ID3 promotes homologous recombination via non-transcriptional and transcriptional mechanisms and its loss confers sensitivity to PARP inhibition.

The inhibitor of DNA-binding 3 (ID3) is a transcriptional regulator that limits interaction of basic helix-loop-helix transcription factors with their target DNA sequences. We previously reported that ID3 loss is associated with mutational signatures linked to DNA repair defects. Here we demonstrate that ID3 exhibits a dual role to promote DNA double-strand break (DSB) repair, particularly homologous recombination (HR). ID3 interacts with the MRN complex and RECQL helicase to activate DSB repair and it facilitates RAD51 loading and downstream steps of HR. In addition, ID3 promotes the expression of HR genes in response to ionizing radiation by regulating both chromatin accessibility and activity of the transcription factor E2F1. Consistently, analyses of TCGA cancer patient data demonstrate that low ID3 expression is associated with impaired HR. The loss of ID3 leads to sensitivity of tumor cells to PARP inhibition, offering new therapeutic opportunities in ID3-deficient tumors.

Replication-dependent cytotoxicity and Spartan-mediated repair of trapped PARP1-DNA complexes.

The antitumor activity of poly(ADP-ribose) polymerase inhibitors (PARPis) has been ascribed to PARP trapping, which consists in tight DNA-protein complexes. Here we demonstrate that the cytotoxicity of talazoparib and olaparib results from DNA replication. To elucidate the repair of PARP1-DNA complexes associated with replication in human TK6 and chicken DT40 lymphoblastoid cells, we explored the role of Spartan (SPRTN), a metalloprotease associated with DNA replication, which removes proteins forming DPCs. We find that SPRTN-deficient cells are hypersensitive to talazoparib and olaparib, but not to veliparib, a weak PARP trapper. SPRTN-deficient cells exhibit delayed clearance of trapped PARP1 and increased replication fork stalling upon talazoparib and olaparib treatment. We also show that SPRTN interacts with PARP1 and forms nuclear foci that colocalize with the replicative cell division cycle 45 protein (CDC45) in response to talazoparib. Additionally, SPRTN is deubiquitinated and epistatic with translesion synthesis (TLS) in response to talazoparib. Our results demonstrate that SPRTN is recruited to trapped PARP1 in S-phase to assist in the excision and replication bypass of PARP1-DNA complexes.

Modulation of PARP activity by Monomethylarsonous (MMA+3) acid and uranium in mouse thymus.

Arsenic exposure is well established to impair the function of zinc finger proteins, including PARP-1. Previous studies from our lab show that early developing T cells in the thymus are very sensitive to arsenite (As+3)-induced genotoxicity mediated through PARP-1 inhibition. Additionally, it has been shown that uranium (in the form of uranyl acetate, UA) also suppresses PARP-1 activity in HEK cells. However, very little is known about whether the As+3 metabolite, monomethylarsonous acid (MMA+3), also inhibits PARP-1 activity and if this is modified by combined exposures with other metals, such as uranium. In the present study, we found that MMA+3 significantly suppressed PARP-1 function, whereas UA at high concentrations significantly increased PARP-1 activity. To evaluate whether the effects on PARP-1 activity were mediated through oxidative stress, we measured the induction of hemoxygenase-1 (Hmox-1) expression by qPCR. MMA+3, but not UA, significantly induced oxidative stress; however, the inhibition of PARP-1 produced by MMA+3 was not reversed by the addition of the antioxidant, Tempol. Further evaluation revealed minimal interactive effects of MMA+3 and UA on PARP-1 function. Collectively, our results show that contrary to As+3, the suppressive effects of MMA+3 on PARP-1 were not substantially driven by oxidative stress. in mouse thymus cells. Results for this study provide important insights into the effects of MMA+3 and uranium exposures on PARP-1 function, which is essential for future studies focused on understanding the effects of complex environmentally relevant metal mixtures.

Inhibition of poly(ADP-ribose)polymerase-1 and DNA repair by uranium.

