Wee-1 Kinase Inhibition Sensitizes High-Risk HPV⁺ HNSCC to Apoptosis Accompanied by Downregulation of MCl-1 and XIAP Antiapoptotic Proteins

Head and neck squamous cell carcinoma (HNSCC) affects more than 500,000 patients worldwide annually and roughly half this number of patients will die from the disease each year (1). Over the past several decades, there has been a rapid emergence of HPV-associated squamous cell carcinoma of the oropharynx (OPSCC), with a concomitant decline in smoking-related OPSCC (1, 2). These distinct types of OPSCC have different clinical presentation and response patterns to the standard treatment approaches, including radiation and chemoradiotherapy, and this has led to different considerations in selecting the most appropriate treatment for HPV⁺ and HPV⁻ patients (3–5). Multiple studies have shown that the overall and disease-free survival for patients with HPV⁺ associated OPSCC is significantly better than for HPV⁻ OPSCC patients (5, 6). However, a group of HPV⁺ OPSCC patients' tumors remain refractory to conventional treatments with radiation given with or without chemotherapy and unusual patterns of recurrence and metastasis are emerging, particularly in patients with T4 and/or N3 staged disease (7–10). Therefore, new approaches to treatment of patients with high-risk HPV⁺ OSCC are needed. Thus, in the present study, we have focused on a strategy to improve treatment of these patients by using a novel molecularly targeted agent to chemosensitize HPV⁺ head and neck cancers.

Wee-1 is a serine/threonine kinase that has been linked to DNA damage induced G₂–M arrest, owing to its ability to inactivate the cyclin-dependent kinase 1 (CDK1), also known as CDC2, through phosphorylation of the Tyr15 residue (11, 12). Therefore, inhibition of the Wee-1 kinase activity can override a G₂ cell-cycle arrest and/or result in DNA damage by disrupting replication of the genome, leading to premature accumulation of cells with extensive DNA damage in mitosis and subsequent cell death through mitotic catastrophe (13, 14). Recent work with AZD-1775 (formerly known as MK-1775), a specific inhibitor of Wee-1, and siRNA-mediated depletion indicates that Wee-1 inhibition abrogates the G₂ checkpoint and selectively sensitizes tumor cells defective of p53 to various DNA-damaging agents, such as gemcitabine, cisplatin, and inhibits tumor growth in in vivo models (15–18). In light of the findings from these preclinical studies, AZD-1775 has entered phase I and II clinical trials as a chemosensitizer in patients with advanced solid tumors and shows good tolerability with minimal collateral cytotoxicity. We have recently demonstrated that MK-1775 can overcome cisplatin resistance in HPV⁻ HNSCC cells expressing high-risk mutant p53 through mitotic arrest followed by senescence rather than apoptosis (19). Although, the status of p53 is predominantly wild-type in HPV-positive HNSCC (20, 21), the high-risk HPV E6 inactivates p53 through proteasomal degradation (22). Therefore, it is possible that the defective p53 status in HPV⁺ HNSCC cells makes these tumors more vulnerable to chemosensitization upon Wee-1 kinase inhibition. Recent in vitro data have shown that HPV⁺ HNSCC cells and ovarian cell lines transfected with E6 oncoprotein are sensitive to Wee-1 kinase inhibition (18, 23, 24). However, to our knowledge, the single-agent efficacy of AZD-1775 or in combination with cisplatin therapy has not been fully evaluated in vivo in high-risk HPV⁺ HNSCC. In addition, it is not clear whether AZD-1775 enhances the sensitivity of HPV⁺ HNSCC cells to cisplatin by mechanism(s) similar to that occurring in HPV⁻ HNSCC cells. In light of this information, we hypothesized that the Wee-1 kinase inhibitor, AZD-1775 will enhance the sensitivity of cisplatin both in vitro and in vivo in preclinical models of HPV⁺ oral cancer.

Our data show that AZD-1775 displays single-agent activity and significantly enhances the response of HPV⁺ HNSCC cells to cisplatin both in vitro and in vivo. Interestingly, unlike the HPV⁻ HNSCC cancer cells, AZD-1775 appears to increase sensitivity of HPV⁺ HNSCC cells to cisplatin therapy through the induction of apoptosis triggered by selective degradation (or cleavage) of the antiapoptotic, MCl-1and XIAP proteins.

