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Oropharyngeal squamous cell carcinoma (OPSCC), largely fueled by the human papillomavirus (HPV), has a complex biological and immunologic phenotype. Although HPV/p16 status can be used to stratify OPSCC patients as a function of survival, it remains unclear what drives an improved treatment response in HPV-associated OPSCC and whether targetable biomarkers exist that can inform a precision oncology approach. We analyzed OPSCC patients treated between 2000 and 2016 and correlated locoregional control (LRC), disease-free survival (DFS) and overall survival (OS) with conventional clinical parameters, risk parameters generated using deep-learning algorithms trained to quantify tumor-infiltrating lymphocytes (TILs) (OP-TIL) and multinucleated tumor cells (MuNI) and targeted transcriptomics. P16 was a dominant determinant of LRC, DFS and OS, but tobacco exposure, OP-TIL and MuNI risk features correlated with clinical outcomes independent of p16 status and the combination of p16, OP-TIL and MuNI generated a better stratification of OPSCC risk compared to individual parameters. Differential gene expression (DEG) analysis demonstrated overlap between MuNI and OP-TIL and identified genes involved in DNA repair, oxidative stress response and tumor immunity as the most prominent correlates with survival. Alteration of inflammatory/immune pathways correlated strongly with all risk features and oncologic outcomes. This suggests that development of OPSCC consists of an intersection between multiple required and permissive oncogenic and immunologic events which may be mechanistically linked. The strong relationship between tumor immunity and oncologic outcomes in OPSCC regardless of HPV status may provide opportunities for further biomarker development and precision oncology approaches incorporating immune checkpoint inhibitors for maximal anti-tumor efficacy.

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Mounting evidence suggests that the Hippo coactivator Yes-associated protein 1 (YAP1) is a major mediator of cancer stem cell (CSC) properties, tumor progression, and therapy resistance as well as often a terminal node of many oncogenic pathways. Thus, targeting YAP1 may be a novel therapeutic strategy for many types of tumors with high YAP1 expression, including esophageal adenocarcinoma. However, effective YAP1 inhibitors are currently lacking. Here, we identify a small molecule (CA3) that not only has remarkable inhibitory activity on YAP1/Tead transcriptional activity but also demonstrates strong inhibitory effects on esophageal adenocarcinoma cell growth especially on YAP1 high–expressing esophageal adenocarcinoma cells both in vitro and in vivo. Remarkably, radiation-resistant cells acquire strong cancer stem cell (CSC) properties and aggressive phenotype, while CA3 can effectively suppress these phenotypes by inhibiting proliferation, inducing apoptosis, reducing tumor sphere formation, and reducing the fraction of ALDH1+ cells. Furthermore, CA3, combined with 5-FU, synergistically inhibits esophageal adenocarcinoma cell growth especially in YAP1 high esophageal adenocarcinoma cells. Taken together, these findings demonstrated that CA3 represents a new inhibitor of YAP1 and primarily targets YAP1 high and therapy-resistant esophageal adenocarcinoma cells endowed with CSC properties. Mol Cancer Ther; 17(2); 443–54. ©2017 AACR.

Supplemental Figure 2. IR triggers a temporary, reversible perturbation in cellular reducing potential.

