltransferase inhibitors (FTI) failed as monotherapies for various cancer types until the discovery of their potency in HRAS mutant human head and neck and bladder cancers. On the other hand, RAS mutant cancers have been a great challenge for cancer research until the discoveries and approvals of several KRAS-G12C allele specific inhibitors. However, G12C inhibitors were effective mainly in lung cancer and de novo and acquired resistance prompted the development of various combinational modalities including immunotherapy as well as SOS- and SHP2-inhibitors. Based on in-silico analysis of drug sensitivities of KRAS mutant cancer cell lines treated with various drugs including FTIs, we have explored the possibility of the combination of FTIs with KRAS inhibitors.Methods. Clinically approved farnesyl-transferase inhibitors (tipifarnib and lonafarnib) were combined with, novel KRAS G12C inhibitors (sotorasib and adagrasib) using human lung-, pancreatic and colorectal- adenocarcinoma cells in vitro and in vivo. Antitumoral effects were evaluated for cell proliferation, apoptosis and migratory activity. Mechanisms of action were investigated by immunoblot analyzes of various farnesylated proteins, RAS activation and signaling, videomicroscopy and also by histopathology of xenograft tumors. Results. Synergistic anticancer effects were observed upon the combination of FTIs with G12C inhibitors in KRAS G12C mutant human cancer cell lines in vitro. We found that the combination interfered with the compensatory re-activation of HRAS, farnesylation of RHEB in the PI3K/mTOR pathway and lamin. Furthermore, we observed enhanced efficacy of sotorasib upon combination with tipifarnib in the xenograft models of lung adenocarcinoma affecting mitotic- and apoptotic rates and inducing necrosis.Discussion. Our findings suggest the potential clinical applicability of the combination of KRAS-G12C inhibitors and farnesyl-transferase inhibitors. Furthermore, our preliminary data suggest that the synergistic effect of FTIs on KRAS-G12C inhibitors can be projected to G12D inhibitors as well. Citation Format: Jozsef Timar, Marcell Baranyi, Eszter Molnar, Ivan Randelovic, Mihaly Cserepes, Jozsef Tovari, Balazs Hegedus. Farnesyltransferase inhibitors show synergistic anticancer effects in combination with novel KRAS G12C inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB220.

Abstract LB366: KRASG12C inhibition enhances immunogenicity in KRASG12C-mutant colorectal cancer

Abstract RAS mutations are among the most prevalent oncogenic mutations, found in approximately 20% of human cancers. KRAS, the predominantly mutated isoform of RAS, is mutated in 40% of colorectal cancer (CRC) and leads to constitutive activation of the MAPK pathway and other downstream effectors. Despite recent FDA approval for KRASG12C selective inhibitor in non-small cell lung cancer, the response rate in CRC is below 20%, posing a challenge for treatment of these tumors. Several studies have implicated oncogenic KRAS signaling in creating an immunosuppressive tumor microenvironment; therefore, we hypothesized that KRAS inhibitors can enhance anti-tumor immunity, providing a rationale for their combination with immune checkpoint blockade. Using a syngeneic KRASG12C CRC mouse model with knockout of APC, TP53, and SMAD4, we examined tumor cell-intrinsic mechanism of cooperativity between KRAS inhibition and immune response. We found that combining KRASG12C inhibition with anti-PD1 produced a more substantial and sustained reduction in tumor volume compared with KRAS or PD-1 inhibition alone. KRASG12C inhibition led to a significant increase in CD3+CD8+ T cell infiltration compared with vehicle control. Single-cell RNA-sequencing corroborated our observations and revealed increased T cell infiltration in KRASG12C tumors following KRASG12C inhibition. Moreover, these T cells upregulated genes involved in interferon gamma production and T cell activation. Importantly, we observed a significant reduction in the Ras pathway genes, specifically in the tumor cell population, along with enrichment of genes involved in abnormal antigen presentation and MHC Class I peptide loading complex. Mechanistically, we found that KRASG12C inhibition activates the innate immune response, which subsequently induce Type I interferons that activate the expression of T cell-recruiting interferon stimulated genes like CXCL9, CXCL10, IFIT1, IFIT2, and IFIT3. Our data presents a connection between KRAS inhibition and anti-tumor immunity, providing insights into novel combination strategies for KRASG12C colorectal cancers. Citation Format: Parasvi S. Patel, Jun Tian, Ferran Fece de la Cruz, Jacquelyn Curtis, Apekshya Panda, Tomonori Oka, Shadmehr Demehri, Moshe Sade-Feldman, Scott Koptez, Ryan B. Corcoran. KRASG12C inhibition enhances immunogenicity in KRASG12C-mutant colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB366.

Abstract LB266: AMG510-mediated inactivation of BCL-xL protein promotes Gasdermin-E-dependent pyroptosis in KRAS-G12C mutant non-small cell lung cancer