The HPV16⁺ HNSCC cell lines used in this study are UMSCC47 from Dr. Thomas Carey (University of Michigan, Michigan, MI) in August 2008, HMS-001 from Dr. James Rocco (Massachusetts General Hospital, Boston, MA) in March 2011, and HB96 (transformed with HPV16 E6/E7) from Dr. Zhi-Yuan Zhang (Ninth People's Hospital School of Medicine, China, Shanghai) in November 2013. The HPV-HNSCC cell lines, HN30 (wtp53) and HN31 (mutp53), were obtained in December 2008 from the laboratory of Dr. John Ensley (Wayne State University, Detroit, MI). The cell lines (HN30, HN31, UMSCC47, and HB96) were maintained in DMEM and the HMS-001 in DMEM/F12 supplemented with 10% FBS, l-glutamine, sodium pyruvate, nonessential amino acids, and a vitamin solution and incubated at 37°C in 5% CO₂ and 95% air. HPV⁺ HNSCC cell lines stably expressing shRNA specific for p53 (shp53) were generated according to protocol described previously (25). HPV⁺ HNSCC cell lines stably expressing MCl-1 were established using a pBabe-Flag h-MCl-1 vector (Addgene) as described previously (26).The identity of all cell lines was authenticated using short tandem repeat testing within 6 months of cell use. The Wee-1 inhibitor, MK-1775, was initially provided by the Merck Corporation through a collaborative agreement arrange by NCI-CTEP. The drug is currently licensed by AstraZeneca and known as AZD-1775, and its chemical structure has been described previously (15). For in vitro studies, AZD-1775 was prepared as 10 mmol/L stock solution in DMSO and stored at −20°C and diluted in culture medium (0.25 μmol/L) immediately before use. The apoptotic agent, staurosporine, was purchased from Sigma and used at 1 μmol/L final concentration. The pan-caspase inhibitor, Z-VAD-FMK, was obtained from R&D System, prepared as 20 mmol/L stock in DMSO, and used at 50 μmol/L final concentration.

The HPV⁺ HNSCC cells were seeded in 6-well plates at predetermined densities, concurrently exposed to different fixed ratio combinations of cisplatin (dose range, 0.01–2 μmol/L) and AZD-1775 (dose range, 0.01–1μmol/L) for 24 hours, and the clonogenic cell survival was determined as previously described (19).

Drug synergy was determined by the combination index (CI) and isobologram analyses, which were generated according to the median-effect method of Chou and Talalay (27) using the CalcuSyn software (Biosoft). The CI is a quantitative representation of the degree of drug interaction. For details, see Supplementary Materials and Methods.

Antibodies used for Western blotting were phospho-γH2AX (Ser139; #2577), phospho-CDC2-Tyr15 (#9111), CDC2 (#9112), cyclin B1 (#4138), PARP-1 (#9542), MCl-1 (#4572), XIAP (#2042), Rad51 (#8875), and caspase-3 (#9661)—all from Cell Signaling Technology; β-actin (#A5316; Sigma-Aldrich); p53(DO-1) (#sc-126), and MCl-1 (#sc-819) are from Santa Cruz; and p21WAFI (Ab-1) (#OP64; Calbiochem); XIAP (#610716; BD Bioscience).

Cells grown on 10-cm plates were treated with clinically relevant doses of cisplatin (1.5 μmol/L), AZD-1775 (0.25 μmol/L) either alone or in combination for 16, 24, or 48 hours. For caspase cleavage detection, cells were pretreated with Z-VAD-FMK for 3 hours before addition of cisplatin and AZD-1775. Whole-cell extracts were prepared and Western blot analysis was conducted with indicated antibodies as described previously (19, 25).

Cells were seeded in 60-mm dishes, treated the next day with 1.5 μmol/L cisplatin, 0.25 μmol/L AZD-1775 either alone or in combination, and then harvested at 24, 48, or 72 hours. The cell-cycle analysis and apoptosis detection were performed as previously described (19).

All animal experimentation was approved by the Institutional Animal Care and use Committee (IACUC) of the University of Texas MD Anderson Cancer Center. Our orthotopic nude mouse tongue model has been previously described in the literature (28). The HPV⁻ HNSCC (HN31) and HPV⁺ HNSCC (UMSCC47, HMS-001) cell lines were injected into the tongues of the male athymic nude mice, and 8 to 10 days after injection, mice were randomized into different groups. Treatment protocol and tumor growth delay measurement were conducted as previously described (19), and a detailed description of the technique is included in the Supplementary Materials and Methods.

Apoptosis was assessed in mice tissue sections with terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay with DeadEnd Fluorometric TUNEL System (Promega) according to the manufacturer's protocol with some modifications and a detailed description is included in the Supplementary Materials and Methods.

Sections were prepared from formalin-fixed, paraffin-embedded mice tumor tissues and subjected to immunohistochemical staining with indicated antibodies according to the protocol as described in Supplementary Materials and Methods.

