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In recent years, various poly(ADP-ribose) polymerase (PARP) inhibitors (PARPis) have been approved for the treatment of several cancers to target the vulnerability of homologous recombination (HR) deficiency (e.g., due to BRCA1/2 dysfunction). In this review we analyze the ongoing debates and recent breakthroughs in the use of PARPis for BRCA1/2-deficient cancers, juxtaposing the 'double-strand break (DSB)' and 'single-stranded DNA (ssDNA) gap' models of synthetic lethality induced by PARPis. We spotlight the complexity of this interaction, highlighting emerging research on the role of DNA polymerase theta (POLθ) and ssDNA gaps in shaping therapy responses. We scrutinize the clinical ramifications of these findings, especially concerning PARPi efficacy and resistance mechanisms, underscoring the heterogeneity of BRCA-mutated tumors and the urgent need for advanced research to bridge the gap between laboratory models and patient outcomes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Currently, poly (ADP-ribose) polymerase inhibitors (PARPi) have been approved by U.S. Food and Drug Administration for BRCA-mutated pancreatic cancer therapy. However, limited indications hinder their further application. Repression of bromodomain-containing protein 4 (BRD4) can block the homologous recombination (HR) repair pathway and has the potential to enhance the response to PARPi in HR-proficient pancreatic cancer therapy. In addition, proteolysis targeting chimeras (PROTACs) can hijack E3 ligase within the cell to ubiquitinate degradation-targeted proteins effectively and quickly, thus enhancing the therapeutic effect on tumors. In the present study, the LB23 compound, which induces PARP1 degradation, was employed in combination with the BRD4 inhibitor JQ1, confirming their synergistic effect in HR-proficient pancreatic cancer through various methods. Moreover, compared to the JQ1 and PARPi olaparib combination, PARP1-PROTAC and JQ1 had more notable synergistic effects. Further research into the synergistic mechanism demonstrated that combination therapy enhanced DNA damage and suppressed DNA repair by inducing cell cycle arrest and cell apoptosis. The present study therefore provides the experimental data for this type of combination therapy, which is expected to be an innovative approach for the treatment of HR-proficient pancreatic cancer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Platinum-based chemotherapy causes genetic damage and induces apoptosis in ovarian cancer cells. Enhancing the ability to resist platinum drug-induced DNA damage and apoptotic stress is critical for tumor cells to acquire drug resistance. Here, we found that Y-box binding protein 1 (YBX1) was highly expressed in cisplatin-resistant patient-derived organoids (PDOs) and was a crucial gene for alleviating platinum-induced stress and maintaining drug resistance characteristics in ovarian cancer cells. Mechanistically, YBX1 recognized m5C modifications in CHD3 mRNA and maintained mRNA stability by recruiting PABPC1 protein. This regulatory process enhanced chromatin accessibility and improved the efficiency of homologous recombination (HR) repair, facilitating tumor cells to withstand platinum-induced apoptotic stress. In addition, SU056, an inhibitor of YBX1, exhibited the potential to reverse platinum resistance in subcutaneous and PDO orthotopic xenograft models. In conclusion, YBX1 is critical for ovarian cancer cells to alleviate the platinum-induced stress and may be a potential target for reversing drug-resistant therapies., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Wenjun Cheng reports financial support was provided by National Natural Science Foundation of China. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Poly(ADP-ribose) polymerase inhibitors (PARPis) exhibit remarkable anticancer activity in tumors with homologous recombination (HR) gene mutations. However, the role of other DNA repair proteins in PARPi-induced lethality remains elusive. Here, we reveal that FANCM promotes PARPi resistance independent of the core Fanconi anemia (FA) complex. FANCM-depleted cells retain HR proficiency, acting independently of BRCA1 in response to PARPis. FANCM depletion leads to increased DNA damage in the second S phase after PARPi exposure, driven by elevated single-strand DNA (ssDNA) gap formation behind replication forks in the first S phase. These gaps arise from both 53BP1- and primase and DNA directed polymerase (PRIMPOL)-dependent mechanisms. Notably, FANCM-depleted cells also exhibit reduced resection of collapsed forks, while 53BP1 deletion restores resection and mitigates PARPi sensitivity. Our results suggest that FANCM counteracts 53BP1 to repair PARPi-induced DNA damage. Furthermore, FANCM depletion leads to increased chromatin bridges and micronuclei formation after PARPi treatment, elucidating the mechanism underlying extensive cell death in FANCM-depleted cells., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The clinical success of PARP1/2 inhibitors (PARPi) prompts the expansion of their applicability beyond homologous recombination deficiency. Here, we demonstrate that the loss of the accessory subunits of DNA polymerase epsilon, POLE3 and POLE4, sensitizes cells to PARPi. We show that the sensitivity of POLE4 knockouts is not due to compromised response to DNA damage or homologous recombination deficiency. Instead, POLE4 loss affects replication speed leading to the accumulation of single-stranded DNA gaps behind replication forks upon PARPi treatment, due to impaired post-replicative repair. POLE4 knockouts elicit elevated replication stress signaling involving ATR and DNA-PK. We find POLE4 to act parallel to BRCA1 in inducing sensitivity to PARPi and counteracts acquired resistance associated with restoration of homologous recombination. Altogether, our findings establish POLE4 as a promising target to improve PARPi driven therapies and hamper acquired PARPi resistance., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: DNA repair allows the survival of cancer cells. Therefore, the development of DNA repair inhibitors is a critical need for sensitizing cancers to chemoradiation. Sae2 CtIP has specific functions in initiating DNA end resection, as well as coordinating cell cycle checkpoints, and it also greatly interacts with the DDR at different levels., Results: In this study, we demonstrated that corylin, a potential sensitizer, causes deficiencies in DNA repair and DNA damage checkpoints in yeast cells. More specifically, corylin increases DNA damage sensitivity through the Sae2-dependent pathway and impairs the activation of Mec1-Ddc2, Rad53-p and γ-H2A. In breast cancer cells, corylin increases apoptosis and reduces proliferation following Dox treatment by inhibiting CtIP. Xenograft assays showed that treatment with corylin combined with Dox significantly reduced tumor growth in vivo., Conclusions: Our findings herein delineate the mechanisms of action of corylin in regulating DNA repair and indicate that corylin has potential long-term clinical utility as a DDR inhibitor., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Homologous recombination (HR)-related gene alterations are present in a significant subset of prostate, breast, ovarian, pancreatic, lung, and colon cancers rendering these tumors as potential responders to specific DNA damaging agents. A small molecule acylfulvene prodrug, LP-184, metabolizes to an active compound by the oxidoreductase activity of enzyme prostaglandin reductase 1 (PTGR1), which is frequently elevated in multiple solid tumor types. Prior work demonstrated that cancer cell lines deficient in a spectrum of DNA damage repair (DDR) pathway genes show increased susceptibility to LP-184. Here, we investigated the potential of LP-184 in targeting multiple tumors with impaired HR function and its mechanism of action as a DNA damaging agent. LP-184 induced elevated DNA double-strand breaks in HR deficient (HRD) cancer cells. Depletion of key HR components BRCA2 or ataxia telangiectasia mutated (ATM) in cancer cells conferred up to 12-fold increased sensitivity to the LP-184. LP-184 showed nanomolar potency in a diverse range of HRD cancer models, including prostate cancer organoids, leiomyosarcoma cell lines, and patient-derived tumor graft models of lung, pancreatic, and prostate cancers. LP-184 demonstrated complete, durable tumor regression in 10 patient-derived xenograft (PDX) models of HRD triple-negative breast cancer (TNBC) including those resistant to PARP inhibitors (PARPi). LP-184 further displayed strong synergy with PARPi in ovarian and prostate cancer cell lines as well as in TNBC PDX models. These preclinical findings illustrate the potential of LP-184 as a pan-HRD cancer therapeutic. Taken together, our results support continued clinical evaluation of LP-184 in a large subset of HRD solid tumors., Significance: New agents with activity against DDR-deficient solid tumors refractory to standard-of-care therapies are needed. We report multiple findings supporting the potential for LP-184, a novel alkylating agent with three FDA orphan drug designations, to fill this void clinically: strong nanomolar potency; sustained, durable regression of solid tumor xenografts; synthetic lethality with HR defects. LP-184 adult phase IA trial to assess safety in advanced solid tumors is ongoing., (© 2024 The Authors; Published by the American Association for Cancer Research.)

