Discovery of novel PARP-1 inhibitors using tandem in silico studies: integrated docking, e-pharmacophore, deep learning based de novo and molecular dynamics simulation approach

PARP inhibitor and chemotherapy combination trials for the treatment of advanced malignancies: does a development pathway forward exist?

Inhibited, trapped or adducted: the optimal selective synthetic lethal mix for BRCAness

Research Highlights

118 Background: Ovarian cancer is the fifth leading cause of cancer-related death among women. Poly adenosine diphosphate ribose polymerase (PARP) inhibitors maintenance has shown to improve survival in patients with recurrent ovarian cancer. Yet, there are notable adverse events which led to treatment discontinuation, interruption, or dose reduction. We conducted a systematic review and meta-analysis of randomized controlled trials (RCT) to determine the risk of PARP inhibitors discontinuation due to adverse events. Methods: MEDLINE, EMBASE databases and meeting abstracts from inception through June 2018 were queried. Phase III RCTs which employed PARP inhibitors maintenance in ovarian cancer and mentioned treatment interruption, dose reduction and treatment discontinuation due to adverse events were included. Mantel-Haenszel (MH) method was used to calculate the estimated pooled risk ratio (RR) with 95% confidence interval (CI). Random effects model was applied. Results: Three phase III RCTs with a total of 1,401 patients with recurrent ovarian cancer were eligible. The study arm used olaparib or niraparib or rucaparib while the control arm utilized placebo. The randomization ratio was 2:1 in all studies. The incidence of treatment interruption due to adverse events was 578 (61.8%) in study group versus 46 (9.8%) in control arm. The relative risk for treatment interruption was statistically significant at 5.87 (95% CI: 2.24 – 15.36, P < 0.001). The reduction in dose was reported in 496 (53.1%) in PARP inhibitors arm versus 37 (7.9%) in control group. The pooled RR for dose reduction was 7.49 (95% CI: 3.44 – 16.29, P < 0.001). The treatment discontinuation rate was 11.4% higher with PARP inhibitors than with control arm (RR - 6.84; 95% CI: 3.51 – 13.34, P < 0.001). Conclusions: Our study showed that patients on PARP inhibitors experienced some adverse events which led to significant drop outs although the definitive advantage to PARP inhibitors is still shown in the studies. Preemptive measures with proper supportive care will aide in reducing those toxicities, improve patients’ quality of life and may probably affect patients’ compliance.

Upon growth factor stimulation or in some EGFR mutant cancer cells, PKM2 translocates into the nucleus to induce glycolysis and cell growth. Here, we report that nuclear PKM2 binds directly to poly-ADP ribose, and this PAR-binding capability is critical for its nuclear localization. Accordingly, PARP inhibition prevents nuclear retention of PKM2 and therefore suppresses cell proliferation and tumor growth. In addition, we found that PAR level correlates with nuclear localization of PKM2 in EGFR mutant brain and lung cancers, suggesting that PAR-dependent nuclear localization of PKM2 likely contributes to tumor progression in EGFR mutant glioblastoma and lung cancers. In addition, some EGFR-inhibitor-resistant lung cancer cells are sensitive to PARP inhibitors. Taken together, our data indicate that suppression of PKM2 nuclear function by PARP inhibitors represents a treatment strategy for EGFR-inhibitor-resistant cancers.

The concept of synthetic lethality is an emerging field in the treatment of cancer and can be applied for new drug development of cancer as already been represented by Poly (ADP-ribose) polymerase (PARPs) inhibitors. In this study, we performed virtual screening of 329 flavonoids obtained from the Naturally Occurring Plant-based Anti-cancer Compound-Activity-Target (NPACT) database to identify novel PARP inhibitors. Virtual screening carried out using different in silico methods which include molecular docking studies, prediction of drug-likeness and in silico toxicity studies. Fifteen out of 329 flavonoids achieved better docking score as compared to rucaparib which is an FDA approved PARP inhibitor. These 15 hits were again rescored using accurate docking mode and drug-likeliness properties were evaluated. The accuracy of the docking method was checked using re-docking. Finally NPACT00183 and NPACT00280 were identified as potential PARP inhibitors with docking score of -139.237 and -129.36, respectively. These two flavonoids also showed no AMES toxicity and no carcinogenicity which was predicted using admetSAR. Our finding suggests that NPACT00183 and NPACT00280 have promising potential to be further explored as PARP inhibitors.

