Dissertations / Theses: 'PARP1 Inhibitors'

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Alkhateeb, Hebah, Gregory A. Ordway, W. Drew Gill, Joshua B. Coleman, Hui Wang-Heaton, Russell W. Brown, Michelle Chandley, et al. "PARP1 inhibition produces unique antidepressant effects in an animal model of treatment-resistant depression." Digital Commons @ East Tennessee State University, 2019. https://dc.etsu.edu/asrf/2019/schedule/49.

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Major depressive disorder (MDD) is a prevalent and enervating mental illness affecting millions globally. Unfortunately, a significant proportion of patients do not receive clinical benefit from existing antidepressant medications. The limited effectiveness of currently available antidepressant drugs emphasizes the need to identify more effective medications for individuals who are treatment-resistant. We have previously reported abnormally elevated poly (ADP-ribose) polymerase-1 (PARP1) gene expression levels in the postmortem brain from MDD brain donors. PARP1 is a DNA damage repair enzyme that is also linked to neuroinflammation through multiple biochemical pathways. PARP1 upregulation in MDD could indicate a role for this enzyme in the etiopathology of MDD, particularly as it relates to neuroinflammation. In fact, we have shown that drugs that inhibit PARP1 produce antidepressant-like properties in two different rodent behavioral models that mimic depressed mood in humans. In the present study, we utilized a unique rodent behavioral model that produces depressive-like behavior by combining psychological stress with stimulation of inflammation. Depressive behavior produced by this experimental paradigm is not reversed by the prototypical antidepressant fluoxetine. This treatment-resistant depression was elicited by treating rats with injections of lipopolysaccharide (LPS; 0.1 ug/kg/day) and daily exposure to chronic unpredictable stress (CUS) for 28 days. Depressive behaviors were measured with sucrose preference and forced swim tests in 5 treatment groups (n=6-8 rats per group) including unstressed rats, CUS rats, CUS+LPS rats, and CUS+LPS rats treated with either the PARP1 inhibitor 3-aminobenzamide (3AB) or the antidepressant fluoxetine. We evaluated the role of neuroinflammation in this model by measuring the amount of microglial activation in several brain regions in rats from all treatment groups. Microglia activation was measured by quantifying the relative amount of expression of the microglia marker protein, IBA1, using an anti-IBA1 antibody. 3AB demonstrated robust and unique antidepressant activity superior to fluoxetine in the treatment-resistant rat model. IBA1-immunoreactivity levels were elevated in brains from CUS and CUS+LPS rats, although there was no evidence that LPS increased IBA1-immunoreactivity above levels found in CUS rats that did not receive LPS. Levels of IBA1-immunoreactivity in the brains from rats treated with either fluoxetine or 3AB trended lower as compared to the CUS and CUS+LPS groups, although this effect did not reach statistical significance. The lack of significant differences is likely related to small sample sizes; experiments are underway to increase the sample sizes of each group. The findings provide further support for the potential of PARP1 inhibitors in treating MDD and suggest that these drugs may be more effective, or more broadly effective than standard antidepressants.

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D'Angeli, Floriana. "Biomolecular effects and bioclinical applications of PARPs inhibitors." Doctoral thesis, Università di Catania, 2017. http://hdl.handle.net/10761/3832.

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Abstract Section I Inhibitors of PARP-1(Poly(ADP-ribose) polymerase-1) act by competing with NAD+, the enzyme physiological substrate, which play a protective role in many pathological conditions characterized by PARP-1 overactivation. It has been shown that PARP-1 also promotes tumor growth and progression through its DNA repair activity. Since angiogenesis is an essential requirement for these activities, we sought to determine whether PARP inhibition might affect rat brain microvascular endothelial cells (GP8.3) migration, stimulated by C6-glioma conditioned medium (CM). Through wound-healing experiments and MTT analysis, we demonstrated that PARP-1 inhibitor PJ-34 [N-(6-Oxo-5,6-dihydrophenanthridin-2-yl)-N,N-dimethylacetamide] abolishes the migratory response of GP8.3 cells and reduces their viability. PARP-1 also acts in a DNA independent way within the Extracellular-Regulated-Kinase (ERK) signaling cascade, which regulates cell proliferation and differentiation. By western analysis and confocal laser scanning microscopy (LSM), we analysed the effects of PJ-34 on PARP-1 expression, phospho-ERK and phospho-Elk-1 activation. The effect of MEK (mitogen-activated-protein-kinase-kinase) inhibitor PD98059 (2-(2-Amino-3-methoxyphenyl)-4H-1-benzopyran-4-one) on PARP-1 expression in unstimulated and in CM-stimulated GP8.3 cells was analyzed by RT-PCR. PARP-1 expression and phospho-ERK activation were significantly reduced by treatment of GP8.3 cells with PJ-34 or PD98059. By LSM, we further demonstrated that PARP-1 and phospho-ERK are coexpressed and share the same subcellular localization in GP8.3 cells, in the cytoplasm as well as in nucleoplasm. Based on these data, we propose that PARP-1 and phospho-ERK interact in the cytosol and then translocate to the nucleus, where they trigger a proliferative response. We also propose that PARP-1 inhibition blocks CM-induced endothelial migration by interfering with ERK signal-transduction pathway.

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Geraets, Liesbeth. "Dietary PARP-1 inhibitors as anti-inflammatory compounds." Maastricht : Maastricht : Universitaire Pers ; University Library, Universiteit Maastricht [host], 2008. http://arno.unimaas.nl/show.cgi?fid=14252.

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Kumpan, Katerina. "Structure-activity studies on inhibitors of the tankyrases." Thesis, University of Bath, 2014. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.619223.

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Tankyrases-1 and -2 (TNKS-1 and -2) are members of the poly(ADP-ribose)polymerase (PARP) enzyme superfamily, which modify and regulate target proteins by addition of multiple (ADP-ribose) units from the substrate NAD+. TNKS-1 and -2 have many cellular roles, including regulation of elongation of telomeres, activation of nuclear mitotic apparatus protein (NuMA) in mitosis and regulation of the Wnt signalling pathway. This makes the tankyrases attractive new targets for design and development of new anti-cancer drugs. 2-(4-Trifluoromethylphenyl)-7,8-dihydro-3H-thiopyranopyrimidin-4-one (XAV939) was one of the few active inhibitors of tankyrases reported until 2013. The aim of this project was to explore the structure-activity relationships towards enhancing potency and selectivity by replacing the saturated sulfur-containing ring with saturated and unsaturated nitrogen heterocycles and by varying the aromatic side-chain. Firstly, ascorbate-modified Sonogashira couplings of bromocyanopyridines and a variety of 4-substituted arylethynes, followed by acidic cyclisation and conversion of the lactone into the lactam, gave differently substituted arylnaphthyridinones. The alternative route used transition-metal-free reaction of bromopyridinecarboxylic acids with symmetrical β-diketones. 7-Phenyl-1,6-naphthyridin-5-one and 7-(4-methylphenyl)-1,6-naphthyridin-5-one were converted to the N1-oxides. Alkylation at 1-N gave 7-aryl-1-methyl-5-oxo-5,6-dihydro-1,6-naphthyridin-1-ium iodides and subsequent reduction gave saturated target 7-aryl-1-methyl-1,2,3,4-tetrahydro-1,6-naphthyridin-5-ones. Other target compounds included pyridopyrimidinones, which were prepared from the corresponding bromopyridinecarboxylic acids by a copper-catalysed reaction with 4-substituted benzamidines. Target tetrahydropyridopyrimidinones, however, were obtained from condensation of 1-benzyl-4-oxopiperidine-3-carboxylic esters with substituted benzamidines. All compounds were evaluated in vitro for inhibition of the catalytic activity of TNKS-2. The best compounds were investigated further, including in vitro TNKS-1 and PARP-1 inhibition and anti-proliferative studies on HT29 and FEK4 cell lines. Notably, 1-methyl-7-(4-trifluoromethylphenyl)-1,2,3,4-tetrahydro-1,6-naphthyridin-5-one and 1-methyl-7-(4-methoxyphenyl)-1,2,3,4-tetrahydro-1,6-naphthyridin-5-one showed 50% inhibition of TNKS-2 at 1.5 nM and 1.1 nM, respectively, showing also high selectivity (IC50 against PARP-1: 4.8 μM and 3.4 μM, respectively). This high potency and selectivity point to potential for development towards therapeutic use in cancer. A patent covering these discoveries has been filed.

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Ahmed, Zina. "Poly-ADP ribos polymeras (PARP) inhibitorers effekt på bröstcancer : Poly-ADP ribos polymeras (PARP) inhibitorers effekt på bröstcancer." Thesis, Umeå universitet, Kemiska institutionen, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-103397.

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Almeida, Gilberto Serrano de. "Pre-clinical imaging evaluation of the PARP inhibitor rucaparib." Thesis, University of Newcastle upon Tyne, 2013. http://hdl.handle.net/10443/2033.

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Poly(ADP-ribose) polymerase 1 (PARP1) is a DNA-binding enzyme involved in DNA repair by the base-excision pathway. The inhibition of PARP1 is being investigated as a cancer treatment. Rucaparib (CO338) is a potent PARP 31 inhibitor currently in Phase II clinical development. In this thesis P in vivo MR Spectroscopy (MRS) and Dynamic Contrast Enhanced (DCE) MRI were used to study acute effects of rucaparib on energy metabolism and tumour vasculature. 1 31 18 18 Ex vivo H and P-MRS, and in vivo [ F]FLT and [ F]FDG-PET, were used to study effects of treatment with rucaparib on tumour metabolism and proliferation. A2780 and SW620 tumours implanted in mice were scanned in a horizontal Varian 7T MR system. Two i.v. injections of the MRI contrast agent gadoteridol were given 90 minutes apart with dynamic phosphorus MRS acquired following the injection of rucaparib, temozolomide or both drugs in combination. The 18 18 same tumours were evaluated by [ F]FLT- and [ F]FDG-PET after 5 daily treatments with rucaparib, temozolomide or the combination, and the livers of PARP1 knock out (KO) and wild type (WT) mice treated in a similar manner 1 31 were analysed by ex vivo H and P-MRS. Tumour uptake of gadoteridol changed significantly after treatment with hydralazine and higher doses of rucaparib in SW620 tumours, and following 31 hydralazine and 1mg/Kg of rucaparib in A2780 tumours. P-MRS studies revealed an increase in the inorganic phosphate (Pi) to β-NTP ratio, consistent with impairment of tumour energy metabolism following hydralazine treatment. 18 [ F]FLT-PET demonstrated a significant reduction in the SUV values in the 18 rucaparib/temozolomide combination group in SW620 tumours, and [ F]FDG- PET revealed a non-significant reduction in tumour metabolism in A2780 1 tumours. H ex vivo MRS demonstrated an increase in the liver NAD concentrations after treatment with rucaparib, but a decrease following the treatment with temozolomide, regardless of the PARP1 status. Together, these pre-clinical imaging studies have shown that MR can be used 18 to investigate the acute anti-vascular effects of rucaparib, that [ F]FLT-PET predicted subsequent changes in tumour volume following combined rucaparib 1 and temozolomide treatment, and that ex vivo H-MRS can be used in mechanistic studies of PARP inhibition. Both MRI/MRS and PET are potential pharmacodynamic and surrogate response imaging biomarkers for PARP inhibitors.

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Hukkanen, M. (Mikko). "DNA damage sensitization of breast cancer cells with PARP10/ARTD10 inhibitor." Master's thesis, University of Oulu, 2019. http://jultika.oulu.fi/Record/nbnfioulu-201909062843.

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Abstract. In this thesis, I studied the DNA damage sensitization of breast cancer cells with PARP10/ARTD10 inhibitor. As the ARTD10 inhibition field is relatively fresh, tests with breast cancer cell lines combined with clinically used chemotherapeutics will elucidate the potential future uses of the inhibitors in a clinically relevant context, directing the future research efforts in the field. To study the link between OUL35 and DNA damage sensitization, cell proliferation experiments were conducted, and to determine whether ARTD10 translocation from cytoplasm into nucleus is enhanced under DNA damaging conditions, western blot assay was performed. Also, the OUL35 potency against full-length ARTD10 was verified to compare it with the catalytic ARTD10 fragment. The determined IC50 of 510 nM suggests that the potency does not differ from studies with catalytic domain. According to the results from ARTD10 translocation studies, it can only be said that DNA damaging agent in general reduces the amount of cytoplasmic ARTD10, whereas I could not confirm the potential translocation to the nucleus. The results of OUL35-mediated sensitization to DNA damaging agents indicates that OUL35 might sensitize breast cancer cells to DNA damage. As a summary, full-length ARTD10 inhibition does not vary from catalytic domain, suggesting that either construct can be used for testing inhibitors, OUL35 may enhance the effect of other DNA damaging agents, and there is a possibility that HU might have an influence on the nuclear translocation of ARTD10. This provides the basis for future evaluation of larger cancer cell panels for sensitization for chemotherapeutics by ARTD10 inhibition.

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Castroviejo, Bermejo Marta. "RAD51 as functional biomarker to select tumors for PARP inhibitor treatment." Doctoral thesis, Universitat Autònoma de Barcelona, 2019. http://hdl.handle.net/10803/667273.

