Biological and therapeutic consequences of RNA dysregulation in cancer immune evasion

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PROJECT SUMMARY Immune checkpoint blockade therapy induces durable anti-tumor immune responses and has transformed care for advanced cancer, including metastatic disease. However, most patients derive little or no clinical benefit from therapy. An important contributor to poor clinical outcomes is an immune-evasive tumor phenotype; in this state, cancer cells remain resistant to clearance even in the presence of anti-tumor immune cells. There is a critical need to (1) identify mechanisms driving an immune-evasive tumor phenotype and (2) therapeutically target these in combination with immune checkpoint inhibitors to stimulate anti-tumor immunity. This proposal is motivated by two unexpected discoveries: First, Muscleblind-like proteins (MBNLs) — a family of RNA-binding proteins — are required for T cell-mediated killing of cancer cells. While MBNL proteins have been exhaustively studied in the context of neuromuscular disease, their contributions to tumor- immune interactions have not been explored. This proposal takes advantage of the wealth of knowledge regarding MBNL-regulated RNA processing to determine novel roles for RNA metabolism in tumor immune evasion. Second, MBNL transcripts are subject to recurrent, pan-cancer mis-splicing that is strongly associated with an immune-evasive phenotype. This splicing-mediated disease mechanism presents a unique opportunity to use splice-switching oligonucleotide therapies, in combination with immune checkpoint inhibitors, to stimulate anti-tumor immunity. Aim 1 uses cellular assays and genomics to determine the mechanistic basis of MBNL’s involvement in interferon signaling, antigen presentation, and gene expression in immune-evasive cancers. Aim 2 uses syngeneic tumor models, high-dimensional flow cytometry, and isoform separation-of- function studies to determine how MBNL splice isoforms regulate RNA metabolism to promote tumor growth and shape the tumor-immune microenvironment. Aim 3 uses preclinical tumor models to test the hypothesis that oligonucleotide-based correction of cancer-associated MBNL mis-splicing will improve tumor response to immune checkpoint inhibitors. While MBNLs and their individual splice isoforms are used as model systems throughout this proposal, the applicant’s long-term goal is to establish his independent research program aimed at defining more general principles of RNA-mediated immune evasion and immunotherapy resistance. To achieve these goals, this proposal outlines a career development plan to augment the applicant’s training in RNA-mediated disease and functional genomics with training in tumor immunology and preclinical therapeutic models. This training will be facilitated by the complementary expertise of the applicant’s mentor (RNA splicing in cancer), co-mentor (tumor immunology), collaborators (preclinical therapeutic studies, MBNL-regulated RNA metabolism), and scientific advisory team (immunology, interferon-signaling).