Most chemotherapeutic agents for leukemia are DNA damaging agents. However, DNA lesions can be repaired by activities of DNA repair systems. Increasing evidence have shown that enhanced DNA damage repair capacity contributes to chemotherapy resistance in leukemia cells. Thus, targeting DNA repair mechanisms is a promising strategy for novel leukemia treatment. SIRT1 expressions were downregulated by lentivirus-delivered SIRT1 shRNA in myeloid leukemia cells. SIRT1 mRNA and protein levels were analyzed by real-time PCR and Western blot, respectively. Flow cytometry was carried out to analyze cell cycle progression, apoptosis and DNA damage repair efficiency. DNA damage levels were assessed by alkaline comet assay, and H2AX phosphorylation was analyzed by immunoblotting and immunofluorescence. A mouse leukemia model was established by transplanting lentivirus-infected K562 cells containing SIRT1 shRNA into sublethally irradiated NOD/SCID mice, and tumorigenesis was evaluated by detecting tumor weights and mice survival. SIRT1 expressions were upregulated in myeloid leukemic patients. Downregulation of SIRT1 by RNAi promoted etoposide-induced DNA damage in myeloid leukemia cells accompanied by reduced NHEJ activity, and increased Ku70 acetylation. Furthermore, SIRT1 knockdown resulted in cell cycle arrest, induction of apoptosis and reduction of K562 cell proliferation accompanied by enhanced p53 and FOXO1 acetylation in K562 cells after etoposide treatment. Importantly, SIRT1 downregulation reduced the tumorigenesis ability of K562 cells in mouse xenografts following chemotherapy treatment. These results revealed that SIRT1 promotes the NHEJ repair pathway by deacetylating Ku70 in K562 cells, suggesting that SIRT1 is a novel therapeutic target for treating myeloid leukemia.

A novel role for HSF1 as an inhibitor of non-homologous end joining (NHEJ) repair activity was identified. HSF1 interacted directly with both of the N-terminal sequences of the Ku70 and Ku86 proteins, which inhibited the endogenous heterodimeric interaction between Ku70 and Ku86. The blocking of the Ku70 and Ku86 interaction by HSF1 induced defective NHEJ repair activity and ultimately activated genomic instability after ionizing radiation (IR), which was similar to effects seen in Ku70 or Ku80 knockout cells. The binding activity between HSF1 and Ku70 or Ku86 was dependent on DNA damage response such as IR exposure, but not on the heat shock mediated transcriptional activation of HSF1. Moreover, the posttranslational modification such as phosphorylation, acetylation and sumoylation of HSF1 did not alter the binding activities of HSF1-Ku70 or HSF1-Ku86. Furthermore, the defect in DNA repair activity by HSF1 was observed regardless of p53 status. Rat mammary tumors derived using dimethylbenz(a)anthracence revealed that high levels of HSF1 expression which correlate with aggressive malignancy, interfered with the binding of Ku70-Ku80. This data suggests that HSF1 interacts with both Ku70 and Ku86 to induce defective NHEJ repair activity and genomic instability, which in turn suggests a novel mechanism of HSF1-mediated cellular carcinogenesis.

End processing at DNA double strand breaks (DSB) is a decisive step in repair pathway selection. Here, we investigated the role of 53BP1/RIF1 in limiting BRCA1/CtIP-mediated end resection to control DSB repair pathway choice. ATM orchestrates this process through 53BP1 phosphorylation to promote RIF1 recruitment. As cells enter S/G2-phase, end resection is activated, which displaces pATM from DSB sites and diminishes 53BP1 phosphorylation and RIF1 recruitment. Consistently, the kinetics of ATM and 53BP1 phosphorylation in S/G2-phase concur. We show that defective 53BP1/RIF1-mediated DSB end-protection in G1-phase stimulates CtIP/MRE11-dependent end-resection, which requires Polo-like kinase 3. This end resection activity in G1 was shown to produce only short tracks of ssDNA overhangs, as evidenced by the findings that in 53BP1 depleted cells, (i) RPA focus intensity was significantly lower in G1 compared to that in S/G2 phase, and (ii) EXO1 knockdown did not alter either number or intensity of RPA foci in G1 but significantly decreased the RPA focus intensity in S/G2 phase. Importantly, we report that the observed DSB end resection in G1 phase inhibits DNA-PK-dependent nonhomologous end joining but is not sufficient to stimulate HR. Instead, it switches the repair to the alternative PARP1-dependent end joining pathway.

