Abstract 3418: Mechanism of cell death induced by the DNA strand-breaking nucleoside analogue CNDAC.
Authors: Liu Xiaojun , Nowak Billie , Jiang Yingjun , Hittelman Walter , Plunkett William 
Published: August 2014

Abstract
The nucleoside analog CNDAC [2’-C-cyano-2’-deoxy-1-β-D-arabino-pentofuranosylcytosine] has a unique action mechanism of inducing single strand breaks following its incorporation into DNA through a β-elimination process. Transcription-coupled nucleotide excision repair (NER) is partially responsible for repair of such nicks [Cancer Res. 2008, 68:3881-89]. However, DNA replication across the unrepaired nicks generates double strand breaks (DSBs) which are mainly repaired by the ATM-dependent homologous recombination (HR) mechanism [Blood, 2010, 116:1737-46]. Both CNDAC (as DFP-10917) and its orally bioavailable prodrug, sapacitabine are in clinical trials in AML/MDS. Our goal was to define the mechanism by which CNDAC-induced DSBs lead to cell death. First, cytogenetic analysis revealed that chromosomal aberrations (breaks and gaps) in cells lacking XPF, the key 5’-NER endonuclease, increased &gt;2 fold and 16 fold after one (15 hr) and two (30 hr) cell cycles of treatment with 1 μM (non-arresting concentration) CNDAC, respectively (N≥60, P&lt;0.01). However, the wild type AA8 cells showed no change after one cell cycle exposure, and only a 5-fold increase in wild types after two cycles (N≥55, P&lt;0.05). Second, diverse cytogenetic changes were detected in AA8-derived cells lacking Rad51D, a paralog of the key recombinase Rad51 in the HR pathway, with only 13% of metaphases (N=106) free of aberration and &gt;70% bearing chromosomal damage too complex to be scored. By contrast, 78% of Rad51D-complemented cells (N=100) had no aberration, with only 1% unscorable. Third, post-mitosis nuclear alterations were further studied in Giemsa-stained AA8 cells after wash out of CNDAC (24 hr). The percentage of multinucleated cells increased 10 fold (1 μM) and 27 fold (10 μM) relative to control 24 hr after wash. Finally, to test the hypothesis that cells may die from mitotic catastrophe, video microscopy was used to track the fate of AA8 cells stably expressing H2B-GFP after CNDAC (0.5, 1 and 10 μM, 6-hr exposure) washout. Cells entering the 1st mitosis within 5 hr after wash presumably had CNDAC incorporation into DNA and therefore were eligible for analysis. There was no difference in the duration of 1st mitosis among all three treatments and control (N≥24, P&gt;0.05). However, 2nd mitosis was significantly prolonged in a concentration-dependent manner in all treatments (N≥37, P&lt;0.05-0.001). The interval between 1st and 2nd mitoses was also prolonged markedly (1 and 10 μM, P&lt;0.01-0.001). A similar trend was seen in H2B-GFP transfected lung cancer line H460. Notably, large numbers of treated AA8 cells became multinucleated upon completion of an aberrant 2nd mitosis. Frequently this was coupled with or followed by onset of apoptosis. These results indicate that CNDAC-induced DSBs, if not repaired, trigger cells to delay progression after 1st mitosis, and to undergo prolonged 2nd mitosis resulting in multinucleation and subsequent apoptosis.
Citation Format: Xiaojun Liu, Billie Nowak, Yingjun Jiang, Walter Hittelman, William Plunkett. Mechanism of cell death induced by the DNA strand-breaking nucleoside analogue CNDAC. [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 3418. doi:10.1158/1538-7445.AM2013-3418