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Robert L Coleman - One of the best experts on this subject based on the ideXlab platform.
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rucaparib in ovarian cancer extending the use of parp inhibitors in the recurrent disease
Future Oncology, 2018Co-Authors: Graziela Dal Z Molin, Shannon Neville Westin, Robert L ColemanAbstract:Rucaparib is a potent inhibitor of poly (ADP-ribose) polymerase (PARP) PARP1, PARP2 and PARP3, and to a lesser extent, PARP4, PARP10, PARP12, PARP15 and PARP16. Study 10 and ARIEL2 evaluated the use of rucaparib as treatment in patients with recurrent high-grade ovarian carcinoma and resulting in approval of rucaparib for patients with both germline and somatic BRCA mutation. Data from the Phase III trial ARIEL3 led to approval in platinum-sensitive disease as maintenance. This article reviews the efficacy, safety, pharmacokinetics and pharmacodynamics of rucaparib as well as future and ongoing trials.
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Rucaparib in ovarian cancer: an update on safety, efficacy and place in therapy:
Therapeutic Advances in Medical Oncology, 2018Co-Authors: Graziela Z Dal Molin, Kohei Omatsu, Anil K Sood, Robert L ColemanAbstract:Rucaparib is a poly (ADP-ribose) polymerase (PARP) inhibitor and potent inhibitor of PARP1, PARP2 and PARP3 enzymes. Phase II and III trials have documented that rucaparib has single-agent antitumo...
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rucaparib in ovarian cancer an update on safety efficacy and place in therapy
Therapeutic Advances in Medical Oncology, 2018Co-Authors: Graziela Dal Z Molin, Kohei Omatsu, Anil K Sood, Robert L ColemanAbstract:Rucaparib is a poly (ADP-ribose) polymerase (PARP) inhibitor and potent inhibitor of PARP1, PARP2 and PARP3 enzymes. Phase II and III trials have documented that rucaparib has single-agent antitumor activity in patients with high-grade ovarian carcinoma, with both BRCA-mutated (germline and somatic) and with homologous recombination deficiency (HRD). Rucaparib as a maintenance treatment showed increased progression-free survival in patients with ovarian carcinoma who achieved a response to platinum-based chemotherapy, with an acceptable safety profile. The approval of this drug, along with the companion diagnostic FoundationFocus CDxBRCA test represents an important new therapeutic option in the treatment of ovarian cancer. This article reviews the mechanisms of action, safety, pharmacokinetics and pharmacodynamics and indications for use of rucaparib as well as future trials.
Françoise Dantzer - One of the best experts on this subject based on the ideXlab platform.
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PARP2 deficiency affects invariant-NKT,-cell maturation and protects mice from ,Concanavalin A-induced liver injury.
AJP - Gastrointestinal and Liver Physiology, 2017Co-Authors: Aveline Filliol, Françoise Dantzer, Claire Piquet-pellorce, Sarah Dion, Valentine Genet, Catherine Lucas-clerc, Michel SamsonAbstract:Excessive or persistent inflammation and hepatocyte death are the key triggers of liver diseases. The poly(ADP-ribose) polymerase (PARP) proteins induce cell death and inflammation. Chemical inhibition of PARP activity protects against liver injury during concanavalin A (ConA)-induced hepatitis. In this mice model, ConA activates immune cells, which promote inflammation and induce hepatocyte death, mediated by the activated invariant natural killer T (iNKT) lymphocyte population. We analyzed immune cell populations in the liver and several lymphoid organs, such as the spleen, thymus, and bone marrow in Parp2-deficient mice to better define the role of PARP proteins in liver immunity and inflammation at steady state and during ConA-induced hepatitis. We show that 1) the genetic inactivation of Parp2, but not PARP1, protected mice from ConA hepatitis without deregulating cytokine expression and leucocyte recruitment; 2) cellularity was lower in the thymus, but not in spleen, liver, or bone marrow of Parp2-/- mice; 3) spleen and liver iNKT lymphocytes, as well as thymic T and NKT lymphocytes were reduced in Parp2 knockout mice. In conclusion, our results suggest that the defect of T-lymphocyte maturation in Parp2 knockout mice leads to a systemic reduction of iNKT cells, reducing hepatocyte death during ConA-mediated liver damage, thus protecting the mice from hepatitis.NEW & NOTEWORTHY The genetic inactivation of Parp2, but not PARP1, protects mice from concanavalin A hepatitis. Immune cell populations are lower in the thymus, but not in the spleen, liver, or bone marrow of Parp2-deficient mice compared with wild-type mice. Spleen and liver invariant natural killer T (NKT) lymphocytes, as well as thymic T and NKT lymphocytes, are reduced in Parp2-deficient mice
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Robust immunoglobulin class switch recombination and end joining in Parp9-deficient mice
Eur J Immunol, 2017Co-Authors: Isabelle Robert, Léa Gaudot, José Yélamos, Aurélia Noll, Heng-kuan Wong, Françoise Dantzer, Valérie Schreiber, Bernardo Reina-san-martinAbstract:To mount highly specific and adapted immune responses, B lymphocytes assemble and diversify their antibody repertoire through mechanisms involving the formation of programmed DNA damage. Immunoglobulin class switch recombination (CSR) is triggered by DNA lesions induced by activation-induced cytidine deaminase, which are processed to double-stranded DNA break (DSB) intermediates. These DSBs activate the cellular DNA damage response and enroll numerous DNA repair factors, involving poly(ADP-ribose) polymerases PARP1, Parp2, and Parp3 to promote appropriate DNA repair and efficient long-range recombination. The macroParp Parp9, which is overexpressed in certain lymphomas, has been recently implicated in DSB repair, acting together with PARP1. Here, we examine the contribution of Parp9 to the resolution of physiological DSBs incurred during V(D)J recombination and CSR by generating Parp9-/- mice. We find that Parp9-deficient mice are viable, fertile, and do not show any overt phenotype. Moreover, we find that Parp9 is dispensable for B-cell development. Finally, we show that CSR and DNA end-joining are robust in the absence of Parp9, indicating that Parp9 is not essential in vivo to achieve physiological DSB repair, or that strong compensatory mechanisms exist.
