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BACKGROUND: Malignant tumours seriously threaten human life and health, and effective treatments for cancer are still being explored. The ability of SHC SH2 domain-binding protein 1 (SHCBP1) to induce cell cycle disturbance and inhibit tumour growth has been increasingly studied, but its dynamic role in the tumour cell cycle and corresponding effects leading to mitotic catastrophe and DNA damage have rarely been studied. RESULTS: In this paper, we found that the nucleoprotein SHCBP1 exhibits dynamic spatiotemporal expression during the tumour cell cycle, and SHCBP1 knockdown slowed cell cycle progression by inducing spindle disorder, as reflected by premature mitotic entry and multipolar spindle formation. This dysfunction was caused by G2/M checkpoint impairment mediated by downregulated WEE1 kinase and NEK7 (a member of the mammalian NIMA-related kinase family) expression and upregulated centromere/kinetochore protein Zeste White 10 (ZW10) expression. Moreover, both in vivo and in vitro experiments confirmed the significant inhibitory effects of SHCBP1 knockdown on tumour growth. Based on these findings, SHCBP1 knockdown in combination with low-dose DNA-damaging agents had synergistic tumouricidal effects on tumour cells. In response to this treatment, tumour cells were forced into the mitotic phase with considerable unrepaired DNA lesions, inducing mitotic catastrophe. These synergistic effects were attributed not only to the abrogation of the G2/M checkpoint and disrupted spindle function but also to the impairment of the DNA damage repair system, as demonstrated by mass spectrometry-based proteomic and western blotting analyses. Consistently, patients with low SHCBP1 expression in tumour tissue were more sensitive to radiotherapy. However, SHCBP1 knockdown combined with tubulin-toxic drugs weakened the killing effect of the drugs on tumour cells, which may guide the choice of chemotherapeutic agents in clinical practice. CONCLUSION: In summary, we elucidated the role of the nucleoprotein SHCBP1 in tumour cell cycle progression and described a novel mechanism by which SHCBP1 regulates tumour progression and through which targeting SHCBP1 increases sensitivity to DNA-damaging agent therapy, indicating its potential as a cancer treatment.

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Homologous recombination (HR) deficiency enhances sensitivity to DNA damaging agents commonly used to treat cancer. In HR-proficient cancers, metabolic mechanisms driving response or resistance to DNA damaging agents remain unclear. Here we identified that depletion of alpha-ketoglutarate (αKG) sensitizes HR-proficient cells to DNA damaging agents by metabolic regulation of histone acetylation. αKG is required for the activity of αKG-dependent dioxygenases (αKGDDs), and prior work has shown that changes in αKGDD affect demethylases. Using a targeted CRISPR knockout library consisting of 64 αKGDDs, we discovered that Trimethyllysine Hydroxylase Epsilon (TMLHE), the first and rate-limiting enzyme in de novo carnitine synthesis, is necessary for proliferation of HR-proficient cells in the presence of DNA damaging agents. Unexpectedly, αKG-mediated TMLHE-dependent carnitine synthesis was required for histone acetylation, while histone methylation was affected but dispensable. The increase in histone acetylation via αKG-dependent carnitine synthesis promoted HR-mediated DNA repair through site- and substrate-specific histone acetylation. These data demonstrate for the first time that HR-proficiency is mediated through αKG directly influencing histone acetylation via carnitine synthesis and provide a metabolic avenue to induce HR-deficiency and sensitivity to DNA damaging agents.

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Since therapy-induced senescence (TIS) can either support or inhibit cancer progression, identifying which types of chemotherapeutic agents can produce the strongest anti-tumor TIS is an important issue. Here, cyclin-dependent kinase4/6 inhibitors (CDK4/6i)-induced senescence was compared to the TIS induced by conventional DNA-damaging agents. Despite both types of agents eliciting a similar degree of senescence, we observed increased expression of the senescence-associated secretory phenotype (SASP) and ligands related to pro-tumor immunity (IL6, CXCL8, TGFß, CD274, and CEACAM1) and angiogenesis (VEGFA) mainly in TIS induced by DNA-damaging agents rather than by CDK4/6i. Additionally, although all agents increased the expression of anti-tumor immunomodulatory proteins related to antigen presentation (MHC-I, B2M) and T cell chemokines (CXCL9, 10, 11), CDK4/6i-induced senescent cells still maintained this expression at a similar or even higher intensity than cells treated with DNA-damaging agents, despite the absence of nuclear factor-kappa-B (NF-κB) and p53 activation. These data suggest that in contrast with DNA-damaging agents, which augment the pro-tumorigenic microenvironment via pro-inflammatory SASP, CDK4/6i can generate TIS only with antitumor immunomodulatory proteins.