In addition to genetic mutations, epigenetic revision plays a major role in the development and progression of cancer; specifically, inappropriate DNA methylation or demethylation of CpG residues may alter the expression of genes that promote tumorigenesis. We hypothesize that DNA repair, specifically the repair of DNA double strand breaks (DSB) by Non-Homologous End Joining (NHEJ) may play a role in this process. Using a GFP reporter system inserted into the genome of HeLa cells, we are able to induce targeted DNA damage that enables the cells, after successfully undergoing NHEJ repair, to express WT GFP. These GFP+ cells were segregated into two expression classes, one with robust expression (Bright) and the other with reduced expression (Dim). Using a DNA hypomethylating drug (AzadC) we demonstrated that the different GFP expression levels was due to differential methylation statuses of CpGs in regions on either side of the break site. Deep sequencing analysis of this area in sorted Bright and Dim populations revealed a collection of different epi-alleles that display patterns of DNA methylation following repair by NHEJ. These patterns differ between Bright and Dim cells which are hypo- and hypermethylated, respectively, and between the post-repair populations and the original, uncut cells. These data suggest that NHEJ repair facilitates a rewrite of the methylation landscape in repaired genes, elucidating a potential source for the altered methylation patterns seen in cancer cells, and understanding the mechanism by which this occurs could provide new therapeutic targets for preventing this process from contributing to tumorigenesis.

Acute myeloid leukemia (AML) with fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) relapses with new chromosome abnormalities following chemotherapy, implicating genomic instability. Error-prone alternative non-homologous end-joining (Alt-NHEJ) DNA double-strand break (DSB) repair is upregulated in FLT3-ITD-expresssing cells, driven by c-Myc. The serine/threonine kinase Pim-1 is upregulated downstream of FLT3-ITD, and inhibiting Pim increases topoisomerase 2 (TOP2) inhibitor chemotherapy drug induction of DNA DSBs and apoptosis. We hypothesized that Pim inhibition increases DNA DSBs by downregulating Alt-NHEJ, also decreasing genomic instability. Alt-NHEJ activity, measured with a green fluorescent reporter construct, increased in FLT3-ITD-transfected Ba/F3-ITD cells treated with TOP2 inhibitors, and this increase was abrogated by Pim kinase inhibitor AZD1208 co-treatment. TOP2 inhibitor and AZD1208 co-treatment downregulated cellular and nuclear expression of c-Myc and Alt-NHEJ repair pathway proteins DNA polymerase θ, DNA ligase 3 and XRCC1 in FLT3-ITD cell lines and AML patient blasts. ALT-NHEJ protein downregulation was preceded by c-Myc downregulation, inhibited by c-Myc overexpression and induced by c-Myc knockdown or inhibition. TOP2 inhibitor treatment increased chromosome breaks in metaphase spreads in FLT3-ITD-expressing cells, and AZD1208 co-treatment abrogated these increases. Thus Pim kinase inhibitor co-treatment both enhances TOP2 inhibitor cytotoxicity and decreases TOP2 inhibitor-induced genomic instability in cells with FLT3-ITD.