The MSH3 gene is one of the DNA mismatch repair (MMR) genes that has undergone somatic mutation frequently in MMR-deficient cancers. MSH3, together with MSH2, forms the MutSβ heteroduplex, which interacts with interstrand cross-links (ICLs) induced by drugs such as cisplatin and psoralen. However, the precise role of MSH3 in mediating the cytotoxic effects of ICL-inducing agents remains poorly understood. In this study, we first examined the effects of MSH3 deficiency on cytotoxicity caused by cisplatin and oxaliplatin, another ICL-inducing platinum drug. Using isogenic HCT116-derived clones in which MSH3 expression is controlled by shRNA expression in a Tet-off system, we discovered that MSH3 deficiency sensitized cells to both cisplatin and oxaliplatin at clinically relevant doses. Interestingly, siRNA-induced down-regulation of the MLH1 protein did not affect MSH3-dependent toxicity of these drugs, indicating that this process does not require participation of the canonical MMR pathway. Furthermore, MSH3-deficient cells maintained higher levels of phosphorylated histone H2AX and 53BP1 after oxaliplatin treatment in comparison with MSH3-proficient cells, suggesting that MSH3 plays an important role in repairing DNA double strand breaks (DSBs). This role of MSH3 was further supported by our findings that MSH3-deficient cells were sensitive to olaparib, a poly(ADP-ribose) polymerase inhibitor. Moreover, the combination of oxaliplatin and olaparib exhibited a synergistic effect compared with either treatment individually. Collectively, our results provide novel evidence that MSH3 deficiency contributes to the cytotoxicity of platinum drugs through deficient DSB repair. These data lay the foundation for the development of effective prediction and treatments for cancers with MSH3 deficiency.

Minocycline protects against asthma independently of its antibiotic function and was recently reported as a potent poly(ADP-ribose) polymerase (PARP) inhibitor. In an animal model of asthma, a single administration of minocycline conferred excellent protection against ovalbumin-induced airway eosinophilia, mucus hypersecretion, and Th2 cytokine production (IL-4/IL-5/IL-12(p70)/IL-13/GM-CSF) and a partial protection against airway hyperresponsiveness. These effects correlated with pronounced reduction in lung and sera allergen-specific IgE. A reduction in poly(ADP-ribose) immunoreactivity in the lungs of minocycline-treated/ovalbumin-challenged mice correlated with decreased oxidative DNA damage. The effect of minocycline on PARP may be indirect, as the drug failed to efficiently block direct PARP activation in lungs of N-methyl-N'-nitro-N-nitroso-guanidine-treated mice or H(2)O(2)-treated cells. Minocycline blocked allergen-specific IgE production in B cells potentially by modulating T cell receptor (TCR)-linked IL-4 production at the mRNA level but not through a modulation of the IL-4-JAK-STAT-6 axis, IL-2 production, or NFAT1 activation. Restoration of IL-4, ex vivo, rescued IgE production by minocycline-treated/ovalbumin-stimulated B cells. IL-4 blockade correlated with a preferential inhibition of the NF-κB activation arm of TCR but not GSK3, Src, p38 MAPK, or ERK1/2. Interestingly, the drug promoted a slightly higher Src and ERK1/2 phosphorylation. Inhibition of NF-κB was linked to a complete blockade of TCR-stimulated GATA-3 expression, a pivotal transcription factor for IL-4 expression. Minocycline also reduced TNF-α-mediated NF-κB activation and expression of dependent genes. These results show a potentially broad effect of minocycline but that it may block IgE production in part by modulating TCR function, particularly by inhibiting the signaling pathway, leading to NF-κB activation, GATA-3 expression, and subsequent IL-4 production.

