PARP inhibitor Olaparib increases the sensitization to radiotherapy in FaDu cells

Radioresistance causes a major problem for improvement of outcomes of patients treated with radiation. Targeting for DNA repair deficient mechanisms is a hallmark of sensitization to resistance. We tested whether Olaparib, a (poly) ADP‐ribose polymerase (PARP) inhibitor, can sensitize the radioresistant FaDu cells to radiotherapy. Radioresistant FaDu cells, called FaDu‐RR cells, were used as the radioresistant hypopharyngeal cancer models. The expression of PARP1 was detected in both FaDu and FaDu‐RR cells. The role of Olaparib in radiosensitization was analysed with several assays including clonogenic cell survival, cell proliferation and cell cycle, and radioresistant xenograft. High expression of PARP1 had a significant effect on enhancing radioresistance in FaDu‐RR cells compared with FaDu cells. After treatment of Olaparib, FaDu‐RR cells showed significantly less and smaller surviving colonies, lower proliferation ability and G2/M arrest than those in the group without treatment. Moreover, Olaparib significantly reduced growth of tumours in FaDu‐RR cell xenografts treated with ionizing radiation. Olaparib can significantly inhibit PARP1 expression and consequently has significant effects on radiosensitization in FaDu‐RR cells. These results indicate that Olaparib may help individualize treatment and improve their outcomes of hypopharyngeal cancer patients treated with radiation.     

Protein Expression of DNA Damage Repair Proteins Dictates Response to Topoisomerase and PARP Inhibitors in Triple-Negative Breast Cancer

Patients with metastatic triple-negative breast cancer (TNBC) have a poor prognosis. New approaches for the treatment of TNBC are needed to improve patient survival. The concept of synthetic lethality, brought about by inactivating complementary DNA repair pathways, has been proposed as a promising therapeutic option for these tumors. The TNBC tumor type has been associated with BRCA mutations, and inhibitors of Poly (ADP-ribose) polymerase (PARP), a family of proteins that facilitates DNA repair, have been shown to effectively kill BRCA defective tumors by preventing cells from repairing DNA damage, leading to a loss of cell viability and clonogenic survival. Here we present preclinical efficacy results of combining the PARP inhibitor, ABT-888, with CPT-11, a topoisomerase I inhibitor. CPT-11 binds to topoisomerase I at the replication fork, creating a bulky adduct that is recognized as damaged DNA. When DNA damage was stimulated with CPT-11, protein expression of the nucleotide excision repair enzyme ERCC1 inversely correlated with cell viability, but not clonogenic survival. However, 4 out of the 6 TNBC cells were synergistically responsive by cell viability and 5 out of the 6 TNBC cells were synergistically responsive by clonogenic survival to the combination of ABT-888 and CPT-11. In vivo, the BRCA mutant cell line MX-1 treated with CPT-11 alone demonstrated significant decreased tumor growth; this decrease was enhanced further with the addition of ABT-888. Decrease in tumor growth correlated with an increase in double strand DNA breaks as measured by γ-H2AX phosphorylation. In summary, inhibiting two arms of the DNA repair pathway simultaneously in TNBC cell lines, independent of BRCA mutation status, resulted in un-repairable DNA damage and subsequent cell death.     

Poly (ADP-ribose) polymerase inhibition synergizes with the NF-κB inhibitor DHMEQ to kill hepatocellular carcinoma cells

Poly (ADP-ribose) polymerase (PARP) enzymes play a key role in the cellular machinery responsible for DNA repair. Dehydroxymethylepoxyquinomicin (DHMEQ), a new inhibitor of NF-κB, induces oxidative stress and DNA damage. The effects of DHMEQ in combination with Olaparib (PARP inhibitor) were studied on hepatocellular carcinoma (HCC) cells. The DHMEQ–Olaparib combination synergistically inhibited cell viability, cell proliferation and colony formation of Hep3B, but had additive effects on Huh7 cells. The synergistic effects of the combination correlated with increased apoptosis, caspase 3/7 activity and PARP cleavage. There was an induction of an endoplasmic reticulum (ER) stress response with significant up-regulation of CHOP and TRB3 genes and splicing of XBP1 mRNA in Hep3B cells but not in Huh7 cells. Silencing of the TRB3 mRNA in Hep3B cells reversed the reduction in viability caused by DHMEQ–Olaparib treatment, while depletion of unspliced XBP1 mRNA in DHMEQ–Olaparib-treated Huh7 cells reduced viability. ROS production was increased after DHMEQ–Olaparib treatment of Hep3B, which caused DNA damage by an accumulation of γH2AX, increased AKT phosphorylation and reduced cell viability. The combination reduced Rad51 nuclear foci in Hep3B cells (not Huh7 cells), and silencing of Rad51 enhanced sensitivity of Huh7 cells to the DHMEQ–Olaparib combination. Knockdown of AKT in Hep3B cells restored the number of Rad51 nuclear foci after DHMEQ–Olaparib treatment. In summary, the DHMEQ–Olaparib combination induced ROS production, which killed HCC cells via DNA damage that could not be repaired by Rad51.SummaryPARPs and NF-κB are frequently deregulated in HCC. The DHMEQ–Olaparib combination exerted synergistic anti-tumour effects on HCC cells through ROS production via DNA damage that could not be repaired by Rad51. This suggested that the DHMEQ–Olaparib combination could be used to treat tumours that were resistant to Olaparib treatment.     

