Abstract 5494: Novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate regioisomers target folate receptor alpha positive ovarian cancer cells via inhibition of de novo purine nucleotide biosynthesis.

Abstract

Abstract Epithelial ovarian cancers (EOC) represent a unique treatment challenge as over 50% of patients are diagnosed with advanced stage disease when surgical and chemotherapeutic interventions prove futile. Approximately 90% of EOC are folate receptor (FR) α positive and FR levels correlate with stage and grade. FRα-targeted therapies represent a rational treatment option for patients with advanced EOC whose treatments fail. There are three primary mechanisms of folate transport in mammalian cells, the reduced folate carrier (RFC), the proton-coupled folate transporter (PCFT) and FRs. RFC is the major transporter for folates and is expressed ubiquitously in normal tissues and tumors. Hence, developing novel antifolates that bypass cellular accumulation by RFC should reduce dose-limiting toxicities. PCFT is expressed in many solid tumors including ovarian cancer and is active at acidic pH. Previous studies established that a novel 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine antifolate with an isosteric thienoyl substitution and a three carbon bridge (AG94) showed selective cellular uptake by FRα and PCFT over RFC. [1,3]Thienoyl regioisomers (AG150 and AG154) of AG94 showed subnanomolar potencies toward both KB nasopharyngeal carcinoma (IC50s = 0.1 and 0.09 nM, respectively) and IGROV1 ovarian carcinoma (IC50s of 0.64 and 0.1nM, respectively). While AG150 and AG154 were substrates for both PCFT and FRs, like AG94, their inhibitory effects at low drug concentrations were mediated primarily via FRα, as indicated by their capacities to bind with high affinities to FRα and protection by excess folic acid. Nucleoside protection assays with adenosine and 5-amino-4-imidazole carboxamide established that the first folate-dependent enzyme in the de novo purine biosynthesis pathway, glycinamide ribonucleotide (GAR) formyltransferase (GARFT), was the primary intracellular target for AG150 and AG154. GARFT targeting was confirmed by in situ metabolic labeling with [14C]glycine and measurement of accumulated [14C]formyl GAR in KB cells, for which IC50s were 1.35 and 1.46 nM, respectively. The purine salvage enzyme methylthioadenosine phosphorylase (MTAP) is frequently co-deleted with CDKN2a in solid tumors including ovarian cancer. Growth inhibitory effects of AG150 and AG154 were reversed in the presence of methylthioadenosine in MTAP-deficient IGROV1 cells transfected with MTAP cDNA. Our results demonstrate potent anti-tumor activities of the 6-pyrrolo[2,3-d]pyrimidine thienoyl regioisomers of AG94, AG150 and AG154, associated with their FRα-mediated cellular uptake and inhibition of GARFT in de novo purine biosynthesis. Preservation of purine salvage including MTAP is likely an important cellular determinant of antitumor efficacy for this class of novel tumor-targeted agents. Citation Format: Shermaine K. Mitchell-Ryan, Lei Wang, Steven Orr, Sita Kugel, Christina Cherian, Aleem Gangjee, Larry H. Matherly. Novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate regioisomers target folate receptor alpha positive ovarian cancer cells via inhibition of de novo purine nucleotide biosynthesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5494. doi:10.1158/1538-7445.AM2013-5494