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Los inhibidores de la enzima Poly (ADP-ribosa) polimerasa (PARPi) son efectivos en el tratamiento de cánceres que presentan defectos en la reparación del ADN por recombinación homóloga (HRR), incluyendo aquellos con mutaciones en BRCA1 y BRCA2 (BRCA1/2). Se han descrito distintos mecanismos de resistencia a PARPi en tumores con mutaciones germinales en BRCA1/2 (gBRCA), y existen otros tumores sin mutaciones en BRCA1/2 (no-BRCA) que responden a PARPi. Existe la necesidad de desarrollar un biomarcador robusto para una mejor selección de tumores deficientes en HRR y extender el uso de PARPi a nuevas indicaciones, así como estudiar terapias en combinación que mejoren la eficacia en la clínica. En este trabajo se evaluó la actividad del PARPi olaparib en xenoimplantes de tumores derivados de pacientes (PDX, patient derived tumor xenografts) con cáncer de mama u ovario, tanto gBRCA como no-gBRCA. Se estudiaron los mecanismos de resistencia y sensibilidad a PARPi in vivo, así como la utilidad de una inmunofluorescencia para detectar focos nucleares de RAD51 como biomarcador de HRR y respuesta a PARPi, tanto en PDXs como en muestras clínicas. Además, se investigó la actividad antitumoral del inhibidor de WEE1 AZD1775 y del inhibidor de ATM AZD0156 en monoterapia y combinación con PARPi. Se investigaron los mecanismos de acción de estas terapias utilizando distintos marcadores de estrés replicativo. Entre los modelos PDX gBRCA resistentes a PARPi no se encontraron mutaciones secundarias en BRCA1/2 pero sí la formación de focos nucleares de BRCA1, en concordancia con la expresión de proteínas BRCA1 hipomórficas. En tres PDX resistentes a PARPi se identificó la pérdida de 53BP1 y FAM35A como mecanismo de resistencia. La única característica común a todos los PDXs resistentes a PARPi, ya sea resistencia primaria o adquirida, fue la capacidad de formación de focos nucleares de la proteína RAD51. De acuerdo con estos resultados, la ausencia de focos de RAD51 se asoció con respuesta clínica a PARPi en muestras de pacientes. Cuando se estudiaron los mecanismos de sensibilidad a PARPi en la colección de PDXs no-gBRCA, se observó la presencia de hipermetilación del promotor de BRCA1 y alteraciones en otros genes relacionados con HRR en modelos sensibles a PARPi. Sin embargo, de nuevo la única característica común a todos los PDXs respondedores fue la ausencia de focos nucleares de RAD51. El ensayo de RAD51 se pudo realizar en muestras no tratadas y mostró ser altamente discriminativo entre sensibilidad y resistencia a PARPi, superando la capacidad predictiva del test genético myChoice® HRD de Myriad. En muestras clínicas de rutina procedentes de pacientes con síndrome de cáncer de mama y ovario hereditario, todos los tumores relacionados con mutaciones en PALB2 se clasificaron como deficientes en HRR. Finalmente, se demostró que la resistencia a la terapia con PARPi en tumores con alteraciones en BRCA1 puede revertirse combinando estos agentes con un inhibidor de WEE1 o de ATM, y en ambas estrategias se reportó una mayor inducción de estrés replicativo en PDX sensibles a la combinación. Los resultados de esta tesis permiten concluir que los tumores gBRCA logran la resistencia a PARPi mediante diferentes mecanismos que restauran HRR y puede detectarse por la formación de focos nucleares de RAD51. Este ensayo funcional permite identificar tumores no-BRCA sensibles a PARPi y representa un biomarcador prometedor para mejorar la selección de pacientes y ampliar la población candidata a recibir esta terapia. Los resultados también impulsan el desarrollo clínico de estrategias terapéuticas que combinen los PARPi con inhibidores de WEE1 y ATM, destacando la inducción de estrés replicativo como principal mecanismo de acción de estos fármacos.
Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) are effective anticancer drugs in cancers with defective homologous recombination DNA repair (HRR), including cancers with mutations in BRCA1 and BRCA2 (BRCA1/2), which also display enhanced sensitivity to DNA damaging chemotherapy such as platinum salts. Several mechanisms of PARPi resistance have been described in tumors with germline mutations in BRCA1/2 (gBRCA) and there are also other tumors with wild type BRCA1/2 (non-BRCA) that benefit from PARPi treatment. Therefore, there is a need to develop robust biomarkers to better select HRR- deficient tumors and extend the use of PARP inhibition in new indications, as well as identify PARPi-resistant tumors and study combination treatment options that enhance clinical efficacy and utility of PARPi. We evaluated the activity of the PARPi olaparib in patient-derived tumor xenografts (PDXs) from patients with breast or ovarian cancer, both with and without gBRCA mutation, exhibiting differential response to PARPi. We studied the in vivo mechanisms of PARPi resistance and sensitivity in these models and tested the formation of RAD51 nuclear foci by immunofluorescence as biomarker of HRR functionality and PARPi response in PDXs and routine clinical samples. We also tested the antitumor activity of the WEE1i AZD1775 and the ATMi AZD0156 as single agent and in combination with PARPi in PDXs. The measurement of replication stress biomarkers was assessed to study the mechanisms of action of these treatment strategies. Within the gBRCA PDXs panel, no BRCA1/2 secondary mutations were found in the PARPi resistant models. BRCA1 nuclear foci were detected in six out of ten PARPi-resistant PDXs, in keeping with expression of hypomorphic BRCA1 isoforms. Loss of 53BP1 and FAM35A were identified in three PDXs, one of which concomitantly expressed an hypomorphic BRCA1 protein. The common feature in all PDXs with primary or acquired PARPi resistance was the formation of RAD51 nuclear foci. Consistently, lack of RAD51 foci was always associated with clinical response to PARPi in patients treated with these agents. When studying the mechanisms of PARPi sensitivity in the non-gBRCA PDX cohort, BRCA1 promoter hypermethylation and alterations in HRR-related genes were found in PARPi- sensitive models. Again, the unique common feature in all PDXs that exhibited tumor regression upon PARPi treatment is the absence of RAD51 nuclear foci. The RAD51 assay could be performed in untreated samples and was highly discriminative of PARPi sensitivity versus PARPi resistance in different PDX cohorts and outperformed the Myriad’s myChoice® HRD genomic test. In routine clinical samples from patients with hereditary breast and ovarian cancer (HBOC) syndrome, all PALB2-related tumors were classified as HRR-deficient by the RAD51 score. In PDXs, PARPi resistance in BRCA1-altered tumors could be reverted upon combination of PARPi with WEE1 or ATM inhibitors and both combination strategies resulted in exacerbated induction of replication stress (RS) in combination- sensitive PDXs. With the results obtained in this thesis, it can be concluded that gBRCA tumors achieve PARPi resistance by several mechanisms that restore HRR function, all detected by the presence of RAD51 nuclear foci. This functional assay also enables the identification of PARPi-sensitive non-gBRCA tumors independently of the mechanisms of HRR-deficiency, thereby being a promising biomarker to better select patients for PARP inhibition and broaden the population who may benefit from this therapy. Our study also supports the clinical development of PARPi combinations such as those with WEE1 and ATM inhibitors and highlighted the induction of RS as the major mechanisms of action of these drugs.

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Löser, Dana A. "Investigating the mechanisms by which PARP inhibitors increase sensitivity to DNA damaging agents." Thesis, University of Sussex, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.505912.

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Damage induced by ionising radiation (IR) is mainly repaired by classical non homologous end joining (D-NHEJ), but a small subset of DSBs is repaired with slow kinetics in an ATM (Ataxia telangiectasia mutated) and Artemis dependent manner. In addition, a PARP-1 dependent NHEJ backup pathway (B-NHEJ) was described, which is thought to function in the absence of D-NHEJ. Using ATM, Artemis or DNA ligase IV deficient mouse embryonic fibroblasts (MEFs) as a model system, the effect of the potent and specific PARP-1/-2 inhibitor KU-0059436 upon clonogenic survival after various types of damage that induce different spectra of SSBs and DSBs was measured. In Artemis or ATM deficient MEFs no sensitising effect of KU-0059436 was detected after neocarzinostatin (NCS) treatment; however PARP inhibition increased sensitivity to IR and methylmethane sulphonate (MMS) markedly. In these cell lines no specific single strand break repair (SSBR) defect was observed, and radio-sensitisation by KU-0059436 was replication dependent. Furthermore PARP inhibition led to increased formation of DSBs, an effect which was augmented in Artemis deficient cells. PARP inhibition in DNA ligase IV deficient cells led to increased sensitivity after damage induction to all agents. However, radiosensitisation by KU-0059436 was replication independent. Results show that PARP inhibition increases the dependence on Artemis and ATM after SSB induction, which is consistent with a model whereby DSBs that arise from SSBs during DNA replication in the presence of a PARP inhibitor require ATM and Artemis for their repair. In cells deficient for DNA ligase IV, PARP inhibition causes additional replication independent sensitisation both by abrogating B-NHEJ and promoting accumulation of replication independent DSBs.

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Guillot, Clément. "Potentiel des inhibiteurs de poly(ADP-ribose) polymérases seuls ou en combinaison avec la radiothérapie comme nouvelle option thérapeutique pour le carcinome hépatocellulaire." Thesis, Lyon 1, 2013. http://www.theses.fr/2013LYO10281.

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Le carcinome hépatocellulaire est l'un des cancers les plus fréquents et des plus sévères à travers le monde. Le diagnostic est souvent tardif et les traitements curatifs ne peuvent être proposés qu'à un nombre limité de patients. Les technologies modernes ont permis le développement de nouvelles méthodes de radiothérapie qui montrent aujourd'hui de bons résultats. Par ailleurs, bien que des déficiences dans les voies de réparation de l'ADN soient associées à une instabilité génomique et une susceptibilité au cancer, une inhibition de ces voies sensibilise les cellules cancéreuses à la chimiothérapie et à la radiothérapie. Dans ce contexte, les inhibiteurs de poly(ADP-ribose) polymérases (PARP) ont déjà montré des résultats prometteurs dans des études pré-cliniques et sont en cours d'évaluation clinique pour de nombreux cancers. Ce travail de thèse a consisté en l'évaluation du potentiel des inhibiteurs de PARP en combinaison avec la radiothérapie comme nouvelle option thérapeutique pour le carcinome hépatocellulaire. La première étape de ce travail a été de caractériser les profils d'expression et d'activité de plusieurs membres de la famille PARP dans des cellules cancéreuses du foie et des hépatocytes primaires humains ainsi que dans des tissus hépatiques. En second lieu, nous avons étudié le potentiel de l'inhibiteur de PARP ABT-888 seul et en combinaison à des radiations ionisantes in vitro. Le traitement par l'inhibiteur de PARP ABT-888 en agent seul a montré une sensibilité variable des différentes lignées cellulaires étudiées à cette drogue. Afin de comprendre la sensibilité variable des cellules cancéreuses hépatiques à l'ABT-888, nous avons analysé leur capacité de réparation des dommages à l'ADN et avons observé des capacités différentes entre les lignées cellulaires. Finalement, nous avons pu montrer que l'ABT-888 sensibilise les cellules cancéreuses hépatiques aux radiations ionisantes. Ce travail de recherche a permis de montrer que les inhibiteurs de PARP ont un fort potentiel pour améliorer les méthodes de radiothérapie utilisées dans la prise en charge du carcinome hépatocellulaire
Hepatocellular carcinoma is the third cause of cancer related death. Due its often late diagnosis and advanced stage, a limited number of patients can benefit from curative treatments. There is thus a constant need for new treatment strategies for patients with hepatocellular carcinoma. Targeting DNA repair pathways to sensitize tumor cells to chemoor radiotherapeutic treatments is now a common strategy under investigation for cancer treatment with inhibitors of poly(ADP-ribose) polymerases (PARP) showing great potential. The aim of this work was to evaluate the potential of PARP inhibitors alone and in combination with radiation therapy as a new strategy for the treatment of hepatocellular carcinoma. We first analyzed the expression and activity of different PARP genes in a panel of liver cancer cell lines and primary human hepatocytes as well as their DNA repair capacity and assess the impact of PARP inhibitors alone and in combination with ionizing radiation in these models on cell survival. A large range in expression of PARP family members, PARP activity and sensitivity to ABT-888 in the panel of liver cells was observed as well as differential excision/synthesis repair capacity. Finally, we showed that ABT-888 sensitizes liver cancer cells to the cell killing effects of ionizing radiation. PARP inhibitors show great potential for improving radiation therapy strategies used in the management of hepatocellular carcinoma

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Michels, Judith. "Les Inhibiteurs de PARP dans le Traitement des Cancers Chimio-Résistants. Etude pré-clinique sur la Dépendance à PARP." Phd thesis, Université Paris Sud - Paris XI, 2013. http://tel.archives-ouvertes.fr/tel-01063796.

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Introduction Le cancer bronchique est un problème de santé publique en étant la première cause de décès par cancer dans le monde. Il reste de mauvais pronostic avec une résistance au Cisplatine qui est inéluctable dans l'histoire naturelle de la maladie. Nous nous sommes intéressés à l'association du CDDP aux inhibiteurs de la Poly(ADP-ribose) polymérase. Les inhibiteurs pharmacologiques de PARP sont source d'optimisme en oncologie clinique en monothérapie pour des tumeurs déficientes pour une voie de réparation de l'ADN et en association aux cytotoxiques classiques.Matériel et méthodes Nous avons généré 9 clones résistants au CDDP après culture de la lignée A549 dans des faibles doses de CDDP. Deux inhibiteurs pharmacologiques de PARP, CEP8983 (CEP) et PJ34 (PJ), ainsi que des siRNA spécifiques de PARP1 sont utilisés pour l'inhibition de PARP. L'apoptose est mesurée en cytométrie de flux par l'intermédiaire du potentiel membranaire de la mitochondrie DiOC6(3) et la perméabilisation de la membrane plasmique est évaluée par l'iodide de propidium. Le test de clonogénicité permet d'évaluer la capacité des cellules à échapper à la mort et à former une colonie. L'activité métabolique des cellules est mesurée par la mesure de clivage du sel de tetrazolium WST-1. L'immunofluorescence sur cellules fixées a permis d'étudier les dommages de l'ADN (γH2AX), la voie intrinsèque de l'apoptose (l'activation de la caspase 3 et la libération du cytochrome c) et la recombinaison homologue (BRCA1, RAD51). En Western Blot nous avons mesuré l'expression et l'activité de PARP (PAR) ainsi que l'expression d'acteurs de la réparation par excision de base (BER) (XRCC1 and polymérase β). Nous avons développé une méthode de détection de PAR en immunohistochimie sur des tissus inclus en paraffine. Résultats Nous avons trouvé un effet synergique pour l'association du CDDP aux inhibiteurs de PARP in vitro. De façon inattendue nous avons observé que les clones résistants au CDDP développent une addiction à PARP et sont spécifiquement tués par l'inhibition de PARP contrairement à la lignée parentale. Ces clones exhibent une hyperexpression et une hyperactivité de PARP. La réponse aux inhibiteurs de PARP corrèle plus précisément avec l'activation plutôt qu'avec l'expression de PARP, pointant que PAR est un biomarqueur plus précis que PARP. Nous avons observé que l'hyperactivation de PARP accompagne une résistance induite au CDDP et prédispose à une sensibilité aux inhibiteurs de PARP dans d'autres lignées de cancer bronchique (H460 et H1650), de mésothéliome (P31), de cancer de l'ovaire (TOV112D) et de col (HeLa). Dans des expériences in vivo nous avons noté que dans les xénogreffes obtenues à partir de clones résistants au CDDP, l'expression de PAR est stablement retrouvée en immunohistochimie. Ces tumeurs répondaient à l'inhibition de PARP par le PJ en diminuant l'expression de PAR. Les clones résistants au CDDP sensibilisés aux I PARP ont une recombinaison homologue conservée, cependant ont un déficit dans les étapes terminales du BER.Conclusion Nous avons identifié un effet synergique pour l'association des inhibiteurs de PARP au CDDP de des lignées de cancer bronchique. Nous avons observé une dépendance à PARP dans des lignées de cancer bronchique résistantes au CDDP et déficientes pour l'élongation du BER. Nous postulant que PAR est un biomarqueur spécifique de la réponse aux inhibiteurs de PARP.

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Michels, Judith. "Les inhibiteurs de PARP dans le traitement des cancers chimio-résistants : étude pré-clinique sur la dépendance à PARP." Thesis, Paris 11, 2013. http://www.theses.fr/2013PA11T049/document.

