Abstract 5305: Tumor cell lineages and immune remodeling of <i>RHOAY42C</i>-driven diffuse gastric cancer

Abstract Diffuse gastric adenocarcinoma (DGAC) is a highly aggressive cancer characterized by poor prognosis and distinct molecular subtypes. Among these, CDH1 inactivation and RHOA mutations, particularly RHOAY42C, define separate oncogenic pathways with distinct biological and clinical implications. To elucidate the functional consequences of RHOAY42C mutation, we established genetically engineered murine gastric organoid models carrying genetic mutations frequently observed in human DGAC. Organoids harboring RhoAY42C in combination with KrasG12D and Trp53 loss (RKP: RHOAY42C, KrasG12D, Trp53-/-) and Cdh1 loss (EKP: Cdh1-/-, KrasG12D, Trp53-/-) exhibited hyperproliferative and multilayered epithelial architectures, indicative of neoplastic cell growth. Transplantation of RKP organoids into immunocompetent mice displayed features of mucinous DGAC, marked by abundant mucin production and signet-ring cell morphology. By contrast, EKP resulted in tumor architectures with invasive features consistent with non-mucinous DGAC.O ur multiomic approaches combining single-cell RNA-sequencing and spatial transcriptomics (Xenium In Situ) revealed divergent tumor cell lineages and transcriptional programs between EKP and RKP models. EKP tumors displayed enrichment of Runx1-dependent epithelial lineages, whereas RKP tumors exhibited Aqp5-driven lineages with high transcriptional plasticity and robust activation of the Hippo signaling pathway. RKP tumors featured prominent MUC1/4+ mucinous cell populations, whose differentiation was mediated by YAP1, the Hippo pathway effector. Pharmacologic inhibition of Hippo signaling effectively abrogated these mucinous cell lineages, highlighting potential therapeutic vulnerabilities in RhoaY42C-driven tumors. Additionally, immune profiling revealed significant differences in the tumor microenvironments (TMEs) of EKP and RKP tumors. RKP tumors exhibited an immune-suppressive microenvironment characterized by peripheral enrichment of exhausted T cells expressing markers such as Pdcd1 and Ctla4. Consistently, cell-to-cell interaction analyses revealed enhanced interactions mediated by PDCD1 and CTLA4. Further analyses revealed that differentially secreted cytokines in EKP and RKP induced immune-suppressive genes expression in T cells of RKP tumor, suggesting that RhoaY42C mutation not only drives tumor-intrinsic changes but also remodels the immune landscape to favor immune evasion. By contrast, the TME of EKP tumors exhibited lower immune suppression, with a reduced presence of T cell exhaustion markers. Together, these findings identified the genotype-specific nature of tumor cell differentiation programs, transcriptional plasticity, and immune microenvironmental remodeling in DGAC. This study highlights Hippo signaling and immune checkpoint pathways as promising therapeutic targets in RHOAY42C-driven mucinous DGAC. Citation Format: Kyung Pil Ko, Jinho Jang, Gengyi Zou, Yuanjian Huang, Jie Zhang, Jae-Il Park. Tumor cell lineages and immune remodeling of RHOA-driven diffuse gastric cancer [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 5305.