Uranium has radiological and non-radiological effects within biological systems and there is increasing evidence for genotoxic and carcinogenic properties attributable to uranium through its heavy metal properties. In this study, we report that low concentrations of uranium (as uranyl acetate; <10 µM) is not cytotoxic to human embryonic kidney cells or normal human keratinocytes; however, uranium exacerbates DNA damage and cytotoxicity induced by hydrogen peroxide, suggesting that uranium may inhibit DNA repair processes. Concentrations of uranyl acetate in the low micromolar range inhibited the zinc finger DNA repair protein poly(ADP-ribose) polymerase (PARP)-1 and caused zinc loss from PARP-1 protein. Uranyl acetate exposure also led to zinc loss from the zinc finger DNA repair proteins Xeroderma Pigmentosum, Complementation Group A (XPA) and aprataxin (APTX). In keeping with the observed inhibition of zinc finger function of DNA repair proteins, exposure to uranyl acetate enhanced retention of induced DNA damage. Co-incubation of uranyl acetate with zinc largely overcame the impact of uranium on PARP-1 activity and DNA damage. These findings present evidence that low concentrations of uranium can inhibit DNA repair through disruption of zinc finger domains of specific target DNA repair proteins. This may provide a mechanistic basis to account for the published observations that uranium exposure is associated with DNA repair deficiency in exposed human populations.

Elucidating the Therapeutic Utility of Olaparib in Sulfatide-Induced Human Astrocyte Toxicity and Neuroinflammation.

Metachromatic leukodystrophy (MLD) is a severe demyelinating, autosomal recessive genetic leukodystrophy, with no curative treatment. The disease is underpinned by mutations in the arylsulfatase A gene (ARSA), resulting in deficient activity of this lysosomal enzyme, and consequential accumulation of galactosylceramide-3-O-sulfate (sulfatide) in the brain. Most of the effects in the brain have been attributed to the accumulation of sulfatides in oligodendrocytes and their cell damage. In contrast, less is known regarding sulfatide toxicity in astrocytes. Poly (ADP-ribose) polymerase (PARP) inhibitors are anti-cancer therapeutics that have proven efficacy in preclinical models of many neurodegenerative and inflammatory diseases, but have never been tested for MLD. Here, we examined the toxic effect of sulfatides on human astrocytes and restoration of this cell damage by the marketed PARP-1 inhibitor, Olaparib. Cultured human astrocytes were treated with increasing concentrations of sulfatides (5-100 µM) with or without Olaparib (100 nM). Cell viability assays were used to ascertain whether sulfatide-induced toxicity was rescued by Olaparib. Immunofluorescence, calcium (Ca2+) imaging, ROS, and mitochondrial damage assays were also used to explore the effects of sulfatides and Olaparib. ELISAs were performed and chemotaxis of peripheral blood immune cells was measured to examine the effects of Olaparib on sulfatide-induced inflammation in human astrocytes. Here, we established a concentration-dependent (EC50∼20 µM at 24 h) model of sulfatide-induced astrocyte toxicity. Our data demonstrate that sulfatide-induced astrocyte toxicity involves (i) PARP-1 activation, (ii) pro-inflammatory cytokine release, and (iii) enhanced chemoattraction of peripheral blood immune cells. Moreover, these sulfatide-induced effects were attenuated by Olaparib (IC50∼100 nM). In addition, sulfatide caused impairments of ROS production, mitochondrial stress, and Ca2+ signaling in human astrocytes, that were indicative of metabolic alterations and that were also alleviated by Olaparib (100 nM) treatment. Our data support the hypothesis that sulfatides can drive astrocyte cell death and demonstrate that Olaparib can dampen many facets of sulfatide-induced toxicity, including, mitochondrial stress, inflammatory responses, and communication between human astrocytes and peripheral blood immune cells. These data are suggestive of potential therapeutic utility of PARP inhibitors in the sphere of rare demyelinating diseases, and in particular MLD. Graphical abstract. Proposed mechanism of action of Olaparib in sulfatide-treated astrocytes. Human astrocytes treated for 24 h with sulfatides increase PARP-1 expression and die. PARP-1 overexpression is modulated by Ca2+ release from the endoplasmic reticulum, thus enhancing intracellular Ca2+ concentration. PARP-1 inhibition with Olaparib reduces Ca2+ influx and cell death. Olaparib also decreases IL-6, IL-8, IL-17, and CX3CL1 release from sulfatide-stimulated astrocytes, suggesting that PARP-1 plays a role in dampening neuroinflammation in MLD. This is confirmed by the reduction of immune cell migration such as lymphocytes, NK cells, and T cells towards sulfatide-treated astrocytes. Moreover, mitochondrial stress and ROS production induced by sulfatides are rescued by PARP-1 inhibition. Future studies will focus on the signaling cascades triggered by PARP-1-mediated currents in reactive astrocytes and Olaparib as a potential therapeutic target for MLD.