Acetaldehyde, a chemical that can cause DNA damage and contribute to cancer, is prevalently present in our environment, e.g. in alcohol, tobacco, and food. Although aldehyde potentially promotes crosslinking reactions among biological substances including DNA, RNA, and protein, it remains unclear what types of DNA damage are caused by acetaldehyde and how they are repaired. In this study, we explored mechanisms involved in the repair of acetaldehyde-induced DNA damage by examining the cellular sensitivity to acetaldehyde in the collection of human TK6 mutant deficient in each genome maintenance system. Among the mutants, mismatch repair mutants did not show hypersensitivity to acetaldehyde, while mutants deficient in base and nucleotide excision repair pathways or homologous recombination (HR) exhibited higher sensitivity to acetaldehyde than did wild-type cells. We found that acetaldehyde-induced RAD51 foci representing HR intermediates were prolonged in HR-deficient cells. These results indicate a pivotal role of HR in the repair of acetaldehyde-induced DNA damage. These results suggest that acetaldehyde causes complex DNA damages that require various types of repair pathways. Mutants deficient in the removal of protein adducts from DNA ends such as TDP1 -/- and TDP2 -/- cells exhibited hypersensitivity to acetaldehyde. Strikingly, the double mutant deficient in both TDP1 and RAD54 showed similar sensitivity to each single mutant. This epistatic relationship between TDP1 -/- and RAD54 -/- suggests that the protein-DNA adducts generated by acetaldehyde need to be removed for efficient repair by HR. Our study would help understand the molecular mechanism of the genotoxic and mutagenic effects of acetaldehyde.

Chromosomal instability (CIN) results in the accumulation of large-scale losses, gains and rearrangements of DNA 1 . The broad genomic complexity caused by CIN is a hallmark of cancer 2 ; however, there is no systematic framework to measure different types of CIN and their effect on clinical phenotypes pan-cancer. Here we evaluate the extent, diversity and origin of CIN across 7,880 tumours representing 33 cancer types. We present a compendium of 17 copy number signatures that characterize specific types of CIN, with putative aetiologies supported by multiple independent data sources. The signatures predict drug response and identify new drug targets. Our framework refines the understanding of impaired homologous recombination, which is one of the most therapeutically targetable types of CIN. Our results illuminate a fundamental structure underlying genomic complexity in human cancers and provide a resource to guide future CIN research., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Three-methyl cytosine (3meC) are toxic DNA lesions, blocking base pairing. Bacteria and humans express members of the AlkB enzymes family, which directly remove 3meC. However, other organisms, including budding yeast, lack this class of enzymes. It remains an unanswered evolutionary question as to how yeast repairs 3meC, particularly in single-stranded DNA. The yeast Shu complex, a conserved homologous recombination factor, aids in preventing replication-associated mutagenesis from DNA base damaging agents such as methyl methanesulfonate (MMS). We found that MMS-treated Shu complex-deficient cells exhibit a genome-wide increase in A:T and G:C substitutions mutations. The G:C substitutions displayed transcriptional and replicational asymmetries consistent with mutations resulting from 3meC. Ectopic expression of a human AlkB homolog in Shu-deficient yeast rescues MMS-induced growth defects and increased mutagenesis. Thus, our work identifies a novel homologous recombination-based mechanism mediated by the Shu complex for coping with alkylation adducts., Competing Interests: BB, AB, SH, KR, DM, CP, AF, TL, RR, RV, TM, HZ, NM, EM, PM, SR, KB No competing interests declared, (© 2021, Bonilla et al.)

The processing of DNA double-strand breaks (DSBs) depends on the dynamic characteristics of chromatin. To investigate how abrupt changes in chromatin compaction alter these dynamics and affect DSB processing and repair, we exposed irradiated cells to hypotonic stress (HypoS). Densitometric and chromosome-length analyses show that HypoS transiently decompacts chromatin without inducing histone modifications known from regulated local chromatin decondensation, or changes in Micrococcal Nuclease (MNase) sensitivity. HypoS leaves undisturbed initial stages of DNA-damage-response (DDR), such as radiation-induced ATM activation and H2AX-phosphorylation. However, detection of ATM-pS1981, γ-H2AX and 53BP1 foci is reduced in a protein, cell cycle phase and cell line dependent manner; likely secondary to chromatin decompaction that disrupts the focal organization of DDR proteins. While HypoS only exerts small effects on classical nonhomologous end-joining (c-NHEJ) and alternative end-joining (alt-EJ), it markedly suppresses homologous recombination (HR) without affecting DNA end-resection at DSBs, and clearly enhances single-strand annealing (SSA). These shifts in pathway engagement are accompanied by decreases in HR-dependent chromatid-break repair in the G 2 -phase, and by increases in alt-EJ and SSA-dependent chromosomal translocations. Consequently, HypoS sensitizes cells to ionizing radiation (IR)-induced killing. We conclude that HypoS-induced global chromatin decompaction compromises regulated chromatin dynamics and genomic stability by suppressing DSB-processing by HR, and allowing error-prone processing by alt-EJ and SSA.

Molecules involved in DNA damage response (DDR) are often overexpressed in cancer cells, resulting in poor responses to chemotherapy and radiotherapy. Although treatment efficacy can be improved with the concomitant use of DNA repair inhibitors, the accompanying side effects can compromise the quality of life of patients. Therefore, in this study, we identified a natural compound that could inhibit DDR, using the single-strand annealing yeast-cell analysis system, and explored its mechanisms of action and potential as a chemotherapy adjuvant in hepatocellular carcinoma (HCC) cell lines using comet assay, flow cytometry, Western blotting, immunofluorescence staining, and functional analyses. We developed a mouse model to verify the in vitro findings. We found that hydroxygenkwanin (HGK) inhibited the expression of RAD51 and progression of homologous recombination, thereby suppressing the ability of the HCC cell lines to repair DNA damage and enhancing their sensitivity to doxorubicin. HGK inhibited the phosphorylation of DNA damage checkpoint proteins, leading to apoptosis in the HCC cell lines. In the mouse xenograft model, HGK enhanced the sensitivity of liver cancer cells to doxorubicin without any physiological toxicity. Thus, HGK can inhibit DDR in liver cancer cells and mouse models, making it suitable for use as a chemotherapy adjuvant.

