Tumor-targeted disruption of mismatch repair in microsatellite stable colorectal cancer

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Colorectal cancer (CRC) is the third most diagnosed cancer in the US with the second highest mortality rate. Locally advanced and metastatic disease have a poor prognosis. A small subset (5-15%) of metastatic CRC patients, who bear tumors deficient in DNA mismatch repair (MMR) that have high microsatellite instability (MSI- high), can respond to immunotherapy with checkpoint blockade (CPI), but most CRC do not. We hypothesize that to induce immune responses in microsatellite stable (MSS) CRC, new strategies that go beyond CPI are needed. In MSI-high CRC tumors, tumor-infiltrating myeloid cells, fibroblasts and the tumors themselves all have inflammatory gene expression signatures and are colocalized into inflammatory hubs with adjacent tumor- reactive CD8 T cells, suggesting that MSI-high CRCs selectively provoke a robust inflammatory multicellular network within the tumor that recruits anti-tumor killer cells. Disruption of MMR in CRC mouse tumor grafts enhances tumor neoantigen expression and recruitment of tumor-specific T cells to trigger immune surveillance. We hypothesize that interventions that convert MSS CRC to an MMR-deficient MSI phenotype could enhance antitumor immunity and make resistant MSS CRC sensitive to CPI. This proposal will investigate whether tumor-targeted gene knockdown can convert MSS CRC to immune responsive MSI-high tumors. There are no known small molecule inhibitors of MMR. We will exploit a method of epithelial cancer-targeted gene knockdown that uses subcutaneous injection of aptamer-small interfering RNAs (AsiCs), which link a high affinity EpCAM aptamer for epithelial tumor targeting to small interfering RNAs, for tumor-selective gene knockdown. EpCAM, the first described tumor antigen, is ~100-1000-fold more highly expressed in epithelial cancers (including virtually all CRC) than normal epithelia, making it attractive for selective CRC tumor targeting. We previously showed that EpCAM-AsiCs that knockdown tumor-dependency genes or genes whose knockdown promotes immune recognition can strongly suppress aggressive breast cancer in orthotopic, metastatic and genetically engineered mouse tumor models and induce an effective immune response in immunologically cold tumors. In this proposal we will evaluate EpCAM-AsiCs to knockdown Mlh1, encoding a key MMR enzyme, in MSS CRC subcutaneous and caecal tumor implants of 4 mouse CRC cell lines and 2 MMR-proficient, MSS organoid lines derived from an aggressive genetically engineered mouse model, which conditionally express mutations in intestinal stem cells of four key genes that are frequently mutated in human CRC – Apc, Kras, Tgfbr2 and Trp53. We will evaluate the effect of MMR disruption on tumor growth in immunodeficient and immunocompetent mice and dissect in detail how microsatellite stability, tumor mutational burden and gene expression in the tumor and the immune response to the tumor are altered. We will also investigate whether CPI enhances the antitumor effect of tumor-targeted Mlh1 knockdown.