Repair Of Single-strand Breaks Research Articles

The TIMELESS and PARP1 interaction suppresses replication-associated DNA gap accumulation.

TIMELESS (TIM) in the fork protection complex acts as a scaffold of the replisome to prevent its uncoupling and ensure efficient DNA replication fork progression. Nevertheless, its underlying basis for coordinating leading and lagging strand synthesis to limit single-stranded DNA (ssDNA) exposure remains elusive. Here, we demonstrate that acute degradation of TIM at ongoing DNA replication forks induces the accumulation of ssDNA gaps stemming from defective Okazaki fragment (OF) processing. Cells devoid of TIM fail to support the poly(ADP-ribosyl)ation necessary for backing up the canonical OF processing mechanism mediated by LIG1 and FEN1. Consequently, recruitment of XRCC1, a known effector of PARP1-dependent single-strand break repair, to post-replicative ssDNA gaps behind replication forks is impaired. Physical disruption of the TIM-PARP1 complex phenocopies the rapid loss of TIM, indicating that the TIM-PARP1 interaction is critical for the activation of this compensatory pathway. Accordingly, combined deficiency of FEN1 and the TIM-PARP1 interaction leads to synergistic DNA damage and cytotoxicity. We propose that TIM is essential for the engagement of PARP1 to the replisome to coordinate lagging strand synthesis with replication fork progression. Our study identifies TIM as a synthetic lethal target of OF processing enzymes that can be exploited for cancer therapy.

Abstract 7112: Preclinical profile of novel and potent small molecule inhibitors of PARG

Abstract Poly(ADP-ribose) glycohydrolase (PARG) is primary enzyme involved in the dePARylation process of DNA repair, specifically in the single strand break repair (SSBRs). PARG is a macrodomain protein with exo- and endo-glycohydrolase activity that liberates free ADP-ribose and PAR chains, respectively. It interacts with various Poly(ADP-ribose) polymerase (PARP) proteins to facilitate appropriate and timely DNA repair. The balance between PARP and PARG activity is essential for efficient DDR. Inhibition of PARG lead to altered DNA repair in cancer cells. We have identified highly potent PARG inhibitors with picomolar IC50 in. PARG is the primary enzyme for dePARylation and accounts for 90% of dePARylation activity. Treatment of HCC-1806 cells with PARG inhibitors showed dose dependent increase of PARylation signature, with sub micromolar IC50. Compounds also demonstrate significant functional, anti-proliferative activity in ovarian and breast cancer cell lines besides a favourable ADME profile. Further pharmacokinetic and pharmacodynamic characterization of the identified hits is currently in progress. Citation Format: Sukanya Patra, Manoj Pothuganti, Githavani Kummari, Venu Sankeshi, Jyothipriya Kapaka, Navnath Karche, Ramamohan Mekala, Srikant Viswanadha. Preclinical profile of novel and potent small molecule inhibitors of PARG [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7112.

Abstract 2083: Discovery of ETX-19477, a novel and selective PARG inhibitor with high potency against tumors with underlying replication stress

Abstract DNA damage response (DDR) is an important cellular process that is regulated by poly ADP ribosylation (PARylation). PARP1 binds to DNA break sites and then catalyzes PARylation, which leads to recruitment of repair factors and initiation of base-excision and single-strand break (SSB) repair. Removal of PAR chains is performed by PARG and is also critical for successful completion of DNA repair. Suppression of PAR chain hydrolysis via PARG depletion or inhibition leads to defective single-strand break repair, reduced kinetics of repair, and hypersensitivity to certain DNA damaging agents. Cancer cells demonstrate high levels of replication stress, characterized as the slowing or stalling of replication forks during DNA replication. Replication stress generates DNA SSBs and exposes unligated Okazaki fragments which activate PARP1. PARP1 catalyzed PARylation slows replication fork progression as a protective mechanism, initiates damage repair, and promotes fork reversal. PAR chain removal, via PARG activity, enables completion of damage repair, replication fork restart, and recovery from replication stress. Under conditions of replication stress, PARP inhibition can result in unrestrained fork progression and the occurrence of DNA double strand breaks from fork runoff. In contrast, PARG inhibition causes prolonged slowing or stalling of replication fork progression, which can lead to replication catastrophe and cancer cell death. Because of these opposing roles, PARG inhibitors show distinct pharmacology as compared to PARP inhibitors in certain cancer cells including cancer cells that are BRCA wild type, homologous recombination proficient, and PARP inhibitor insensitive or resistant. ETX-19477 is a novel inhibitor of PARG that demonstrates low nM, on-target activity in cells as measured by the accumulation of PAR chains. ETX-19477 induces death and/or inhibits the proliferation of multiple cancer cell types including ER+HER2- breast, serous and mucinous ovarian, lung, and gastric cancers and induces pan-nuclear γH2AX expression, a hallmark of replication catastrophe. Preclinical studies demonstrate that ETX-19477 is orally bioavailable with favorable pharmacokinetics. Oral treatment of mice with ETX-19477 is well tolerated and shows robust dose-dependent in vivo anti-tumor activity in breast and ovarian cell-line derived and patient-derived xenograft models. Efficacy is observed with either once or twice daily dosing and with a discontinuous dosing regimen. In mouse xenograft models, induction of γH2AX expression in tumor tissue correlates with tumor growth inhibition. Preclinical data show ETX-19477 demonstrates potent and specific anti-proliferative effects by exposing underlying existing replication stress vulnerabilities in cancer cells across multiple tumor types. ETX-19477 will be advanced into clinical trials in 2024. Citation Format: John P. Holleran, Tamara S. Rodems, Shweta Sharma, Angelina M. Santini, Cindy Wuerz, Juping Liu, Lu Tian, Joseph Dosch, Joy Nader, Richard Rivera, Shyama Herath, Joseph Marakovits, Christopher McBride, Ryan Stansfield, Amy Salyards, James M. Veal, Jeffrey A. Stafford, Gretchen Bain. Discovery of ETX-19477, a novel and selective PARG inhibitor with high potency against tumors with underlying replication stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2083.