Mutations in BRCA1 or BRCA2 (BRCA) is synthetic lethal with poly(ADP-ribose) polymerase inhibitors (PARPi). Lethality is thought to derive from DNA double-stranded breaks (DSBs) necessitating BRCA function in homologous recombination (HR) and/or fork protection (FP). Here, we report instead that toxicity derives from replication gaps. BRCA1- or FANCJ-deficient cells, with common repair defects but distinct PARPi responses, reveal gaps as a distinguishing factor. We further uncouple HR, FP, and fork speed from PARPi response. Instead, gaps characterize BRCA-deficient cells, are diminished upon resistance, restored upon resensitization, and, when exposed, augment PARPi toxicity. Unchallenged BRCA1-deficient cells have elevated poly(ADP-ribose) and chromatin-associated PARP1, but aberrantly low XRCC1 consistent with defects in backup Okazaki fragment processing (OFP). 53BP1 loss resuscitates OFP by restoring XRCC1-LIG3 that suppresses the sensitivity of BRCA1-deficient cells to drugs targeting OFP or generating gaps. We highlight gaps as a determinant of PARPi toxicity changing the paradigm for synthetic lethal interactions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

DNA double-strand breaks (DSBs) are repaired mainly by non-homologous end joining (NHEJ) or homologous recombination (HR). RIF1 negatively regulates resection through the effector Shieldin, which associates with a short 3' single-stranded DNA (ssDNA) overhang by the MRN (MRE11-RAD50-NBS1) complex, to prevent further resection and HR repair. In this study, we show that RIF1, but not Shieldin, inhibits the accumulation of CtIP at DSB sites immediately after damage, suggesting that RIF1 has another effector besides Shieldin. We find that protein phosphatase 1 (PP1), a known RIF1 effector in replication, localizes at damage sites dependent on RIF1, where it suppresses downstream CtIP accumulation and limits the resection by the MRN complex. PP1 therefore acts as a RIF1 effector distinct from Shieldin. Furthermore, PP1 deficiency in the context of Shieldin depletion elevates HR immediately after irradiation. We conclude that PP1 inhibits resection before the action of Shieldin to prevent precocious HR in the early phase of the damage response., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Defects in the DNA damage response (DDR) can lead to genome instability, producing mutations or aberrations that promote the development and progression of cancer. But it also confers such cells vulnerable to cell death when they inhibit DNA damage repair. Poly (ADP-ribose) polymerase (PARP) plays a central role in many cellular processes, including DNA repair, replication, and transcription. PARP induces the occurrence of poly (ADP-ribosylation) (PARylation) when DNA single strand breaks (SSB) occur. PARP and various proteins can interact directly or indirectly through PARylation to regulate DNA repair. Inhibitors that directly target PARP have been found to block the SSB repair pathway, triggering homologous recombination deficiency (HRD) cancers to form synthetic lethal concepts that represent an anticancer strategy. It has therefore been investigated in many cancer types for more effective anti-cancer strategies, including gastric cancer (GC). This review describes the antitumor mechanisms of PARP inhibitors (PARPis), and the preclinical and clinical progress of PARPis as monotherapy and combination therapy in GC.

To identify approaches to target DNA repair vulnerabilities in cancer, we discovered nanomolar potent, selective, low molecular weight (MW), allosteric inhibitors of the polymerase function of DNA polymerase Polθ, including ART558. ART558 inhibits the major Polθ-mediated DNA repair process, Theta-Mediated End Joining, without targeting Non-Homologous End Joining. In addition, ART558 elicits DNA damage and synthetic lethality in BRCA1- or BRCA2-mutant tumour cells and enhances the effects of a PARP inhibitor. Genetic perturbation screening revealed that defects in the 53BP1/Shieldin complex, which cause PARP inhibitor resistance, result in in vitro and in vivo sensitivity to small molecule Polθ polymerase inhibitors. Mechanistically, ART558 increases biomarkers of single-stranded DNA and synthetic lethality in 53BP1-defective cells whilst the inhibition of DNA nucleases that promote end-resection reversed these effects, implicating these in the synthetic lethal mechanism-of-action. Taken together, these observations describe a drug class that elicits BRCA-gene synthetic lethality and PARP inhibitor synergy, as well as targeting a biomarker-defined mechanism of PARPi-resistance.

Targeting DNA repair proteins with small-molecule inhibitors became a proven anti-cancer strategy. Previously, we identified an inhibitor of a major protein of homologous recombination (HR) RAD51, named B02. B02 inhibited HR in human cells and sensitized them to chemotherapeutic drugs in vitro and in vivo . Here, using a medicinal chemistry approach, we aimed to improve the potency of B02. We identified the B02 analog, B02-isomer, which inhibits HR in human cells with significantly higher efficiency. We also show that B02-iso sensitizes triple-negative breast cancer MDA-MB-231 cells to the PARP inhibitor (PARPi) olaparib.

Purpose: Genotoxic chemotherapy and radiotherapy can cause DNA double stranded breaks (DSBs) in primordial follicle (PMF) oocytes, which then undergo apoptosis. The development of effective new fertility preservation agents has been hampered, in part, by a limited understanding of DNA repair in PMF oocytes. This study investigated the induction of classical DSB repair pathways in the follicles of wild type (WT) and apoptosis-deficient Puma -/- mice in response to DSBs caused by the chemotherapy agent cisplatin., Methods: Adult C57BL/6 WT and Puma -/- mice were injected i.p. with saline or cisplatin (5 mg/kg); ovaries were harvested at 8 or 24 h. Follicles were counted, and H2A histone family member (γH2AX) immunofluorescence used to demonstrate DSBs. DNA repair protein RAD51 homolog 1 (RAD51) and DNA-dependent protein kinase, catalytic subunit (DNA-PKcs) immunofluorescence were used to identify DNA repair pathways utilised., Results: Puma -/- mice retained 100% of follicles 24 h after cisplatin treatment. Eight hours post-treatment, γH2AX immunofluorescence showed DSBs across follicular stages in Puma -/- mice; staining returned to control levels in PMFs within 5 days, suggesting repair of PMF oocytes in this window. RAD51 immunofluorescence eight hours post-cisplatin was positive in damaged cell types in both WT and Puma -/- mice, demonstrating induction of the homologous recombination pathway. In contrast, DNA-PKcs staining were rarely observed in PMFs, indicating non-homologous end joining plays an insignificant role., Conclusion: PMF oocytes are able to conduct high-fidelity repair of DNA damage accumulated during chemotherapy. Therefore, apoptosis inhibition presents a viable strategy for fertility preservation in women undergoing treatment.

Competing Interests: The authors declare no competing interest.

Synthetic lethality is a successful strategy employed to develop selective chemotherapeutics against cancer cells. Inactivation of RAD52 is synthetically lethal to homologous recombination (HR) deficient cancer cell lines. Replication protein A (RPA) recruits RAD52 to repair sites, and the formation of this protein-protein complex is critical for RAD52 activity. To discover small molecules that inhibit the RPA:RAD52 protein-protein interaction (PPI), we screened chemical libraries with our newly developed Fluorescence-based protein-protein Interaction Assay (FluorIA). Eleven compounds were identified, including FDA-approved drugs (quinacrine, mitoxantrone, and doxorubicin). The FluorIA was used to rank the compounds by their ability to inhibit the RPA:RAD52 PPI and showed mitoxantrone and doxorubicin to be the most effective. Initial studies using the three FDA-approved drugs showed selective killing of BRCA1-mutated breast cancer cells (HCC1937), BRCA2-mutated ovarian cancer cells (PE01), and BRCA1-mutated ovarian cancer cells (UWB1.289). It was noteworthy that selective killing was seen in cells known to be resistant to PARP inhibitors (HCC1937 and UWB1 SYr13). A cell-based double-strand break (DSB) repair assay indicated that mitoxantrone significantly suppressed RAD52-dependent single-strand annealing (SSA) and mitoxantrone treatment disrupted the RPA:RAD52 PPI in cells. Furthermore, mitoxantrone reduced radiation-induced foci-formation of RAD52 with no significant activity against RAD51 foci formation. The results indicate that the RPA:RAD52 PPI could be a therapeutic target for HR-deficient cancers. These data also suggest that RAD52 is one of the targets of mitoxantrone and related compounds., Competing Interests: The authors have declared that no competing interests exist.