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Common and unique genetic interactions of the poly(ADP-ribose) polymerases PARP1 and PARP2 with DNA double-strand break repair pathways
DNA Repair, 2016Co-Authors: Rajib Ghosh, Françoise Dantzer, Sanchita Roy, Johan Kamyab, Sonia FrancoAbstract:In mammalian cells, chromatin poly(ADP-ribos)ylation (PARylation) at sites of DNA Double-Strand Breaks (DSBs) is mediated by two highly related enzymes, PARP1 and PARP2. However, enzyme-specific genetic interactions with other DSB repair factors remain largely undefined. In this context, it was previously shown that mice lacking PARP1 and H2AX, a histone variant that promotes DSB repair throughout the cell cycle, or the core nonhomologous end-joining (NHEJ) factor Ku80 are not viable, while mice lacking PARP1 and the noncore NHEJ factor DNA-PKcs are severely growth retarded and markedly lymphoma-prone. Here, we have examined the requirement for PARP2 in these backgrounds. We find that, like PARP1, PARP2 is essential for viability in mice lacking H2AX. Moreover, treatment of H2AX-deficient primary fibroblasts or B lymphocytes with PARP inhibitors leads to activation of the G2/M checkpoint and accumulation of chromatid-type breaks in a lineage- and gene-dose dependent manner. In marked contrast to PARP1, loss of PARP2 does not result in additional phenotypes in growth, development or tumorigenesis in mice lacking either Ku80 or DNA-PKcs. Altogether these findings highlight specific nonoverlapping functions of PARP1 and PARP2 at H2AX-deficient chromatin during replicative phases of the cell cycle and uncover a unique requirement for PARP1 in NHEJ-deficient cells.
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Parp3 negatively regulates immunoglobulin class switch recombination
PLoS Genetics, 2015Co-Authors: Isabelle Robert, Vincent Heyer, Léa Gaudot, Aurélia Noll, Françoise Dantzer, Mélanie Rogier, Bernardo Reina-san-martinAbstract:To generate highly specific and adapted immune responses, B cells diversify their antibody repertoire through mechanisms involving the generation of programmed DNA damage. Somatic hypermutation (SHM) and class switch recombination (CSR) are initiated by the recruitment of activation-induced cytidine deaminase (AID) to immunoglobulin loci and by the subsequent generation of DNA lesions, which are differentially processed to mutations during SHM or to double-stranded DNA break intermediates during CSR. The latter activate the DNA damage response and mobilize multiple DNA repair factors, including PARP1 and Parp2, to promote DNA repair and long-range recombination. We examined the contribution of Parp3 in CSR and SHM. We find that deficiency in Parp3 results in enhanced CSR, while SHM remains unaffected. Mechanistically, this is due to increased occupancy of AID at the donor (Sμ) switch region. We also find evidence of increased levels of DNA damage at switch region junctions and a bias towards alternative end joining in the absence of Parp3. We propose that Parp3 plays a CSR-specific role by controlling AID levels at switch regions during CSR.
