Abstract LB-126: Replication-associated DNA double-strand breaks and checkpoint abrogation promote gene amplification.

Abstract

Abstract Gene amplification, a copy number increase of genomic segments through DNA rearrangements, drives cancer progression and acquired resistance to anti-cancer drugs. Understanding the cellular processes involved in gene amplification is important to rationally develop management strategies for cancer patients with such aggressive tumors. DNA double-strand breaks (DSBs) are the most dangerous lesions of DNA and often initiate gene amplification. A main cellular process giving rise to DSBs in cancer cells is DNA replication, as cancer cells are under strong replication stress. We hypothesized that failure to repair replication-associated DSBs would result in DNA rearrangements and gene amplification. To test this hypothesis, we suppressed the expression of DNA repair protein MRE11 in mammalian cells. MRE11 is a component of the MRN (Mre11-Rad50-NBS1) complex that repairs DSBs of different origin including those arising during DNA replication. Indeed, knocking down MRE11 resulted in the 10-fold increase in the frequency of gene amplification. DSBs were markedly increased in the MRE11 knockdown cells in the S phase of the cell cycle, suggesting a DNA replication-associated origin of DSBs. Importantly, DSBs continued to be abundant in the M phase, indicating that MRE11 knockdown cells lack both intra-S and G2/M checkpoints. The possibility that checkpoint abrogation could also promote gene amplification was further tested in the I-SceI endonuclease-induced gene amplification system, where checkpoint abrogation by caffeine resulted in the 4-fold increase in amplification frequency. These results provide a proof-of-concept that pharmacological modulation of cell cycle checkpoints can alternate the gene amplification frequency, and may be used to prevent proliferation of cells with gene amplification. Citation Format: Anna Kondratova, Michael Marotta, Takaaki Watanabe, Hisashi Tanaka. Replication-associated DNA double-strand breaks and checkpoint abrogation promote gene amplification. [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 LB-126. doi:10.1158/1538-7445.AM2013-LB-126