Defective DNA damage repair, leading to genomic instability, is a common event during tumorigenesis. Despite enabling the persistence of mutations, any of which can confer a growth advantage to the nascent tumor, these defects place an Achilles Heel reliance on remaining repair pathways for survival from DNA damage. The protein kinases ataxia telangiectasia mutated (ATM) and ATM and Rad3 related (ATR) are key mediators of a DNA damage response activated by DNA damage during the S and G2 phases of cell cycle. Together they signal a series of cellular responses including activation of checkpoints and repair by homologous recombination. Loss of ATM pathway function frequently occurs in cancer, commonly from loss of function mutations in the tumor suppressor, p53, a substrate of ATM. This leads to a reliance on ATR that can be exploited for therapeutic benefit. Activation of ATR, by generation of S-phase DNA damage (replication stress, [RS]), can arise from treatment with DNA damaging drugs and certain targeted therapies such as inhibitors of poly ADP ribose polymerase (PARP). PARP is an enzyme involved in several DNA repair pathways, including base excision repair. Some PARP inhibitors have been shown to form an irreversible complex with DNA, potentially generating a direct RS lesion. While initial data indicates that inhibition of ATR and PARP is synergistic in some cancer cells, a comprehensive assessment has not been reported. Inhibition of ATR was cytotoxic in combination with PARP inhibitors against many cancer, but not non-cancer, cells. This effect was observed with multiple PARP inhibitors irrespective of their potential to form a DNA complex. In large cell panels of over 100 cancer cell lines, greater synergy was observed for the combination of an ATR and PARP inhibitor in cell lines with mutation of the TP53 gene. This was confirmed in isogenic cell lines depleted for ATM or p53, and is consistent with the profile of ATR and DNA damaging drug combinations. Furthermore, a triple combination of a PARP inhibitor, ATR inhibitor and the DNA damaging drug, cisplatin, retained cancer cell specific cytotoxic activity. In vitro dose-scheduling studies with the doublet of a PARP and ATR inhibitor showed optimal activity was achieved with transient concurrent exposure to both agents. This schedule contrasts with that for ATR inhibitors in combination with DNA damaging drugs where sequential dosing was optimal. In a mouse xenograft model concurrent dosing on a twice-weekly schedule was effective and well-tolerated. These data demonstrate the potential of combining ATR and PARP inhibitors in patients with p53 defective tumors. An optimal dose schedule was defined from cell and mouse studies. Together the data support clinical evaluation of ATR and PARP inhibitor combinations. Citation Format: John Pollard, Phil Reaper, Adele Peek, Stuart Hughes, Hakim Djeha, Steven Cummings, Karen Larbi, Marina Penney, Jim Sullivan, Darin Takemoto, Chris DeFranco. Pre-clinical combinations of ATR and PARP inhibitors: Defining target patient populations and dose schedule. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3711.

