Search

Purpose: Cisplatin (CDDP)-based chemotherapy is a first-line treatment for patients with advanced head and neck squamous cell carcinomas (HNSCC), despite a high rate of treatment failures, acquired resistance, and subsequent aggressive behavior. The purpose of this study was to study the mechanism of CDDP resistance and metastasis in HNSCC. We investigated the role of NRF2 pathway activation as a driven event for tumor progression and metastasis of HNSCC. Experimental Design: Human HNSCC cell lines that are highly resistant to CDDP were generated. Clonogenic survival assays and a mouse model of oral cancer were used to examine the impact of NRF2 activation in vitro and in vivo on CDDP sensitivity and development of metastasis. Western blotting, immunostaining, whole-exome sequencing, single-cell transcriptomic and epigenomic profiling platforms were performed to dissect clonal evolution and molecular mechanisms. Results: Implantation of CDDP-resistant HNSCC cells into the tongues of nude mice resulted in a very high rate of distant metastases. The CDDP-resistant cells had significantly higher expression of NRF2 pathway genes in the presence of newly acquired KEAP1 mutations, or via epigenomic activation of target genes. Knockdown of NRF2 or restoration of the wild-type KEAP1 genes resensitized resistant cells to CDDP and decreased distant metastasis (DM). Finally, treatment with inhibitor of glutaminase-1, a NRF2 target gene, alleviated CDDP resistance. Conclusions: CDDP resistance and development of DM are associated with dysregulated and epigenetically reprogrammed KEAP1–NRF2 signaling pathway. A strategy targeting KEAP1/NRF2 pathway or glutamine metabolism deserves further clinical investigation in patients with CDDP-resistant head and neck tumors.

Purpose: In oropharyngeal squamous cell carcinoma (OPC), high CD8+ tumor-infiltrating lymphocyte (CD8+TIL) density confers improved prognosis. We compared neoadjuvant durvalumab (PD-L1 inhibitor) with durvalumab + tremelimumab (CTLA-4 inhibitor) in terms of impact on CD8+TIL density, safety, and efficacy in patients with OPC. Patients and Methods: Patients with newly diagnosed stage II–IVA OPC or locoregionally recurrent OPC amenable to resection were included. Patients were randomized to two cycles of durvalumab or durvalumab + tremelimumab before surgery. The primary endpoint was change between baseline and resection specimen in CD8+TIL density between arms. Secondary endpoints included safety, response rate per RECIST, major pathologic response (MPR; ≤10% viable tumor cells) rate, and patient-reported outcomes. Results: Of 28 eligible patients (14/arm), 20 (71%) had newly diagnosed OPC, and 24 (86%) were p16-positive. The posttreatment to pretreatment median CD8+TIL density ratio was 1.31 for durvalumab and 1.15 for combination treatment (P = 0.97; 95% CI: −1.07–2.28). In each group, 6 patients (43%, 95% CI: 17.66–71.14) had a response. Eight patients (29%) had a MPR at the primary tumor and/or nodal metastases. Neither baseline CD8+TIL density nor PD-L1 expression level correlated with overall response, but a trend toward greater CD8+TIL change in patients with a MPR was seen (P = 0.059; 95% CI: −0.33–3.46). Four patients (14%) had grade ≥3 adverse events. At median follow-up time of 15.79 months, all patients were alive, and one had an additional recurrence. Conclusions: Durvalumab + tremelimumab did not increase CD8+TIL density more than durvalumab alone did. The observed safety and activity support further investigation of neoadjuvant checkpoint inhibitor for OPC.

Purpose: TP53 mutations are highly prevalent in head and neck squamous cell carcinoma (HNSCC) and associated with increased resistance to conventional treatment primarily consisting of chemotherapy and radiation. Restoration of wild-type p53 function in TP53-mutant cancer cells represents an attractive therapeutic approach and has been explored in recent years. In this study, the efficacy of a putative p53 reactivator called COTI-2 was evaluated in HNSCC cell lines with different TP53 status. Experimental Design: Clonogenic survival assays and an orthotopic mouse model of oral cancer were used to examine in vitro and in vivo sensitivity of HNSCC cell lines with either wild-type, null, or mutant TP53 to COTI-2 alone, and in combination with cisplatin and/or radiation. Western blotting, cell cycle, live-cell imaging, RNA sequencing, reverse-phase protein array, chromatin immunoprecipitation, and apoptosis analyses were performed to dissect molecular mechanisms. Results: COTI-2 decreased clonogenic survival of HNSCC cells and potentiated response to cisplatin and/or radiation in vitro and in vivo irrespective of TP53 status. Mechanistically, COTI-2 normalized wild-type p53 target gene expression and restored DNA-binding properties to the p53-mutant protein in HNSCC. In addition, COTI-2 induced DNA damage and replication stress responses leading to apoptosis and/or senescence. Furthermore, COTI-2 lead to activation of AMPK and inhibition of the mTOR pathways in vitro in HNSCC cells. Conclusions: COTI-2 inhibits tumor growth in vitro and in vivo in HNSCC likely through p53-dependent and p53-independent mechanisms. Combination of COTI-2 with cisplatin or radiation may be highly relevant in treating patients with HNSCC harboring TP53 mutations.

