Pharmacological Inhibition of PARP6 Triggers Multipolar Spindle Formation and Elicits Therapeutic Effects in Breast Cancer Zebin Wang1, Shaun E
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Published OnlineFirst October 8, 2018; DOI: 10.1158/0008-5472.CAN-18-1362 Cancer Translational Science Research Pharmacological Inhibition of PARP6 Triggers Multipolar Spindle Formation and Elicits Therapeutic Effects in Breast Cancer 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 Abstract PARP proteins represent a class of post-translational mod- subset of breast cancer cells in vitro and antitumor effects in vivo. ification enzymes with diverse cellular functions. Targeting In addition, Chk1 was identified as a specific substrate of PARPs has proven to be efficacious clinically, but exploration PARP6 and was further confirmed by enzymatic assays and of the therapeutic potential of PARP inhibition has been by mass spectrometry. Furthermore, when modification of limited to targeting poly(ADP-ribose) generating PARP, Chk1 was inhibited with AZ0108 in breast cancer cells, we including PARP1/2/3 and tankyrases. The cancer-related func- observed marked upregulation of p-S345 Chk1 accompanied tions of mono(ADP-ribose) generating PARP, including by defects in mitotic signaling. Together, these results establish PARP6, remain largely uncharacterized. Here, we report a proof-of-concept antitumor efficacy through PARP6 inhibi- novel therapeutic strategy targeting PARP6 using the first tion and highlight a novel function of PARP6 in maintaining reported PARP6 inhibitors. By screening a collection of PARP centrosome integrity via direct ADP-ribosylation of Chk1 and compounds for their ability to induce mitotic defects, we modulation of its activity. uncovered a robust correlation between PARP6 inhibition and induction of multipolar spindle (MPS) formation, which was Significance: These findings describe a new inhibitor of phenocopied by PARP6 knockdown. Treatment with AZ0108, PARP6 and identify a novel function of PARP6 in regulating a PARP6 inhibitor with a favorable pharmacokinetic profile, activation of Chk1 in breast cancer cells. Cancer Res; 78(23); potently induced the MPS phenotype, leading to apoptosis in a 6691–702. Ó2018 AACR. Introduction spontaneous tumorigenesis, although the typical consequence of multipolar mitosis is often detrimental to dividing cells (5). The presence of supernumerary centrosomes is a common To ensure successful mitosis, aneuploid cancer cells can evolve feature of solid malignant tumors, differentiating them from several mechanisms to avoid chromosome segregation errors (6). normal cells. Centrosomes normally function as the main micro- One major mechanism involves clustering and assembling mul- tubule-organizing centers (MTOC) and play an essential role tiple centrosomes into pseudo-bipolar spindles through a tightly during cellular mitosis (1). Evidence suggests that centrosome regulated process (6, 7). Disruption of this process results in abnormalities contribute to a high degree of aneuploidy in cancer multipolar mitosis and has been shown to be a therapeutic cells and are associated with advanced tumor grade (2–4). A recent strategy to specifically kill cancer cells with amplified centrosomes report indicates centrosome amplification is sufficient to promote 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 1 Oncology, IMED Biotech Unit, AstraZeneca R&D Boston, Waltham, Massachu- (10–12). Through unbiased siRNA screens, members of the setts. 2Institute of Chemical Biology, Department of Chemistry, Imperial College therapeutically tractable PARPs enzyme family have also been London, London, United Kingdom. implicated in centrosome clustering and bipolar spindle forma- Note: Supplementary data for this article are available at Cancer Research tion, providing the preliminary evidence to further investigate Online (http://cancerres.aacrjournals.org/). the pharmacological inhibition of PARPs to specifically perturb Z. Wang and S.E. Grosskurth contributed equally to this article and share first mitosis in cancer cells (8, 13). authorship. PARPs are a family of 17 enzymes that catalyze the transfer of þ Corresponding Author: Huawei Chen, AstraZeneca R&D Boston, 35 Gatehouse the ADP-ribose from NAD to post-translationally modify accep- Drive, Waltham, MA, 02451. Phone: 781-839-4417; Fax. 781-839-4200; E-Mail: tor proteins. Depending on their catalytic activity, PARPs can be [email protected] further divided into poly(ADP-ribose) generating PARPs such as doi: 10.1158/0008-5472.CAN-18-1362 