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Introduction Le cancer bronchique est un problème de santé publique en étant la première cause de décès par cancer dans le monde. Il reste de mauvais pronostic avec une résistance au Cisplatine qui est inéluctable dans l’histoire naturelle de la maladie. Nous nous sommes intéressés à l’association du CDDP aux inhibiteurs de la Poly(ADP-ribose) polymérase. Les inhibiteurs pharmacologiques de PARP sont source d’optimisme en oncologie clinique en monothérapie pour des tumeurs déficientes pour une voie de réparation de l’ADN et en association aux cytotoxiques classiques.Matériel et méthodes Nous avons généré 9 clones résistants au CDDP après culture de la lignée A549 dans des faibles doses de CDDP. Deux inhibiteurs pharmacologiques de PARP, CEP8983 (CEP) et PJ34 (PJ), ainsi que des siRNA spécifiques de PARP1 sont utilisés pour l’inhibition de PARP. L’apoptose est mesurée en cytométrie de flux par l’intermédiaire du potentiel membranaire de la mitochondrie DiOC6(3) et la perméabilisation de la membrane plasmique est évaluée par l’iodide de propidium. Le test de clonogénicité permet d’évaluer la capacité des cellules à échapper à la mort et à former une colonie. L’activité métabolique des cellules est mesurée par la mesure de clivage du sel de tetrazolium WST-1. L’immunofluorescence sur cellules fixées a permis d’étudier les dommages de l’ADN (γH2AX), la voie intrinsèque de l’apoptose (l’activation de la caspase 3 et la libération du cytochrome c) et la recombinaison homologue (BRCA1, RAD51). En Western Blot nous avons mesuré l’expression et l’activité de PARP (PAR) ainsi que l’expression d’acteurs de la réparation par excision de base (BER) (XRCC1 and polymérase β). Nous avons développé une méthode de détection de PAR en immunohistochimie sur des tissus inclus en paraffine. Résultats Nous avons trouvé un effet synergique pour l’association du CDDP aux inhibiteurs de PARP in vitro. De façon inattendue nous avons observé que les clones résistants au CDDP développent une addiction à PARP et sont spécifiquement tués par l’inhibition de PARP contrairement à la lignée parentale. Ces clones exhibent une hyperexpression et une hyperactivité de PARP. La réponse aux inhibiteurs de PARP corrèle plus précisément avec l’activation plutôt qu’avec l’expression de PARP, pointant que PAR est un biomarqueur plus précis que PARP. Nous avons observé que l’hyperactivation de PARP accompagne une résistance induite au CDDP et prédispose à une sensibilité aux inhibiteurs de PARP dans d’autres lignées de cancer bronchique (H460 et H1650), de mésothéliome (P31), de cancer de l’ovaire (TOV112D) et de col (HeLa). Dans des expériences in vivo nous avons noté que dans les xénogreffes obtenues à partir de clones résistants au CDDP, l’expression de PAR est stablement retrouvée en immunohistochimie. Ces tumeurs répondaient à l’inhibition de PARP par le PJ en diminuant l’expression de PAR. Les clones résistants au CDDP sensibilisés aux I PARP ont une recombinaison homologue conservée, cependant ont un déficit dans les étapes terminales du BER.Conclusion Nous avons identifié un effet synergique pour l’association des inhibiteurs de PARP au CDDP de des lignées de cancer bronchique. Nous avons observé une dépendance à PARP dans des lignées de cancer bronchique résistantes au CDDP et déficientes pour l’élongation du BER. Nous postulant que PAR est un biomarqueur spécifique de la réponse aux inhibiteurs de PARP
Introduction Driven by the facts that non small cell lung cancer (NSCLC) is the leading cause of cancer-related morbidity and mortality worldwide and that NSCLC patients often develop resistance against Cisplatin (CDDP)-based therapies, we addressed the question of the combination therapy of CDDP with poly(ADP-ribose) polymerases (PARP) inhibitors. Inhibitors of PARP have raised great expectations for the treatment of a variety of cancers, either as monotherapeutic agent against DNA repair-deficient tumours or combined to DNA-damaging compounds.Material and methods We generated nine CDDP-resistant clones by prolonged exposure to low dose CDDP of the A549 NSCLC parental cell line. Two distinct PARP inhibitors, CEP8983 (CEP) and PJ34 (PJ) as well as PARP1 knockdown with small interfering RNAs (siRNAs) were used for PARP inhibition. Apoptosis was measured by the simultaneous assessment for the loss of the mitochondrial transmembrane potential (m) and the breakdown of the plasma membrane using the m-sensitive fluorochrome DiOC6(3) and the vital dye propidium iodide, respectively. Moreover clonogenic survival was assessed. In vitro assessments of the enzymatic activity of cells were based on the reduction of the colorless tetrazolium salt. Immunofluorescence microscopy determinations were performed with antibodies specific for DNA damage (γH2AX), intrinsic apoptosis (cleaved Caspase-3 and cytochrome c), and homologous recombination (RAD51 and BRCA1). Immunoblotting was assed for PARP1 expression and activity (PAR) and base excision repair (BER) effectors (XRCC1 and polymerase β). We developed an immunohistochemical staining method that specifically detects PAR on paraffin-embedded cell pellets and tissue sections.Results We found that PARP inhibitors and PARP1 siRNAs synergized with CDDP in the killing of NSCLC cells in vitro. Unexpectedly, CDDP-resistant NSCLC cell clones developed addiction to PARP hyperactivation, thereby becoming susceptible to apoptosis induction by PARP inhibition. We showed that these cisplatin-resistant clones, exhibited high PARP protein levels and increased PARP activity, leading to an increased poly-ADP ribosylation of cellular proteins, as compared to their parental, cisplatin-sensitive counterparts. These cisplatin-resistant cells become susceptible to cell death as induced by PARP inhibition, correlating with the hyperactivity of PARP (elevated PAR levels) more accuratly than with the overexpression of PARP. Suggesting that PAR levels may constitute a more accurate biomarker than PARP to predict the sensitivity of cells to PARP inhibition. We expanded the observation that cisplatin resistance causes PARP upregulation and hyperactivation and subsequent sensitization to PARP inhibition to additional five human cancer cell lines including two NSCLC (H1650 and H460), one mesothelioma (P31), one ovarian (TOV112D) and one cervical cancer (HeLa) cell line. To get further insight into this issue, we generated in vivo experiments. Tumors derived from CDDP-resistant cells were characterized by elevated levels of PAR suggesting that PAR levels are preserved during tumor formation. Those PAR-overexpressing tumors responded to the administration of PJ in vivo with a consistent reduction in PAR immunoreactivity. CDDP resistant clones that are specifically killed by PARP inhibitors assessed efficient homologous recombination repair however deficient BER elongation.Conclusion We showed a beneficial effect for the association therapy of PARP inhibitors with CDDP in several NSCLC cell lines. We have identified an addiction to PARP in CDDP resistant cell lines with deficient BER elongation. We postulate that PAR is a specific predictive biomarker for the response to PARP inhibitors

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Raithel, Kerstin. "Effekt von S-Lost in HaCaT-Zellkulturen : Induktion der Apoptose und Effekt eines PARP-Inhibitors /." München, 2008. http://opac.nebis.ch/cgi-bin/showAbstract.pl?sys=000254372.

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Ordway, Gregory A., W. D. Gill, J. B. Coleman, Hui Wang-Heaton, and Russell W. Brown. "Anti-Inflammatory PARP Inhibitor Demonstrates Antidepressant Activity in Animal Model of Treatment Resistant Depression." Digital Commons @ East Tennessee State University, 2019. https://dc.etsu.edu/etsu-works/8643.

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Background: Major depressive disorder is associated with elevated levels of DNA oxidation, DNA damage, and gene expression of DNA repair enzymes including poly (ADP-ribose) polymerase-1 (PARP1). Elevated PARP1 activity is directly linked to neuroinflammation and PARP inhibitors are anti-inflammatory and neuroprotective. We previously showed that PARP inhibitors produce antidepressant-like effects equivalent to fluoxetine in rodent models. Here, we examined whether the PARP inhibitor 3-aminobenzamide (3AB) is effective in a rat model of treatment-resistant depression. Methods: Treatment-resistant depression was modeled with injections of lipopolysaccharide (LPS; 0.1 ug/kg/day) and daily chronic unpredictable stress (CUS) for 28 days. Anhedonia and helplessness were indexed with sucrose preference and forced swim tests, respectively, in 5 groups of rats (n¼6-8 rats/group) including unstressed, CUS, and CUS+LPS rats treated with saline, and CUS+LPS rats treated with either 3AB or fluoxetine. Results: Anhedonia induced by CUS+LPS was significantly attenuated by 3AB (p¼0.01), while fluoxetine failed to do so. Likewise, 3AB was superior to fluoxetine in reducing helplessness, where latency to immobility times were significantly lower in CUS+LPS rats treated with fluoxetine (p¼0.001) compared to unstressed rats, but not significantly different for 3AB-treated CUS+LPS rats. Conclusions: The PARP inhibitor 3AB demonstrated robust and unique antidepressant activity superior to fluoxetine in the TRD rat model. PARP is linked to neuroinflammation through release of microglia-activating factors including poly (ADP-ribose) and HMGB1, and through NF-kB activation, pathways under investigation by our lab. PARP inhibitors are currently used clinically to facilitate cytotoxicity of DNA-damaging anti-cancer treatments. Further research could implicate re-purposing non-cytotoxic PARP inhibitors for treatment-resistant depression.

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Maier, Christian. "Auswirkungen des PARP-1 Inhibitors INO-1001 auf Ischämie-Reperfusionsbedingte Organschädigungen nach thorakalem Aortencrossclamping am Schwein." [S.l. : s.n.], 2008. http://nbn-resolving.de/urn:nbn:de:bsz:289-vts-64137.

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Ordway, Gregory A., Attila Szebeni, Liza J. Hernandez, Jessica D. Crawford, Katalin Szebeni, Michelle J. Chandley, Katherine C. Burgess, Corwin Miller, Erol Bakkalbasi, and Russell W. Brown. "Antidepressant-Like Actions of Inhibitors of Poly(ADP-Ribose) Polymerase in Rodent Models." Digital Commons @ East Tennessee State University, 2017. https://dc.etsu.edu/etsu-works/2768.

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Many patients suffering from depressive disorders are refractory to treatment with currently available antidepressant medications, while many more exhibit only a partial response. These factors drive research to discover new pharmacological approaches to treat depression. Numerous studies demonstrate evidence of inflammation and elevated oxidative stress in major depression. Recently, major depression has been shown to be associated with elevated levels of DNA oxidation in brain cells, accompanied by increased gene expression of the nuclear base excision repair enzyme, poly(ADP-ribose) polymerase-1. Given these findings and evidence that drugs that inhibit poly(ADP-ribose) polymerase-1 activity have antiinflammatory and neuroprotective properties, the present study was undertaken to examine the potential antidepressant properties of poly(ADP-ribose) polymerase inhibitors.

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Morel, Daphné. "Identifying Synthetic Lethal and Selective Approaches to Target PBRM1-Deficiency in Clear Cell Renal Cell Carcinoma PBRM1 Deficiency in Cancer is Synthetic Lethal with DNA Repair Inhibitors Exploiting Epigenetic Vulnerabilities in Solid Tumors: Novel Therapeutic Opportunities in the Treatment of SWI/SNF-Defective Cancers Combining Epigenetic Drugs with other Therapies for Solid Tumours — Past Lessons and Future Promise Targeting Chromatin Defects in Selected Solid Tumors Based on Oncogene Addiction, Synthetic Lethality and Epigenetic Antagonism." Thesis, université Paris-Saclay, 2020. http://www.theses.fr/2020UPASL017.

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L’inactivation de polybromo-1 (PBRM1) est un évènement fréquent dans de nombreux cancers. En particulier, les carcinomes rénaux à cellules claires présentent une déficience en PBRM1 dans 40 à 50% des cas. A ce jour, il n’existe pas d’approche de médecine précision connue capable de cibler spécifiquement les cellules tumorales déficientes en PBRM1.Pour identifier des cibles de létalité synthétique associées à la perte de PBRM1, nous avons (i) réalisé un criblage pharmacologique à haut débit évaluant la sensibilité à 167 molécules dans un modèle cellulaire isogénique pour PBRM1, et (ii) étudié l’impact transcriptomique et protéomique de la perte de PBRM1 dans ce même modèle.Nous avons ensuite caractérisé les mécanismes sous-jacents à la relation de létalité synthétique découverte.Nous avons identifié et validé une relation de létalité synthétique existante entre la perte tumorale de PBRM1 et l’inhibition pharmacologique de PARP, pouvant être potentialisée par l’ajout d’un inhibiteur d’ATR.Cette relation de létalité synthétique était caractérisée par un niveau basal élevé de stress cellulaire chez les cellules déficientes en PBRM1, associant anomalies mitotiques, stress transcriptionnel et stress réplicatif – tous ces phénomènes étant exacerbés à l’ajout d’inhibiteurs de PARP, jusqu’à dépasser les capacités cellulaires à maintenir un phénotype compatible avec la survie.Ces observations apportent la preuve de concept préclinique que les inhibiteurs de PARP sont de potentiels candidats thérapeutiques pour cibler spécifiquement les tumeurs déficientes en PBRM1
Polybromo-1 (PBRM1) inactivation occurs in multiple malignancies and is of particular importance in clear cell renal cell carcinomas (ccRCC), as it drives 40 to 50% of cases. Currently, no precision-medicine approach uses PBRM1 deficiency to specifically target tumour cells. To uncover novel synthetic lethal approaches to treat PBRM1-defective cancers, we performed (i) a high-throughput pharmacological screening, evaluating the sensitivity to 167 small molecules in a PBRM1-isogenic cellular model, and the (ii) systematic mapping of the whole transcriptomic and proteomic profiles associated with PBRM1 loss-of-function within this model. We further investigated the mechanism underlying this synthetic lethal relationship.We identified and validated synthetic lethal effects between PBRM1 loss and both PARP and ATR inhibition. Combinatorial use of PARP with ATR inhibitors exerted additive cytotoxic effects in PBRM1-defective tumor cells. These synthetic lethal relationships were characterized by a pre-existing replication stress in PBRM1-deficient cells associated with mitosis and DNA damage repair abnormalities, which were exacerbated upon PARP inhibition selectively in PBRM1-defective cells.These data provide the preclinical basis for evaluating PARP inhibitors as a monotherapy or in combination in patients with PBRM1-deficient ccRCC

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Block, Katherine M. "Design of Novel Cancer Therapeutics Through The Validation of PARG as a Therapeutic Target and the Evaluation of Small Molecule Inhibitors of Hypoxia-Induced Transcription." Diss., The University of Arizona, 2010. http://hdl.handle.net/10150/194826.

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Because of the severe toxicity and limiting side effects of traditional chemotherapy, there exists a critical need to develop better-tolerated, safer drugs to treat cancer. Recent advances in our understanding of the molecular mechanisms governing carcinogenesis have ushered in a new age in drug discovery and have enabled the design of much more sophisticated agents to treat cancer. This work describes two approaches to the development of novel, specifically targeted cancer therapeutics.The first approach involves the synthesis of a class of a new class of small molecules called epidithiodiketopiperazines (ETPs) designed to inhibit hypoxia-induced transcription. Specifically, these agents block the interaction of the transcription factor HIF-1α (hypoxia inducible factor-1α) and its required coactivator p300/CBP by inducing a structural change in p300 that renders it incapable of binding to HIF-1α. Preventing hypoxia-mediated transcription has the potential to stop the process of angiogenesis that is critical for sustained tumor growth and metastasis. Moreover, because HIF-1α also controls genes for energy production and matrix remodeling, ETPs may also halt metabolic adaptation and tumor progression. Our results show that ETPs prevent the association of HIF-1α and p300 and abrogate hypoxia signaling on both the transcriptional and translational levels in endogenous systems. In addition, they do not exhibit broad-spectrum cytotoxicity or global inhibition of the transcriptional response.The second approach addresses the validation of poly(ADP-ribose) glycohydrolase (PARG) as a new therapeutic target. This project describes studies aimed to further our understanding of the interaction between poly(ADP-ribose) polymerases (PARPs) and PARG with the ultimate goal of using this knowledge to design novel therapeutics. This portion of the dissertation involves a series of studies in mouse embryonic fibroblasts (MEFs) with genetic mutations in their PARP and PARG function. MEF cell lines containing a truncated form of PARG lacking the regulatory domain demonstrate over-activation of PARP-1, but not PARP-2. Additionally, deletion of the PARG regulatory domain impairs the DNA damage response to SSBs and DSBs and significantly increases cell death resulting from genotoxic stress. Taken together, these studies suggest a specific interaction between PARP-1 and the regulatory domain of PARG that is critical for proper PARP-1 function.