Supplemental figure legends - detailed text

Ionizing radiation is the primary nonsurgical treatment modality for solid tumors. Its effectiveness is impacted by temporal constraints such as fractionation, hypoxia, and development of radioresistant clones. Biomarkers of acute radiation response are essential to developing more effective clinical algorithms. We hypothesized that acute perturbations in tumor lactate levels act as a surrogate marker of radiation response. In vitro experiments were carried out using validated human-derived cell lines from three histologies: anaplastic thyroid carcinoma (ATC), head and neck squamous cell carcinoma (HNSCC), and papillary thyroid carcinoma (PTC). Cellular metabolic activity was measured using standard biochemical assays. In vivo validation was performed using both an orthotopic and a flank derivative of a previously established ATC xenograft murine model. Irradiation of cells and tumors triggered a rapid, dose-dependent, transient decrease in lactate levels that was reversed by free radical scavengers. Acute lactate perturbations following irradiation could identify hypoxic conditions and correlated with hypoxia-induced radioresistance. Mutant TP53 cells and cells in which p53 activity was abrogated (shRNA) demonstrated a blunted lactate response to irradiation, consistent with a radioresistant phenotype. Lactate measurements therefore rapidly detected both induced (i.e., hypoxia) and intrinsic (i.e., mutTP53-driven) radioresistance. We conclude that lactate is a quantitative biomarker of acute genotoxic stress, with a temporal resolution that can inform clinical decision making. Combined with the spatial resolution of newly developed metabolic imaging platforms, this biomarker could lead to the development of truly individualized treatment strategies. Mol Cancer Ther; 14(12); 2901–8. ©2015 AACR.

Supplemental Figure 6. Loss of p53 function results in decreased radiosensitivity in HNSCC cell lines.

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Anaplastic thyroid carcinoma (ATC) accounts for more than 50% of thyroid cancer mortality and is generally refractory to conventional treatment. On the basis of recent studies, we hypothesized that ATC metabolism can be targeted to improve response to chemoradiotherapy. Eight established and authenticated ATC cell lines were sequenced at 140 sites contained within 26 commonly mutated genes to identify novel potential therapeutic targets. Cellular proliferation, energy, and reducing potential stores were measured under conditions of specific nutrient deprivation. Tumor metabolism was evaluated using hyperpolarized 13C MRI in a murine orthotopic xenograft model of ATC. Sensitivity to chemotherapeutic agents and radiation (XRT) was assayed using cytotoxicity assays. We identified mutations in BRAF, NRAS, and KIT but failed to identify generalized novel targets for therapeutic intervention. ATC cell lines exhibited a mesenchymal phenotype and generalized dependence on glucose for energy, reducing potential and survival. Glycolytic inhibition using 2-deoxyglucose (2-DG) sensitized ATC cells to conventional chemotherapy and external beam radiation. In vivo, 2-DG induced a transient, but significant reduction in ATC metabolic activity. Generalized dependence of ATC cells on glucose catabolism makes them susceptible to the sensitizing effects of 2-DG for radiation therapy and chemotherapy. Under in vivo conditions, 2-DG can inhibit ATC metabolism. However, the modest magnitude and transient nature of this effect suggest the need for antimetabolic agents with more favorable pharmacodynamics to achieve therapeutic effects. Mol Cancer Ther; 11(6); 1373–80. ©2012 AACR.

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Chemical structures for novel YAP1 inhibitors

Supplemental Figure 1. Metabolic inhibition and exogenous ROS perturb cellular reducing potential.

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Supplemental Figure 5. Acute IR effects on reducing potential and lactate can be used to detect intrinsic radioresistance driven by loss of p53 activity.

Supplemental Figure 4. Lactate accumulates in extra-cellular media as a function of cellular metabolism over time.

Supplemental Figure 3. IR effects on cellular lactate and clonogenic survival are driven by ROS.

Supplemental Figure 2. Association of YAP1 and CDK6 in chemo/radiation resistant tissues and cells.

Supplementary Figure S5. Downregulation of MCl-1, XIAP and their cleavage by AZD-1775 in HPV+ HNSCC cells are caspase-dependent;and Supplementary Figure S6. mRNA levels of MCl-1, XIAP and stable forced expression of MCl-1 in HPV+ HNSCC cells.

AZD-1775 as a single agent shows antitumor activity in HPV+ patient derived xenografts xenograft (PDX).

Supplementary Data from p16 Represses DNA Damage Repair via a Novel Ubiquitin-Dependent Signaling Cascade

Supplemental Figure 1. Correlation of YAP1 and CDK6 in 78 cases of ESCC and 71 cases of EAC from TCGA database.