Purpose: Development of extranodal extension (ENE) has been associated with poor survival in patients with oral cavity squamous cell carcinoma (OSCC). Here, we sought to confirm the role of ENE as a poor prognostic factor, and identify genomic and epigenetic markers of ENE in order to develop a predictive model and improve treatment selection. Experimental Design: An institutional cohort (The University of Texas MD Anderson Cancer Center) was utilized to confirm the impact of ENE on clinical outcomes and evaluate the genomic signature of primary and ENE containing tissue. OSCC data from The Cancer Genome Atlas (TCGA) were analyzed for the presence of molecular events associated with nodal and ENE status. Results: ENE was associated with decreased overall and disease-free survival. Mutation of the TP53 gene was the most common event in ENE+ OSCC. The frequency of TP53 mutation in ENE+ tumors was higher compared with ENE− tumors and wild-type (WT) TP53 was highly represented in pN0 tumors. pN+ENE+ patients had the highest proportion of high-risk TP53 mutations. Both primary tumors (PT) and lymph nodes with ENE (LN) exhibited a high rate of TP53 mutations (58.8% and 58.8%, respectively) with no significant change in allele frequency between the two tissue sites. Conclusions: ENE is one of the most significant markers of OSCC OS and DFS. There is a shift toward a more aggressive biological phenotype associated with high-risk mutations of the TP53 gene. Prospective clinical trials are required to determine whether TP53 mutational status can be used for personalized treatment decisions. Clin Cancer Res; 24(7); 1727–33. ©2018 AACR.

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: 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.

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.

Background: Sinonasal undifferentiated carcinoma (SNUC) is a rare, highly aggressive cancer that arises in the nasal cavity and paranasal sinuses. Despite aggressive multimodal therapy the prognosis remains poor. Because of the locally advanced nature of SNUC, we have used cisplatin-based induction chemotherapy before definitive surgery or radiation therapy in our institution. Unfortunately, about 30% of SNUC patients do not respond to this treatment, and the lack of response is associated with poor survival. Therefore, developing strategies to overcome resistance to induction chemotherapy is crucial. Our current gene expression analysis of SNUC specimens indicated that the TGFβ signaling pathway might be activated in nonresponders. Using the MDA8788-6 SNUC cell line established from a nonresponder, we found that TGFβ signaling is upregulated in an autocrine manner in the SNUC cell line. Since inhibition of the TGFβ pathway overcame cisplatin resistance in this cell line, we decided to examine the effects of TGFβ inhibition in vivo. Materials and Methods: The MDA8788-6 SNUC cell line were cultured under serum-free condition overnight to induce TGFβ signaling. The cells were then resuspended in the serum-free conditioned medium collected from the same cell line to maintain activation of the pathway. Two million MDA8788-6 cells in the conditioned medium were inoculated into the flank of mice subcutaneously. On the same day of the inoculation, we started treatment with i) vehicle control, ii) cisplatin only (4 mg/kg, once per week, intravenously), iii) EW-7197 only (TGFβRI specific inhibitor, 40 mg/kg, five times per week, oral gavage), and iv) cisplatin plus EW-7197. After 5 courses of treatments, the mice were sacrificed. Average tumor volume of each treatment group 31 days after the cell inoculation was compared by Tukey’s multiple comparisons test. Area under the curve (AUC) was calculated according to the tumor growth curve of each mouse from 0 to 31 days after the cell inoculation. AUC of CDDP group and CDDP plus EW-7197 group was compared by unpaired, two-tailed t test with Welch’s correction. Results: The EW-7197 treated group itself did not show antitumor growth effect compared to the control group. Comparison of AUC of the tumor volume showed significant difference between the cisplatin alone and cisplatin plus EW-7197; the combination of cisplatin and EW-7197 had better antitumor growth effect compared to cisplatin alone. Conclusions: Our finding suggests that TGFβ inhibition may offer hope to SNUC patients struggling with chemoresistance. Citation Format: Yoko Takahashi, Hideaki Takahashi, Moran Amit, Frederico O. Gleber-Netto, Diana Bell, Tong-Xin Xie, Dianna Roberts, Ahmed S. Abdelmeguid, Curtis Pickering, Jeffrey N. Myers, Ehab Y. Hanna. Overcoming cisplatin resistance in sinonasal undifferentiated carcinoma by inhibiting the TGFβ signaling pathway in vivo [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr B08.

Background: Standard treatment for head and neck squamous cell carcinoma (HNSCC) includes platinum-based therapy given in combination with radiation. Although platinum has been a key drug of HNSCC treatment for decades, many patients develop resistance and the prognosis is unsatisfactory, and therefore development of novel treatment strategies is needed. Targeting ATR has been shown to be effective as a monotherapy in various type of cancers. Thus, inhibition of ATR could be a new approach when combined with DNA damaging agents to kill cancer cells. Here, we hypothesize that addition of ATR inhibitor AZD6738 can enhance the antitumor effects of cisplatin in HNSCC cells and result in better survival in preclinical models of HNSCC. Materials and Methods: Clonogenic survival assays, reverse phase protein array (RPPA), Western blotting, cell cycle analysis, and apoptosis assay were used to evaluate the in vitro sensitivity of HNSCC cells to AZD6738 and cisplatin and the molecular mechanisms associated with antitumor response. An orthotopic mouse model of oral cancer was used to explore in vivo efficacy of the combination treatment. Results: HNSCC cells treated with various concentrations of AZD6738 showed an IC50 value of 0.24-3.00 μmol/L and no differential sensitivity was observed among cells with different TP53 mutational status. Treatment with constant drug concentration ratios of AZD6738 and cisplatin (2:1) resulted in a combination index of Conclusions: Addition of AZD6738 synergizes with cisplatin in vitro and potentiates cisplatin response in vivo. Mechanistically, combination of cisplatin and AZD6738 induces DNA damage and replication stress, resulting in apoptotic cell death. Interestingly, proteins essential for the origin licensing as part of the DNA replication initiation machinery were decreased following combination treatment, offering a potential mechanistic insight and warranting further investigation. Citation Format: Antje Lindemann, Hideaki Takahashi, Ameeta A. Patel, Walter N. Hittelman, Abdullah A. Osman, Jeffrey N. Myers. Inhibition of ATR as a therapeutic strategy to overcome cisplatin resistance in HNSCC [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr PR08.

