Abstract 4794: Targeting mitochondrial and cytosolic one carbon metabolism of pancreatic adenocarcinoma via the proton-coupled folate transporter with novel 5-substituted pyrrolo[3,2-d]pyrimidine analogs
Authors: Ning Changwen , Dekhne Aamod , Nayeen Md. Junayed , Katinas Jade M. , Wong Jennifer , Frühauf Josephine , Bao Xun , O’Connor Carrie , Wallace-Povirk Adrianne , Li Jing , Dann Charles E. , Gangjee Aleem , Matherly Larry H. , Hou Zhanjun 
Published: August 2019

Abstract
Pancreatic cancer (PaC) represents the 4th leading cause of cancer-related deaths in the US with a mortality rate of 99%. The 5-year overall survival rate for PaC is currently 8%. One-carbon (C1) metabolism is frequently altered in cancer. For PaC, TCGA data sets show that elevated expression of key enzymes involved in cytosolic [5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFTase) and serine hydroxymethyltransferase (SHMT)1] and mitochondrial [SHMT2 and methylene tetrahydrofolate dehydrogenase 2 (MTHFD2)] C1 metabolism is associated with poor survival. Antifolate therapeutics disrupt cytosolic C1 pathways required for syntheses of thymidylate, purines, and certain amino acids, and are a mainstay for therapy of several cancers. Antifolate uptake into tumors and tissues involves the reduced folate carrier, the major tissue folate transporter, and the proton-coupled folate transporter (PCFT), which shows a more limited tissue distribution but is widely expressed in human solid tumors and is active only at acidic pHs characterizing the tumor microenvironment. We discovered novel 5-substituted pyrrolo[3,2-d]pyrimidine analogs (AGF347, AGF359) with PCFT transport that potently inhibited proliferation of PaC cell lines (AsPC-1, BxPC-3, CFPAC-1, HPAC, MIA PaCa-2 and PANC-1), of which HPAC (KRAS mutant) and BxPC-3 (KRAS wild-type) cells were most sensitive. The PaC cell lines all expressed PCFT transcripts and proteins that were active for PCFT transport with 3H-AGF347 at acid pH. When HPAC cells were incubated with 3H-AGF347 over 48 h, drug accumulated in both cytosol and mitochondria. 3H-AGF347 was extensively metabolized to polyglutamates. Treatment of PaC cells with AGF347 and AGF359 inhibited proliferation by inducing glycine and adenosine auxotrophy that was rescued by excess glycine and adenosine. This implied that both mitochondria and cytosolic C1 metabolism was inhibited. Inhibition of mitochondrial SHMT2 and cytosolic SHMT1, glycinamide ribonucleotide formyltransferase and/or AICARFTase was confirmed by in vitro targeted metabolomics and assays with purified enzymes. Tumor cell killing was confirmed (with HPAC and BxPC-3) by colony-forming assays with AGF347 and AGF359 and drug-induced apoptosis with AGF347 was demonstrated (with HPAC) by annexin V-PI staining and flow cytometry. AGF347 and AGF359 depleted purine nucleotides and inhibited mTOR signaling via S6K1 at least in part (for BxPC-3) via activation of AMPK, likely due to elevated ZMP accompanying suppression of AICARFTase. Collectively, our studies identify first-in-class inhibitors and establish the considerable therapeutic potential of dual-targeting mitochondrial and cytosolic C1 metabolism in PaC independent of KRAS mutation status and reflecting cellular uptake by PCFT.
Citation Format: Changwen Ning, Aamod Dekhne, Md. Junayed Nayeen, Jade M. Katinas, Jennifer Wong, Josephine Frühauf, Xun Bao, Carrie O’Connor, Adrianne Wallace-Povirk, Jing Li, Charles E. Dann, Aleem Gangjee, Larry H. Matherly, Zhanjun Hou. Targeting mitochondrial and cytosolic one carbon metabolism of pancreatic adenocarcinoma via the proton-coupled folate transporter with novel 5-substituted pyrrolo[3,2-d]pyrimidine analogs [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 4794.