DNA damage and up-regulation of PARP-1 induced by columbin in vitro and in vivo.

Columbin (CLB) is the most abundant (>1.0%) furan-containing diterpenoid lactone in herbal medicine Tinospora sagittate (Oliv.) Gagnep. The furano-terpenoid was found to be hepatotoxic, but the exact mechanisms remain unknown. The present study demonstrated that administration of CLB at 50 mg/kg induced hepatotoxicity, DNA damage and up-regulation of PARP-1 in vivo. Exposure to CLB (10 µM) induced GSH depletion, over-production of ROS, DNA damage, up-regulation of PARP-1 and cell death in cultured mouse primary hepatocytes in vitro. Co-treatment of mouse primary hepatocytes with ketoconazole (10 µM) or glutathione ethyl ester (200 µM) attenuated the GSH depletion, over-production of ROS, DNA damage, up-regulation of PARP-1, and cell death induced by CLB, while co-exposure to L-buthionine sulfoximine (BSO, 1000 µM) intensified such adverse effects resulting from CLB exposure. These results suggest that the metabolic activation of CLB by CYP3A resulted in the depletion of GSH and increase of ROS formation. The resultant over-production of ROS subsequently disrupted the DNA integrity and up-regulated the expression of PARP-1 in response to DNA damage, and ROS-induced DNA damage was involved in the hepatotoxicity of CLB.

DNA damage triggers an interplay between wtp53 and c-Myc affecting lymphoma cell proliferation and Kaposi sarcoma herpesvirus replication.

The induction of DNA damage together with the interference with DNA repair represents a promising strategy in cancer treatment. Here we show that the PARP-1/2/3 inhibitor AZD2461 in combination with the CHK1 inhibitor UCN-01 altered the DNA damage response and reduced cell proliferation in PEL cells, an aggressive B cell lymphoma highly resistant to chemotherapies. AZD2461/UCN-01 combination activated p53/p21 and downregulated c-Myc in these cells, leading to a reduced expression level of RAD51, molecule involved in DNA repair. The effect of AZD2461/UCN-01 on c-Myc and p53/p21 was inter-dependent and, besides impairing cell proliferation, contributed to the activation of the replicative cycle of KSHV, carried in a latent state in PEL cells. Finally, we found that the pharmacological or genetic inhibition of p21 counteracted the viral lytic cycle activation and further reduced PEL cell proliferation, suggesting that it could induce a double beneficial effect in this setting. This study unveils that, therapeutic approaches, based on the induction of DNA damage and the reduction of DNA repair, could be used to successfully treat this malignant lymphoma.

In Silico Discovery of Novel Flavonoids as Poly ADP Ribose Polymerase (PARP) Inhibitors.

BACKGROUND: 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. OBJECTIVE: 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. MATERIALS AND METHODS: Virtual screening carried out using different in silico methods which include molecular docking studies, prediction of drug-likeness and in silico toxicity studies. RESULTS: 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. CONCLUSION: Our finding suggests that NPACT00183 and NPACT00280 have promising potential to be further explored as PARP inhibitors.