Abstract 7131: Base excision repair regulation of replication stress induced poly(ADP-ribose) checkpoints

Abstract PARP-inhibitors (PARPi) have been developed to treat homology-directed repair deficient cancers such as BRCA1/2-defective breast and ovarian tumors. However, acquired resistance to PARPi is now common, suggesting the need to identify new protein targets. To that end, a missed opportunity is found in the PARP1/PARP2 activation mechanism in response to DNA damage and replication stress and the resulting protein complexes and enzymes that depend on and coordinate PARP-mediated DNA repair processes. We have found that PARP1/PARP2 activation, whether from acute DNA damage, from replication stress or both triggers a temporal and coordinated interplay of base excision repair and single-strand break repair (BER/SSBR) proteins and enzymes. In turn, the recruitment of these BER and SSBR proteins suppresses PARP1/PARP2 activation, thereby functioning as a network of PARP1/PARP2 regulators, modulating the PARP-mediated response to replication stress. To that end, we find that loss of expression of these key BER/SSBR proteins releases a break on replication-stress induced PARP1/PARP2 activation, triggering un-restrained PARP1/PARP2 activation and elevated levels of replication-dependent poly(ADP-ribose) (PAR). We next confirmed that the loss of these downstream BER enzymes render cancer cells responsive to PARPi’s when the BRCA1/2 pathways are restored. Further, we show that BER expression regulates the cellular response to inhibitors of the PAR degrading enzyme PARG, suggesting that XRCC1-dependent BER proteins regulate replication-stress dependent PAR accumulation. The increase in PAR due to PARG inhibition, that is further regulated by BER, drives a strong intra-S phase response, indicated by activation of ATR and CHK1 signaling. As the checkpoint governed by ATR and CHK1 functions as a protective mechanism, we show that PARG inhibition is synthetically lethal with ATR and CHK1 inhibition and this may be explained by our demonstration of PARGi-mediated trapping of essential BER proteins at the site of damage and PAR accumulation. Together, these studies reveal a significant role for BER/SSBR proteins in regulating replication stress induced PARylation and the resulting intra-S phase checkpoint. Citation Format: Md Ibrahim, Md Maruf Khan, Rasha Q. Al-Rahaleh, Jennifer Clark, Faisal Hayat, Qingming Fang, Christopher A. Koczor, Marie E. Migaud, Robert W. Sobol. Base excision repair regulation of replication stress induced poly(ADP-ribose) checkpoints [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7131.

