The DNA damage response and repair (DDR/R) network, a sum of hierarchically structured signaling pathways that recognize and repair DNA damage, and the immune response to endogenous and/or exogenous threats, act synergistically to enhance cellular defense. On the other hand, a deregulated interplay between these systems underlines inflammatory diseases including malignancies and chronic systemic autoimmune diseases, such as systemic lupus erythematosus, systemic sclerosis, and rheumatoid arthritis. Patients with these diseases are characterized by aberrant immune response to self-antigens with widespread production of autoantibodies and multiple-tissue injury, as well as by the presence of increased oxidative stress. Recent data demonstrate accumulation of endogenous DNA damage in peripheral blood mononuclear cells from these patients, which is related to (a) augmented DNA damage formation, at least partly due to the induction of oxidative stress, and (b) epigenetically regulated functional abnormalities of fundamental DNA repair mechanisms. Because endogenous DNA damage accumulation has serious consequences for cellular health, including genomic instability and enhancement of an aberrant immune response, these results can be exploited for understanding pathogenesis and progression of systemic autoimmune diseases, as well as for the development of new treatments.

Background: Genomic instability promotes evolution and heterogeneity of tumors. Unraveling its mechanistic basis is essential for the design of appropriate therapeutic strategies. In a previous study, we reported an unexpected oncogenic property of p21^WAF1/Cip1, showing that its chronic expression in a p53-deficient environment causes genomic instability by deregulation of the replication licensing machinery. Results: We now demonstrate that p21^WAF1/Cip1 can further fuel genomic instability by suppressing the repair capacity of low- and high-fidelity pathways that deal with nucleotide abnormalities. Consequently, fewer single nucleotide substitutions (SNSs) occur, while formation of highly deleterious DNA double-strand breaks (DSBs) is enhanced, crafting a characteristic mutational signature landscape. Guided by the mutational signatures formed, we find that the DSBs are repaired by Rad52-dependent break-induced replication (BIR) and single-strand annealing (SSA) repair pathways. Conversely, the error-free synthesis-dependent strand annealing (SDSA) repair route is deficient. Surprisingly, Rad52 is activated transcriptionally in an E2F1-dependent manner, rather than post-translationally as is common for DNA repair factor activation. Conclusions: Our results signify the importance of mutational signatures as guides to disclose the repair history leading to genomic instability. We unveil how chronic p21^WAF1/Cip1 expression rewires the repair process and identifies Rad52 as a source of genomic instability and a candidate therapeutic target.

Background: Excessive autoantibody production characterizing systemic lupus erythematosus (SLE) occurs irrespective of the disease’s clinical status and is linked to increased lymphocyte apoptosis. Herein, we tested the hypothesis that defective DNA damage repair contributes to increased apoptosis in SLE. Methods: We evaluated nucleotide excision repair at the N-ras locus, DNA double-strand breaks repair and apoptosis rates in peripheral blood mononuclear cells from anti-dsDNA autoantibody-positive patients (six with quiescent disease and six with proliferative nephritis) and matched healthy controls following ex vivo treatment with melphalan. Chromatin organization and expression levels of DNA repair- and apoptosis-associated genes were also studied in quiescent SLE. Results: Defective nucleotide excision repair and DNA double-strand breaks repair were found in SLE, with lupus nephritis patients showing higher DNA damage levels than those with quiescent disease. Melphalan-induced apoptosis rates were higher in SLE than control cells and correlated inversely with DNA repair efficiency. Chromatin at the N-ras locus was more condensed in SLE than controls, while treatment with the histone deacetylase inhibitor vorinostat resulted in hyperacetylation of histone H4, chromatin decondensation, amelioration of DNA repair efficiency and decreased apoptosis. Accordingly, genes involved in DNA damage repair and signaling pathways, such as DDB1, ERCC2, XPA, XPC, MRE11A, RAD50, PARP1, MLH1, MLH3, and ATM were significantly underexpressed in SLE versus controls, whereas PPP1R15A, BARD1 and BBC3 genes implicated in apoptosis were significantly overexpressed. Conclusions: Epigenetically regulated functional abnormalities of DNA repair machinery occur in SLE, regardless of clinical disease activity, and may promote lymphocyte apoptosis. Approaches to correct these abnormalities may be of therapeutic value in SLE.

