Homologous Recombination-mediated DNA Repair Research Articles

LncRNA LINC01664 promotes cancer resistance through facilitating homologous recombination-mediated DNA repair

The intracellular responses to DNA double-strand breaks (DSB) repair are crucial for genomic stability and play an essential role in cancer resistance. In addition to canonical DSB repair proteins, long non-coding RNAs (lncRNAs) have been found to be involved in this sophisticated network. In the present study, we performed a loss-of-function screen for a customized siRNA Premix Library to identify lncRNAs that participate in homologous recombination (HR) process. Among the candidates, we identified LINC01664 as a novel lncRNA required for HR repair. Furthermore, LINC01664 knockdown significantly increased the sensitivity of cancer cells to DNA damage agents such as ionizing radiation and genotoxic drugs. Mechanistically, LINC01664 interacted with Sirt1 promoter and then activated Sirt1 transcription, which contributed to HR-mediated DNA damage repair. In summary, our findings revealed a new mechanism of LINC01664 in DNA damage repair, providing evidence for a potential therapeutic strategy for eliminating the treatment bottlenecks caused by cancer resistance to chemotherapy and radiotherapy.

DNA damage-induced proteasome phosphorylation controls substrate recognition and facilitates DNA repair

Upon DNA damage, numerous proteins are targeted for ubiquitin-dependent proteasomal degradation, which is an integral part of the DNA repair program. Although details of the ubiquitination processes have been intensively studied, little is known about whether and how the 26S proteasome is regulated in the DNA damage response (DDR). Here, we show that human Rpn10/PSMD4, one of the three ubiquitin receptors of the 26S proteasome, is rapidly phosphorylated in response to different types of DNA damage. The phosphorylation occurs at Rpn10-Ser266 within a conserved SQ motif recognized by ATM/ATR/DNA-PK. Blockade of S266 phosphorylation attenuates homologous recombination-mediated DNA repair and sensitizes cells to genotoxic insults. In vitro and in cellulo experiments indicate that phosphorylation of S266, located in the flexible linker between the two ubiquitin-interacting motifs (UIMs) of Rpn10, alters the configuration of UIMs, and actually reduces ubiquitin chain (substrate) binding. As a result, essential DDR proteins such as BRCA1 are spared from premature degradation and allowed sufficient time to engage in DNA repair, a scenario supported by proximity labeling and quantitative proteomic studies. These findings reveal an inherent self-limiting mechanism of the proteasome that, by controlling substrate recognition through Rpn10 phosphorylation, fine-tunes protein degradation for optimal responses under stress.

NSUN6-mediated 5-methylcytosine modification of NDRG1 mRNA promotes radioresistance in cervical cancer

BackgroundRadioresistance is the leading cause of death in advanced cervical cancer (CC). Dysregulation of RNA modification has recently emerged as a regulatory mechanism in radiation and drug resistance. We aimed to explore the biological function and clinical significance of 5-methylcytosine (m5C) in cervical cancer radiosensitivity.MethodsThe abundance of RNA modification in radiotherapy-resistant and sensitive CC specimens was quantified by liquid chromatography-tandem mass spectrometry. The essential RNA modification-related genes involved in CC radiosensitivity were screened via RNA sequencing. The effect of NSUN6 on radiosensitivity was verified in CC cell lines, cell-derived xenograft (CDX), and 3D bioprinted patient-derived organoid (PDO). The mechanisms of NSUN6 in regulating CC radiosensitivity were investigated by integrative m5C sequencing, mRNA sequencing, and RNA immunoprecipitation.ResultsWe found a higher abundance of m5C modification in resistant CC samples, and NSUN6 was the essential m5C-regulating gene concerning radiosensitivity. NSUN6 overexpression was clinically correlated with radioresistance and poor prognosis in cervical cancer. Functionally, higher NSUN6 expression was associated with radioresistance in the 3D PDO model of cervical cancer. Moreover, silencing NSUN6 increased CC radiosensitivity in vivo and in vitro. Mechanistically, NDRG1 was one of the downstream target genes of NSUN6 identified by integrated m5C-seq, mRNA-seq, and functional validation. NSUN6 promoted the m5C modification of NDRG1 mRNA, and the m5C reader ALYREF bound explicitly to the m5C-labeled NDRG1 mRNA and enhanced NDRG1 mRNA stability. NDRG1 overexpression promoted homologous recombination-mediated DNA repair, which in turn led to radioresistance in cervical cancer.ConclusionsAberrant m5C hypermethylation and NSUN6 overexpression drive resistance to radiotherapy in cervical cancer. Elevated NSUN6 expression promotes radioresistance in cervical cancer by activating the NSUN6/ALYREF-m5C-NDRG1 pathway. The low expression of NSUN6 in cervical cancer indicates sensitivity to radiotherapy and a better prognosis.