Abstract 538: Establishment of PARP inhibitor-induced resistant patient-derived ovarian cancer xenograft models

Abstract Introduction: Poly (ADP-ribose) polymerases (PARPs) can recruit DNA repair effectors and cope with DNA single-strand breaks. For DNA double-strand breaks (DSB), there are two main repair mechanisms: non-homologous end joining repair (NHEJ) which is prone to error due to infidelity events, and BRCA1/2 mediated homologous recombination repair (HR) restoring the original sequence. Therefore, inhibition of PARPs may cause both single stranded break repair deficiency and HR deficiency in BRCA1/2 deficient patients, leading to cell death. The concept of “synthetic lethality” has led to the development of PARP inhibitors, including Olaparib, Niraparib and Rucaparib in clinical practice. Despite encouraging efficacy of these PARP inhibitors, acquired resistance, such as HR repair restoration, drug efflux pump upregulation and replication fork protection, has limited their clinical benefits. Therefore, to investigate novel therapeutic strategies which can overcome the acquired resistance, we established patient-derived xenograft (PDX) ovarian cancer models with PARP inhibitor-induced resistance. Methods: OV9402, an ovarian cancer PDX model with BRCA 1 c.3155del, was implanted subcutaneously and treated with Niraparib at 35 mg/kg, daily, for 129 days. Relapse tumors (tumor #1 and tumor #2) were isolated and inoculated into naïve mice. Then the 2nd round of treatment (Niraparib, 35 mg/kg, daily) was given when the mean tumor volume reached 140 mm3. The tumors showing no significant response to Niraparib were collected on either day 32 (#1 derived tumor model) and day 22 (#2 derived tumor model) after randomization and their gene changes were analyzed by RNA sequencing (RNA-seq). Results: Niraparib significantly inhibited OV9402 tumor growth, with complete regression observed in nine out of ten mice on day 54 after the 1st round of treatment. However, tumor regrowth was observed in two out of the nine mice from day 68 onwards. The relapsed tumors (tumor #1 and tumor #2) from these two mice were collected and re-inoculated into 5 naïve mice to establish tumor models for the 2nd round of Niraparib treatment, separately. For #1 derived tumor model, all the tumors presented complete resistance to the Niraparib treatment. RNA-seq data showed that the gene expression of ABCB1, which encodes for efflux pumps, was upregulated. In parallel, complete resistance to Niraparib was observed in all the tumors derived from 2# tumor bearing mice. The RNA-seq data demonstrated that an acquired secondary genetic deletion on BRCA1 genes, which restored the functional protein expression loss in the parental line and restored HR repair, was found in the tumors. Conclusion: Ovarian cancer PDX models that have effectively developed resistance to PARP inhibitors have the potential to serve as valuable preclinical tools to evaluate potential next-generation PARP inhibitors and innovative combination strategies. Citation Format: Li Hua, Shuang Li, Chenpan Nie, Xiaobo Chen, Guo Sheng, Ludovic Bourre, Jingjing Wang. Establishment of PARP inhibitor-induced resistant patient-derived ovarian cancer xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 538.

Abstract 5858: Systematic identification of pathways associated with antibody drug conjugate sensitivity in breast cancer

Abstract Antibody drug conjugates (ADCs) are revolutionizing precision therapeutics in oncology. Sacituzumab Govitecan (SG), combining a Topoisomerase I (TOP1) inhibitor payload (SN38) and the tumor-selective antigen TROP2-targeting antibody hRS7, has demonstrated efficacy in challenging cancers including triple negative breast cancer (TNBC). Despite its promising clinical efficacy, the intricacies of sensitivity and resistance mechanisms to SG remain elusive. To systematically interrogate ADC sensitivity mechanisms, we employed genome-wide CRISPR-Cas9 knockout screening to discover novel regulators influencing SG sensitivity in human TNBC cells. Interactome analysis of top SG sensitizing hits showed clustered pathways of interest, including DNA repair/replication, mTORC1/metabolism, ubiquitin/proteolysis and endosome trafficking/sorting. A top druggable hit sensitizing to SG was PARP1, encoding the poly (ADP-ribose) polymerase 1 that is a key enzyme required for single-strand break repair pathways and for DNA replication fork stability. Furthermore, the synergistic lethality between SG and PARP1 was confirmed in experiments combining SG with PARP inhibitor treatment of TNBC cell lines. To selectively identify SG/ADC-specific genes and pathways, we performed a secondary round of CRISPR knockout and activation screens using a custom library comprising top hits discovered in the primary screen, treating cells in parallel with either SG or cytotoxic payload SN38 alone. Pathway analysis of genes selectively modulating sensitivity to SG but not SN38 revealed novel mediators involved in TROP2 turnover and recycling. We then performed validation and mechanistic studies to elucidate how specific genetic permutations regulate antibody delivery and sensitivity to SG. In summary, employing a systematic CRISPR screening method, we uncovered numerous novel pathways associated with both resistance and sensitization, playing a role in ADC delivery and target protein turnover. Our current focus involves prioritizing druggable genes and pathways, laying the foundation for proof-of-concept studies that will combine ADCs with specific targeted therapeutics, aiming to enhance tumor-killing efficacy. Citation Format: Bogang Wu, Sheng Sun, Nayana Thimmiah, Aiko Nagayama, James T. Coates, Ting Liu, Win Thant, Elena Bitman, David Li, John Doench, Aditya Bardia, Leif W. Ellisen. Systematic identification of pathways associated with antibody drug conjugate sensitivity in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5858.