Three dimensional and microphysiological bone marrow models detect in vivo positive compounds.

Micronucleus (MN) assessment is a valuable tool in safety assessment. However, several compounds are positive in the in vivo bone marrow (BM) MN assay but negative in vitro, reflecting that BM complexity is not recapitulated in vitro. Importantly, these compounds are not genotoxic; rather, drug-driven pharmacological-effects on the BM increase MN, however, without mechanistic understanding, in vivo positives stop drug-progression. Thus, physiologically-relevant BM models are required to bridge the gap between in vitro and in vivo. The current study aimed to investigate the utility of two human 3D BM models (fluidic and static) for MN assessment. MN induction following treatment with etoposide and Poly-ADP Ribose Polymerase inhibitor (PARPi) and prednisolone (negative in vitro, positive in vivo) was determined in 2D L5178Y and human BM cells, and the 3D BM models. Etoposide (0-0.070 µM) and PARPi (0-150 µM) induced MN in both 3D BM models indicating their utility for genotoxicity testing. Interestingly, PARPi treatment induced a MN trend in 3D more comparable to in vivo. Importantly, prednisolone (0-1.7 mM) induced MN in both 3D BM models, suggesting recapitulation of the in vivo microenvironment. These models could provide a valuable tool to follow up, and eventually predict, suspected pharmacological mechanisms, thereby reducing animal studies.

Evaluation of a Low-Toxicity PARP Inhibitor as a Neuroprotective Agent for Parkinson's Disease.

Repurposing PARP-1 inhibitors (PARPi) for non-oncological applications offers an attractive therapeutic strategy for pathological conditions characterized by PARP-1 hyperactivity. In the context of Parkinson's disease (PD), PARP-1 hyperactivity has been linked to neuronal death and disease progression. From a therapy perspective, the evaluation of PARPi as neuroprotective agents may offer a new therapeutic alternative for neurodegenerative disorders. An ideal PARPi needs to inhibit PARP-1 hyperactivity while also limiting downstream DNA damage and cellular toxicity-an effect that is attractive in cancer but far from ideal in neurological disease applications. Consequently, in this study, we set out to evaluate the neuroprotective properties of a previously reported low-toxicity PARPi (10e) using in vitro neuronal models of PD. 10e is a structural analogue of FDA-approved PARPi olaparib, with high PARP-1 affinity and selectivity. Our studies revealed that 10e protects neuronal cells from oxidative stress and DNA damage. In addition, 10e exhibits neuroprotective properties against α-synuclein pre-formed fibrils (αSyn PFF) mediated effects, including reduction in the levels of phosphorylated αSyn and protection against abnormal changes in NAD+ levels. Our in vitro studies with 10e provide support for repurposing high-affinity and low-toxicity PARPi for neurological applications and lay the groundwork for long-term therapeutic studies in animal models of PD.

Specific Toxicity of Maintenance Olaparib Versus Placebo in Advanced Malignancies: A Systematic Review and Meta-analysis.

BACKGROUND/AIM: We performed a systematic review and meta-analysis to investigate the safety of maintenance with olaparib after platinum-based chemotherapy in cancer patients. MATERIALS AND METHODS: Eligible studies included randomized controlled trials (RCTs) regarding the clinical role of olaparib maintenance therapy versus placebo in BRCA-mutated, advanced cancers. Safety profile from each selected study was investigated for all-grade and G3-G4 haematological and non-haematological adverse drug events (ADEs). RESULTS: Four RTCs that involved 1099 patients were included in the analysis. Overall incidences of all-grade and G3-4 ADEs in olaparib group were 97.6% and 41%, respectively. Patients treated with maintenance olaparib showed higher risk of all-grade and G3-G4 anaemia, all-grade neutropenia and thrombocytopenia. Moreover, all-grade and G3-G4 fatigue, all-grade vomiting, diarrhoea, nausea and decreased appetite were more common in the olaparib group compared to placebo. CONCLUSION: Despite an increased risk and incidence of several haematological and non-haematological toxicities, olaparib is a relatively safe agent for the treatment of advanced solid tumors. Prompt identification of ADEs is mandatory to avoid therapy discontinuation and optimize treatment.