Homologous recombination proficient subtypes of high-grade serous ovarian cancer: treatment options for a poor prognosis group.

Approximately 50% of tubo-ovarian high-grade serous carcinomas (HGSCs) have functional homologous recombination-mediated (HR) DNA repair, so-called HR-proficient tumors, which are often associated with primary platinum resistance (relapse within six months after completion of first-line therapy), minimal benefit from poly(ADP-ribose) polymerase (PARP) inhibitors, and shorter survival. HR-proficient tumors comprise multiple molecular subtypes including cases with CCNE1 amplification, AKT2 amplification or CDK12 alteration, and are often characterized as "cold" tumors with fewer infiltrating lymphocytes and decreased expression of PD-1/PD-L1. Several new treatment approaches aim to manipulate these negative prognostic features and render HR-proficient tumors more susceptible to treatment. Alterations in multiple different molecules and pathways in the DNA damage response are driving new drug development to target HR-proficient cancer cells, such as inhibitors of the CDK or P13K/AKT pathways, as well as ATR inhibitors. Treatment combinations with chemotherapy or PARP inhibitors and agents targeting DNA replication stress have shown promising preclinical and clinical results. New approaches in immunotherapy are also being explored, including vaccines or antibody drug conjugates. Many approaches are still in the early stages of development and further clinical trials will determine their clinical relevance. There is a need to include HR-proficient tumors in ovarian cancer trials and to analyze them in a more targeted manner to provide further evidence for their specific therapy, as this will be crucial in improving the overall prognosis of HGSC and ovarian cancer in general.

ZNF827 is a single-stranded DNA binding protein that regulates the ATR-CHK1 DNA damage response pathway

The ATR-CHK1 DNA damage response pathway becomes activated by the exposure of RPA-coated single-stranded DNA (ssDNA) that forms as an intermediate during DNA damage and repair, and as a part of the replication stress response. Here, we identify ZNF827 as a component of the ATR-CHK1 kinase pathway. We demonstrate that ZNF827 is a ssDNA binding protein that associates with RPA through concurrent binding to ssDNA intermediates. These interactions are dependent on two clusters of C2H2 zinc finger motifs within ZNF827. We find that ZNF827 accumulates at stalled forks and DNA damage sites, where it activates ATR and promotes the engagement of homologous recombination-mediated DNA repair. Additionally, we demonstrate that ZNF827 depletion inhibits replication initiation and sensitizes cancer cells to the topoisomerase inhibitor topotecan, revealing ZNF827 as a therapeutic target within the DNA damage response pathway.

Genetic analysis of cervical cancer with lymph node metastasis.

To find out the differences in gene characteristics between cervical cancer patients with and without lymph node metastasis, and to provide reference for therapy. From January 2018 to June 2022, recurrent cervical cancer patients 39 cases with lymph node metastasis and 73 cases without lymph node metastasis underwent testing of 1,021 cancer-related genes by next-generation sequencing. Maftools software was used to analyze somatic single nucleotide/insertion-deletion variation mutation, co-occurring mutation, cosmic mutation characteristics, oncogenic signaling pathways. EP300 and FBXW7 were significantly enriched in lymph node-positive patients. Lymph node-positive patients with EP300 or FBXW7 mutations had lower overall survival (OS) after recurrence. Both lymph node-positive and -negative patients had plenty of co-occurring mutations but few mutually exclusive mutations. Lymph node-positive co-occurring mutation number ≥6 had lower OS, while lymph node-negative co-occurring mutation number ≥3 had lower OS after recurrence. The etiology of SBS3 was defects in DNA double strand break repair by homologous recombination, which exclusively exist in lymph node-positive patients. There was no difference in median tumor mutation burden (TMB) between positive and negative lymph nodes, but TMB was significantly associated with PIK3CA mutation. The somatic SNV/Indels of EP300 and FBXW7, SBS3 homologous recombination-mediated DNA repair defect were enriched in lymph node-positive patients. For lymph node-positive patients, EP300 or FBXW7 mutations predicted poor prognosis. No matter lymph node-positive or negative, more co-occurring mutation number predicted poor prognosis. PIK3CA mutation may account for the higher TMB and help identify patients who benefit from immunotherapy.

