基于小分子化合物库筛选协同多腺苷二磷酸核糖聚合酶抑制剂杀伤卵巢癌细胞的药物

多腺苷二磷酸核糖聚合酶(poly(ADP-ribose) polymerase, PARP)抑制剂是一类靶向 DNA 修复缺陷癌细胞的新型药物。早期研究表明 PARP 抑制剂取得了令人满意的结果,然而药物治疗后出现的耐药机制尚未完全揭露。因此,有必要寻找更多的靶向药物与PARP 抑制剂联用,以达到杀伤肿瘤细胞的目的。本文基于379种小分子化合物和PARP抑制剂尼拉帕尼(Niraparib)的联合用药筛选,通过细胞增殖实验、克隆存活实验和免疫荧光染色等方法筛选潜在的具有协同PARP抑制剂杀伤卵巢癌细胞的药物。结果表明,其中有8种小分子化合物具有较好的联合用药效果,包括2种已经报道的与PARP抑制剂具有联用效果的小分子化合物STF-118804和Disulfiram。我们从中选取原肌球蛋白受体激酶 A (tropomyosin receptor kinase A,TrKA)的抑制剂GW441756,进行了多种肿瘤细胞的验证以及初步机制的探究。Niraparib和TrKA抑制剂的联合用药显著增加肿瘤细胞对PARP抑制剂的敏感性(P<0.05)。从机制上分析,联合用药组细胞内γH2AX foci的数目显著增加(P<0.05),说明TrKA抑制剂阻碍损伤后细胞的DNA损伤修复能力;同时,联合用药显著降低细胞内同源重组修复(homologous recombination repair,HRR)标志物RAD51 foci(P<0.05)的形成,说明TrKA抑制剂可能通过抑制细胞的HRR效率阻碍细胞的DNA损伤修复。本研究的结果提示,TrKA抑制剂可以作为一种与PARP抑制剂联用杀伤卵巢癌细胞的潜在药物。     

Targeted therapies in epithelial ovarian cancer: Molecular mechanisms of action

Ovarian cancer is the leading cause of death in women with gynecological cancer. Most patients are diagnosed at an advanced stage and have a poor prognosis. Currently, surgical tumor debulking, followed by platinum- and taxane-based chemotherapy is the standard treatment for advanced ovarian cancer. However, these patients are at great risk of recurrence and emerging drug resistance. Therefore, novel treatment strategies are required to improve outcomes for women with advanced ovarian cancer. A variety of molecular targeted agents, the majority of which are monoclonal antibodies and small-molecule protein-kinase inhibitors, have been explored in the management of ovarian cancer. The targets of these agents include angiogenesis, the human epidermal growth factor receptor family, ubiquitin-proteasome pathway, epigenetic modulators, poly(ADP-ribose) polymerase (PARP), and mammalian target of rapamycin (mTOR) signaling pathway, which are aberrant in tumor tissue. The antiangiogenic agent, bevacizumab, has been reported as the most effective targeted agent and should be included in the standard chemotherapeutic regimen for advanced ovarian cancer. PARP inhibitors, which are mainly used in breast and ovarian cancer susceptibility gene-mutated patients, and mTOR inhibitors are also attractive treatment strategies, either alone or combination with chemotherapy, for ovarian cancer. Understanding the tumor molecular biology and identification of predictive biomarkers are essential steps for selection of the best treatment strategies. This article reviews the molecular mechanisms of the most promising targeted agents that are under early phase clinical evaluation for ovarian cancer.     

