Immune determinants modulating cancer development in Lynch Syndrome

Lynch Syndrome (LS) represents a hereditary predisposition syndrome associated with DNA mismatch repair (MMR) pathway impairment. LS-associated tumors demonstrate a high level of microsatellite instability (MSI-H), and consequently a high mutational and neoantigen burden, which results in improved response to treatment with immune checkpoint inhibitors (CPI). We previously showed that frameshift (fs) mutations encoding neoantigens in MSI-H tumors may control CPI efficacy and have quantifiably distinct characteristics from point mutations such as (i) major divergence from self, leading to differential immunogenicity; (ii) shared expression in MSI-H CRC tumors across patients; and (iii) expression of highly immunogenic epitopes that can elicit neoantigen-reactive CD8+ T cells detectable in blood, a proxy for intratumoral activity. Pre-malignant LS lesions are infiltrated with T cells expressing proinflammatory cytokines (TNF, IL-12), and CTLA-4, LAG3 and PD-L1 checkpoints suggesting early immune activation and recognition of tumor antigens. LS is therefore an ideal setting for design of cancer prevention strategies, including vaccination, and identification of antigen-specific T cell responses involved in immune surveillance and escape. We hypothesize that high quality shared neoantigen expression and a functional T cell repertoire capable of trafficking to and clearing MMRd lesions, deters progression of premalignant polyps to overt cancer in Lynch Syndrome. The goal of this application is to dissect the landscape, quality, and evolution of neoantigens expressed within pre-malignant colorectal polyps of LS patients, to track and identify immunogenic antigens that can be targets for prevention of tumor development. The UG3 aims are: 1: Identification of neoantigens expressed in premalignant colon polyps of Lynch Syndrome patients. We will map the spectrum of novel and shared neoantigen-expressing frameshift mutations in pre-cancerous colorectal polyps. 2: Identification of fs-specific TCRs capable of recognizing quality fs-neoantigens. We will confirm the immunogenicity of shared neoantigens expressed in precancerous polyps and identify fs-specific T cell receptors (TCRs), including shared TCRs, by barcoded peptide-MHC tetramers and single cell (sc)RNA/TCR sequencing from LS patient peripheral blood. The UH3 aims are to 1: Validate fs-neoantigen expression and tissue trafficking of fs-specific TCRs in a separate validation cohort. 2: Assess the influence of prior MSI-H cancer on shared fs-neoantigen repertoire and T cell recognition in subsequent colon lesions. We will determine whether prior exposure to shared MSI-H fsneoantigens through their expression in non-colonic sites shapes new colonic lesions and T cell surveillance, as a model of “pre-vaccination”. 3: Assess fs-specific T cell exhaustion and co-localization with immunosuppressive hubs in advanced precancerous lesions. Using bulk/scRNAseq, spatial transcriptomics, and multiplexed immunohistochemistry we will determine mechanisms of immune escape in the PME to identify checkpoints for future immunoprevention strategies.