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Poly(ADP-ribose) polymerases in double-strand break repair: focus on PARP1, PARP2 and PARP3
Exp Cell Res, 2014Co-Authors: Carole Beck, Bernardo Reina-san-martin, Isabelle Robert, Valérie Schreiber, Françoise DantzerAbstract:Poly(ADP-ribosyl)ation (PARylation) is a post-translational modification of proteins catalysed by Poly(ADP-ribose) polymerases (PARP). A wealth of recent advances in the biochemical and functional characterization of the DNA-dependent PARP family members have highlighted their key contribution in the DNA damage response network, the best characterized being the role of PARP1 and PARP2 in the resolution of single-strand breaks as part of the BER/SSBR process. How PARylation contributes to the repair of double-strand breaks is less well defined but has become recently the subject of significant research in the field. The aim of this review is to provide an overview of the current knowledge concerning the role of the DNA-activated PARP1, PARP2 and PARP3 in cellular response to double-strand breaks (DSB). In addition, we outline the biological significance of these properties in response to programmed DNA lesions formed during physiological processes such as antibody repertoire assembly and diversification.
Junko Murai - One of the best experts on this subject based on the ideXlab platform.
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resistance to parp inhibitors by slfn11 inactivation can be overcome by atr inhibition
Oncotarget, 2016Co-Authors: Junko Murai, Ying Feng, Guoying K Yu, Yuanbin Ru, Saiwen Tang, Yuqiao Shen, Yves PommierAbstract:// Junko Murai 1 , Ying Feng 2 , Guoying K. Yu 2 , Yuanbin Ru 2 , Sai-Wen Tang 1,3 , Yuqiao Shen 2 and Yves Pommier 1 1 Developmental Therapeutics Branch and Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA 2 BioMarin Pharmaceutical Inc., Novato, CA, USA 3 Current affiliation: Division of Blood and Marrow Transplantation, Department of Medicine, Stranford University School of Medicine, Stanford, CA, USA Correspondence to: Yves Pommier, email: // Keywords : PARP-trapping, ATR, PARP inhibitor, BRCA, homologous recombination Received : August 25, 2016 Accepted : August 26, 2016 Published : September 27, 2016 Abstract Poly(ADP-ribose) polymerase inhibitors (PARPIs) kill cancer cells by trapping PARP1 and PARP2. Talazoparib, the most potent PARPI inhibitor (PARPI), exhibits remarkable selectivity among the NCI-60 cancer cell lines beyond BRCA inactivation. Our genomic analyses reveal high correlation between response to talazoparib and Schlafen 11 ( SLFN11 ) expression. Causality was established in four isogenic SLFN11 -positive and -negative cell lines and extended to olaparib. Response to the talazoparib-temozolomide combination was also driven by SLFN11 and validated in 36 small cell lung cancer cell lines, and in xenograft models. Resistance in SLFN11 -deficient cells was caused neither by impaired drug penetration nor by activation of homologous recombination. Rather, SLFN11 induced irreversible and lethal replication inhibition, which was independent of ATR-mediated S-phase checkpoint. The resistance to PARPIs by SLFN11 inactivation was overcome by ATR inhibition, mechanistically because SLFN11 -deficient cells solely rely on ATR activation for their survival under PARPI treatment. Our study reveals that SLFN11 inactivation, which is common (~45%) in cancer cells, is a novel and dominant resistance determinant to PARPIs.
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stereospecific parp trapping by bmn 673 and comparison with olaparib and rucaparib
Molecular Cancer Therapeutics, 2014Co-Authors: Sharyin N Huang, Amelie Renaud, Joel Morris, Junko Murai, Jiuping Ji, Beverly A Teicher, Shunichi Takeda, Yiping Zhang, James H DoroshowAbstract:Anti-poly(ADP-ribose)polymerase (PARP) drugs were initially developed as catalytic inhibitors to block the repair of DNA single-strand breaks. We recently reported that several PARP inhibitors have an additional cytotoxic mechanism by trapping PARP-DNA complexes, and that both olaparib and niraparib act as PARP poisons at pharmacological concentrations. Therefore, we have proposed that PARP inhibitors should be evaluated based both on catalytic PARP inhibition and PARP-DNA trapping. Here, we evaluated the novel PARP inhibitor, BMN 673, and compared its effects on PARP1 and PARP2 with two other clinical PARP inhibitors, olaparib and rucaparib, using biochemical and cellular assays in genetically-modified chicken DT40 and human cancer cell lines. Although BMN 673, olaparib and rucaparib are comparable at inhibiting PARP catalytic activity, BMN 673 is ~100-fold more potent at trapping PARP-DNA complexes and more cytotoxic as single agent than olaparib, while olaparib and rucaparib show similar potencies in trapping PARP-DNA complexes. The high level of resistance of PARP1/2 knockout cells to BMN 673 demonstrates the selectivity of BMN 673 for PARP1/2. Moreover, we show that BMN 673 acts by stereospecific binding to PARP1 as its enantiomer, LT674, is several orders of magnitude less efficient. BMN 673 is also ~100-fold more cytotoxic than olaparib and rucaparib in combination with the DNA alkylating agents methyl methane sufonate (MMS) and temozolomide. Our study demonstrates that BMN 673 is the most potent clinical PARP inhibitor tested to date with the highest efficiency at trapping PARP-DNA complexes.