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Cherry, K. E. "The poly (ADP-ribose) polymerase (PARP) inhibitors AG14361 and AG014699 : mechanisms of action and implications for clinical application." Thesis, Queen's University Belfast, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.546027.

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Chabanon, Roman. "Exploiting DNA Repair Vulnerabilities to Modulate Anti-Cancer Immunity : a Study of the Immunological Potential of PARP inhibitors." Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLS007.

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Les inhibiteurs de poly(ADP-ribose) polymérase (PARPi) ciblent sélectivement les cellules porteuses de défauts des voies de réparation de l’ADN tels que les mutations de BRCA1/2 et les défauts d’ERCC1. Sur le plan clinique, plusieurs PARPi ont été approuvés pour le traitement des cancers BRCA-mutés ou platine-sensibles du sein et de l’ovaire, et des essais cliniques sont en cours pour évaluer l’efficacité des PARPi dans le cancer bronchique non-à-petites cellules (CBNPC) platine-sensible. Alors que les PARPi ont un fort potentiel thérapeutique dans les cancers comportant des défauts de réparation de l’ADN, de plus en plus d’essais cliniques évaluent également l’efficacité de ces médicaments en combinaison avec les « inhibiteurs d’immune checkpoints » (ICI) dans diverses populations de patients. Dans ce contexte, il est essentiel de mieux comprendre comment les PARPi modulent la réponse immunitaire anti-tumorale, et d’étudier le potentiel immunologique inhérent de ces médicaments.Dans cette étude, nous avons établi que les cellules de CBNPC déficientes en ERCC1 expriment fortement la signature interféron (IFN) de type I, et que les tumeurs de CBNPC ayant une faible expression d’ERCC1 ont un infiltrat lymphocytaire renforcé. En utilisant des lignées cellulaires isogéniques et des xénogreffes dérivées de patients, nous avons montré que plusieurs PARPi, notamment l’olaparib et le rucaparib, ont des propriétés immunomodulatrices dans les modèles de CBNPC ERCC1-déficients et de cancers du sein triple-négatifs (CSTN) BRCA1-mutés. D’un point de vue mécanistique, les PARPi génèrent des fragments d’ADN cytoplasmiques ayant les caractéristiques de micronoyaux ; ceux-ci activent la voie cGAS/STING et déclenchent une réponse IFN de type I, associée à la sécrétion de la cytokine CCL5. De manière importante, ces effets sont largement diminués dans les cellules de CSTN BRCA1-révertantes et les cellules de CBNPC ré-exprimant ERCC1, ce qui suggère que les défauts de réparation de l’ADN amplifient les phénotypes immunitaires associés au traitement par PARPi. En outre, ces effets sont totalement abrogés dans les cellules de CSTN PARP1-neutralisées, ce qui confirme que les phénotypes observés dépendent d’un effet spécifique des PARPi sur leur cible.Au-delà de leur potentiel d’activation d’une immunité spécifique des cellules cancéreuses via cGAS/STING et la signalisation IFN de type I, nous avons également constaté que les PARPi potentialisent les effets inducteurs de l‘IFN de type II sur l’expression de PD-L1 dans des lignées cellulaires et cellules tumorales fraîches de patients CBNPC, surtout en présence de défauts d’ERCC1. De plus, nous avons montré que certains PARPi, utilisés à des concentrations létales, activent de manière indépendante les éléments moléculaires clés de la mort cellulaire immunogénique, dont l’exposition de la calréticuline à la surface des cellules cancéreuses, la sécrétion d’ATP et le relargage d’HMGB1 en grandes quantités dans le milieu extracellulaire.Dans l’ensemble, ces données précliniques suggèrent que les PARPi ont des propriétés immunomodulatrices intrinsèques qui participent à l’activation de réponses immunitaires anti-tumorales ; ce potentiel pourrait être exploité cliniquement en combinaison avec les ICI dans des populations adéquatement sélectionnées au plan moléculaire
Poly(ADP-ribose) polymerase inhibitors (PARPi) selectively target cancer cells with DNA repair deficiencies such as BRCA1/2 mutations or ERCC1 defects. Clinically, several PARPi are currently approved for the treatment of BRCA-mutant or platinum-sensitive advanced ovarian and breast cancers, and ongoing clinical trials are investigating the efficacy of PARPi in platinum-sensitive Non-Small Cell Lung Cancer (NSCLC). While PARPi constitute potent targeted therapies for the treatment of DNA repair-deficient malignancies, an increasing number of clinical trials are also evaluating their efficacy in combination with immune checkpoint inhibitor (ICI) in various populations. In this context, it is of critical importance to better understand how PARPi might modulate immune responses against cancer, and to investigate the inherent immunological potential of these agents.In this study, we show that ERCC1-defective NSCLC cells exhibit an enhanced type I interferon (IFN) transcriptomic signature and that low ERCC1 expression correlates with increased lymphocytic infiltration in human NSCLC tumours. Using isogenic cell lines and patient-derived xenografts, we further demonstrate that several clinical PARPi, including olaparib and rucaparib, display cell-autonomous immunomodulatory properties in ERCC1-defective NSCLC and BRCA1-mutant triple-negative breast cancer (TNBC) models. Mechanistically, PARPi generate cytoplasmic chromatin fragments with micronuclei characteristics; this activates the cGAS/STING pathway and elicits downstream type I IFN signalling and CCL5 secretion. Importantly, these effects are suppressed in BRCA1-reverted TNBC cells and ERCC1-rescued NSCLC cells, suggesting that DNA repair defects exacerbate the innate immunity-related phenotypes triggered by PARPi. Similarly, these effects are totally abrogated in PARP1-null TNBC cells, supporting the on-target effect of PARPi in mediating such phenotypes. Besides this potential to activate tumour cell-autonomous immunity through cGAS/STING and type I IFN signalling, we also observed that PARPi synergize with type II IFN to induce PD-L1 expression in NSCLC cell lines and fresh patient tumour cells, especially in the ERCC1-deficient setting. Moreover, we show that lethal concentrations of some PARPi independently activate the key damage-associated molecular patterns dictating the immunogenicity of cancer cell death, including calreticulin exposure at the tumour cell surface, ATP secretion and HMGB1 release in the extracellular compartment.Together, these preclinical data suggest that PARPi have intrinsic immunomodulatory properties that activate anti-cancer immune responses; this could be exploited clinically in combination with ICI in appropriately molecularly-selected populations

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Kohl, Vanessa [Verfasser], and Alice [Akademischer Betreuer] Fabarius. "Synthetische Letalität von PARP- und APE1-Inhibitoren bei hämatologischen Neoplasien / Vanessa Kohl ; Betreuer: Alice Fabarius." Heidelberg : Universitätsbibliothek Heidelberg, 2021. http://d-nb.info/1237750598/34.

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Harand, Kristina Marie. "Assessment of Acrolein-induced Toxicity Using In-vitro Modeling to Evaluate the Role of PARP Inhibitors in Reducing Cytotoxicity." Scholar Commons, 2016. http://scholarcommons.usf.edu/etd/6091.

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Acrolein is an electrophilic α, β-unsaturated aldehyde. Additionally, acrolein is a metabolite of the antineoplastic alkylating agent cyclophosphamide and is implicated in off-target effects, including to bladder hemorrhagic cystitis and cyclophosphamide-induced cardiotoxicity, both of which have led to serious secondary iatrogenic injury during and following chemotherapy. At low concentrations acrolein inhibits cell proliferation without inducing apoptosis, while at high concentrations may result in secondary apoptosis promotion. This investigation assessed the role of the enzyme poly (ADP-ribose) polymerase (PARP) in acrolein induced toxicity using the established toxicological H9c2 (2-1) cardiomyoblast in vitro model. H9c2 (2-1) cells were plated in 24-well plates at 75,000 cells per well three days prior to testing, followed by acrolein dosing at concentrations between 10 µM and 1000µM for either 30 or 55 minutes. PARP activity was quantitatively measured in total cell lysates using a biotin-avidin-conjugated horseradish peroxidase-TMB reporter system in a 96-well microplate formate. The lowest effective dose of toxicity at 30 minute dosing was found at 25 μM (PARP Activity 1.65-fold control) which returned to baseline at 100 μM; concentrations at or above 250 μM results in significant PARP activity reductions (≤ 0.46-fold control). Biomarkers were further characterized for cytotoxicity (AST presence), and viability (MTT reduction) in order to facilitate mechanistic characterization of PARP-mediated acrolein cardiotoxicity. Investigation of a PARP inhibitor was assessed to explore the intervention for acrolein induced cardiac tissue damage.

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Drew, Yvette Claire. "The potential of the PARP-1 inhibitor, AGO14699, in human cancers defective in homologous recombination DNA repair." Thesis, University of Newcastle upon Tyne, 2012. http://hdl.handle.net/10443/1551.

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The aims of this study were to undertake the first comprehensive in vitro, in vivo and clinical investigation into the effects of the PARP-1 inhibitor, AG014699, in human cancers defective in homologous recombination (HR) DNA double strand break (DSB) repair. HR deficient cells were 9-fold more sensitive to AG014699 than HR proficient cells (mean LC50 = 3.26 μM vs. 29.68; P < 0.0001), confirming the theory of synthetic lethality. BRCA1 methylated UACC3199 breast cancer cells were also sensitive to AG014699 with mean LC50 significantly lower than the HR proficient cells (7.6 μM vs. 29.68; P = 0.002). AG014699 inhibited PARP activity by > 95% and induced DNA DSBs in all 11 cell lines studied. Evidence of HR (by Rad51 foci) was observed only in cells with functional BRCA1/2. A prolonged schedule of AG014699 (10 mg/kg daily for five days of a seven-day cycle for six cycles) more effectively delayed the growth of BRCA2 mutated xenografts than a ten day AG014699 schedule (tumour growth delay (TGD) = 27.5 vs. 12.5 days; P = 0.02). AG014699 significantly delayed UACC3199 tumour growth compared to untreated controls (mean time to relative tumour volume 5 = 35.8 vs. 25.2 days; P = 0.05); confirming in vitro findings that BRCA1 methylated cancer cells are sensitive to PARP inhibition. Clinical trial data from 38 patients demonstrated that AG014699 is non-toxic and efficacious with a clinical benefit rate of 34%. Higher baseline PARP-1 activity was associated with response to AG014699. The major findings of these studies are: the confirmation of the selective cytotoxicity of PARP inhibitors in BRCA mutated cancers; the results in UACC3199 cells which suggest that cancers with other HR defects could benefit from single agent PARP inhibitors, and finally the concept that length of exposure to (not just degree of) PARP inhibition is important for single agent anti-tumour activity. Furthermore, these data have formed the basis for a major amendment to the clinical trial; the result of which is eagerly awaited.

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Jdey, Wael. "Mécanismes de sensibilité/résistance des cellules tumorales aux inhibiteurs de réparation de l'ADN Dbait." Thesis, Université Paris-Saclay (ComUE), 2016. http://www.theses.fr/2016SACLS463/document.

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Les défauts dans les voies de réparation de l’ADN sont aujourd’hui largement exploités pour le traitement du cancer. En effet, la capacité des tumeurs à réparer les lésions induites par les traitements génotoxiques (chimio- et radiothérapie) leur confère une résistance intrinsèque ou acquise à ces traitements. Développer des inhibiteurs de réparation de l’ADN permettrait de contrecarrer cette résistance et de sensibiliser les tumeurs à ces thérapies conventionnelles. Les inhibiteurs de la Poly(ADP-ribose) polymérase (PARPi), premiers candidats de cette famille d’inhibiteurs de réparation de l’ADN, ont montré des résultats encourageants mais sont néanmoins restreints à une sous-population de tumeurs avec une déficience dans la voie de réparation par recombinaison homologue (DRH). De plus, des résistances à ces PARPi ont été constatées suite à la réactivation de la voie RH ou de voies alternatives. Il est donc urgent de développer des agents plus efficaces qui permettraient de limiter la problématique de résistance. Dans le laboratoire, nous avons identifié une nouvelle classe d’inhibiteurs de réparation de l’ADN, les Dbait, consistant en une petite molécule d’ADN double-brin qui miment une cassure double-brin (CDB). AsiDNA, une molécule de la famille Dbait, agit en séquestrant et hyper activant la protéine PARP et ses partenaires, ainsi que la protéine DNA-PK qui modifie la chromatine, inhibant ainsi le recrutement au niveau du site du dommage de plusieurs protéines de réparation des voies RH ou NHEJ. Dans ce manuscrit, nous avons étudié la question des mécanismes de sensibilité à AsiDNA, et nous avons identifié l’instabilité génétique, générée essentiellement par des défauts dans les voies de réparation des CDBs, comme caractéristique majeure pour être sensible à AsiDNA dans différents modèles de cellules et de xénogreffes. De façon intéressante, l’instabilité génétique ne corrélait pas avec la sensibilité aux PARPi, qui présentaient également un profil d’action différent d’AsiDNA. En se basant sur ces différences, et sur le mode d’action d’AsiDNA agissant en tant qu’inhibiteur de la voie RH, la combinaison de ces deux molécules permettrait de s’affranchir de la restriction génétique (DRH) essentielle pour l’efficacité des PARPi. Pour valider cette hypothèse, nous avons montré par des analyses moléculaires que l’olaparib, un PARPi, et AsiDNA préviennent le recrutement au niveau des sites des dommages de XRCC1 et de RAD51/53BP1, respectivement. La combinaison de ces deux inhibiteurs permettait l’accumulation des dommages non réparés résultant en une augmentation de la mort de cellules tumorales de différentes origines, et un retard significatif de la croissance des xénogreffes. Cependant, les cellules non tumorales ne présentaient ni une augmentation des dommages ni de la mort cellulaire. Ces résultats soulignent l’intérêt thérapeutique de la combinaison d’AsiDNA avec les PARPi qui permettrait de s’affranchir de la dépendance au statut DRH et d’élargir leur champ d’application. Dans cette thèse, nous avons également traité la question de la résistance acquise à AsiDNA. En effet, contrairement à l’imatinib et au 6-thioguanine, nous n’avons pas isolé de clones résistants à AsiDNA après des expériences de mutagénèse ou après des traitements répétés sur différents modèles cellulaires. Un tel comportement défie notre acceptation commune de la théorie Darwinienne pour expliquer la résistance des cellules tumorales aux traitements
Defects in the DNA repair pathways are now widely exploited for the treatment of cancer. Indeed, the ability of tumors to repair the damage induced by genotoxic treatments (chemotherapy and radiotherapy) gives them an intrinsic or acquired resistance to these treatments. Developing DNA repair inhibitors would help to counteract this resistance and sensitize tumors to these conventional therapies. Poly(ADP-ribose) polymerase inhibitors (PARPi), first candidates for this family of DNA repair inhibitors, have shown encouraging results but are nevertheless restricted to a tumor subpopulation with Deficiencies in the Homologous Recombination repair pathway (HRD). In addition, resistances to these PARPi were observed following the reactivation of the HR pathway or alternative pathways. It is therefore urgent to develop more effective agents to limit the resistance problem. In the laboratory, we have identified a new class of DNA repair inhibitors, Dbait, consisting of a small double-stranded DNA molecule that mimics a double-strand break (DSB). AsiDNA, a molecule of the Dbait family, acts by hijacking and hyper activating the PARP protein and its partners, as well as DNA-PK protein that modifies chromatin, thereby inhibiting recruitment at the damage site of several DNA repair proteins. In this manuscript, we studied the issue of mechanisms of sensitivity to AsiDNA, and we identified the genetic instability, generated mainly by defects in the DSBs’ repair, as major feature to be sensitive to AsiDNA in different models of tumor cells and xenografts. Interestingly, genetic instability does not correlate with sensitivity to PARPi, which also had a different action profile than AsiDNA. Based on these differences, and on the mode of action of AsiDNA acting as an inhibitor of the HR pathway, the combination of these two molecules would allow bypassing the genetic restriction (HRD) essential for PARPi efficiency. To validate this hypothesis, we have shown by molecular analyzes that olaparib, a PARPi, and AsiDNA prevent the recruitment at damage sites of the repair proteins XRCC1 and RAD51 / 53BP1, respectively. The combination of these two inhibitors allowed the accumulation of unrepaired damage resulting in an increase of tumor cells’ death, and a significant delay in the growth of xenografts. However, non-tumor cells were not sensitive to this combined treatment. These results highlight the therapeutic interest of combining AsiDNA with PARPi to recapitulate synthetic lethality in all tumors independently of their HR status. In this thesis, we also addressed the issue of acquired resistance to AsiDNA. Indeed, contrary to imatinib and 6-thioguanine, we didn’t recover resistant clones to AsiDNA after mutagenesis or after repeated cycles of treatment on different cell models. Such behavior challenges our common acceptation of a Darwin evolution theory to explain tumor cells resistance to treatment

