Title

Identification of Tumor Immune Regulatory Factors Using High Throughput CRISPR Screening

Date of Award

Spring 2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Genetics

First Advisor

Chen, Sidi

Abstract

Cancer is a broad umbrella term that covers hundreds of diseases, each with their own unique molecular pathologies and genetic legions. The field of cancer biology has strived to uncover the significance of this vast array of mutations, hoping to translate basic science into clinically meaningful biomarkers, useful for diagnosis, prognosis, and in recent years treatment. This process has only become more relevant with the adoption of targeted molecular medicine, whether it be tyrosine kinase inhibitors (TKIs) or immune therapies targeting checkpoint blockade molecules.Immune therapies in particular, have the potential to revolutionize cancer management. Therapies such as checkpoint blockade inhibitors (CPIs) have been nothing short of miraculous for a small subset of cancer patients. These therapies work by blocking inhibitory molecules on the surface of cancer cells, or lymphocytes, to restore tumor-immune reactivity. While immune therapies represent a powerful emergent tool in our arsenal to combat cancer, they are not without their drawbacks. Currently only a minority of tumors types respond to CPI therapy, and the majority of those who do respond are likely to develop resistance. Combination trials are underway in multiple cancer types where immune therapies are used. The goal is to identify novel combination partners that can overcome acquired or innate resistance to immune therapy. These combinations are often not rationally designed however, given the field’s poor understanding of the mechanisms that underlie resistance. Against this backdrop, understanding the genetic basis of tumor-intrinsic regulatory factors that mediate immune evasion has never been more important. To this end, we have employed the use of CRISPR Cas9 screening technology to efficiently screen for these factors in two distinct models of cancer. First, I present a genome wide screen to uncover tumor-intrinsic regulators that modulate the tumor-immune microenvironment. Our genome-scale in vivo CRISPR screen robustly identified multiple tumor intrinsic regulators that alter the ability of cells to grow across different levels of immunocompetence. Of these Prkar1a, a negative regulator of cyclic AMP signaling, was identified as a powerful suppressor of tumor growth. Prkar1a loss of function (LOF) mutations allowed non-tumorigenic hepatocytes to form tumors in fully immune competent mice. Moreover, Prkar1a deficient tumor cells lead to drastic alterations in the phosphoproteome and transctriptome, corresponding to pro-inflammatory changes in the tumor microenvironment and differential tumor lymphocyte infiltration. Second, I present a novel asymmetric double knockout CRISPR Cas9 screen to identify genetic interactions that mediate resistance (or sensitization of resistance) to cytotoxic CD8+ T-cell killing in an in vitro model of melanoma. Our screen confirmed the well reported ability of Jak1 LOF mutations to confer resistance to immune destruction. In this background of Jak1 mediated resistance we identified a genetic interaction partner, Kmt2d, that when co-mutated sensitized the cells to CD8+ T cell killing. This finding mirrors previous work in our lab that independently identified Kmt2d as a novel regulator of immune responses in the context of checkpoint blockade therapy. We expand on this finding, confirming Kmt2d LOF mutations’ ability to sensitize Jak1 pathway mutations, including Jak1-/- and IFNgR1-/- backgrounds. In these works, I identify genetic regulators of both the tumor-immune microenvironment, and resistance to CD8+ T-cell killing of cancer cells. These findings provide additional insight into the mechanisms cancer cells use to avoid immune destruction, and how to overcome them.

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