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abstract a257 stereospecific trapping of parp dna complexes by bmn 673 and comparison with olaparib and rucaparib
Molecular Cancer Therapeutics, 2013Co-Authors: Junko Murai, Sharyin N Huang, Amelie Renaud, Joel Morris, Jiuping Ji, James H Doroshow, Beverly A Teicher, Shunichi Takeda, Yiping Zhang, Yves PommierAbstract:Anti-poly(ADP-ribose)polymerase (PARP) drugs were initially developed as catalytic inhibitors to block the repair of DNA single-strand breaks. Yet, several PARP inhibitors have an additional cytotoxic mechanism by trapping PARP-DNA complexes; both olaparib and niraparib act as PARP poisons at pharmacological concentrations (Murai et al., Cancer Res, 2012). Here, we evaluate the novel PARP inhibitor, BMN 673, and compare its effects on PARP1 and PARP2 with two other clinical PARP inhibitors, olaparib and rucaparib, using biochemical and cellular assays in genetically-modified chicken DT40 and human cancer cell lines. We show that BMN 673, olaparib, and rucaparib are similarly potent at inhibiting PARP catalytic activity. At the same time, BMN 673 is ∼100-fold more potent at trapping PARP-DNA complexes and more cytotoxic as a single agent than olaparib, while olaparib and rucaparib show similar potencies in trapping PARP-DNA complexes. The high level of resistance of PARP1/2 knockout cells to BMN 673 demonstrates the selectivity of BMN 673 for PARP1/2. Moreover, we show that BMN 673 acts by stereospecific binding to PARP1 as its enantiomer, LT674, is several orders of magnitude less efficient, and that BMN 673 is more cytotoxic than olaparib and rucaparib in combination with the DNA alkylating agents methyl methane sulfonate (MMS) and temozolomide. Our study demonstrates that BMN 673 is the most potent clinical PARP inhibitor to date with the highest efficiency at trapping PARP-DNA complexes. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A257. Citation Format: Junko Murai, Shar-yin N. Huang, Amelie Renaud, Yiping Zhang, Jiuping Ji, Shunichi Takeda, Joel Morris, Beverly Teicher, James H. Doroshow, Yves Pommier. Stereospecific trapping of PARP-DNA complexes by BMN 673 and comparison with olaparib and rucaparib. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A257.
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trapping of PARP1 and parp2 by clinical parp inhibitors
Cancer Research, 2012Co-Authors: Junko Murai, Sharyin N Huang, Amelie Renaud, James H Doroshow, Shunichi Takeda, Yiping Zhang, Benu Brata Das, Yves PommierAbstract:Small-molecule inhibitors of PARP are thought to mediate their antitumor effects as catalytic inhibitors that block repair of DNA single-strand breaks (SSB). However, the mechanism of action of PARP inhibitors with regard to their effects in cancer cells is not fully understood. In this study, we show that PARP inhibitors trap the PARP1 and PARP2 enzymes at damaged DNA. Trapped PARP-DNA complexes were more cytotoxic than unrepaired SSBs caused by PARP inactivation, arguing that PARP inhibitors act in part as poisons that trap PARP enzyme on DNA. Moreover, the potency in trapping PARP differed markedly among inhibitors with niraparib (MK-4827) > olaparib (AZD-2281) >> veliparib (ABT-888), a pattern not correlated with the catalytic inhibitory properties for each drug. We also analyzed repair pathways for PARP-DNA complexes using 30 genetically altered avian DT40 cell lines with preestablished deletions in specific DNA repair genes. This analysis revealed that, in addition to homologous recombination, postreplication repair, the Fanconi anemia pathway, polymerase β, and FEN1 are critical for repairing trapped PARP-DNA complexes. In summary, our study provides a new mechanistic foundation for the rational application of PARP inhibitors in cancer therapy.
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trapping of PARP1 and parp2 by clinical parp inhibitors
Cancer Research, 2012Co-Authors: Junko Murai, Sharyin N Huang, Amelie Renaud, Jiuping Ji, James H Doroshow, Shunichi Takeda, Yiping Zhang, Yves PommierAbstract:Abstract Small-molecule inhibitors of PARP are thought to mediate their antitumor effects as catalytic inhibitorsthat block repair of DNA single-strand breaks (SSB). However, the mechanism of action of PARP inhibi-tors with regard to their effects in cancer cells is not fully understood. In this study, we show that PARPinhibitors trap the PARP1 and PARP2 enzymes at damaged DNA. Trapped PARP–DNA complexes were morecytotoxic than unrepaired SSBs caused by PARP inactivation, arguing that PARP inhibitors act in part aspoisons that trap PARP enzyme on DNA. Moreover, the potency in trapping PARP differed markedly amonginhibitors with niraparib (MK-4827)> olaparib (AZD-2281)>> veliparib (ABT-888), a pattern not correlatedwith the catalytic inhibitory properties for each drug. We also analyzed repair pathways for PARP–DNAcomplexes using 30 genetically altered avian DT40 cell lines with preestablished deletions in specificDNArepair genes. This analysis revealed that, in addition to homologous recombination, postreplication repair,the Fanconi anemia pathway, polymerase b, and FEN1 are critical for repairing trapped PARP–DNAcomplexes. In summary, our study provides a new mechanistic foundation for the rational application ofPARP inhibitors in cancer therapy. Cancer Res; 72(21); 5588–99. 2012 AACR.