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Coleman, Joshua B., Wesley Drew Gill, Allee C. Maxwell, and Russell W. Brown. "Analysis of a Poly(ADP-ribose) Polymerase (PARP) Inhibitor in a Treatment-resistant Depression Model in the Rat." Digital Commons @ East Tennessee State University, 2020. https://dc.etsu.edu/asrf/2020/presentations/53.

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Over 16 million people in the US suffer from major depressive disorder (MDD) each year. Approximately 1/3rd of MDD patients (~5 million) obtain only partial remission or no benefit after trials with multiple drugs or drug combinations. Recently, Ordway and colleagues have reportedelevated levels of DNA oxidation and upregulated gene expression of the base excision repair enzyme poly(ADP-ribose) polymerase-1 (PARP1) in postmortem brain from donors who had MDD at the time of death, as compared to age-matched psychiatrically normal control donors. This study was designed to test whether an inhibitor of PARP, 3-aminobenzamide (3-AB), may be effective to alleviate depressive-like behaviors in a rodent model of treatment-resistant depression. Male rats were ip administered lipopolysaccharide (LPS;100ug/kg) daily for 28 days, and administered a chronic unpredictable stressor on each day. All rats were also administered saline, 3-AB (40 mg/kg), or the serotonin-reuptake inhibitor (SSRI) fluoxetine (trade name: Prozac; 10 mg/kg) on each day, approximately 30 min after LPS treatment. During the 28 day period of LPS treatment, animals were behaviorally tested 5 times on sucrose preference (a test of anhedonia). At the end of the 28 day period, rats were behaviorally tested on a test of acute stress, the Porsolt swim test. Results revealed that 3-AB alleviated anhedonia and the response to acute stress in the Porsolt swim test superior to the fluoxetine group, demonstrating the utility of a PARP inhibitor to alleviate depressive-like behavior in this model. In addition, fluoxetine produced a loss of weight which recovered over days, but not to control levels, and 3-AB did not produce this effect. This study shows that PARP inhibitors may be effective in treatment-resistant depression.

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Jewett, Benjamin E., Merry N. Miller, Libby A. Ligon, Zachary Carter, Ibrahim Mohammad, and Gregory A. Ordway. "Rapid and Temporary Improvement of Depression and Anxiety Observed Following Niraparib Administration: A Case Report." Digital Commons @ East Tennessee State University, 2020. https://dc.etsu.edu/etsu-works/8592.

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Background: Cancer patients are disproportionately affected by generalized anxiety and major depression. For many, current treatments for these conditions are ineffective. In this case report, we present a serendipitous case of anxiety and depression improvement following administration of the poly (ADP-ribose) polymerase (PARP) inhibitor niraparib. Case presentation: A 61-year old woman with a 20-year history of mild depression developed recurrent ovarian carcinoma and was placed on niraparib for maintenance chemotherapy. With the original onset of ovarian cancer, she experienced an episode of major depression that was resolved with sertraline. After recurrence of ovarian cancer, she experienced a recurrence of major depression and a new onset of generalized anxiety that failed to completely respond to multiple medications. After beginning niraparib therapy the patient noticed a rapid resolution of the symptoms of her anxiety and depression, an effect that was limited to 10-14 days. Due to bone marrow suppression, the patient was taken off and restarted on niraparib several times. Each discontinuation of niraparib resulted in return of her depression and anxiety, while each recontinuation of niraparib resulted in an improvement in her mood and anxiety. Conclusions: This case demonstrates rapid and temporary improvement of anxiety and depression following niraparib administration. There is ample preclinical data that PARP signaling may play a role in psychiatric illness. A small amount of indirect data from clinical trials also shows that niraparib may have psychiatric benefits. Further research on PARP inhibition and its potential psychoactive effects is sorely needed.

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Cartwright, Luke. "The potential of poly (ADP-ribose) polymerase (PARP) inhibitors to improve plant growth and yield : novel crop protection agents under stressed conditions." Thesis, University of Sheffield, 2017. http://etheses.whiterose.ac.uk/19289/.

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Chemical inhibition of the activity of poly (ADP-ribose) polymerases (PARPs) is associated with enhanced stress tolerance and growth in response to a broad range of abiotic plant stressors. This led to the suggestion that PARP inhibitors might have application in future crop protection strategies. However, the vast majority of studies to date have involved short-term, in vitro assays which are not representative of the conditions crop plants experience in the field. This work aimed to quantify the impact of chemical PARP inhibitor application on photosynthesis, growth and yield in planta, under well-watered and droughted conditions. In Chapter 2, a protocol for the quantification of the impact of drought stress on photosynthesis was developed, mainly using chlorophyll fluorescence imaging. In Chapter 3, the impacts of PARP inhibitors on photosynthesis, growth and survival in response to drought were measured. PARP inhibitors enhanced survival to severe stress but there was a cost associated with application under well-watered and moderate drought conditions. Chlorophyll fluorescence and growth measurements indicated that PARP inhibitors also had a damaging effect. Results from Chapter 4 suggested that PARP and PSII inhibitors had broadly negative impacts on yield. There was a strong relationship between growth (maximum plant object sum area) and yield under stress, which enabled the effects that compounds had on yield to be predicted approximately 40 days earlier than measuring at harvest. By fitting a model to the growth data it was possible to predict the impacts even earlier still. In Chapter 5, application of a PARP inhibitor reduced stomatal conductance but did not alter opening/closing kinetics, indicating the compound had an anti-transpirant effect. The enhanced stress tolerance of PARP-deficient plants likely protected against severe drought. However, a trade-off arises because of the costs associated with application under more moderate conditions. If PARP inhibitors are to be used in agriculture the cost/benefit balance will have to be carefully considered.

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Burckel, Hélène. "Synthèse et évaluation de molécules bifonctionnelles alkylantes de l’ADN et inhibitrices de la PARP pour la radiochimiothérapie concomitante." Thesis, Strasbourg, 2012. http://www.theses.fr/2012STRAJ092.

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Cette étude a consisté en le développement et l’évaluation de nouvelles molécules pour la radiochimiothérapie concomitante. Ce travail a abouti à la conception de nouveaux agents chimiothérapeutiques duaux basés sur la combinaison covalente de deux radiosensibilisateurs: un inhibiteur de la PARP d’une part, et un alkylant de l’ADN (complexe de platine ou témozolomide) d’autre part. Les évaluations biologiques ont permis de mettre en évidence l’intérêt d’une molécule duale inhibiteur de la PARP/platine. Parallèlement à ce projet, le développement d’outils moléculaires pour l’étude d’inhibiteurs de la PARP a été entrepris. Ainsi, plusieurs sondes d’affinité ont été conçues pour une étude d’inhibiteurs de la PARP par protéomique chimique. Ce travail a permis de valider la spécificité d’une sonde d’affinité pour la PARP1 et la PARP2. Finalement, des molécules fluorescentes inhibitrices de la PARP ont été développées dans l’objectif d’un criblage d’inhibiteurs de PARP3 par anisotropie de fluorescence
The main topic of this work was the development and biological evaluation of dual molecules for concomitant chemoradiotherapy. To this end, new dual chemotherapeutic agents were designed by linking covalently two radiosensitizers: a PARP inhibitor and an alkylating agent (platinum complex or temozolomide). This study led to an efficient PARP inhibitor/platinum dual molecule. A complementary approach was to develop affinity probes to study PARP inhibitors by a chemical proteomic method. This study permitted to validate the selectivity of an affinity probe for PARP1 and PARP2. Finally, fluorescent PARP inhibitor probes were synthesised and evaluated for a PARP3 screening by fluorescence anisotropy

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Weigert, Verena [Verfasser], Rainer [Akademischer Betreuer] Fietkau, and Luitpold [Gutachter] Distel. "PARP inhibitors combined with ionizing radiation induce different effects in melanoma cells and healthy fibroblasts / Verena Weigert ; Gutachter: Luitpold Distel ; Betreuer: Rainer Fietkau." Erlangen : Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), 2021. http://d-nb.info/1231077956/34.

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Morice, Pierre-Marie. "Evaluation de la déficience de la recombinaison homologue et de la réponse des tumeurs ovariennes aux inhibiteurs de PARP grâce à l'utilisation de modèles de culture 3D en vue du développement d'un test prédictif Identifying eligible patients to PARP inhibitors: from NGS-based tests to promising 3D functional assays Automated scoring for assessment of RAD51-mediated homologous recombination in patient-derived tumor organoids of ovarian cancers Risk of myelodysplastic syndrome and acute myeloid leukemia related to PARP inhibitors: a combined approach using a safety meta-analysis of placebo randomized controlled trials and the World Health Organization's pharmacovigilance database The long non-coding RNA ‘UCA1’ modulates the response to chemotherapy of ovarian cancer through direct binding to miR-27a-5p and control of UBE2N levels." Thesis, Normandie, 2020. http://www.theses.fr/2020NORMC414.

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Chaque année, plus de 150 000 décès sont associés aux cancers épithéliaux de l’ovaire dans le monde, notamment en raison du développement d’une résistance à la chimiothérapie. Environ la moitié de ces cancers présentent des altérations moléculaires provoquant une déficience de la réparation de l’ADN par recombinaison homologue (HRD) qui les sensibilise à l’action des inhibiteurs de la protéine PARP (PARPi). A ce jour, il n’existe pas de test capable d’appréhender le phénotype HRD dans sa globalité, limitant ainsi l’accès à ces traitements. Dans ce contexte, nous avons entrepris de mettre au point des tests fonctionnels basés sur l’utilisation d’explants tumoraux tranchés puis sur l’utilisation d’organoïdes tumoraux dérivés de tumeurs ovariennes de patientes chimio-naïves ou antérieurement traitées. La culture d’explants s’est révélée inappropriée pour la réalisation de ces tests et nous avons alors focalisé nos travaux sur les organoïdes tumoraux. Ces derniers ont été exposés au carboplatine (traitement de 1e ligne) et à deux inhibiteurs de PARP (l’olaparib et le niraparib) utilisés en traitement d’entretien. En parallèle, nous avons collecté les données cliniques des patientes (survie, intervalle sans platine, RECIST, traitements) afin d’évaluer le potentiel prédictif de ces modèles. Les organoïdes tumoraux établis ont répondu de façon hétérogène aux différents médicaments testés, et nos résultats montrent que les tests réalisés sur les organoïdes sont capables d’identifier des patientes présentant un niveau de résistance élevé au carboplatine, suggérant que ce test fonctionnel pourrait présenter un intérêt prédictif vis-à-vis de ce médicament. Concernant le potentiel prédictif des organoïdes vis-à-vis des PARPi, des profils de sensibilité variés ont été identifiés, mais la corrélation avec la réponse clinique reste à établir par des études menées sur des échantillons de tumeurs issus de patientes traitées par ces médicaments
Worldwide each year, more than 150 000 women die from epithelial ovarian cancer largely due to emergence of resistance to chemotherapy. Approximately half of these cancers display molecular alterations that cause deficiency of DNA repair via homologous recombination (HRD), which confer sensitivity to PARP protein inhibitors (PARPi). To date, there is no test capable of fully identifying the HRD phenotype, thus limiting access to these treatments. In this context, we are developing functional assays based on the use of tumor explant slices and then, on the use of tumor organoids derived from ovarian tumors of chemotherapy-naive or previously treated patients. The culture of explants was unsuitable for this application and we then focused our work on tumor organoids. Tumor organoids were exposed to carboplatin (first-line treatment) and two PARP inhibitors (olaparib and niraparib) used for maintenance therapy. In parallel, we collected clinical data from patients (survival, platinum-free interval, RECIST, treatments) to evaluate the predictive potential of these models. The established tumor organoids responded heterogeneously to different drugs, and our results show that the organoid-based assay is capable of identifying patients highly resistant to carboplatin, suggesting that this functional assay could have a predictive value for patients treated with carboplatin. Regarding the potential of organoids in predicting PARPi response, multiple sensitivity profiles have been identified, but the correlation with clinical response has yet to be determined by studies conducted on tumor samples from patients treated with these drugs

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Sulier, Kiaya Minh-Li. "Developing 1,2,3,4-tetrahydro-5H-aryl[1,4]diazepin-5-ones and Related Scaffolds as Poly-(ADP-ribosyl) Polymerase (PARP) Inhibitors and Exploring Their Targeted Polypharmacology with Kinases." Thesis, Virginia Tech, 2017. http://hdl.handle.net/10919/86200.

