Abstract 1260: Polymerase kappa determines the sensitivity of MTH1 inhibitors to cisplatin-resistant cell
Kumar SanjivHelge GadSean G. RuddRachel M. HurleyPatric HerrJosé Manuel Calderón‐MontañoOliver MortusewiczTobias KoolmeisterSylvain JaquesEstefanía Burgos‐MorónAndreas HöglundTe‐Chang LeeMartin ScobieScott H. KaufmannS. John WerohaUlrika Warpman BerglundAndrea Wahner HendricksonThomas Helleday
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Abstract Resistance is one the main reason for overall decrease in survival of cancer patient treated with cisplatin in different types of cancer. Cisplatin kills cancer cells by various mechanisms, but mainly through formation of inter- and intra stand crosslinks of DNA. Different types of translesion polymerase including Polymerase kappa (POLK) are involved in repair of DNA lesions. We observed high expression levels of POLK in cisplatin resistant bladder and ovarian cancer cells compared to parental cells. Due to its low proof-reading activity POLK can incorporate 8-oxo-dGTP into DNA. The MTH1 protein (Nudix hydrolase- NUDT1) sanitizes oxidized dNTP pools to prevent incorporation of damaged bases during DNA replication. Recently we have generated MTH1 inhibitors that damage the DNA and induce cancer specific cell death through incorporation of more oxidized dNTPs. We found cisplatin resistant bladder cancer cells (NTUB1/P) were more sensitive to MTH1 inhibitors in comparison to parental NTUB1 cells. As POLK is involved in incorporation of 8-oxo-dGTP into DNA, we hypothesized that high expression levels of POLK in cisplatin resistant cells make them more sensitive to MTH1 inhibitors as more 8-oxo-dGTP would be incorporated into DNA, resulting in more DNA damage and cell death in comparison to parental cells. Indeed, we observes higher induction of cleaved-PARP, γH2AX, cleaved-Caspase 3 and more annexin v positive cells in cisplatin resistant NTUB1/P cells in comparison to parental NTUB1 cells upon treatment with MTH1 inhibitors. MTH1 inhibitor also significantly delays the NTUB1/P xenograft tumor growth in comparison to vehicle treatment in immunosuppressive mice. Knocking down POLK in cisplatin resistant NTUB1/P cells by siRNA resulted in decreased incorporation of 8-oxo-dGTP and sensitivity to MTH1 inhibitors compared to non target control cells. Overexpression of POLK in NTUB1 and NTUB1/P cells results in further sensitization to MTH1 inhibitors. In conclusion elevated levels of POLK in cisplatin resistance cells determines increased sensitivity towards MTH1 inhibitors. Thus MTH1 inhibitors can be a potential promising therapy for the treatment of cisplatin resistant tumors in patients. Citation Format: Kumar Sanjiv, Helge Gad, Sean Rudd, Rachel Hurley, Patric Herr, José Manuel Calderón Montaño, Oliver Mortusewicz, Tobias Koolmeister, Sylvain Jaques, Estefanía Burgos Morón, Andreas Hoglund, Te-Chang Lee, Martin Scobie, Scott Kaufmann, John Weroha, Ulrika Warpman Berglund, Andrea Wahner Hendrickson, Thomas Helleday. Polymerase kappa determines the sensitivity of MTH1 inhibitors to cisplatin-resistant cell. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1260.Keywords:
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Abstract We have characterized the biochemical association of two DNA damage‐dependent enzymes, poly(ADP‐ribose) polymerase‐1 (PARP‐1) [EC 2.4.2.30] and DNA polymerase β (pol β ) [2.7.7.7]. We reproducibly observed that pol β is an efficient covalent target for ADP‐ribose polymers under standard conditions of enzymatically catalyzed ADP‐ribosylation of β NAD + as a substrate. The efficiency of poly(ADP‐ribosyl)ation increased as a function of the pol β and β NAD + concentrations. To further characterize the molecular interactions between these two unique polymerases, we also subjected human recombinant PARP‐1 to peptide‐specific enzymatic degradation with either caspase‐3 or caspase‐7 in vitro. This proteolytic treatment, commonly referred to as ‘a hallmark of apoptosis’, generated the two physiologically relevant peptide fragments of PARP‐1, e.g. , a 24‐kDa amino‐terminus and an 89‐kDa carboxy‐terminal domain. Interestingly, co‐incubation of the two peptide fragments of PARP‐1 with full‐length pol β resulted in their domain‐specific molecular association as determined by co‐immunoprecipitation and reciprocal immunoblotting. Therefore, our data strongly suggest that, once PARP‐1 is proteolyzed by either caspase‐3 or caspase‐7 during cell death, the specific association of its apoptotic fragments with DNA repair enzymes, such as pol β , may serve a regulatory molecular role in the execution phase of apoptosis.