Background: Concurrent thoracic radiation plus chemotherapy is the mainstay of first-line treatment for limited-stage small cell lung cancer (LS-SCLC). Despite initial high responsiveness to combined chemo- and radiotherapy, SCLC almost invariably relapses and develops resistance within one year, leading to poor prognosis in patients with LS-SCLC. Developing new chemical agents that increase ionizing radiation's cytotoxicity against SCLC is urgently needed., Results: Dual histone deacetylase (HDAC) and PI3K inhibitor FK228 not only displayed potent anticancer activity, but also enhanced the therapeutic effects of radiotherapy in SCLC cells. Mechanistically, radioresistant SCLC cells exhibit a lower level of histone H3K9 acetylation and a higher expression level of the MRE11-RAD50-NBS1 (MRN) complex and show more efficient and redundant DNA damage repair capacities than radiosensitive SCLC cells. FK228 pretreatment resulted in marked induction of H3k9 acetylation, attenuated homologous recombination (HR) repair competency and impaired non-homologous end joining (NHEJ) repair efficacy, leading to the accumulation of radiation-induced DNA damage and radiosensitization., Conclusion: The study uncovered that FK228 sensitized human radioresistant SCLC cells to radiation mainly through induction of chromatin decondensation and suppression of DNA damage signaling and repair. Our study provides a rational basis for a further clinical study to test the potential of FK228 as a radiosensitizing agent to increase the radiation-induced tumor cell kill in LS-SCLC patients.

Chromatin is a barrier to efficient DNA repair, as it hinders access and processing of certain DNA lesions. ALC1/CHD1L is a nucleosome-remodeling enzyme that responds to DNA damage, but its precise function in DNA repair remains unknown. Here we report that loss of ALC1 confers sensitivity to PARP inhibitors, methyl-methanesulfonate, and uracil misincorporation, which reflects the need to remodel nucleosomes following base excision by DNA glycosylases but prior to handover to APEX1. Using CRISPR screens, we establish that ALC1 loss is synthetic lethal with homologous recombination deficiency (HRD), which we attribute to chromosome instability caused by unrepaired DNA gaps at replication forks. In the absence of ALC1 or APEX1, incomplete processing of BER intermediates results in post-replicative DNA gaps and a critical dependence on HR for repair. Hence, targeting ALC1 alone or as a PARP inhibitor sensitizer could be employed to augment existing therapeutic strategies for HRD cancers., Competing Interests: Declaration Of Interests G.H. and S.J.B are inventors on a patent derived from this work. S.J.B. is also scientific co-founder and VP Science Strategy at Artios Pharma Ltd., Babraham Research Campus, Cambridge, UK. The authors declare no other competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Monotherapy with poly ADP-ribose polymerase (PARP) inhibitors results in a limited objective response rate (≤60% in most cases) in patients with homologous recombination repair (HRR)-deficient cancer, which suggests a high rate of resistance in this subset of patients to PARP inhibitors (PARPi). To overcome resistance to PARPi and to broaden their clinical use, we performed high-throughput screening of 99 anticancer drugs in combination with PARPi to identify potential therapeutic combinations. Here, we found that GSK3 inhibitors (GSK3i) exhibited a strong synergistic effect with PARPi in a panel of colorectal cancer (CRC) cell lines with diverse genetic backgrounds. The combination of GSK3β and PARP inhibition causes replication stress and DNA double-strand breaks, resulting in increased anaphase bridges and abnormal spindles. Mechanistically, inhibition or genetic depletion of GSK3β was found to impair the HRR of DNA and reduce the mRNA and protein level of BRCA1. Finally, we demonstrated that inhibition or depletion of GSK3β could enhance the in vivo sensitivity to simmiparib without toxicity. Our results provide a mechanistic understanding of the combination of PARP and GSK3 inhibition, and support the clinical development of this combination therapy for CRC patients.

Xanthatin (Xa) is a bicyclic sesquiterpene lactone identified from the plant Xanthium L. with impressive antitumor activity, but the role of Xa in non-small cell lung cancer (NSCLC) is not known. Here we found that Xa inhibits proliferation, migration, invasion and induces apoptosis in NSCLC cells. RNA sequencing and Gene set enrichment analysis revealed that Xa significantly activates p53 pathway and suppresses E2F targets, G2M checkpoint and MYC targets in A549 cells. Among these changed genes, the down-regulated gene BARD1 triggered by Xa was identified as a candidate involved in Xa's antitumor effect because of its vital role in homologous recombination (HR). Further studies demonstrated that Xa inhibits HR through the BARD1/BRCA1/RAD51 axis, which enhances cell sensitivity to cisplatin. Mechanistic studies showed that Xa inhibits BARD1 through the JAK2/STAT4 pathway. Our study revealed that Xa is a promising drug to treat NSCLC, especially in combination with conventional chemotherapy., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Targeted cancer therapies represent a milestone towards personalized treatment as they function via inhibition of cancer-specific alterations. Polymerase θ (POLQ), an error-prone translesion polymerase, also involved in DNA double-strand break (DSB) repair, is often upregulated in cancer. POLQ is synthetic lethal with various DNA repair genes, including known cancer drivers such as BRCA1/2, making it essential in homologous recombination-deficient cancers. Thus, POLQ represents a promising target in cancer therapy and efforts for the development of POLQ inhibitors are actively underway with first clinical trials due to start in 2021. This review summarizes the journey of POLQ from a backup DNA repair enzyme to a promising therapeutic target for cancer treatment., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

The response to poly(ADP-ribose) polymerase inhibitors (PARPi) is dictated by homologous recombination (HR) DNA repair and the abundance of lesions that trap PARP enzymes. It remains unclear, however, if the established role of PARP in promoting chromatin accessibility impacts viability in these settings. Using a CRISPR-based screen, we identified the PAR-binding chromatin remodeller ALC1/CHD1L as a key determinant of PARPi toxicity in HR-deficient cells. ALC1 loss reduced viability of breast cancer gene (BRCA)-mutant cells and enhanced sensitivity to PARPi by up to 250-fold, while overcoming several resistance mechanisms. ALC1 deficiency reduced chromatin accessibility concomitant with a decrease in the association of base damage repair factors. This resulted in an accumulation of replication-associated DNA damage, increased PARP trapping and a reliance on HR. These findings establish PAR-dependent chromatin remodelling as a mechanistically distinct aspect of PARPi responses and therapeutic target in HR-deficient cancers.

Covalent linkage between DNA and proteins produces highly toxic lesions and can be caused by commonly used chemotherapeutic agents, by internal and external chemicals and by radiation. In this study, using Escherichia coli, we investigate the consequences of 5-azacytidine (5-azaC), which traps covalent complexes between itself and the Dcm cytosine methyltransferase protein. DNA protein crosslink-dependent effects can be ascertained by effects that arise in wild-type but not in dcmΔ strains. We find that 5-azaC induces the bacterial DNA damage response and stimulates homologous recombination, a component of which is Dcm-dependent. Template-switching at an imperfect inverted repeat ("quasipalindrome", QP) is strongly enhanced by 5-azaC and this enhancement was entirely Dcm-dependent and independent of double-strand break repair. The SOS response helps ameliorate the mutagenic effect of 5-azaC but this is not a result of SOS-induced DNA polymerases since their induction, especially PolIV, seems to stimulate QP-associated mutagenesis. Cell division regulator SulA was also required for recovery of QP mutants induced by 5-azaC. In the absence of Lon protease, Dcm-dependent QP-mutagenesis is strongly elevated, suggesting it may play a role in DPC tolerance. Deletions at short tandem repeats, which occur likewise by a replication template-switch, are elevated, but only modestly, by 5-azaC. We see evidence for Dcm-dependent and-independent killing by 5-azaC in sensitive mutants, such as recA, recB, and lon; homologous recombination and deletion mutations are also stimulated in part by a Dcm-independent effect of 5-azaC. Whether this occurs by a different protein/DNA crosslink or by an alternative form of DNA damage is unknown., (Copyright © 2021. Published by Elsevier B.V.)