Purpose:Esophageal cancer is a lethal disease that is often resistant to therapy. Alterations of YAP1 and CDK6 are frequent in esophageal cancer. Deregulation of both molecules may be responsible for therapy resistance.Experimental Design:Expressions of YAP1 and CDK6 were examined in esophageal cancer cells and tissues using immunoblotting and immunohistochemistry. YAP1 expression was induced in esophageal cancer cells to examine YAP1-mediated CDK6 activation and its association with radiation resistance. Pharmacologic and genetic inhibitions of YAP1 and CDK6 were performed to dissect the mechanisms and assess the antitumor effects in vitro and in vivo.Results:YAP1 expression was positively associated with CDK6 expression in resistant esophageal cancer tissues and cell lines. YAP1 overexpression upregulated CDK6 expression and transcription, and promoted radiation resistance, whereas treatment with the YAP1 inhibitor, CA3, strongly suppressed YAP1 and CDK6 overexpression, reduced Rb phosphorylation, as well as sensitized radiation-resistant/YAP1high esophageal cancer cells to irradiation. CDK4/6 inhibitor, LEE011, and knock down of CDK6 dramatically inhibited expression of YAP1 and sensitized resistant esophageal cancer cells to irradiation indicating a positive feed-forward regulation of YAP1 by CDK6. In addition, suppression of both the YAP1 and CDK6 pathways by the combination of CA3 and LEE011 significantly reduced esophageal cancer cell growth and cancer stem cell population (ALDH1+ and CD133+), sensitized cells to irradiation, and showed a strong antitumor effect in vivo against radiation-resistant esophageal cancer cells.Conclusions:Our results document that a positive crosstalk between the YAP1 and CDK6 pathways plays an important role in conferring radiation resistance to esophageal cancer cells. Targeting both YAP1 and CDK6 pathways could be a novel therapeutic strategy to overcome resistance in esophageal cancer.

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Purpose: This study examined the potential role of the nuclear deubiquitinating enzyme BRCA1-associated protein-1 (BAP1) in radioresistance in head and neck squamous cell cancer (HNSCC).Experimental Design: We overexpressed, knocked down, and rescued BAP1 expression in six HNSCC cell lines, three human papillomavirus (HPV)–negative and three HPV-positive, and examined the effects on radiosensitivity in vitro and in an HNSCC mouse xenograft model. Radiosensitivity was assessed by clonogenic cell survival and tumor growth delay assays; changes in protein expression were analyzed by immunofluorescence staining and Western blotting. We also analyzed The Cancer Genome Atlas HNSCC database to test for associations between BAP1 expression and outcome in patients.Results: Overexpression of BAP1 induced radioresistance in both cell lines and xenograft models; conversely, BAP1 knockdown led to increased ubiquitination of histone H2A, which has been implicated in DNA repair. We further found that BAP1 depletion suppressed the assembly of constitutive BRCA1 foci, which are associated with homologous recombination (HR), but had minimal effect on γ-H2AX foci and did not affect proteins associated with nonhomologous end joining, suggesting that BAP1 affects radiosensitivity in HNSCC by modifying HR. Finally, in patients with HNSCC, overexpression of BAP1 was associated with higher failure rates after radiotherapy.Conclusions: BAP1 can induce radioresistance in HNSCC cells, possibly via deubiquitination of H2Aub and modulation of HR, and was associated with poor outcomes in patients with HNSCC. BAP1 may be a potential therapeutic target in HNSCC. Clin Cancer Res; 24(3); 600–7. ©2017 AACR.

Supplemental Figure 4. Anti-tumor effects of combination inhibition of YAP1 and CDK6 as well as radiation therapy in mice bearing radiation resistant tumors.

