Date of Award

Spring 4-17-2023

Embargo Period

4-24-2023

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular and Cellular Biology and Pathobiology

Additional Department

Microbiology and Immunology

College

College of Graduate Studies

First Advisor

Eric Bartee

Second Advisor

Patrick Woster

Third Advisor

Jessica Thaxton

Fourth Advisor

Chrystal Paulos

Fifth Advisor

Anand Mehta

Abstract

Oncolytic virotherapy (OV) is a class of immunotherapy for treatment of malignancy. Using viruses that exhibit natural coincidental tropisms for cancer, or others that have been engineered to the same effect, intentional infection of lesions leads to two therapeutically beneficial effects: (1) direct destruction of the infected tumor through virally-mediated cell lysis, and (2) recruitment of an otherwise blunted or absent anti-cancer immune response to affect both local and disseminated disease. A surfeit of cancer-specific changes are accumulated during progression from first genetic insult to clinical detection, presenting a dramatically altered underlying biology of cell and tissue. The viruses employed within OV have been characterized over decades, however, all largely within the context of normal and otherwise-healthy host cells bearing infections. As such, these disparities between cancerous tissues and their normal counterparts may pose barriers to viral infection not encountered or compensated for.

Dysregulations within cellular metabolism are a hallmark of cancer, and the replication of all viruses – oncolytic or not – is contingent on access to host metabolites. Despite this, no research has been conducted evaluating how metabolic changes within tumors may lead to resistance to OV infection. One such dysregulated metabolic pathway is synthesis and consumption of L-arginine (Arg), a semi-essential amino acid whose bioavailability is required for the in vitro replication of several oncolytic viruses. Cancer types such as hepatocellular carcinoma, sarcoma, and melanoma often clinically present as functionally auxotrophic for this amino acid due to epigenetic silencing of argininosuccinate synthetase 1 (ASS1), an enzyme responsible for the conversion of citrulline and aspartate into the Arg precursor argininosuccinate (AS). Additionally, the recruitment of Arginase-1 (ARG1) positive myeloid derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) further insult Arg availability within the TME.

Here, we show that the in vitro replication of oncolytic myxoma virus (MYXV) is dependent on the presence of bioavailable Arg, with insight towards several stages within the viral life cycle. We demonstrate that the presence of MDSCs negatively reduces viral burden within infected tumors in a B16F10 model of murine melanoma, with their depletion capable of reducing initial required MYXV dose to elicit a therapeutic response, and these effects possibly attributable to an expression of ARG1 as evidenced within cocultures ex vivo. Secondly, we determine the role of ASS1 in mediating tumoral capacity for viral replication in vitro and in vivo using CRISPR/Cas9 generated ASS1KO cell lines. Here, we find tumors formed from functionally ASS1KO B16F10 melanoma cells display multi-log reductions in MYXV replication during oncolytic virotherapy (OV) as well as significantly poorer therapeutic responses. Lastly, we demonstrate that reconstitution of Arg biosynthesis through ASS1-armed MYXV constructs at least partially rescues these effects. Collectively, these studies demonstrate an Arg-dependent replication of MYXV that may be affected by cancer-specific changes within Arg metabolism and consumption. This work is the first to characterize a metabolic barrier to achieving optimal viral replication within tumors, and indicates that consideration towards tumoral metabolism may improve replication and therapeutic efficacy of OV agents.

Rights

Copyright is held by the author. All rights reserved.

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