Metabolic Stability Assessment of New PARP Inhibitor Talazoparib Using Validated LC-MS/MS Methodology: In silico Metabolic Vulnerability and Toxicity Studies.

BACKGROUND: Talazoparib (BMN673) is a new poly(ADP-ribose) polymerase inhibitor that has been FDA approved for patients suffering from metastatic breast cancer with germline BRCA mutations. METHOD AND RESULTS: In the current study, an accurate and efficient liquid chromatography-tandem mass spectrometry (LC-MS/MS) analytical methodology was developed for TZB estimation in addition to its metabolic stability assessment. TZB and lapatinib (LAP) (which is chosen as an internal standard; IS) were separated using reversed phase elution system (Hypersil C18 column) with an isocratic mobile phase. The linearity range of the established method was 5-500 ng/mL (r2 ≥ 0.999) in the human liver microsomes (HLMs) matrix. Different parameters were calculated to confirm the method sensitivity (limit of quantification was 2.0 ng/mL), and reproducibility (intra- and inter-day precision and accuracy were below 3.1%) of our methodology. For evaluation of TZB metabolic stability in HLM matrix, intrinsic clearance (9.59 µL/min/mg) and in vitro half-life (72.7 mins) were calculated. TZB treatment discontinuations were reported due to adverse events and dose accumulation, so in silico metabolic vulnerability (experimental and in silico) and toxicity assessment (in silico) of TZB were performed utilizing P450 Metabolism and DEREK modules of StarDrop software. CONCLUSION: TZB is slowly metabolized by the liver. TZB was reported to be minimally metabolized by the liver that approved our outcomes. We do recommend that plasma levels be monitored in cases when talazoparib is used for a long period of time, since it is possible for TZB to bioaccumulate after multiple doses to toxic levels. According to our knowledge, the current method is considered the first LC-MS/MS methodology for evaluating TZB metabolic stability. Further drug discovery studies can be done depending on this concept allowing the designing of new series of compounds with more safety profile through reducing side effects and improving metabolic behavior.

Structure based pharmacophore study to identify possible natural selective PARP-1 trapper as anti-cancer agent.

Inhibition of poly(ADP-ribose) polymerase-1 (PARP-1) has turned out an innovative approach for cancer therapy due to its involvement in DNA repair pathways. Although several potent PARP-1 inhibitors have been identified, they exhibit high toxicity, resistivity and diverse pharmacological profile in clinical trials, which necessitate for extensive investigation and development of selective inhibitors. Therefore, the study aimed to identify selective natural PARP-1 inhibitors to reduce toxicity and resistivity with high potency. Accordingly, the combined approach of structure-based pharmacophore and molecular docking study was performed. Hence, the two hits (SN00167272 and STOCK1N-92279) were identified to have all the pharmacophoric features that showed interaction with key residues (Gly863, Ser904, Tyr896, and Tyr907) and least conserved residues (Tyr889 and Asp766). Additionally, these inhibitors represented interactions with unique selective residues (Asp756, Val762, Glu763 and Val886) and exhibited strong interaction with PARP-1 through binding free energy and molecular dynamics study. Hence, the identified hits could further considered for experimental investigations as they may reduce off-target and resistivity of currently available inhibitors and developed as potential anti-cancer agents in the future. Also, the study provides a specific structural insight which could further help to design selective and potent PARP-1 inhibitors.

Phase 1 study of veliparib (ABT-888), a poly (ADP-ribose) polymerase inhibitor, with carboplatin and paclitaxel in advanced solid malignancies.

