Abstract 3101: Multitargeting of SHMT2 and purine biosynthesis is an effective antitumor treatment under hypoxia

Abstract These studies investigate the antitumor effects of targeting serine hydroxymethyltransferase (SHMT) 2 under hypoxia, the downstream metabolic consequences, and how to exploit unique metabolic vulnerabilities for cancer therapy. Mitochondrial one-carbon (C1) flux through SHMT2 and 5, 10-methylene tetrahydrofolate (THF) dehydrogenase 2 (MTHFD2) is the major source of C1 units in cancer cells. In mitochondria, THF serves as a cofactor for the catabolism of serine by SHMT2 to produce glycine and 5, 10-methylene-THF which is subsequently metabolized by MTHFD2 and MTHFD1L. The major products of this mitochondrial C1 flux include formate and NADH, along with glycine for protein, purine and glutathione (GSH) synthesis. Thus, mitochondrial C1 metabolism is important for maintaining redox homeostasis and scavenging reactive oxygen species (ROS). Under hypoxic conditions, the flux through SHMT2 and MTHFD2 is the major source of NADH; therefore, inhibition of SHMT2 under hypoxia may increase oxidative stress resulting in cell death. However, inhibition of SHMT2 under hypoxia may facilitate cell growth as a result of aspartate accumulation due to depletion of NADH and suppression of the TCA cycle. Aspartate accumulation preserves de novo purine biosynthesis under these conditions via donation of an amine group to IMP. We discovered novel 5-substituted pyrrolo[3, 2-d]pyrimidine multitargeted antifolates (e.g., AGF347) which inhibit SHMT2 in the mitochondria in addition to cytosolic targets (SHMT1 and folate-dependent enzymes in purine biosynthesis). We hypothesized that these agents may overcome inherent challenges of targeting SHMT2 under hypoxia, reflecting their inhibition of purine biosynthetic targets to preserve antitumor activity. Further, since all current SHMT2 inhibitors also inhibit SHMT1, to uncover the respective impacts of inhibiting these homologous enzymes, we utilized tetracycline-inducible SHMT1 or SHMT2 HCT116 tumor cell models to simulate selective inhibition of these targets by removal of doxycycline. We demonstrate that the pyrrolo[3, 2-d]pyrimidine compounds retain potent antitumor efficacy under hypoxia, as reflected in inhibition of cell proliferation and colony formation. We assessed the metabolic consequences of antifolate treatment under hypoxia and normoxia by measuring malate, aspartate, and purine levels by LC-MS/MS in addition to effects on ROS and GSH levels. We also demonstrated that loss of SHMT2 activity alone is sufficient for anticancer effects under both normoxia and hypoxia, whereas loss of SHMT1 alone had no effect. These studies demonstrate the considerable promise of multitargeted pyrrolo[3, 2-d]pyrimidine antifolates for cancer therapy under conditions under which standard-of-care agents may be less effective. Citation Format: Mathew Joseph Schneider, Carrie OConnor, Xun Bao, Md. Junayed Nayeen, Zhanjun Hou, Jing Li, Aleem Gangjee, Larry Matherly. Multitargeting of SHMT2 and purine biosynthesis is an effective antitumor treatment under hypoxia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3101.