Locally advanced and metastatic breast cancer remains a challenge to treat. With emerging study results, it is important to interpret the available clinical data and apply the evidence offering the most effective treatment to the right patient. Poly(ADP Ribose) Polymerase (PARP) inhibitors are a new class of drug and their role in the treatment of locally advanced and metastatic breast cancer is being established. To determine the efficacy, safety profile, and potential harms of Poly(ADP-Ribose) Polymerase (PARP) inhibitors in the treatment of patients with locally advanced or metastatic breast cancer. The primary outcome of interest was overall survival; secondary outcomes included progression-free survival, tumour response rate, quality of life, and adverse events. On 8 June 2020, we searched the Cochrane Breast Cancer Group Specialised Register, the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE via OvidSP, Embase via OvidSP, World Health Organization International Clinical Trials Registry Platform (WHO ICTRP) search portal and ClinicalTrials.gov. We also searched proceedings from the major oncology conferences as well as scanned reference lists from eligible publications and contacted corresponding authors of trials for further information, where needed. We included randomised controlled trials on participants with locally advanced or metastatic breast cancer comparing 1) chemotherapy in combination with PARP inhibitors, compared to the same chemotherapy without PARP inhibitors or 2) treatment with PARP inhibitors, compared to treatment with other chemotherapy. We included studies that reported on our primary outcome of overall survival and secondary outcomes including progression-free survival, tumour response rate, quality of life, and adverse events. We used standard methodological procedures defined by Cochrane. Summary statistics for the endpoints used hazard ratios (HR) with 95% confidence intervals (CI) for overall survival and progression-free survival, and odds ratios (OR) for response rate (RR) and toxicity. We identified 49 articles for qualitative synthesis, describing five randomised controlled trials that were included in the quantitative synthesis (meta-analysis). A sixth trial was assessed as eligible but had ended prematurely and no data were available for inclusion in our meta-analysis. Risk of bias was predominately low to unclear across all studies except in regards to performance bias (3/5 high risk) and detection bias for the outcomes of quality of life (2/2 high risk) and reporting of adverse events (3/5 high risk). High-certainty evidence shows there may be a small advantage in overall survival (HR 0.87, 95% CI 0.76 to 1.00; 4 studies; 1435 patients). High-certainty evidence shows that PARP inhibitors offer an improvement in PFS in locally advanced/metastatic HER2-negative, BRCA germline mutated breast cancer patients (HR 0.63, 95% CI 0.56 to 0.71; 5 studies; 1474 patients). There was no statistical heterogeneity for these outcomes. Subgroup analyses for PFS outcomes based on trial level data were performed for triple-negative breast cancer, hormone-positive and/or HER2-positive breast cancer, BRCA1 and BRCA2 germline mutations, and patients who had received prior chemotherapy for advanced breast cancer or not. The subgroup analyses showed a persistent PFS benefit regardless of the subgroup chosen. Pooled analysis shows PARP inhibitors likely result in a moderate improvement in tumour response rate compared to other treatment arms (66.9% vs 48.9%; RR 1.39, 95% CI 1.24 to 1.54; 5 studies; 1185 participants; moderate-certainty evidence). The most common adverse events reported across all five studies included neutropenia, anaemia and fatigue. Grade 3 or higher adverse events probably occur no less frequently in patients receiving PARP inhibitors (59.4% for PARP arm versus 64.5% for non-PARP arm, RR 0.98, 95% CI 0.91 to 1.04; 5 studies; 1443 participants; moderate-certainty evidence). Only two studies reported quality of life outcomes so this was not amenable to meta-analysis. However, both studies that did assess quality of life showed PARP inhibitors were superior compared to physician's choice of chemotherapy in terms of participant-reported outcomes. In people with locally advanced or metastatic HER2-negative, BRCA germline mutated breast cancer, PARP inhibitors offer an improvement in progression-free survival, and likely improve overall survival and tumour response rates. This systematic review provides evidence supporting the use of PARP inhibitors as part of the therapeutic strategy for breast cancer patients in this subgroup. The toxicity profile for PARP inhibitors is probably no worse than chemotherapy but more information is required regarding quality of life outcomes, highlighting the importance of collecting such data in future studies. Future studies should also be powered to detect clinically important differences in overall survival and could focus on the role of PARP inhibitors in other relevant breast cancer populations, including HER2-positive, BRCA-negative/homologous recombination repair-deficient and Programmed Death-Ligand 1 (PDL1) positive.