Small molecule nitroalkenes inhibit RAD51-mediated homologous recombination and amplify triple-negative breast cancer cell killing by DNA-directed therapies

Nitro fatty acids (NO2-FAs) are endogenously generated lipid signaling mediators from metabolic and inflammatory reactions between conjugated diene fatty acids and nitric oxide or nitrite-derived reactive species. NO2-FAs undergo reversible Michael addition with hyperreactive protein cysteine thiolates to induce posttranslational protein modifications that can impact protein function. Herein, we report a novel mechanism of action of natural and non-natural nitroalkenes structurally similar to (E) 10-nitro-octadec-9-enoic acid (CP-6), recently de-risked by preclinical Investigational New Drug-enabling studies and Phase 1 and Phase 2 clinical trials and found to induce DNA damage in a TNBC xenograft by inhibiting homologous-recombination (HR)-mediated repair of DNA double-strand breaks (DSB). CP-6 specifically targets Cys319, essential in RAD51-controlled HR-mediated DNA DSB repair in cells. A nitroalkene library screen identified two structurally different nitroalkenes, a non-natural fatty acid [(E) 8-nitro-nonadec-7-enoic acid (CP-8)] and a dicarboxylate ester [dimethyl (E)nitro-oct-4-enedioate (CP-23)] superior to CP-6 in TNBC cells killing, synergism with three different inhibitors of the poly ADP-ribose polymerase (PARP) and γ-IR. CP-8 and CP-23 effectively inhibited γ-IR-induced RAD51 foci formation and HR in a GFP-reported assay but did not affect benign human epithelial cells or cell cycle phases. In vivo, CP-8 and CP-23's efficacies diverged as only CP-8 showed promising anticancer activities alone and combined with the PARP inhibitor talazoparib in an HR-proficient TNBC mouse model. As preliminary preclinical toxicology analysis also suggests CP-8 as safe, our data endorse CP-8 as a novel anticancer molecule for treating cancers sensitive to homologous recombination-mediated DNA repair inhibitors.

FBL promotes cancer cell resistance to DNA damage and BRCA1 transcription via YBX1.

Fibrillarin (FBL) is a highly conserved nucleolar methyltransferase responsible for methylation of ribosomal RNA and proteins. Here, we reveal a role for FBL in DNA damage response and its impact on cancer proliferation and sensitivity to DNA-damaging agents. FBL is highly expressed in various cancers and correlates with poor survival outcomes in cancer patients. Knockdown of FBL sensitizes tumor cells and xenografts to DNA crosslinking agents, and leads to homologous recombination-mediated DNA repair defects. We identify Y-box-binding protein-1 (YBX1) as a key interacting partner of FBL, and FBL increases the nuclear accumulation of YBX1 in response to DNA damage. We show that FBL promotes the expression of BRCA1 by increasing the binding of YBX1 to the BRCA1 promoter. Our study sheds light on the regulatory mechanism of FBL in tumorigenesis and DNA damage response, providing potential therapeutic targets to overcome chemoresistance in cancer.

DNA Repair and Therapeutic Strategies in Cancer Stem Cells.

First-line cancer treatments successfully eradicate the differentiated tumour mass but are comparatively ineffective against cancer stem cells (CSCs), a self-renewing subpopulation thought to be responsible for tumour initiation, metastasis, heterogeneity, and recurrence. CSCs are thus presented as the principal target for elimination during cancer treatment. However, CSCs are challenging to drug target because of numerous intrinsic and extrinsic mechanisms of drug resistance. One such mechanism that remains relatively understudied is the DNA damage response (DDR). CSCs are presumed to possess properties that enable enhanced DNA repair efficiency relative to their highly proliferative bulk progeny, facilitating improved repair of double-strand breaks induced by radiotherapy and most chemotherapeutics. This can occur through multiple mechanisms, including increased expression and splicing fidelity of DNA repair genes, robust activation of cell cycle checkpoints, and elevated homologous recombination-mediated DNA repair. Herein, we summarise the current knowledge concerning improved genome integrity in non-transformed stem cells and CSCs, discuss therapeutic opportunities within the DDR for re-sensitising CSCs to genotoxic stressors, and consider the challenges posed regarding unbiased identification of novel DDR-directed strategies in CSCs. A better understanding of the DDR mediating chemo/radioresistance mechanisms in CSCs could lead to novel therapeutic approaches, thereby enhancing treatment efficacy in cancer patients.