Variations in the molecular function of human papillomavirus (HPV) have been associated with outcome in HPV-positive oropharyngeal squamous cell carcinomas (HPV+OPSCC). HPV-mediated carcinogenesis is initiated by the E6 and E7 proteins, which promotes the inhibition of the p53 and pRb proteins by targeting them for degradation by the proteasome system. Considering that the proteasome system is an important mediator of HPV molecular activity, we hypothesize that its activity may be an important modifier of HPV+OPSCC phenotypes. To test this hypothesis, we analyzed the in silico expression of proteasome genes among HPV+ oropharyngeal squamous cell carcinoma cases from The Cancer Genome Atlas (TCGA) and explored the proteasome activity of HPV+ squamous cell carcinoma cell lines in vitro. Higher expression of proteasome genes was significantly associated with worse 5-year overall survival (p=0.0003), lower expression levels of E1^E4 gene (p = 0.032), and more HPV integration (p = 0.028). In vitro analysis indicated that proteasome activity was distinct between E1^E4 negative and E1^E4 positive HPV+ cell lines. Interestingly, E1^E4 negative cell lines, which exhibited higher levels of some proteasome genes, were more sensitive to proteasome inhibition than E1^E4 positive cells. These results indicate that proteasome activity may vary significantly among HPV+ OPSCC cases and may represent an important modulator of tumor phenotypes. Expression of proteasome genes and also proteasome activity was highly correlated with expression of HPV genes and may function as a potential predictive biomarker. Finally, proteasome targeting may represent a potential therapeutic alternative for a subgroup of HPV+ OPSCC patients. Citation Format: Frederico O. Gleber-Netto, Meng Gao, Li Shen, Jing Wang, Jeffrey N. Myers, Faye M. Johnson, Curtis R. Pickering. Variations in the proteasome activity are associated with human papilloma virus (HPV) gene expression and patient survival in HPV+ oropharyngeal tumors [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr B14.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer in the world, with 500,000 new diagnoses and 300,000 cancer-related deaths worldwide annually. Despite 50 years of basic, clinical, and translational research, survival rates for patients with HNSCC have not remarkably improved, indicating a need for identification of new targets and biomarkers to guide therapy. Recent sequencing data from our group and others have identified Caspase-8 (CASP8) as one of the most frequently mutated genes in HNSCCs, with CASP8 mutations occurring in ~10% of the primary tumors. Genomics analysis using The Cancer Genome Atlas (TCGA) HNSCC dataset revealed that CASP8 mutations are associated with poorer overall patient survival. CASP8 belongs to the caspase family of proteases and has important roles in both apoptotic and necroptotic death. Our goal was to understand CASP8 signaling in HNSCC and examine whether those pathways could be exploited therapeutically. CASP8 serves as the apical-most caspase for the extrinsic apoptosis pathway, initiating programmed cell death following death receptor ligation. Cells deficient in Caspase-8 show resistance to death-receptor mediated killing in vitro and in vivo. Intriguingly, however, knockout of CASP8 in some tissues promotes cell death through induction of necroptosis, a.k.a programmed necrosis. Necroptosis is mediated by receptor-interacting protein kinases RIP1, RIP3 and their substrate mixed lineage kinase-like (MLKL) and often occurs following activation of tumor necrosis factor alpha (TNF-alpha) signaling. CASP8 has been shown to cleave and inhibit RIP1 and RIP3, thereby suppressing necroptosis. We hypothesized that mutation or loss of CASP8 could promote necroptosis and modulation of the pathway with small-molecule agents might have the potential for clinical utility. We found that deletion of CASP8 rendered HNSCCs susceptible to necroptosis induced by second mitochondria-derived activator of caspase (SMAC) mimetic treatment (TL-32711) when combined with TNF-alpha and Z-VAD-FMK, a pancaspase inhibitor, in vitro. Inhibition of Caspase-8 function was also associated with enhanced necroptotic killing by ionizing radiation in combination with SMAC mimetic treatment in vitro. We further demonstrated that the level of RIP3 expression determines susceptibility to SMAC mimetic-induced necroptosis in HNSCCs. In conclusion, CASP8 is an important regulator of cell death pathways in HNSCC. Modulation of these pathways can enhance the efficacy of radiotherapy and has the potential for clinical translation. Ongoing studies will give further insight into how CASP8 regulates necroptosis in HNSCCs and provide preclinical justification for use of these pathways as a therapeutic target in HNSCC patients. Citation Format: Burak Uzunparmak, Jeffrey N. Myers, Curtis R. Pickering. Loss of caspase-8 renders head and neck squamous cell carcinomas susceptible to necroptosis [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr B18.