Squamous cell carcinoma driven by human papillomavirus (HPV) is more sensitive to DNA-damaging therapies than its HPV-negative counterpart. Here, we show that p16, the clinically used surrogate for HPV positivity, renders cells more sensitive to radiotherapy via a ubiquitin-dependent signaling pathway, linking high levels of this protein to increased activity of the transcription factor SP1, increased HUWE1 transcription, and degradation of ubiquitin-specific protease 7 (USP7) and TRIP12. Activation of this pathway in HPV-positive disease led to decreased homologous recombination and improved response to radiotherapy, a phenomenon that can be recapitulated in HPV-negative disease using USP7 inhibitors in clinical development. This p16-driven axis induced sensitivity to PARP inhibition and potentially leads to “BRCAness” in head and neck squamous cell carcinoma (HNSCC) cells. Thus, these findings support a functional role for p16 in HPV-positive tumors in driving response to DNA damage, which can be exploited to improve outcomes in both patients with HPV-positive and HPV-negative HNSCC.Significance:In HPV-positive tumors, a previously undiscovered pathway directly links p16 to DNA damage repair and sensitivity to radiotherapy via a clinically relevant and pharmacologically targetable ubiquitin-mediated degradation pathway.

Purpose: Mortality of patients with head and neck squamous cell carcinoma (HNSCC) is primarily driven by tumor cell radioresistance leading to locoregional recurrence (LRR). In this study, we use a classification of TP53 mutation (disruptive vs. nondisruptive) and examine impact on clinical outcomes and radiation sensitivity.Experimental Design: Seventy-four patients with HNSCC treated with surgery and postoperative radiation and 38 HNSCC cell lines were assembled; for each, TP53 was sequenced and the in vitro radioresistance measured using clonogenic assays. p53 protein expression was inhibited using short hairpin RNA (shRNA) and overexpressed using a retrovirus. Radiation-induced apoptosis, mitotic cell death, senescence, and reactive oxygen species (ROS) assays were carried out. The effect of the drug metformin on overcoming mutant p53-associated radiation resistance was examined in vitro as well as in vivo, using an orthotopic xenograft model.Results: Mutant TP53 alone was not predictive of LRR; however, disruptive TP53 mutation strongly predicted LRR (P = 0.03). Cell lines with disruptive mutations were significantly more radioresistant (P < 0.05). Expression of disruptive TP53 mutations significantly decreased radiation-induced senescence, as measured by SA-β-gal staining, p21 expression, and release of ROS. The mitochondrial agent metformin potentiated the effects of radiation in the presence of a disruptive TP53 mutation partially via senescence. Examination of our patient cohort showed that LRR was decreased in patients taking metformin.Conclusions: Disruptive TP53 mutations in HNSCC tumors predicts for LRR, because of increased radioresistance via the inhibition of senescence. Metformin can serve as a radiosensitizer for HNSCC with disruptive TP53, presaging the possibility of personalizing HNSCC treatment. Clin Cancer Res; 18(1); 290–300. ©2011 AACR.

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Purpose: The primary cause of death due to head and neck squamous cell carcinoma (HNSCC) is local treatment failure. The goal of this study was to examine this phenomenon using an unbiased approach.Experimental Design: We utilized human papilloma virus (HPV)-negative cell lines rendered radiation-resistant (RR) via repeated exposure to radiation, a panel of HPV-negative HNSCC cell lines and three cohorts of HPV-negative HNSCC tumors (n = 68, 97, and 114) from patients treated with radiotherapy and subjected to genomic, transcriptomic, and proteomic analysis.Results: RR cell lines exhibited upregulation of several proteins compared with controls, including increased activation of Axl and PI3 kinase signaling as well as increased expression of PD-L1. Additionally, inhibition of either Axl or PI3 kinase led to decreased PD-L1 expression. When clinical samples were subjected to RPPA and mRNA expression analysis, PD-L1 was correlated with both Axl and PI3K signaling as well as dramatically associated with local failure following radiotherapy. This finding was confirmed examining a third cohort using immunohistochemistry. Indeed, tumors with high expression of PD-L1 had failure rates following radiotherapy of 60%, 70%, and 50% compared with 20%, 25%, and 20% in the PD-L1–low expression group (P = 0.01, 1.9 × 10−3, and 9 × 10−4, respectively). This finding remained significant on multivariate analysis in all groups. Additionally, patients with PD-L1 low/CD8+ tumor-infiltrating lymphocytes high had no local failure or death due to disease (P = 5 × 10−4 and P = 4 × 10−4, respectively).Conclusions: Taken together, our data point to a targetable Axl–PI3 kinase–PD-L1 axis that is highly associated with radiation resistance. Clin Cancer Res; 23(11); 2713–22. ©2017 AACR.