Mutations in genes encoding DNA double-strand break (DSB) repair components, especially homologous recombination (HR) proteins, were found to predispose to breast and ovarian cancer. Beyond high penetrance risk gene mutations underlying monogenic defects, low risk gene mutations generate polygenic defects, enlarging the fraction of individuals with a predisposing phenotype. DSB repair dysfunction opens new options for targeted therapies; poly (ADP-ribose) polymerase (PARP) inhibitors have been approved for BRCA-mutated and platinum-responsive ovarian cancers. In this work, we performed functional analyses in peripheral blood lymphocytes (PBLs) using a case-control design. We examined 38 women with familial history of breast and/or ovarian cancer, 40 women with primary ovarian cancer and 34 healthy controls. Using a GFP-based test we analyzed error-prone DSB repair mechanisms which are known to compensate for HR defects and to generate chromosomal instabilities. While non-homologous end-joining (NHEJ) did not discriminate between cases and controls, we found increases of single-strand annealing (SSA) in women with familial risk vs. controls (P=0.016) and patients with ovarian cancer vs. controls (P=0.002). Consistent with compromised HR we also detected increased sensitivities to carboplatin in PBLs from high-risk individuals (P<0.0001) as well as patients (P=0.0011) compared to controls. Conversely, neither PARP inhibitor responses nor PARP activities were altered in PBLs from the case groups, but PARP activities increased with age in high-risk individuals, providing novel clues for differential drug mode-of-action. Our findings indicate the great potential of detecting SSA activities to deliver an estimate of ovarian cancer susceptibility and therapeutic responsiveness beyond the limitations of genotyping.

Ovarian cancer is usually diagnosed at late stages when cancer has spread beyond the ovary and patients ultimately succumb to the development of drug-resistant disease. There is an urgent and unmet need to develop therapeutic strategies that effectively treat ovarian cancer and this requires a better understanding of signaling pathways important for ovarian cancer progression. Aurora A kinase (AURKA) plays an important role in ovarian cancer progression by mediating mitosis and chromosomal instability. In the current study, we investigated the role of AURKA in regulating the DNA damage response and DNA repair in ovarian carcinoma cells. We discovered that AURKA modulated the expression and activity of PARP, a crucial mediator of DNA repair that is a target of therapeutic interest for the treatment of ovarian and other cancers. Further, specific inhibition of AURKA activity with the small molecule inhibitor, alisertib, stimulated the non-homologous end-joining (NHEJ) repair pathway by elevating DNA-PKcs activity, a catalytic subunit required for double-strand break (DSB) repair, as well as decreased the expression of PARP and BRCA1/2, which are required for high-fidelity homologous recombination-based DNA repair. Further, AURKA inhibition stimulates error-prone NHEJ repair of DNA double-strand breaks with incompatible ends. Consistent with in vitro findings, alisertib treatment increased phosphorylated DNA-PKcs(pDNA-PKcsT2609) and decreased PARP levels in vivo. Collectively, these results reveal new non-mitotic functions for AURKA in the regulation of DNA repair, which may inform of new therapeutic targets and strategies for treating ovarian cancer.

Mutations in the breast cancer susceptibility 1 (BRCA1) gene are catalysts for breast and ovarian cancers. Most mutations are associated with the BRCA1 N- and C-terminal domains linked to DNA double-strand break (DSB) repair. However, little is known about the role of the intervening serine-glutamine (SQ) - cluster in the DNA damage response beyond its importance in regulating cell cycle checkpoints. We show that serine-to-alanine alterations at critical residues within the SQ-cluster known to be phosphorylated by ATM and ATR result in reduced homologous recombination repair (HRR) and aberrant mitosis. While a S1387A BRCA1 mutant - previously shown to abrogate S-phase arrest in response to radiation - resulted in only a modest decrease in HRR, S1387A together with an additional alteration, S1423A (BRCA12P), reduced HRR to vector control levels and similar to a quadruple mutant also including S1457A and S1524A (BRCA14P). These effects appeared to be independent of PALB2. Furthermore, we found that BRCA14P promoted a prolonged and struggling HRR late in the cell cycle and shifted DSB repair from HRR to non-homologous end joining which, in the face of irreparable chromosomal damage, resulted in mitotic catastrophe. Altogether, SQ-cluster phosphorylation is critical for allowing adequate time for completing normal HRR prior to mitosis and preventing cells from entering G1 prematurely resulting in gross chromosomal aberrations.

