Strategies for Targeted Therapy in Head and Neck Squamous Cell Carcinoma Using WEE1 Inhibitor AZD1775 | Clinical Pharmacy and Pharmacology | JAMA Otolaryngology–Head & Neck Surgery | JAMA Network
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Figure 1.  Markers of DNA Damage and Replication Stress After AZD1775 Therapy in a p53 Wild-Type vs Knockdown Isogenic Cell Line Model
Markers of DNA Damage and Replication Stress After AZD1775 Therapy in a p53 Wild-Type vs Knockdown Isogenic Cell Line Model

A, Western blot of UM-SCC-74A demonstrating knockdown of the p53 protein (UM-SCC-74A p53kd). B, Flow cytometry of UM-SCC-74A pBabe vector control (UMSCC-74A pB) and UM-SCC-74A p53kd showing staining of phosphorylated histone H2AX (γ-H2AX) with WEE1 inhibitor treatment (first columns); corresponding immunofluorescent staining of cells with phosphorylated replication protein A–positive (p-RPA+) (green) and 5-ethynyl-2-deoxyuridine–positive (EdU+) (red) (second columns). DAPI indicates 4′,6-diamidino-2-phenylindole.

Figure 2.  Flow Cytometry After 24-Hour Treatment With AZD1775
Flow Cytometry After 24-Hour Treatment With AZD1775

Premature mitosis (histone H3S10P+ [HH3S10P+], <4N DNA content) is seen only in the p53-deficient line. DAPI indicates 4′,6-diamidino-2-phenylindole.

1.
Moser  R, Xu  C, Kao  M,  et al.  Functional kinomics identifies candidate therapeutic targets in head and neck cancer.  Clin Cancer Res. 2014;20(16):4274-4288.PubMedGoogle ScholarCrossref
2.
Aarts  M, Sharpe  R, Garcia-Murillas  I,  et al.  Forced mitotic entry of S-phase cells as a therapeutic strategy induced by inhibition of WEE1.  Cancer Discov. 2012;2(6):524-539.PubMedGoogle ScholarCrossref
3.
Domínguez-Kelly  R, Martín  Y, Koundrioukoff  S,  et al.  Wee1 controls genomic stability during replication by regulating the Mus81-Eme1 endonuclease.  J Cell Biol. 2011;194(4):567-579.PubMedGoogle ScholarCrossref
4.
Sirbu  BM, Couch  FB, Feigerle  JT, Bhaskara  S, Hiebert  SW, Cortez  D.  Analysis of protein dynamics at active, stalled, and collapsed replication forks.  Genes Dev. 2011;25(12):1320-1327.PubMedGoogle ScholarCrossref
5.
clinicaltrials.gov. WEE1 Inhibitor MK-1775, Docetaxel, and Cisplatin Before Surgery in Treating Patients With Borderline Resectable Stage III-IVB Squamous Cell Carcinoma of the Head and Neck. NCT02508246. https://clinicaltrials.gov/ct2/show/NCT02508246. Accessed January 25, 2016.
Research Letter
June 2017

Strategies for Targeted Therapy in Head and Neck Squamous Cell Carcinoma Using WEE1 Inhibitor AZD1775

Author Affiliations
  • 1Department of Otolaryngology–Head and Neck Surgery, University of Washington, Seattle
  • 2Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington
  • 3Department of Pathology, University of Washington, Seattle
JAMA Otolaryngol Head Neck Surg. 2017;143(6):631-633. doi:10.1001/jamaoto.2016.4563

We previously reported on the use of the high-throughput RNA interference kinome screen to identify genes required for the survival of p53 mutant head and neck squamous cell carcinoma (HNSCC) cells but not that of normal cells.1 We identified p53 synthetic lethal interactions with several G2/M checkpoint regulators, including WEE1 and CHK1, and found HNSCC killing by the WEE1 inhibitor AZD1775.1 During DNA damage, WEE1 initiates G2/M arrest via inhibition of cyclin-dependent kinase 1 to prevent mitosis and allow DNA repair. WEE1 also stabilizes DNA replication forks during S phase via a cyclin-dependent kinase 2 mechanism. Thus, WEE1 inhibition likely leads to unrestrained or premature mitosis and replication stress.2,3

To more precisely map the cellular response to the WEE1 inhibitor and explore the contributions of p53 to its effect, we used flow cytometry and immunohistochemistry in HNSCC cell lines with defined p53 status. Understanding the mechanism of WEE1 inhibition and its dependence on p53 will help us optimize drug combination strategies for targeted therapy against p53-negative cancers.

Methods

To pinpoint p53-specific effects, we generated an isogenic cell line pair (UM-SCC-74A pBabe vector control and UM-SCC-74A p53 knockdown) by transducing a retroviral shp53 vector to the p53 wild-type parental line UM-SCC-74A (Figure 1A). Cells were treated with 500 nM of AZD1775 alone from 0 to 32 hours in a pulse chase experiment with 5-ethynyl-2-deoxyuridine (EdU) to label cells undergoing replication. Fixation and intracellular staining were performed to measure cell cycle, DNA damage, premature mitosis, and DNA content using antibodies for EdU, phosphorylated histone H2AX (γ-H2AX), histone H3S10P+ (HH3S10P+), and 4′,6-diamidino-2-phenylindole dye. Flow cytometry was performed on a BD Canto II system (BD Biosciences) with a minimum of 10 000 events captured. In conjunction, we performed immunofluorescence in situ against 4′,6-diamidino-2-phenylindole, EdU, and phosphorylated replication protein A (S4/S8). Phosphorylated replication protein A is a specific marker to quantify response to stalled and collapsed replication forks as a marker of replication-related stress.4 This study was approved by the institutional review board at the Fred Hutchinson Research Cancer Center.