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Cellular sensitivity is an approach to inhibit the growth of certain cells in response to any non-permissible conditions, as the presence of a cytotoxic agent or due to changes in growth parameters such as temperature, salt, or media components. Sensitivity tests are easy and informative assays to get insight into essential gene functions in various cellular processes. For example, cells having any functionally defective genes involved in DNA replication exhibit sensitivity to non-permissive temperatures and to chemical agents that block DNA replication fork movement. Here, we describe a sensitivity test for multiple strains of Saccharomyces cerevisiae and Candida albicans of diverged genetic backgrounds subjected to several genotoxic chemicals simultaneously. We demonstrate it by testing the sensitivity of DNA polymerase defective yeast mutants by using spot analysis combined with colony forming unit (CFU) efficiency estimation. The method is very simple and inexpensive, does not require any sophisticated equipment, can be completed in 2-3 days, and provides both qualitative and quantitative data. We also recommend the use of this reliable methodology for assaying the sensitivity of these and other fungal species to antifungal drugs and xenobiotic factors.

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Antibody‒drug conjugates (ADCs), which combine the advantages of monoclonal antibodies with precise targeting and payloads with efficient killing, show great clinical therapeutic value. The ADCs' payloads play a key role in determining the efficacy of ADC drugs and thus have attracted great attention in the field. An ideal ADC payload should possess sufficient toxicity, low immunogenicity, high stability, and modifiable functional groups. Common ADC payloads include tubulin inhibitors and DNA damaging agents, with tubulin inhibitors accounting for more than half of the ADC drugs in clinical development. However, due to clinical limitations of traditional ADC payloads, such as inadequate efficacy and the development of acquired drug resistance, novel highly efficient payloads with diverse targets and reduced side effects are being developed. This perspective summarizes the recent research advances of traditional and novel ADC payloads with main focuses on the structure-activity relationship studies, co-crystal structures, and designing strategies, and further discusses the future research directions of ADC payloads. This review also aims to provide valuable references and future directions for the development of novel ADC payloads that will have high efficacy, low toxicity, adequate stability, and abilities to overcome drug resistance.

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A colorimetric assay of DNA cleavage by bleomycin (BLM) derivatives was developed utilizing high colloidal stability on double-stranded (ds) DNA-modified gold nanoparticles (dsDNA-AuNPs) possessing a cleavage site. The assay was performed using dsDNA-AuNPs treated with inactive BLM or activated BLM (Fe(II)⋅BLM). A 10-min exposure in dsDNA-AuNPs with inactive BLM treatment resulted in a rapid color change from red to purple because of salt-induced non-crosslinking aggregation of dsDNA-AuNPs. In contrast, the addition of active Fe(II)⋅BLM retained the red color, probably because of the formation of protruding structures at the outermost phase of dsDNA-AuNPs caused by BLM-mediated DNA cleavage. Furthermore, the results of our model experiments indicate that oxidative base release and DNA-cleavage pathways could be visually distinguished with color change. The present methodology was also applicable to model screening assays using several drugs with different mechanisms related to antitumor activity. These results strongly suggest that this assay with a rapid color change could lead to simple and efficient screening of potent antitumor agents.

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Antibody‒drug conjugates (ADCs), which combine the advantages of monoclonal antibodies with precise targeting and payloads with efficient killing, show great clinical therapeutic value. The ADCs' payloads play a key role in determining the efficacy of ADC drugs and thus have attracted great attention in the field. An ideal ADC payload should possess sufficient toxicity, low immunogenicity, high stability, and modifiable functional groups. Common ADC payloads include tubulin inhibitors and DNA damaging agents, with tubulin inhibitors accounting for more than half of the ADC drugs in clinical development. However, due to clinical limitations of traditional ADC payloads, such as inadequate efficacy and the development of acquired drug resistance, novel highly efficient payloads with diverse targets and reduced side effects are being developed. This perspective summarizes the recent research advances of traditional and novel ADC payloads with main focuses on the structure-activity relationship studies, co-crystal structures, and designing strategies, and further discusses the future research directions of ADC payloads. This review also aims to provide valuable references and future directions for the development of novel ADC payloads that will have high efficacy, low toxicity, adequate stability, and abilities to overcome drug resistance.

