Decoding the metabolic routes of purine nucleotides in cancer - Resubmission - 1 - SUMMARY ̶Purine nucleotides are vital for RNA and DNA synthesis, signaling, metabolism, and energy homeostasis. The first chemotherapy drug ever developed, methotrexate, targeted purine synthesis and subsequently led to the development of numerous purine antimetabolites for cancer treatment. Still, our understanding of how tumors maintain and regulate their purine pools in vivo remains poorly understood. To synthesize purines, cells use two principal routes: the de novo and salvage pathways. The de novo pathway uses amino acids and small molecules to assemble the purine ring. This pathway is highly regulated through multiple pro- growth and oncogenic signaling pathways, including mTORC1, RAS, and MYC. The salvage pathway operates alongside the de novo pathway by recycling existing nucleobases from the diet or from nucleotide catabolism to generate purine nucleotides in an energy-efficient manner. However, to date, the contribution of specific purine synthesis pathways to nucleotide supply in tumors in vivo remains undescribed. Here, we established new methods using stable isotope infusions and metabolomics to determine the contribution of the salvage and de novo purine synthesis pathways to nucleotide supply in tumors in vivo. Through quantitative metabolic analysis, we show that de novo synthesis and salvage pathways contribute similarly to the maintenance of purine nucleotide pools in tumors. Our data also indicate remarkable coordination and flexibility between the de novo and salvage pathways to meet their purine needs. Furthermore, feeding mice nucleotides led to enhanced tumor growth while inhibiting purine salvage reduced tumor growth, indicating a critical role for the salvage pathway in tumor metabolism. This proposal aims to unveil critical insight into purine nucleotide metabolism, with the goal of identifying rational strategies for effectively targeting this pathway in cancer. In Aim 1, we will investigate the mechanisms by which major oncogenic signaling and their downstream signaling components regulate flux through the salvage pathway both in cell culture and in tumor models. In Aim 2, we will elucidate the influence of dietary nucleotides on metabolic reprogramming and tumor cell growth, and evaluate the requirement for the purine salvage pathway in mediating these effects. In Aim 3, we will delineate the mechanisms that mediate the metabolic flexibility between the de novo and salvage pathways and assess whether suppressing these mechanisms renders tumor cells more susceptible to purine-based treatments. We will also identify metabolic vulnerabilities that can be co-targeted with the salvage pathway to achieve effective anti-tumor effects. This proposal will expand our mechanistic understanding of the purine synthesis pathways in vivo. Additionally, it will pave the way for the identification of new strategies to target purine supply mechanisms in tumors to effectively eradicate cancer growth.