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Poly(ADP-ribose) polymerase-1 consumes NAD+ to catalyze poly(ADP-ribosyl)ation of target proteins, which modulates various biological functions. However, excessive poly(ADP-ribose) polymerase-1 activation mediates oxidative cell death. Our recent studies have indicated that NAD+ can enter into astrocytes to prevent poly(ADP-ribose) polymerase-1 cytotoxicity. In this study, we show that NADH can also enter into astrocytes, which can significantly decrease poly(ADP-ribose) polymerase-1-induced astrocyte death even when applied 3–4 h after poly(ADP-ribose) polymerase-1 activation. The protective effects can be produced by 10 μM NADH, which is significantly lower than that required for NAD+ to be protective. These results provide novel information suggesting that NADH can be used for decreasing poly(ADP-ribose) polymerase-1 toxicity, and extracellular NADH can enter into astrocytes to influence cellular functions.
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Single-strand breaks are the commonest lesions arising in cells, and defects in their repair are implicated in neurodegenerative disease. One of the earliest events during single-strand break repair (SSBR) is the rapid synthesis of poly(ADP-ribose) (PAR) by poly(ADP-ribose) polymerase (PARP), followed by its rapid degradation by poly(ADP-ribose) glycohydrolase (PARG). While the synthesis of poly(ADP-ribose) is important for rapid rates of chromosomal SSBR, the relative importance of poly(ADP-ribose) polymerase 1 (PARP-1) and PARP-2 and of the subsequent degradation of PAR by PARG is unclear. Here we have quantified SSBR rates in human A549 cells depleted of PARP-1, PARP-2, and PARG, both separately and in combination. We report that whereas PARP-1 is critical for rapid global rates of SSBR in human A549 cells, depletion of PARP-2 has only a minor impact, even in the presence of depleted levels of PARP-1. Moreover, we identify PARG as a novel and critical component of SSBR that accelerates this process in concert with PARP-1.
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Metabolic stability
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ADP-RIBOSE POLYMER METABOLISM POLY ADP-RIBOSE POLYMERASE: STRUCTURE AND FUNCTION MOLECULAR AND GENETIC APPROACHES: BIOLOGICAL SIGNIFICANCE OF POLY ADP-RIBOSYLATION REACTIONS POLY ADP-RIBOSE POLYMERASE AND CELL DEATH ACTIVATION OF POLY ADP-RIBOSE POLYMERASE IN THE PATHOGENESIS OF ISCHAEMIA REPERFUSION INJURY NEW POLY ADP-RIBOSE POLYMERASE INHIBITORS FOR RADIOTHERAPY AND CHEMOTHERAPY OF CANCER PERSPECTIVES
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Poly( ADP-ribose) polymerase 1 (PARP-1) plays an essential role in DNA repair and maintenance of homeostasis and genome stability. Increased PARP-1 activity has been reported in various forms of cancer. Thus the absence of PARP-1 or the use of its inhibitors depresses DNA repair function, sensitizes cancer cells to DNA damage and enhances the therapeutic effect of radio-or chemotherapy. PARP-1 is potentially a therapeutic target of malignant tumors.
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Poly(ADP-ribose) polymerases; DNA repair enzymes; Neoplasms; Poly (ADP-ribose) polymerases inhibitors
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