The Student t and a one-way ANOVA tests were carried out to analyze in vitro data. For mouse studies, a 2-way ANOVA test was used to compare tumor volumes between control and treatment groups. For immunohistochemical analyses, a χ² test was used to compare immunostaining between control and treatment groups. All data were expressed as mean ± standard error, and P < 0.05 was considered significant.

To explore sensitivity of HPV⁺ HNSCC cells to cisplatin and AZD-1775 as single agents, we performed dose–response curves with each drug alone in HPV⁺ HNSCC cell lines (UMSCC47, HB96, HMS-001) using standard clonogenic survival assays. Compared with the relative cisplatin resistance that we previously reported in HPV⁻ HNSCC cells (ref. 19; e.g., average IC₅₀ ≥ 0.77 μmol/L), there was a clear trend toward increased cisplatin sensitivity in HPV⁺ cells with IC₅₀ values ranging between 0.3 and 0.5 μmol/L. Similarly, HPV⁺ cells were more sensitive to AZD-1775 as a single agent (e.g., IC₅₀, 0.09–0.24 μmol/L; Fig. 1A and B). Compared with IC₅₀ values, we previously reported for HPV⁻ HNSCC cell lines which ranged from 0.25 to 0.375 μmol/L (19). Representative images of clonogenic survival assays following single agent AZD-1775 are shown in Fig. 1C, demonstrating the relative sensitivity of HPV⁺ HNSCC cells treated with various doses of AZD-1775.

We next investigated whether Wee-1 kinase inhibition was synergistic with cisplatin treatment in the HPV⁺ HNSCC cell lines, using the combination index and fraction affected (Fa) method of Chou and Talalay (27). Addition of AZD-1775 significantly enhanced the cytotoxic effect of cisplatin in these cells and the combination effect reveals strong synergism manifested by the shift of cisplatin response curves and the CI values < 1 (Fa, 0.5, ±SD) of 0.14 ± 0.09, 0.15 ± 0.13, and 0.073 ± 0.07 (Fig. 1D–1F, top plots) for UMSCC47, HB96, and HMS-001, respectively. The CI plots (Fig. 1D–1F, bottom plots) in all HPV⁺ HNSCC cells show a clear strong synergistic effect at the more relevant FA values (≥50%). In addition, conservative isobologram plots of effective dose ED50, ED75, and ED90 were generated and further confirmed synergism of the drug combination in all HPV⁺ HNSCC cells examined (Supplementary Fig. S1A–S1C). Because HMS-001 shows greater sensitivity to AZD-1775, it was used for further experiments. The data clearly demonstrate that AZD-1775 has single-agent activity and synergizes with cisplatin to inhibit in vitro tumor growth in HPV⁺ HNSCC.

To confirm that AZD-1775 affects its downstream target, the HPV⁺ HNSCC cells were treated with clinically relevant doses of the drugs as indicated previously, and phosphorylation of CDK1 was examined by Western blotting (Fig. 1G). Following cisplatin alone, increased phosphorylation of CDK1 was apparent in all 3 HPV⁺ HNSCC cells. AZD-1775 alone or in combination with cisplatin caused substantial suppression of CDK1 phosphorylation accompanied by decreased protein levels of its protein partner, cyclin B1 (Fig. 1G), indicating an effective engagement of downstream targets and inhibition of the Wee-1 activity. Furthermore, the combination of cisplatin and AZD-1775 significantly enhanced levels of the DNA damage marker, γH2AX (phospho-ser139) in HMS001 (Fig. 1H) as well as the 2 other HPV⁺ cell lines (data not shown), indicating an increase and persistence of unrepaired DNA damage in all the HPV⁺ HNSCC cancer cells tested. The increase in the phosphorylated γH2AX level was evident as early as 8 hours (data not shown), peaked gradually, and remained elevated at 48 hours (Fig. 1H). Although AZD-1775 treatment alone triggered elevated γH2AX compared with cisplatin or untreated cells by Western blotting, levels were highest with the combination. Also, evident was an increase in γH2AX foci intensity in the HPV⁺ HNSCC cells (Supplementary Fig. S2A and S2B) 4 hours following treatment with AZD-1775 alone or in combination with cisplatin.

On the basis of our finding that inhibition of Wee-1 results in persistent cisplatin-induced DNA damage, and previous studies demonstrating a role for Wee-1 in homologous recombination (HR; refs. 29, 30), we examined alteration in the expression level of the HR competent marker, Rad51 in HPV⁺ HNSCC cells following treatment with the drugs. Significant decrease in the Rad51 protein level was observed at 16 hours and declined by 48 hours after treatment with AZD-1775 alone or in combination with cisplatin compared with cisplatin alone or untreated control cells (Supplementary Fig. S2C). Correspondingly, AZD-1775 treatment led to a decrease in cisplatin-induced Rad51 focus formation in HPV⁺ HNSCC cells (Supplementary Fig. S2D and S2E). Taken together, these data suggest that Wee-1 inhibition sensitizes HPV⁺ HNSCC cells to cisplatin through inhibition of Rad51-mediated HR.

