Abstract A100: Pemetrexed-induced uracil accumulation is an importatn source of DNA damage in UNG deficient cells, contributing to hematologic toxicity.

Abstract

Abstract Pemetrexed is a multi-target antifolate that has proven activity against several solid tumors. Dose limiting myelosuppression and acquired resistance are major challenges to pemetrexed and antifolate chemotherapy regimens. We have previously shown that resistance to pemetrexed in human cancer cells can be overcome via inhibition of DNA base excision repair (BER), the major pathway responsible for the resolution of uracil in DNA. To understand the implications of UNG deficiency in vivo, we have investigated the impact of UNG deficiency on whole animal response to pemetrexed. Here, we illustrate that pemetrexed treatment induces more significant reduction in bone marrow cell numbers and relatively low peripheral lymphocyte counts in UNG−/− mice compared to UNG+/+ littermates. Compared to wild-type cells, UNG−/− MEFs also show reduced viability and increased cell death in response to pemetrexed. Hematopoietic cell sensitivity was mirrored by increased pemetrexed cell killing in UNG mutant human lymphoblastoid cells, derived from patients with biallelic inactivating mutations in the UNG gene. Cellular sensitivity is characterized by reduced nucleotide incorporation, arrest in S-phase of the cell cycle, and induction of apoptosis. Pemetrexed treatment of UNG−/− cells also causes robust induction of γ-H2AX, a surrogate marker of DNA double strand breaks. Through γ-H2AX ChIP-sequencing we provide the first direct evidence that DNA double strand breaks are located specifically at AT(U) rich sequences, directly implicating uracil accumulation in the formation of double strand breaks following pemetrexed treatment. UNG deficient cells showed no differential sensitivity to cisplatin or temozolomide, two drugs unrelated to uracil accumulation. Moreover, through thymidine supplementation, we were able to partially reverse the pemetrexed-mediated induction of DNA damage markers, including γ-H2AX and protect UNG−/− cells from cell death, further supporting a uracil-mediated mechanism of cytotoxicity in UNG−/− cells. In sum, these data suggest that the accumulation of uracil in the DNA of hematopoietic cells drives myelosuppression and lymphocyte toxicity. Mechanistically, our data support a previously underappreciated role for uracil accumulation, UNG activity and the BER pathway in the determination of cellular sensitivity to pemetrexed and suggest UNG expression may be an important predictor of adverse events, such as myelosuppression in individuals receiving pemetrexed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A100.