Abstract 4531: Molecular determinants of sensitivity to pralatrexate in a panel of human cancer cell lines

Abstract

Abstract Background: Pralatrexate (FOLOTYN®), an antifolate inhibiting dihydrofolate reductase (DHFR), is a high affinity substrate for cellular uptake via reduced folate carrier-1 (RFC-1) and polyglutamation by folyl-polyglutamyl synthetase (FPGS). This study evaluated the sensitivity of human cancer cell lines to pralatrexate and methotrexate, and investigated molecular determinants of primary sensitivity and acquired resistance. Materials and Methods: Anti-proliferative activity was evaluated in a panel (N=15) of human cancer cell lines using the MTT assay. mRNA expression was analyzed by qRT-PCR and protein expression by Western blot. Results: Two groups of cell lines with at least 100-fold difference in sensitivity to pralatrexate were identified. Pralatrexate was 3- to 19-fold (mean: 9-fold) more potent than methotrexate in the sensitive lines (N=9). Sensitivity to pralatrexate correlated with higher FPGS mRNA expression. Two pralatrexate-resistant cell lines (DU-PDX & HEP-PDX) and two methotrexate-resistant cell lines (DU-MTX & HEP-MTX) were established from DU-145 (prostate) and HEP2 (head & neck) cell lines. DU-PDX and HEP-PDX cells were cross-resistant to methotrexate. DU-MTX and HEP-MTX cells showed partial cross-resistance to pralatrexate but remained more sensitive to pralatrexate compared to methotrexate (7.5- and 11-fold, resp.). Acquired resistance to pralatrexate was associated with decreased RFC-1 expression in both DU-PDX and HEP-PDX cell lines, suggesting that reduced pralatrexate uptake may be an important mechanism of acquired resistance. No change in DHFR mRNA expression was found in the pralatrexate-resistant cells. In contrast, significant increases in DHFR mRNA and protein expression were seen in the methotrexate-resistant HEP-MTX cells, suggesting different mechanisms of resistance for these two antifolates. Conclusions: Consistent with prior findings, pralatrexate was more potent than methotrexate against a panel of human cancer cell lines. Primary sensitivity to pralatrexate was associated with higher FPGS expression whereas acquired resistance was associated with decreased RFC-1 expression. Increased DHFR expression correlated with resistance to methotrexate, but not pralatrexate. The marked differences in potency and molecular determinants of tumor response observed in these preclinical models provide rationale for development of pralatrexate in methotrexate-sensitive cancers as well as in settings of acquired methotrexate resistance. 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 4531. doi:10.1158/1538-7445.AM2011-4531