IKZF1 selectively enhances homologous recombination repair by interacting with CtIP and USP7 in multiple myeloma

Rationale: In multiple myeloma (MM), the activities of non-homologous end joining (NHEJ) and homologous recombination repair (HR) are increased compared with healthy controls. Whether and how IKZF1 as an enhancer of MM participates in the DNA repair pathway of tumor cells remains elusive.Methods: We used an endonuclease AsiSI-based system and quantitative chromatin immunoprecipitation assay (qChIP) analysis to test whether IKZF1 is involved in DNA repair. Immunopurification and mass spectrometric (MS) analysis were performed in MM1.S cells to elucidate the molecular mechanism that IKZF1 promotes DNA damage repair. The combination effect of lenalidomide or USP7 inhibitor with PARP inhibitor on cell proliferation was evaluated using MM cells in vitro and in vivo.Results: We demonstrate that IKZF1 specifically promotes homologous recombination DNA damage repair in MM cells, which is regulated by its interaction with CtIP and USP7. In this process, USP7 could regulate the stability of IKZF1 through its deubiquitinating activity. The N-terminal zinc finger domains of IKZF1 and the ubiquitin-like domain of USP7 are necessary for their interaction. Furthermore, targeted inhibition IKZF1 or USP7 could sensitize MM cells to PARP inhibitor treatment in vitro and in vivo.Conclusions: Our findings identify USP7 as a deubiquitinating enzyme for IKZF1 and uncover a new function of IKZF1 in DNA damage repair. In translational perspective, the combination inhibition of IKZF1 or USP7 with PARP inhibitor deserves further evaluation in clinical trials for the treatment of MM.     

DNA damage invokes mismatch repair-dependent cyclin D1 attenuation and retinoblastoma signaling pathways to inhibit CDK2.

DNA-damage evokes cell cycle checkpoints, which function to maintain genomic integrity. The retinoblastoma tumor suppressor (RB) and mismatch repair complexes are known to contribute to the appropriate cellular response to specific types of DNA damage. However, the signaling pathways through which these proteins impact the cell cycle machinery have not been explicitly determined. RB-deficient murine embryo fibroblasts continued a high degree of DNA replication following the induction of cisplatin damage, but were inhibited for G(2)/M progression. This damage led to RB dephosphorylation/activation and subsequent RB-dependent attenuation of cyclin A and CDK2 activity. In both Rb+/+ and Rb -/- cells, cyclin D1 expression was attenuated following DNA damage. As cyclin D1 is a critical determinant of RB phosphorylation and cell cycle progression, we probed the pathway through which cyclin D1 degradation occurs in response to DNA damage. We found that attenuation of endogenous cyclin D1 is dependent on multiple mismatch repair proteins. We demonstrate that the mismatch repair-dependent attenuation of endogenous cyclin D1 is critical for attenuation of CDK2 activity and induction of cell cycle checkpoints. Together, these studies couple the activity of the retinoblastoma and mismatch repair tumor suppressor pathways through the degradation of cyclin D1 and dual attenuation of CDK2 activity.     

Behind the wheel and under the hood: Functions of cyclin-dependent kinases in response to DNA damage

Cell division and the response to genotoxic stress are intimately connected in eukaryotes, for example, by checkpoint pathways that signal the presence of DNA damage or its ongoing repair to the cell cycle machinery, leading to reversible arrest or apoptosis. Recent studies reveal another connection: the cyclin-dependent kinases (CDKs) that govern both DNA synthesis (S) phase and mitosis directly coordinate DNA repair processes with progression through the cell cycle. In both mammalian cells and yeast, the two major modes of double strand break (DSB) repair – homologous recombination (HR) and non-homologous end joining (NHEJ) – are reciprocally regulated during the cell cycle. In yeast, the cell cycle kinase Cdk1 directly promotes DSB repair by HR during the G2 phase. In mammalian cells, loss of Cdk2, which is active throughout S and G2 phases, results in defective DNA damage repair and checkpoint signaling. Here we provide an overview of data that implicate CDKs in the regulation of DNA damage responses in yeast and metazoans. In yeast, CDK activity is required at multiple points in the HR pathway; the precise roles of CDKs in mammalian HR have yet to be determined. Finally, we consider how the two different, and in some cases opposing, roles of CDKs – as targets of negative regulation by checkpoint signaling and as positive effectors of repair pathway selection and function – could be balanced to produce a coordinated and effective response to DNA damage.     