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Poly-(ADP-ribsoyl) Polymerases (PARPs) are a superfamily of enzymes comprised of 17 known isoforms. PARP inhibitors (PARPi) have shown success in clinical trials for the treatment of homologous recombination-deficient cancers. Though proven effective initially, tumors treated with PARPi eventually develop resistance. Combinatorial therapeutics targeting PARP and other pathways that may re-sensitize tumors to PARP inhibition, including PI3K/AKT/mTor pathway, and cell-cycle checkpoints (such as CDKs, CHK, and Wee) are being tested. In this context, the synthetic lethality of cyclin-dependent kinase 1 (CDK1) and PARP1 is known. Evaluation of PARP1 and CDK1 pharmacophores led to the development of the tetrahydro-arylazepinone (TAAP) scaffold as a potential dual PARP1/CDK1 inhibitor. We screened a handful of TAAP analogs against PARP1 in a cell-free assay that identified the low micromolar PARP1 inhibitor 1,2,3,4-tetrahydro-5H-benzo[e][1,4]-diazepin-5-one (TBAP), which served as the lead compound. The analogous 1,2,3,4-tetrahydro-5H-pyrido[2,3-e][1,4]-diazepin-5-one (TPAP) series showed a similar bioactivity profile. Satisfyingly, the N1-benzyl TPAP analogue showed activity in the low nanomolar range. The TAAP series (i.e., 6/7-membered scaffold) unfortunately lacked CDK1 inhibitory activity. Finally, many PARPi's show poor isoform-selectivity. The development of isoform-selective PARPi can clarify the specific function of each PARP isoform and may reduce the adverse side effects shown by PARPi. A handful of TAAP analogs were screened against 13 PARP isoforms, where some compounds demonstrated exquisite PARP1/2 selectivity. Concurrently, we discovered an inhibitor for PARP11, an isoform that lacks any known synthetic ligand. Future directions are suggested towards fine-tuning the structure-activity relationship of TAAP-isoform selective PARPi as well as developing a dual PARP1/CDK1 inhibitor.
Master of Science

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Chuang, Hsiao-Ching. "Mechanistic Validation of Potential Anti-Breast Cancer Therapeutics." The Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1338213365.

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Grivas, Paul Christopher. "The role of poly (ADP-ribose) polymerase-1 inhibitors : prevention of non glutathione-dependent carbon tetrachloride-induced hepatotoxicity." [Tampa, Fla] : University of South Florida, 2007. http://purl.fcla.edu/usf/dc/et/SFE0001953.

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Engert, Florian [Verfasser], Volker Gutachter] Dötsch, and Simone [Gutachter] [Fulda. "PARP inhibitors in combination with chemotherapeutics target the underlying genetic phenotype of Ewing’s sarcoma and osteosarcoma to induce cell death or synthetic lethality / Florian Engert. Gutachter: Volker Dötsch ; Simone Fulda." Frankfurt am Main : Universitätsbibliothek Johann Christian Senckenberg, 2016. http://d-nb.info/1112601538/34.

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Engert, Florian Verfasser], Volker [Gutachter] Dötsch, and Simone [Gutachter] [Fulda. "PARP inhibitors in combination with chemotherapeutics target the underlying genetic phenotype of Ewing’s sarcoma and osteosarcoma to induce cell death or synthetic lethality / Florian Engert. Gutachter: Volker Dötsch ; Simone Fulda." Frankfurt am Main : Universitätsbibliothek Johann Christian Senckenberg, 2016. http://d-nb.info/1112601538/34.

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Steinbichler, Teresa Bernadette [Verfasser], Heinz Karl [Akademischer Betreuer] Höfler, and Irene [Akademischer Betreuer] Esposito. "Der Einfluss des PARP Inhibitors AZD2281 (Olaparib) auf das Wachstum von Mantelzelllymphomen in Abhängigkeit vom ATM und p53 Mutationsstatus / Teresa Bernadette Steinbichler. Gutachter: Irene Esposito ; Heinz Karl Höfler. Betreuer: Heinz Karl Höfler." München : Universitätsbibliothek der TU München, 2015. http://d-nb.info/1070624195/34.

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Kwan, Jair Chau Clinical School St Vincent's Hospital Faculty of Medicine UNSW. "Pharmacological activation of pro-survival pathways as a strategy for improving donor heart preservation." Awarded by:University of New South Wales. Clinical School - St Vincent's Hospital, 2009. http://handle.unsw.edu.au/1959.4/44663.

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Despite the development and use of specialised cardiac preservation solutions, the quality of the donor heart may still be compromised by its obligatory exposure to periods of ischaemia (both cold and warm) followed by reperfusion upon reintroduction of the recipient circulation. This is reflected in Transplant Registry data showing increased primary allograft failure as a function of increasing ischaemic time. The research described in this thesis is designed to further the understanding of the mechanisms by which the donor heart may be adapted to these prolonged periods of ischaemia and reperfusion by the activation of endogenous pro-survival signalling pathways by the addition of pharmacological agents to Celsior, a clinical preservation solution. Studies were conducted in an isolated working rat heart model of donor heart preservation. The first study investigated the cardioprotective effects of a novel inhibitor of poly(ADP-ribose) polymerase 1, INO-1153. Maximum protective effect (after a 6 hour storage period) was observed when the PARP inhibitor was administered prior to cardiac arrest and storage and when the agent was added to the Celsior cardioplegic / storage solution. This protective affect was associated with activation of the Akt signalling pathway and could be prevented by inhibition of Akt phosphorylation and activation. The second study examined functional protection and pro-survival signalling pathway activation in hearts arrested and stored for 6 hours in Celsior supplemented with glyceryl trinitrate (an exogenous source of nitric oxide) and Cariporide (an inhibitor of sodium hydrogen exchange). Here, cardiac protection was accompanied by activation of the ERK 1/2 pro-survival pathway as well as a decrease in apoptosis. The third study examined the cardioprotective effect of supplementation of Celsior with all three agents after an extended (10 hour) period of hypothermic storage. Significant recovery of function was only observed in the triply supplemented hearts, being accompanied by activation of both the Akt and ERK pathways. These studies demonstrate for the first time the feasibility of recruitment of endogenous pro-survival pathways as an approach to increasing the post-storage function of the donor heart. Importantly, for the logistics of clinical transplantation, these pathways can be recruited by addition of appropriate pharmacological agents to the arresting and storage solution.

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Croset, Amélie. "Identification et caractérisation des mécanismes d'action des molécules appats, les SiDNA, dans l'inhibition des voies de réparation des cassures simple-brin." Thesis, Paris 11, 2013. http://www.theses.fr/2013PA11T018.

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La plupart des traitements anticancéreux, comme la chimiothérapie ou la radiothérapie, sont cytotoxiques et causent des dommages à l'ADN dans le but d’induire la mort des cellules tumorales. Cependant, l’efficacité d’activité de réparation de l'ADN des tumeurs entraine des résistances intrinsèques et acquises aux traitements. L'une des étapes précoces de la réparation de l’ADN est le recrutement de protéines au niveau du site de dommage. Ce recrutement est coordonné par une cascade de modifications et est contrôlé par des protéines senseurs telles que la protéine kinase ADN dépendante (DNA-PK) et / ou la poly (ADP- ribose) polymérase (PARP). Dans ce manuscrit, nous avons identifié et caractérisé le mécanisme d'action de petites molécules d'ADN (les siDNA), mimant des cassures double brin (appelé Dbait) ou simple brin (appelé Pbait), dans l’inhibition des voies de réparation des cassures simple brin (SSBR/BER). Nous démontrons que les molécules Dbait recrutent et activent à la fois PARP et DNA-PK, contrairement aux molécules Pbait qui ne recrutent que la PARP. L'étude comparative de ces deux molécules permet d'analyser les rôles respectifs des deux voies de signalisation: les deux molécules recrutent les protéines impliquées dans la voie de réparation des cassures simple brin (comme PARP, PCNA et XRCC1) et empêchent leurs recrutements aux niveaux des lésions chromosomiques. Les molécules Dbait inhibent par ailleurs le recrutement des protéines impliquées dans la voie de réparation des cassures double brin (NHEJ et HR). Pbait et Dbait désorganisent la réparation de l’ADN et sensibilisent les cellules tumorales aux traitements. L’inhibition de la réparation des cassures simple brin semble dépendre d’un piégeage des protéines directement sur les siDNA ou indirectement sur les polymères PAR. L’inhibition des voies de réparation des cassures double brin (DSB) semble par contre se faire de façon indirecte ; cette inhibition résulterait plutôt de la phosphorylation des protéines de réparation des DSB de part l’activation de DNA-PK. Les molécules Dbait et Pbait induisent un effet de létalité synthétique des cellules tumorales BRCA mutées. Cependant, la mutation BRCA semble être suffisante mais non nécessaire pour induire la sensibilité des cellules tumorales aux traitements Dbait. En effet, nous avons démontré que les molécules Dbait peuvent aussi sensibiliser les cellules ne présentant pas de mutation BRCA mais ayant toutefois une forte instabilité génétique. Nous avons trouvé une corrélation entre le niveau basal de protéines de réparation de l'ADN (ɣH2AX, PARP et PAR), le taux basal de cassures à l’ADN, la présence de micronoyaux (MN) et la sensibilité des cellules tumorales au traitement Dbait. Nous avons émis l’hypothèse que cette instabilité génétique, déterminé par la quantification de MN dans des biopsies tumorales, pourrait être un biomarqueur prédictif de l’effet du Dbait, non seulement dans les cancers du sein, mais aussi dans les glioblastomes, les mélanomes, les mélanomes uvéaux et les cancers du côlon
Most conventional cancer treatments, such as chemotherapy or radiotherapy, are cytotoxic and cause DNA damages in the tumoral treated cells, which ultimately lead to their death. However, several intrinsic and acquired resistances of tumors to these treatments are due to the tumor efficient DNA repair activities. One of the major early steps of DNA repair is the recruitment of repair proteins at the damage site and this is coordinated by a cascade of modifications controlled by sensor proteins such as DNA-dependent protein kinase (DNA-PK) and/or poly (ADP-ribose) polymerase (PARP). In this manuscript, we identify and characterize the mechanism of action of short interfering DNA molecules (siDNA), mimicking double-strand breaks (called Dbait) or single-strand breaks (called Pbait) in Single Strand Break Repair pathway (SSBR/BER) inhibition. We demonstrate that Dbait bound and induced both PARP and DNA-PK activities, whereas Pbait acts only on PARP. The comparative study of the two molecules allows analysis of the respective roles of the two signaling pathways: both molecules recruit proteins involved in single-strand break repair (such as PARP, XRCC1 and PCNA) and prevent their recruitment at chromosomal damage. Dbait, but not Pbait, also inhibits recruitment of proteins involved in double-strand break (DSB) repair. By these ways, Pbait and Dbait disorganized DNA repair, thereby sensitizing cells to treatments. SSB repair inhibition depends upon a direct trapping of the main proteins on both molecules and an indirect trapping in PAR polymers. DSB repair inhibition may be indirect, resulting from the phosphorylation of DSB repair proteins by activated DNA-PK. The DNA repair inhibition by both molecules is confirmed by their synthetic lethality with BRCA mutations tumoral cell lines. However, BRCA mutation could be sufficient but not necessary to induce breast cancer cell lines and tumors sensitivity to Dbait treatment. In fact, we demonstrate that Dbait molecules could also have a stand-alone effect in BRCA wild type cells with a high genetic instability. We found a correlation between DNA repair proteins basal level (ɣH2AX, PARP and PAR), DNA break basal level, presence of micronucleus (MN) and tumoral cell lines sensitivity to Dbait treatment. We hypothesis that this genetic instability, determined by MN in tumor biopsies, could be a predictive biomarker of Dbait stand-alone effect, not only in breast cancer treatment, but also in glioblastoma, melanoma, uveal melanoma and colon cancer treatment

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Billaud, Amandine. "Analyse moléculaire, enjeux et limites des thérapies ciblées en oncologie : extension des sensibilités aux anti-PARP dans les cancers ovariens par caractérisation de variants non annotés et nouveaux mécanismes de résistance dans les cancers bronchiques. Caractérisation moléculaire de l’EGFR dans les cancers bronchiques non à petites cellules : étude prospective comparative des technologies NGS et automate Idylla Somatic mRNA analysis of BRCA1 splice variants provides a direct theranostic impact on PARP inhibitors." Thesis, Angers, 2020. http://www.theses.fr/2020ANGE0003.

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Malgré les bénéficies cliniques considérables de la prise en considération du contexte moléculaire tumoral, les thérapies ciblées présentent certaines limitations majeures. La première partie de ces travaux se concentre sur l’étude des inhibiteurs de tyrosine kinase ciblant l’EGFR dans les cancers bronchiques non à petites cellules. L’amélioration des méthodes de détection des biomarqueurs a été complétée par la caractérisation in vitro d’un mécanisme de résistance acquise non précédemment identifié. L’exposition de cellules pulmonaires à un agent mutagène puis une pression de sélection a ainsi mis en évidence la signature TBK1 et l’effet synergique de la co-inhibition s’est révélé intéressant. Le deuxième aspect concernent les inhibiteurs de PARP, incontournables dans la prise en charge des cancers de la sphère gynécologique. Ces thérapies reposant sur le principe de létalité synthétique, la présence de mutations pathogènes de BRCA1/2 est un pré-requis, illustrant la problématique des variants de signification inconnue. Face à la nécessité de leur caractérisation fonctionnelle, une étude transcriptionnelle des variants d’épissage par extraction de l’ARNm depuis les prélèvements FFPE a d’abord été réalisée. Puis, afin d’évaluer tous les variants, l’édition génomique a été développée comparant les efficacités d’édition d’un variant d’intérêt et d’un variant silencieux dans un contexte haploïde où ces gènes sont essentiels. La signification biologique de variants de BRCA1/2, et la généralisation de notre approche à l’ensemble des gènes suppresseurs de tumeur essentiels de nos cellules, peut ainsi être évaluée en 3 semaines, délai compatible avec les impératifs cliniques
Despite significant clinical benefit from the consideration of molecular context, targeted therapies are still challenging. First part of this work focused on tyrosine kinase inhibitors targeting EGFR in non small cell lung cancers. Thus, improvement of biomarkers detection methods was completed by in vitro characterization of an unreported mechanism of acquired resistance. Briefly, pulmonary cells were exposed to a mutagen agent and a selection pressure was applied with EGFR inhibitors allowing the detection of TBK1 signature. Finally, synergic effect of that co-inhibition was highlighted. Now essentials in gynaecological cancers management, PARP inhibitors represent the second part of that work. Those targeted therapies are based on synthetic lethality. Consequently, BRCA1/2 pathogenic mutations are required for their administration, illustrating the issue of variants of uncertain significance. Toward their functional characterization necessity, a transcriptional analysis of splicing variant was first conducted on mRNA extracted from FFPE samples. Then, to evaluate functional signification of all types of variants, genomic edition was developed. Editing efficiencies of the unknown variant and a silent control one were compared in a haploid model where those genes are essentials. Functional signification of BRCA1/2 variants, and thereby mutations from all essential tumor suppressor genes in our model, can be evaluated in three weeks which is compatible with clinical management

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Martin, Nadine. "Rôle de la SUMO E3 ligase PIASy dans les mécanismes de contrôle de la prolifération cellulaire et de réponse aux dommages." Paris 6, 2007. http://www.theses.fr/2007PA066243.