XRCC1 Polymorphisms p.Arg194Trp, p.Arg280His, and p.Arg399Gln, Polycyclic Aromatic Hydrocarbons, and Infertility: A Case-Control and In Silico Study.

XRCC1 is involved in repair of single-strand breaks generated by mutagenic exposure. Polymorphisms within XRCC1 affect its ability to efficiently repair DNA damage. Polycyclic aromatic hydrocarbons or PAHs are genotoxic compounds which form bulky DNA adducts that are linked with infertility. Few reports suggest combined role of XRCC1 polymorphisms and PAHs in infertility. Present study investigates association of three XRCC1 polymorphisms (p.Arg194Trp, p.Arg280His, p.Arg399Gln) with male and female infertility in a North-West Indian population using case-control approach. Additionally, in silico approach has been used to predict whether XRCC1 polymorphisms effect interaction of XRCC1 with different PAHs. For case-control study, XRCC1 polymorphisms were screened in peripheral blood samples of age- and gender-matched 201 infertile cases (♂-100, ♀-101) and 201 fertile controls (♂-100, ♀-101) using PCR-RFLP method. For in silico study, AutoDock v4.2.6 was used for molecular docking of B[a]P, BPDE-I, ( ±)-anti-BPDE, DB[a,l]P, 1-N, 2-N, 1-OHP, 2-OHF with XRCC1 and assess effect of XRCC1 polymorphisms on their interaction. In case-control study, statistical analysis showed association of XRCC1 p.Arg280His GA genotype (p = 0.027), A allele (p = 0.019) with reduced risk of male infertility. XRCC1 p.Arg399Gln AA genotype (p = 0.021), A allele (p = 0.014) were associated with reduced risk for female primary infertility. XRCC1 p.Arg194Trp T allele was associated with increased risk for female infertility (p = 0.035). In silico analysis showed XRCC1-PAH interaction with non-significant effect of XRCC1 polymorphisms on predicted binding. Therefore, present study concludes that XRCC1 polymorphism-modified risk for male and female infertility in North-West Indians without significant effect on predicted XRCC1-PAH interactions. This is the first report on XRCC1 in female infertility.

PARG-deficient tumor cells have an increased dependence on EXO1/FEN1-mediated DNA repair

Targeting poly(ADP-ribose) glycohydrolase (PARG) is currently explored as a therapeutic approach to treat various cancer types, but we have a poor understanding of the specific genetic vulnerabilities that would make cancer cells susceptible to such a tailored therapy. Moreover, the identification of such vulnerabilities is of interest for targeting BRCA2;p53-deficient tumors that have acquired resistance to poly(ADP-ribose) polymerase inhibitors (PARPi) through loss of PARG expression. Here, by performing whole-genome CRISPR/Cas9 drop-out screens, we identify various genes involved in DNA repair to be essential for the survival of PARG;BRCA2;p53-deficient cells. In particular, our findings reveal EXO1 and FEN1 as major synthetic lethal interactors of PARG loss. We provide evidence for compromised replication fork progression, DNA single-strand break repair, and Okazaki fragment processing in PARG;BRCA2;p53-deficient cells, alterations that exacerbate the effects of EXO1/FEN1 inhibition and become lethal in this context. Since this sensitivity is dependent on BRCA2 defects, we propose to target EXO1/FEN1 in PARPi-resistant tumors that have lost PARG activity. Moreover, EXO1/FEN1 targeting may be a useful strategy for enhancing the effect of PARG inhibitors in homologous recombination-deficient tumors.

Site-specific acetylation of polynucleotide kinase 3'-phosphatase regulates its distinct role in DNA repair pathways.