Results

We observed that AZD1775 triggered DNA damage as measured by γ-H2AX, particularly in mid S phase of the cell cycle (Figure 1B). In addition, cells treated with AZD1775 stained double positive with EdU and phosphorylated replication protein A by immunofluorescence (Figure 1B). These 2 observations suggest that replication stress represents one specific mechanism of cellular response to WEE1 inhibition. Although there was increased overall DNA damage in p53-deficient lines, the percentage of cells positive for replication stress was equivalent in p53 functional and p53-deficient cell lines (18.2% vs 18.6%). The replication stress response induced by WEE1 inhibition represents a pathway independent of p53 status.

In later phases of the cell cycle, treatment with AZD1775 markedly (≥3-fold) upregulated mitosis (HH3S10P+), including premature mitosis (HH3S10P+, <4N DNA content) only in the UM-SCC74A p53-deficient line (Figure 2). This abrogation of the G2/M checkpoint by the WEE1 inhibitor represents a pathway to mitotic catastrophe that is specific to p53 status.

Discussion

Our data suggest that even as a single-agent AZD1775 is significantly more cytotoxic to p53 mutated than to p53 wild-type HNSCC cell lines. We characterize 2 separate mechanisms of action of AZD1775 in HNSCC cell lines: DNA replication stress and DNA damage independent of p53 status and abrogation of the G2/M checkpoint, which resulted in premature mitosis in p53-deficient lines. Elucidation of the mechanisms by which AZD1775 induces replication stress and abrogates the G2/M checkpoint is needed to maximize the effects of WEE1 therapy in p53-deficient HNSCC. We have already begun clinical translation of these findings, launching a phase 1 clinical trial with AZD1775 given neoadjuvantly to patients with HNSCC.5

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Article Information

Corresponding Authors: Eduardo Méndez, MD, MS, Clinical Research Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, Mailstop D5-390, Seattle, WA 98109 (edmendez@uw.edu).

Accepted for Publication: December 3, 2016.

Published Online: February 16, 2017. doi:10.1001/jamaoto.2016.4563

Author Contributions: Drs Sidorova and Méndez contributed equally to this work, had full access to all the data in the study, and take responsibility for the integrity of the data and the accuracy of the data analysis.

Study concept and design: Kao, Sidorova, Méndez.

Acquisition, analysis, or interpretation of data: All authors.

Drafting of the manuscript: Kao, Méndez.

Critical revision of the manuscript for important intellectual content: All authors.

Statistical analysis: Kao.

Obtained funding: Kao, Méndez.

Administrative, technical, or material support: Kao, Green, Sidorova.

Study supervision: Sidorova, Méndez.

Conflict of Interest Disclosures: All authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest and none were reported.

Funding/Support: This work was supported by grant U01 CA176303 from the National Cancer Institute, grant RSG TBG-123653 from the American Cancer Society, grant NIH R01 GM115482 from the University of Washington Royalty Research Fund, Research Training in Otolaryngology NRSA Institutional Training Grant T32DC00018 from the National Institute on Deafness and Other Communication Disorders, and center funds from the Department of Otolaryngology–Head and Neck Surgery, University of Washington.

Role of the Funder/Sponsor: The funding sources had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

Additional Contributions: Carla Grandori, MD, PhD, and Charles de Leeuw, PhD, assisted in synthesizing the retroviral construct for short hairpin RNA p53 and generation of the isogenic paired cell lines.

References
1.
Moser  R, Xu  C, Kao  M,  et al.  Functional kinomics identifies candidate therapeutic targets in head and neck cancer.  Clin Cancer Res. 2014;20(16):4274-4288.PubMedGoogle ScholarCrossref
2.
Aarts  M, Sharpe  R, Garcia-Murillas  I,  et al.  Forced mitotic entry of S-phase cells as a therapeutic strategy induced by inhibition of WEE1.  Cancer Discov. 2012;2(6):524-539.PubMedGoogle ScholarCrossref
3.
Domínguez-Kelly  R, Martín  Y, Koundrioukoff  S,  et al.  Wee1 controls genomic stability during replication by regulating the Mus81-Eme1 endonuclease.  J Cell Biol. 2011;194(4):567-579.PubMedGoogle ScholarCrossref
4.
Sirbu  BM, Couch  FB, Feigerle  JT, Bhaskara  S, Hiebert  SW, Cortez  D.  Analysis of protein dynamics at active, stalled, and collapsed replication forks.  Genes Dev. 2011;25(12):1320-1327.PubMedGoogle ScholarCrossref
5.
clinicaltrials.gov. WEE1 Inhibitor MK-1775, Docetaxel, and Cisplatin Before Surgery in Treating Patients With Borderline Resectable Stage III-IVB Squamous Cell Carcinoma of the Head and Neck. NCT02508246. https://clinicaltrials.gov/ct2/show/NCT02508246. Accessed January 25, 2016.
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