Colorectal cancer displays three patterns of tumorspecific MHC-II inducibility and dynamics, with implications for combining epigenetic therapy with immunotherapy

Abstract Introduction: Tumor specific MHC class II (tsMHC-II) expression impacts tumor microenvironmental immunity. tsMHC-II positive cancer cells act as surrogate antigen presenting cells and targets for CD4+ T cell-mediated lysis. In colorectal cancer (CRC), tsMHC-II negativity is common and in cell lines is attributable to CIITA promoter methylation. To clarify mechanisms of tsMHC-II repression in CRC we analyzed CRC organoids which are epigenetically faithful to tissue of origin. Methods: 15 primary CRC organoids were treated with IFNγ +/- epigenetic modifiers. Flow cytometry was used to assess for tsMHC-II inducibility. RT-qPCR, total RNAseq, nanopore sequencing, bisulfite conversion/pyrosequencing and western blotting were used to quantitate CIITA, STAT1, IRF1 and JAK1 expression, gene structure and promoter methylation and ChIP-PCR to quantitate H3K9ac, H3K9Me2 and EZH2 occupancy at CIITA. Results: We define three types of tsMHC-II response to IFNγ in CRC: strong- (≥50%), delayed/weak- (11-49%) and non-inducibility (≤10%). 8/15 demonstrated strong expression, 4/15 delayed/weak: although it was possible to overcome this weak expression with prolonged IFNγ exposure in 3/15 without additional pharmacological treatment. In 1/15 organoid, expression was restricted even with prolonged exposure due to IFNγ-mediated EZH2 occupancy at CIITA: tsMHC-II expression was enhanced by EZH2 and HDAC inhibition. Non-inducibility is seen in 3 CMS1 organoids due to JAK1 mutation. These demonstrated low level (11%) JAK1 promoter methylation, but tsMHC-II inducibility was not rescued with DNMT inhibition. No organoid demonstrated CIITA promoter methylation, despite confirming the presence in previously described 2D cell lines, suggesting this is acquired through culture methods and not reflective of disease biology. Conclusion: We demonstrate using epigenetically appropriate models the individual variation in pharmacologic tractability of epigenetic modification in CRC. Providing IFNγ signaling is intact, most CRC organoids are class II inducible. Up-regulation of restricted tsMHC-II through targeted epigenetic therapy is seen in 1/15 organoid. These data may explain some of the disappointing results of immuno-epigenetic approaches in the clinic. Citation Format: Oliver J. Pickles, Kasun Wanigasooriya, Anetta Ptasinska, Akshay J. Patel, Helen L. Robbins, Claire Bryer, Celina M. Whalley, Louise Tee, Neeraj Lal, Maria Pinna, Nahla Elzefzafy, Phillipe Taniere, Andrew D. Beggs, Gary W. Middleton. Colorectal cancer displays three patterns of tumor-specific MHC-II inducibility and dynamics, with implications for combining epigenetic therapy with immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2989.