Abstract 1596: Tumor-derived RAC1A159V gain-of-function mutation promotes an immunosuppressive tumor microenvironment and represses response to immune checkpoint inhibitors

Immune checkpoint inhibitors (ICIs) specifically target immune checkpoint molecules and activate cytotoxic T lymphocytes (CTLs) to kill cancer cells and have seen great success in various solid tumors. However, the overall response rate of ICI monotherapy in patients is only ∼15% due to an immunosuppressive microenvironment of many tumors, and understanding the mechanism of ICI therapy resistance is urgently needed to improve the outcomes. RAC1, a member of Rho GTPase family, regulates multiple cellular processes in cancer, including vesicle trafficking, glycolysis, gene transcription, and exocytosis/endocytosis, all of which are tightly associated with the creation of a tumor immune microenvironment (TIME). RAC1A159V is a gain-of-function mutation found in several cancer types including colon cancer, head and neck cancer, and lung cancer, and is related to poor prognosis. Mammalian target of rapamycin (mTOR) signaling, one of the downstream effectors of RAC1, promotes tumor progression via diverse mechanisms including glycolytic metabolism and tumor cell proliferation. To define the role of RAC1A159V in cancer immune therapy, we have generated RAC1A159V knock-in MC38 colon cancer cells by CRISPR/Cas9 gene editing. PAK-PBD pull-down assay and western-blotting confirmed that RAC1A159V mutation resulted in upregulated RAC1 activity and mTOR signaling in vitro. In subcutaneous MC38 tumor xenograft models we found that while MC38 RAC1A159V tumor grew similarly as RAC1WT tumor cells in immunodeficient mouse recipients, it grew faster than RAC1WT tumors and are highly resistant to anti-PD1 therapy in immune competent mice. Flow cytometry analysis revealed that the RAC1A159V tumors have an immunosuppressive TIME with a significantly higher Treg/CTL ratio than RAC1WT tumors. ScRNA-seq of the whole tumors showed that RAC1A159V tumor is a “cold” tumor with suppressed tumor-immune cell interactions. Deep RNA-seq analysis of the tumor cells further indicated that RAC1A159V activates mTORC1 signaling in tumor cells, which in turn increases tumor cell glycolysis and inhibits chemokine production. Rapamycin, a mTORC1 inhibitor, could sensitize RAC1A159V tumors to anti-PD1 treatment by inhibiting tumor glycolysis and promoting chemokine production in tumor cells. RT-qPCR studies further indicated that inhibiting RAC1A159V tumor cell glycolysis by 2-DG also enhanced chemokine production in vitro, similar to the effect observed with rapamycin. Overall, our studies demonstrate that tumor-derived RAC1A159V mutation turns the TIME to “cold” and renders the tumor resistant to ICIs via mTOR mediated changes in glycolysis and chemotaxis. The results provide mechanistic insights of RAC1 mutation-mediated TIME and suggest a combination therapeutic approach for “cold” tumors by combining RAC1 signaling inhibition with ICIs. Citation Format: Mingjun Cai, Mike Adam, Fukun Guo, Yi Zheng. Tumor-derived RAC1A159V gain-of-function mutation promotes an immunosuppressive tumor microenvironment and represses response to immune checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1596.