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To maintain genome fidelity and prevent diseases such as cancer, our cells must constantly detect, and efficiently and precisely repair, DNA damage. Paradoxically, DNA-damaging agents in the form of radiation and chemotherapy are also used to treat cancer. Olivieri et al. used a CRISPR-based screen to identify genes that, when disrupted, lead to sensitivity or resistance to 27 different DNA-damaging agents used in the lab and/or in the clinic to treat cancer patients1. Their results reveal multiple new genes and connections that regulate these critical DNA damage repair pathways, with implications for basic and clinical research as well as cancer therapy.

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Platinum-based chemotherapy has been the cornerstone of systemic treatment in ovarian cancer. Since no validated molecular predictive markers have been identified yet, the response to platinum-based chemotherapy has been evaluated clinically, based on platinum-free interval. The new promising marker Schlafen 11 seems to correlate with sensitivity or resistance to DNA-damaging agents, including platinum compounds or PARP inhibitors in various types of cancer. We provide background information about the function of Schlafen 11, its evaluation in tumor tissue, and its prevalence in ovarian cancer. We discuss the current evidence of the correlation of Schlafen 11 expression in ovarian cancer with treatment outcomes and the potential use of Schlafen 11 as the key predictive and prognostic marker that could help to better stratify ovarian cancer patients treated with platinum-based chemotherapy or PARP inhibitors. We also provide perspectives on future directions in the research on this promising marker.

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BACKGROUND: Increased radioresistance due to previous irradiation or radiosensitivity due to human papilloma virus (HPV) infection can be observed in head and neck squamous cell carcinoma (HNSCC). The DNA-damage response of cells after exposure to DNA-damaging agents plays a crucial role in determining the fate of exposed cells. Tightly regulated and interconnected signaling networks are activated to detect, signal the presence of and repair the DNA damage. Novel therapies targeting the DNA-damage response are emerging; however, an improved understanding of the complex signaling networks involved in tumor radioresistance and radiosensitivity is needed. MATERIALS AND METHODS: In this study, we exposed isogenic human HNSCC cell lines with altered radiosensitivity to DNA-damaging agents: radiation, cisplatin and bleomycin. We investigated transcriptional alterations in the DNA-damage response by using a pathway-focused panel and reverse-transcription quantitative PCR. RESULTS: In general, the isogenic cell lines with altered radiosensitivity significantly differed from one another in the expression of genes involved in the DNA-damage response. The radiosensitive (HPV-positive) cells showed overall decreases in the expression levels of the studied genes. In parental cells, upregulation of DNA-damage signaling and repair genes was observed following exposure to DNA-damaging agents, especially radiation. In contrast, radioresistant cells exhibited a distinct pattern of gene downregulation after exposure to cisplatin, whereas the levels in parental cells were unchanged. Exposure of radioresistant cells to bleomycin did not significantly affect the expression of DNA-damage signaling and repair genes. CONCLUSIONS: Our analysis identified several possible targets: NBN, XRCC3, ATR, GADD45A and XPA. These putative targets should be studied and potentially exploited for sensibilization to ionizing radiation and/or cisplatin in HNSCC. The use of predesigned panels of DNA-damage signaling and repair genes proved to offer a convenient and quick approach to identify possible therapeutic targets.