Low ATM protein expression and depletion of p53 correlates with olaparib sensitivity in gastric cancer cell lines

Small-molecule inhibitors of poly (ADP-ribose) polymerase (PARP) have shown considerable promise in the treatment of homologous recombination (HR)-defective tumors, such as BRCA1- and BRCA2-deficient breast and ovarian cancers. We previously reported that mantle cell lymphoma cells with deficiency in ataxia telangiectasia mutated (ATM) are sensitive to PARP-1 inhibitors in vitro and in vivo. Here, we report that PARP inhibitors can potentially target ATM deficiency arising in a solid malignancy. We show that ATM protein expression varies between gastric cancer cell lines, with NUGC4 having significantly reduced protein levels. Significant correlation was found between ATM protein expression and sensitivity to the PARP inhibitor olaparib, with NUGC4 being the most sensitive. Moreover, reducing ATM kinase activity using a small-molecule inhibitor (KU55933) or shRNA-mediated depletion of ATM protein enhanced olaparib sensitivity in gastric cancer cell lines with depletion or inactivation of p53. Our results demonstrate that ATM is a potential predictive biomarker for PARP-1 inhibitor activity in gastric cancer harboring disruption of p53, and that combined inhibition of ATM and PARP-1 is a rational strategy for expanding the utility of PARP-1 inhibitors to gastric cancer with p53 disruption.     

HMGB3 promotes PARP inhibitor resistance through interacting with PARP1 in ovarian cancer

Poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi) resistance remains a therapeutic challenge in ovarian cancer. High-mobility group box 3 (HMGB3) plays significant roles in the development of drug resistance of many cancers. However, the function of HMGB3 in PARPi resistance is poorly understood. In the current study, we clarified that HMGB3 was aberrantly overexpressed in high-grade serous ovarian carcinoma (HGSOC) tissues, and high HMGB3 levels indicated shorter overall survival and drug resistance in HGSOC. The overexpression of HMGB3 increased the insensitivity of ovarian cancer to PARPi, whereas HMGB3 knockdown reduced PARPi resistance. Mechanistically, PARP1 was identified as a novel interaction partner of HMGB3, which could be blocked using olaparib and was enhanced upon DNA damage conditions. We further showed that loss of HMGB3 induced PARP1 trapping at DNA lesions and inhibited the PARylation activity of PARP1, resulting in an increased DNA damage response and cell apoptosis. The PARPi-resistant role of HMGB3 was also verified in a xenograft mouse model. In conclusion, HMGB3 promoted PARPi resistance via interacting with PARP1, and the targeted inhibition of HMGB3 might overcome PARPi resistance in ovarian cancer therapy.Subject terms: Chemotherapy, Ovarian cancer, Ovarian cancer, Cancer therapeutic resistance     

Synergistic effect of PARP inhibitor and BRD4 inhibitor in multiple models of ovarian cancer

Ovarian cancer has the highest facility rate among gynaecological tumours. Current therapies including PARP inhibitors have a defect that ovarian tumour is easy to recurrent and become resistant to therapy. To solve this problem, we found that BRD4 inhibitor AZD5153 and PARP inhibitor olaparib had a widespread synergistic effect in multiple models with different gene backgrounds. AZD5153 sensitizes cells to olaparib and reverses the acquired resistance by down-regulating PTEN expression levels to destabilize hereditary materials. In this study, we used the following multiple ovarian cancer models PDX, PDO and 3D/2D cell lines to elucidate the co-effect of AZD5153 and olaparib in vivo and in vitro. The similar results of these models further proved that the mechanism identified was consistent with the biological process occurring in ovarian cancer patients after drug treatment. This consistency between the results of different models suggests the possibility of translating these laboratory research findings into clinical studies towards developing treatments.     

Cationic liposome codelivering PI3K pathway regulator improves the response of BRCA1-deficient breast cancer cells to PARP1 inhibition

