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Pharmacological Inhibition of PARP6 Triggers Multipolar Spindle Formation and Elicits Therapeutic Effects in Breast Cancer

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Pharmacological Inhibition of PARP6 Triggers Multipolar Spindle Formation and Elicits Therapeutic Effects in Breast Cancer Author Manuscript Published OnlineFirst on October 8, 2018; DOI: 10.1158/0008-5472.CAN-18-1362 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. TITLE: Pharmacological inhibition of PARP6 triggers multipolar spindle formation and elicits therapeutic effects in breast cancer AUTHORS AND AFFILIATIONS: Zebin Wang1*, Shaun E. Grosskurth1*, Tony Cheung1, Philip Petteruti1, Jingwen Zhang1, Xin Wang1, Wenxian Wang1, Farzin Gharahdaghi1, Jiaquan Wu1, Nancy Su1, Ryan T. Howard2, Michele Mayo1, Dan Widzowski1, David A. Scott1, Jeffrey W. Johannes1, Michelle L. Lamb1, Deborah Lawson1, Jonathan R. Dry1, Paul D. Lyne1, Edward W. Tate2, Michael Zinda1, Keith Mikule1, Stephen E. Fawell1, Corinne Reimer1 and Huawei Chen1 Oncology, IMED Biotech Unit, AstraZeneca R&D Boston, Waltham, MA, 02451, USA1 Institute of Chemical Biology, Department of Chemistry, Imperial College London, London SW7 2AZ, UK2 RUNNING TITLE: Targeting PARP6 in breast cancer KEYWORDS: PARP6; PARP inhibitors; PARylation; ADP‐ribosylation; Chk1, multipolar spindle formation (MPS) Significance: Findings describe a new inhibitor of PARP6 and identify a novel function of PARP6 in regulating activation of Chk1 in breast cancer cells CORRESPONDING AUTHOR: Huawei Chen, Oncology, IMED Biotech Unit, AstraZeneca R&D Boston, Waltham, MA, 02451 Tel. (781)‐839‐4417; Fax. (781)‐839‐4500; E‐Mail: [email protected] * These authors contributed equally to this work and share first authorship. POTENTIAL CONFLICTS OF INTEREST: 1 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 8, 2018; DOI: 10.1158/0008-5472.CAN-18-1362 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. R.T. Howard and E. W. Tate are collaborators of AstraZeneca and receive funding through the EPSRC Centre for Doctoral Training in Chemical Biology (Imperial College London) and the rest of the authors were employees and/or shareholders of AstraZeneca. 2 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 8, 2018; DOI: 10.1158/0008-5472.CAN-18-1362 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. ABSTRACT PARP (poly ADP‐ribose polymerase) proteins represent a class of post‐translational modification enzymes with diverse cellular functions. Targeting PARPs has proven to be efficacious clinically, but exploration of the therapeutic potential of PARP inhibition has been limited to targeting poly(ADP‐ribose) (PAR) generating PARP including PARP1/2/3 and tankyrases. The cancer‐related functions of mono(ADP‐ribose) (MAR) generating PARP, including PARP6, remain largely uncharacterized. Here, we report a novel therapeutic strategy targeting PARP6 using the first reported PARP6 inhibitors. By screening a collection of PARP compounds for their ability to induce mitotic defects, we uncovered a robust correlation between PARP6 inhibition and induction of multipolar spindle (MPS) formation, which was phenocopied by PARP6 knockdown. Treatment with AZ0108, a PARP6 inhibitor with a favorable pharmacokinetic profile, potently induced the MPS phenotype, leading to apoptosis in a subset of breast cancer cells in vitro and antitumor effects in vivo. In addition, Chk1 was identified as a specific substrate of PARP6 and was further confirmed by enzymatic assays and by mass spectrometry. Furthermore, when modification of Chk1 was inhibited with AZ0108 in breast cancer cells, we observed marked upregulation of p‐S345 Chk1 accompanied by defects in mitotic signaling. Together these results establish proof‐of‐concept antitumor efficacy through PARP6 inhibition and highlight a novel function of PARP6 in maintaining centrosome integrity via direct ADP‐ribosylation of Chk1 and modulation of its activity. 