We have previously shown that inhibition of Wee-1 abrogates cisplatin-induced G₂ checkpoint in HNSCC cells harboring high-risk mutant p53 (19). To confirm this takes place in HPV⁺ HNSCC cells, we performed cell-cycle analysis in unsynchronized cells (UMSCC47, HMS-001) treated as previously described. In response to cisplatin alone, UMSCC47 and HMS-001 HPV⁺ cells were arrested at the G₂–M phase (64.45%, and 49.27%, respectively) 48 hours after treatment (Fig. 2A and B and Supplementary Fig. S3A and S3B). Addition of AZD-1775 abrogated the cisplatin-induced G₂ checkpoint arrest as manifested by reduction in the fraction of cells in G₂ phase from 49.27% to 11.05% within 48 hours in HMS-001 cells and from 60.71% to 31.48% within 72 hours in UMSCC47 cells (Fig. 2A and B; Supplementary Fig. S3A and S3B). Abrogation of cisplatin-induced G₂ checkpoint arrest and unscheduled mitosis were confirmed by the presence of increased phospho-H3 protein levels on Western blotting (data not shown). Treatment with either AZD-1775 alone or AZD-1775 plus cisplatin led to an increase in the percentage of cells with sub-G₁ DNA content at 48 and 72 hours, when compared with cisplatin alone for both UMSCC47 and HMS-001 cells (Fig. 2A and B; Supplementary Fig. S3A and S3B). By 72 hours, the percentage of UMSCC47 with sub-G₁ DNA content following treatment with AZD-1775 alone or the combination was 44% and 38%, respectively, compared with just 10% in cisplatin-treated cells. In HMS-001 cells, the highest percentage of sub-G₁ DNA occurred after 72-hour combination treatment (71%), compared with AZD-1775 alone (54%) or cisplatin alone (36%). No polyploidy or micronuclei were observed in these cells upon the combination treatment, indicating an absence of mitotic catastrophe (data not shown). To investigate whether cells were dying through apoptosis, HPV⁺ (UMSCC47, HB96, HMS-001) and HPV- (HN30, HN31) HNSCC cells were treated with the drugs as previously indicted and cell lysates examined for PARP-1 protein cleavage 48 hours later. Consistent with our previous findings (19), no PARP-1 cleavage was found in the HPV⁻ HNSCC cell lines regardless of the drug treatments (Fig. 2C). However, in both HPV⁺ cell lines, AZD-1775 alone induced PARP-1 cleavage that was even greater when combined with cisplatin (Fig. 2C). The presence of apoptosis in HPV⁺ HNSCC cells was further confirmed with the positive APO-BrdUrd TUNEL staining (Fig. 2D and E). In UMSCC47, the percentage of apoptotic cells was greatly enhanced by the combination treatment compared with AZD-1775 or cisplatin alone (69% vs. 23% and 9.4%). However, HMS-001 cells were exquisitely sensitive to single-agent AZD-1775, which induced apoptosis in greater than 90% of the population. This was consistent with high levels of PARP-1 cleavage observed in HMS-001 treated with AZD-1775 alone. Increased caspase-3/7 activity was observed at 24 hours in all HPV⁺ HNSCC cells following all treatments but was significantly greater in the combination of cisplatin and AZD-1775 versus single agents (Supplementary Fig. S4A and S4B). No increase in caspase-3/7 activity was observed in the HPV⁻ HNSCC cell lines (Supplementary Fig. S4C and S4D). Absence of caspase-dependent apoptosis in HPV⁻ HNSCC cells was confirmed with Western blotting (Supplementary Fig. S4E). The increased caspase-dependent activity could be blocked by the pan-caspase inhibitor Z-VAD-FMK, demonstrating the specificity of the assay and the efficacy of the inhibitor. Treatment with Z-VAD-FMK profoundly inhibited the PARP-1 cleavage observed in HMS-001 exposed to AZD-1775 alone or in combination with cisplatin, confirming that PARP-1 cleavage was a consequence of drug-induced caspase activation (Supplementary Fig. S4F). Collectively, these data indicate that AZD-1775 shows single-agent activity and enhances cisplatin sensitivity in HPV⁺ HNSCC through caspase-mediated apoptosis.