Purpose: Epidemiologic studies have identified an increasing incidence of squamous cell carcinoma of the oral tongue (SCCOT) in younger patients. Experimental Design: DNA isolated from tongue tumors of young (45 years) patients at was subjected to whole-exome sequencing and copy-number analysis. These data were compared with data from similar patients in the TCGA (The Cancer Genome Atlas) project. Results: In this study, we found that gene-specific mutation and copy-number alteration frequencies were similar between young and old patients with SCCOT in two independent cohorts. Likewise, the types of base changes observed in the young cohort were similar to those in the old cohort even though they differed in smoking history. TCGA data also demonstrate that the genomic effects of smoking are tumor site–specific, and we find that smoking has only a minor impact on the types of mutations observed in SCCOT. Conclusions: Overall, tumors from young patients with SCCOT appear genomically similar to those of older patients with SCCOT, and the cause for the increasing incidence of young SCCOT remains unknown. These data indicate that the functional impact of smoking on carcinogenesis in SCCOT is still poorly understood. Clin Cancer Res; 20(14); 3842–8. ©2014 AACR.

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.

Purpose: We investigated whether vandetanib, an inhibitor of the tyrosine kinase activities of vascular endothelial growth factor receptor-2 (VEGFR-2), epidermal growth factor receptor (EGFR), and rearranged during transfection (RET), could augment the antitumor activity of radiation with or without cisplatin in preclinical in vitro and in vivo models of human head and neck squamous cell carcinoma (HNSCC). Experimental Design: OSC-19 and HN5 HNSCC cells that were cisplatin and radioresistant were treated with vandetanib, cisplatin, and radiation alone or in combination in vitro and in vivo using an orthotopic nude mouse model. Treatment effects were assessed using clonogenic survival assay, tumor volume, bioluminescence imaging, tumor growth delay, survival, microvessel density, tumor and endothelial cell apoptosis, and EGFR and Akt phosphorylation data. Results: Vandetanib plus cisplatin radiosensitized HNSCC cells in vitro and in vivo. The combination treatment with vandetanib, cisplatin, and radiation was superior to the rest of treatments (including the double combinations) in antitumoral effects, prolonging survival, decreasing cervical lymph node metastases in vivo. It also increased both tumor and tumor-associated endothelial cell apoptosis and decreased microvessel density in vivo. An analysis of tumor growth delay data revealed that vandetanib plus cisplatin enhanced radioresponse in vivo. All vandetanib-containing treatments inhibited EGFR and Akt phosphorylation in vitro and in vivo. Conclusion: The addition of vandetanib to combination therapy with cisplatin and radiation was able to effectively overcome cisplatin and radioresistance in in vitro and in vivo models of HNSCC. Further study of this regimen in clinical trials may be warranted. Clin Cancer Res; 17(7); 1815–27. ©2011 AACR.

Purpose: Aberrant expression of inflammatory molecules, such as inducible nitric oxide (NO) synthase (iNOS), has been linked to cancer, suggesting that their inhibition is a rational therapeutic approach. Whereas iNOS expression in melanoma and other cancers is associated with poor clinical prognosis, in vitro and in vivo studies suggest that iNOS and NO can have both protumor and antitumor effects. We tested the hypothesis that targeted iNOS inhibition would interfere with human melanoma growth and survival in vivo in a preclinical model. Experimental Design: We used an immunodeficient non-obese diabetic/severe combined immunodeficient xenograft model to test the susceptibility of two different human melanoma lines to the orally-given iNOS-selective small molecule antagonist N6-(1-iminoethyl)-l-lysine-dihydrochloride (L-nil) with and without cytotoxic cisplatin chemotherapy. Results: L-nil significantly inhibited melanoma growth and extended the survival of tumor-bearing mice. L-nil treatment decreased the density of CD31+ microvessels and increased the number of apoptotic cells in tumor xenografts. Proteomic analysis of melanoma xenografts with reverse-phase protein array identified alterations in the expression of multiple cell signaling and survival genes after L-nil treatment. The canonical antiapoptotic protein Bcl-2 was downregulated in vivo and in vitro after L-nil treatment, which was associated with increased susceptibility to cisplatin-mediated tumor death. Consistent with this observation, combination therapy with L-nil plus cisplatin in vivo was more effective than either drug alone, without increased toxicity. Conclusions: These data support the hypothesis that iNOS and iNOS-derived NO support tumor growth in vivo and provide convincing preclinical validation of targeted iNOS inhibition as therapy for solid tumors. Clin Cancer Res; 16(6); 1834–44

Head and neck squamous cell carcinoma (HNSCC) is a leading cause of cancer deaths worldwide, and resistance to standard treatments with radiation/chemotherapy accounts for the majority of HNSCC deaths. Mutation of TP53 occurs in 60-85% of non-HPV-associated HNSCC cases, and some mutant p53s display oncogenic properties, termed “gain-of-function (GOF),” which are independent of wild-type (WT) p53 functions. These GOF p53 mutant proteins can enhance cell transformation, increase tumor formation, and confer cellular resistance to chemo- and/or radio-therapy. Therefore, we are interested in the development of innovative therapeutic approaches to overcome this resistance. Pre-clinical studies both in vitro and in vivo have shown the promise of some novel targeted agents including Chk-1, Wee-1, and PARP inhibitors used alone or in combination with HDAC inhibition. In addition, COTI-2 is a novel drug with striking single-agent and radio-/chemo-sensitizing activity in pre-clinical models of HNSCC bearing TP53 mutations. Our preliminary data demonstrate that COTI-2 markedly decreases cell growth in vitro and induces p21, noxa, and puma expression in HNSCC cells. COTI-2 also negatively modulates the PI3K/AKT/mTOR survival pathway, which is constitutively activated in many types of cancer including HNSCC. In vitro studies in our laboratory have shown that COTI-2 has a nanomolar (IC50: 1.3-13.2 nm) activity in nine human HNSCC cell lines. Additionally, GOF mutant p53-bearing HNSCC cells are significantly delayed in outgrowth in an orthotopic nude mouse model of tongue cancer when treated with COTI-2. Current clinical studies are evaluating the safety and efficacy of the combination of COTI-2 alone and in combination with cisplatin in phase I/II studies of patients with refractory HNSCC and whether response is associated with TP53 mutational status of the tumor determined from sequencing of circulating cell free DNA. If this combination is found to safely provide sensitization of HNSCC to cisplatin-based chemotherapy in the majority of human patients who have TP 53 mutant tumors, this should make a major impact on the survival of individuals suffering from HNSCC, and potentially other p53 driven epithelial malignancies. Citation Format: Jeffrey Myers. Personalizing HNSCC treatment based on TP53 mutational status [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr IA08.