Inhibitors of poly-ADP-ribose polymerase 1 (PARPi) are highly effective in killing cells deficient in homologous recombination (HR); thus, PARPi have been clinically utilized to successfully treat BRCA2-mutant tumors. However, positive response to PARPi is not universal, even among patients with HR-deficiency. Here, we present the results of genome-wide CRISPR knockout and activation screens which reveal genetic determinants of PARPi response in wildtype or BRCA2-knockout cells. Strikingly, we report that depletion of the ubiquitin ligase HUWE1, or the histone acetyltransferase KAT5, top hits from our screens, robustly reverses the PARPi sensitivity caused by BRCA2-deficiency. We identify distinct mechanisms of resistance, in which HUWE1 loss increases RAD51 levels to partially restore HR, whereas KAT5 depletion rewires double strand break repair by promoting 53BP1 binding to double-strand breaks. Our work provides a comprehensive set of putative biomarkers that advance understanding of PARPi response, and identifies novel pathways of PARPi resistance in BRCA2-deficient cells.

Purpose: Pancreatic ductal adenocarcinoma (PDAC) arising in patients with a germline BRCA1 or BRCA2 (g BRCA ) mutation may be sensitive to platinum and PARP inhibitors (PARPi). However, treatment stratification based on g BRCA mutational status alone is associated with heterogeneous responses., Experimental Design: We performed a seven-arm preclinical trial consisting of 471 mice, representing 12 unique PDAC patient-derived xenografts, of which nine were g BRCA mutated. From 179 patients whose PDAC was whole-genome and transcriptome sequenced, we identified 21 cases with homologous recombination deficiency (HRD), and investigated prognostic biomarkers., Results: We found that biallelic inactivation of BRCA1 / BRCA2 is associated with genomic hallmarks of HRD and required for cisplatin and talazoparib (PARPi) sensitivity. However, HRD genomic hallmarks persisted in xenografts despite the emergence of therapy resistance, indicating the presence of a genomic scar. We identified tumor polyploidy and a low Ki67 index as predictors of poor cisplatin and talazoparib response. In patients with HRD PDAC, tumor polyploidy and a basal-like transcriptomic subtype were independent predictors of shorter survival. To facilitate clinical assignment of transcriptomic subtype, we developed a novel pragmatic two-marker assay (GATA6:KRT17)., Conclusions: In summary, we propose a predictive and prognostic model of g BRCA -mutated PDAC on the basis of HRD genomic hallmarks, Ki67 index, tumor ploidy, and transcriptomic subtype., (©2020 American Association for Cancer Research.)

In the present day, it is possible to incorporate targeted mutations or replace a gene using genome editing techniques such as customisable CRISPR/Cas9 system. Although induction of DNA double-strand breaks (DSBs) by genome editing tools can be repaired by both non-homologous end joining (NHEJ) and homologous recombination (HR), the skewness of the former pathway in human and other mammals normally result in imprecise repair. Scientists working at the crossroads of DNA repair and genome editing have devised new strategies for using a specific pathway to their advantage. Refinement in the efficiency of precise gene editing was witnessed upon downregulation of NHEJ by knockdown or using small molecule inhibitors on one hand, and upregulation of HR proteins and addition of HR stimulators, other hand. The exploitation of cell cycle phase differences together with appropriate donor DNA length/sequence and small molecules has provided further improvement in precise genome editing. The present article reviews the mechanisms of improving the efficiency of precise genome editing in several model organisms and in clinics.

Background: PARP inhibitors niraparib and talazoparib are FDA approved for special cases of breast cancer. PARP is an interesting repair protein which is frequently affected in cancer cells. We studied the combined action of talazoparib or niraparib with ionizing radiation in melanoma cells and healthy fibroblasts., Methods: Homologous recombination (HR) status in six different melanoma cell lines and healthy fibroblasts was assessed. Cell cultures were treated with PARP inhibitors talazoparib or niraparib and ionizing radiation (IR). Apoptosis, necrosis and cell cycle distribution was analyzed via flow cytometry. Cell migration was studied by scratch assays., Results: Studied melanoma cell cultures are HR deficient. Studied healthy fibroblasts are HR proficient. Talazoparib and niraparib have congruent effects within the same cell cultures. In all cell cultures, combined treatment increases cell death and G2/M arrest compared to IR. Combined treatment in melanoma cells distinctly increases G2/M arrest. Healthy fibroblasts are less affected by G2/M arrest. Treatment predominantly decelerates or does not modify migration. In two cell cultures migration is enhanced under the inhibitors., Conclusions: Although the two PARP inhibitors talazoparib and niraparib appear to be suitable for a combination treatment with ionizing radiation in our in vitro studies, a combination treatment cannot generally be recommended. There are clear interindividual differences in the effect of the inhibitors on different melanoma cells. Therefore, the effect on the cancer cells should be studied prior to a combination therapy. Since melanoma cells increase more strongly than fibroblasts in G2/M arrest, the fractional application of combined treatment should be further investigated.

Synthetic lethality strategies for cancer therapy exploit cancer-specific genetic defects to identify targets that are uniquely essential to the survival of tumor cells. Here we show RAD27/FEN1 , which encodes flap endonuclease 1 (FEN1), a structure-specific nuclease with roles in DNA replication and repair, and has the greatest number of synthetic lethal interactions with Saccharomyces cerevisiae genome instability genes, is a druggable target for an inhibitor-based approach to kill cancers with defects in homologous recombination (HR). The vulnerability of cancers with HR defects to FEN1 loss was validated by studies showing that small-molecule FEN1 inhibitors and FEN1 small interfering RNAs (siRNAs) selectively killed BRCA1 - and BRCA2 -defective human cell lines. Furthermore, the differential sensitivity to FEN1 inhibition was recapitulated in mice, where a small-molecule FEN1 inhibitor reduced the growth of tumors established from drug-sensitive but not drug-resistant cancer cell lines. FEN1 inhibition induced a DNA damage response in both sensitive and resistant cell lines; however, sensitive cell lines were unable to recover and replicate DNA even when the inhibitor was removed. Although FEN1 inhibition activated caspase to higher levels in sensitive cells, this apoptotic response occurred in p53-defective cells and cell killing was not blocked by a pan-caspase inhibitor. These results suggest that FEN1 inhibitors have the potential for therapeutically targeting HR-defective cancers such as those resulting from BRCA1 and BRCA2 mutations, and other genetic defects., Competing Interests: Competing interest statement: R.D.K. and J.H.J.P. are both listed as authors on an upcoming review article based on meeting proceedings of talks presented at the 10th Weinman Symposium held in 2018. R.D.K. and J.H.J.P. did not directly collaborate on work presented at the symposium that is described in the review article., (Copyright © 2020 the Author(s). Published by PNAS.)

Poly(ADP ribose) polymerase inhibitors (PARPi) have efficacy in triple negative breast (TNBC) and ovarian cancers (OCs) harboring BRCA mutations, generating homologous recombination deficiencies (HRDs). DNA methyltransferase inhibitors (DNMTi) increase PARP trapping and reprogram the DNA damage response to generate HRD, sensitizing BRCA-proficient cancers to PARPi. We now define the mechanisms through which HRD is induced in BRCA-proficient TNBC and OC. DNMTi in combination with PARPi up-regulate broad innate immune and inflammasome-like signaling events, driven in part by stimulator of interferon genes (STING), to unexpectedly directly generate HRD. This inverse relationship between inflammation and DNA repair is critical, not only for the induced phenotype, but also appears as a widespread occurrence in The Cancer Genome Atlas datasets and cancer subtypes. These discerned interactions between inflammation signaling and DNA repair mechanisms now elucidate how epigenetic therapy enhances PARPi efficacy in the setting of BRCA-proficient cancer. This paradigm will be tested in a phase I/II TNBC clinical trial., Competing Interests: Competing interest statement: F.V.R. and S.B.B. are co-inventors on US Provisional Patent Application Number 61/929,680 for the concept of the combinatorial therapy.