A 1,2,3-Triazole Derivative of Quinazoline Exhibits Antitumor Activity by Tethering RNF168 to SQSTM1/P62.

Quinazoline and its derivatives have drawn much attention in the development of potential antitumor agents. Here, we synthesized a series of 1,2,3-triazole derivatives of quinazoline at the C6 position and evaluated for their cytotoxic activity in various human cancer cell lines. We found that compound 5a was the most cytotoxic to HCT-116 cells (IC50, 0.36 μM). Target profiling found that 5a directly binds to both the autophagy-associated protein SQSTM1/P62 and the E3 ligase RNF168, promoting their interaction. Consistently, 5a treatment induces a decrease in RNF168-mediated H2A ubiquitination and compromises homologous recombination-mediated DNA repair, thus increasing the sensitivity of HCT-116 to X-ray radiation. Moreover, 5a suppressed xenografted tumor growth in mice in a dose-dependent manner. Taken together, the 1,2,3-triazole derivative of quinazoline 5a may serve as a novel compound for tumor therapy based on its role in promoting a P62/RNF168 interaction.

Therapeutic targeting of ATR in alveolar rhabdomyosarcoma

Despite advances in multi-modal treatment approaches, clinical outcomes of patients suffering from PAX3-FOXO1 fusion oncogene-expressing alveolar rhabdomyosarcoma (ARMS) remain dismal. Here we show that PAX3-FOXO1-expressing ARMS cells are sensitive to pharmacological ataxia telangiectasia and Rad3 related protein (ATR) inhibition. Expression of PAX3-FOXO1 in muscle progenitor cells is not only sufficient to increase sensitivity to ATR inhibition, but PAX3-FOXO1-expressing rhabdomyosarcoma cells also exhibit increased sensitivity to structurally diverse inhibitors of ATR. Mechanistically, ATR inhibition leads to replication stress exacerbation, decreased BRCA1 phosphorylation and reduced homologous recombination-mediated DNA repair pathway activity. Consequently, ATR inhibitor treatment increases sensitivity of ARMS cells to PARP1 inhibition in vitro, and combined treatment with ATR and PARP1 inhibitors induces complete regression of primary patient-derived ARMS xenografts in vivo. Lastly, a genome-wide CRISPR activation screen (CRISPRa) in combination with transcriptional analyses of ATR inhibitor resistant ARMS cells identifies the RAS-MAPK pathway and its targets, the FOS gene family, as inducers of resistance to ATR inhibition. Our findings provide a rationale for upcoming biomarker-driven clinical trials of ATR inhibitors in patients suffering from ARMS.

Abstract 2658: NOS inhibition augments PI3K inhibitor-induced DNA damage and mesenchymal-to-epithelial transition in metaplastic breast cancer