Integrated genomic analysis of head and neck squamous cell carcinoma (HNSCC) has identified a heterogeneous spectrum of alterations dominated by tumor suppressor genes. An interesting new candidate driver gene is Caspase-8 (CASP8), which is mutated in 18% of oral cavity HPV-negative tumors. CASP8 is not obviously a part of any well-characterized driver pathway in HNSCC, and its role in this tumor type is unknown. In TCGA genomic data CAPS8 mutant tumors have a unique spectrum of alterations and expression changes, including a dramatic increase in markers of inflammation, which suggest they are a genomic subtype of HNSCC. CAPS8 is a key component of the extrinsic apoptotic pathway, but it also has a role in necrotic death, inflammation, and other pathways. We have characterized human and mouse CASP8 knock-out cell lines in vitro and in vivo in order to begin to understand the function of this gene in HNSCC. Loss of CASP8 function prevents extrinsic apoptosis and alters proliferation and tumorigenicity. It may also regulate the antitumor immune response. CASP8 is an exciting new candidate driver gene in HNSCC that may offer new opportunities for therapeutic intervention. Citation Format: Burak Uzunparmak, Antje Lindemann, Mitchell J. Frederick, Jeffrey N. Myers, Curtis R. Pickering. Caspase-8 is a novel candidate driver gene in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 04.

Comprehensive characterization of genomic alterations prevalent in head and neck squamous cell carcinoma (HNSCC) has been performed by our group and others, and more recently by The Cancer Genome Atlas (TCGA) project. A significant barrier towards targeting the genomic alterations collectively found in HNSCC is that the genomic landscape of this disease is dominated by mutations to tumor suppressor genes that are inherently difficult to target, with very few mutated oncogenes present. Although potentially targetable oncogenic drivers may exist within chromosomal regions of copy number gain, the large number of genes within these regions makes identifying the precise targets challenging. To overcome some of these difficulties, we have been leveraging high throughput loss of function shRNA screens performed in vivo in order to identify co-dependencies and potential ways to target genomic alterations in HNSCC. Using a large panel of genomically characterized established HNSCC cell lines in conjunction with several different shRNA libraries, including ones that target druggable genes (i.e. FDAome), genes involved in DNA repair, and candidate HNSCC driver genes, we hope to link shRNA targets that are vital for in vivo growth and survival to specific genomic subtypes present within HNSCC. Following pooled in vitro infection with these libraries, HNSCC cell lines are injected into the flanks of mice where in vivo selection takes place, and dropout of barcoded shRNAs targeting genes advantageous for tumor growth is subsequently analyzed from harvested tumors by next generation sequencing. The FDAome library contains shRNAs to nearly 200 genes immediately targetable by FDA approved drugs, allowing identification of druggable co-dependent pathways through simple lethality. By comparing dropout of shRNAs from tumors of mice treated with or without carboplatin, genes that chemosensitize tumors in vivo are also being identified within the FDAome and DNA repair shRNA libraries. Screens performed with our custom HNSCC shRNAs library targeting many genes from regions recurrently amplified in large subsets of patient tumors should identify which genes within these regions are true drivers and potentially interesting drug candidates. Thus far, we have completed in vivo screens employing the FDAome and DNA damage libraries on four different HNSCC cell lines, comprised of 3 different NOTCH1 mutants and one NOTCH1 wild type cell line, which collectively include two HPV-positive and two HPV-negative cell lines. Data analysis has identified a number of druggable targets that inhibit the growth of NOTCH1 mutant cell lines or that seem to be specific for HPV+ tumors. Some of these targets have been further validated through in vivo experiments employing single shRNAs to genes of interest, employing targeting sequences distinct from the libraries. The growth inhibitory effects for some of the hits are apparent in vivo, but not in vitro, highlighting the ability of this unique platform to identify genes or pathways that drive the three-dimensional growth of tumors in the microenvironment of the host. We have also completed an in vivo screen on two of the cell lines using the custom shRNA library that targets genes recurrently amplified in HNSCC, and are currently analyzing data. Preliminary analysis indicates that this high throughput in vivo shRNA screening platform is a promising and relevant approach for identifying co-dependencies and ways to target genomic alterations in cancer. Citation Format: Tongxin Xie, Ramya L. Parimi, Manish F. Kumar, Liang P. Yang, Jiping Wang, Sahil Seth, Carugo Alessandro, Christopher A. Bristow, Frederick Scott Robinson, Michael D. Peoples, Heath Skinner, Jeffrey N. Myers, Curtis R. Pickering, Mitchell J. Frederick. An in vivo high throughput shRNA screening platform for identifying ways to target genomic alterations [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr IA12.

