Abstract 5403: Targeted therapy of non-small cell lung cancer (NSCLC) by novel 6-pyrrolo[2,3-d]pyrimidine thienoyl antifolates with selective transport by the proton-coupled folate transporter (PCFT)
Authors: Wilson Mike R. , Hou Zhanjun , Wang Lei , Wang Si , Ratnam Manohar , Gangjee Aleem , Matherly Larry 
Published: August 2015

Pemetrexed (PMX) is a 5-substituted pyrrolo[2,3-d]pyrimidine antifolate used for first-line and maintenance therapy of NSCLC. Whereas correlations were reported between clinical responses in NSCLC and levels of folate receptor (FR) α, FRα is not a pharmacologically relevant PMX transporter. Rather, PMX is primarily transported by the reduced folate carrier (RFC) and PCFT. RFC is ubiquitously expressed, whereas PCFT shows more limited expression in normal tissues, although both are broadly expressed in tumor cells. Unlike RFC, PCFT is active at acidic pH, with significant levels at pH 6.8. Using patient cDNAs from normal lung (n = 8) and NSCLC samples (n = 37), we measured transcripts for RFC and PCFT, and for intracellular targets for PMX, including thymidylate synthase (TS) and glycinamide ribonucleotide formyltransferase (GARFTase), by real-time RT-PCR (qPCR). PCFT was detected at similar levels in normal lung and NSCLC specimens, while RFC was decreased in tumors compared to normal lung. Expression of TS and GARFTase were increased in NSCLC samples, compared to normal tissue. In NSCLC cell lines A549, CRL5872, H460, H1650 and H2030, PCFT and RFC, but not FRα, were detected by qPCR. We synthesized novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl regioisomers related to PMX [AGF94 (2′,5′) and AGF154 (2′,4′)] as potential antitumor agents. Based on patterns of inhibition of proliferation of engineered Chinese hamster ovary cells expressing human RFC or PCFT, AGF94 and AGF154, unlike PMX, are not transported by RFC but are excellent substrates for PCFT. AGF94 and AGF154 showed potencies comparable to PMX toward all the NSCLC cell lines. We found that [3H]PMX and [3H]AGF154 were transported by PCFT into the NSCLC cell lines at pH 5.5 and pH 6.8 and uptake could be blocked by non-radioactive AGF94. AGF154 accumulation over sustained exposure at pH 5.5 was greater than that of PMX, whereas accumulations of AGF154 and PMX were nearly identical at pH 6.8. In H460 cells treated with PMX, thymidine (10 μM) partially protected cells from growth inhibition, whereas neither adenosine (10 μM) nor 5-aminoimidazole-4-carboxamide (AICA) (320 μM) were protective. In cells treated with AGF94 or AGF154, adenosine and AICA, but not thymidine, were completely protective, consistent with inhibition of de novo purine biosynthesis at GARFTase. Potent GARFTase inhibition by AGF94 and AGF154 was confirmed by an in situ assay which measures incorporation of [14C]glycine into formyl GAR (FGAR), the GARFTase product. Our results suggest that novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates may offer significant promise for treating NSCLC, reflecting their selective membrane transport by PCFT over RFC and potent inhibition of de novo purine biosynthesis.

Citation Format: Mike R. Wilson, Zhanjun Hou, Lei Wang, Si Wang, Manohar Ratnam, Aleem Gangjee, Larry Matherly. Targeted therapy of non-small cell lung cancer (NSCLC) by novel 6-pyrrolo[2,3-d]pyrimidine thienoyl antifolates with selective transport by the proton-coupled folate transporter (PCFT). [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 5403. doi:10.1158/1538-7445.AM2015-5403