Abstract 4836: Re-expression of MHCII in colorectal organoids: Investigating the novel role of EHMT1 in regulating MHCII expression

Background: Major histocompatibility complex class II is expressed by mature professional antigen-presenting cells, forming a critical part of the innate immune response. However, other cell types, including tumour (tsMHCII), can be induced to express MHCII in response to inflammatory signalling by IFN-g, leading to increased tumour killing. TsMHCII-II expression has been associated with a higher number of tumour-infiltrating CD4 and CD8, with improved progression-free survival (PFS) and overall survival (OS). These observations suggested that increased tsMHCII expression is associated with increased tumour recognition by T cells and enhanced antitumor immunity. However, treatment with IFN-g is not a practical solution due to patient side effects and the finding that a high percentage of tumours have no upregulation of MHCII in response to IFNg stimulation. We have recently shown that EHMT1 is a potential target to upregulate MHCII in a genome wide CRISPR/Cas9 screen. In this study, we aim to 1) investigate a potential non-canonical relationship between EHMT1 and MHC-II expression in a microsatellite stable (MSS) colorectal organoid model 2) Evaluate the ability of organoids that constitutively express MHC-II to prime naïve T cells; and 3) explore pharmacological EHMT1 inhibitors as treatments to enhance MHC-II expression in tumour cells Methods: CRISPR/Cas9 was used to knock down the EHMT1 gene in colorectal organoids, confirmed with Sanger sequencing and Western blot analysis. To investigate the mechanistic differences between these clones and the WT, we carried out a differential gene expression analysis using RNAseq and ChIP-sequencing, focusing on the impact of EHMT1 knockout on whole genomic methylation signatures. Furthermore, we investigated the efficacy of targeting EHMT1 pharmacologically, utilising a selection of EHMT1 inhibitors. Finally, we co-cultured EHMT1-/- clones with allogenic T cells in a mixed lymphocyte reaction (MLR) assay. T cell stimulation was evaluated by assessing CD25, CD69, CD107a, and CD137 Results: In contrast to the wild-type (WT) organoid that showed no response to IFNg stimulation, four EHMT1-/- knockout clones demonstrated upregulated levels of MHCII expression, independent of IFNg. The level of MHCII expression by the clones was correlated with the level of methylated H3K9. Additionally, EHMT inhibitors showed variable efficacy in enhancing MHCII expression in wild-type organoids. Analysis is ongoing to investigate the capability of these clones with constitutively expressed MHCII to process and present tumor antigens to stimulate naïve CD4+ T cells Conclusion: This study reveals a potentially novel role for EHMT1 in modulating the tumour immune response by controlling the expression of MHC-II in colorectal organoids. Future work will focus on assessing the efficacy of combining immune checkpoint inhibitors with EHMT1 inhibitors Citation Format: Nahla A. Elzefzafy, Louise Tee, Maria Pinna, Neeraj Lal, Gary M. M Middleton, Andrew Beggs. Re-expression of MHCII in colorectal organoids: Investigating the novel role of EHMT1 in regulating MHCII expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular s); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1): nr 4836.