Characterization of shared immunogenic poly-epitope frameshift mutations in microsatellite-instability-high (MSI-H) colorectal cancer

Lynch Syndrome (LS) is a hereditary condition associated with a predisposition to microsatellite-Instability-High (MSI-H) colorectal cancer. LS exhibits a distinctive genomic profile marked by the accumulation of frameshift mutations. The syndrome is caused by inherited mutations in the DNA Mismatch Repair (MMR) machinery, resulting in the accumulation of mutations in microsatellite regions of the genome. Germline mutations in the MMR complex genes, including MLH1, MSH2/6, and PMS2, define Lynch Syndrome, with the most common mutations observed in MLH1 and MSH2 (70%- 90%) and MSH6 and PMS2 (10%-30). These mutations contribute to the accumulation of shared frameshift mutations, emphasizing the need to investigate the specific immunogenic properties of such mutations and their potential implications for immune-surveillance. To begin to address this, our group aims to comprehensively characterize shared immunogenic poly-epitope frameshift mutations within MSI-H colorectal cancer, with a particular focus on their relevance to Lynch Syndrome. By leveraging genomic sequencing and bioinformatic analyses we identified Lynch Syndrome specific frameshift mutations and evaluated their potential immunogenicity using in silico prediction and in vitro antigen priming immunological assays. Using an APC/T-cell assay we characterized LS frameshift mutation (Fsmut) immunoreactivity across 12 donors by testing peptide pools specific to each individual frameshift mutation. The screening involved a comprehensive analysis of 47Fsmut peptide pools. Additionally, peptides specific to 14 single nucleotide variants (SNVs) that are highly shared amongst MSI-H CRC patients were also evaluated. Ultimately these data can be leveraged to support generation of targeted immunotherapeutic strategies for the development "off the shelf" vaccines without the need for patient selection or de novo vaccine manufacturing.. The data reveal immunoreactivity across multiple frameshift mutations and human HLAs, emphasizing the potential for enhanced T cell responses against tumor neoantigens. The study not only identifies immunogenic frameshift neoantigens but also explores strategies to optimize T cell responses, considering factors like specificity, immunodominance, and overall immune system efficacy. In addition, CRISPR knockout (KO) experiments targeting MLH1 and MSH6 genes in SW620 and HT55 CRC cell lines were undertaken to investigate the specific roles of these genes in DNA repair mechanisms and genomic stability, particularly in the context of mismatch repair (MMR) deficiency and its implications for frameshift development. Successful disruption of MLH1 and MSH6 at mRNA and protein levels was confirmed using Sanger sequencing, Western blotting, and qPCR analyses. Whole exome sequencing (WES) revealed an increased number of frameshift mutations (FSmut) following MLH1 and MSH6 knockout, highlighting the critical role of MMR genes in maintaining mismatch repair (MMR) activity and genomic integrity. This research contributes valuable insights for developing targeted approaches to harness the immune response against Lynch Syndrome-associated frameshift mutations.