Ataxia-telangiectasia mutated (ATM) kinase orchestrates nuclear DNA damage responses but is proposed to be involved in other important and clinically relevant functions. Here, we provide evidence for what we believe are 2 novel and intertwined roles for ATM: the regulation of ribonucleotide reductase (RR), the rate-limiting enzyme in the de novo synthesis of deoxyribonucleoside triphosphates, and control of mitochondrial homeostasis. Ataxia-telangiectasia (A-T) patient fibroblasts, wild-type fibroblasts treated with the ATM inhibitor KU-55933, and cells in which RR is inhibited pharmacologically or by RNA interference (RNAi) each lead to mitochondrial DNA (mtDNA) depletion under normal growth conditions. Disruption of ATM signaling in primary A-T fibroblasts also leads to global dysregulation of the R1, R2, and p53R2 subunits of RR, abrogation of RRdependent upregulation of mtDNA in response to ionizing radiation, high mitochondrial transcription factor A (mtTFA)/mtDNA ratios, and increased resistance to inhibitors of mitochondrial respiration and translation. Finally, there are reduced expression of the R1 subunit of RR and tissue-specific alterations of mtDNA copy number in ATM null mouse tissues, the latter being recapitulated in tissues from human A-T patients. Based on these results, we propose that disruption of RR and mitochondrial homeostasis contributes to the complex pathology of A-T and that RR genes are candidate disease loci in mtDNA-depletion syndromes.

High-grade serous ovarian carcinoma (HGSOC), the most lethal gynecological cancer, often leads to chemoresistant diseases. The p53 protein is a key transcriptional factor regulating cellular homeostasis. A majority of HGSOCs have inactive p53 because of genetic mutations. However, genetic mutation is not the only cause of p53 inactivation. The aggregation of p53 protein has been discovered in different types of cancers and may be responsible for impairing the normal transcriptional activation and pro-apoptotic functions of p53. We demonstrated that in a unique population of HGSOC cancer cells with cancer stem cell properties, p53 protein aggregation is associated with p53 inactivation and platinum resistance. When these cancer stem cells differentiated into their chemosensitive progeny, they lost tumor-initiating capacity and p53 aggregates. In addition to the association of p53 aggregation and chemoresistance in HGSOC cells, we further demonstrated that the overexpression of a p53-positive regulator, p14ARF, inhibited MDM2-mediated p53 degradation and led to the imbalance of p53 turnover that promoted the formation of p53 aggregates. With in vitro and in vivo models, we demonstrated that the inhibition of p14ARF could suppress p53 aggregation and sensitize cancer cells to platinum treatment. Moreover, by two-dimensional gel electrophoresis and mass spectrometry we discovered that the aggregated p53 may function uniquely by interacting with proteins that are critical for cancer cell survival and tumor progression. Our findings help us understand the poor chemoresponse of a subset of HGSOC patients and suggest p53 aggregation as a new marker for chemoresistance. Our findings also suggest that inhibiting p53 aggregation can reactivate p53 pro-apoptotic function. Therefore, p53 aggregation is a potential therapeutic target for reversing chemoresistance. This is paramount for improving ovarian cancer patients' responses to chemotherapy, and thus increasing their survival rate.