Deregulation of metabolism and disruption of genome integrity are hallmarks of cancer 1 . Increased levels of the metabolites 2-hydroxyglutarate, succinate and fumarate occur in human malignancies owing to somatic mutations in the isocitrate dehydrogenase-1 or -2 (IDH1 or IDH2) genes, or germline mutations in the fumarate hydratase (FH) and succinate dehydrogenase genes (SDHA, SDHB, SDHC and SDHD), respectively 2-4 . Recent work has made an unexpected connection between these metabolites and DNA repair by showing that they suppress the pathway of homology-dependent repair (HDR) 5,6 and confer an exquisite sensitivity to inhibitors of poly (ADP-ribose) polymerase (PARP) that are being tested in clinical trials. However, the mechanism by which these oncometabolites inhibit HDR remains poorly understood. Here we determine the pathway by which these metabolites disrupt DNA repair. We show that oncometabolite-induced inhibition of the lysine demethylase KDM4B results in aberrant hypermethylation of histone 3 lysine 9 (H3K9) at loci surrounding DNA breaks, masking a local H3K9 trimethylation signal that is essential for the proper execution of HDR. Consequently, recruitment of TIP60 and ATM, two key proximal HDR factors, is substantially impaired at DNA breaks, with reduced end resection and diminished recruitment of downstream repair factors. These findings provide a mechanistic basis for oncometabolite-induced HDR suppression and may guide effective strategies to exploit these defects for therapeutic gain.

Luteolin (3',4',5,7-tetrahydroxyflavone), a naturally occurring flavonoid, has been shown to have anticancer activity in many types of cancer cell lines. The anticancer capacity of luteolin may be related to its ability to induce DNA double-strand breaks (DSBs). Here, we used DT40 cells to determine whether nonhomologous end joining (NHEJ) and homologous recombination (HR) are involved in the repair mechanism of luteolin-induced DNA damage. Cells defective in Ku70 (an enzyme associated with NHEJ) or Rad54 (an enzyme essential for HR) were hypersensitive and presented more apoptosis in response to luteolin. Moreover, the sensitivity and apoptosis of Ku70 -/- and Rad54 -/- cells were associated with increased DNA damage when the numbers of γ-H2AX foci and chromosomal aberrations (CAs) were compared with those from WT cells. Additionally, after treatment with luteolin, Ku70 -/- cells presented more Top2 covalent cleavage complexes (Top2cc). These results indicated that luteolin induced DSBs in DT40 cells and demonstrated that both NHEJ and HR participated in the repair of luteolin-induced DSBs, which might be related to the inhibition of topoisomerases. These results imply that simultaneous inhibition of NHEJ and HR with luteolin treatment would provide a powerful protocol in cancer chemotherapy., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

PARP1 and PARP2 play critical roles in regulating DNA repair and PARP inhibitors have been approved for the treatment of BRCA1/2-mutated ovarian and breast cancers. It has long been known that PARP inhibition sensitizes cancer cells to DNA-damaging cytotoxic agents independent of BRCA status, however, clinical use of PARP inhibitors in combination with DNA-damaging chemotherapy is limited by the more-than-additive cytotoxicity. The natural compound alantolactone (ATL) inhibits the thioredoxin reductase to induce ROS accumulation and oxidative DNA damage selectively in cancer cells. Here, we showed that nontoxic doses of ATL markedly synergized with the PARP inhibitor olaparib to result in synthetic lethality irrespective of homologous recombination status. Synergistic cytotoxicity was seen in cancer but not noncancerous cells and was reduced by the ROS inhibitor NAC or knockdown of OGG1, demonstrating that the cytotoxicity resulted from the repair of ATL-induced oxidative DNA damage. PARP1 knockdown suppressed the synergistic lethality and olaparib was much more toxic than veliparib when combined with ATL, suggesting PARP-trapping as the primary inducer of cytotoxicity. Consistently, combined use of ATL and olaparib caused intense signs of replication stress and formation of double strand DNA breaks, leading to S and G 2 arrest followed by apoptosis. In vivo, the combination effectively induced regression of tumor xenografts, while either agent alone had no effect. Hence, PARP trapping combined with specific pro-oxidative agents may provide safe and effective ways to broaden the therapeutic potential of PARP inhibitors.

Purpose: We postulate that the deoxyguanosine analogue CNDAG [9-(2-C-cyano-2-deoxy-1-β-D-arabino-pentofuranosyl)guanine] likely causes a single-strand break after incorporation into DNA, similar to the action of its cytosine congener CNDAC, and that subsequent DNA replication across the unrepaired nick would generate a double-strand break. This study aimed at identifying cellular responses and repair mechanisms for CNDAG prodrugs, 2-amino-9-(2-C-cyano-2-deoxy-1-β-D-arabino-pentofuranosyl)-6-methoxy purine (6-OMe) and 9-(2-C-cyano-2-deoxy-1-β-D-arabino-pentofuranosyl)-2,6-diaminopurine (6-NH 2 ). Each compound is a substrate for adenosine deaminase, the action of which generates CNDAG., Methods: Growth inhibition assay, clonogenic survival assay, immunoblotting, and cytogenetic analyses (chromosomal aberrations and sister chromatid exchanges) were used to investigate the impact of CNDAG on cell lines., Results: The 6-NH 2 derivative was selectively potent in T cell malignant cell lines. Both prodrugs caused increased phosphorylation of ATM and its downstream substrates Chk1, Chk2, SMC1, NBS1, and H2AX, indicating activation of ATM-dependent DNA damage response pathways. In contrast, there was no increase in phosphorylation of DNA-PKcs, which participates in repair of double-strand breaks by non-homologous end-joining. Deficiency in ATM, RAD51D, XRCC3, BRCA2, and XPF, but not DNA-PK or p53, conferred significant clonogenic sensitivity to CNDAG or the prodrugs. Moreover, hamster cells lacking XPF acquired remarkably more chromosomal aberrations after incubation for two cell cycle times with CNDAG 6-NH 2 , compared to the wild type. Furthermore, CNDAG 6-NH 2 induced greater levels of sister chromatid exchanges in wild-type cells exposed for two cycles than those for one cycle, consistent with increased double-strand breaks after a second S phase., Conclusion: CNDAG-induced double-strand breaks are repaired mainly through homologous recombination.

This study was initiated to examine the effects of caffeine on the DNA damage response (DDR) and homologous recombination (HR) in mammalian cells. A 5 mM caffeine treatment caused the cell cycle to stall at G2/M and cells eventually underwent apoptosis. Caffeine exposure also induced a strong DDR along with subsequent activation of wildtype p53 protein. An unexpected observation was the caffeine-induced depletion of Rad51 (and Brca2) proteins. Consequently, caffeine-treated cells were expected to be inefficient in HR. However, a dichotomy in the HR response of cells to caffeine treatment was revealed. Caffeine treatment rendered cells significantly better at performing the nascent DNA synthesis that accompanies the early strand invasion steps of HR. Additionally, caffeine treatment increased chromatin accessibility and elevated the efficiency of illegitimate recombination. Conversely, the increase in nascent DNA synthesis did not translate into a higher number of gene targeting events. Thus, prolonged caffeine exposure stalls the cell cycle, induces a p53-mediated apoptotic response and a down-regulation of critical HR proteins, and for reasons discussed, stimulates early steps of HR, but not the formation of complete recombination products., Competing Interests: Declaration of Competing Interest There are no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