PURPOSE: Veliparib is an oral inhibitor of poly (ADP-ribose) polymerase (PARP)-1 and -2. PARP-1 expression may be increased in cancer, and this increase confers resistance to cytotoxic agents. We aimed to determine the recommended phase 2 dose (RP2D), maximum tolerated dose (MTD), dose-limiting toxicity (DLT), and pharmacokinetics (PK) of veliparib combined with paclitaxel and carboplatin. METHODS: Eligibility criteria included patients with advanced solid tumors treated with ≤ 3 prior regimens. Paclitaxel and carboplatin were administered on day 3 of a 21-day cycle. Veliparib was given PO BID days 1-7, except for cycle 1 in the first 46 patients to serve as control for toxicity and PK. A standard "3 + 3" design started veliparib at 10 mg BID, paclitaxel at 150 mg/m2, and carboplatin AUC 6. The pharmacokinetic (PK) disposition of veliparib, paclitaxel, and carboplatin was determined by LC-MS/MS and AAS during cycles 1 and 2. RESULTS: Seventy-three patients were enrolled. Toxicities were as expected with carboplatin/paclitaxel chemotherapy, including neutropenia, thrombocytopenia, and peripheral neuropathy. DLTs were seen in two of seven evaluable patients at the maximum administered dose (MAD): veliparib 120 mg BID, paclitaxel 200 mg/m2, and carboplatin AUC 6 (febrile neutropenia, hyponatremia). The MTD and RP2D were determined to be veliparib 100 mg BID, paclitaxel 200 mg/m2, and carboplatin AUC 6. Median number of cycles of the three-agent combination was 4 (1-16). We observed 22 partial and 5 complete responses. Veliparib did not affect paclitaxel or carboplatin PK disposition. CONCLUSION: Veliparib, paclitaxel, and carboplatin were well tolerated and demonstrated promising antitumor activity.

Sequential Therapy with PARP and WEE1 Inhibitors Minimizes Toxicity while Maintaining Efficacy.

We demonstrate that concurrent administration of poly(ADP-ribose) polymerase (PARP) and WEE1 inhibitors is effective in inhibiting tumor growth but poorly tolerated. Concurrent treatment with PARP and WEE1 inhibitors induces replication stress, DNA damage, and abrogates the G2 DNA damage checkpoint in both normal and malignant cells. Following cessation of monotherapy with PARP or WEE1 inhibitors, effects of these inhibitors persist suggesting that sequential administration of PARP and WEE1 inhibitors could maintain efficacy while ameliorating toxicity. Strikingly, while sequential administration mirrored concurrent therapy in cancer cells that have high basal replication stress, low basal replication stress in normal cells protected them from DNA damage and toxicity, thus improving tolerability while preserving efficacy in ovarian cancer xenograft and patient-derived xenograft models.

Influence of PARP-1 inhibition in the cardiotoxicity of the topoisomerase 2 inhibitors doxorubicin and mitoxantrone.

Doxorubicin (DOX) and Mitoxantrone (MTX) are very effective drugs for a range of tumors despite being highly cardiotoxic. DNA topoisomerase 2 beta (Top2ß) was revealed as key mediator of DOX-induced cardiotoxicity, although ROS generation is also an important mechanism. Oxidative stress is also an important issue in MTX-induced cardiotoxicity that is manifested by mitochondrial dysfunction. Studies have demonstrated the relationship between PARP-1 overactivation and cell viability in DOX-treated cardiomyocytes. In reference of MTX, data regarding PARP-1 overactivation as the mechanism responsible for cardiotoxicity is difficult to find. The aim of this study was to evaluate the influence of PARP-1 inhibitor DPQ on DOX- and MTX-mediated cardiotoxicity. Cells were exposed for 24 h to DOX or MTX in the presence or absence of DPQ. Viability, apoptosis, and genotoxicity assays were carried out. Immunofluorescence of phosphorylated histone H2AX was analyzed in H9c2 cells and cardiomyocytes from neonatal rats. Results demonstrated that DPQ co-treatment increases DOX-induced apoptosis in H9c2 cells. DPQ also prevents DOX and MTX-ROS generation in part by increasing SOD and CAT activities. Furthermore, DPQ co-treatment increased the generation of DNA strand breaks by DOX and MTX whilst also inducing phosphorylation of H2AX, MRE11, and ATM in H9c2 cells. Our results demonstrated that as well as increasing DNA damage and inducing apoptotic cell death, DPQ enhances DOX- and MTX-mediated cytotoxicity in H9c2.

