Abstract 5490: Mice deficient in uracil DNA glycosylase (UDG) display increased sensitivity to the antifolate pemetrexed

Abstract

Abstract Pemetrexed, a multi-target antifolate, causes genomic uracil incorporation through inhibition of the enzymes thymidylate synthetase (TS) and dihydrofolate reductase (DHFR). Base excision repair (BER), initiated by uracil DNA glycosylase (UDG), actively recognizes and removes misincorporated uracil from the genome. Mice deficient in UDG have been described and display increased genomic uracil accumulation, elevated spontaneous mutation frequency, and higher incidence of B-cell lymphoma. We have previously reported the in vitro induction of UDG in H460 human lung cancer cells after pemetrexed exposure and have illustrated hypersensitivity to pemetrexed in human colon cancer cell lines lacking UDG expression (DLD1 UDG-/-). Here, we have investigated the role of UDG expression on pemetrexed sensitivity in vivo using gene-targeted UDG-/- mice. Mice received either a single 150mg/kg dose of pemetrexed on day 1 or received 3 consecutive 150 mg/kg doses of pemetrexed on days 1-3 and were sacrificed on day 4 or 5, respectively. Compared to UDG+/+ and UDG+/- littermates, UDG-/- mice were markedly myelosuppressed following pemetrexed treatment, as evidenced by a ∼50% reduction in bone marrow cell numbers and a ∼70% reduction in total colony forming units (CFU). Moreover, marrow from pemetrexed-treated UDG-/- mice expressed higher levels of DNA damage markers γ-H2AX and cleaved-PARP than control samples suggesting the formation of DNA double strand breaks and activation of apoptosis in UDG-/- marrow following pemetrexed exposure. Western blot analysis further evinced Chk-1 phosphorylation (p-Chk1, Ser345) in marrow from UDG-/- mice but not UDG+/+ or UDG+/- samples, suggesting inadequate removal of pemetrexed-induced genomic uracil may stall DNA replication and contribute to the cytotoxicity observed. These data in addition with our novel observations of enhanced pemetrexed sensitivity in UDG-/- mouse embryonic fibroblast (MEFs), UDG mutant human lymphoblastoid cell lines (LCLs), and DLD1 UDG-/- cells, strongly suggest that UDG deficiency potentiates pemetrexed cytotoxicity in in vitro as well as in in vivo models. Additionally, our findings indicate a role for genomic uracil accumulation in the mechanism of cytotoxicity for pemetrexed. Furthermore, our data illustrate a synthetic lethal interaction between TS/DHFR inhibition and UDG deficiency and promote UDG as a target for improving pemetrexed efficacy. 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 5490. doi:10.1158/1538-7445.AM2011-5490