Mammalian polynucleotide kinase 3'-phosphatase (PNKP), a DNA end-processing enzyme with 3'-phosphatase and 5'-kinase activities, is involved in multiple DNA repair pathways, including base excision (BER), single-strand break (SSBR), and double-strand break repair (DSBR). However, little is known as to how PNKP functions in such diverse repair processes. Here we report that PNKP is acetylated at K142 (AcK142) by p300 constitutively but at K226 (AcK226) by CBP, only after DSB induction. Co-immunoprecipitation analysis using AcK142 or AcK226 PNKP-specific antibodies showed that AcK142-PNKP associates only with BER/SSBR, and AcK226 PNKP with DSBR proteins. Despite the modest effect of acetylation on PNKP's enzymatic activity in vitro, cells expressing non-acetylable PNKP (K142R or K226R) accumulated DNA damage in transcribed genes. Intriguingly, in striatal neuronal cells of a Huntington's Disease (HD)-based mouse model, K142, but not K226, was acetylated. This is consistent with the reported degradation of CBP, but not p300, in HD cells. Moreover, transcribed genomes of HD cells progressively accumulated DSBs. Chromatin-immunoprecipitation analysis demonstrated the association of Ac-PNKP with the transcribed genes, consistent with PNKP's role in transcription-coupled repair. Thus, our findings demonstrate that acetylation at two lysine residues, located in different domains of PNKP, regulates its distinct role in BER/SSBR versus DSBR.

Chromosomal single-strand break repair and neurological disease: Implications on transcription and emerging genomic tools

Cells are constantly exposed to various sources of DNA damage that pose a threat to their genomic integrity. One of the most common types of DNA breaks are single-strand breaks (SSBs). Mutations in the repair proteins that are important for repairing SSBs have been reported in several neurological disorders. While several tools have been utilised to investigate SSBs in cells, it was only through recent advances in genomics that we are now beginning to understand the architecture of the non-random distribution of SSBs and their impact on key cellular processes such as transcription and epigenetic remodelling. Here, we discuss our current understanding of the genome-wide distribution of SSBs, their link to neurological disorders and summarise recent technologies to investigate SSBs at the genomic level.

Cisplatin and Procaterol Combination in Gastric Cancer? Targeting Checkpoint Kinase 1 for Cancer Drug Discovery and Repurposing by an Integrated Computational and Experimental Approach.

Checkpoint kinase 1 (CHK1), a serine/threonine kinase, plays a crucial role in cell cycle arrest and is a promising therapeutic target for drug development against cancers. CHK1 coordinates cell cycle checkpoints in response to DNA damage, facilitating repair of single-strand breaks, and maintains the genome integrity in response to replication stress. In this study, we employed an integrated computational and experimental approach to drug discovery and repurposing, aiming to identify a potent CHK1 inhibitor among existing drugs. An e-pharmacophore model was developed based on the three-dimensional crystal structure of the CHK1 protein in complex with CCT245737. This model, characterized by seven key molecular features, guided the screening of a library of drugs through molecular docking. The top 10% of scored ligands were further examined, with procaterol emerging as the leading candidate. Procaterol demonstrated interaction patterns with the CHK1 active site similar to CHK1 inhibitor (CCT245737), as shown by molecular dynamics analysis. Subsequent in vitro assays, including cell proliferation, colony formation, and cell cycle analysis, were conducted on gastric adenocarcinoma cells treated with procaterol, both as a monotherapy and in combination with cisplatin. Procaterol, in synergy with cisplatin, significantly inhibited cell growth, suggesting a potentiated therapeutic effect. Thus, we propose the combined application of cisplatin and procaterol as a novel potential therapeutic strategy against human gastric cancer. The findings also highlight the relevance of CHK1 kinase as a drug target for enhancing the sensitivity of cytotoxic agents in cancer.

Polyadenosine diphosphate-ribose polymerase inhibitors: advances, implications, and challenges in tumor radiotherapy sensitization.

Polyadenosine diphosphate-ribose polymerase (PARP) is a key modifying enzyme in cells, which participates in single-strand break repair and indirectly affects double-strand break repair. PARP inhibitors have shown great potential in oncotherapy by exploiting DNA damage repair pathways, and several small molecule PARP inhibitors have been approved by the U.S. Food and Drug Administration for treating various tumor types. PARP inhibitors not only have significant antitumor effects but also have some synergistic effects when combined with radiotherapy; therefore they have potential as radiation sensitizers. Here, we reviewed the advances and implications of PARP inhibitors in tumor radiotherapy sensitization. First, we summarized the multiple functions of PARP and the mechanisms by which its inhibitors exert antitumor effects. Next, we discuss the immunomodulatory effects of PARP and its inhibitors in tumors. Then, we described the theoretical basis of using PARP inhibitors in combination with radiotherapy and outlined their importance in oncological radiotherapy. Finally, we reviewed the current challenges in this field and elaborated on the future applications of PARP inhibitors as radiation sensitizers. A comprehensive understanding of the mechanism, optimal dosing, long-term safety, and identification of responsive biomarkers remain key challenges to integrating PARP inhibition into the radiotherapy management of cancer patients. Therefore, extensive research in these areas would facilitate the development of precision radiotherapy using PARP inhibitors to improve patient outcomes.