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La modification des protéines par SUMO joue un rôle important dans de nombreuses fonctions cellulaires. Le travail présenté dans cette thèse vise à analyser l'action des protéines PIAS, régulateurs transcriptionnels et SUMO E3 ligases, et en particulier de PIASy dans le contexte de l'oncogenèse. Nous avons montré que PIASy induit la sénescence des fibroblastes primaires, en activant les voies Rb et p53, ou l'apoptose si la voie Rb est déficiente. Nous avons parallèlement identifié de nouveaux partenaires protéiques de PIASy. PIASy induit l'accumulation de FIP200 dans le noyau, ce qui inhibe l'action de FIP200 dans la voie mTOR. FIP200 coopère avec PIASy pour activer l'expression du gène p21. Par ailleurs, la modification de PARP-1 par SUMO stimulée par PIASy régule la réponse au choc thermique. Ainsi, ce travail a mis en évidence un rôle important de PIASy dans le contrôle de la prolifération cellulaire et la réponse aux dommages, et a précisé son action au niveau moléculaire

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Hassanein, Mohamed. "Facteurs prédictifs de mutation germinale BRCA1 dans le cancer du sein héréditaire." Thesis, Aix-Marseille 2, 2010. http://www.theses.fr/2010AIX20714.

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En France, le cancer de sein héréditaire représente environ 2500 nouveaux cas par an, dont prés de la moitié est attribuée à la mutation du gène BRCA1.La recherche de la mutation par biologie moléculaire est un travail fastidieux, coûteux et long (8 mois d’attente environ actuellement).Pour trouver une solution à ce délai, nous avons étudié en immunohistochimie une série initiale de 21 anticorps répartis en 5 groupes : anticorps antiBrca1 du commerce, liés à la perte de l’inactivation de l’X, liés à la signature basale ou myoépithéliale, anticorps dits classiques du cancer de sein et finalement dérivés de signatures établies par cDNAarray.Nous avons utilisé la technique de’ tissue microarrays’ en utilisant de manière comparative une population de 27 cas de cancer de sein présentant une mutation germinale de BRCA1, et 81 cas témoins de cancer de sein sporadiques appariés à l’âge, ainsi qu’à des lignées cellulaires d’origine mammaires. Dans une deuxième série indépendante de validation nous avons appliqué les résultats obtenus de la première série sur 28 cas de cancer mammaire muté, et 28 cas du cancer mammaire sporadique dans les mêmes conditions initiales.Nos résultats montrent pour la première fois sur des tissus tumoraux une probabilité forte d’une association entre la mutation Brca1 et la perte de l’inactivation de l’X ; confirment la valeur de MS110 comme un bon anticorps prédictif d’une mutation de Brca1 ; apportent un argument pour une participation myoépithéliale dans l’oncogenèse de cancer mammaire Brca1 muté; appuient la relation entre ce dernier et les récepteurs RE,RP ainsi que P53 , Bcl2,Ki67 et valident en protéomique la valeur discriminant de CDC47 correspondant à un des gènes de la signature génomique.Après confirmation des mêmes résultats dans la série de validation, nous soutenons en analyses multivariés un modèle qui comprend seulement Grade 3, MS110, Lys27H3 négative, Vimentine et KI67 positive. Cette équation correspond à une sensibilité de 82% et spécificité de 81% et propose une approche rapide économique de pré- ciblage de la mutation Brca1 ; ce qui améliorait la prise en charge préventive, thérapeutique et globale des patients et leurs familles
Family structure, lack of reliable information, cost and delay are usual concerns faced with when deciding to perform BRCA analyses. Testing the breast cancer tissues with four antibodies (MS110, lys27H3, Vimentin, KI67) in addition to grade evaluation enabled to rapidly select patients to carry out genetic testing identification. We constituted an initial breast cancer tissue micro-array, considered as a learning set comprising 27 BRCA1 and 81 sporadic tumours. A second independent validation set of 28 BRCA1 tumours was matched to 28 sporadic tumours using the same original conditions.We have investigated morphological parameters and 21 markers by immunohistochemistry.A logistic regression model was used to select the minimal number of markers providing the best model to predict BRCA1 status. The model was applied to the validation set to estimate specificity and sensibility.In the initial set, the univariate analysis identified 11 markers significantly associated with BRCA1 status. Then the best multivariate model comprised only Grade 3, MS110, Lys27H3, Vimentin and KI67. When applied to the validation set, BRCA1 tumours were correctly classified with a sensitivity of 83% and a specificity of 81%. The performance of this model was superior when compared to other profiles.This work offers a new rapid and economic method for the pre-screening of patients at high risk of being BRCA1mutation carriers, then to guide genetic testing, and finally to provide appropriate preventive measure, advices and treatments including targeted therapy to patients and their families

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Magro, Tatiana Natália Tavares. "Testing the combinatory use of PARP1 and RPA inhibitors in breast cancer cell lines." Master's thesis, 2019. http://hdl.handle.net/10773/29527.

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Loss of tumor suppressor BRCA2 is strongly associated to breast cancer. BRCA2 is essential to homologous recombination (HR), which is a DNA repair pathway crucial to genomic stability. When cells become mutant for BRCA2, they cannot repair DNA damage through homologous recombination, which is an accurate repair pathway, and instead, rely on alternative error-prone pathways for DNA repair. Tumor cells mutant for BRCA2 become therefore dependent on these alternative repair pathways for survival. The most recent generation of targeted therapies for breast cancer is PARP1 inhibitors. PARP1 is a protein essential to the initiation of several DNA repair pathways, including those alternative repair pathways, like the non-homologous end joining (NHEJ). The use of these inhibitors compromises the alternative pathways leading to tumor cell death. Though tumor cells mutant for BRCA2 are particularly sensitive to PARP1 inhibitors, the onset of tumor resistance has been frequently observed after long-term treatments. This motivated us to find alternative proteins whose inhibition specifically impaired, similar to PARP1, tumor cells viability and/or growth. It was recently reported that BRCA2 regulates RNA polymerase II transcription and prevents the formation of R-loops, which are 3-strand nucleic acid structures composed of DNA:RNA hybrids. Accumulation of these R-loops is implicated in the process of carcinogenesis due to the accumulation of single-stranded DNA (ssDNA) and increased genomic instability. RPA is a ssDNA-binding protein whose function is crucial to protect ssDNA and avoid forming secondary structures, being crucial to DNA replication and DNA repair. Our objective is to identify novel druggable targets whose inhibition can be used in combination or as an alternative to PARP1 inhibitors, minimizing tumor resistance risk. Our working hypothesis is that combinatory inhibition of PARP1 and RPA will specifically increase the loss of breast cancer cells viability.
A perda do supressor tumoral BRCA2 está fortemente associada ao cancro da mama. O BRCA2 é essencial para a recombinação homóloga, que é uma via de reparação de ADN crucial para a estabilidade genómica. Quando as células sofrem mutações neste gene, elas não conseguem reparar os danos no ADN através da recombinação homóloga, que é uma via de reparação precisa, e em vez disso, tornam-se dependentes de vias alternativas, propensas a erros, para reparação de ADN. Deste modo, as células tumorais mutantes para BRCA2 tornam-se dependentes destas vias alternativas para sobreviverem. A geração mais recente de terapias direcionadas são os inibidores da PARP1. A PARP1 é uma proteína essencial para a iniciação de várias vias de reparação de ADN, incluindo as tais vias de reparação alternativas, como a non-homologous end joining (NHEJ). A utilização destes inibidores compromete as vias alternativas levando à morte das células tumorais. Embora as células tumorais mutantes para BRCA2 sejam particularmente sensíveis aos inibidores da PARP1, o aparecimento da resistência tumoral tem sido observado frequentemente após tratamentos de longo-prazo. Este problema motivou-nos a procurar proteínas alternativas cuja inibição prejudicasse especificamente, à semelhança da PARP1, a viabilidade e/ou crescimento das células tumorais. Foi recentemente reportado que o BRCA2 regula a transcrição da ARN polimerase II e previne a formação de R-loops, que são estruturas de ácidos nucleicos de 3 cadeias compostas por híbridos de ADN:ARN. A acumulação destes R-loops está implicada no processo de carcinogénese devido à acumulação de ADN de cadeia simples (do inglês ssDNA) e ao aumento de instabilidade genómica. O RPA é uma proteína que se liga ao ssDNA e evita que se formem estruturas secundárias, sendo crucial para a replicação e reparação de ADN. O nosso objetivo é identificar novos alvos terapêuticos cuja inibição possa ser usada em combinação ou como alternativa aos inibidores da PARP1, minimizando o risco de resistência tumoral. A nossa hipótese de trabalho é que a inibição combinatória de PARP1 e RPA aumentará especificamente a perda de viabilidade das células de cancro da mama.
Mestrado em Biomedicina Molecular

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43

Dziaková, Lucia. "Studium interakcí PARP inhibitorů s ABC lékovými efluxními transportéry." Master's thesis, 2020. http://www.nusl.cz/ntk/nusl-411910.

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Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Lucia Dziaková Supervisor: RNDr. Jakub Hofman, Ph.D. Title of diploma thesis: Study on interactions of PARP inhibitors with ABC drug efflux transporters. ATP-binding cassette (ABC) transporters are integral membrane proteins that use the energy obtained from ATP to carry transport of numerous endogenous substrances out of the cells, but attention is drawn primarily to the fact that they transfer also xenobiotics. Their overexpression in tumor tissue contributes to multidrug resistance (MDR), which in most cases leads to therapy failure. Poly(ADP-ribose)polymerase inhibitors (PARPi) represent a promising therapeutic approach in the treatment of cancers that exhibit defects in homologous recombination (HR). This work focuses on four selected PARPi (olaparib, rucaparib, niraparib, veliparib) and their interaction potential towards ABC drug efflux transporters (ABCB, ABCC1, ABCG2). In our work, we worked with MDCKII cells (parent, transduced by the transporters of interest) and utilized the principle of accumulation studies based on the measurement of fluorescence intensity of specific model substrates (hoechst33342, calcein AM, daunorubicin, mitoxantrone). We used established inhibitors of studied...

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44

Aubert-Jürgens, Ana. "STAT3 inhibitors for cancer treatment." Phd thesis, 2005. https://tuprints.ulb.tu-darmstadt.de/563/1/aubert-jurgens-part1.pdf.

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The critical role of the activation of signal transducer and activator of transcription 3 (STAT3) in the growth and survival of human tumor cells was one of the subjects of this thesis. It was found that the stable incorporation of v-Src into MCF10A cells, a human immortalized breast epithelial cell line, induced constitutive phosphorylation of STAT3 in these cells. MCF10A-v-Src cells displayed growth factor independence for proliferation and survival, and anchorage independence. However, these cells did not form tumors in nude mice, indicating that they were not fully transformed. Furthermore, inhibition of activated STAT3 in 293 cells decreased growth and induced apoptosis in these cells. The same results were obtained in 293 cells stably transfected with tetracycline-inducible dominant negative (DN) STAT3, and in 293 cells transiently expressing STAT3 RNAi. Since STAT3 promises to be a good target for cancer treatment, it was attempted to develop inhibitors through rational design. Within STAT3, the SH2-domain was chosen as being the best target site. One limitation of this site turned out to be its similarity to family members in this region, and even to other SH2-domain containing proteins, such as Src, all of which present a highly conserved pocket to bind phosphotyrosines. A medium-throughput assay to measure STAT3 dimerization in vitro was established to determine the affinity of compounds to the SH2-domain of STAT3. Peptides of different length derived from the phosphotyrosine-containing motif of STAT3 (AAPY*LKTKF) were tested in the assay. Y*L was found to be the minimal sequence to block dimerization, and out of the tested peptides, PY*LKT had the highest affinity. This was consistent with the observations made on the crystal structure of STAT3, which indicated that besides the phosphotyrosine-binding site, position + 1 and + 3 contributed most importantly to binding. The relevance of position + 1 was further confirmed by substitution of Leu+ 1 by Ala in the peptide Y*LKT, which decreased the affinity more than ten times. Finally, one non-peptidic small molecule inhibitor identified by virtual ligand screening (VLS) proved to be a STAT3 dimerization inhibitor in vitro. This is the first small molecule inhibitor of STAT3 identified so far, and might serve to study structure activity relationships (SAR) to optimize the structure and find more potent and bioavailable compounds.

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Sohail, Honeah. "PARP inhibitor ABT-888 as potentiating agent for topoisomerase inhibitor SN-38." 2009. http://hdl.rutgers.edu/1782.2/rucore10001600001.ETD.000051407.

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Allison, Simon J., Maria Sadiq, Efstathia Baronou, Patricia A. Cooper, C. Dunnill, N. T. Georgopoulos, A. Latif, et al. "Preclinical anti-cancer activity and multiple mechanisms of action of a cationic silver complex bearing N-heterocyclic carbene ligands." 2017. http://hdl.handle.net/10454/11960.

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Yes
Organometallic complexes offer the prospect of targeting multiple pathways that are important in cancer biology. Here, the preclinical activity and mechanism(s) of action of a silver-bis(N-heterocyclic carbine) complex (Ag8) were evaluated. Ag8 induced DNA damage via several mechanisms including topoisomerase I/II and thioredoxin reductase inhibition and induction of reactive oxygen species. DNA damage induction was consistent with cytotoxicity observed against proliferating cells and Ag8 induced cell death by apoptosis. Ag8 also inhibited DNA repair enzyme PARP1, showed preferential activity against cisplatin resistant A2780 cells and potentiated the activity of temozolomide. Ag8 was substantially less active against non-proliferating non-cancer cells and selectively inhibited glycolysis in cancer cells. Ag8 also induced significant anti-tumour effects against cells implanted intraperitoneally in hollow fibres but lacked activity against hollow fibres implanted subcutaneously. Thus, Ag8 targets multiple pathways of importance in cancer biology, is less active against non-cancer cells and shows activity in vivo in a loco-regional setting.
RMP and MS funded by Yorkshire Cancer Research (pump priming grant BPP 046). IJS and AL funded by NIHR Research & Innovation Division, Strategic Project Funding 2013 and Manchester Pharmacy School Fellowship.

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Nikoloska, Irena. "Towards the Synthesis of Novel PARP Inhibitors through Diels-Alder Chemistry." Thesis, 2012. http://hdl.handle.net/10214/3561.

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Poly(ADP-ribose) polymerase (PARP) represents a large family of enzymes that are activated upon DNA breakage, which are then involved in a cascade of reactions that eventually lead to cell death. PARPs, known to cause a variety of damage to the human body, are targets of many researches’ investigating potential inhibition mechanisms. To this point, plenty of PARP inhibitors have been synthesized and tested for potency against many diseases. Some have great potency against particular diseases, while others are already being used as drugs. The current research suggests a potential synthesis of novel PARP inhibitors, to be accomplished through Diels-Alder chemistry. The proposed compounds consist of an isoindolinone core bearing different functional groups. Three different strategies are described for the synthesis of the novel PARP inhibitors and all protocols involve sulfur dioxide extrusion chemistry to create a diene, the desired transient starting material. The diene is envisioned further to be reacted with a variety of dienophiles to give possibly potential PARP inhibitors.
NSERC

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48

Mishra, Anup. "Targeting RAD51C Pathological Mutants by Synthetic Lethality and Extended Functions of RAD51C/XRCC3 in Mitochondrial Genome Maintenance." Thesis, 2017. http://etd.iisc.ac.in/handle/2005/4155.

