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CDKN2A/P16 Deletion in Head and Neck Cancer Cells Is Associated with Cdk2 Activation, Replication Stress, and Vulnerability to Chk1 Inhibition

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CDKN2A/P16 Deletion in Head and Neck Cancer Cells Is Associated with Cdk2 Activation, Replication Stress, and Vulnerability to Chk1 Inhibition Author Manuscript Published OnlineFirst on December 11, 2017; DOI: 10.1158/0008-5472.CAN-17-2802 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. CDKN2A/p16 deletion in head and neck cancer cells is associated with Cdk2 activation, replication stress, and vulnerability to Chk1 inhibition Mayur A. Gadhikar1, Jiexin Zhang2, Li Shen2, Xiayu Rao2, Jing Wang2, Mei Zhao1, Nene N. Kalu3, Faye M. Johnson3, Lauren A. Byers3, John Heymach3, Walter N. Hittelman6, Durga Udayakumar4,5, Raj K. Pandita4, Tej K. Pandita4, Curtis R. Pickering1, Abena B. Redwood7, Helen Piwnica-Worms7, Katharina Schlacher8 , Mitchell J. Frederick9*, Jeffrey N. Myers1*. 1, Department of Head and Neck Surgery, University of Texas MD Anderson Cancer Center, Houston, Texas, USA 2 Department of Biostatistics and Computational Biology, University of Texas MD Anderson Cancer Center, Houston, Texas, USA 3 Thoracic Head and Neck Medical Oncology, University of Texas MD Anderson Cancer Center, Houston, Texas, USA 4 Department of Radiation Oncology, Institute for Academic Medicine, Houston Methodist 6 Department of Experimental Therapeutics, University of Texas, MD Anderson Cancer Center, University of Texas MD Anderson Cancer Center, Houston, TX , USA 7 Department of Experimental Radiation Oncology, University of Texas MD Anderson Cancer Center, Houston, TX, USA 8 Department of Cancer Biology, University of Texas MD Anderson Cancer Center, Houston, TX, USA 9 Department of Otolaryngology - Head and Neck Surgery, Baylor College of Medicine, Houston, TX, USA Present address: 5Department of Radiology, University of Texas Southwestern Medical Center, Dallas, TX, USA Running Title: p16 deletion associates with Chk1i sensitivity in HNSCC Keywords: CDKN2A/p16 deletions, prexasertib, replication stress, HNSCC Grant support: This work was supported by NIH/NIDCR grants P50 CA97007, R01 DE024601, and R01 DE014613 to J.N. Myers (PI) and 1U01DE025181 to J. N. Myers (PI) and M.J. Frederick (PI). T.K.Pandita was supported by NIH grants CA129537 and GM109768. * Corresponding Authors: Dr. Jeffrey N. Myers, MD, PhD, FACS, Professor and Director of Research, Department of Head and Neck Surgery, MD Anderson Cancer Center, PO Box 301402, Unit 1445, Houston, TX 77230-1402, USA. Phone: (713) 745-2667; Fax: (713) 794-4662; E-mail: [email protected] Dr. Mitchell J Frederick, PhD, Associate Professor, Department of Otolaryngology - Head and Neck Surgery, Baylor College of Medicine, 6501 Fannin Street, Room NA514, Houston, TX 77030, USA. Phone: (713) 7798-1263; E-mail: [email protected] 1 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on December 11, 2017; DOI: 10.1158/0008-5472.CAN-17-2802 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Checkpoint kinase inhibitors (CHKi) exhibit striking single agent activity in certain tumors, but the mechanisms accounting for hypersensitivity are poorly understood. We screened a panel of 49 established human head and neck squamous cell carcinoma (HNSCC) cell lines and report that nearly 20% are hypersensitive to CHKi monotherapy. Hypersensitive cells underwent early S- phase arrest at drug doses sufficient to inhibit greater than 90% of Chk1 activity. Reduced rate of DNA replication fork progression and chromosomal shattering were also observed, suggesting replication stress as a root causative factor in CHKi hypersensitivity. To explore genomic underpinnings of CHKi hypersensitivity, comparative genomic analysis was performed between hypersensitive cells and cells categorized as least sensitive because they showed drug IC50 value greater than the cell panel median and lacked early S phase arrest. Novel association between CDKN2A/p16 copy number loss, Cdk2 activation, replication stress and hypersensitivity of HNSCC cells to CHKi monotherapy was found. Restoring p16 in cell lines harboring CDKN2A/p16 genomic deletions alleviated Cdk2 activation and replication stress, attenuating CHKi hypersensitivity. Taken together, our results suggest a biomarker-driven strategy for selecting HNSCC patients who may benefit the most from CHKi therapy. 