Purpose: Although the majority of patients with HPV+ oropharyngeal cancers have a favorable prognosis, there are some patients with tumors that are resistant to aggressive chemoradiotherapy with unusual patterns of locoregional and systemic recurrences. Therefore, more effective therapies are needed. In this study, we investigated the chemosensitizing efficacy of the selective Wee-1 kinase inhibitor, AZD-1775, in HPV+ head and neck squamous cell carcinoma (HNSCC).Experimental Design: Clonogenic survival assays and an orthotopic mouse model of HPV+ oral cancer were used to examine the in vitro and in vivo sensitivity of HPV+ HNSCC cell lines to AZD-1775 in combination with cisplatin, respectively. Cell-cycle analysis, DNA damage (γH2AX), homologous recombination (HR), and apoptosis were examined to dissect molecular mechanisms.Results: We found that AZD-1775 displays single-agent activity and enhances the response of HPV+ HNSCC cells to cisplatin both in vitro and in vivo. The sensitivity of the HPV+ HNSCC cells to AZD-1775 alone or in combination with cisplatin was associated with G2 checkpoint abrogation, persistent DNA damage, and apoptosis induction. This finding of AZD-1775 increasing the sensitivity of HPV+ HNSCC cells to cisplatin through apoptosis was not seen previously in the HPV− HNSCC cancer cells and is accompanied by a decreased expression of the antiapoptotic proteins, MCl-1and XIAP, which appear to be cleaved following AZD-1775 treatment.Conclusions: AZD-1775 selectively sensitizes HPV+ HNSCC cells and orthotopic oral xenografts to cisplatin through apoptosis and support the clinical investigation of AZD-1775 in combination with cisplatin particularly in patients with advanced and recurrent metastatic HPV+ HNSCC tumors. Clin Cancer Res; 21(21); 4831–44. ©2015 AACR.

Supplemental data

Purpose: Head and neck squamous cell carcinoma (HNSCC) is commonly treated with radiotherapy, and local failure after treatment remains the major cause of disease-related mortality. To date, human papillomavirus (HPV) is the only known clinically validated, targetable biomarkers of response to radiation in HNSCC.Experimental Design: We performed proteomic and transcriptomic analysis of targetable biomarkers of radioresistance in HPV-negative HNSCC cell lines in vitro, and tested whether pharmacologic blockade of candidate biomarkers sensitized cells to radiotherapy. Candidate biomarkers were then investigated in several independent cohorts of patients with HNSCC.Results: Increased expression of several targets was associated with radioresistance, including FGFR, ERK1, EGFR, and focal adhesion kinase (FAK), also known as PTK2. Chemical inhibition of PTK2/FAK, but not FGFR, led to significant radiosensitization with increased G2–M arrest and potentiated DNA damage. PTK2/FAK overexpression was associated with gene amplification in HPV-negative HNSCC cell lines and clinical tumors. In two independent cohorts of patients with locally advanced HPV-negative HNSCC, PTK2/FAK amplification was highly associated with poorer disease-free survival (DFS; P = 0.012 and 0.034). PTK2/FAK mRNA expression was also associated with worse DFS (P = 0.03). Moreover, both PTK2/FAK mRNA (P = 0.021) and copy number (P = 0.063) were associated with DFS in the Head and Neck Cancer subgroup of The Cancer Genome Atlas.Conclusions: Proteomic analysis identified PTK2/FAK overexpression is a biomarker of radioresistance in locally advanced HNSCC, and PTK2/FAK inhibition radiosensitized HNSCC cells. Combinations of PTK2/FAK inhibition with radiotherapy merit further evaluation as a therapeutic strategy for improving local control in HPV-negative HNSCC. Clin Cancer Res; 22(18); 4643–50. ©2016 AACR.