High-grade serous ovarian carcinoma (HGSOC) is the most common subtype of ovarian cancer with low 5-year survival rates. Inhibitors of poly (ADP-ribose) polymerase (PARP) are promising novel agents that uniquely target HGSOCs with DNA repair deficiencies. The majority of patients with DNA repair proficient HGSOCs do not respond to PARP inhibitor monotherapy, highlighting the need for novel treatment approaches for these women. The goal of this study was to expand the patient population that could benefit from PARP inhibition by identifying the off-target effects that are favorable in combination with CHK1 inhibitors among the five most clinically advanced PARP inhibitors. Cell cycle and western blot analysis revealed that the four most potent PARP trappers (rucaparib, olaparib, niraparib, and talazoparib) have a strong effect on the cell cycle. The PARP trappers in particular activated CHK1 through phosphorylation at the serine 345 residue, decreased total protein levels of CDC25C, and arrested cells in the S/G2 phase of the cell cycle. These results were not observed after treatment with veliparib, the least potent PARP trapper. We created phospo-CHK1 mutants to confirm CHK1 activation at serine 345 and to further investigate whether potent PARP trappers activate CHK1 on serine 317, another residue that is also phosphorylated in response to replication stress. The data suggest that PARP trappers activate CHK1 at both the serine 317 and 345 phosphorylation sites. Next, we examined whether PARP inhibition-induced activation of CHK1 is a determinant of synergy between PARP and CHK1 inhibitors. We exposed one of our established BRCA wild-type, TP53 mutant HGSOC patient-derived xenograft models to a PARP1 inhibitor (veliparib or talazoparib) or to the CHK1 inhibitor (MK-8776) or a combination of PARP and CHK1 inhibitors. CHK1 inhibition slightly increased sensitivity to talazoparib compared to single agent therapy. However, the tumor volume was significantly reduced by the combination of CHK1 inhibition and veliparib treatment compared to either single agent alone. This study suggests that there are off-target cell cycle effects that vary among the five most clinically-advanced PARP inhibitors that likely influence the response to combinatorial treatment. We propose the use of PARP and CHK1 inhibition as a strategy for HGSOC patients who would otherwise have minimal benefit to PARP inhibitor monotherapy. Citation Format: Monica E. Wielgos, Jay Patibandla, Michelle Firlit, Elke Van Oudenhove, Paulina Cybulska, Petar Jelinic, Douglas A. Levine. Variable off-target effects of clinically advanced PARP inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 335.

Background Aberrant or impaired repair of double-strand DNA breaks is a common feature of de novo acute myeloid leukemia and myelodysplastic syndromes. Since poly (ADP-ribose) polymerase (PARP) inhibitors have been recently shown to selectively target cells with defects in double-strand DNA repair, the aim of this study was to explore the possibility of exploiting defects in DNA repair in leukemic cells using PARP inhibitors. Leukemic cell lines were exposed to various PARP inhibitors alone and in combination with non-cytotoxic concentrations of DNA methyltransferase inhibitor, 5' aza-2'-deoxycytidine and/or the histone deacetylase inhibitor, MS275, to test for potentiation of apoptosis with these agents. PARP inhibitors, KU-0058948 and PJ34, induced cell cycle arrest and apoptosis of primary myeloid leukemic cells and myeloid leukemic cell lines in vitro. Immunofluorescence analysis also revealed that PARP inhibitor sensitivity in these leukemic cells was due to a defect in homologous recombination DNA repair. Addition of 5' aza-2'-deoxycytidine failed to increase the cytotoxicity of PARP inhibitors. In contrast, MS275 potentiated the cytotoxic effect of KU-0058948 and PJ34 in all PARP inhibitor-sensitive leukemic cells. Immunofluorescence analysis supported the idea that histone deacetylase inhibitors potentiate cytotoxicity by inhibiting DNA repair processes. Conclusions On the basis of the data presented here, we suggest that PARP inhibitors can potentially exploit defects in double-strand DNA break repair in leukemic cells, paving the way for testing the therapeutic potential of these agents in myelodysplastic syndromes and acute myeloid leukemia.