Alkylating agents are frequently used as first-line chemotherapeutics for various newly diagnosed cancers. Disruption of genome integrity by such agents can lead to cell lethality if DNA lesions are not removed. Several DNA repair mechanisms participate in the recovery of mono- or bi-functional DNA alkylation. Thus, DNA repair capacity is correlated with the therapeutic response. Here, we assessed the function of novel water-soluble N-mustard BO-1055 (ureidomustin) in DNA damage response and repair mechanisms. As expected, BO-1055 induces ATM and ATR-mediated DNA damage response cascades, including downstream Chk1/Chk2 phosphorylation, S/G2 cell-cycle arrest, and cell death. Further investigation revealed that cell survival sensitivity to BO-1055 is comparable to that of mitomycin C. Both compounds require nucleotide excision repair and homologous recombination, but not non-homologous end-joining, to repair conventional cross-linking DNA damage. Interestingly and unlike mitomycin C and melphalan, MGMT activity was also observed in BO-1055 damage repair systems, which reflects the occurrence of O-alkyl DNA lesions. Combined treatment with ATM/ATR kinase inhibitors significantly increases BO-1055 sensitivity. Our study pinpoints that BO-1055 can be used for treating tumors that with deficient NER, HR, and MGMT DNA repair genes, or for synergistic therapy in tumors that DNA damage response have been suppressed.

Radiotherapy is based on the induction of lethal DNA damage, primarily DNA double-strand breaks (DSB). Efficient DSB repair via Non-Homologous End Joining or Homologous Recombination can therefore undermine the efficacy of radiotherapy. By suppressing DNA-DSB repair with hyperthermia (HT) and DNA-PKcs inhibitor NU7441 (DNA-PKcsi), we aim to enhance the effect of radiation.The sensitizing effect of HT for 1 hour at 42°C and DNA-PKcsi [1 μM] to radiation treatment was investigated in cervical and breast cancer cells, primary breast cancer sphere cells (BCSCs) enriched for cancer stem cells, and in an in vivo human tumor model. A significant radio-enhancement effect was observed for all cell types when DNA-PKcsi and HT were applied separately, and when both were combined, HT and DNA-PKcsi enhanced radio-sensitivity to an even greater extent. Strikingly, combined treatment resulted in significantly lower survival rates, 2 to 2.5 fold increase in apoptosis, more residual DNA-DSB 6 h post treatment and a G2-phase arrest. In addition, tumor growth analysis in vivo showed significant reduction in tumor growth and elevated caspase-3 activity when radiation was combined with HT and DNA-PKcsi compared to radiation alone. Importantly, no toxic side effects of HT or DNA-PKcsi were found.In conclusion, inhibiting DNA-DSB repair using HT and DNA-PKcsi before radiotherapy leads to enhanced cytotoxicity in cancer cells. This effect was even noticed in the more radio-resistant BCSCs, which are clearly sensitized by combined treatment. Therefore, the addition of HT and DNA-PKcsi to conventional radiotherapy is promising and might contribute to more efficient tumor control and patient outcome.

DNA methyltransferase inhibitors (DNMTi) and histone deacetylase inhibitors (HDACi) are under investigation for the treatment of cancer, including the plasma cell malignancy multiple myeloma (MM). Evidence exists that DNA damage and repair contribute to the cytotoxicity mediated by the DNMTi decitabine. Here, we investigated the DNA damage response (DDR) induced by decitabine in MM using 4 human MM cell lines and the murine 5T33MM model. In addition, we explored how the HDACi JNJ-26481585 affects this DDR. Decitabine induced DNA damage (gamma-H2AX foci formation), followed by a G0/G1- or G2/M-phase arrest and caspase-mediated apoptosis. JNJ-26481585 enhanced the anti-MM effect of decitabine both in vitro and in vivo. As JNJ-26481585 did not enhance decitabine-mediated gamma-H2AX foci formation, we investigated the DNA repair response towards decitabine and/or JNJ-26481585. Decitabine augmented RAD51 foci formation (marker for homologous recombination (HR)) and/or 53BP1 foci formation (marker for non-homologous end joining (NHEJ)). Interestingly, JNJ-26481585 negatively affected basal or decitabine-induced RAD51 foci formation. Finally, B02 (RAD51 inhibitor) enhanced decitabine-mediated apoptosis. Together, we report that decitabine-induced DNA damage stimulates HR and/or NHEJ. JNJ-26481585 negatively affects RAD51 foci formation, thereby providing an additional explanation for the combinatory effect between decitabine and JNJ-26481585.