Bernhard Lüscher - One of the best experts on this subject based on the ideXlab platform.
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ARTD10/PARP10 Induces ADP-Ribosylation of GAPDH and Recruits GAPDH into Cytosolic Membrane-Free Cell Bodies When Overexpressed in Mammalian Cells
MDPI AG, 2018Co-Authors: Emilia Mayo, Bernhard Lüscher, Henning Kleine, Gaia Fabrizio, Emanuele Salvatore Scarpa, Annalisa Stilla, Nadia Dani, Fulvio Chiacchiera, Francesca Attanasio, Maria Di GirolamoAbstract:Protein ADP-ribosylation is a reversible post-translational modification of cellular proteins that is catalysed by enzymes that transfer one (mono) or several (poly) units of ADP-ribose from β-NAD+ to a specific amino acid of the target protein. The most studied member of the ADP-ribosyltransferase family is PARP1 (also known as ADP-ribosyltransferase diphtheria toxin-like 1, ARTD1), which is directly activated by DNA strand breaks and is involved in DNA damage repair, chromatin remodelling and transcriptional regulation. Much less is known about the further 16 members of this family. Among these, ARTD10/PARP10 has been previously characterised as a mono-ADP-ribosyltransferase with a role in cell proliferation and in NF-kB signalling. In the present study, we identified the glycolytic enzyme GAPDH as an interactor and a novel cellular target for ARTD10/PARP10. Moreover, we detected the co-localisation of GAPDH and ARTD10/PARP10 in well-defined cytosolic bodies, which we show here to be membrane-free, rounded structures using immunogold labelling and electron microscopy. Using the cognitive binding module macro domain to visualise ADP-ribosylated proteins by immunofluorescence microscopy in cells over-expressing the ARTD10/PARP10 enzyme, we show that the staining of the ARTD10/PARP10-dependent cytosolic bodies was lost when the cells were treated with compounds that inhibit ARTD10/PARP10, either by directly inhibiting the enzyme or by reducing the cellular NAD+ levels. In parallel, the same treatment affected the co-localisation of GAPDH and ARTD10/PARP10, as GAPDH disappeared from the cytosolic cell bodies, which indicates that its presence there depends on the catalytic activity of ARTD10/PARP10. In line with this, in cells over-expressing the ARTD10/PARP10 catalytic domain alone, which we show here to form stress granules, GAPDH was recruited into stress granules. These data identify ARTD10/PARP10 as the enzyme that modifies and recruits GAPDH into cytosolic structures
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The conserved macrodomains of the non-structural proteins of Chikungunya virus and other pathogenic positive strand RNA viruses function as mono-ADP-ribosylhydrolases
Scientific Reports, 2017Co-Authors: Laura Eckei, Sarah Krieg, Mareike Bütepage, Anne Lehmann, Annika Gross, Barbara Lippok, Alexander R. Grimm, Beate M. Kümmerer, Giulia Rossetti, Bernhard LüscherAbstract:Human pathogenic positive single strand RNA ((+)ssRNA) viruses, including Chikungunya virus, pose severe health problems as for many neither efficient vaccines nor therapeutic strategies exist. To interfere with propagation, viral enzymatic activities are considered potential targets. Here we addressed the function of the viral macrodomains, conserved folds of non-structural proteins of many (+)ssRNA viruses. Macrodomains are closely associated with ADP-ribose function and metabolism. ADP-ribosylation is a post-translational modification controlling various cellular processes, including DNA repair, transcription and stress response. We found that the viral macrodomains possess broad hydrolase activity towards mono-ADP-ribosylated substrates of the mono-ADP-ribosyltransferases ARTD7, ARTD8 and ARTD10 (aka PARP15, PARP14 and PARP10, respectively), reverting this post-translational modification both in vitro and in cells. In contrast, the viral macrodomains possess only weak activity towards poly-ADP-ribose chains synthesized by ARTD1 (aka PARP1). Unlike poly-ADP-ribosylglycohydrolase, which hydrolyzes poly-ADP-ribose chains to individual ADP-ribose units but cannot cleave the amino acid side chain - ADP-ribose bond, the different viral macrodomains release poly-ADP-ribose chains with distinct efficiency. Mutational and structural analyses identified key amino acids for hydrolase activity of the Chikungunya viral macrodomain. Moreover, ARTD8 and ARTD10 are induced by innate immune mechanisms, suggesting that the control of mono-ADP-ribosylation is part of a host-pathogen conflict.