Although some progresses have been made in breast cancer therapy, effective treatment for BRCA1-deficient breast cancer remains to be a great challenge. It has been demonstrated that the PI3K pathway is inappropriately activated in BRCA1-deficient breast cancers which can be downregulated by microRNA 451 (miR-451). In addition, although PARP1 inhibitors showed relatively positive results in both preclinical and clinical studies, additional efforts to decrease drug resistance as well as reduce systematic toxicity need to be addressed. To this end, by encapsulating the miR-451 mimic and PARP1 inhibitor in the same cationic liposome, we examined the potential of enhancing the response of PARP1 inhibition on BRCA1-deficient breast cancer by regulating the PI3K pathway. Our results revealed that in BRCA1-deficient human breast cancer cell line, PARP1 inhibition resulted in DNA damage with viability decrease, G2/M arrest as well as apoptosis. In contrast, single PI3K inhibition induced G1 arrest along with retarded cell proliferation. However, it was noted that combination of PARP inhibitor and PI3K regulator could exert synergetic function to evidently decrease cell proliferation compared with PARP inhibition alone, which was also confirmed by in vivo antitumor assay using xenograft tumor models. Collectively, our results offer an alternative but superior strategy for the therapy of BRCA1-deficient human breast cancers which may benefit the clinical applications.     

MET inhibition enhances PARP inhibitor efficacy in castration-resistant prostate cancer by suppressing the ATM/ATR and PI3K/AKT pathways

Up to 30% of patients with metastatic castration-resistant prostate cancer (CRPC) patients carry altered DNA damage response genes, enabling the use of poly adenosine diphosphate–ribose polymerase (PARP) inhibitors in advanced CRPC. The proto-oncogene mesenchymal–epithelial transition (MET) is crucial in the migration, proliferation, and invasion of tumour cells. Aberrant expression of MET and its ligand hepatocyte growth factor is associated with drug resistance in cancer therapy. Here, we found that MET was highly expressed in human CRPC tissues and overexpressed in DU145 and PC3 cells in a drug concentration-dependent manner and is closely related to sensitivity to PARP inhibitors. Combining the PARP inhibitor olaparib with the MET inhibitor crizotinib synergistically inhibited CRPC cell growth both in vivo and in vitro. Further analysis of the underlying molecular mechanism underlying the MET suppression-induced drug sensitivity revealed that olaparib and crizotinib could together downregulate the ATM/ATR signaling pathway, inducing apoptosis by inhibiting the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) pathway, enhancing the olaparib-induced antitumour effect in DU145 and PC3 cells. In conclusion, we demonstrated that MET inhibition enhances sensitivity of CRPC to PARP inhibitors by suppressing the ATM/ATR and PI3K/AKT pathways and provides a novel, targeted therapy regimen for CRPC.     

Evaluation of candidate biomarkers to predict cancer cell sensitivity or resistance to PARP-1 inhibitor treatment

Impaired DNA damage response pathways may create vulnerabilities of cancer cells that can be exploited therapeutically. One such selective vulnerability is the sensitivity of BRCA1- or BRCA2-defective tumors (hence defective in DNA repair by homologous recombination, HR) to inhibitors of the poly(ADP-ribose) polymerase-1 (PARP-1), an enzyme critical for repair pathways alternative to HR. While promising, treatment with PARP-1 inhibitors (PARP-1i) faces some hurdles, including (1) acquired resistance, (2) search for other sensitizing, non-BRCA1/2 cancer defects and (3) lack of biomarkers to predict response to PARP-1i. Here we addressed these issues using PARP-1i on 20 human cell lines from carcinomas of the breast, prostate, colon, pancreas and ovary. Aberrations of the Mre11-Rad50-Nbs1 (MRN) complex sensitized cancer cells to PARP-1i, while p53 status was less predictive, even in response to PARP-1i combinations with camptothecin or ionizing radiation. Furthermore, monitoring PARsylation and Rad51 foci formation as surrogate markers for PARP activity and HR, respectively, supported their candidacy for biomarkers of PARP-1i responses. As to resistance mechanisms, we confirmed the role of the multidrug resistance efflux transporters and its reversibility. More importantly, we demonstrated that shRNA lentivirus-mediated depletion of 53BP1 in human BRCA1-mutant breast cancer cells increased their resistance to PARP-1i. Given the preferential loss of 53BP1 in BRCA-defective and triple-negative breast carcinomas, our findings warrant assessment of 53BP1 among candidate predictive biomarkers of response to PARPi. Overall, this study helps characterize genetic and functional determinants of cellular responses to PARP-1i and contributes to the search for biomarkers to exploit PARP inhibitors in cancer therapy.     