Abstract Purpose: Metaplastic breast cancer (MpBC) is a highly chemoresistant and aggressive breast cancer variant with limited therapeutic options. Most MpBCs harbor a triple-negative breast cancer (TNBC) phenotype, yet have a worse prognosis compared to TNBC. We and others discovered two key molecular alterations in MpBC associated with poor overall survival: 1) hyperactivation of the phosphoinositide 3-kinase (PI3K) signaling pathway and 2) enhanced inducible nitric oxide synthase (iNOS) signaling. Nitric oxide produced from iNOS is able to activate various oncogenic pathways, such as PI3K signaling. In MpBC, both PI3K and NOS signaling pathways may synergistically work to enhance chemoresistance. We propose to evaluate whether pan-NOS inhibitor NG-monomethyl-l-arginine (L-NMMA) augments the efficacy of alpha isoform-specific PI3K inhibitor alpelisib in MpBC in vitro and in vivo models. Methods: MpBC cell lines (Hs578T, BT549, SUM159) and MpBC Patient-Derived Xenograft (PDX) models were used in this study. Cell viability was determined with SRB Assay and the combination index was evaluated using Chou-Talalay Method. Flow cytometry was conducted to evaluate cellular apoptosis and cell cycle distribution analysis. In vivo efficacy of NOS and PI3K inhibition was assessed using MpBC PDX models. Transcriptional analysis of PDX tumors treated with vehicle control, single-agent (L-NMMA or alpelisib), or combination therapy (L-NMMA+alpelisib) was conducted to reveal top enriched pathways. Immunoblotting and immunofluorescence analyses of treated MpBC cell lines and PDX tissues were performed. Results: Combination of L-NMMA and alpelisib synergistically reduced cellular proliferation (CI<1), enhanced apoptosis, depleted nucleotide pools, and arrested MpBC cell lines in G2/M phase of cell cycle relative to single-agent treatment. Furthermore, NOS+PI3K inhibition significantly suppressed tumor volume in MpBC PDX models with PIK3CA mutations, although less significantly in MpBC PIK3CA-wild type PDX models, augmented efficacy of taxane chemotherapy, and improved overall survival. Transcriptional analysis of MpBC PDX tumors treated with combination therapy versus vehicle control revealed that epithelial-to-mesenchymal transition, DNA repair, and E2F targets were among the top enriched pathways, suggesting their plausible involvement in the mechanism of response. Immunoblotting/immunofluorescence analyses of cell lines/PDX models confirmed that combination therapy enhances DNA damage, reduces expression of homologous recombination-mediated DNA repair proteins, and induces mesenchymal-to-epithelial transition, rendering MpBC cells potentially more chemosensitive. Conclusion: Combined NOS and PI3K inhibition is a novel therapeutic strategy that may improve the efficacy of chemotherapy in patients with MpBC. Citation Format: Tejaswini P. Reddy, Akshjot Puri, Liliana Guzman-Rojas, Bijan Mahboubi, Wei Qian, Jianying Zhou, Baek Kim, Stacy Moulder, Helen Piwnica-Worms, Roberto Rosato, Jenny C. Chang. NOS inhibition augments PI3K inhibitor-induced DNA damage and mesenchymal-to-epithelial transition in metaplastic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2658.

BRCA1 and Metastasis: Outcome of Defective DNA Repair.

Simple SummaryBRCA1 has critical functions in accurately repairing double stand breaks in the DNA through a process known as homologous recombination. BRCA1 also has various functions in other cellular processes that safeguard the genome. Thus, mutations or silencing of this tumor suppressor significantly increases the risk of developing breast, ovarian, and other cancers. Metastasis refers to the spread of cancer to other parts of the body and is the leading cause of cancer-related deaths. In this review, we discuss the mechanisms by which BRCA1 mutations contribute to the metastatic and aggressive nature of the tumor cells.Heritable mutations in BRCA1 and BRCA2 genes are a major risk factor for breast and ovarian cancer. Inherited mutations in BRCA1 increase the risk of developing breast cancers by up to 72% and ovarian cancers by up to 69%, when compared to individuals with wild-type BRCA1. BRCA1 and BRCA2 (BRCA1/2) are both important for homologous recombination-mediated DNA repair. The link between BRCA1/2 mutations and high susceptibility to breast cancer is well established. However, the potential impact of BRCA1 mutation on the individual cell populations within a tumor microenvironment, and its relation to increased aggressiveness of cancer is not well understood. The objective of this review is to provide significant insights into the mechanisms by which BRCA1 mutations contribute to the metastatic and aggressive nature of the tumor cells.

HMOB2 deficiency impairs homologous recombination-mediated DNA repair and sensitises cancer cells to PARP inhibitors

Monopolar spindle-one binder (MOBs) proteins are evolutionarily conserved and contribute to various cellular signalling pathways. Recently, we reported that hMOB2 functions in preventing the accumulation of endogenous DNA damage and a subsequent p53/p21-dependent G1/S cell cycle arrest in untransformed cells. However, the question of how hMOB2 protects cells from endogenous DNA damage accumulation remained enigmatic. Here, we uncover hMOB2 as a regulator of double-strand break (DSB) repair by homologous recombination (HR). hMOB2 supports the phosphorylation and accumulation of the RAD51 recombinase on resected single-strand DNA (ssDNA) overhangs. Physiologically, hMOB2 expression supports cancer cell survival in response to DSB-inducing anti-cancer compounds. Specifically, loss of hMOB2 renders ovarian and other cancer cells more vulnerable to FDA-approved PARP inhibitors. Reduced MOB2 expression correlates with increased overall survival in patients suffering from ovarian carcinoma. Taken together, our findings suggest that hMOB2 expression may serve as a candidate stratification biomarker of patients for HR-deficiency targeted cancer therapies, such as PARP inhibitor treatments.

