Abstract 847: Suppression of p53R2 but not R2 radiosensitizes mutant p53 tumor cells
Authors: Sheryl A. Flanagan, Jeffrey J. Ackroyd, Sudha Mannava, Mikhail A. Nikiforov, Donna S. Shewach
Journal: Cancer Research (2014)

Abstract Gemcitabine (2’,2’-difluroro-2’-deoxycytidine;dFdCyd) is a potent radiosensitizer in tumor cells in vitro and in vivo. dFdCyd elicits cytotoxicity primarily via incorporation of its triphosphate, dFdCTP, into DNA, whereas inhibition of ribonucleotide reductase (RR) by dFdCDP produces a profound depletion of dATP which correlates to radiosensitization. We have demonstrated that dNTP imbalances generated by dFdCyd produce mismatches in DNA, which augment sensitivity to subsequent ionizing radiation (IR) but are not required to elicit cytotoxicity. We have proposed that RR suppression would be as effective as dFdCyd for radiosensitization. RR is a heterodimeric tetramer composed of the regulatory and active site subunit R1 paired with either R2 or its p53-inducible homolog, p53R2, as the catalytic and rate-limiting subunit. We used a RNAi approach to suppress either R2 or p53R2 (≥ 90% suppression of either R2 or p53R2, with little effect (≤20%) on expression of its homolog) in two p53 wild type cell lines, MCF7 breast carcinoma and A549 lung carcinoma. This approach produced equivalent radiosensitization, dATP depletion, cytotoxicity and increase in DNA mismatches compared to inactivation of RR by dFdCyd (IC50). These results reinforce our prior finding that radiosensitization with dFdCyd is the result of inhibition of RR and not incorporation into DNA or cytotoxicity. We then proposed that R2 but not p53R2 suppression would radiosensitize mutant p53 tumor cells. Interestingly, R2 shRNA suppression did not radiosensitize mutant p53 MCF7/ADR cells (radiation enhancement ratio (RER) = 1.07 ± 0.06). Despite the mutant p53 status of MCF7/ADR cells, p53R2 was elevated (≥ 50%) following suppression of R2 compared to untreated cells (no shRNA). We hypothesized that this increase in p53R2 permits RR to continue producing dNTPs for DNA replication and repair in the absence of R2, thus preventing radiosensitization. Indeed, simultaneous suppression of R2 and p53R2 produced excellent radiosensitization (RER = 1.68 ± 0.04 (shRNAs) vs. 1.6 ± 0.01 (dFdCyd (IC50)), and similar cytotoxicity (surviving fraction (SF) = 76.5 ± 8.3% (shRNA) vs. 71.3 ± 6.7% (dFdCyd)). Impressively, suppression of p53R2 alone produced excellent radiosensitization (RER= 1.76 ± 0.30) with similar cytotoxicity (SF = 84 ± 20%) compared to the double knockdown or dFdCyd. Considering that most solid tumors express mutant p53, suppression of p53R2 instead of R2 may be a more effective method for radiosensitization. These studies suggest that deoxynucleotide biosynthesis is regulated differently in p53 wild type compared to mutant p53 tumor cells, and may be used to maintain DNA replication and repair and prevent anticancer efficacy after damage induced by IR or other DNA damaging agents. The mechanism by which mutant p53 tumor cells upregulate p53R2 warrants further investigation. Note: This abstract was not presented at the meeting. Citation Format: Sheryl A. Flanagan, Jeffrey J. Ackroyd, Sudha Mannava, Mikhail A. Nikiforov, Donna S. Shewach. Suppression of p53R2 but not R2 radiosensitizes mutant p53 tumor cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 847. doi:10.1158/1538-7445.AM2014-847