Abstract B024: RAS(ON) G12D- and Multi-selective inhibitor doublet drives complete responses in combination with anti-PD-1 in a preclinical model of MSS KRAS G12D mutant CRC

Abstract Colorectal cancer (CRC) is the third most commonly diagnosed cancer in the United States. About 40% of colorectal cancers have mutations in KRAS, with KRAS G12D representing the largest population. Targeted therapies, as well as immune checkpoint inhibitor therapies, have limited efficacy as monotherapies for patients with microsatellite stable (MSS) CRC. However, recent clinical evidence in BRAF mutant MSS CRC showed that BRAF inhibitors in combination with MEK or EGFR inhibitors and anti-PD-1 resulted in significantly longer overall survival and a higher response rate than targeted therapy. CRC is a heterogeneous disease, and the availability of preclinical models that recapitulate the complexity of human tumors is limited. Here we developed a syngeneic preclinical model representative of MSS RAS mutant CRC tumors to investigate if RAS pathway inhibition with direct RAS inhibitors can sensitize to immune checkpoint inhibition. CMT93 is a murine cell line derived from a polypoid carcinoma of the rectum that harbors a Ctnnb1 (gene that encodes B-catenin) gain of function mutation and a Tp53 loss of function mutation and doesn’t harbor any mutations in mismatch repair genes. We engineered the eCMT93 model using CRISPR-Cas9 editing to express homozygous Kras G12D and confirmed that the proliferative capacity was completely dependent of Kras G12D based on the sensitivity to a RAS(ON) G12D-selective inhibitor in vitro. Following in vivo implantation subcutaneous, into the colon or into the liver, eCMT93 tumors showed glandular morphology and budding typical of well differentiated invasive CRC; in addition, they retained their potential to metastasize and expressed CRC histology markers, like CK7, CD19 and CK20. The RAS(ON) G12D-selective inhibitor RMC-9805 and the RAS(ON) multi-selective inhibitor RMC-6236 induced transient tumor regressions in the eCMT93 subcutaneous model. The combination of RMC-6236 with RMC-9805 substantially improved the depth and durability of response, achieving 60% complete regressions (CRs) on treatment. Transcriptomic and histological analyses showed that only the RAS(ON) doublet increased T cell infiltration significantly, potentiated T cell function by enhancing cytokine signaling, and upregulated the antigen presentation machinery. While anti-PD-1 alone achieved 20% CRs, the combination of anti-PD-1 with the RAS(ON) doublet, RMC-9805 and RMC-6236, achieved 100% CRs. In summary, in this syngeneic model of MSS RAS mutant CRC, the RAS(ON) doublet of RMC-6236 and RMC-9805 favorably modulated the tumor environment by increasing T cell infiltration and functionality and showed combinatorial activity. These preclinical studies support clinical evaluation to determine whether effective targeting of oncogenic RAS, with for example a RAS(ON) doublet, may enhance responses to immunologic therapies in RAS mutant MSS CRC. Citation Format: Avery J Salmon, Cristina Blaj, Linh Tran, Enrico Payson, Philip Wig, Rich Liang, Paola Soto-Perez, Vivian Morton, Miguel Sandoval, Jacqueline A.M. Smith, Elsa Quintana. RAS(ON) G12D- and Multi-selective inhibitor doublet drives complete responses in combination with anti-PD-1 in a preclinical model of MSS KRAS G12D mutant CRC [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr B024.