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To counteract the potentially calamitous effects of genomic instability in the form of double-strand breaks (DSBs), cells have evolved with two major mechanisms. First, DNA non¬homologous end joining (NHEJ) which requires no significant homology, and second, homologous recombination (HR) that uses intact sequences on the sister chromatid or homologous chromosome as a template to repair the broken DNA. Although NHEJ repairs DSBs in all stages of cell cycle; it is generally error-prone due to insertions or deletions of few nucleotides at the breakpoint. In contrast, DSBs that are generated during S and G2 phase of the cell are preferentially repaired by HR that utilizes neighboring sister chromatid as a template. A central role in the HR reaction is promoted by the RAD51 recombinase which polymerizes onto single-stranded DNA (ssDNA), catalyzes pairing and strand invasion with homologous DNA molecule. Assembly of RAD51 monomers onto ssDNA is a relatively slow process and is facilitated by several mediator proteins. The tumor suppressor protein BRCA2 is the best-characterized RAD51 mediator in DSB repair by HR. Many reports in the past two decades have established that RAD51 recruitment at break sites also depends on the RAD51 paralogs. Mammalian cells encode five RAD51 paralogs; RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3 which share 20–30% identity at amino acid level with RAD51 and with each other. In addition to their role in HR, RAD51 paralogs have been identified to be involved in DNA damage signaling and replication fork maintenance. In addition, mouse knockout of RAD51 paralogs causes early embryonic lethality. Recent studies show that germline mutations in all five RAD51 paralogs cause various types of cancer including breast and ovarian cancers. Pedigree analyses revealed that similar to BRCA1 and BRCA2, pathological missense mutants of RAD51C were of high penetrance. Historically, defects in the DNA repair pathways have been exploited for cancer chemo-and radiotherapy. In an attempt to develop better cancer therapeutic approaches, the concept of synthetic lethality for cancer therapy has been recently proposed. One such example is the use of PARP1 inhibitors to treat tumors carrying mutations in HR genes, such as BRCA1 and BRCA2. Inhibition of PARP1 compromises single-strand break repair (SSBR) pathway. Upon replication fork collision, the accumulated SSBs are converted to one-ended DSBs, which are efficiently repaired by the HR for cell survival. As a result, HR-deficient tumors with BRCA1-or BRCA2-deficiency exhibit extreme sensitivity to PARP-1 inhibition resulting in cell death. This approach was highly successful in targeting tumors with severe defects in Fanconi anemia (FA)-BRCA proteins which led to PARP inhibitors being tested in clinical trials. However, targeting cancer cells that express hypomorphic mutants of HR proteins is highly challenging since such partially functional mutants require a high dosage of PARP inhibitors for effective sensitization which renders normal cells toxic and can also lead to tumor resistance. The pathological RAD51C mutants that were identified in breast and ovarian cancer patients are hypomorphic with partial repair function. The first part of my Ph.D. thesis is aimed at developing an effective strategy to target cells that express hypomorphic RAD51C mutants. To this end, we used RAD51C deficient CL-V4B hamster cells and expressed the pathological RAD51C mutants associated with breast and ovarian cancers. Cells expressing RAD51C mutants that were severely defective for HR function exhibited high sensitivity to low doses of PARP1 inhibitor (4-ANI). These cells also accumulated in G2/M and displayed chromosomal aberrations. However, RAD51C mutants that were hypomorphic were partially sensitized even at higher concentrations of PARP inhibitor. RAD51C/ CL-V4B cells displayed higher PARP activity compared WT V79B cells. Notably, PARP activity was directly proportional to the sensitivity of RAD51C mutants towards 4-ANI where highly sensitive RAD51C mutants showed higher PARP activity and vice versa. Increased PARP activity was associated with replication stress as confirmed by an increase of PARP activity in cells treated with replication stress inducer, hydroxyurea (HU). Notably, treatment of CL-V4B cells with PARP1 inhibitor (4-ANI) resulted in the accumulation of PARP1 onto the chromatin which eventually led to the formation of DSBs which suggests that PARP1 entrapment triggers replication fork collapse leading to one-ended DSBs in S-phase. To further understand the molecular mechanism of PARP inhibitor-induced toxicity of RAD51C deficient cells, we carried out chromatin fractionation from V79B and CL-V4B cells at varying time points of 4-ANI treatment. Surprisingly, there was an enhanced loading of NHEJ proteins on chromatin in CL-V4B compared to V79B cells. Consistently, an increased error-prone NHEJ was observed in CL-V4B cells which resulted in increased chromosomal aberrations and cell death. Furthermore, inhibition of DNA-PKcs or depletion of KU70 or Ligase IV restored this phenotype. Thus, error-prone NHEJ in collaboration with PARP inhibition sensitizes RAD51C deficient cells. Since ionizing radiation (IR) is known to stimulate NHEJ activity, we hypothesized that irradiation in combination with PARP inhibitor would further sensitize the RAD51C deficient tumors. Strikingly, stimulation of NHEJ by a low dose of IR in the PARP inhibitor-treated RAD51C deficient cells and cells expressing pathological RAD51C mutants induced enhanced toxicity ‘synergistically’. These results demonstrate that cancer cells arising due to hypomorphic mutations in RAD51C can be specifically targeted by a ‘synergistic approach’ and imply that this strategy can be potentially applied to cancers with hypomorphic mutations in other HR pathway genes. In addition to nuclear functions, RAD51 paralogs RAD51C and XRCC3 have been shown to localize to mitochondria and contribute to mitochondrial genome stability. However, the molecular mechanism by which RAD51 and RAD51 paralogs carry out this function is unclear. The second part of my thesis was dedicated to studying whether RAD51C/XRCC3 facilitates mitochondrial DNA replication and the underlying mechanism by which RAD51C/XRCC3 participate in mitochondrial genome maintenance during unperturbed conditions. Using mitochondrial subfractionation we show that RAD51 and RAD51 paralogs (RAD51C and XRCC3) are an integral part of mitochondrial nucleoid and absence of RAD51C/XRCC3 and RAD51 prevents the restoration of mtDNA upon depletion of mtDNA. This suggests that RAD51 and RAD5C/XRCC3 participate in mtDNA replication. To determine whether this function of RAD51C is exclusive to mitochondria we expressed NLS mutant of RAD51C which was defective for nuclear functions. Interestingly, cells expressing RAD51C R366Q were able to efficiently repopulate the depleted mtDNA after EtBr stress similar to that of WT RAD51C expressing cells, suggesting a nuclear independent function of RAD51C in mitochondrial genome maintenance. mtDNA-IP analysis revealed that RAD51 and RAD51C/XRCC3 are recruited to the mtDNA control regions spontaneously along with mitochondrial polymerase POLG. Moreover, RAD51 was found to associate with TWINKLE helicase and this association was required for the recruitment of RAD51 and RAD51C/XRCC3 at the D-loop. As in nucleus, mtDNA replisome also encounters replication stresses like altered dNTP pools, a collision between replication and transcription machinery, rNTP incorporation, oxidative stress which hampers replication fork progression. Using Dideoxycytidine (ddC) as replication stress inducer in mitochondria, we observed nearly 3-4 fold enrichment of RAD51, RAD51C, XRCC3 and POLG at the mtDNA mutation hotspot region D310. Notably, RAD51C/XRCC3 deficient cells exhibited increased lesions in the mitochondrial genome spontaneously, pointing towards the importance of RAD51C/XRCC3 in the prevention of mtDNA lesions. Moreover, RAD51C/XRCC3 deficiency prevented the repair of ddC induced mtDNA lesions. Given that RAD51C/XRCC3 and RAD51 are localized to mtDNA control regions along with POLG and their deficiency affects mtDNA replication we were curious to learn the effect of RAD51C/XRCC3 deficiency on the recruitment of POLG in mtDNA. To test this we performed a mtDNA-IP assay of POLG in RAD51C deficient cells which revealed that deficiency of RAD51C/XRCC3 and RAD51 affected the recruitment of POLG on mtDNA control regions. As a consequence RAD51C/XRCC3 deficient cells exhibit aberrant mitochondrial functions. These findings propose a mechanism for a direct role of RAD51C/XRCC3 in maintaining the mtDNA integrity under replication stress conditions.

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Wang, Wei-Jie, and 王為婕. "Mechanisms of SAHA and PARP Inhibitors Induced Cell Death in Cancer Cells." Thesis, 2015. http://ndltd.ncl.edu.tw/handle/34790258374946152730.

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碩士
國立陽明大學
生命科學系暨基因體科學研究所
103
I. Combination therapy of SAHA and PARP inhibitors in HCC treatment Genomic instability is one of the cancer hallmarks. Because severe DNA damage results in cell death, inhibitors of the DNA repair system can be used as anticancer drugs. An inhibitor of histone deacetylases (HDACs) - suberoylanilide hydroxamic acid (SAHA), is being evaluated for phase2/3 clinical trial to treat several types of cancers. Mechanism of SAHA-induced cell death is not totally clear, but reactive oxygen species (ROS) release and double strand break (DSB) may play a role in apoptosis of SAHA-treated cells. Upon DNA damage, p53 activates its downstream target genes and triggers DNA repair or apoptosis. In our previous study, mutation of TP53 and down-regulation of PARP4 were found to be associated with chromosome 4q loss of heterozygosity in hepatocellular carcinoma (HCC), implicating that sensitivity to DNA-damaging agents in HCC cells might depend on the TP53 status and PARP4 expression level. A pan-PARP inhibitor 3-aminobenzamide (3-ABA), known to inhibit the single strand break repairing system by targeting PARP1/2, was chosen as a candidate to be combined with SAHA for treating HCC cells with TP53 mutation. As a result of impaired DNA repair, DSB and apoptosis were enhanced, as measured by γ-H2AX staining and caspase 3/PARP1 cleavage, respectively. Sensitivity to SAHA and 3-ABA was reduced in TP53 knocked down or mutant TP53 stably transfected HepG2 cells. Depending on genetic background, SAHA/3-ABA-induced cytotoxicity was variable and could be attributed to cell cycle arrest and cell death. In HepG2 cells, SAHA-induced up-regulation of CDKN1A was enhanced by 3-ABA. In Hep3B cells, TXNIP level was further elevated by 3-ABA, contributing to ROS release and DSB. Consistently, N-acetylcysteine, a ROS inhibitor, reduced the expression of the targeted genes, GRP94 and CHOP, of unfolded protein response, and blunted SAHA/3-ABA induced cell death. Another PARP inhibitor rucaparib, when combining with SAHA, induced prominent cell death, and it was accompanied by up-regulation of p21 (WAF1/CIP). Unlike 3-ABA, rucaparib did not enhance SAHA-induced DSB. Thus our results support that multiple pathways and effectors are involved in SAHA-induced cancer cell death, and, contingent on TP53, a new strategy of therapeutic development can be designed by adding PARP inhibitors, to achieve personalized medicine for HCC. II. Cellular characterization of PARP4 Poly(ADP-ribose) polymerase family, member 4 (PARP4) catalyzes poly(ADP-ribosyl)ation PARsylation of numerous proteins localized in cytosol and nuclei. In previous studies, PARsylation of targeted proteins were known to be involved in regulating DNA damage detection, DNA repair, and cell death pathways. To investigate whether PARP4 also participates in the maintenance of genome integrity, subcellular localization of PARP4 was analyzed. Nuclear translocation of PARP4 was observed with DSBs induced by various DNA-damaging agents. In H2O2-treated HeLa cells, PARP4 localized with γ-H2AX, a DSB marker, while in etoposide-treated HEK293T cells, co-localization of PARP4 and PAR was observed. Notably, PARP4 nuclear localization is an early event and it became detectable by fluorescence microscopy two hours after addition of 4 μM SAHA. PARP4 knockdown enhanced DSBs and cell death in SAHA/3-ABA-treated cancer cells. Finally, SAHA targeted on PARP4 by down-regulation of mRNA and protein expression, suggesting that enhancement of cell death by combining SAHA with PARP inhibitors might work through their effects on the PARP4 level and DNA repair. Taken together, our data support that PARP4 may function in the maintenance of genome integrity, and it could be served as a target to design anticancer drugs.

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Meyer, Stephanie C. "Synergistic effects of combining PARP inhibitor (AZD2281) and ATR inhibitor (AZD6738) in Ewing Sarcoma cell lines." Thesis, 2018. https://hdl.handle.net/2144/30818.

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Abstract:

Ewing Sarcoma (ES) is an aggressive pediatric solid tumor. Even though overall-survival for localized patients is approximately 70%, the overall-survival for high risk ES patients has not improved in the last 20 years. Therefore, there is a need for exploration of new therapeutic agents in ES. Recent evidence has demonstrated that ES cells behave like BRCA-deficient tumor types which renders them sensitive to PARP inhibitors in vitro and in vivo. However, a phase II study of the efficacy of single-agent PARP inhibition in patients with relapsed ES did not significantly improve outcome. As single-agent therapy is rarely expected to result in significant clinical responses, in this study, we plan to validate potential targeted combination therapies with PARP inhibitors in ES. Since ES appears to demonstrated BRCA-deficient biology with impaired homologous recombination, cells are expected to be sensitive to both PARP inhibitors and ATR inhibitors, drugs which have a role in regulating DNA damage and impairing homologous recombination. In breast cancer and ovarian cell lines with genetic BRCA-deficiency, PARP and ATR inhibitors have synergistic activity. We hypothesize that these inhibitors will also have synergistic anti-Ewing activity. Furthermore, we recognize that ES cells demonstrate remarkably quiet genomes suggesting that there is minimal ongoing DNA-damage when cells are growing unperturbed. Therefore, we also plan to test the effect of adding low-dose genotoxic chemotherapy to induce additional sensitivity to the combination of PARP and ATR inhibitors in ES. The specific aims of this study were to explore the possible anti-tumor effect of PARP inhibitors combined with ATR inhibitors in ES cell lines, and to explore whether low dose genotoxic chemotherapy with SN38 can potentiate the anti-tumor effect of combined PARP and ATR inhibition in ES cell lines. We studied the anti-Ewing Sarcoma effect of the combination of a PARP inhibitor, AZD2281, and an ATR inhibitor, AZD6738, across a range of doses with and without low doses of a DNA damaging agent, SN38 (irinotecan metabolite), in two ES cell lines. We analyzed synergy by determining the Combination Index (CI) and Fractional Inhibition (FA) of each combination. We found that the ATR inhibitor, AZD6738, was synergistic across large range of concentrations when combined with the PARP inhibitor, AZD2281, in ES cell lines. We also found that treatment of cells with low doses of SN38 increases ES cell sensitivity to treatment with the PARP inhibitor and ATR inhibitor combination. This study provides preclinical support for additional studies exploring these combinations in ES. Given the low number of pediatric patients with ES compared to adult cancer patients, there will be limited attempts in combining these agents in clinical trials. Therefore, the development of an in vivo trial testing the safety and efficacy of this combination in ES mouse models is proposed.
2020-07-03T00:00:00Z

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