Supplementary Figure 1 HN31 cells with BAP1 vector control, knockdown, and wild-type variants were immunoblotted for the expression of various proteins associated with the DNA damage response (both homologous recombination [BAP1] and non-homologous end-rejoining [CHK1, CHK2, p53]) after a 4Gy dose of radiation. BAP1 knockdown did not affect levels of NHEJ proteins. Supplementary Table 1: Antibody information Supplementary Table 2: Cell line information

Supplemental Figure 3. Combination inhibition of YAP1 and CDK6 eradicate CSCs population in radiation resistant cells A&B.

Supplementary Figure Legends

Analysis of combined drug effects; Immunofluorescence; Caspase-Glo 3/7 activity assay; Quantitative reverse-transcriptase PCR (qRT-PCR); Orthotopic Mouse Model of Oral Cavity Cancer and Tumor Growth Delay; Generation of patient-derived HNSCC xenografts and treatment with AZD-1775; Immunohistochemistry; and In vivo TUNEL assay.

Supplemental methods Supplementary Figure 1. Total and phospho-PTK2/FAK is overexpressed in selected radioresistant HNSCC cells compared to radiosensitive cell lines via immunoblot. Supplementary Figure 2. A.) Inhibition of PTK2/FAK expression via stable expression of shRNA to PTK2/FAK in HN31 HNSCC cells. B.) Stable inhibition of PTK2/FAK leads to radiosensitization in vitro. C & D.) The combination of PF562271 and radiation leads to C.) increased γ-H2AX expression and D.) higher levels of G2/M arrest and sub-G1 staining in Fadu HNSCC cells. Supplementary Figure 3. A & B.) PTK2/FAK is highly expressed in a large public database of HNSCC cell lines (www.oncomine.com) (A.) and tumor specimens (TCGA) (B.) compared to other tumor types. Supplementary Table 1. HNSCC cell lines included in RPPA. Supplementary Table 2. Tumor characteristics of the institutional cohorts. Supplementary Table 3. Univariate and multivariate analysis of PTK2/FAK copy number and disease free survival (DFS) in the two institutional cohorts and the Head and Neck Cancer TCGA cohort. NA - not included in the model. HR - Hazard ratio. 95% CI- 95% Confidence Interval. Supplementary Table 4. Tumor characteristics of the HNSCC TCGA cohort.

Supplemental Methods. Description of additional methods and procedures used in the study.

TP53 is the most frequently altered gene in head and neck squamous cell carcinoma (HNSCC), with mutations occurring in over two thirds of cases; however, the predictive response of these mutations to cisplatin-based therapy remains elusive. In the current study, we evaluate the ability of the Evolutionary Action score of TP53-coding variants (EAp53) to predict the impact of TP53 mutations on response to chemotherapy. The EAp53 approach clearly identifies a subset of high-risk TP53 mutations associated with decreased sensitivity to cisplatin both in vitro and in vivo in preclinical models of HNSCC. Furthermore, EAp53 can predict response to treatment and, more importantly, a survival benefit for a subset of head and neck cancer patients treated with platinum-based therapy. Prospective evaluation of this novel scoring system should enable more precise treatment selection for patients with HNSCC. Cancer Res; 75(7); 1205–15. ©2015 AACR.

Supplementary Table 1: TP53 mutations scored and stratified by EAp53 and Poeta classification in the platinum based induction chemotherapy cohort.