PARPs 1-5, catalytically inactive PARPs such as PARPs 9 and 13, Ó2018 American Association for Cancer Research. and mono(ADP-ribose) generating PARPs such as PARPs 6-8, www.aacrjournals.org 6691 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst October 8, 2018; DOI: 10.1158/0008-5472.CAN-18-1362 Wang et al. 10-12, and 14-15 (14, 15). The post-translational modification alone positive control—negative control). IC50 values were cal- of substrate proteins by PARPs has been demonstrated, for culated by fitting the percentage inhibition to a four-parameter example, by PARP1-3 to regulate numerous signaling cascades, logistic regression equation with an AstraZeneca in-house calcu- including DNA damage response, chromatin remodeling, and lation engine. transcriptional regulation, as well as by PARP5a-5b for telomere maintenance, spindle assembly, vesicular movement, and degra- Immunofluorescent multipolar spindle assay dation of the beta-catenin destruction complex (16, 17). Given Indicated cell lines were plated in 96-well plates at 7,000 cells that PARPs have functional roles in mitosis and that PARP1/2 per well and incubated at 37C overnight. The cells were treated inhibition is a successful therapeutic approach to treat homolo- with compounds ranging from 0 to 11 mmol/L for 48 hours. gous recombination defective tumors in the clinic (18, 19), The cells were fixed with 4% formaldehyde at room temperature this prompted us to perform a cell-based MPS phenotypic for 10 minutes followed with ice-cold methanol fixation for screen with compounds that have structural similarity to PARP another 10 minutes. After four washes in PBS, the cells were inhibitor scaffolds. incubated in blocking buffer for 1 hour at room temperature to Here, we report the characterization of novel small molecule reduce non-specific binding. The cells were labeled with primary PARP inhibitors that induce MPS formation in cancer cells and antibodies, 1:2,000 dilution of anti-cyclin B antibody (Thermo provide evidence that this phenotype is due to PARP6 inhibition. Fisher) and 1:4,000 dilution of anti-pericentrin antibody PARP6 is a mono (ADP-ribose) generating PARP with little (Abcam), for 16 hours at 4C. After washing with PBS four times, biological characterization. Pharmacological inhibition of PARP6 the cells were labeled with secondary antibodies, 1:200 Alexa in breast cancer cells using AZ0108 (20), an optimized inhibitor Fluor 488 anti-rabbit antibody and Alexa Fluor 594 anti-mouse developed from phenotypic screen hits, resulted in MPS forma- antibody, for 1 hour at room temperature. After washing with PBS tion, impaired cell growth, and induction of apoptosis in vitro and twice, the cells were stained with Hoechst dye for 10 minutes at in vivo. Using a high-density protein array–based ADP-ribosyla- room temperature. The cells were washed twice with PBS and then tion assay, we identified Chk1 kinase within a subset of proteins images were acquired by ImageXpress Micro (Molecular Devices) involved in regulating centrosome function that were enriched as or Operatte (PerkinElmer) High Content Screening System. direct PARP6 substrates. We confirmed PARP6 ADP-ribosylates Cyclin B label was used for scoring the mitotic cells and pericen- Chk1 directly and demonstrated Chk1 S345 phosphorylation was trin was used for scoring the centrosome number in each mitotic significantly upregulated upon PARP6 inhibition, which was cell. The percentage of increase of mitotic cells with greater than accompanied by de-activation of other mitotic proteins during 2 centrosomes compared with the DMSO control was used to the G2–M transition. Taken together, our studies demonstrate a calculate EC50 values. For CHK1 and pericentrin staining, CHK1 critical role for PARP6 in ensuring the integrity of mitosis and antibody (1:100, Cell Signaling Technology) and pericentrin provide preclinical proof-of-concept for inhibiting PARP6 as a (Abcam) antibodies were used to stain HCC1806 cells after novel cancer therapeutic strategy. AZ0108 treatment following the same protocol. Images were taken with Operetta (PerkinElmer) High Content Screening Sys- tem, and CHK1 intensity was analyzed with Harmony analysis Materials and Methods software (PerkinElmer). Chemicals and cell lines AZD2281/olaparib, AZ9482, and AZ0108 were synthesized Breast cancer cell line proliferation assays by AstraZeneca (20–22) and diluted in dimethyl sulfoxide Cell lines were sourced as previously described (23, 24) and (Sigma-Aldrich). Majority of cell lines were purchased from were cultured