Elevated expression of antiapoptotic proteins has been reported in many tumors (31–33). In addition, several studies have shown that activation of the Wee-1 primary target, CDK1, plays a key role in mitotic arrest–induced apoptosis by decreasing stability and expression of several antiapoptotic proteins through proteasomal degradation (34–36). Thus, we examined whether the Wee-1 inhibition–induced apoptosis in HPV⁺ HNSCC cells (UMSCC47, HB96, HMS-001) exposed to cytotoxic treatment with cisplatin is associated with decreased levels of the antiapoptotic proteins, including Bcl-2, Bcl-xL,MCl-1, XIAP, c-IAP-1, and c-IAP-2. AZD-1775 and the combination with cisplatin selectively decreased the expression of apoptosis inhibitors MCl-1 and XIAP in HPV⁺ HNSCC but not in HPV⁻ HNSCC cells (Fig. 3A and B, respectively). The decrease in protein expression was time-dependent observed at 24 hours and remain suppressed 48 hours after treatment. No changes in Bcl-2, Bcl-xL, c-IAP-1, or c-IAP-2 protein expression were found following all treatments (data not shown).

MCl-1 and XIAP are highly regulated antiapoptotic proteins and inactivated through cleavage by caspase activation either in spontaneous apoptosis or in drug-induced programmed cell death (37, 38). To test for such possibility, the most sensitive HPV⁺ cells (HMS-001, HB96) were treated with cisplatin, AZD-1775 alone, or in combination, and MCl-1 and XIAP protein cleavage was examined by Western blotting. Appearance of MCl-1 and XIAP full-length and cleaved fragments was evident in these cells (Fig. 3C–E). In addition, the decrease in both MCl-1 and XIAP protein induced by treatment with AZD-1775 or AZD-1775 plus cisplatin could be prevented in HMS-001 cells by adding Z-VAD-FMK (Supplementary Fig. 5SA), indicating that the reduction of these antiapoptotic proteins was most likely due to their cleavage following caspase activation. Further supporting this, appearance of cleaved MCl-1 bands on Western blotting after exposure to AZD-1775 alone or AZD-1775 combined with cisplatin was also inhibited by the pan-caspase inhibitor (Supplementary Fig. S5B). Expression of MCl-1 and XIAP can be regulated at both the transcriptional and/or translational levels (37, 39, 40). No changes in mRNA levels of MCl-1 and XIAP determined by qRT-PCR analysis were observed in HPV⁺ HNSCC cells treated with cisplatin, AZD-1775, or in combination (Supplementary Fig. S6A and S6B), suggesting that AZD-1775 enhances the ability of cisplatin to regulate antiapoptotic proteins levels through posttranslational rather than transcriptional regulation.

To investigate the role of decreased MCl-1 in apoptosis induced by exposure to AZD-1775 or its combination with cisplatin, Flag-tagged MCl-1 protein was overexpressed in HPV⁺ HNSCC (HB96) cells (Supplementary Fig. S6C). Overexpression of MCl-1 inhibited PARP-1 cleavage induced by either AZD-1775 or the combination with cisplatin (Fig. 3F), demonstrating that downregulation of the MCl-1 has a critical role in AZD-1775–induced apoptosis in HPV⁺ HNSCC tumors treated with cisplatin.

A recent study reported that low levels of normally functioning wild-type p53 remain in HPV⁺ HNSCC cells despite downregulation by the viral protein E6 and that this p53 can be activated by radiation therapy, leading to cell death through apoptosis (41).Therefore, we examined whether cisplatin alone or in combination with AZD-1775 sensitizes HPV⁺ HNSCC cells by reactivating p53 and its immediate primary target gene p21, leading to cell death. While treatment with cisplatin alone increased p53 and p21 protein levels, addition of AZD-1775 did not further augment their protein levels in any of the HPV⁺ HNSCC cells tested (Fig. 4A). Use of a TP53-specific shRNA, but not control shRNA, led to significant reduction in p53 expression levels in UMSCC47 and undetectable in HB96 cell lines (Fig. 4B). Therefore, the HB96 HPV⁺ HNSCC cell line was subsequently used for examining the role of wild-type p53 on sensitivity to cisplatin in these cells. Using clonogenic survival assays, treatment of HB96 cells with cisplatin alone resulted in survival fractions that were similar between the control shRNA (IC₅₀: 0.34 μmol/L) and shp53RNA (IC₅₀: 0.31 μmol/L; Fig. 4C). HPV⁺ HNSCC cells expressing shp53RNA showed survival fractions that were not different from those observed with control shRNA upon treatment with cisplatin and/or in combination with AZD-1775 (Fig. 4D). Finally, we examined the effect of TP53-knockdown on apoptosis by detection of PARP-1 cleavage on Western blot analysis. No difference in PARP-1 cleavage was observed in control shRNA or shp53RNA HB96 cells following cisplatin alone or in combination with AZD-1775 (Fig. 4E). These results suggest that the sensitization of HPV⁺ HNSCC to cisplatin alone or in combination with AZD-1775 is not mediated through the p53 reactivation.