Strigolactones are a novel class of plant hormones produced in roots that regulate shoot and root development. We previously reported that strigolactone analogs (SLs) induce G2/M cell cycle arrest and apoptosis in a variety of human cancer cells and inhibit tumor growth of human breast cancer xenografts in mice. SLs had no significant influences on non-transformed cells. Here we report for the first time that SLs induce DNA damage in the form of DNA double-strand breaks (DSBs) and activate the DNA damage response signaling by inducing phosphorylation of ATM, ATR and DNA-PKcs and co-localization of the DNA damage signaling protein, 53BP1, with γH2AX nuclear foci. We further report that in addition to DSBs induction, SLs simultaneously impair DSBs repair, mostly homology-directed repair (HDR) and to a lesser extent non-homologous end joining (NHEJ). In response to SLs, RAD51, the homologous DSB repair protein, is ubiquitinated and targeted for proteasomal degradation and it fails to co-localize with γH2AX foci. Interestingly, SLs synergize with DNA damaging agents-based therapeutics. The combination of PARP inhibitors and SLs showed an especially potent synergy, but only in BRCA1-proficient cells. No synergy was observed between SLs and PARP inhibitors in BRCA1-deficient cells, supporting a role for SLs in HDR impairment. Together, our data suggest that SLs increase genome instability and cell death by a unique mechanism of inducing DNA damage and inhibiting DNA repair.

Glioblastoma multiforme (GBM) is the most aggressive and lethal type of brain tumor. Standard treatment for GBM patients is surgery followed by radiotherapy and/or chemotherapy, but tumors always recur. Traditional therapies seem to fail because they eliminate only the bulk of the tumors and spare a population of stem-like cells termed tumor-initiating cells. The stem-like state and preferential activation of DNA damage response in the GBM tumor-initiating cells contribute to their radio-resistance and recurrence. The molecular mechanisms underlying this efficient activation of damage response and maintenance of stem-like state remain elusive. Here we show that RBM14 controls DNA repair pathways and also prevents cell differentiation in GBM spheres, causing radio-resistance. Knockdown of RBM14 affects GBM sphere maintenance and sensitizes radio-resistant GBM cells at the cellular level. We demonstrate that RBM14 knockdown blocks GBM regrowth after irradiation in vivo. In addition, RBM14 stimulates DNA repair by controlling the DNA-PK-dependent non-homologous end-joining (NHEJ) pathway. These results reveal unexpected functions of the RNA-binding protein RBM14 in control of DNA repair and maintenance of tumor-initiating cells. Targeting the RBM14-dependent pathway may prevent recurrence of tumors and eradicate the deadly disease completely.

Acquisition of BCR-ABL mutations underlies drug resistance of chronic myeloid leukemia (CML) to tyrosine kinase inhibitors, but the molecular mechanisms of mutation acquisition are poorly understood. We previously showed that lysine deacetylase sirtuin 1, SIRT1, promotes acquisition of BCR-ABL mutations in association with enhancing KU70 mediated non-homologous end joining DNA repair. In this study, we demonstrate that lysine specific demethylase 1 (LSD1) plays an opposite role to SIRT1 in regulating DNA repair and mutation acquisition. In response to therapeutic stress and DNA damage, LSD1 and SIRT1 compete for binding to KU70 on DNA damage foci globally and on the ABL locus. The recruitment of SIRT1 or LSD1 to KU70 impacts chromatin structure but does not correlate well with their direct histone modification functions, and SIRT1 helps maintain histone H4K16 acetylation and open chromatin for repair. The competitive KU70 binding by these proteins affects cancer cells' ability to repair broken DNA and acquire resistant genetic mutations in CML and prostate cancer cells. We identify that the core domain of KU70 binds both LSD1 and SIRT1, forming a molecular basis for the competition. The C-terminal SAP motif of KU70 mediates LSD1/SIRT1 competitive interaction by suppressing LSD1 binding to KU70 and ectopic expression of SAP-deleted KU70 to CML cells compromises their ability to acquire BCR-ABL mutations. Our study reveals a novel cellular stress response mechanism in cancer cells and a key role of LSD1/SIRT1/KU70 dynamic interaction in regulating DNA repair and mutation acquisition.