Abstract LB_A19: Genome-wide CRISPR/Cas9 screens for the identification of targeted vulnerabilties in PARP inhibitor-resistant PARG;BRCA2-deficient tumor cells: A focus on EXO1/FEN1-mediated DNA repair

Abstract Targeting poly(ADP-ribose) glycohydrolase (PARG) is currently explored as a therapeutic approach to treat various cancer types, but we have a poor understanding of the specific genetic vulnerabilities that would make cancer cells susceptible to such a tailored therapy. Moreover, the identification of such vulnerabilities is of interest to target BRCA2;p53-deficient tumors that have acquired resistance to poly(ADP-ribose) polymerase inhibitors (PARPi), through loss of PARG expression. Here, by performing whole genome CRISPR/Cas9 drop-out screens we identify various genes involved in DNA repair to be essential for the survival of PARG;BRCA2;p53-deficient cells. In particular, our findings reveal EXO1 and FEN1 as major synthetic lethal interactors to PARG loss. We provide evidence that PARG;BRCA2;p53-deficient cells exhibit compromised replication fork progression, DNA single-strand break repair and Okazaki fragment processing, alterations that exacerbate and become lethal upon EXO1/FEN1 inhibition. Since this sensitivity is dependent on BRCA2 defects, we propose EXO1/FEN1 targeting in PARPi-resistant tumors that have lost PARG activity. Moreover, EXO1/FEN1 targeting may be a useful strategy to enhance the effect of PARG inhibitors in HR-deficient tumors. Citation Format: Christina Andronikou, Kamila Burdova, Diego Dibitetto, Cor Lieftink, Roderick L. Beijersbergen, Hana Hanzlikova, Jos Jonkers, Sven Rottenberg. Genome-wide CRISPR/Cas9 screens for the identification of targeted vulnerabilties in PARP inhibitor-resistant PARG;BRCA2-deficient tumor cells: A focus on EXO1/FEN1-mediated DNA repair [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_A19.

Analysis of the Relationship between PARP1 and BRCA1 Suggests PARP1 Gene Has a Role in Breast Cancer

Poly [ADP-ribose] polymerase 1 (PARP1), one of the genes in the PARP family, is mainly involved in the detection and repair of DNA damage in cells, and its upregulation has been associated with tumorigenesis. PARP1 is essential to all cells in the body as it ensures that DNA is replicated correctly. The PARP1 protein addresses repair of single-stranded breaks (SSBs) in DNA by initially binding near the point of break in the DNA. While it is bound to the region of the SSB, PARP1 transfers ADP ribosyl moiety from NAD+ to acceptor proteins. This leads to the recruitment of DNA repair proteins to the region of DNA breaks. The PARP1 protein also aids the double-stranded break (DSB) repair of DNA by recruiting the homologous recombination (HR) pathway proteins. Due to this role of PARP1 in DNA repair, it has been associated with cancer growth. Using databases, including UniProt, Xena Browser, and CBioPortal, this article explores the relationship between PARP1 mutations and BRCA1-mutated breast cancer. BRCA1 is a tumor suppressor gene, which is involved in DNA repair through the HR pathway. Therefore, in BRCA1-mutated cells, the lack of tumor suppressor factors leads to cancer growth. However, in a cell that has both BRCA1 mutations and PARP1 inhibition, DNA damage cannot be repaired, leading to apoptosis. Hence, PARP1 inhibitors have become essential in BRCA1-mutated breast cancers. There are several PARP1 inhibitors that are currently being used to treat breast cancer, which work using several mechanisms of action, including PARP1 trapping, which prevents the repair, transcription, and replication of DNA. This article also focuses on the clinical trials of three specific drugs, Olaparib, Iniparib, and Veliparib, which are used to treat breast cancer. However, due to the lack of understanding surrounding PARP1 inhibition mechanisms and potential drug combinations, more research needs to be done to understand potential biomarker targets and PARP1 inhibitor resistance.

