Abstract 4483: Impact of loss of folate receptor alpha on antitumor effects of novel 6-substituted pyrrolo[2,3-d]pyrimidine antifolates with substrate activities for both folate receptors and the proton-coupled folate transporter
Authors: Hou Zhanjun , Orr Steve , George Christina , Wallace Adrianne , Matherly Larry H. , Wang Lei , Yang Si , Gandjee Aleem 
Published: August 2015

Abstract
Major folate uptake mechanisms include the reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT), and folate receptors α and β. Antifolates such as methotrexate or pemetrexed are excellent substrates for human RFC (hRFC) and variously for human PCFT (hPCFT) and human folate receptor α (hFRα). Recent attention has focused on developing tumor-targeted folate-based therapies, reflecting tumor-selective uptake by hFRα and/or hPCFT. Novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl regioisomers AGF94 (2′,5′) and AGF154 (2′,4′) were synthesized and characterized as potent inhibitors of cell proliferation of engineered Chinese hamster ovary (CHO) cells expressing hPCFT or hFRα, but not for CHO cells expressing hRFC. In 41 ovarian cancer specimens from patients, hFRα, hPCFT and hRFC were all expressed. FRα transcript levels increased with stage of disease, although hPCFT levels were constant. IGROV1 is a clinically relevant ovarian cancer cell line that expresses both hFRα and hPCFT. IGROV1 cells were highly sensitive to both AGF94 and AGF154, as reflected in inhibition of cell proliferation in vitro. Growth inhibition by AGF94 or AGF154 in IGROV1 cells was partially reversed by excess folic acid, likely reflecting drug uptake by hFRα, as well as by hPCFT. To evaluate the impact of loss of hFRα on AGF94 and AGF154 in vitro inhibition of IGROV1 cell proliferation, FRα was knocked down (&gt;95%) by lentivirus shRNAs. Clonal isolates were generated (n = 17) and profiled by RT-PCR for expression of the folate transporters and key enzymes likely to impact antifolate sensitivities (e.g., folylpolyglutamyl synthetase, β-glycinamide ribonucleotide formyltransferase), relative to a non-targeted control (NTC) cells. Gene expression profiles were similar for IGROV1 hFRα knockdown (KD) clones and NTC cells. Two representative hFRα KD clones were assayed for inhibition of cell proliferation by AGF94 and AGF154, with IC50s for IGROV1 KD clones of 26±1.8 and 26±9 nM for AGF94, and 65±1.2 and 81±2.4 nM for AGF154. For comparison, IC50s in the IGROV1 NTC cells expressing both hFRα and hPCFT were 0.6±0.1 nM for AGF94 and 2.2±0.8 nM for AGF154. In contrast, the hFRα-specific antifolate, AGF102 showed IC50s of &gt; 1000 nM and ∼2.5 nM for KD and NTC cells. The preservation of substantial inhibitory activity for AGF94 and AGF154, accompanying the nearly complete loss of FRα expression in the KD clones is likely due to the sustained uptake of these novel antifolates by hPCFT. For AGF154, this was confirmed by direct uptake assays with [3H]-AGF154. Our results suggest that 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates with substantial substrate activities for hFRα and hPCFT may offer significant advantages in treating tumors that express both these transport systems.
Citation Format: Zhanjun Hou, Steve Orr, Christina George, Adrianne Wallace, Larry H. Matherly, Lei Wang, Si Yang, Aleem Gandjee. Impact of loss of folate receptor alpha on antitumor effects of novel 6-substituted pyrrolo[2,3-d]pyrimidine antifolates with substrate activities for both folate receptors and the proton-coupled folate transporter. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4483. doi:10.1158/1538-7445.AM2015-4483