YTHDF1 promotes breast cancer cell growth,DNA damage repair and chemoresistance

Chemoresistance represents a major obstacle to the treatment of human cancers. Increased DNA repair capacity is one of the important mechanisms underlying chemoresistance. In silico analysis indicated that YTHDF1, an m6A binding protein, is a putative tumor promoter in breast cancer. Loss of function studies further showed that YTHDF1 promotes breast cancer cell growth in vitro and in vivo. YTHDF1 facilitates S-phase entry, DNA replication and DNA damage repair, and accordingly YTHDF1 knockdown sensitizes breast cancer cells to Adriamycin and Cisplatin as well as Olaparib, a PARP inhibitor. E2F8 is a target molecule by YTHDF1 which modulates E2F8 mRNA stability and DNA damage repair in a METTL14-dependent manner. These data demonstrate that YTHDF1 has a tumor-promoting role in breast cancer, and is a novel target to overcome chemoresistance.Subject terms: Breast cancer, Breast cancer     

Combination of the 6-thioguanine and disulfiram/Cu synergistically inhibits proliferation of triple-negative breast cancer cells by enhancing DNA damage and disrupting DNA damage checkpoint

Because of the lack of specific molecular targeted therapies, triple-negative breast cancer (TNBC) has high tumour recurrence and metastasis rates. It is urgent to develop novel chemotherapeutic strategies to improve patient survival. DNA damaging agents have been shown to sensitize cancer to genotoxic chemotherapies. We first found that 6-thioguanine (6-TG) can activate the NF-кB signalling pathway. Our results showed that NF-кB signalling was reduced when cells were treated with 6-TG/disulfiram (DSF)/Cu. DSF/Cu enhanced the 6-TG-mediated inhibition of proliferation. 6-TG/DSF/Cu inhibited cell cycle progression, causing cell cycle arrest in the S phase and G2/M phase. Moreover, the combined effect of 6-TG and DSF/Cu induced apoptosis, and either agent alone was able to induce apoptosis. The accumulation of γH2A indicated that DSF/Cu increased the DNA damage induced by 6-TG. Combined treatment with 6-TG and DSF/Cu synergistically reduced the levels of both phosphorylated and total ataxia-telangiectasia-mutated-and-Rad3-related kinase (ATR), suggesting that DSF/Cu promoted 6-TG-induced DNA damage by suppressing ATR protein kinases, therefore enhancing cell apoptosis. In conclusion, we demonstrate that the combination of 6-TG and DSF/Cu exerted a significant synergistic antitumour effect on human TNBC in vitro and in vivo by enhancing DNA damage and disrupting DNA damage checkpoints. We propose that this combination therapy could be a novel strategy for the treatment of TNBC.     

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.     

聚腺苷二磷酸-核糖聚合酶1功能与其抑制剂的作用及耐药机制

聚腺苷二磷酸-核糖聚合酶1(poly ADP-ribose polymerase-1,PARP1)是细胞中重要的修饰酶,其最广为人知的作用是通过自身PAR修饰,募集以XRCC1为首的多种DNA损伤修复效应蛋白质,参与DNA单、双链损伤修复。PARP1还能通过促进复制叉停滞与核小体解聚,为DNA损伤修复提供有利条件,维持基因组稳定性。近年来,除DNA损伤修复方面的作用,还发现PARP1能影响细胞凋亡、自噬与炎症通路,与神经退行性疾病的发生发展密切相关。而PARP抑制剂(PARP inhibitor,PARPi)是一种靶向PARP1,与细胞同源重组(homologous recombination,HR)缺陷表型共同作用,产生合成致死效应的抗肿瘤药物。该药物可捕获PARP1并抑制其活性,一方面直接干扰PARP1参与的DNA损伤修复通路,另一方面也抑制了PARP1介导的DNA损伤修复通路选择和复制叉停滞,使细胞基因组不稳定。然而,在临床治疗中常发现肿瘤细胞对PARPi不敏感。肿瘤细胞对PARPi耐药与自身基因突变高度相关,这些基因分别作用于细胞HR修复途径、PARP1循环途径、复制叉稳定性和药物主动外排等方面,在耐药肿瘤患者中确定具体的突变位点,将为临床治疗提供帮助。本文旨在对PARP1的功能作一综述,并重点介绍PARPi的作用机制和与肿瘤耐药相关的突变基因及其耐药机制,以期加深对细胞中PARP1介导的DNA损伤修复通路的认识,并为将来的临床治疗提供新思路。     