Synthesis and Evaluation of a Mitochondria-Targeting Poly(ADP-ribose) Polymerase-1 Inhibitor.

The poly(ADP-ribose) polymerase (PARP) family of enzymes plays a crucial role in cellular and molecular processes including DNA damage detection and repair and transcription; indeed, PARP inhibitors are under clinical evaluation as chemotherapeutic adjuncts given their capacity to impede genomic DNA repair in tumor cells. Conversely, overactivation of PARP can lead to NAD+ depletion, mitochondrial energy failure, and cell death. Since PARP activation facilitates genomic but impedes mitochondrial DNA repair, nonselective PARP inhibitors are likely to have opposing effects in these cellular compartments. Herein, we describe the synthesis and evaluation of the mitochondria-targeting PARP inhibitor, XJB-veliparib. Attachment of the hemigramicidin S pentapeptide isostere for mitochondrial targeting using a flexible linker at the primary amide site of veliparib did not disrupt PARP affinity or inhibition. XJB-veliparib was effective at low nanomolar concentrations (10-100 nM) and more potent than veliparib in protection from oxygen-glucose deprivation (OGD) in primary cortical neurons. Both XJB-veliparib and veliparib (10 nM) preserved mitochondrial NAD+ after OGD; however, only XJB-veliparib prevented release of NAD+ into cytosol. XJB-veliparib (10 nM) appeared to inhibit poly(ADP-ribose) polymer formation in mitochondria and preserve mitochondrial cytoarchitecture after OGD in primary cortical neurons. After 10 nM exposure, XJB-veliparib was detected by LC-MS in mitochondria but not nuclear-enriched fractions in neurons and was observed in mitoplasts stripped of the outer mitochondrial membrane obtained from HT22 cells. XJB-veliparib was also effective at preventing glutamate-induced HT22 cell death at micromolar concentrations. Importantly, in HT22 cells exposed to H2O2 to produce DNA damage, XJB-veliparib (10 µM) had no effect on nuclear DNA repair, in contrast to veliparib (10 µM) where DNA repair was retarded. XJB-veliparib and analogous mitochondria-targeting PARP inhibitors warrant further evaluation in vitro and in vivo, particularly in conditions where PARP overactivation leads to mitochondrial energy failure and maintenance of genomic DNA integrity is desirable, e.g., ischemia, oxidative stress, and radiation exposure.

Colorectal Cancer Chemotherapy: The Evolution of Treatment and New Approaches.

Colorectal cancer (CRC) is one the greatest contributors to cancer related mortality. Although 5 year survival rate for patients at the early stage of CRC (stages I and II) is above 60%, more than 50% of patients are diagnosed at or beyond stage III when distant metastasis has already occurred, in which case 5 year survival rate drops to 10%. Chemotherapeutic intervention coupled with surgery is the backbone of metastatic CRC treatment and the only means of enhanced survival. For decades following its discovery, an antimetabolite 5- fluorouracil (5-FU) was the only chemotherapeutic agent available to successfully improve 12 month survival in CRC patients. Treatment of metastatic CRC has been considered palliative for many years; aiming to increase the duration and quality of the patient&#039;s remaining life, with little hope of cure, highlighting the need for novel DNA and RNA targeted therapies in the treatment of CRC. Over the last several decades, combinations of several chemotherapeutic agents have been incorporated into routine clinical practice. Combination regimes incorporating irinotecan, a semisynthetic inhibitor of topoisomerase, oxaliplatin, a third-generation platinum compound that causes mitotic arrest via the formation of DNA adducts, and capecitabine, a 5-FU prodrug, are now all established options for use as first-line, second-line and sequential treatment of CRC. This review provides a brief overview of the evolution of CRC chemotherapy as well as new and emerging treatment options.