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The emergence of drug resistance in pathogens leads to a loss of effectiveness of antimicrobials and complicates the treatment of bacterial infections. Quinoxaline 1,4-dioxides represent a prospective scaffold for search of new compounds with improved chemotherapeutic characteristics. Novel 2-acyl-3-trifluoromethylquinoxaline 1,4-dioxides with alteration of substituents at position 2 and 6 were synthesized via nucleophilic substitution with piperazine moiety and evaluated against a broad panel of bacteria and fungi by measuring their minimal inhibitory concentrations. Their mode of action was assessed by whole-genomic sequencing of spontaneous drug-resistant Mycobacterium smegmatis mutants, followed by comparative genomic analysis, and on an original pDualrep2 system. Most of the 2-acyl-3-trifluoromethylquinoxaline 1,4-dioxides showed high antibacterial properties against Gram-positive strains, including mycobacteria, and the introduction of a halogen atom in the position 6 of the quinoxaline ring further increased their activity, with 13c being the most active compound. The mode of action studies confirmed the DNA-damaging nature of the obtained quinoxaline 1,4-dioxides, while drug-resistance may be provided by mutations in redox homeostasis genes, encoding enzymes potentially involved in the activation of the compounds. This study extends views about the antimicrobial and antifungal activities of the quinoxaline 1,4-dioxides and can potentially lead to the discovery of new antibacterial drugs.

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DNA lesions arise from a combination of physiological/metabolic sources and exogenous environmental influences. When left unrepaired, these alterations accumulate in the cells and can give rise to mutations that change the function of important proteins (i.e. tumor suppressors, oncoproteins), or cause chromosomal rearrangements (i.e. gene fusions) that also result in the deregulation of key cellular molecules. Progressive acquisition of such genetic changes promotes uncontrolled cell proliferation and evasion of cell death, and hence plays a key role in carcinogenesis. Another less-studied consequence of DNA damage accumulating in the host genome is the integration of oncogenic DNA viruses such as Human papillomavirus, Merkel cell polyomavirus, and Hepatitis B virus. This critical step of viral-induced carcinogenesis is thought to be particularly facilitated by DNA breaks in both viral and host genomes. Therefore, the impact of DNA damage on carcinogenesis is magnified in the case of such oncoviruses via the additional effect of increasing integration frequency. In this review, we briefly present the various endogenous and exogenous factors that cause different types of DNA damage. Next, we discuss the contribution of these lesions in cancer development. Finally, we examine the amplified effect of DNA damage in viral-induced oncogenesis and summarize the limited data existing in the literature related to DNA damage-induced viral integration. To conclude, additional research is needed to assess the DNA damage pathways involved in the transition from viral infection to cancer. Discovering that a certain DNA damaging agent increases the likelihood of viral integration will enable the development of prophylactic and therapeutic strategies designed specifically to prevent such integration, with an ultimate goal of reducing or eliminating these viral-induced malignancies.

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OBJECTIVE: To investigate whether the inhibitions of ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) kinases by their specific inhibitors, KU-55933 and VE-821, respectively, are able to promote the cytotoxic activity of genotoxic agents including gemcitabine, 5-Fluorouracil, cisplatin and doxorubicin, in cholangiocarcinoma (CCA) and immortalized cholangiocyte cell lines. METHODS: Cell viability of cells treated with DNA damaging agents, alone and in combination with KU-55933 and VE-821, was determined by MTT assay. The changes of cell cycle distribution were evaluated by flow cytometry analysis. Colony formation was conducted to assess the effects of KU-55933 and VE-821 on cell proliferation. The levels of protein expression and phosphorylation were examined by western blot analysis. KEY FINDINGS: The cytotoxic effects of DNA damaging agents varied among CCA cell lines. Each DNA damaging drug induced different phases of the cell cycle in CCA cells. The combinations of both KU-55933 and VE-821 with DNA damaging agents promoted more cytotoxic activity than single inhibition in some CCA cell lines. ATM and ATR inhibitors decreased the effects of DNA damaging agent-induced ATM-Chk2 and ATR-Chk1 activations in CCA cells. CONCLUSIONS: Inhibitions of ATM and ATR potentiated the cytotoxic effects of DNA damaging agents in CCA cells, especially p53 defective HuCCA1 and RMCC1 cell lines.