Evaluation of candidate biomarkers to predict cancer cell sensitivity or resistance to PARP-1 inhibitor treatment

Impaired DNA damage response pathways may create vulnerabilities of cancer cells that can be exploited therapeutically. One such selective vulnerability is the sensitivity of BRCA1- or BRCA2-defective tumors (hence defective in DNA repair by homologous recombination, HR) to inhibitors of the poly(ADP-ribose) polymerase-1 (PARP-1), an enzyme critical for repair pathways alternative to HR. While promising, treatment with PARP-1 inhibitors (PARP-1i) faces some hurdles, including (1) acquired resistance, (2) search for other sensitizing, non-BRCA1/2 cancer defects and (3) lack of biomarkers to predict response to PARP-1i. Here we addressed these issues using PARP-1i on 20 human cell lines from carcinomas of the breast, prostate, colon, pancreas and ovary. Aberrations of the Mre11-Rad50-Nbs1 (MRN) complex sensitized cancer cells to PARP-1i, while p53 status was less predictive, even in response to PARP-1i combinations with camptothecin or ionizing radiation. Furthermore, monitoring PARsylation and Rad51 foci formation as surrogate markers for PARP activity and HR, respectively, supported their candidacy for biomarkers of PARP-1i responses. As to resistance mechanisms, we confirmed the role of the multidrug resistance efflux transporters and its reversibility. More importantly, we demonstrated that shRNA lentivirus-mediated depletion of 53BP1 in human BRCA1-mutant breast cancer cells increased their resistance to PARP-1i. Given the preferential loss of 53BP1 in BRCA-defective and triple-negative breast carcinomas, our findings warrant assessment of 53BP1 among candidate predictive biomarkers of response to PARPi. Overall, this study helps characterize genetic and functional determinants of cellular responses to PARP-1i and contributes to the search for biomarkers to exploit PARP inhibitors in cancer therapy.     

Therapeutic applications of PARP inhibitors: Anticancer therapy and beyond

The aim of this article is to describe the current and potential clinical translation of pharmacological inhibitors of poly(ADP-ribose) polymerase (PARP) for the therapy of various diseases. The first section of the present review summarizes the available preclinical and clinical data with PARP inhibitors in various forms of cancer. In this context, the role of PARP in single-strand DNA break repair is relevant, leading to replication-associated lesions that cannot be repaired if homologous recombination repair (HRR) is defective, and the synthetic lethality of PARP inhibitors in HRR-defective cancer. HRR defects are classically associated with BRCA1 and 2 mutations associated with familial breast and ovarian cancer, but there may be many other causes of HRR defects. Thus, PARP inhibitors may be the drugs of choice for BRCA mutant breast and ovarian cancers, and extend beyond these tumors if appropriate biomarkers can be developed to identify HRR defects. Multiple lines of preclinical data demonstrate that PARP inhibition increases cytotoxicity and tumor growth delay in combination with temozolomide, topoisomerase inhibitors and ionizing radiation. Both single agent and combination clinical trials are underway. The final part of the first section of the present review summarizes the current status of the various PARP inhibitors that are in various stages of clinical development. The second section of the present review summarizes the role of PARP in selected non-oncologic indications. In a number of severe, acute diseases (such as stroke, neurotrauma, circulatory shock and acute myocardial infarction) the clinical translatability of PARP inhibition is supported by multiple lines of preclinical data, as well as observational data demonstrating PARP activation in human tissue samples. In these disease indications, PARP overactivation due to oxidative and nitrative stress drives cell necrosis and pro-inflammatory gene expression, which contributes to disease pathology. Accordingly, multiple lines of preclinical data indicate the efficacy of PARP inhibitors to preserve viable tissue and to down-regulate inflammatory responses. As the clinical trials with PARP inhibitors in various forms of cancer progress, it is hoped that a second line of clinical investigations, aimed at testing of PARP inhibitors for various non-oncologic indications, will be initiated, as well.     