The most frequent rearrangement of the human MLL gene fuses MLL to AF4 resulting in high-risk infant B-cell acute lymphoblastic leukemia (B-ALL). MLL fusions are also hallmark oncogenic events in secondary acute myeloid leukemia. They are a direct consequence of mis-repaired DNA double strand breaks (DNA-DSBs) due to defects in the DNA damage response associated with exposure to topoisomerase-II poisons such as etoposide. It has been suggested that MLL fusions render cells susceptible to additional chromosomal damage upon exposure to etoposide. Conversely, the genome-wide mutational landscape in MLL-rearranged infant B-ALL has been reported silent. Thus, whether MLL fusions compromise the recognition and/or repair of DNA damage remains unanswered. Here, the fusion proteins MLL-AF4 (MA4) and AF4-MLL (A4M) were CRISPR/Cas9-genome edited in the AAVS1 locus of HEK293 cells as a model to study MLL fusion-mediated DNA-DSB formation/repair. Repair kinetics of etoposide- and ionizing radiation-induced DSBs was identical in WT, MA4- and A4M-expressing cells, as revealed by flow cytometry, by immunoblot for γH2AX and by comet assay. Accordingly, no differences were observed between WT, MA4- and A4M-expressing cells in the presence of master proteins involved in non-homologous end-joining (NHEJ; i.e.KU86, KU70), alternative-NHEJ (Alt-NHEJ; i.e.LigIIIa, WRN and PARP1), and homologous recombination (HR, i.e.RAD51). Moreover, functional assays revealed identical NHEJ and HR efficiency irrespective of the genotype. Treatment with etoposide consistently induced cell cycle arrest in S/G2/M independent of MA4/A4M expression, revealing a proper activation of the DNA damage checkpoints. Collectively, expression of MA4 or A4M does neither influence DNA signaling nor DNA-DSB repair.

CC-115, a selective dual inhibitor of the mammalian target of rapamycin (mTOR) kinase and DNA-dependent protein kinase (DNA-PK), is undergoing Phase 1 clinical studies. Here we report the characterization of DNA-PK inhibitory activity of CC-115 in cancer cell lines. CC-115 inhibits auto-phosphorylation of the catalytic subunit of DNA-PK (DNA-PKcs) at the S2056 site (pDNA-PK S2056), leading to blockade of DNA-PK-mediated non-homologous end joining (NHEJ). CC-115 also indirectly reduces the phosphorylation of ataxia-telangiectasia mutated kinase (ATM) at S1981 and its substrates as well as homologous recombination (HR). The mTOR kinase and DNA-PK inhibitory activity of CC-115 leads to not only potent anti-tumor activity against a large panel of hematopoietic and solid cancer cell lines but also strong induction of apoptosis in a subset of cancer lines. Mechanistically, CC-115 prevents NHEJ by inhibiting the dissociation of DNA-PKcs, X-ray repair cross-complementing protein 4 (XRCC4), and DNA ligase IV from DNA ends. CC-115 inhibits colony formation of ATM-deficient cells more potently than ATM-proficient cells, indicating that inhibition of DNA-PK is synthetically lethal with the loss of functional ATM. In conclusion, CC-115 inhibits both mTOR signaling and NHEJ and HR by direct inhibition of DNA-PK. The mechanistic data not only provide selection of potential pharmacodynamic (PD) markers but also support CC-115 clinical development in patients with ATM-deficient tumors.

