Cancer cell proliferation and survival are dependent upon on a sufficient supply of deoxyribonucleotide triphosphates (dNTPs), the substrates for DNA replication. dNTPs are produced by convergent intracellular metabolic pathways, either by the de novo pathway which utilizes glucose and amino acid precursors or by the salvage pathway which allows for the scavenging of preformed nucleosides from the environment. Despite its fundamental nature, the regulation of nucleotide biosynthesis by intracellular signaling networks in cancer cells is poorly defined and the implications of this control are unclear. Here we systematically evaluate the regulation of cancer cell nucleotide metabolism by two signaling networks, the replication stress response pathway and signaling elicited by the cytokine interferon. Armed with new insights into the regulation of nucleotide metabolism in cancer we develop new, rationally designed therapeutic strategies to be translated for the treatment of precisely defined patient populations.
Chapter 1 provides an introduction to nucleotide metabolism function and regulation in cancer.
Chapter 2 concerns the investigation of nucleotide metabolism regulation by the replication stress response signaling pathway. Here we define a link between the proximal replication stress response kinase ataxia telangiectasia and Rad3-related protein (ATR) and rate-limiting enzymes in de novo and salvage nucleotide biosynthetic pathways: ribonucleotide reductase (RNR) and deoxycytidine kinase (dCK). In cancer cells, ATR inhibition produces a collateral dependency on RNR and dCK activity. We demonstrate that simultaneously targeting ATR, RNR and dCK using small molecule inhibitors effectively eradicates acute lymphoblastic leukemia cells in vitro and in vivo.
In Chapter 3 we report an investigation of nucleotide metabolism regulation by the pleiotropic cytokine type I interferon. We determine that interferon signaling is constitutive in pancreatic ductal adenocarcinoma (PDAC) patient tumors and xenograft models and is driven by the cGAS/STING pathway. In PDAC cells, interferon signaling triggers a decrease in dNTP pool abundance and up-regulates dNTP phosphohydrolysis mediated by sterile alpha-motif and histidine-aspartate domain-containing protein 1 (SAMHD1). Activation of ATR is a compensatory response to this stress and we find that interferon signaling triggers a dependency on ATR activity in a subset of PDAC models.
Chapter 4 concerns the identification of a cancer cell metabolic vulnerability that results from impaired dNTP catabolism. Here we show that inhibition of purine nucleoside phosphorylase (PNP) selectively induces dNTP pool imbalance and cell death in cancer cells lacking the phosphohydrolase SAMHD1. In this chapter we define a new synthetic lethal interaction in cancer and demonstrate that SAMHD1 deficiency is a requisite biomarker for PNP inhibitor activity.
In Chapter 5 we present the development and application of a cell-based metabolic modifier screening platform that leverages the redundancy in pyrimidine metabolism for the discovery of specific modulators of convergent nucleotide biosynthetic pathways. Here we report the identification and characterization of selective small molecule inhibitors of the pyrimidine de novo pathway enzyme dihydroorotate dehydrogenase and of nucleoside transporters which are essential for nucleoside salvage pathway activity.