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Zebrafish are widely used for detecting toxic agents because of their unique advantages. The conventional zebrafish-based tests use lethal rates and morphological changes as criteria to evaluate the toxicity. To increase the sensitivity of using zebrafish to detect toxic agents, a fluorescence resonance energy transfer-based apoptotic biosensor was introduced into zebrafish genome to generate transgenic sensor zebrafish. Seven chemicals including heavy metals, nanomaterials and DNA-damaging agents were used to treat the sensor zebrafish to determine the sensitivity of the sensor zebrafish. The results showed that sensor zebrafish can detect the toxicity of the tested agents with single-cell sensitivity. Using the sensor zebrafish, we found that, at 100 nM, heavy metal cadmium (Cd) induced apoptosis of zebrafish cells, while no obvious morphological or behavioral changes were observed from the sensor zebrafish. Even at 44.5 nM (the maximum allowable concentration in drinking water), Cd induced a significant increase of apoptosis in sensor zebrafish. ZnO nanoparticles caused apoptosis in sensor zebrafish at a very low concentration of 100 ng/mL. DNA-damaging agents induced the apoptosis of many cells in sensor zebrafish. The sensor zebrafish are much more sensitive than the conventional zebrafish-based tests and can serve as a powerful tool for detecting toxic agents.

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Cancer cells can evade immune recognition by losing major histocompatibility complex (MHC) class I. Hence, MHC class I-negative cancers represent the most challenging cancers to treat. Chemotherapeutic drugs not only directly kill tumors but also modulate the tumor immune microenvironment. However, it remains unknown whether chemotherapy-treated cancer cells can activate CD8 T cells independent of tumor-derived MHC class I and whether such MHC class I-independent CD8 T-cell activation can be exploited for cancer immunotherapy. Here, we showed that chemotherapy-treated cancer cells directly activated CD8 T cells in an MHC class I-independent manner and that these activated CD8 T cells exhibit virtual memory (VM) phenotypes. Consistently, in vivo chemotherapeutic treatment preferentially increased tumor-infiltrating VM CD8 T cells. Mechanistically, MHC class I-independent activation of CD8 T cells requires cell-cell contact and activation of the PI3K pathway. VM CD8 T cells contribute to a superior therapeutic effect on MHC class I-deficient tumors. Using humanized mouse models or primary human CD8 T cells, we also demonstrated that chemotherapy-treated human lymphomas activated VM CD8 T cells independent of tumor-derived MHC class I. In conclusion, CD8 T cells can be directly activated in an MHC class I-independent manner by chemotherapy-treated cancers, and these activated CD8 T cells may be exploited for developing new strategies to treat MHC class I-deficient cancers.

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Basal-like breast cancer is an incurable disease with limited therapeutic options, mainly due to the frequent development of anti-cancer drug resistance. Therefore, identification of druggable targets to improve current therapies and overcome these resistances is a major goal. Targeting DNA repair mechanisms has reached the clinical setting and several strategies, like the inhibition of the CHK1 kinase, are currently in clinical development. Here, using a panel of basal-like cancer cell lines, we explored the synergistic interactions of CHK1 inhibitors (rabusertib and SAR020106) with approved therapies in breast cancer and evaluated their potential to overcome resistance. We identified a synergistic action of these inhibitors with agents that produce DNA damage, like platinum compounds, gemcitabine, and the PARP inhibitor olaparib. Our results demonstrated that the combination of rabusertib with these chemotherapies also has a synergistic impact on tumor initiation, invasion capabilities, and apoptosis in vitro. We also revealed a biochemical effect on DNA damage and caspase-dependent apoptosis pathways through the phosphorylation of H2AX, the degradation of full-length PARP, and the increase of caspases 3 and 8 activity. This agent also demonstrated synergistic activity in a platinum-resistant cell line, inducing an increase in cell death in response to cisplatin only when combined with rabusertib, while no toxic effect was found on non-tumorigenic breast tissue-derived cell lines. Lastly, the combination of CHK1 inhibitor with cisplatin and gemcitabine resulted in more activity than single or double combinations, leading to a higher apoptotic effect. In conclusion, in our study we identify therapeutic options for the clinical development of CHK1 inhibitors, and confirm that the inhibition of this kinase can overcome acquired resistance to cisplatin.

