Abstract 2992: Cellular pharmacodynamics of mitochondrial one-carbon metabolism-targeting 5-substituted pyrrolo[3,2-d]pyrimidine antifolates

Abstract

Abstract Folate-dependent one-carbon metabolism (1CM) is compartmentalized in the mitochondria and cytosol and generates a number of metabolites critical to tumor propagation. Folates are taken up by the plasma membrane facilitative transporters, reduced folate carrier (RFC; major tissue transporter) and proton-coupled folate transporter (PCFT; narrow physiological niche, but commonly expressed in solid tumors), and then transported into mitochondria via the mitochondrial folate transporter (MFT; SLC25A32). Although drug-targeting of cytosolic 1CM remains a clinical mainstay for a variety of cancers, development of clinically-useful agents targeting mitochondrial 1CM remains elusive. Of particular pharmacological interest is the mitochondrial 1CM enzyme, serine hydroxymethyltransferase2 (SHMT2). SHMT2 expression correlates with the oncogenic phenotype in a host of different cancers and, overall, SHMT2 is the fifth-most differentially expressed metabolic enzyme in cancer versus normal tissues. Despite its unequivocal oncogenic importance and therapeutic potential, there are no clinically relevant inhibitors of SHMT2. In this study, we characterized cellular pharmacodynamics of novel 5-substituted pyrrolo[3,2-d]pyrimidine antifolates (AGF291, AGF320, and AGF347) which show in vitroantitumor efficacy toward H460 lung, HCT-116 colon, and MIA PaCa-2 pancreatic cancer cells. Inhibition of mitochondrial SHMT2 and cytosolic 1CM at the purine nucleotide biosynthesis enzymes glycinamide ribonucleotide formyltransferase (GARFTase) and/or 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFTase) by this series was established by in vitro targeted metabolomics in H460, HCT-116, and MIA PaCa-2 cells and in vitro cell-free assays with purified enzymes. By depleting SHMT2-derived formate, these compounds potentiate their own direct inhibition of GARFTase and AICARFTase. Depletion of adenine nucleotide pools in vitro by all compounds led to inhibition of mTOR signaling to S6K1 in HCT116 cells. Subcellular fractionation of MIA PaCa-2 and MFT-null and human MFT-transfected glyB Chinese hamster ovary cells confirmed synthesis of polyglutamyl forms of AGF347 in both cytosol and mitochondria with mitochondrial uptake of AGF347 in part mediated by MFT. Treatment by all compounds decreased the cellular GSH/GSSG ratio, indicating depleted ability to combat oxidative stress. In vivo, AGF347 demonstrated potent antitumor efficacy against MIA PaCa-2 xenografts in SCID mice (n=5) with median tumor growth delay (T-C) in 4 mice >38 days and 1 of 5 tumor-free survivors (cured). In vivo metabolomics on tumor xenografts confirmed inhibition of serine catabolism and purine biosynthesis. Collectively, our studies establish the exceptional therapeutic potential of inhibitors dual-targeting mitochondrial and cytosolic 1CM. Citation Format: Aamod Dekhne, Khushbu Shah, Gregory S. Ducker, Md. Junayed Nayeen, Jade M. Katinas, Jennifer Wong, Arpit Doshi, Xun Bao, Hasini Kalpage, Jenney Liu, Seongho Kim, Adrianne Wallace-Povirk, Changwen Ning, Carrie O'Connor, Zhanjun Hou, Lisa Polin, Jing Li, Maik Hüttemann, Joshua D. Rabinowitz, Charles E. Dann, Aleem Gangjee, Larry Matherly. Cellular pharmacodynamics of mitochondrial one-carbon metabolism-targeting 5-substituted pyrrolo[3,2-d]pyrimidine antifolates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2992.