Meiotic Cohesin and Variants Associated With Human Reproductive Aging and Disease.

Successful human reproduction relies on the well-orchestrated development of competent gametes through the process of meiosis. The loading of cohesin, a multi-protein complex, is a key event in the initiation of mammalian meiosis. Establishment of sister chromatid cohesion via cohesin rings is essential for ensuring homologous recombination-mediated DNA repair and future proper chromosome segregation. Cohesin proteins loaded during female fetal life are not replenished over time, and therefore are a potential etiology of age-related aneuploidy in oocytes resulting in decreased fecundity and increased infertility and miscarriage rates with advancing maternal age. Herein, we provide a brief overview of meiotic cohesin and summarize the human genetic studies which have identified genetic variants of cohesin proteins and the associated reproductive phenotypes including primary ovarian insufficiency, trisomy in offspring, and non-obstructive azoospermia. The association of cohesion defects with cancer predisposition and potential impact on aging are also described. Expansion of genetic testing within clinical medicine, with a focus on cohesin protein-related genes, may provide additional insight to previously unknown etiologies of disorders contributing to gamete exhaustion in females, and infertility and reproductive aging in both men and women.

Helicobacter pylori CagA elicits BRCAness to induce genome instability that may underlie bacterial gastric carcinogenesis

Infection with CagA-producing Helicobacter pylori plays a causative role in the development of gastric cancer. Upon delivery into gastric epithelial cells, CagA deregulates prooncogenic phosphatase SHP2 while inhibiting polarity-regulating kinase PAR1b through complex formation. Here, we show that CagA/PAR1b interaction subverts nuclear translocation of BRCA1 by inhibiting PAR1b-mediated BRCA1 phosphorylation. It hereby induces BRCAness that promotes DNA double-strand breaks (DSBs) while disabling error-free homologous recombination-mediated DNA repair. The CagA/PAR1b interaction also stimulates Hippo signaling that circumvents apoptosis of DNA-damaged cells, giving cells time to repair DSBs through error-prone mechanisms. The DSB-activated p53-p21Cip1 axis inhibits proliferation of CagA-delivered cells, but the inhibition can be overcome by p53 inactivation. Indeed, sequential pulses of CagA in TP53-mutant cells drove somatic mutation with BRCAness-associated genetic signatures. Expansion of CagA-delivered cells with BRCAness-mediated genome instability, from which CagA-independent cancer-predisposing cells arise, provides a plausible "hit-and-run mechanism" of H.pylori CagA for gastric carcinogenesis.

ASPM promotes homologous recombination-mediated DNA repair by safeguarding BRCA1 stability

SummaryDNA double-strand break (DSB) repair by homologous recombination (HR) is essential for ensuring genome stability. Abnormal spindle-like microcephaly-associated (ASPM) gene encodes a spindle protein that is commonly implicated in primary microcephaly. We found that ASPM is recruited to sites of DNA damage in a PARP2-dependent manner. ASPM interacts with BRCA1 and its E3 ligase HERC2, preventing HERC2 from accessing to BRCA1 and ensuring BRCA1 stability. Inhibition of ASPM expression promotes HERC2-mediated BRCA1 degradation, compromises HR repair efficiency and chromosome stability, and sensitizes cancer cells to ionizing radiation. Moreover, we observed a synergistic effect between ASPM and PARP inhibition in killing cancer cells. This research has uncovered a novel function for ASPM in facilitating HR-mediated repair of DSBs by ensuring BRCA1 stability. ASPM might constitute a promising target for synthetic lethality-based cancer therapy.

Enhanced Efficacy of Combined Therapy with Checkpoint Kinase 1 Inhibitor and Rucaparib via Regulation of Rad51 Expression in BRCA Wild-Type Epithelial Ovarian Cancer Cells.

