Abstract 4791: Impact of folate transport redundancies on the therapy with tumor-targeted and untargeted antifolates
Authors: Hou Zhanjun , O’Connor Carrie , Ning Changwen , Wallace-Povirk Adrianne , Frühauf Josephine , Nayeen Md. Junayed , Gangjee Aleem , Matherly Larry H. 
Published: August 2019

Abstract
Folates participate in one-carbon (C1) transfer reactions in normal and cancer cells. Antifolate therapeutics disrupt cytosolic C1 metabolism, and have long been a mainstay for the therapy of a number of cancers. There are three major folate transporter systems in human tissues and tumors, including the reduced folate carrier (RFC), folate receptors (FRs) and proton-coupled folate transporter (PCFT). RFC is broadly expressed in tissues and tumors and is characterized by a neutral pH optimum. PCFT has more limited tissue distribution but is widely expressed in human solid tumors and exhibits an acidic pH optimum. FRα is expressed in a subset of solid tumors including epithelial ovarian cancer (EOC) but shows limited expression in most normal tissues. We previously discovered novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl compounds typified by AGF94 which show selective cellular uptake by FRα and/or PCFT over RFC, in contrast to the classical antifolates methotrexate (MTX) and pemetrexed (PMX) which are transported by both PCFT and RFC, and to a lesser extent by FRα. We showed that AGF94 had enhanced antitumor activities by targeting cytosolic C1 metabolism to PCFT-expressing tumors but lacking RFC, reflecting contraction of cellular tetrahydrofolate pools. In EOC cells, AGF94 showed substantial in vitro inhibition of cell proliferation independent of FRα expression, as long as PCFT was present. We systematically explored the impact of folate transporter redundancies among PCFT, FRα and RFC in determining anti-tumor efficacy of novel tumor-targeted and classical antifolates. Towards this goal, we engineered cell line models from PCFT-, FR-, and RFC-null HeLa cells to express doxycycline (DOX)-inducible FRα or RFC. We used these models to constitutively express PCFT together with DOX-inducible FRα or RFC. These were characterized for transporter expression and function, as well as intracellular folate levels, with/without DOX induction. The relative contributions of RFC, PCFT and FRα to transport function were evaluated from pH 5.5 to 7.4, with radiolabeled MTX and AGF347 which targets both cytosolic and mitochondrial C1 metabolism. We assessed antiproliferative activities of classical antifolates (PMX, MTX, PT523) versus tumor-targeted compounds (e.g., AGF94, AGF102, AGF347) in these models with/without DOX. Our results establish that co-expression of the major folate transporters can have variable and surprisingly disparate and substantial impacts on anti-tumor efficacies of both classical and tumor-targeted antifolates. Our study identified critical determinants of anti-tumor activity with classical and tumor-targeted antifolates, including relative levels of folate transporter expression and transporter specificity, and the impact of intracellular folate levels and extracellular pH.
Citation Format: Zhanjun Hou, Carrie O’Connor, Changwen Ning, Adrianne Wallace-Povirk, Josephine Frühauf, Md. Junayed Nayeen, Aleem Gangjee, Larry H. Matherly. Impact of folate transport redundancies on the therapy with tumor-targeted and untargeted 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 4791.