Combination Platinum-based and DNA Damage Response-targeting Cancer Therapy: Evolution and Future Directions.

Maintenance of genomic stability is a critical determinant of cell survival and is necessary for growth and progression of malignant cells. Interstrand crosslinking (ICL) agents, including platinum-based agents, are first-line chemotherapy treatment for many solid human cancers. In malignant cells, ICL triggers the DNA damage response (DDR). When the damage burden is high and lesions cannot be repaired, malignant cells are unable to divide and ultimately undergo cell death either through mitotic catastrophe or apoptosis. The activities of ICL agents, in particular platinum-based therapies, establish a "molecular landscape," i.e., a pattern of DNA damage that can potentially be further exploited therapeutically with DDR-targeting agents. If the molecular landscape created by platinum-based agents could be better defined at the molecular level, a systematic, mechanistic rationale(s) could be developed for the use of DDR-targeting therapies in combination/maintenance protocols for specific, clinically advanced malignancies. New therapeutic drugs such as poly(ADP-ribose) polymerase (PARP) inhibitors are examples of DDR-targeting therapies that could potentially increase the DNA damage and replication stress imposed by platinum-based agents in tumor cells and provide therapeutic benefit for patients with advanced malignancies. Recent studies have shown that the use of PARP inhibitors together with platinum-based agents is a promising therapy strategy for ovarian cancer patients with "BRCAness", i.e., a phenotypic characteristic of tumors that not only can involve loss-of-function mutations in either BRCA1 or BRCA2, but also encompasses the molecular features of BRCA-mutant tumors. On the basis of these promising results, additional mechanism-based studies focused on the use of various DDR-targeting therapies in combination with platinum-based agents should be considered. This review discusses, in general, (1) ICL agents, primarily platinum-based agents, that establish a molecular landscape that can be further exploited therapeutically; (2) multiple points of potential intervention after ICL agent-induced crosslinking that further predispose to cell death and can be incorporated into a systematic, therapeutic rationale for combination/ maintenance therapy using DDR-targeting agents; and (3) available agents that can be considered for use in combination/maintenance clinical protocols with platinum-based agents for patients with advanced malignancies.

Small molecule inhibitors uncover synthetic genetic interactions of human flap endonuclease 1 (FEN1) with DNA damage response genes.

Flap endonuclease 1 (FEN1) is a structure selective endonuclease required for proficient DNA replication and the repair of DNA damage. Cellularly active inhibitors of this enzyme have previously been shown to induce a DNA damage response and, ultimately, cell death. High-throughput screens of human cancer cell-lines identify colorectal and gastric cell-lines with microsatellite instability (MSI) as enriched for cellular sensitivity to N-hydroxyurea series inhibitors of FEN1, but not the PARP inhibitor olaparib or other inhibitors of the DNA damage response. This sensitivity is due to a synthetic lethal interaction between FEN1 and MRE11A, which is often mutated in MSI cancers through instabilities at a poly(T) microsatellite repeat. Disruption of ATM is similarly synthetic lethal with FEN1 inhibition, suggesting that disruption of FEN1 function leads to the accumulation of DNA double-strand breaks. These are likely a result of the accumulation of aberrant replication forks, that accumulate as a consequence of a failure in Okazaki fragment maturation, as inhibition of FEN1 is toxic in cells disrupted for the Fanconi anemia pathway and post-replication repair. Furthermore, RAD51 foci accumulate as a consequence of FEN1 inhibition and the toxicity of FEN1 inhibitors increases in cells disrupted for the homologous recombination pathway, suggesting a role for homologous recombination in the resolution of damage induced by FEN1 inhibition. Finally, FEN1 appears to be required for the repair of damage induced by olaparib and cisplatin within the Fanconi anemia pathway, and may play a role in the repair of damage associated with its own disruption.