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artd10 substrate identification on protein microarrays regulation of gsk3β by mono adp ribosylation
Cell Communication and Signaling, 2013Co-Authors: Karla L. H. Feijs, Patricia Verheugd, Henning Kleine, Anne K Braczynski, Alexandra H Forst, Nicolas Herzog, Ulrike Linzen, Elisabeth Kremmer, Bernhard LüscherAbstract:Although ADP-ribosylation has been described five decades ago, only recently a distinction has been made between eukaryotic intracellular poly- and mono-ADP-ribosylating enzymes. Poly-ADP-ribosylation by ARTD1 (formerly PARP1) is best known for its role in DNA damage repair. Other polymer forming enzymes are ARTD2 (formerly PARP2), ARTD3 (formerly PARP3) and ARTD5/6 (formerly Tankyrase 1/2), the latter being involved in Wnt signaling and regulation of 3BP2. Thus several different functions of poly-ADP-ribosylation have been well described whereas intracellular mono-ADP-ribosylation is currently largely undefined. It is for example not known which proteins function as substrate for the different mono-ARTDs. This is partially due to lack of suitable reagents to study mono-ADP-ribosylation, which limits the current understanding of this post-translational modification. We have optimized a novel screening method employing protein microarrays, ProtoArrays®, applied here for the identification of substrates of ARTD10 (formerly PARP10) and ARTD8 (formerly PARP14). The results of this substrate screen were validated using in vitro ADP-ribosylation assays with recombinant proteins. Further analysis of the novel ARTD10 substrate GSK3β revealed mono-ADP-ribosylation as a regulatory mechanism of kinase activity by non-competitive inhibition in vitro. Additionally, manipulation of the ARTD10 levels in cells accordingly influenced GSK3β activity. Together these data provide the first evidence for a role of endogenous mono-ADP-ribosylation in intracellular signaling. Our findings indicate that substrates of ADP-ribosyltransferases can be identified using protein microarrays. The discovered substrates of ARTD10 and ARTD8 provide the first sets of proteins that are modified by mono-ADP-ribosyltransferases in vitro. By studying one of the ARTD10 substrates more closely, the kinase GSK3β, we identified mono-ADP-ribosylation as a negative regulator of kinase activity.
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ARTD10 substrate identification on protein microarrays: regulation of GSK3β by mono-ADP-ribosylation
Cell Communication and Signaling, 2013Co-Authors: Karla L. H. Feijs, Patricia Verheugd, Henning Kleine, Anne K Braczynski, Alexandra H Forst, Nicolas Herzog, Ulrike Linzen, Elisabeth Kremmer, Bernhard LüscherAbstract:Background Although ADP-ribosylation has been described five decades ago, only recently a distinction has been made between eukaryotic intracellular poly- and mono-ADP-ribosylating enzymes. Poly-ADP-ribosylation by ARTD1 (formerly PARP1) is best known for its role in DNA damage repair. Other polymer forming enzymes are ARTD2 (formerly PARP2), ARTD3 (formerly PARP3) and ARTD5/6 (formerly Tankyrase 1/2), the latter being involved in Wnt signaling and regulation of 3BP2. Thus several different functions of poly-ADP-ribosylation have been well described whereas intracellular mono-ADP-ribosylation is currently largely undefined. It is for example not known which proteins function as substrate for the different mono-ARTDs. This is partially due to lack of suitable reagents to study mono-ADP-ribosylation, which limits the current understanding of this post-translational modification. Results We have optimized a novel screening method employing protein microarrays, ProtoArrays®, applied here for the identification of substrates of ARTD10 (formerly PARP10) and ARTD8 (formerly PARP14). The results of this substrate screen were validated using in vitro ADP-ribosylation assays with recombinant proteins. Further analysis of the novel ARTD10 substrate GSK3β revealed mono-ADP-ribosylation as a regulatory mechanism of kinase activity by non-competitive inhibition in vitro . Additionally, manipulation of the ARTD10 levels in cells accordingly influenced GSK3β activity. Together these data provide the first evidence for a role of endogenous mono-ADP-ribosylation in intracellular signaling. Conclusions Our findings indicate that substrates of ADP-ribosyltransferases can be identified using protein microarrays. The discovered substrates of ARTD10 and ARTD8 provide the first sets of proteins that are modified by mono-ADP-ribosyltransferases in vitro . By studying one of the ARTD10 substrates more closely, the kinase GSK3β, we identified mono-ADP-ribosylation as a negative regulator of kinase activity.