To determine whether AZD-1775 can sensitize HPV⁺ HNSCC cells to cisplatin in vivo, UMSCC47 and HMS-001 HPV⁺ cells were injected into the tongues of nude mice as previously described (28). Mice were treated with the drugs as described in Materials and Methods, and effects on tumor growth were examined. Cisplatin-treated mice showed modest suppressive effects on tumor growth compared with untreated control groups (Fig. 5A and B). AZD-1775 alone showed significant growth inhibition compared with untreated mice (Fig. 5A and B; P < 0.001, P < 0.0001 for UMSCC47 and HMS-001, respectively), but the difference from cisplatin-treated mice was only significant in HMS-001- but not UMSCC47-bearing tumors (Fig. 5A and B, P < 0.05). The combination of cisplatin and AZD-1775 significantly inhibited growth more than cisplatin treatment alone or control in tumors of mice bearing UMSCC47 and HMS-001 HPV⁺ cells (Fig. 5A and B). Moreover, combination treatment showed modest effect on tumor growth inhibition over AZD-1775 alone in mice tumors carrying UMSCC47 cells (Fig. 5A, P = 0.07), but this effect was significant in mice bearing tumors with HMS-001 cells (Fig. 5B, P < 0.0001). In addition, AZD-1775 monotherapy produced greater tumor growth inhibition in mice injected subcutaneously with HOSC19 HPV⁺ patient-derived tumor xenografts (PDX; Supplementary Fig. S7A). To confirm that the enhancement of cisplatin antitumor efficacy by AZD-1775 was associated with engagement of downstream targets in vivo, phospho-CDK1 was evaluated in tissue sections obtained from tongue tumor xenografts. Consistent with earlier in vitro studies, AZD-1775 or its combination with cisplatin significantly decreased in vivo phospho-CDK1 immunostaining levels in orthotopic tongue tumors compared with cisplatin or untreated tumors (Fig. 5C–F). A decrease in phospho-CDK1 immunostaining was also observed in PDX tumors (HOSC19; Supplementary Fig. S7B and S7C). In addition, proliferating cell nuclear antigen (PCNA), a marker of proliferation, was significantly reduced in the UMSCC47 orthotopic tongue tumors from mice treated with either AZD-1775 or its combination with cisplatin compared to cisplatin alone or untreated control tumors (Supplementary Fig. S8A and S8B, χ², P < 0.0001). Immunostaining with the p16 antibody confirmed that HPV⁺ HNSCC cells maintained HPV positivity in vivo (Supplementary Fig. S8C). Collectively, the results suggest that tumor growth inhibition is associated with decreased cellular proliferation in vivo in mice tongues bearing HPV⁺ HNSCC tumors treated with either AZD-1775 alone or in combination with cisplatin.

The occurrence of apoptosis in tongue tumor xenografts bearing HPV⁺ HNSCC (UMSCC47, HMS-001) cells was examined using the TUNEL assay. TUNEL-positive apoptotic cells were increased in tissue sections obtained from HPV⁺ tumor xenografts of mice treated with AZD-1775 alone or in combination with cisplatin compared with untreated or cisplatin-treated mice (Fig. 6A and B), indicative of apoptosis and consistent with our in vitro results. Quantification of TUNEL-positive apoptotic cells shown in Fig. 6A and B was presented as apoptotic index (Fig. 6C and D). Furthermore, MCl-1 and XIAP expression in tumor sections of UMSCC47 oral xenograft mice significantly decreased upon therapy with combination of cisplatin and AZD-1775 or AZD-1775 alone, compared with cisplatin-treated and control groups (Fig. 6E and F). TUNEL positivity was also observed in the PDX tumors (HOSC19) treated with AZD-1775 (Supplementary Fig. S7D and S7E). Neither TUNEL positivity nor reduction in expression of MCl-1 and XIAP was detected in mice tumors bearing HPV⁻ HNSCC (mutp53 HN31) cells following cisplatin and AZD-1775 or AZD-1775 alone treatment, indicating minimal induction of apoptosis in these tumors (Supplementary Fig. S9A–S9C). These results suggest that AZD-1775 synergistic interaction with cisplatin therapy in vivo in HPV⁺ oral cancer results from apoptotic death associated with the downregulation of MCl-1 and XIAP antiapoptotic proteins.