BRCAness is considered a predictive biomarker to platinum and poly(ADP-ribose) polymerase (PARP) inhibitors. However, recent trials showed that its predictive value was limited in triple-negative breast cancer (TNBC) treated with platinum. Moreover, tumors with mutations of DNA damage response (DDR) genes, such as homologous recombination (HR) genes, could be sensitive to platinum and PARP inhibitors. Thus, we aim to explore the relationship between mutation status of DDR genes and BRCAness in TNBC. We sequenced 56 DDR genes in 120 TNBC and identified BRCAness by array comparative genomic hybridization. The sequencing results showed that 13, 14, and 14 patients had BRCA, non-BRCA HR, and non-HR DDR gene mutations, respectively. Array comparative genomic hybridization revealed that BRCA-mutated and HR gene-mutated TNBC shared similar BRCAness features, both having higher numbers and longer length of large-scale structural aberration (LSA, >10 Mb) and similar altered chromosomal regions of LSA. These suggested non-BRCA HR gene-mutated TNBC shared similar characteristics with BRCA-mutated TNBC, indicating non-BRCA HR gene-mutated TNBC sensitive to platinum and PARP inhibitors. Among tumors with mutation of non-HR DDR genes, 3 PTEN and 1 MSH6 mutation also contained significant LSAs (BRCAness); however, they had different regions of genomic alteration to BRCA and HR gene-mutated tumors, might explain prior findings that PTEN- and MSH6-mutated cancer cells not sensitive to PARP inhibitors. Therefore, we hypothesize that the heterogeneous genomic background of BRCAness indicates different responsiveness to platinum and PARP inhibitors. Direct sequencing DDR genes in TNBC should be applied to predict their sensitivity toward platinum and PARP inhibitors., (© 2020 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Dysfunction of p53 and resistance to cancer drugs can arise through mutually exclusive overexpression of MDM2 or MDM4. Cisplatin-resistant cells, however, can demonstrate increased binding of both MDM2 and MDM4 to p53 but in absence of cellular overexpression. Whether MDM2 inhibitors alone can activate p53 in these resistant cells was investigated with the goal to establish the mechanism for potential synergy with cisplatin. Thus, growth inhibition by individual drugs and combinations was assessed by a colorimetric assay. Drug-treated parental A2780 and resistant tumor cells were also examined for protein expression using immunoblot and reverse phase protein array (RPPA) and then subjected to Ingenuity Pathway Analysis (IPA). Gene expression was assessed by real-time polymerase chain reaction, DNA damage by confocal microscopy, cell cycle by flow cytometry, and homologous recombination (HR) by a GFP reporter assay. Our results demonstrate that Nutlin-3 but not RITA (reactivation of p53 and induction of tumor cell apoptosis) effectively disrupted the p53-MDM2-MDM4 complex to activate p53, which increased robustly with cisplatin/Nutlin-3 combination and enhanced antitumor effects more than either agent alone. RPPA, IPA, and confocal microscopy provided evidence for an "apparent" increase in DNA damage resulting from HR inhibition by cisplatin/Nutlin-3. Molecularly, the specific HR protein Rad51 was severely downregulated by the combination via two mechanisms: p53-dependent transrepression and p53/MDM2-mediated proteasomal degradation. In conclusion, Nutlin-3 fully destabilizes the p53-MDM2-MDM4 complex and synergizes with cisplatin to intensify p53 function, which then downregulates Rad51 through a bimodal mechanism. As a result, HR is inhibited and antitumor activity enhanced in otherwise HR-proficient sensitive and resistant tumor cells. SIGNIFICANCE STATEMENT: Rad51 downregulation by the combination of cisplatin and Nutlin-3 inhibits homologous recombination (HR), which leads to persistence in DNA damage but not an increase. Thus, inhibition of HR enhances antitumor activity in otherwise HR-proficient sensitive and resistant tumor cells., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

Purpose: Although EGFR inhibitor (EGFRi) is used in cancer therapy to suppress tumor growth and resistance to treatment including radiotherapy, EGFRi resistance frequently developed, which significantly reduced treatment outcomes. Therefore, developing alternative approaches for EGFRi is of great importance. Based on our recent observation that ATM inhibitor (ATMi) efficiently inhibited ionizing radiation (IR)-induced EGFR activation in mouse embryo fibroblasts (MEF), the main purpose of this study is to determine whether ATMi could inhibit IR-induced EGFR activation in human tumor cell lines and explore its potential in EGFRi-alternative therapies. Materials and methods: We compared the effects of ATMi, EGFRi individually or in combination on IR-induced EGFR phosphorylation, cell growth and radio-sensitization in nine human tumor cell lines including lung adenocarcinoma (A549 and H358), glioblastoma (LN229), cervical cancer (HeLa), colorectal carcinoma (SW480 and HCT116) and nasopharygeal carcinoma (5-8 F, 6-10B and HK1) cell lines. In addition, we detected the effects of ATMi, EGFRi alone or both on the efficiency of non-homologous end-joining (NHEJ) and homologous recombination (HR) using I-SceI -GFP based NHEJ or HR reporter cell lines. Results: Compared to EGFRi treatment, ATMi treatment decreased IR-induced EGFR phosphorylation, suppressed growth and increased IR sensitization in tested cell lines at a similar or even more efficient level. Combining ATMi and EGFRi did not significantly increased the effects on these phenotypes as ATMi treatment alone. Also, similar to ATMi, EGFRi mainly reduced the efficiency of HR but not NHEJ although combining ATMi and EGFRi further inhibited the HR efficiency. Conclusions: Our study demonstrates that ATMi can function like EGFRi in human tumor cells to inhibit tumor cell growth and sensitize the tumor cells to IR, suggesting that ATMi treatment as an alternative approach may exert anticancer effects on EGFRi-resistant tumor cells and facilitate radiotherapy.

Inhibition of the cell-cycle kinases CDK4 and CDK6 is now part of the standard treatment in advanced breast cancer. CDK4/6 inhibitors, however, are not expected to cooperate with DNA-damaging or antimitotic chemotherapies as the former prevent cell-cycle entry, thus interfering with S-phase- or mitosis-targeting agents. Here, we report that sequential administration of CDK4/6 inhibitors after taxanes cooperates to prevent cellular proliferation in pancreatic ductal adenocarcinoma (PDAC) cells, patient-derived xenografts, and genetically engineered mice with Kras G12V and Cdkn2a-null mutations frequently observed in PDAC. This effect correlates with the repressive activity of CDK4/6 inhibitors on homologous recombination proteins required for the recovery from chromosomal damage. CDK4/6 inhibitors also prevent recovery from multiple DNA-damaging agents, suggesting broad applicability for their sequential administration after available chemotherapeutic agents., Competing Interests: Declaration of Interests D.J.S., T.X., and T.V.A. are employees of Pfizer and hold shares in the company. M.H. declares stock and ownership interests (Champions Oncology, Pharmacyte Biotech, BioOncotech, Nelum, Eng T Cells), received honoraria (Pfizer, Novartis, MSD Oncology, Celgene, BiolineRx, Champions Oncology, Roche, SOBI, Agenus, Erytech Pharma, Pharmacyte Biotech), had consulting or advisory roles (Oncomatryx Biopharma, Novartis, Pfizer, Celgene, Merck, Champions Oncology, Pharmacyte Biotech, SOBI, Roche, BiolineRx, Erytech Pharma, Agenus, Bayer, Bristol-Myers Squibb, Nelum, Eng T Cells, InxMed; Bioncotech, Takeda), received research funding (Berg, Oncomatryx Biopharma, Pfizer, Celgene, Bicycle Therapeutics, BiolineRx, Asana Biosciences), and participates in patents, royalties, and other Intellectual Property from Myriad Genetics. M.M. has had advisory or consulting relationships, or research agreements with Pfizer, Eli Lilly, and Novartis. The other authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Synthetic lethality is an innovative framework for discovering novel anticancer drug candidates. One example is the use of PARP inhibitors (PARPi) in oncology patients with BRCA mutations. Here, we exploit a new paradigm based on the possibility of triggering synthetic lethality using only small organic molecules (dubbed "fully small-molecule-induced synthetic lethality"). We exploited this paradigm to target pancreatic cancer, one of the major unmet needs in oncology. We discovered a dihydroquinolone pyrazoline-based molecule ( 35d ) that disrupts the RAD51-BRCA2 protein-protein interaction, thus mimicking the effect of BRCA2 mutation. 35d inhibits the homologous recombination in a human pancreatic adenocarcinoma cell line. In addition, it synergizes with olaparib (a PARPi) to trigger synthetic lethality. This strategy aims to widen the use of PARPi in BRCA -competent and olaparib-resistant cancers, making fully small-molecule-induced synthetic lethality an innovative approach toward unmet oncological needs.

