Abstract 2528: Therapeutic targeting of a novel 6-substituted pyrrolo[2,3-<i>d</i>]pyrimidine thienoyl antifolate to human solid tumors based on selective uptake by PCFT
Authors: Sita Kugel Desmoulin, Lei Wang, Larry Tait, Lisa Polin, Zhanjun Hou, Christina Cherian, Aleem Gangjee, Larry H. Matherly
Journal: Cancer Research (2011)

Abstract The proton-coupled folate transporter (PCFT) is a proton-folate symporter with an acidic pH optimum, such that PCFT may provide a mechanism for targeting cytotoxic antifolates to tumors, based on their acidic microenvironments (pH 6.2-6.8). PCFT transcripts and protein are high in human solid tumor tissues and cell lines (especially hepatic and ovarian tumors) and limited in most human normal tissues. We identified a novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate (AG71) that is a selective transport substrate for PCFT over the ubiquitously expressed reduced folate carrier (RFC) and a potent inhibitor of the de novo purine nucleotide biosynthetic enzyme glycinamide ribonucleotide (GAR) formyl transferase (GARFTase). Both AG71 and the clinically relevant antifolate pemetrexed (Pmx) were potently inhibitory toward proliferation of a HeLa subline (R1-11-PCFT4) that lacked RFC expression and was engineered to ectopically express physiologically relevant PCFT levels. Unlike Pmx, AG71 did not inhibit proliferation of R1-11-RFC6 cells which express physiologically relevant RFC levels but no PCFT. In colony-forming assays at pH 6.8, AG71 inhibited colony formation of R1-11-PCFT4 cells in a dose- and time-dependent manner, comparable to Pmx. We measured uptake of [3H]AG71 in R1-11-PCFT4 cells and found that transport was optimal at pH 5.5 but still significant at pH 6.8. At pH 6.8, AG71 was metabolized by folylpoly-γ-glutamate synthase to AG71 polyglutamates, as detected by HPLC. GARFTase was validated as a major intracellular target with an in situ GARFTase activity assay at pH 6.8 involving [14C]glycine incorporation into [14C]formyl GAR. In HepG2 human hepatocellular carcinoma cells, PCFT levels were elevated and [3H]AG71 was transported at pH 6.8, whereupon it was extensively metabolized to polyglutamates. By proliferation assays, AG71 was potently inhibitory (IC50 of 227.5 nM) toward HepG2 cells. To test in vivo efficacy of AG71, SCID mice were implanted with subcutaneous HepG2 tumors and treated with AG71 (180 mg/kg × 3d). Compound AG71 was highly active against HepG2 tumors. In conclusion, we have established the expression patterns and pH-specific transport for PCFT in human solid tumor cells and demonstrated that the novel PCFT-targeted antifolate AG71 can be internalized into tumor cells by PCFT leading to significant growth delay of HepG2 xenografts. Our findings offer exciting new therapeutic possibilities for selectively targeting chemotherapy to tumors with novel antifolates based on their acidic microenvironments. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2528. doi:10.1158/1538-7445.AM2011-2528