Proteomic analysis of ionomycin-treated and untreated mammary epithelial MCF10A cells elucidated differences in Ku80 cleavage. Ku80, a subunit of the Ku protein complex, is an initiator of the non-homologous, end-joining (NHEJ), double-strand breaks (DSBs) repair pathway. The nuclear Ku80 was cleaved in a calcium concentration-dependent manner by m-calpain but not by m-calpain. The cleavage of nuclear Ku80 at its α/β domain was validated by Western blotting analysis using flag-tagged expression vectors of truncated versions of Ku80 and a flag antibody and was confirmed in m-calpain knock-down cells and in vitro cell-free evaluation with recombinant proteins of calpains, Ku70, and Ku80. In addition, the cleaved Ku80 still formed a Ku heterodimer and promoted DNA DSB repair activity. Taken together, these findings indicate that translocated m-calpain enhances the NHEJ pathway through the cleavage of Ku80. Based on the present study, m-calpain in DNA repair pathways might be a novel anticancer drug target, or its mechanism might be a possible route for resistance acquisition of DNA damage-inducing chemotherapeutics.

Ku70-dependent canonical nonhomologous end-joining (c-NHEJ) DNA repair system is fundamental to the genome maintenance and B-cell lineage. c-NHEJ is upregulated and error-prone in incurable forms of chronic lymphocytic leukemia which also displays telomere dysfunction, multiple chromosomal aberrations and the resistance to DNA damage-induced apoptosis. We identify in these cells a novel DNA damage inducible form of phospho-Ku70. In vitro in different cancer cell lines, Ku70 phosphorylation occurs in a heterodimer Ku70/Ku80 complex within minutes of genotoxic stress, necessitating its interaction with DNA damage-induced kinase pS2056-DNA-PKcs and/or pS1981-ATM. The mutagenic effects of phospho-Ku70 are documented by a defective S/G2 checkpoint, accelerated disappearance of γ-H2AX foci and kinetics of DNA repair resulting in an increased level of genotoxic stress-induced chromosomal aberrations. Together, these data unveil an involvement of phospho-Ku70 in fast but inaccurate DNA repair; a new paradigm linked to both the deregulation of c-NHEJ and the resistance of malignant cells.

Patients with human papillomavirus-positive (HPV+) head and neck squamous cell carcinomas (HNSCCs) have increased response to radio- and chemotherapy and improved overall survival, possibly due to an impaired DNA damage response. Here, we investigated the correlation between HPV status and repair of DNA damage in HNSCC cell lines. We also assessed in vitro and in vivo sensitivity to the PARP inhibitor veliparib (ABT-888) in HNSCC cell lines and an HPV+ patient xenograft. Repair of DNA double strand breaks (DSBs) was significantly delayed in HPV+ compared to HPV- HNSCCs, resulting in persistence of γH2AX foci. Although DNA repair activators 53BP1 and BRCA1 were functional in all HNSCCs, HPV+ cells showed downstream defects in both non-homologous end joining and homologous recombination repair. Specifically, HPV+ cells were deficient in protein recruitment and protein expression of DNA-Pk and BRCA2, key factors for non-homologous end joining and homologous recombination respectively. Importantly, the apparent DNA repair defect in HPV+ HNSCCs was associated with increased sensitivity to the PARP inhibitor veliparib, resulting in decreased cell survival in vitro and a 10-14 day tumor growth delay in vivo. These results support the testing of PARP inhibition in combination with DNA damaging agents as a novel therapeutic strategy for HPV+ HNSCC.

Ionizing radiation (IR) induces highly cytotoxic double-strand breaks (DSBs) and also clustered oxidized bases in mammalian genomes. Base excision repair (BER) of bi-stranded oxidized bases could generate additional DSBs as repair intermediates in the vicinity of direct DSBs, leading to loss of DNA fragments. This could be avoided if DSB repair via DNA-PK-mediated nonhomologous end joining (NHEJ) precedes BER initiated by NEIL1 and other DNA glycosylases (DGs). Here we show that DNA-PK subunit Ku inhibits DGs via direct interaction. The scaffold attachment factor (SAF)-A, (also called hnRNP-U), phosphorylated at Ser59 by DNA-PK early after IR treatment, is linked to transient release of chromatin-bound NEIL1, thus preventing BER. SAF-A is subsequently dephosphorylated. Ku inhibition of DGs in vitro is relieved by unphosphorylated SAF-A, but not by the phosphomimetic Asp59 mutant. We thus propose that SAF-A, in concert with Ku, temporally regulates base damage repair in irradiated cell genome.