Synthesis of novel dual target inhibitors of PARP and HSP90 and their antitumor activities

Poly (ADP-ribose) polymerase (PARP) inhibitors have achieved great success in clinical application, especially for the prolonged survival of cisplatin-sensitive ovarian cancer patients. However, there are still many patients who do not respond to PARP inhibitors. Novel PARP inhibitors with higher activity are urgently needed. Herein we report a series of compounds by molecular hybridization PARP-1 inhibitor Olaparib (Ola) with HSP90 inhibitor C0817 (one curcumin derivative). All synthesized compounds were evaluated for their antiproliferative activity in vitro, and some were further assessed for their inhibitory activities of the PARP enzyme and HSP90 affinity. Our results indicated that compound 4 could bind to HSP90 and cause static quenching, indicating that compound 4 was able to bind to HSP90, moreover, downstream molecular breast cancer 1 (BRAC-1) was reduced. In conclusion, dual target inhibitors of PARP and HSP90 exhibited stronger selective cytotoxicities against cancer.     

Machine-learning aided in situ drug sensitivity screening predicts treatment outcomes in ovarian PDX tumors

Long-term treatment outcomes for patients with high grade ovarian cancers have not changed despite innovations in therapies. There is no recommended assay for predicting patient response to second-line therapy, thus clinicians must make treatment decisions based on each individual patient. Patient-derived xenograft (PDX) tumors have been shown to predict drug sensitivity in ovarian cancer patients, but the time frame for intraperitoneal (IP) tumor generation, expansion, and drug screening is beyond that for tumor recurrence and platinum resistance to occur, thus results do not have clinical utility. We describe a drug sensitivity screening assay using a drug delivery microdevice implanted for 24 h in subcutaneous (SQ) ovarian PDX tumors to predict treatment outcomes in matched IP PDX tumors in a clinically relevant time frame. The SQ tumor response to local microdose drug exposure was found to be predictive of the growth of matched IP tumors after multi-week systemic therapy using significantly fewer animals (10 SQ vs 206 IP). Multiplexed immunofluorescence image analysis of phenotypic tumor response combined with a machine learning classifier could predict IP treatment outcomes against three second-line cytotoxic therapies with an average AUC of 0.91.     

miR-155-5p增强卵巢癌细胞UWB1.289对PARP抑制剂的敏感性

摘要 目的：探讨卵巢癌细胞UWB1.289中miR-155-5p对PARP抑制剂敏感性的影响及可能涉及的分子机制研究。方法：采用qRT-PCR技术检测miR-155-5p在有BRCA1/2突变和无BRCA1/2突变的卵巢癌组织及卵巢癌细胞中的表达情况。利用细胞转染、qRT-PCR以及Western Blot技术检测转染miR-155-5p模拟物和抑制剂的卵巢癌细胞UWB1.289中miR-155-5p的表达以及同源重组修复相关基因SIRT1、BRG1的表达。通过双荧光素酶报告基因实验验证miR-155-5p与SIRT1、BRG1之间的靶向性。运用CCK-8检测卵巢癌细胞UWB1.289中miR-155-5p对PARP抑制剂敏感性的影响。结果：与无BRCA1/2突变的卵巢癌组织及卵巢癌细胞相比,miR-155-5p在有BRCA1/2突变的卵巢癌组织及卵巢癌细胞中低表达。转染miR-155-5p模拟物可增加卵巢癌细胞UWB1.289中miR-155-5p的表达,同时降低同源重组修复相关基因SIRT1、BRG1的表达;转染miR-155-5p抑制剂可下调卵巢癌细胞UWB1.289中miR-155-5p的表达,同时增加SIRT1、BRG1的表达,进一步通过双荧光素酶报告基因实验证实miR-155-5p与SIRT1、BRG1存在特异性靶向结合序列。与对照组相比,干扰同源重组修复相关基因以及miR-155-5p过表达均可增强卵巢癌细胞UWB1.289对PARP抑制剂的敏感性。结论：miR-155-5p可能通过影响同源重组修复基因增强卵巢癌细胞UWB1.289对PARP抑制剂的敏感性。     