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Exogenous and endogenous damage to the DNA is inevitable. Several DNA repair pathways including base excision, nucleotide excision, mismatch, homologous and non-homologous recombinations are conserved across all organisms to faithfully maintain the integrity of the genome. The base excision repair (BER) pathway functions to repair single-base DNA lesions and during the process creates the premutagenic apurinic/apyrimidinic (AP) sites. In this review, we discuss the components of the BER pathway in the nematode Caenorhabditis elegans and delineate the different phenotypes caused by the deletion or the knockdown of the respective DNA repair gene, as well as the implications. To date, two DNA glycosylases have been identified in C. elegans, the monofunctional uracil DNA glycosylase-1 (UNG-1) and the bifunctional endonuclease III-1 (NTH-1) with associated AP lyase activity. In addition, the animal possesses two AP endonucleases belonging to the exonuclease-3 and endonuclease IV families and in C. elegans these enzymes are called EXO-3 and APN-1, respectively. In mammalian cells, the DNA polymerase, Pol beta, that is required to reinsert the correct bases for DNA repair synthesis is not found in the genome of C. elegans and the evidence indicates that this role could be substituted by DNA polymerase theta (POLQ), which is known to perform a function in the microhomology-mediated end-joining pathway in human cells. The phenotypes observed by the C. elegans mutant strains of the BER pathway raised many challenging questions including the possibility that the DNA glycosylases may have broader functional roles, as discuss in this review.

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BACKGROUND: The prognosis of gastric cancer (GC) is poor with a median overall survival (OS) of less than 12 months in advanced-stage disease. The search for distinct genetic subgroups of GC patients and predictive biomarkers is ongoing. While BRCA1 or BRCA2 germline mutations (gBRCAm) have potential therapeutic implications in ovarian, breast and pancreatic cancers, their significance in GC patients has not been established. PATIENTS AND METHODS: A retrospective multi-center data analysis of GC patients with gBRCAm was conducted, detailing the clinical characteristics and disease course in this unique subset of patients. RESULTS: Ten GC patients with gBRCAm were identified, six of them with metastatic disease. The median OS of all ten GC patients was 47.5 (13-192) months. Median OS for patients diagnosed with operable disease was 55.5 (13-192) months and of the patients with metastatic disease (calculated from metastatic disease diagnosis) 32 (15-52) months with an exceptional 1-, 2- and 3-year survival rate of 100%, 83.3% and 50%, respectively. CONCLUSION: These preliminary data suggest that gBRCAm in GC patients are associated with a favorable prognosis. Furthermore, gBRCAm might be a predictive biomarker to DNA-damaging agents response in GC patients, similarly to its established role in other malignancies. Further research is needed to confirm our findings.

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Epithelial ovarian cancer (EOC) is very sensitive to upfront chemotherapy. This condition is attributable to defects in the DNA damage repair system. Agents that damage DNA are the main drugs used for its treatment. Many EOC cells have DNA repair deficiencies that confer susceptibility to these agents. Platinum is the most important agent for first-line and also for relapses, together with other drugs that can be given as monotherapy or along with platinum or other drugs. Lately, the emerging role of PARP inhibitors has changed the landscape of opportunities for patients with EOC. All these strategies will be reviewed in this article.

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The DNA damage response (DDR) is necessary to maintain genome integrity and prevent the accumulation of oncogenic mutations. Consequently, proteins involved in the DDR often serve as tumor suppressors, carrying out the crucial task of keeping DNA fidelity intact. Mediator of DNA damage checkpoint 1 (MDC1) is a scaffold protein involved in the early steps of the DDR. MDC1 interacts directly with γ-H2AX, the phosphorylated form of H2AX, a commonly used marker for DNA damage. It then propagates the phosphorylation of H2AX by recruiting ATM kinase. While the function of MDC1 in the DDR has been reviewed previously, its role in cancer has not been reviewed, and numerous studies have recently identified a link between MDC1 and carcinogenesis. This includes MDC1 functioning as a tumor suppressor, with its loss serving as a biomarker for cancer and contributor to drug sensitivity. Studies also indicate that MDC1 operates outside of its traditional role in DDR, and functions as a co-regulator of nuclear receptor transcriptional activity, and that mutations in MDC1 are present in tumors and can also cause germline predisposition to cancer. This review will discuss reports that link MDC1 to cancer and identify MDC1 as an important player in tumor formation, progression, and treatment. We also discuss mechanisms by which MDC1 levels are regulated and how this contributes to tumor formation.

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