This is the first study to investigate the chemosensitizing effects of a highly selective inhibitor of the Wee-1 kinase, AZD-1775, in high-risk HPV⁺ HNSCC tumor cells treated with cisplatin. This drug combination was chosen because platinum-based chemotherapy plays a key role in the management of patients with HPV⁺ HNSCC tumors. AZD-1775 shows single-agent antitumor activity synergizes with cisplatin and induces apoptosis in vitro in all HPV⁺ HNSCC cell lines tested. Interestingly, cisplatin arrests HPV⁺ HNSCC cells at the G₂ phase and that addition of AZD-1775 abrogates the G₂ block and pushes the cells rapidly into apoptosis. Collectively, these data suggest that AZD-1775 may be used as single-agent and in combination with cisplatin therapy in HPV⁺ HNSCC. No apoptotic death was found in the HPV⁻ HNSCC cells with addition of AZD-1775 or in combination with cisplatin, consistent with our recently published data (19) in which we showed that mitotic catastrophe associated with senescence, rather than apoptosis is the major mechanism of synergistic interaction between cisplatin and AZD-1775 in high-risk p53-mutant HPV⁻ HNSCC cells. Although we did not observe apoptosis associated with AZD-1775 single-agent treatment in our HPV⁻ p53-mutant HNSCC cell lines, Moser and colleagues (18) have recently shown that apoptosis can occur in vitro in HPV⁻ cells treated with higher doses of MK-1775. Most likely, the difference between their studies and ours can be attributed to different concentrations of this agent used in our versus their experiments. We used a dose of 250 nmol/L which has been shown to achieve greater than 80% reduction in phosphorylation of the Wee-1 downstream target CDK1 (15, 19, 24, 42, 43); however, we have also observed apoptosis at much higher concentrations of the drug in p53-mutant HPV⁻ HNSCC cells.

The combination of cisplatin and AZD-1775 significantly produced high level of unrepaired and persistent DNA damage in all the HPV⁺ HNSCC cancer cells tested. It is possible that such high level of unrepaired DNA damage may have triggered replication stress associated with extreme lethality and unscheduled premature mitosis leading to induction of apoptosis in HPV⁺ tumor cells. This possibility is supported by published data showing that Wee-1 inhibition by AZD-1775 caused replication stress resulting from deregulated CDK1 activity and increased origin firing in pancreatic cancer (30). To our knowledge, this is the first report to demonstrate that the induction of apoptosis by AZD-1775 in HPV⁺ HNSCC tumors is accompanied by significant decrease in MCl-1 and XIAP protein levels. Because MCl-1 and XIAP are known substrates of caspase-3 and -7 (37, 38), it is likely that the observed decrease in MCl-1 and XIAP was due to caspase-mediated processing after cisplatin and AZD-1775 treatment in HPV⁺ HNSCC. It has been previously shown that cleavage of MCl-1 and XIAP interferes with their antiapoptotic function in Jurkat and HeLa cell lines (44, 45). Correspondingly, we have demonstrated that both MCl-1 and XIAP are cleaved in response to treatment with AZD-1775 alone or in combination with cisplatin in HPV⁺ HNSCC cells. Furthermore, the downregulation of MCl-1 and its cleavage by AZD-1775 alone or in combination with cisplatin were completely attenuated by caspase inhibition, suggesting that MCl-1 and XIAP are targets of inactivation and degradation by caspase-3/7 in these cells. In addition, caspase inhibition prevented apoptosis in these cells. We are not certain whether the degradation of Mcl-1 and XIAP we observed is a consequence of apoptosis or the cause of cell death phenotype. However, in our study, it is significant that enforced expression of MCl-1 markedly diminished AZD-1775–mediated PARP1 cleavage and apoptosis in HPV⁺ HNSCC (HB96) cells, suggesting that downregulation of antiapoptotic proteins is sufficient to trigger apoptosis. The mechanism by which AZD-1775 triggers the caspase-mediated apoptosis in these cells is not clear. Evidence exists that in response to DNA damage, activation of several pro-caspases is required before mitochondrial permeabilization and apoptosis can take place (46, 47). In light of this evidence, it is possible that Wee-1 inhibition further augments cisplatin-mediated DNA damage response, followed by caspase activation, cleavage of MCl-1 and XIAP, leading to apoptotic cell death in HPV⁺ HNSCC. This possibility is supported by our time course experiments, which revealed that the DNA damage occurred at 8 hours and preceded caspase-3/7 activation observed at 24 hours after treatment. Alternatively, the primary downstream target of Wee-1, the CDK1, may directly induce caspase activation in HPV⁺ HNSCC cells.