Abstract Introduction: Studies have shown that molecular targeted therapy can mediate Gasdermin-E (GSDME) dependent pyroptosis. However, GSDME expression is suppressed in many mutated patients, including those with KRAS-G12C-mutated non-small-cell lung cancer (NSCLC). The efficacy of AMG510 against KRAS-G12C inhibition (KRAS-G12Ci) is constrained by tumor recurrence. Pyroptosis is widely involved in the occurrence and development of tumors. However, the role and molecular mechanism of AMG510 in pyroptosis have not been well identified. Here, we demonstrate that inactivation of anti-apoptotic Bcl-2 family members during KRAS-G12Ci treatment enhances pyroptosis, which then promotes the sensitivity of AMG510 in NSCLC. Methods: We assessed pyroptosis using live cells imaging, LDH release assays, ELISA, Western blotting, and flow cytometry. In vitro assays were used to investigate the function of GSDME. Co-culture between peripheral blood mononuclear cells (PBMCs) and KRAS-G12C mutant lung cancer cell lines explored the anti-tumor immunity of cytotoxic T lymphocytes affected by knockdown of GSDME. The molecular mechanism was determined using bulk RNA sequencing, molecular cloning technology, and Western blotting. Results: We demonstrated that KRAS G12Ci treatment promotes cleavage of GSDME, release of high mobility group protein B1 (HMGB1), and LDH, markers of pyroptotic cell death. Additionally, GSDME was significantly downregulated in KRAS-mutated NSCLC compared with patients of KRAS wild type in TCGA cohort, while significantly upregulated after AMG510 treatment and downregulated after AMG510 resistance in vitro assay. We identified that KRAS-G12Ci resulted in the inactivation of BCL-xL, leading to caspase-3-dependent cleavage of gasdermin E. Conversely, amplification of BCL-xL downregulated the expression of GSDME, therefore suppressing the process of pyroptosis. GSDME knockdown in KRAS-G12C mutant lung cancer cell lines showed defective HMGB1 release, reduced caspase-3 viability, and decreased granzyme B and IFN-γ secretion by CD8+ T cells in response to KRAS-G12Ci, and more frequent tumor cell growth. Conclusion: Our findings indicate that GSDME-dependent pyroptosis is a critical determinant of KRAS-G12C-targeted inhibitor AMG510, and through the modulation exerted by the Bcl-2 family protein BCL-xL upon GSDME. These findings suggest that activation of GSDME may provide an effective strategy to sensitize cancer cells to KRAS-G12Ci treatment. Citation Format: Linfeng Luo, Anlin Li, Wei Du, Maojian Chen, JIng Li, Yixin Zhou, Li Zhang, Shaodong Hong. AMG510-mediated inactivation of BCL-xL protein promotes Gasdermin-E-dependent pyroptosis in KRAS-G12C mutant non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB266.

Abstract LB165: GFH547: An orally bioavailable, cyclophilin A-hijacking panRAS(on) inhibitor with broad spectrum anti-tumor activities

Abstract KRAS has recently become druggable with the approval of two KRAS G12C inhibitors - sotorasib and adagrasib. However, current SIIP-based KRAS inhibitors preferentially target the inactive, GDP-bound KRAS and thus are susceptible to adaptive resistance due to MAPK pathway reactivation. Furthermore, RTK pathway alterations frequently emerge to cause acquired resistance. These drawbacks limit the rate and duration of anti-tumor response. Developing more robust small molecule RAS (on) inhibitors that target active, GTP-bound RAS proteins is a promising new approach to enhance clinical benefits. Here we report preclinical characterization of a novel RAS (on) inhibitor - GFH547, designed to treat cancers carrying multiple RAS mutations. In biochemical assays, GFH547 disrupted the binding of GMPPNP-bound G12V, G12D or wild type KRAS to RAF1 in a CypA-dependent manner, as the inhibitory effect was significantly abolished in the absence of CypA. GFH547 suppressed phosphor-ERK1/2 (p-ERK) levels in KRAS G12V- and G12D-mutated cells with IC50s &amp;lt; 1 nM, and demonstrated potent tumor-suppressive effect in 29 cancer cell lines carrying diverse KRAS (G12A, G12C, G12V, G12R, G12S or G13D), NRAS (G12D or Q61L) or HRAS (Q61H) mutations, with IC50s of 0.04-1.9 nM. In addition, GFH547 was also cytotoxic in 9 cancer cell lines carrying EGFR or FGFR2/3 amplification, mutation or fusion, with IC50s of 0.12-198 nM. Conversely, GFH547 was inactive against 4 BRAF V600E-mutated, RAS-independent cancer cell lines. In KRAS-mutated xenograft models, oral administration of GFH547 elicited rapid and potent inhibition of intratumoral p-ERK levels, and led to tumor regression at dose levels as low as 1 mg/kg QD. In contrast to sotorasib and adagrasib, GFH547 was not susceptible to RTK-reactivation following EGF stimulation, and remained effective against cell lines carrying KRAS mutations that confer acquired resistance to SIIP-based KRAS inhibitors. At 10 μM, GFH547 showed no off-target activity in 72-kinase selectivity and SafetyScreen44 panels. GFH547 has a bioavailability of 76% and was well tolerated in mice, with an estimated therapeutic index &amp;gt;20. Taken together, by hijacking CypA to block oncogenic RAS proteins broadly in their active forms, GFH547 represents a new generation of RAS inhibitors that are superior to the mainstream SIIP-based KRAS inhibitors in overcoming adaptive and acquired resistance. The compound demonstrated strong therapeutic activities in tumors with activating RAS and RTK mutations/alterations. Based on these promising results, we are advancing a CypA-binding panRAS(on) inhibitor into the clinic as targeted therapy for cancers addicted to RAS pathways. Citation Format: Feng Yan, Jichen Zhao, Tao Liang, Tao Jiang, Chonglan Lin, Ling Peng, Kai Ma, Huabin Yang, Wan He, Zhen Li, Leitao Zhang, Xiaoming Xu, Yanhui Zhao, Xiaohui Zhang, Yonghong Ni, Qiang Liu, Jinting Gao, Fubo Xie, Xueyan Gao, Xiaoling Lan, Li Wang, Jingyang Zhang, Hongcan Ren, Dong Liu, Siyuan Le, Fusheng Zhou, Jiong Lan, Qiang Lu. GFH547: An orally bioavailable, cyclophilin A-hijacking panRAS(on) inhibitor with broad spectrum anti-tumor activities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB165.