Date of Award

Fall 10-1-2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Genetics

First Advisor

Chen, Sidi

Abstract

Over the past decade, tremendous resources have been devoted to sequencing the genomes of patient cancers. But while the molecular portraits of cancer are now in higher resolution than ever before, it has remained challenging to derive clinically actionable insights from these data. A central issue is that tumor genome sequencing often can only tell us what mutations are present, not which ones are functionally important. Towards this end, I sought to build an improved toolkit for functional cancer genomics, with the overarching goal of creating experimental platforms that are practical, scalable, and flexible. In collaboration with several colleagues, I developed autochthonous AAV-mediated CRISPR/Cas9 screens to quantitatively interrogate the contributions of specific mutations towards tumorigenesis in the murine liver and brain. Importantly, this experimental system is well suited for studies in tumor immunology, as it preserves the native tissue microenvironment in the context of an immunocompetent host. I subsequently utilized the AAV-CRISPR tumor model to further investigate the genetic determinants of response to PD-1 checkpoint blockade immunotherapy. In addition to recovering known regulators of immunotherapy response, I found that deficiency of the histone modifier KMT2D sensitizes diverse tumor types to immune checkpoint blockade by promoting the generation of immunogenic neoantigens. I further sought to comprehensively dissect the genetic regulation of anti-tumor immunity responses from both sides of the aisle, interrogating the immune cells that react against tumors as well as the cancer cells themselves. As a foray into the roles of noncoding RNAs in anti-tumor immunity, I mapped the landscape of sdRNAs expressed in human cancers, finding numerous transcripts that are associated with signatures of anti-tumor immune infiltration and survival across multiple cancer types. My colleagues and I also performed genome-scale CRISPR screens in primary CD8+ T cells to identify genetic regulators of T cell degranulation and tumor infiltration. These studies identified that Dhx37 knockout enhances NF-kB signaling in CD8+ T cells, leading to enhanced anti-tumor function in vitro and in vivo. Using genome-scale CRISPR activation (CRISPRa) screens, I further sought to identify genes that could promote tumorigenesis in immunocompetent hosts when overexpressed. In pursuing this line of study, my colleagues and I unexpectedly found that CRISPRa could be repurposed as a new immunotherapy modality, an approach we termed MAEGI (multiplexed activation of endogenous genes as immunotherapy). By delivering CRISPRa systems through AAVs to forcibly overexpress mutated genes directly in tumors, MAEGI enhanced immune recognition of tumor neoantigens, thereby eliciting robust and long-lasting anti-tumor immunity. The CRISPR screens employed in the aforementioned studies have an important limitation, however. Cancers arise from the sequential acquisition of genetic or epigenetic alterations, and the unique combinations of these alterations can interact in complex ways. To more precisely study these genetic interactions, I established a strategy for in vivo combinatorial knockout screening using massively-parallel CRISPR-Cas12a array profiling. I applied this technique to pinpoint mutation combinations that synergistically promote lung metastasis, demonstrating the utility of this technology to dissect genetic interactions in cancer. Finally, I devised a method for programmable sequential mutagenesis by combining CRISPR-Cas12a arrays with Cre recombination cassettes. I employed this approach to model the stepwise acquisition of resistance mutations against immunotherapy, thus providing a controlled experimental system for exploring strategies to overcome immunotherapy resistance.

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