Abstract A117: Chk1-dependent up-regulation of ribonucleotide reductase R2 in response to camptothecin-induced DNA damage

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Abstract To investigate drug mechanisms of action and identify molecular targets for the development of rational drug combinations, we are conducting synthetic siRNA-based RNAi screens to identify genes whose silencing affects anti-cancer drug responses. One such screen indicated that the silencing of RRM1 and RRM2, which encode the large and small subunits of the human ribonucleotide reductase (RNR) complex respectively, markedly enhances the cytotoxicity of the Topoisomerase 1 (Top1) inhibitor camptothecin (CPT). Silencing of RRM2 was also found to enhance DNA damage as measured by -H2AX. Further studies showed that CPT up-regulates both RRM1 and RRM2 mRNA and protein levels, and induces the nuclear translocation of RRM2. The Checkpoint kinase 1 (Chk1) was also up-regulated and activated in response to CPT. CHEK1 down-regulation by siRNA and small molecule inhibition of Chk1 activity blocked induction of RRM2 by CPT. CHEK1 siRNA also suppressed E2F1 up-regulation by CPT, and silencing of E2F1 suppressed the up-regulation of RRM2. Silencing of ATR or ATM, and inhibition of ATM activity by KU-55933 blocked Chk1 activation and RRM2 up-regulation. This study links the function of known components of CPT-induced DNA damage response with proteins required for the synthesis of dNTPs, presumably in preparation for DNA repair. Specifically, we propose that, upon DNA damage, Chk1 activation, mediated by ATM and ATR, regulates RRM2 expression through the E2F1 transcription factor. Up-regulation in RRM2 levels, coupled with its nuclear recruitment suggests an active role for RNR in the cellular response to CPT-mediated DNA damage that could potentially be exploited as strategy for enhancing the efficacy of Top 1inhibitors. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A117.