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substrate assisted catalysis by PARP10 limits its activity to mono adp ribosylation
Molecular Cell, 2008Co-Authors: Henning Kleine, Elzbieta Poreba, Krzysztof Lesniewicz, Paul O Hassa, Michael O Hottiger, David W Litchfield, Brian H Shilton, Bernhard LüscherAbstract:ADP-ribosylation controls many processes, including transcription, DNA repair, and bacterial toxicity. ADP-ribosyltransferases and poly-ADP-ribose polymerases (PARPs) catalyze mono- and poly-ADP-ribosylation, respectively, and depend on a highly conserved glutamate residue in the active center for catalysis. However, there is an apparent absence of this glutamate for the recently described PARP6-PARP16, raising questions about how these enzymes function. We find that PARP10, in contrast to PARP1, lacks the catalytic glutamate and has transferase rather than polymerase activity. Despite this fundamental difference, PARP10 also modifies acidic residues. Consequently, we propose an alternative catalytic mechanism for PARP10 compared to PARP1 in which the acidic target residue of the substrate functionally substitutes for the catalytic glutamate by using substrate-assisted catalysis to transfer ADP-ribose. This mechanism explains why the novel PARPs are unable to function as polymerases. This discovery will help to illuminate the different biological functions of mono- versus poly-ADP-ribosylation in cells.
James H Doroshow - One of the best experts on this subject based on the ideXlab platform.
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stereospecific parp trapping by bmn 673 and comparison with olaparib and rucaparib
Molecular Cancer Therapeutics, 2014Co-Authors: Sharyin N Huang, Amelie Renaud, Joel Morris, Junko Murai, Jiuping Ji, Beverly A Teicher, Shunichi Takeda, Yiping Zhang, James H DoroshowAbstract:Anti-poly(ADP-ribose)polymerase (PARP) drugs were initially developed as catalytic inhibitors to block the repair of DNA single-strand breaks. We recently reported that several PARP inhibitors have an additional cytotoxic mechanism by trapping PARP-DNA complexes, and that both olaparib and niraparib act as PARP poisons at pharmacological concentrations. Therefore, we have proposed that PARP inhibitors should be evaluated based both on catalytic PARP inhibition and PARP-DNA trapping. Here, we evaluated the novel PARP inhibitor, BMN 673, and compared its effects on PARP1 and PARP2 with two other clinical PARP inhibitors, olaparib and rucaparib, using biochemical and cellular assays in genetically-modified chicken DT40 and human cancer cell lines. Although BMN 673, olaparib and rucaparib are comparable at inhibiting PARP catalytic activity, BMN 673 is ~100-fold more potent at trapping PARP-DNA complexes and more cytotoxic as single agent than olaparib, while olaparib and rucaparib show similar potencies in trapping PARP-DNA complexes. The high level of resistance of PARP1/2 knockout cells to BMN 673 demonstrates the selectivity of BMN 673 for PARP1/2. Moreover, we show that BMN 673 acts by stereospecific binding to PARP1 as its enantiomer, LT674, is several orders of magnitude less efficient. BMN 673 is also ~100-fold more cytotoxic than olaparib and rucaparib in combination with the DNA alkylating agents methyl methane sufonate (MMS) and temozolomide. Our study demonstrates that BMN 673 is the most potent clinical PARP inhibitor tested to date with the highest efficiency at trapping PARP-DNA complexes.
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abstract a257 stereospecific trapping of parp dna complexes by bmn 673 and comparison with olaparib and rucaparib
Molecular Cancer Therapeutics, 2013Co-Authors: Junko Murai, Sharyin N Huang, Amelie Renaud, Joel Morris, Jiuping Ji, James H Doroshow, Beverly A Teicher, Shunichi Takeda, Yiping Zhang, Yves PommierAbstract:Anti-poly(ADP-ribose)polymerase (PARP) drugs were initially developed as catalytic inhibitors to block the repair of DNA single-strand breaks. Yet, several PARP inhibitors have an additional cytotoxic mechanism by trapping PARP-DNA complexes; both olaparib and niraparib act as PARP poisons at pharmacological concentrations (Murai et al., Cancer Res, 2012). Here, we evaluate the novel PARP inhibitor, BMN 673, and compare its effects on PARP1 and PARP2 with two other clinical PARP inhibitors, olaparib and rucaparib, using biochemical and cellular assays in genetically-modified chicken DT40 and human cancer cell lines. We show that BMN 673, olaparib, and rucaparib are similarly potent at inhibiting PARP catalytic activity. At the same time, BMN 673 is ∼100-fold more potent at trapping PARP-DNA complexes and more cytotoxic as a single agent than olaparib, while olaparib and rucaparib show similar potencies in trapping PARP-DNA complexes. The high level of resistance of PARP1/2 knockout cells to BMN 673 demonstrates the selectivity of BMN 673 for PARP1/2. Moreover, we show that BMN 673 acts by stereospecific binding to PARP1 as its enantiomer, LT674, is several orders of magnitude less efficient, and that BMN 673 is more cytotoxic than olaparib and rucaparib in combination with the DNA alkylating agents methyl methane sulfonate (MMS) and temozolomide. Our study demonstrates that BMN 673 is the most potent clinical PARP inhibitor to date with the highest efficiency at trapping PARP-DNA complexes. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A257. Citation Format: Junko Murai, Shar-yin N. Huang, Amelie Renaud, Yiping Zhang, Jiuping Ji, Shunichi Takeda, Joel Morris, Beverly Teicher, James H. Doroshow, Yves Pommier. Stereospecific trapping of PARP-DNA complexes by BMN 673 and comparison with olaparib and rucaparib. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A257.