Small cell lung cancer (SCLC) is a highly malignant cancer type with a 5-year survival rate of less than 10%. Different from non-small cell lung cancer (NSCLC), no effective target therapies have been approved to treat SCLC. Poly (ADP) ribose polymerase (PARP) overexpression in SCLC has prompted great efforts to evaluate the role of PARP inhibitors in clinic. To maximize their therapeutic value it is urgent in need to explore the best combination strategies between PARP inhibitors and other pathway modulators. In this study we identified a SCLC patient-derived xenograft (PDX) model with high PARP level and low nicotinamide phosphoribosyltransferase (NAMPT) level. Furthermore, synergistic effect of PARP and NAMPT dual inhibition was demonstrated, supporting translational research of PARP inhibitors and NAMPT inhibitors in this model. The mRNA expression of PARP as well as its 30 most-studied synthetic lethality genes was compared between SCLC and NSCLC in both PDXs and cancer cell lines. We found that NAMPT levels decreased most significantly in SCLC compared with NSCLC across the 30 synthetic lethality genes of PARP. As NAMPT is the rate-limiting enzyme for the synthesis of the PARP substrate β-NAD+, we hypothesized that low β-NAD+ level due to low NAMPT level might render SCLC sensitive to PARP inhibition. LU-01-0547, a SCLC PDX model with high PARP and low NAMPT expression, was identified here. To test our hypothesis, we investigated the preclinical efficacy of ABT888, a PARP inhibitor, with or without FK866, a NAMPT inhibitor, in this subcutaneous xenograft model. The single-agent efficacy of ABT888 was demonstrated (TGI=96%@100 mg/kg, BID). At a sub-optimal dose (50 mg/kg, BID), treatment of ABT888 alone produced little activity in the same model, while its combination with a NAMPT inhibitor, FK866, significantly boosted the antitumor response indicating the synergistic effect (TGI=117%, ORR=6/6, CR=4/6). When nicotinic acid (NA) was administered to promote β-NAD+ biosynthesis via the de novo pathway other than the NAMPT-mediated transformation, the PARP inhibitor’s effectiveness was greatly antagonized (TGI=70% vs. 13%), suggesting β-NAD+ level might correlate with PARP inhibitor sensitivity. In conclusion, this study identified a SCLC PDX model with high PARP and low NAMPT expression. Synergistic inhibition of PARP and NAMPT in this model was demonstrated superior to either treatment alone in efficacy, which warranted its future application in drug discovery. To our knowledge, this was the first in vivo evidence of synthetic lethality in SCLC PDX, supporting future clinical test of combination of PARP and NAMPT inhibitors in SCLC patients. Citation Format: Zhixiang Zhang, Dongfang Li, Chen Chen, Bo Zhang, Xuzhen Tang, Hao Ye, Qingyang Gu, Qunsheng Ji. Validation of synthetic lethality of PARP and NAMPT dual inhibition in a small cell lung cancer PDX model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3852. doi:10.1158/1538-7445.AM2017-3852

Poly (ADP-ribose) polymerase (PARP) inhibitors are the first clinically approved drugs designed to exploit synthetic lethality, and were first introduced as a cancer-targeting strategy in 2005. They have led to a major change in the treatment of advanced ovarian cancer, and altered the natural history of a disease with extreme genetic complexity and defective DNA repair via homologous recombination (HR) pathway. Furthermore, additional mechanisms apart from breast related cancer antigens 1 and 2 (BRCA1/2) mutations can also result in HR pathway alterations and consequently lead to a clinical benefit from PARP inhibitors. Novel combinations of PARP inhibitors with other anticancer therapies are challenging, and better understanding of PARP biology, DNA repair mechanisms, and PARP inhibitor mechanisms of action is crucial. It seems that PARP inhibitor and biologic agent combinations appear well tolerated and clinically effective in both BRCA-mutated and wild-type cancers. They target differing aberrant and exploitable pathways in ovarian cancer, and may induce greater DNA damage and HR deficiency. The input of immunotherapy in ovarian cancer is based on the observation that immunosuppressive microenvironments can affect tumour growth, metastasis, and even treatment resistance. Several biologic agents have been studied in combination with PARP inhibitors, including inhibitors of vascular endothelial growth factor (VEGF; bevacizumab, cediranib), and PD-1 or PD-L1 (durvalumab, pembrolizumab, nivolumab), anti-CTLA4 monoclonal antibodies (tremelimumab), mTOR-(vistusertib), AKT-(capivasertib), and PI3K inhibitors (buparlisib, alpelisib), as well as MEK 1/2, and WEE1 inhibitors (selumetinib and adavosertib, respectively). Olaparib and veliparib have also been combined with chemotherapy with the rationale of disrupting base excision repair via PARP inhibition. Olaparib has been investigated with carboplatin and paclitaxel, whereas veliparib has been tested additionally in combination with temozolomide vs. pegylated liposomal doxorubicin, as well as with oral cyclophosphamide, and topoisomerase inhibitors. However, overlapping myelosuppression observed with PARP inhibitor and chemotherapy combinations requires further investigation with dose escalation studies. In this review, we discuss multiple clinical trials that are underway examining the antitumor activity of such combination strategies.

Genetic and Epigenetic Defects in DNA Repair Lead to Synthetic Lethality of Poly (ADP-Ribose) Polymerase (PARP) Inhibitors in Aggressive Myeloproliferative Disorders