Effects of the PARP Inhibitor Niraparib on the Radiosensitivity of Human Lung and Cervical Cancer Cells

DNA damage repair (DDR) initiated after DNA damage may lead to a decreased radiosensitivity of tumor cells or even radiation resistance. Previous studies indicate that PARP inhibitors block the repair of DNA single-strand breaks (SSBs), and unrepaired SSBs may continuously accumulate and form double-strand breaks (DSBs) that promotes tumor cell death. Studies have also shown that PARP inhibitors act synergistically with RT to inhibit DDR. In this study, we aim to investigate the effect of Niraparib, a clinically approved PARP inhibitor on the radiosensitivity of human lung and cervical cancer cells, and preliminarily explore the underlying mechanism. The human lung cancer cell line A549 and human cervical cancer cell line Siha were both treated with niraparib (for 1h), or X-ray (4Gy), or Niraparib (for 1h) combined with X-ray (4Gy). The effect was examined via measurements of cell proliferation by the cell counting kit-8 assay, and cell viability was detected by clone formation assays. Cell apoptosis and cell cycle distribution were investigated by flow cytometry. In human lung and cervical cancer cell lines, Niraparib combined with radiation therapy significantly inhibited cell proliferation. The proportion of apoptotic cells in cell lines treated with Niraparib plus radiation (the combination group) was significantly higher compared with control, radiation-only, and Niraparib-only groups. Additionally, the proportion of A549 cells in the G2/M phase was significantly increased in the combination group compared with the radiation-only group. The results support that PARP inhibitor Niraparib increases the radiosensitivity of tumor cells, promotes their apoptosis, and induces cell cycle redistribution. The possible mechanism is associated with the inhibition of radiation-induced DNA damage repair.

Double trouble for prostate cancer: synergistic action of AR blockade and PARPi in non-HRR mutated patients.

Prostate cancer (PCa) is the most common cancer in men worldwide. Despite better and more intensive treatment options in earlier disease stages, a large subset of patients still progress to metastatic castration-resistant PCa (mCRPC). Recently, poly-(ADP-ribose)-polymerase (PARP)-inhibitors have been introduced in this setting. The TALAPRO-2 and PROpel trials both showed a marked benefit of PARPi in combination with an androgen receptor signaling inhibitor (ARSI), compared with an ARSI alone in both the homologous recombination repair (HRR)-mutated, as well as in the HRR-non-mutated subgroup. In this review, we present a comprehensive overview of how maximal AR-blockade via an ARSI in combination with a PARPi has a synergistic effect at the molecular level, leading to synthetic lethality in both HRR-mutated and HRR-non-mutated PCa patients. PARP2 is known to be a cofactor of the AR complex, needed for decompacting the chromatin and start of transcription of AR target genes (including HRR genes). The inhibition of PARP thus reinforces the effect of an ARSI. The deep androgen deprivation caused by combining androgen deprivation therapy (ADT) with an ARSI, induces an HRR-like deficient state, often referred to as "BRCA-ness". Further, PARPi will prevent the repair of single-strand DNA breaks, leading to the accumulation of DNA double-strand breaks (DSBs). Due to the induced HRR-deficient state, DSBs cannot be repaired, leading to apoptosis.

Parp Inhibitors and Radiotherapy: A New Combination for Prostate Cancer (Systematic Review).

PARPi, in combination with ionizing radiation, has demonstrated the ability to enhance cellular radiosensitivity in different tumors. The rationale is that the exposure to radiation leads to both physical and biochemical damage to DNA, prompting cells to initiate three primary mechanisms for DNA repair. Two double-stranded DNA breaks (DSB) repair pathways: (1) non-homologous end-joining (NHEJ) and (2) homologous recombination (HR); and (3) a single-stranded DNA break (SSB) repair pathway (base excision repair, BER). In this scenario, PARPi can serve as radiosensitizers by leveraging the BER pathway. This mechanism heightens the likelihood of replication forks collapsing, consequently leading to the formation of persistent DSBs. Together, the combination of PARPi and radiotherapy is a potent oncological strategy. This combination has proven its efficacy in different tumors. However, in prostate cancer, there are only preclinical studies to support it and, recently, an ongoing clinical trial. The objective of this paper is to perform a review of the current evidence regarding the use of PARPi and radiotherapy (RT) in PCa and to give future insight on this topic.

The CSB chromatin remodeler regulates PARP1- and PARP2-mediated single-strand break repair at actively transcribed DNA regions.

Efficient repair of oxidized DNA is critical for genome-integrity maintenance. Cockayne syndrome protein B (CSB) is an ATP-dependent chromatin remodeler that collaborates with Poly(ADP-ribose) polymerase I (PARP1) in the repair of oxidative DNA lesions. How these proteins integrate during DNA repair remains largely unknown. Here, using chromatin co-fractionation studies, we demonstrate that PARP1 and PARP2 promote recruitment of CSB to oxidatively-damaged DNA. CSB, in turn, contributes to the recruitment of XRCC1, and histone PARylation factor 1 (HPF1), and promotes histone PARylation. Using alkaline comet assays to monitor DNA repair, we found that CSB regulates single-strand break repair (SSBR) mediated by PARP1 and PARP2. Strikingly, CSB's function in SSBR is largely bypassed when transcription is inhibited, suggesting CSB-mediated SSBR occurs primarily at actively transcribed DNA regions. While PARP1 repairs SSBs at sites regardless of the transcription status, we found that PARP2 predominantly functions in actively transcribed DNA regions. Therefore, our study raises the hypothesis that SSBR is executed by different mechanisms based on the transcription status.