PurposeThis study aimed to evaluate anticancer effects of combination treatment with poly(ADP-ribose) polymerase (PARP) and checkpoint kinase 1 (Chk1) inhibitors in BRCA wild-type ovarian cancer. PARP inhibitors can function as DNA-damaging agents in BRCA wild-type cancer, even if clinical activity is limited. Most epithelial ovarian cancers are characterized by a TP53 mutation causing dysfunction at the G1/S checkpoint, which makes tumor cells highly dependent on Chk1-mediated G/M phase cell-cycle arrest for DNA repair.Materials and MethodsWe investigated the anticancer effects of combination treatment with prexasertib (LY2606368), a selective ATP competitive small molecule inhibitor of Chk1 and Chk2, and rucaparib, a PARP inhibitor, in BRCA wild-type ovarian cancer cell lines (OVCAR3 and SKOV3).ResultsWe found that combined treatment significantly decreased cell viability in all cell lines and induced greater DNA damage and apoptosis than in the control and/or using monotherapies. Moreover, we found that prexasertib significantly inhibited homologous recombination–mediated DNA repair and thus showed a marked anticancer effect in combination treatment with rucaparib. The anticancer mechanism of prexasertib and rucaparib was considered to be caused by an impaired G2/M checkpoint due to prexasertib treatment, which forced mitotic catastrophe in the presence of rucaparib.ConclusionOur results suggest a novel effective therapeutic strategy for BRCA wild-type epithelial ovarian cancer using a combination of Chk1 and PARP inhibitors.

Inhibition of ELF3 confers synthetic lethality of PARP inhibitor in non-small cell lung cancer

Background E74 Like ETS Transcription Factor 3 (ELF3) functions as a transcriptional factor to regulate non-small cell lung cancer (NSCLC) differentiation and progression. Poly(ADP-ribose) polymerase (PARP) inhibitors demonstrate anti-tumor effect in NSCLC. This study aimed to investigate whether ELF3 confers synthetic lethal with PARP inhibitor in NSCLC. Materials and methods The sensitivity of PARP inhibitor, Olaparib, to different NSCLC cell lines was determined by half maximal inhibitory concentration (IC50). Expression of ELF3 in NSCLC cell lines was evaluated by western blot. The effects of ELF3 on cytotoxicity of Olaparib to NSCLC were investigated by MTT (3-(4,5- di methyl thiazol −2-yl)-2,5-di phenyl tetrazolium bromide) and colony formation assays. The underlying mechanism involved in synthetic lethality with ELF3 and PARP inhibitors in NSCLC were detected by immunofluorescence and Western blot. Results ELF3 was up-regulated in NSCLC cell lines exhibiting resistance to PARP inhibitor, Olaparib. Knock down of ELF3 decreased the sensitivity and enhanced cytotoxicity of Olaparib to NSCLC cells. Moreover, knock down of ELF3 increased S139 phosphorylated histone H2AX (γH2AX), and inhibited homologous recombination activity via down-regulation of DNA repair protein RAD51 homolog 1 (RAD51), thus showing deficiency in DNA damage repair. Over-expression of ELF3 could up-regulate phosphorylation of AKT (Protein kinase B), while knock down of ELF3 regulated homologous recombination-mediated DNA repair via down-regulation of phosphorylation of AKT. Conclusion Knock down of ELF3 revealed homologous recombination deficiency via AKT signaling pathway, and synthetic lethality with ELF3 inhibition and PARP inhibitor indicated the clinical significance of PARP inhibitor in ELF3-deficient NSCLC.

Global crotonylome reveals CDYL-regulated RPA1 crotonylation in homologous recombination-mediated DNA repair.

Previously, we reported that chromodomain Y-like (CDYL) acts as a crotonyl-coenzyme A hydratase and negatively regulates histone crotonylation (Kcr). However, the global CDYL-regulated crotonylome remains unclear. Here, we report a large-scale proteomics analysis for protein Kcr. We identify 14,311 Kcr sites across 3734 proteins in HeLa cells, providing by far the largest crotonylome dataset. We show that depletion of CDYL alters crotonylome landscape affecting diverse cellular pathways. Specifically, CDYL negatively regulated Kcr of RPA1, and mutation of the Kcr sites of RPA1 impaired its interaction with single-stranded DNA and/or with components of resection machinery, supporting a key role of RPA1 Kcr in homologous recombination DNA repair. Together, our study indicates that protein crotonylation has important implication in various pathophysiological processes.