To uncover genomic alterations that can be used as molecular markers for oral cavity squamous cell carcinoma (OSCC) in Taiwan, we recruited patients who were ethnically Taiwanese and admitted to Chang Gung Memorial Hospital at Taipei. We performed whole-exome sequencing (WES) of matched tumor/PBMC DNAs from 50 OSCC patients (75-Mbp target region, mean depth = 244 ± 54), and RNA sequencing of matched tumor/adjacent normal tissues from 39 of the 50 patients (mean read count = 38.7 ± 15.2 million). A subset of the identified somatic mutations was validated in 49 tumor tissues using an IonAmpliSeqTM Comprehensive Cancer Panel (CCP; 409 cancer-related genes). In total, our exome sequencing analyses uncovered 33,160 somatic single nucleotide variants (SNVs) and InDels. The targeted approach of CCP enabled us to independently validate 84.91% of the SNVs identified by our whole-exome sequencing. We cross-referenced our data with that of 172 oral cavity cancer samples (OSCC-TCGA) from the Head-Neck Squamous Cell Carcinoma (HNSC) project of TCGA (HNSC-TCGA) and 106 from India (OSCC-India), as annotated in the International Cancer Genome Consortium (ICGC). We found that our patient cohort was characteristic of OSCC with regard to frequently mutated genes, such as CDKN2A, TP53, and NOTCH1. Interestingly, however, our analysis further identified mutations in seven other genes that turned out to be unique, locally prevalent (>10% of patients) alterations. We further used our exome sequencing data to examine copy number alterations in our samples and our findings largely recapitulated the profile found in OSCC-TCGA, which included significantly amplified/deleted regions encompassing genes such as EGFR, FGFR1, FADD, FAT1 and CDKN2A. Comparative analysis of the transcriptomes of tumor and normal tissues allowed us to identify 3,548 genes as being differentially expressed in OSCC-Taiwan; these included 1,220 up-regulated and 2,328 down-regulated genes. Notably, 91.3% of these differentially expressed genes were similarly identified in the OSCC-TCGA data, indicating that the overall expression profile of OSCC is highly similar across ethnic groups. In the meeting, I will report the unique genomic alterations with the potential biomarkers in OSCC, and these findings might introduce a hitherto unknown genetic component of this disease. Citation Format: Kai-Ping Chang, Ting-Wen Chen, Curtis R. Pickering, Jeffrey N. Myers, Yu-Sun Chang. Integrated omics analyses identify prognostic biomarkers of oral cavity squamous cell carcinoma in Taiwan [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr IA10.

Proton radiotherapy (PRT) has shown to be less toxic in the treatment of head and neck cancers (HNSCC) than photon radiotherapy (XRT). XRT causes multiple types of cancer cell death by inducing DNA double-strand breaks. The DNA damage induced by PRT is more complex and has more severe biological consequences versus XRT. The present study was undertaken to uncover the cell death caused by PRT versus XRT in human papillomavirus positive (HPV+, frequently p53 wild-type, p16 positive; p53, p16 are key factors for cell death) and HPV-negative (HPV-, frequently p53 mutant, p16 negative) HNSCC cells. HNSCC cell lines HN5, SqCC/Y1 (HPV-) and UMSCC-47, UPCI-SCC-154 (HPV+) were used. Single doses of 4 Gy irradiation induced cell apoptosis, necrosis, mitotic catastrophe, and senescence were determined. Clinical 200-MeV proton beams (18 cm × 18 cm field) or 6-MV X-ray beams (25 cm × 25 cm field) were used. Cells were positioned in the centers of the irradiation fields (in the middle of the spread-out Bragg peak for PRT). At 4 hours (h), 24h and 48h after PRT or XRT, cell necrosis and apoptosis were determined by Annexin VFITC-conjugated staining. XRT and PRT caused increased cell necrosis in the HPV– SqCC/Y1 (moderate at 24h, significant at 48h [p all < 0.05]) and HN5 (moderate at 24h, 48h) cells, at slightly higher rates in PRT versus XRT treated cells; and in the HPV+ UPCI-SCC-154 (moderate at 4h) and UMSCC-47 (moderate at 48h) cells, with no difference between PRT versus XRT treatment groups. XRT and PRT induced a small percentage of cell apoptosis (range: 1.1 ± 0.5 to 7.6 ± 2.9). Cell apoptosis was only moderately increased in the HPV+ UMSCC-47 (at 4h, 24h, 48h) and HPV– SqCC/Y1 (at 24h, 48h) cells, with slightly more apoptosis seen in the XRT versus the PRT group. Cell senescence was assessed using a well-established senescence-associated biomarker (SA-β-gal). XRT and PRT induced significantly increased cellular senescence at both 4 and 6 days in all four cell lines (p all < 0.01), with PRT treatment leading to a significantly higher percentage of senescence in cells than XRT (p all < 0.01). Mitotic catastrophe, an important cell death mechanism by which solid tumors respond to clinical radiotherapy, was determined using co-staining for cytoplasm with γ- tubulin (with Texas red) and for nucleus with Dapi (blue). XRT and PRT both led to a significantly increased number of cells undergoing mitotic catastrophe at 4h, 24h, 48h and 72h (p all < 0.05) in all four cell lines, with significantly higher rates seen in PRT treated versus XRT treated (p all < 0.05). Moreover, the peak of cell mitotic catastrophe induced by PRT occurred at 48h in the HPV– cells, versus at 72h in the HPV+ cells; while no obvious peak was observed in the XRT treated group. In conclusion, mitotic catastrophe and senescence are the major types of cell death induced by both XRT and PRT, in which PRT inducing higher levels of both types of cell death as compared to XRT. Citation Format: Li Wang, Shichao Han, Jinming Zhu, Xiaochun Wang, Yuting Li, Zeming Wang, Eric Lin, Xiaofang Wang, David P. Molkentine, Pierre Blanchard, Yining Yang, Ruiping Zhang, Narayan Sahoo, Michael Gillin, Xiaorong Ronald Zhu, Xiaodong Zhang, Jeffrey N. Myers, Steven J. Frank. Proton versus photon irradiation induced cell death in head and neck cancer cells with different human papillomavirus status [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 69.