2 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on December 11, 2017; DOI: 10.1158/0008-5472.CAN-17-2802 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Introduction Therapeutic advances in head and neck squamous cell carcinoma (HNSCC) have been stymied by resistance to conventional therapies and lack of molecular targets. Inhibition of critical components in the DNA Damage response (DDR) pathway, such as cell cycle checkpoint kinases (Chk1/2), potentiates genotoxic therapies in cancer cell lacking wild type p53 function [1-5]. While early stage clinical trials are currently exploring the utility of combining Chk inhibitors with chemotherapy in various cancers, single agent sensitivity to chk1 inhibition has been sporadically reported for a limited number of tumor cell lines derived from various cancers [4, 6-9]. Whether susceptibility to Chk1 inhibition monotherapy is confined to a few cancer types or could represent a broader phenomenon remains unclear. Importantly, the genomic or molecular factor(s) influencing susceptibility to Chk1 inhibition monotherapy in any given tumor type has remained elusive. In melanoma cells, high basal levels of DNA damage were associated with vulnerability to Chk1 inhibition [8]; whereas in ovarian tumors, subtypes bearing high endogenous Chk1 activity exhibited greatest sensitivity to Chk1 inhibition [9]. Elevated levels of endogenous DNA damage resulting from transgenic overexpression of c-myc in murine lymphoma models has also been associated with Chk1 inhibition sensitivity [7]. More recently, sensitivity to Chk1 inhibition in tumor cells has been ascribed to Cdk2 activation in S phase and associated with an elevation of DNA damage[10]. In all of these studies, compared to checkpoint kinase 1 inhibitor (Chk1i) resistant cancers, much higher levels of DNA damage arose in susceptible tumors post Chk1 inhibition, which was sometimes associated with an abnormally long S-phase [4, 7-9]. These findings suggest that Chk1 inhibition sensitivity could be related to the induction of replication stress. In support of this idea, Chk1 plays a vital role during S-phase in the suppression of abnormal replication origin firings and in the maintenance of fork stability to aid proper completion of DNA replication [11, 12]. Tumor cell resistance to Chk1 inhibition can be overcome by artificial induction of replication stress which triggers replication checkpoint and promotes cell reliance on Chk1 function [13]. Even so, it has not been firmly established why certain tumor cells are innately sensitive to Chk1 inhibition and others are not. Moreover, direct experimental demonstration of replication stress in tumor cells acutely sensitive to Chk1 inhibition has been lacking. Instead, most studies have relied on 3 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on December 11, 2017; DOI: 10.1158/0008-5472.CAN-17-2802 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. surrogate markers linked to replication stress or focused more on downstream events preceding cell death, such as replication catastrophe, caspase activation, apoptosis, and abnormal mitosis [4, 7-9]. In HNSCC, the efficacy of Chk inhibition monotherapy was reported in a Phase I dose escalation study (Cancer Res 2013; 73(8 Suppl): Abstract nr LB-200). We screened 49 HNSCC cell lines for their sensitivity to a Chk inhibitor, prexasertib (LY2606368 mesylate monohydrate) and found that 9 out of 49 cell lines (18%) were hypersensitive and exhibited an early S-phase cell cycle arrest at or below drug doses that inhibited greater than 90% of Chk1 activity. Knockdown studies with siRNA revealed that Chk1, not Chk2, mediates survival in hypersensitive cells. Chk1 inhibition also led to reduction in the replication fork progression rate, DNA breaks, and chromosomal shattering, directly demonstrating replication stress as the underlying upstream cause of acute hypersensitivity to Chk1 inhibition in a subset of HNSCC. Importantly, through comprehensive integrated genomic analysis, we identify high level copy number losses, low RNA and protein expression of CDKN2A/p16 in hypersensitive cells and propose it as a major deterministic factor associated with Cdk2 activation, replication stress and hypersensitivity to Chk1 inhibition in HNSCC. Further validation of these findings could enable the possibility of treatment selection of patients with CDKN2A/p16 deletion for treatment with Chk1 inhibition. Materials and Methods Please refer to the supplemental data for detailed experimental procedures on immunoblotting, DNA fiber assay, and exome-sequencing and mutational calling. Cell Culture and Reagents: HNSCC cell lines for this study were obtained from an established cell repository in the laboratory of Dr. Jeffrey N. Myers (University of Texas MD Anderson Cancer Center, Houston, TX) under approved institutional protocols. HNSCC cells were authenticated against the parental cell lines using short-tandem repeat analysis
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