Combinations of PARP Inhibitors with Temozolomide Drive PARP1 Trapping and Apoptosis in Ewing’s Sarcoma

Ewing’s sarcoma is a malignant pediatric bone tumor with a poor prognosis for patients with metastatic or recurrent disease. Ewing’s sarcoma cells are acutely hypersensitive to poly (ADP-ribose) polymerase (PARP) inhibition and this is being evaluated in clinical trials, although the mechanism of hypersensitivity has not been directly addressed. PARP inhibitors have efficacy in tumors with BRCA1/2 mutations, which confer deficiency in DNA double-strand break (DSB) repair by homologous recombination (HR). This drives dependence on PARP1/2 due to their function in DNA single-strand break (SSB) repair. PARP inhibitors are also cytotoxic through inhibiting PARP1/2 auto-PARylation, blocking PARP1/2 release from substrate DNA. Here, we show that PARP inhibitor sensitivity in Ewing’s sarcoma cells is not through an apparent defect in DNA repair by HR, but through hypersensitivity to trapped PARP1-DNA complexes. This drives accumulation of DNA damage during replication, ultimately leading to apoptosis. We also show that the activity of PARP inhibitors is potentiated by temozolomide in Ewing’s sarcoma cells and is associated with enhanced trapping of PARP1-DNA complexes. Furthermore, through mining of large-scale drug sensitivity datasets, we identify a subset of glioma, neuroblastoma and melanoma cell lines as hypersensitive to the combination of temozolomide and PARP inhibition, potentially identifying new avenues for therapeutic intervention. These data provide insights into the anti-cancer activity of PARP inhibitors with implications for the design of treatment for Ewing’s sarcoma patients with PARP inhibitors.     

Adenosine receptor A2b confers ovarian cancer survival and PARP inhibitor resistance through IL-6-STAT3 signalling

Ovarian cancer is the deadliest gynecologic cancer worldwide, and the therapeutic options are limited. PARP inhibitor (PARPi) represents an effective therapeutic strategy and has been approved for maintenance therapy. However, the intrinsic or acquired resistance to PARPi becomes a big challenge. To investigate the mechanisms for PARPi resistance, we analysed public databases and established Olaparib-resistant ovarian cancer cells for exploration. Our results showed that the inflammatory pathway and adenosine receptor A2b (Adora2b/A_2B) expression were significantly increased in Olaparib-resistant cells. A_2B was highly expressed in recurrent ovarian tumours and negatively correlated with the clinical outcomes in cancer patients. Olaparib treatment enhanced A_2B expression through NF-κB activation. The elevated A_2B contributed to Olaparib resistance by sensing adenosine signal and promoting tumour cell survival, growth and migration via IL-6-STAT3 signalling. Therefore, inhibition of A_2B-IL-6-STAT3 axis could overcome Olaparib resistance and synergize with Olaparib to reduce cancer cell growth and lead to cell death. Our findings reveal a critical role of A_2B signalling in mediating PARPi resistance independent of DNA damage repair, providing insights into developing novel therapies in ovarian cancers.     

CDK1 inhibition sensitizes normal cells to DNA damage in a cell cycle dependent manner

Cyclin-dependent kinase 1 (CDK1) orchestrates the transition from the G2 phase into mitosis and as cancer cells often display enhanced CDK1 activity, it has been proposed as a tumor specific anti-cancer target. Here we show that the effects of CDK1 inhibition are not restricted to tumor cells but can also reduce viability in non-cancer cells and sensitize them to radiation in a cell cycle dependent manner.

Radiosensitization by the specific CDK1 inhibitor, RO-3306, was determined by colony formation assays in three tumor lines (HeLa, T24, SQ20B) and three non-cancer lines (HFL1, MRC-5, RPE). Initial results showed that CDK1 inhibition radiosensitized tumor cells, but did not sensitize normal fibroblasts and epithelial cells in colony formation assays despite effective inhibition of CDK1 signaling. Further investigation showed that normal cells were less sensitive to CDK1 inhibition because they remained predominantly in G1 for a prolonged period when plated in colony formation assays. In contrast, inhibiting CDK1 a day after plating, when the cells were going through G2/M phase, reduced their clonogenic survival both with and without radiation. Our finding that inhibition of CDK1 can damage normal cells in a cell cycle dependent manner indicates that targeting CDK1 in cancer patients may lead to toxicity in normal proliferating cells. Furthermore, our finding that cell cycle progression becomes easily stalled in non-cancer cells under normal culture conditions has general implications for testing anti-cancer agents in these cells.