In response to DNA double-strand breaks, MAD2L2-containing shieldin complex plays a critical role in the choice between homologous recombination (HR) and non-homologous end-joining (NHEJ)-mediated repair. Here we show that EZH2 inhibition upregulates MAD2L2 and sensitizes HR-proficient epithelial ovarian cancer (EOC) to poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitor in a CARM1-dependent manner. CARM1 promotes MAD2L2 silencing by driving the switch from the SWI/SNF complex to EZH2 through methylating the BAF155 subunit of the SWI/SNF complex on the MAD2L2 promoter. EZH2 inhibition upregulates MAD2L2 to decrease DNA end resection, which increases NHEJ and chromosomal abnormalities, ultimately causing mitotic catastrophe in PARP inhibitor treated HR-proficient cells. Significantly, EZH2 inhibitor sensitizes CARM1-high, but not CARM-low, EOCs to PARP inhibitors in both orthotopic and patient-derived xenografts., Competing Interests: Declaration of Interests S.K. and R.Z. are co-inventors on a patent application covering the use of EZH2 inhibitors in CARM1-expressing cancers. All other authors declare no competing interests., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Purpose of Review: To summarize recently discovered PARP inhibitor resistance mechanisms and highlight the clinical relevance of these findings to date., Recent Findings: A predominant mechanism of acquired PARP inhibitor resistance in homologous recombination-deficient cancers is the acquisition of homologous recombination proficiency as a consequence of secondary genetic or epigenetic events, such as secondary mutations in BRCA1 or BRCA2, or reversal of BRCA1 promoter methylation that restores homologous recombination and leads to PARP inhibitor resistance. Multiple other potential mechanisms of acquired resistance to PARP inhibitors including loss of DNA end resection inhibition (53BP1/REV7/RIF1/Sheldin) or DNA replication fork protection (PTIP/EZH2), but also increased drug efflux or induction of a reversible senescent or mesenchymal cell state have been described in ovarian cancer models. However, only few of these mechanisms have been identified in clinical samples., Summary: Multiple adaptive responses following PARP inhibitor treatment have been identified. Further research is needed to better understand what role these mechanisms play for clinical PARP inhibitor resistance and how these mechanisms may render ovarian cancer cells susceptible to subsequent novel combination therapies.

Lung cancer is the leading cause of cancer death worldwide. PARP inhibitors have become a new line of cancer therapy and a successful demonstration of the synthetic lethality concept. The mechanism and efficacy of PARP inhibitors have been well studied in some cancers, especially homologous recombination (HR)-deficient ovarian cancer and breast cancer, yet such studies are still relatively fewer in lung cancer. Here we found that HR genes are frequently mutated in lung cancer patients, exposing a window for targeted therapies by PARP inhibitors. We depleted BRCA1 and BRCA2 in non-small cell lung cancer (NSCLC) cancer cells and found these cells are hypersensitive to the PARP inhibitor olaparib in cell viability and clonogenic survival assays. Olaparib specifically induces apoptosis in A549 cells with BRCA1 or BRCA2 depletion, as determined by positive Annexin-V staining. In addition, we show that A549 cells with ATM shRNA knockdown are also hypersensitive to Olaparib. In summary, our data support the potential use of PARP inhibitors in NSCLC with HR deficiency., (Copyright © 2019. Published by Elsevier Inc.)

Chromosomal instability not only has a negative effect on survival in triple-negative breast cancer, but also on the well treatable subgroup of luminal A tumors. This suggests a general mechanism independent of subtypes. Increased chromosomal instability (CIN) in triple-negative breast cancer (TNBC) is attributed to a defect in the DNA repair pathway homologous recombination. Homologous recombination (HR) prevents genomic instability by repair and protection of replication. It is unclear whether genetic alterations actually lead to a repair defect or whether superior signaling pathways are of greater importance. Previous studies focused exclusively on the repair function of HR. Here, we show that the regulation of HR by the intra-S-phase damage response at the replication is of overriding importance. A damage response activated by Ataxia telangiectasia and Rad3 related-checkpoint kinase 1 (ATR-CHK1) can prevent replication stress and leads to resistance formation. CHK1 thus has a preferred role over HR in preventing replication stress in TNBC. The signaling cascade ATR-CHK1 can compensate for a double-strand break repair error and lead to resistance of HR-deficient tumors. Established methods for the identification of HR-deficient tumors for Poly(ADP-Ribose)-Polymerase 1 (PARP1) inhibitor therapies should be extended to include analysis of candidates for intra-S phase damage response.

DNA repair inhibition has been described as an essential event leading to the initiation of carcinogenesis. In a previous study, we observed that the exposure to metal mixture induces changes in the miR-nome of the cells that was correlated with the sub-expression of mRNA involved in processes and diseases associated with metal exposure. From this analysis, one of the miRNAs that shows changes in its expression is miR-222, which is overexpressed in various cancers associated with exposure to metals. In silico studies showed that a possible target for the microRNA-222 could be Rad 51c, a gene involved in the double-stranded DNA repair. We could appreciate that up-regulation of miR-222 reduces the expression both gene and as a protein expression of Rad51c by RT-PCR and immunoblot, respectively. A luciferase assay was performed to validate Rad51c as miR-222 target. Neutral comet assay was performed in order to evaluate DNA double-strand breaks under experimental conditions. Here, we demonstrate that miR-222 up-regulation, directly regulates Rad51c expression negatively, and impairs homologous recombination of double-strand break DNA repair during the initiation stage of cell transformation. This inhibition triggers morphological transformation in a two-stage Balb/c 3T3 cell assay, suggesting that this small RNA acts as an initiator of the carcinogenesis process., Competing Interests: The authors have declared that no competing interests exist.

53BP1 activity drives genome instability and lethality in BRCA1-deficient mice by inhibiting homologous recombination (HR). The anti-recombinogenic functions of 53BP1 require phosphorylation-dependent interactions with PTIP and RIF1/shieldin effector complexes. While RIF1/shieldin blocks 5'-3' nucleolytic processing of DNA ends, it remains unclear how PTIP antagonizes HR. Here, we show that mutation of the PTIP interaction site in 53BP1 (S25A) allows sufficient DNA2-dependent end resection to rescue the lethality of BRCA1 Δ11 mice, despite increasing RIF1 "end-blocking" at DNA damage sites. However, double-mutant cells fail to complete HR, as excessive shieldin activity also inhibits RNF168-mediated loading of PALB2/RAD51. As a result, BRCA1 Δ11 53BP1 S25A mice exhibit hallmark features of HR insufficiency, including premature aging and hypersensitivity to PARPi. Disruption of shieldin or forced targeting of PALB2 to ssDNA in BRCA1 D11 53BP1 S25A cells restores RNF168 recruitment, RAD51 nucleofilament formation, and PARPi resistance. Our study therefore reveals a critical function of shieldin post-resection that limits the loading of RAD51., (Copyright © 2019 Elsevier Inc. All rights reserved.)