Functional Relevance of Pyrimidine Sensing and Response Pathways in Tumor Metabolism and Cancer Therapy

Nucleotide metabolism is effectively targeted by anti-cancer agents, but their use is limited by toxicity to non- transformed cells, especially anti-cancer immune cells. Our overarching hypothesis is that a more complete understanding of nucleotide metabolism—from sensing mechanisms to the response to nucleotide depletion—

will enable sophisticated anti-cancer strategies that target cancer cells while sparing normal and immune cells. In data that forms the premise of our application: (1) We identify a molecular mechanism by which the rate limiting step of pyrimidine synthesis, catalyzed by the enzyme CAD, is allosterically regulated by UTP. We

engineer constitutively active CAD mutants to assess the importance of this regulatory activity. (2) We determined that a p53-dependent ribosome biogenesis checkpoint (RBC) is triggered by pyrimidine ribonucleotide insufficiency, a feature that we propose underlies a key metabolic difference between cancer and

normal cells. (3) We discovered two methods for perturbing pyrimidine metabolism that induce a 10-30 fold increase in sensitivity to clinically relevant cytosine analogs. Our objective is to understand how perturbations to pyrimidine sensing and the response to pyrimidine depletion impact cancer metabolism, anti-tumor immune

cell function, and the response to therapy. Our rationale is that disrupting CAD pyrimidine sensing will result in pyrimidine overflow, and perturbation of the RBC will cause reversible cell cycle arrest, each of which will differentially affect cancer and tumor immune cell metabolism and function. Our specific aims test the following

hypotheses: (1) Pyrimidine overflow will limit cancer cell function while promoting anti-cancer T cell activation, (2) Uridine analogs modulate pyrimidine salvage pathway activity, promoting sensitivity to pyrimidine-based chemotherapeutics, (3) Chronic pyrimidine synthesis inhibition induces a GEM-sensitive cancer cell state, (4)

The RBC restrains tumor immune cell function and limits p53-WT cancer cell toxicity, (5) Bypass of the RBC potentiates the effects of immunotherapy and chemotherapeutics targeting pyrimidine biosynthesis. This contribution is significant because pyrimidine metabolism is a major cancer target that impacts both cancer and

anti-tumor immune cell function, but whose efficient targeting needs improvement. This research is innovative because (1) we utilize novel CAD hyperactive mutants based on our work identifying CAD allosteric regulation mechanisms, (2) we use separation of function MDM2 mutants to interrogate the impact of the RBC on cancer

and immune cell function in culture and tumors, (3) we leverage our extensive experience in cellular metabolism to understand the metabolic impact of the perturbed pyrimidine sensing and response pathways and pyrimidine- based chemotherapeutics. Completion of these aims will fundamentally advance our understanding of pyrimidine

synthesis regulation and cell cycle control mechanisms that respond to nucleotide limitation. The information gained will elucidate strategies to target cancer cells while sparing normal immune cell function, informing future approaches to inhibit cancer intrinsic targets and promote anti-tumor immunity.