Ribonucleotide reductase catalyzes the production of deoxyribonucleoside diphosphates, the precursors of deoxyribonucleoside triphosphates for DNA synthesis. Mammalian ribonucleotide reductase (RNR) is a tetramer consisting of two non-identical homodimers, R1 and either R2 or p53R2, which are considered to be involved in DNA replication and repair, respectively. We have demonstrated that DNA damage by doxorubicin and cisplatin caused a steady elevation of the R2 protein in p53(؊/؊) HCT-116 human colon carcinoma cells but induced degradation of the protein in p53(؉/؉) cells. To evaluate the involvement of R2 in response to DNA damage, p53(؊/؊) HCT-116 cells were stably transfected with an expression vector transcribing short hairpin/short interference RNA directed against R2 mRNA. Stably transfected clones exhibited a pronounced reduction of the R2 protein with no change in the cellular growth rate. Furthermore, short interference RNA-mediated reduction of the R2 protein caused a marked increase in sensitivity to the DNA-damaging agent cisplatin as well as to the RNR inhibitors Triapine® and hydroxyurea. Ectopic expression of p53R2 partially reversed the cytotoxicity of cisplatin but not that of RNR inhibitors to R2 knockdown cells. The increase in sensitivity to cisplatin and RNR inhibitors was correlated with the suppression of dATP and dGTP levels caused by stable expression of R2-targeted short interference RNA. These results indicated that DNA damage resulted in elevated levels of the R2 protein and dNTPs and, consequently, enhanced the survival of p53(؊/؊) HCT-116 cells. The findings provide evidence that R2-RNR can be employed to supply dNTPs for the repair of DNA damage in cells with an impaired p53-dependent induction of p53R2. Ribonucleotide reductase (RNR)1 catalyzes a rate-limiting reaction in which ribonucleoside diphosphates are converted to their corresponding deoxyribonucleoside diphosphates, the precursors of deoxyribonucleoside triphosphates (dNTPs) required for DNA synthesis and repair (1). In mammalian cells, the catalytically active RNR is considered to be an ␣ 2 ␤ 2 heterotetramer consisting of two large R1 subunits and two small R2 subunits. To maintain a balanced size of dNTP pools (2), the enzymatic activity of RNR is tightly regulated by the binding of nucleoside triphosphates (ATP, dATP, dGTP, and dTTP) to allosteric sites in the R1 subunit (1). In proliferating cells, the level of the R2 protein is low in the G 1 phase of the cell cycle but accumulates and reaches maximal levels during the S phase, followed by degradation when passing through the G 2 /M phase (3-6). Thus, the activity of RNR is additionally controlled by cell cycle-specific availability of the R2 subunit, whereas the R1 protein level remains relatively constant throughout the cell cycle (3, 4). When cells undergo G 1 arrest following DNA damage, the supply of dNTPs for DNA repair is, in turn, provided by transcriptional activation of the recently identified R2 homologue, p53R2 (7, 8). Thus, DNA damage causes p53-depend...

Poly(ADP-ribose) polymerase (PARP) inhibitors exploit synthetic lethality to target epithelial ovarian cancer (EOC) with hereditary BRCA mutations and defects in homologous recombination repair (HRR). However, such an approach is limited to a small subset of EOC patients and compromised by restored HRR due to secondary mutations in BRCA genes. Here, it was demonstrated that triapine, a small molecule inhibitor of ribonucleotide reductase, enhances the sensitivity of BRCA wild-type EOC cells to the PARP inhibitor olaparib and the topoisomerase II inhibitor etoposide. Triapine abolishes olaparib-induced BRCA1 and Rad51 foci, and disrupts BRCA1 interaction with the Mre11-Rad50-Nbs1 (MRN) complex in BRCA1 wild-type EOC cells. It has been shown that phosphorylation of CtIP (RBBP8) is required for interaction with BRCA1 and with MRN to promote DNA double-strand break (DSB) resection during S- and G2-phases of the cell cycle. Mechanistic studies within reveal that triapine inhibits CDK activity and blocks olaparib-induced CtIP phosphorylation through Chk1 activation. Furthermore, triapine abrogates etoposide-induced CtIP phosphorylation and DSB resection as evidenced by marked attenuation of RPA32 phosphorylation. Concurrently, triapine obliterates etoposide-induced BRCA1 foci and sensitizes BRCA1 wild-type EOC cells to etoposide. Using a GFP-based HRR assay, it was determined that triapine suppresses HRR activity induced by an I-SceI-generated DSB. These results suggest that triapine augments the sensitivity of BRCA wild-type EOC cells to drug-induced DSBs by disrupting CtIP-mediated HRR.