Abstract #875: DNA strand breaks induced by the nucleoside analogue CNDAG are repaired by homologous recombination
Authors: Xiaojun Liu, Akira Matsuda, William Plunkett
Journal: Cancer Research (2009)

AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO

The deoxyguanosine analogue 9-(2- C -cyano-2-deoxy-1-\#946;-D- arabino -pentofuranosyl) guanine (CNDAG) likely causes a single-strand break subsequent to its incorporation into DNA, similar to the action of its cytosine congener, CNDAC, which is currently undergoing clinical investigation. We postulate that subsequent processing or DNA replication across the unrepaired nick would generate a double strand break. This study was aimed at identifying cellular response and repair mechanisms for two CNDAG prodrugs, 2-amino-9-(2- C -cyano-2-deoxy-1-\#946;-D- arabino -pentofuranosyl)-6-methoxy purine (6-OMe) and 9-(2- C -cyano-2-deoxy-1-\#946;-D- arabino -pentofuranosyl)-2,6-diaminopurine (6-NH2). Each compound is a substrate for adenosine deaminase, the action of which generates CNDAG. First, growth inhibition of CCRF-CEM T-lymphoblasts was evident at 1 \#956;M of each compound, although this was dependent upon both concentration and time of exposure. Second, 24-hr exposure to 40 \#956;M 6-OMe or 6-NH2 arrested CCRF-CEM cells in S phase, but had no cell cycle effect on other cell lines of hematological origin (myeloid, ML-1 and OCI-AML3; lymphoma, Raji and Granta 519). This could be attributed to relatively low activity of deoxycytidine kinase in the latter cell lines. Third, p53 knocked-out HCT116 cells were equally sensitive to CNDAG 6-NH2 as were parental cells expressing wild type p53 (p>0.05) in a clonogenic assay, indicating that cytotoxicity of CNDAG is independent of p53 status. Fourth, both CNDAG prodrugs activated repair proteins in multiple DNA damage response pathways, as revealed by immunoblotting. 24-hr incubation of CCRF-CEM cells with 50 \#956;M 6-OMe or 6-NH2 increased the phosphorylation of Ser-1981 on ATM (3.3 or 9.5-fold relative to control), Ser-345 on Chk1 (8.0 or 5.4-fold), Thr-68 on Chk2 (2.2 or 1.6-fold), Ser-966 on SMC1 (8.4 or 7.5-fold), Ser-343 on Nbs1 (3.4 or 3.0-fold) and \#947;-H2AX (50%). In contrast, there was no increase in phosphorylation of two other sensor kinases, DNA-PKcs (Ser-2056) which participates in repair of double strand breaks by non-homologous end-joining, and ATR (Ser-428) which senses stalled DNA replication forks. Finally, we investigated the role of components of homologous recombination in CNDAG-induced DNA damage repair. Chinese hamster cells deficient in either of the two Rad51-interacting proteins, Xrcc3 and Brca2, conferred greater than 30-fold sensitivity to CNDAG 6-OMe and 6-NH2 relative to their wild type lines. These results demonstrate that DNA damage caused by CNDAG activates ATM-dependent signaling pathways and is repaired through homologous recombination. Because cytosine and guanine nucleoside congeners have remarkably different clinical activities, e.g., cytarabine and nelarabine, it will be useful to pursue investigations to more fully characterize the metabolism and actions of CNDAG prodrugs.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 875.