Abstract 3825: The impact of the reduced folate carrier on the collateral sensitivity of novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates with selective uptake by the proton-coupled folate transporter
Authors: Desmoulin Sita Kugel , Wang Lei , Polin Lisa , White Kathryn , Kushner Juiwanna , Stout Mark , Hou Zhanjun , Cherian Christina , Gangjee Aleem , Matherly Larry H. 
Published: August 2012

Abstract
The 6-substituted pyrrolo[2,3-d]pyrimidine thienonyl antifolates with 3 (AG94) or 4 (AG71) carbon bridge lengths represent a new class of agents that inhibit α-glycinamide ribonucleotide (GAR) formyltransferase (GARFTase). AG71 and AG94 are selectively transported by the proton-coupled folate transporter (PCFT) over the reduced folate carrier (RFC), the major facilitative folate transporter in tissues and tumors. PCFT functions optimally at acidic pHs. Transport of AG71 and AG94 by PCFT and the acidic tumor microenvironment may further increase tumor selectivity. Although AG71 and AG94 are not RFC substrates, levels of RFC could nonetheless impact their anti-proliferative effects, via expansion or contraction of intracellular tetrahydrofolate (THF) pools. Studies were performed with wild-type (WT) and methotrexate resistant R5 HeLa cells in which RFC is deleted. In proliferation assays with AG71 and AG94, R5 cells were more sensitive than were WT cells (3.9- and 2.4-fold, respectively). RFC-transfected R5 cells had IC50s for growth inhibition similar to WT cells. To determine the mechanisms of the collateral sensitivities to AG71 and AG94, we measured steady-state intracellular [3H]THF accumulations at a physiologic concentration of [3H]5-formyl THF in WT and R5 cells. R5 cells had contracted intracellular [3H]THF pools compared to WT cells. The collateral antifolate sensitivities in R5 cells were independent of PCFT transport, since there were no differences in transport for AG71 and AG94 at any pH between the sublines. Intracellular THF cofactors may compete at the level of polyglutamylation by folylpolyglutamate synthetase and/or GARFTase binding. When incubated with radiolabeled AG71 and AG94, WT cells accumulated slightly lesser amounts of total drug forms (84 and 76% for AG71 and AG94, respectively) than did R5 cells, most of these as polyglutamates. This difference was greatest for the longest chain-length (Glu4-Glu6) polyglutamates (increased ∼2-fold for both AG71 and AG94). GARFTase activity was measured in WT and R5 cells by an in situ metabolic assay which measures incorporation of [14C]glycine into [14C]formyl GAR. R5 and WT cells were differentially sensitive to GARFTase inhibition by AG71 and AG94 and these differences were lost when RFC expression was restored to R5 cells. In an in vivo efficacy trial with subcutaneous WT and R5 cells in SCID mice, antitumor efficacies of both AG71 and AG94 were increased in R5 cells relative to WT cells and this effect was greater for AG71. These findings suggest that novel antifolates with selective membrane transport by PCFT over RFC would have enhanced activities toward tumors that have lost RFC function, and that levels of RFC may be an important biomarker of chemotherapy efficacy with this new class of solid tumor-selective drugs.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3825. doi:1538-7445.AM2012-3825