Abstract 3826: Therapeutic targeting malignant mesothelioma with a novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate via its selective uptake by the proton-coupled folate transporter
Authors: Cherian Christina , Desmoulin Sita Kugel , Wang Lei , Polin Lisa , White Kathryn , Kushner Juiwanna , Stout Mark , Hou Zhanjun , Gangjee Aleem , Matherly Larry H. 
Published: August 2012

Abstract
Pemetrexed (Pmx; Alimta) is among the most active agents for treating malignant pleural mesothelioma (MPM). Pmx is transported into cells by 3 systems, the reduced folate carrier (RFC), folate receptor (FR) β, and proton-coupled folate transporter (PCFT). RFC is present in all tissues and tumors, whereas PCFT and FRα are more narrowly expressed. By real-time RT-PCR, clinically-relevant MPM cell lines express both RFC and PCFT but not FRα. The reported clinical efficacy of Pmx toward MPM may partly reflect high levels of PCFT in this tumor. Novel 6-substituted pyrrolo[2,3-d]pyrimidine antifolates with significant PCFT substrate activity but with greater specificities toward PCFT over RFC than Pmx, might offer significant advantages for selective therapeutic targeting MPM with reduced toxicity. We directly tested this notion for AG94, a 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate with a 3 carbon bridge. In HeLa sublines expressing moderate PCFT (R1-11-PCFT4) or RFC (R1-11-RFC6) levels, AG94 selectively inhibited (&gt;35-fold) proliferation of PCFT-expressing R1-11-PCFT4 cells, in contrast to the nearly identical inhibitions with Pmx toward these sublines. H2452 human MPM cells showed similar sensitivities to the anti-proliferative effects of AG94 and Pmx (IC50s of 72 nM and 64 nM, respectively). Analogous results were obtained by colony-forming assays. Growth inhibition by AG94 was reversed with excess adenosine and 5-amino-4-imidazolecarboxamide but not thymidine. Potent inhibition of glycinamide ribonucleotide formyltransferase (GARFTase), the first folate-dependent step in de novo purine biosynthesis, was confirmed by an in situ GARFTase assay in both R-1-11-PCFT and H2452 cells. Assays with [3H]AG94 with engineered Chinese hamster ovary cells confirmed selective PCFT transport, characterized by an acidic pH optimum and transport superiority over Pmx at both pH 5.5 and pH 6.8. Analogous results were seen with R1-11-PCFT4 and H2452 cells. In both R1-11-PCFT4 and H2452 cells, [3H]Pmx and [3H]AG94 were metabolized to polyglutamates, including di- to heptaglutamyl forms. Efficacy trials with subcutaneous early and upstage H2452 tumors were performed in SCID mice administered AG94, compared to gemcitabine and cisplatin. AG94 showed antitumor efficacies superior to gemcitabine and cisplatin. All mice tolerated the treatment regimens well. Our results demonstrate potent antitumor efficacies of AG94 toward H2452 MPM cells in vitro and in vivo, reflecting its efficient membrane transport by PCFT over RFC, synthesis of polyglutamate conjugates, and inhibition of GARFTase. Selectivity for non-RFC cellular uptake processes by potent antifolate drugs such as AG94 presents an exciting new opportunity for treating solid tumors including MPM over chemotherapy agents currently available.
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 3826. doi:1538-7445.AM2012-3826