Background: Cancers arise from an accumulation of somatic mutations. Numerous intrinsic and extrinsic mutagenic sources exist, yet our understanding of which processes are active in a given cancer genome is limited. Specific mutagenic processes have mutational preferences, called signatures. Large-scale analyses of cancer genomes/exomes from The Cancer Genome Atlas (TCGA) have identified 30 unique signatures. APOBEC is a family of cytosine deaminases which function in the immune response to viral infection. A signature for APOBEC3 activity has been identified in a subset of cancers, including head and neck squamous cell carcinoma (HNSCC). The specifics of APOBEC activity in HNSCC remain poorly understood. Methods: We utilized bioinformatic approaches to explore the role of APOBEC mutations in tumor exomes, transcriptomes and germline exomes from 511 HNSCC patients in TCGA. Results: 58% of HNSCC are statically enriched for the APOBEC3 signature compared to all other cancers in TCGA. HPV+ HNSCC are statistically enriched for the APOBEC3 signature compared to HPV- samples (p < 0.0013). MutSig was used to test the prevalence of APOBEC3 mutations in known driver genes compared to passengers. While the mean rate of APOBEC3 mutations was similar between MuSig genes and all other genes (19.4% vs 21.9%), PIK3CA demonstrated a 20-fold increase above all other genes. When evaluated by HPV status, 87% of PIK3CA mutations in HPV+ samples were APOBEC3 mutations compared to 55% of mutations in HPV- samples (p Conclusions: APOBEC3 is a major source of mutations in HNSCC. HPV+ HNSCC have the highest enrichment of APOBEC3 mutations. APOBEC3 mutations are strongly overrepresented in PIK3CA at two known hotspots, suggesting these mutations are of functional importance. APOBEC3A is the most likely APOBEC to be active in HPV+ HNSCC; however, expression levels of APOBEC isoforms do not correlate well with APOBEC mutational rates, suggesting a temporal dissonance between enzyme activity and tumor sample harvest. APOBEC3 activity appears to correlate with upregulation of immune signaling pathways, supporting the hypothesis that APOBEC is acting as part of the innate response to viral infection, although its activity in HPV- cancer suggests other mechanisms of upregulation as well. Citation Format: Daniel L. Faden, Sean Thomas, Jeffrey Myers, Nishant Agrawal, Joseph DeRisi. APOBEC is a major source of mutations in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 11.

Purpose: Although the treatment of locally advanced head and neck cancer (HNSCC) has evolved in recent years, 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 protein p53, is by far the most commonly mutated gene found in HNSCC, and is in turn associated with treatment failure and poor survival outcomes. Although HPV+ HNSCC has a good prognosis, a group of HPV+ patients' tumors remain refractory to conventional treatments and unusual patterns of recurrence and metastasis are emerging. Therefore, more effective therapies are urgently needed. Our recent work has demonstrated that increased DNA damage response associated induction of replication stress and impaired Rad51-mediated homologous recombination (HR) sensitizes HNSCC tumor cells to WEE1 kinase inhibition with AZD1775. While the use of single molecularly targeted agents has demonstrated limited therapeutic benefits, there is growing interest in targeting multiple pathways or multiple steps within a single pathway to achieve even more effective cancer treatments. In this context, combinations of agents that target the DNA damage and HR responses are an exciting new area of investigation. Given the synthetic lethality of PARP inhibitor, olaparib in BRCA1/2-mutant or HR repair-defective cancers, combining PARP inhibitors with agents that inhibit HR is another interesting area of intensive investigation. Therefore, we hypothesize that simultaneous targeting of PARP and WEE1 will decrease the clonogenic survival in vitro and inhibit tumor growth and prolong animal survival in treated mice in an in vivo orthotopic nude mouse model of oral cancer. 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 and HPV+ HNSCC cell lines to olaparib in combination with AZD1775 respectively. Cell cycle analysis, DNA damage (γH2AX), HR, live cell imaging, and apoptosis were performed to dissect molecular mechanisms. Results: We found that olaparib synergized with AZD1775 in vitro to inhibit growth of HNSCC cells irrespective of p53 status or HPV positivity (median CI = 0.07-0.19). Mechanistically, these drugs interact synergistically to induce DNA damage, replication stress, and impaired Rad51-mediated HR through activation of CDK1 and increased Chk1 phosphorylation preceded by enhanced activity of ATR, culminating in aberrant mitosis associated with apoptotic cell death. Assessment of olaparib and AZD1775 combination therapy in vivo in an orthotopic mouse model of oral cancer is currently underway. Conclusions: Olaparib synergizes with AZD1775 in HNSCC cells in vitro through apoptosis. Our data provide an early-preclinical evidence for the rational combination of WEE1 and PARP inhibitors in the treatment of advanced HNSCC. Citation Format: Hideaki Takahashi, Antje Lindemann, Ameeta A. Patel, Weiwei Liu, Noriaki Tanaka, Lin Tang, Mei Zhao, Walter N. Hittelman, Jeffrey N. Myers, Abdullah A. Osman. Synergistic antitumor activity of olaparib combined with AZD1775 in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 44.