A phase 1/2 study of combination olaparib and radium-223 in men with metastatic castration-resistant prostate cancer with bone metastases (COMRADE): A trial in progress.

TPS5110 Background: Radium-223, an α-emitting radioisotope inducing DNA double-stranded breaks leading to cell death has demonstrated improvement in overall survival (OS) in men with metastatic castration-resistant prostate cancer (mCRPC) with bone metastases. PARP inhibitors prevent the repair of DNA single-stranded breaks and have demonstrated clinical efficacy as monotherapy in mCRPC patients harboring alterations in the homologous recombination repair (HRR) pathway and in combination with androgen receptor signaling inhibitors irrespective of DNA repair mutation status. In preclinical models, PARP inhibitors have shown efficacy as radio sensitizing agents. We designed a phase 1/2 trial to identify the recommended phase 2 dose (RPD2) of olaparib and radium-223 and test the hypothesis that radium-223 + olaparib will demonstrate anti-tumor activity in mCRPC irrespective of homologous recombination repair deficiency (HRD) status (NCT03317392). Methods: This open-label, multicenter, phase 1/2 study evaluates the dosing, safety and efficacy of olaparib with radium-223 in men with mCRPC with bone metastases. The phase 1 component used a 3+3 dose escalation design to determine the olaparib recommended phase 2 dose (RP2D) + fixed dose radium-223 (55 kBq/kg IV every 4 weeks x 6). The phase 1 portion has completed accrual (n = 12) and demonstrated that the RP2D of olaparib is 200 mg by mouth twice daily when combined with standard dose radium-223. The phase 2 component of the study which was activated in 1/2021 is an open-label, randomized trial (1:1) evaluating olaparib + radium-223 compared to radium-223 alone. The inclusion criteria include patients with mCRPC with any number of prior therapy lines for mCRPC and ≥2 bone metastases by imaging and at least 1 lesion which has not been treated with prior radiation. Patients with visceral metastases or malignant lymphadenopathy ( > 4 cm in short diameter) are excluded. The primary endpoint for phase 2 is radiographic progression-free survival (rPFS) per PCWG3 guidelines for bone metastases and RECIST v1.1 for non-bone disease. The integrated biomarker is Oncopanel assay to determine HRR status. A key secondary endpoint will evaluate rPFS in biomarker positive and negative patients. Other secondary endpoints include time to PSA progression, PSA response, time to subsequent therapy, time to first skeletal event, and OS. Exploratory endpoints include WES/WTS of baseline biopsy tissue, assessment of RAD51 IHC and correlation with outcomes, ctDNA assessment and correlation with responses, evaluation of changes in the tumor immune microenvironment, and quality of life assessment. Target accrual is 133 patients. Clinical trial information: NCT03317392 .

The Fate of Oxidative Strand Breaks in Mitochondrial DNA.

Mitochondrial DNA (mtDNA) is particularly vulnerable to somatic mutagenesis. Potential mechanisms include DNA polymerase γ (POLG) errors and the effects of mutagens, such as reactive oxygen species. Here, we studied the effects of transient hydrogen peroxide (H2O2 pulse) on mtDNA integrity in cultured HEK 293 cells, applying Southern blotting, ultra-deep short-read and long-read sequencing. In wild-type cells, 30 min after the H2O2 pulse, linear mtDNA fragments appear, representing double-strand breaks (DSB) with ends characterized by short GC stretches. Intact supercoiled mtDNA species reappear within 2-6 h after treatment and are almost completely recovered after 24 h. BrdU incorporation is lower in H2O2-treated cells compared to non-treated cells, suggesting that fast recovery is not associated with mtDNA replication, but is driven by rapid repair of single-strand breaks (SSBs) and degradation of DSB-generated linear fragments. Genetic inactivation of mtDNA degradation in exonuclease deficient POLG p.D274A mutant cells results in the persistence of linear mtDNA fragments with no impact on the repair of SSBs. In conclusion, our data highlight the interplay between the rapid processes of SSB repair and DSB degradation and the much slower mtDNA re-synthesis after oxidative damage, which has important implications for mtDNA quality control and the potential generation of somatic mtDNA deletions.