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trapping of PARP1 and parp2 by clinical parp inhibitors
Cancer Research, 2012Co-Authors: Junko Murai, Sharyin N Huang, Amelie Renaud, James H Doroshow, Shunichi Takeda, Yiping Zhang, Benu Brata Das, Yves PommierAbstract:Small-molecule inhibitors of PARP are thought to mediate their antitumor effects as catalytic inhibitors that block repair of DNA single-strand breaks (SSB). However, the mechanism of action of PARP inhibitors with regard to their effects in cancer cells is not fully understood. In this study, we show that PARP inhibitors trap the PARP1 and PARP2 enzymes at damaged DNA. Trapped PARP-DNA complexes were more cytotoxic than unrepaired SSBs caused by PARP inactivation, arguing that PARP inhibitors act in part as poisons that trap PARP enzyme on DNA. Moreover, the potency in trapping PARP differed markedly among inhibitors with niraparib (MK-4827) > olaparib (AZD-2281) >> veliparib (ABT-888), a pattern not correlated with the catalytic inhibitory properties for each drug. We also analyzed repair pathways for PARP-DNA complexes using 30 genetically altered avian DT40 cell lines with preestablished deletions in specific DNA repair genes. This analysis revealed that, in addition to homologous recombination, postreplication repair, the Fanconi anemia pathway, polymerase β, and FEN1 are critical for repairing trapped PARP-DNA complexes. In summary, our study provides a new mechanistic foundation for the rational application of PARP inhibitors in cancer therapy.
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trapping of PARP1 and parp2 by clinical parp inhibitors
Cancer Research, 2012Co-Authors: Junko Murai, Sharyin N Huang, Amelie Renaud, Jiuping Ji, James H Doroshow, Shunichi Takeda, Yiping Zhang, Yves PommierAbstract:Abstract Small-molecule inhibitors of PARP are thought to mediate their antitumor effects as catalytic inhibitorsthat block repair of DNA single-strand breaks (SSB). However, the mechanism of action of PARP inhibi-tors with regard to their effects in cancer cells is not fully understood. In this study, we show that PARPinhibitors trap the PARP1 and PARP2 enzymes at damaged DNA. Trapped PARP–DNA complexes were morecytotoxic than unrepaired SSBs caused by PARP inactivation, arguing that PARP inhibitors act in part aspoisons that trap PARP enzyme on DNA. Moreover, the potency in trapping PARP differed markedly amonginhibitors with niraparib (MK-4827)> olaparib (AZD-2281)>> veliparib (ABT-888), a pattern not correlatedwith the catalytic inhibitory properties for each drug. We also analyzed repair pathways for PARP–DNAcomplexes using 30 genetically altered avian DT40 cell lines with preestablished deletions in specificDNArepair genes. This analysis revealed that, in addition to homologous recombination, postreplication repair,the Fanconi anemia pathway, polymerase b, and FEN1 are critical for repairing trapped PARP–DNAcomplexes. In summary, our study provides a new mechanistic foundation for the rational application ofPARP inhibitors in cancer therapy. Cancer Res; 72(21); 5588–99. 2012 AACR.
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advances in using parp inhibitors to treat cancer
BMC Medicine, 2012Co-Authors: Shivaani Kummar, Alice Chen, Ralph E Parchment, Robert J Kinders, Jay Ji, Joseph E Tomaszewski, James H DoroshowAbstract:The poly (ADP-ribose) polymerase (PARP) family of enzymes plays a critical role in the maintenance of DNA integrity as part of the base excision pathway of DNA repair. PARP1 is overexpressed in a variety of cancers, and its expression has been associated with overall prognosis in cancer, especially breast cancer. A series of new therapeutic agents that are potent inhibitors of the PARP1 and PARP2 isoforms have demonstrated important clinical activity in patients with breast or ovarian cancers that are caused by mutations in either the BRCA1 or 2 genes. Results from such studies may define a new therapeutic paradigm, wherein simultaneous loss of the capacity to repair DNA damage may have antitumor activity in itself, as well as enhance the antineoplastic potential of cytotoxic chemotherapeutic agents.