3 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 8, 2018; DOI: 10.1158/0008-5472.CAN-18-1362 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. INTRODUCTION The presence of supernumerary centrosomes is a common feature of solid malignant tumors, differentiating them from normal cells. Centrosomes normally function as the main microtubule‐organizing centers (MTOCs) and play an essential role during cellular mitosis [1]. Evidence suggests that centrosome abnormalities contribute to a high degree of aneuploidy in cancer cells and are associated with advanced tumor grade [2‐4]. A recent report indicates centrosome amplification is sufficient to promote spontaneous tumorigenesis, although the typical consequence of multipolar mitosis is often detrimental to dividing cells [5]. To ensure successful mitosis, aneuploid cancer cells can evolve several mechanisms to avoid chromosome segregation errors [6]. One major mechanism involves clustering and assembling multiple centrosomes into pseudo‐bipolar spindles through a tightly regulated process [6, 7]. Disruption of this process results in multipolar mitosis and has been shown to be a therapeutic strategy to specifically kill cancer cells with amplified centrosomes while sparing normal cells [8, 9]. The tubulin‐stabilizing agent griseofulvin and its derivative GF‐15, as well as the HSET/KIFC1 inhibitor AZ82, have been reported to induce formation of multipolar spindles (MPS) to selectively kill cancer cells [10‐12]. Through unbiased siRNA screens, members of the therapeutically tractable poly(ADP‐ribose) polymerases (PARPs) enzyme family have also been implicated in centrosome clustering and bipolar spindle formation, providing the preliminary evidence to further investigate the pharmacological inhibition of PARPs to specifically perturb mitosis in cancer cells [8, 13]. PARPs are a family of 17 enzymes that catalyze the transfer of the ADP‐ribose from NAD+ to post‐translationally modify acceptor proteins. Depending on their catalytic activity, PARPs can 4 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 8, 2018; DOI: 10.1158/0008-5472.CAN-18-1362 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. be further divided into poly(ADP‐ribose) generating PARPs such as PARPs 1‐5, catalytically inactive PARPs such as PARPs 9 and 13, and mono(ADP‐ribose) generating PARPs such as PARPs 6‐8, 10‐12, and 14‐15 [14, 15]. The post‐translational modification of substrate proteins by PARPs has been demonstrated, for example, by PARP1‐3 to regulate numerous signaling cascades including DNA damage response, chromatin remodeling, and transcriptional regulation, as well as by PARP5a‐5b for telomere maintenance, spindle assembly, vesicular movement, and degradation of the beta‐catenin destruction complex [16, 17]. Given that PARPs have functional roles in mitosis and that PARP1/2 inhibition is a successful therapeutic approach to treat homologous recombination defective tumors in the clinic [18, 19], this prompted us to perform a cell‐based MPS phenotypic screen with compounds that have structural similarity to PARP inhibitor scaffolds. Here, we report the characterization of novel small molecule PARP inhibitors that induce MPS formation in cancer cells and provide evidence that this phenotype is due to PARP6 inhibition. PARP6 is a mono (ADP‐ribose) generating PARP with little biological characterization. Pharmacological inhibition of PARP6 in breast cancer cells using AZ0108 [20], an optimized inhibitor developed from phenotypic screen hits, resulted in MPS formation, impaired cell growth, and induction of apoptosis in vitro and in vivo. Using a high‐density protein array‐based ADP‐ ribosylation assay, we identified Chk1 kinase within a subset of proteins involved in regulating centrosome function that were enriched as direct PARP6 substrates. We confirmed PARP6 ADP‐ ribosylates Chk1 directly and demonstrated Chk1 S345 phosphorylation was significantly upregulated upon PARP6 inhibition, which was accompanied by de‐activation of other mitotic proteins during the G2‐M transition. Taken together, our studies demonstrate a critical role for 5 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 8, 2018; DOI: 10.1158/0008-5472.CAN-18-1362 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. PARP6 in ensuring the integrity of mitosis and provide preclinical proof‐of‐concept for inhibiting PARP6 as a novel cancer therapeutic strategy. MATERIALS AND METHODS Chemicals and cell lines AZD2281/olaparib, AZ9482, and AZ0108 were synthesized by AstraZeneca [20‐22] and diluted in dimethyl sulfoxide (Sigma‐Aldrich). Majority of cell lines were purchased from ATCC or some were obtained from DSMZ and cultured according to providers’ instructions (detailed information in Supplemental Table S1). Cell lines were generally maintained at low passages before use. PARP protein enzymatic assays All assays were performed following the BPS PARP assay kit protocols. In brief, PARP enzymatic reactions were conducted in duplicate at room temperature for 1 hour in a 96 well plate
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