The nucleoside analog 2'-C-cyano-l-beta-D-arabinofuranosylcytosine (CNDAC) is currently being evaluated in clinical trials. Earlier studies supported by this grant elucidated the cellular metabolism of CNDAC and demonstrated a unique mechanism of action. Once the CNDAC nucleotide is incorporated into DNA and a subsequent nucleotide is added, the electrophilic properties of the cyano group induce the analog to undergo an electronic rearrangement (beta elimination), that causes a break in the 3'-phophodiester linkage. This renders a permanent single strand break in the DNA, as this action is also accompanied by a molecular rearrangement of the analog to form a 2'-3'-dideoxyribose sugar. This is a de facto chain terminator that also serves as the molecular signature of these reactions. Although CNDAC-treated cells are able to continue through S phase, these molecular events cause cell cycle progression to arrest in G2. With respect to nucleoside analogs, these events are entirely unique. We hypothesize that DNA strand breaks that arise after incorporation of CNDAC nucleotide into DNA are recognized by DNA integrity surveillance mechanisms, and that a G2 DNA damage checkpoint is activated. If so, this may be a protective mechanism by which cells in part resist the potential lethality of CNDAC. We further postulate that dysregulation of this mechanism may accelerate or potentiate cell death. As such, the constituents of the checkpoint pathway are novel targets for the development of new therapeutics. It is a major goal of this application to elucidate the sensing and regulatory mechanisms that cause cells to arrest in G2 following treatment with CNDAC. Further, we will use small molecule inhibitors, siRNA knockdown approaches, and genetic mutations to disrupt specific components of this checkpoint pathway in order to evaluate the importance of each to enforcing the checkpoint. Finally, we will investigate the consequences of incorporation of CNDAC nucleotide into the repair patch generated by activation of nucleotide excision repair in quiescent cells. Thus, in addition to providing a basic understanding of the novel actions of this nucleoside analog, we expect that the results of these studies will be applicable to mechanism-based rationales for the use of CNDAC in combinations with other anticancer agents. ? ?
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