Abstract 4893: Role of MGMT expression in the genomic landscape and immunogenicity in human colorectal carcinomas

Abstract Introduction: Colorectal cancer (CRC) is second most common cause of cancer-related mortality in the USA and most patients with advanced tumors lack effective therapeutic options. Epigenetic silencing resulting in reduced expression of O6-methylguanine-DNA methyltransferase (MGMT) has been described in diverse tumor types including CRC and it is linked with reduced DNA repair capability and increased sensitivity to alkylating agents such as temozolomide. However, the potential role and clinical significance of MGMT overexpression in cancer cells has not been studied. Methods: We used spatially resolved multiplex quantitative immunofluorescence (mQIF) analysis to concurrently measure the levels of MGMT protein, γH2AX and CD8+ tumor infiltrating lymphocytes (TILs) in a cohort of 111 CRCs and paired non-tumor colorectal mucosa from the same patient represented in tissue microarray format. To assess the genomic and immunologic context of MGMT expression, we studied the methylome, mutation profiling and mRNA expression of 526 CRCs from TCGA. The immunologic landscape of tumors was studied using GSEA and multiple transcriptomic-based tumor immune infiltration scores such as TIMER, CIBERSORT, etc. Finally, the genomic alterations and immunogenicity of SW620 human CRC cells (with baseline MGMT hypermethylation) with or without MGMT transfection were studied using in vitro co-culture preparations coupled to ultra-deep DNAseq and flow cytometry analysis. Results: On mQIF analysis, cancer-cell MGMT protein upregulation was identified in 73.9% of CRCs and was associated with reduced levels of γH2Ax in malignant cells and lower CD8+ TILs. 26.1% of cases showed MGMT protein downregulation. The genomic analysis of the TCGA cohort revealed that the mean nonsynonymous mutational load was significantly lower in MGMT-high than low tumors with marked differences in the type of variants and frequency of oncogenic mutations. The transcriptomic analysis revealed that MGMT overexpressing tumors showed lower immune infiltration than the MGMT-low group and reduced representation of immune associated signatures including interferon gamma response and TNF-alpha signaling. In contrast, DNA repair, TP53 pathways, were enriched. The exogenous expression of MGMT in SW620 cells reduced the number of nonsynonymous mutations by 15.6% (141 vs 119 mean mutations) and was also associated with low T-cell activation (CD69+/CD8+ T-cells) and cancer-cell elimination on allogenic co-cultures with human PBMCs. Conclusion: MGMT protein upregulation is common in human CRCs and is associated with reduced DNA damage response, distinct genomic features, and adaptive immune evasion. To our knowledge, this study is the first to report the biological and clinical characteristics of CRCs with MGMT overexpression that could support diagnostic and therapeutic developments. Citation Format: Barani Kumar Rajendran, Janie Zhang, Shruti Desai, Joanna Gibson, Jassim DiPalermo, Patricia LoRusso, Yong Kong, Hongyu Zhao, Michael Cecchini, Kurt Alex Schalper. Role of MGMT expression in the genomic landscape and immunogenicity in human colorectal carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4893.