Ovarian carcinoma (OC) is the most lethal gynecological malignancy. Despite the advances in the treatment of OC with combinatorial regimens, including surgery and platinum-based chemotherapy, patients generally exhibit poor prognosis due to high chemotherapy resistance. Herein, we tested the hypothesis that DNA damage response (DDR) pathways are involved in resistance of OC patients to platinum chemotherapy. Selected DDR signals were evaluated in two human ovarian carcinoma cell lines, one sensitive (A2780) and one resistant (A2780/C30) to platinum treatment as well as in peripheral blood mononuclear cells (PBMCs) from OC patients, sensitive (n = 7) or resistant (n = 4) to subsequent chemotherapy. PBMCs from healthy volunteers (n = 9) were studied in parallel. DNA damage was evaluated by immunofluorescence γH2AX staining and comet assay. Higher levels of intrinsic DNA damage were found in A2780 than in A2780/C30 cells. Moreover, the intrinsic DNA damage levels were significantly higher in OC patients relative to healthy volunteers, as well as in platinum-sensitive patients relative to platinum-resistant ones (all P<0.05). Following carboplatin treatment, A2780 cells showed lower DNA repair efficiency than A2780/C30 cells. Also, following carboplatin treatment of PBMCs ex vivo, the DNA repair efficiency was significantly higher in healthy volunteers than in platinum-resistant patients and lowest in platinum-sensitive ones (t_1/2 for loss of γH2AX foci: 2.7±0.5h, 8.8±1.9h and 15.4±3.2h, respectively; using comet assay, t_1/2 of platinum-induced damage repair: 4.8±1.4h, 12.9±1.9h and 21.4±2.6h, respectively; all P<0.03). Additionally, the carboplatin-induced apoptosis rate was higher in A2780 than in A2780/C30 cells. In PBMCs, apoptosis rates were inversely correlated with DNA repair efficiencies of these cells, being significantly higher in platinum-sensitive than in platinum-resistant patients and lowest in healthy volunteers (all P<0.05). We conclude that perturbations of DNA repair pathways as measured in PBMCs from OC patients correlate with the drug sensitivity of these cells and reflect the individualized response to platinum-based chemotherapy.

DNA double-strand breaks (DSBs) lead to mutations, genomic instability and apoptotic death, whereas accumulation of apoptotic cells results in excessive autoantigen presentation and autoantibody formation. We aimed to measure DSB levels in lupus nephritis, a severe complication of the prototypic systemic autoimmune disease. The intrinsic DNA damage and the apoptosis induction/DSB levels were evaluated in peripheral blood mononuclear cells of six patients and 10 healthy controls following exposure to genotoxic agents (melphalan, cisplatin) ex vivo. DSBs were assessed using immunofluorescence quantification of γH2AX foci and comet assay. Intrinsic DNA damage was increased in lupus versus control cells in both assays (Olive Tail Moment units of 15.8 ± 2.3 versus 3.0 ± 1.4 in comet, p < 0.01; % γH2AX-positive cells: 13.6 ± 1.8 versus 4.6 ± 0.9, p < 0.01, respectively). Melphalan or cisplatin doses as low as 9.9 ± 4.8 or 29.8 ± 8.3 µg/ml, respectively, were sufficient to induce apoptosis in lupus cells; control cells required doses of 32.3 ± 7.7 and 67.7 ± 5.5 µg/ml, respectively. Drug-induced DSB levels were increased in lupus versus control cells, with the area under the curve (AUC) for melphalan-induced DSBs being 3050 ± 610 (% γH2AX-positive staining cells) × (drug dose) in patients and 1580 ± 350 in controls (p < 0.05); the corresponding values for cisplatin-induced AUC were 13900 ± 1800 for lupus and 4500 ± 750 for controls (p < 0.01). Interestingly, within either lupus patients or controls examined, the accumulation of DSBs correlated with apoptosis degrees (all p < 0.01). Results in lupus cells were not associated with individual disease activity level or treatment modalities at the time of the study. These findings suggest a novel mechanism by which increased accumulation of DSBs may render cells more sensitive to apoptosis, thus contributing to the induction of systemic autoimmunity.

We sought to determine whether DNA damage response (DDR)-related aberrations predict therapeutic benefit in cisplatin-treated head and neck squamous cell carcinoma (HNSCC) patients and how DDR pathways are modulated after treatment with olaparib alone or in combination with cisplatin or durvalumab. Oxidative stress, abasic sites and DDR-related parameters, including endogenous DNA damage, DNA repair mechanisms and apoptosis rates, were evaluated in HNSCC cell lines and peripheral blood mononuclear cells from 46 healthy controls (HC) and 70 HNSCC patients at baseline and following treatment with cisplatin-containing chemoradiation or nivolumab or enrolled in the OPHELIA phase II trial (NCT02882308; olaparib alone, olaparib plus cisplatin, olaparib plus durvalumab). HNSCC patients at diagnosis exhibited deregulated DDR-related parameters and higher levels of oxidative stress and abasic sites compared with HC (all P < 0.05). Accordingly, nucleotide excision repair (NER; ERCC1, ERCC2/XPD, XPA, XPC) and base excision repair (APEX1, XRCC1) genes were downregulated in patients versus HC whereas double-strand breaks repair (MRE11A, RAD50, RAD51, XRCC2) and mismatch repair (MLH1, MSH2, MSH3) genes were overexpressed. Corresponding results were obtained in cell lines (all P < 0.001). Excellent correlations were observed between individual ex vivo and in vivo/therapeutic results, with cisplatin non-responders showing higher levels of endogenous DNA damage, augmented oxidative stress and abasic sites, increased NER capacities and reduced apoptosis than responders (all P < 0.05). Also, longer progression-free survival correlated with lower NER capacity (P = 0.037) and increased apoptosis (P = 0.029). Interestingly, treatment with olaparib-containing regimens results in the accumulation of cytotoxic DNA damage and exerts an extra antitumor effect by elevating oxidative stress (all P < 0.05). Nivolumab induced no significant changes in the DDR parameters examined. Aberrations in DDR signals are implicated in the response to HNSCC chemotherapy and can be exploited as novel therapeutic targets, sensitive/effective non-invasive biomarkers as well as for the design of novel clinical trials.