Enhanced self-renewal of hematopoietic stem/progenitor cells mediated by the stem cell gene Sall4

We reviewed preclinical data and clinical development of MDM2 (murine double minute 2), ALK (anaplastic lymphoma kinase) and PARP (poly [ADP-ribose] polymerase) inhibitors. MDM2 binds to p53, and promotes degradation of p53 through ubiquitin-proteasome degradation. JNJ-26854165 and RO5045337 are 2 small-molecule inhibitors of MDM2 in clinical development. ALK is a transmembrane protein and a member of the insulin receptor tyrosine kinases. EML4-ALK fusion gene is identified in approximately 3-13% of non-small cell lung cancer (NSCLC). Early-phase clinical studies with Crizotinib, an ALK inhibitor, in NSCLC harboring EML4-ALK have demonstrated promising activity with high response rate and prolonged progression-free survival. PARPs are a family of nuclear enzymes that regulates the repair of DNA single-strand breaks through the base excision repair pathway. Randomized phase II study has shown adding PARP-1 inhibitor BSI-201 to cytotoxic chemotherapy improves clinical outcome in patients with triple-negative breast cancer. Olaparib, another oral small-molecule PARP inhibitor, demonstrated encouraging single-agent activity in patients with advanced breast or ovarian cancer. There are 5 other PARP inhibitors currently under active clinical investigation.     

ATM-Deficient Colorectal Cancer Cells Are Sensitive to the PARP Inhibitor Olaparib

The ataxia telangiectasia mutated (ATM) protein kinase plays a central role in the cellular response to DNA damage. Loss or inactivation of both copies of the ATM gene (ATM) leads to ataxia telangiectasia, a devastating childhood condition characterized by neurodegeneration, immune deficiencies, and cancer predisposition. ATM is also absent in approximately 40% of mantle cell lymphomas (MCLs), and we previously showed that MCL cell lines with loss of ATM are sensitive to poly-ADP ribose polymerase (PARP) inhibitors. Next-generation sequencing of patient tumors has revealed that ATM is altered in many human cancers including colorectal, lung, prostate, and breast. Here, we show that the colorectal cancer cell line SK-CO-1 lacks detectable ATM protein expression and is sensitive to the PARP inhibitor olaparib. Similarly, HCT116 colorectal cancer cells with shRNA depletion of ATM are sensitive to olaparib, and depletion of p53 enhances this sensitivity. Moreover, HCT116 cells are sensitive to olaparib in combination with the ATM inhibitor KU55933, and sensitivity is enhanced by deletion of p53. Together our studies suggest that PARP inhibitors may have potential for treating colorectal cancer with ATM dysfunction and/or colorectal cancer with mutation of p53 when combined with an ATM kinase inhibitor.     

Proteomics for identifying mechanisms and biomarkers of drug resistance in cancer

A major problem in chemotherapy of cancer patients is drug resistance as well as unpredictable response to treatment. During chemotherapy, multiple alterations of genetics and epigenetics that contribute to chemoresistance take place, eventually impacting on disease outcome. A more complex picture of the mechanisms of drug resistance is now emerging through application of high-throughput proteomics technology. We have entered an exciting time where proteomics are being applied to characterize the mechanisms of drug resistance, and to identify biomarkers for predicting response to chemotherapy, thereby leading to personalized therapeutic strategies of cancer patients. Comparative proteomics have identified a large number of differentially expressed proteins associated with chemoresistance. Although roles and mechanisms of such proteins in chemoresistance need to be further proved, at least some of them may be potential biomarkers for predicting chemotherapeutic response. Herein, we review the recent advancements on proteomic investigation of chemoresistance in human cancer, and emphasize putative biomarkers for predicting chemotherapeutic response and possible mechanisms of chemoresistance identified through proteomic approaches. Suggested avenues for future work are discussed.