Functionally, the E6 viral oncoprotein of HPV inactivates wild-type p53 and inhibits apoptosis (48). Our data revealed that complete knockdown of TP53 by shRNA did not affect clonogenic cell survival and did not block apoptosis in HPV⁺ HNSCC treated with cisplatin or in combination with AZD-1775. This finding indicates that reactivation of p53 is not sufficient to sensitize HPV⁺ HNSCC to cisplatin treatment combined with Wee-1 kinase inhibition and is not in agreement with recent published data which showed that knockdown of p53 resulted in radiation resistance in HPV⁺ HNSCC (41). It is possible that reactivation of p53 has different roles depending on the type of genotoxic therapy. Potentially, HPV E6 has additional effects other than inactivation of p53 that augment sensitivity to cisplatin and AZD-1775 in HPV⁺ HNSCC. Probably, HPV⁺ cells have greater deficiency repairing the DNA damage upon cytotoxic treatment, predisposing them to cell death by apoptosis. Interestingly, both E6 and E7 are involved in activation and repression of DNA damage response pathways to support viral genome maintenance and amplification in the normal viral life cycle (49). A recent study has also shown that E6 and E7 activate caspases-3/-7 and -9 upon differentiation to induce viral genome amplification (50). Understanding of how these alterations contribute to DNA damage response to cisplatin and Wee-1 kinase inhibition will warrant future investigations.

As preclinical evaluation, we demonstrated that oral administration of AZD-1775 as single-agent inhibited in vivo growth of HPV⁺ HNSCC oral xenografts and also synergizes with cisplatin to induce tumor regression, confirming our in vitro data. Furthermore, in vivo tumor growth inhibition was associated with an induction of apoptosis and decreased level of MCl-1 and XIAP immunostaining in the combination of cisplatin- and AZD-1775–treated HPV⁺ HNSCC tumor xenografts compared with the control groups consistent with the in vitro results. Neither TUNEL positivity nor reduced immunostaining of MCl-1 and XIAP were detected in the HPV⁻ HNSCC tumor sections, confirming in vitro findings and suggesting different mechanism of response to cisplatin and AZD-1775 in HPV⁻ head and neck cancers as reported in our previous publication (19).

In conclusion, unlike HPV⁻ HNSCC cells, those derived from the HPV⁺ HNSCC show enhanced sensitivity to cisplatin with AZD-1775 treatment through apoptosis associated with reduced expression of MCl-1 and XIAP both in vitro and in vivo. It seems that inactivation of these antiapoptotic proteins via posttranslational cleavage induced by AZD-1775 treatment plays an important role in modulating the response of the HPV⁺ HNSCC tumors to chemotherapy. Our study could have implications for the use of MCl-1 and XIAP expression as surrogate end biomarkers in future clinical trials combining cisplatin and Wee-1 kinase inhibition. In addition, our findings further suggest that MCl-1 and XIAP could be potential therapeutic targets in HPV⁺ HNSCC tumors. Our preclinical data on Wee-1 kinase inhibition demonstrate the susceptibility of HPV⁺ HNSCC tumors to deregulation of the G₂–M checkpoint and support initiation of clinical trials with AZD-1775 in combination with cisplatin particularly in patients with advanced and recurrent metastatic HPV⁺ HNSCC tumors (18).

No potential conflicts of interest were disclosed.

Conception and design: N. Tanaka, A.A. Patel, M.J. Frederick, A.L. Fitzgerald, T.-x. Xie, C. Caulin, G. Zhou, J.N. Myers, A.A. Osman

Development of methodology: N. Tanaka, J. Wang, H.D. Skinner, A.A. Osman

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): N. Tanaka, A.A. Patel, N.N. Kalu, N.L. Silver, H.D. Skinner, F.M. Johnson, A.A. Osman

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): N. Tanaka, M.J. Frederick, T.-x. Xie, N.L. Silver, C. Caulin, J.N. Myers, A.A. Osman

Writing, review, and/or revision of the manuscript: N. Tanaka, M.J. Frederick, N.L. Silver, C. Caulin, H.D. Skinner, F.M. Johnson, J.N. Myers, A.A. Osman

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): A.A. Patel, M. Zhao, A.L. Fitzgerald, N.L. Silver, A.A. Osman

Study supervision: N. Tanaka, A.A. Osman

This work was supported by the NIH Specialized Program of Research Excellence Grant P50CA097007 (J.N. Myers), the NIH/NIDCR R01DE024601 (J.N. Myers), the Cancer Prevention and Research Institute of Texas (CPRIT) RP12025802 (J.N. Myers), the Stiefel Oropharyngeal Cancer Program, and the UTMDACC HPV⁺ HNSCC Moonshot Program (A.A Osman and J.N Myers).

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