Author Manuscript Published OnlineFirst on August 8, 2017; DOI: 10.1158/1078-0432.CCR-17-0947 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Replication stress leading to apoptosis within the S-phase contributes to synergism between vorinostat and AZD1775 in HNSCC harboring high risk TP53 mutation Noriaki Tanaka1*, Ameeta A. Patel1*, Lin Tang1, Natalie L. Silver2, Antje Lindemann1, Hideaki Takahashi1, Roman Jaksik3, Xiayu Rao4, Nene N. Kalu5, Tseng-Cheng Chen7, Jiping Wang1, Mitchell J. Frederick8, Faye Johnson5, Frederico O. Gleber-Netto1, Siqing Fu9, Marek Kimmel3, Jing Wang4, Walter N. Hittelman6, Curtis R. Pickering1, Jeffrey N. Myers1*, Abdullah A. Osman1* *Authors contributed equally to this work 1Department of Head and Neck Surgery, The University of Texas MD Anderson Cancer Center, Houston, Texas. 2Department of Otolaryngology, Division of Head and Neck Oncologic Surgery, University of Florida College of Medicine, Gainesville, Florida. 3Department of Statistics, Rice University, Houston, Texas. 4Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas. 5Department of Thoracic/Head and Neck Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas. 6Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer, Houston, Texas. 1 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 8, 2017; DOI: 10.1158/1078-0432.CCR-17-0947 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 7Department of Otolaryngology, National Taiwan University Hospital, Taipei, Taiwan. 8Department of Otolaryngology/Head and Neck Surgery, Baylor College of Medicine, Houston, Texas. 9Department of Investigational Cancer Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, Texas. Funding: This work was supported by the National Institute of Health/NIDCR R01DE024601 (A.A. Osman and J.N. Myers), and The German Research Foundation (A. Lindemann). Corresponding Authors: Abdullah A. Osman, PhD, Assistant Professor, and Jeffrey N. Myers, MD, PhD, Professor of Surgery and Deputy Chair Department of Head and Neck Surgery, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030-4009 Phone: (713) 745-2667 Fax: (713) 794-4662 E-mail: [email protected]; and [email protected] Running Title: Vorinostat synergizes with AZD1775 in HNSCC Keywords: HNSCC cells, high-risk mutant p53, WEE1, AZD1775, vorinostat, DNA damage, 2 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 8, 2017; DOI: 10.1158/1078-0432.CCR-17-0947 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. CDK1, Chk1, RRM2, replication stress, Rad51, homologous recombination, CD31, apoptosis Abbreviations: HNSCC, head and neck squamous cell carcinoma; CDK1, cyclin dependent kinase 1; HDACs, Histone deacetylases; RRM2, ribonucleotide reductase enzyme-2; EdU, 5- ethynyl-2-deoxyuridine; DIC, differential interference contrast; HR, homologous recombination; RPPA, Reverse phase protein array Conflicts of Interest: The authors have no potential conflicts of interest to disclose Abstract Word Count: 250 Total Number of Figures and Tables: Six Total Number of Supplementary Figures and Tables: 8 3 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 8, 2017; DOI: 10.1158/1078-0432.CCR-17-0947 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Purpose: The cure rate for patients with advanced head and neck squamous cell carcinoma (HNSCC) remains poor due to resistance to standard therapy primarily consisting of chemoradiation. Since mutation of TP53 in HNSCC occurs in 60-80% of non-HPV associated cases and is in turn associated with resistance to these treatments, more effective therapies are needed. In this study, we evaluated the efficacy of a regimen combining vorinostat and AZD1775 in HNSCC cells with a variety of p53 mutations. Experimental Design: Clonogenic survival assays and an orthotopic mouse model of oral cancer were used to examine the in vitro and in vivo sensitivity of high-risk mutant p53 HNSCC cell lines to vorinostat in combination with AZD1775. Cell cycle, replication stress, homologous recombination (HR), live cell imaging, RNA-sequencing, and apoptosis analyses were performed to dissect molecular mechanisms. Results: We found that vorinostat synergizes with AZD1775 in vitro to inhibit growth of HNSCC cells harboring high-risk mutp53. These drugs interact synergistically to induce DNA damage, replication stress associated with impaired Rad51-mediated homologous recombination through activation of CDK1 and inhibition of Chk1 phosphorylation, culminating in an early apoptotic cell death during the S-phase of the cell cycle. The combination of vorinostat and AZD1775 inhibits tumor growth and angiogenesis in vivo in an orthotopic mouse model of oral cancer and prolongs animal survival. Conclusions: Vorinostat synergizes with AZD1775 in HNSCC cells with mutant p53 in vitro and in vivo. A strategy combining HDAC and WEE1 inhibition deserves further clinical investigation in patients with advanced HNSCC. 4 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 8, 2017; DOI: 10.1158/1078-0432.CCR-17-0947 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Translational Relevance Although the treatment of locally advanced head and neck cancer has evolved recently, the cure rate for patients with aggressive tumors and high-risk of failure remains poor due to resistance to standard chemoradiotherapy. Since TP53, the gene that encodes the p53 protein, is by far the most commonly mutated gene found in head and neck cancer and is, in turn, associated with treatment failure and poor survival outcomes, more effective therapies are urgently needed. In this study, we evaluate the efficacy of combining the HDAC inhibitor, vorinostat, and the WEE1 inhibitor, AZD1775, in head and neck tumor cells expressing high-risk mutant p53. Our data demonstrate the vulnerability of HNSCC to vorinostat and AZD1775 through induction of replication stress associated with decreased Rad51-mediated homologous recombination. Thus, we provide preclinical foundation for initiation of clinical trials using combination of HDACs and WEE1 inhibitors particularly for patients with advanced and recurrent metastatic HNSCC tumors. 5 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 8, 2017; DOI: 10.1158/1078-0432.CCR-17-0947 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Introduction Head and neck squamous cell carcinoma (HNSCC) is the seventh most common cancer worldwide (1). The cure rate for patients with advanced HNSCC remains poor due to resistance to standard therapy primarily consisting of cisplatin and radiation. Mutation of TP53 in HNSCC occurs in 60-80% of HPV-negative cases (2,3) and is associated with resistance to these treatments. Recently, we developed a novel computational approach termed evolutionary action (EAp53), which can stratify patients with tumors harboring TP53 mutations as high or low risk. Patients with high-risk TP53 mutations had the poorest survival outcomes, shortest time to the development of distant metastases, and increased resistance to cisplatin- based therapy (4,5). Since the group of tumors with this high-risk mutations show resistance to cisplatin or radiation, we are interested in the development of novel therapeutic approaches to overcome this resistance and improve treatment outcomes. WEE1 is a kinase that has been linked to DNA damage-induced S-phase replication stress and G2/M arrest, owing to its ability to inactivate cyclin dependent kinase 1 (CDK1) through phosphorylation of the Tyr15 residue (6-10). Recently, we demonstrated that the WEE1 kinase inhibitor, AZD1775 sensitizes HNSCC cells harboring high-risk TP53 mutations to cisplatin both in vitro and in vivo through induction of persistent DNA damage response associated with mitotic delay and subsequent senescence (11). Modulation of the acetylation status of histones and transcription factors is an essential mechanism for regulating gene expression (12,13). Histone acetylation is generally associated with elevated transcription, whereas deacetylated histones are often linked to repressed transcription (14). Histone deacetylases (HDACs) act enzymatically to remove the acetyl group 6 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 8, 2017; DOI: 10.1158/1078-0432.CCR-17-0947 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. from histones and silence gene expression (14). Elevated activities of histone deacetylases