Many mutant p53 proteins exert oncogenic gain-of-function (GOF) properties in promoting cancer cell invasive growth and metastasis, but the mechanisms mediating these functions largely remain elusive. We show here that overexpression of the GOF mutant p53 G245D and other GOF p53 mutants enhances invasive cell growth of p53-deficient head and neck squamous cell carcinoma (HNSCC) UM-SCC-1 cells both in vitro 3D culture and in vivo orthotopic tumor mouse model. We demonstrate that the expression of the oncogenic forkhead transcription factor FOXM1 is upregulated by GOF mutant p53s in UM-SCC-1 cells and tumors. Moreover, we show that overexpression of GOF mutant p53 G245D decreases the inhibitory phosphorylation of FOXO3a, a tumor suppressive forkhead transcription factor, leading to its cytoplasmic accumulation. Consistent with the observation that FOXO3a negatively regulates FOXM1 expression, we demonstrate that overexpression FOXO3a decreases GOF mutant p53-mediated FOXM1 expression, whereas downregulation of FOXO3a partially restores decreased FOXM1 expression due to loss of GOF mutant p53. In support of the roles of FOXO3a-FOXM1 signaling axis in mutant p53's GOF signaling, we further show that overexpression of FOXO3a or downregulation of FOXM1 impairs GOF mutant p53-mediated cell invasion, whereas downregulation of FOXO3a, in part, rescues impaired cell invasion due to downregulated GOF mutant p53. Finally, given that AMP-activated protein kinase (AMPK) directly phosphorylates and promotes FOXO3a function, and that our prior demonstration that GOF mutant p53s inhibit AMPK, our current study establishes that GOF mutant p53-AMPK-FOX3a-FOXM1 signaling is one of important mechanisms through which mutant p53s gain oncogenic functions in HNSCCs. Citation Format: Noriaki Tanaka, Mei Zhao, Jiexin Zhang, Jing Wang, Ge Zhou, Jeffrey N. Myers. Gain-of-function mutant p53 promotes the oncogenic potential of head and neck squamous cell carcinoma cell by targeting forkhead transcription factors FOXO3a and FOXM1 [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 73.

Background: Sinonasal undifferentiated carcinoma (SNUC) is a rare, highly aggressive cancer that arises in the nasal cavity and paranasal sinuses. Despite aggressive multimodal therapy the prognosis remains poor, and the median survival time is less than 18 months. Because of its locally advanced nature, cisplatin-based induction chemotherapy has been used in our institution to downstage unresectable tumors before definitive surgery or radiation therapy. Interestingly, our previous study showed that response to induction chemotherapy is a prognostic indicator and may be used to select patients for definitive local treatment. In this study, we performed gene expression analysis on SNUC specimens to identify molecular markers to predict/distinguish between responders and non-responders to induction chemotherapy. Materials and Methods: Twenty-seven formalin-fixed paraffin-embedded samples from 24 patients treated at MD Anderson Cancer Center between 1999 to 2016 were used in this study. Among them, 12 specimens were treatment-naïve. Gene expression analysis was performed on an HTG EdgeSeq Oncology Biomarker Panel. Unsupervised clustering analysis and Mann-Whitney test were performed using JMP software and IBM SPSS Statistics 23, previously. Pathway analysis was performed using Ingenuity Pathway Analysis. Results: First, we attempted to identify a set of genes which could predict response to induction chemotherapy. Unfortunately, unsupervised clustering analysis on treatment-naïve specimen was incapable of distinguishing between responders and non-responders. However, using discriminant analysis we succeeded in finding genes classifying patients into responders and non-responders. Then we studied the tumor specimens harvested after induction chemotherapy; differentially expressed genes between the two groups (Mann-Whitney test; p Conclusions: Our study revealed the possibility that cell survival and proliferation pathways are upregulated in non-responders to induction chemotherapy in SNUC. This finding suggests that non-responders to induction chemotherapy might be treated by inhibiting those pathways. Our comprehensive gene expression study is a significant step towards developing new therapeutic approaches in SNUC. Citation Format: Yoko Takahashi, Diana Bell, Frederico O. Gleber-Netto, Tong-Xin Xie, Dianna Roberts, Curtis Pickering, Jeffrey N. Myers, Ehab Y. Hanna. Identification of gene expression patterns distinguishing responders and non-responders to induction chemotherapy in sinonasal undifferentiated carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 42.

Purpose: Adenoid cystic carcinoma (ACC) can often be controlled with surgery and postoperative adjuvant radiotherapy but is also characterized by late local recurrence and distant metastasis. No effective systemic therapeutic agents have been found to alter the natural history of ACC. Therefore, new therapeutic approaches are needed. In this study, we evaluated whether vandetanib (Zactima), a potent inhibitor of vascular endothelial growth factor receptor-2 (VEGFR-2) and epidermal growth factor receptor (EGFR) tyrosine kinases, had antitumor efficacy in vitro and in an orthotopic nude mouse model of human ACC.Experimental Design: The in vitro effects of vandetanib were assessed in three ACC cell lines on cell growth, apoptosis, and VEGFR-2 and EGFR phosphorylation levels. The in vivo antitumor activity of vandetanib was examined in nude mice bearing parotid gland ACC tumors. The mice were treated for 4 weeks with vandetanib (50 mg/kg/d) or placebo (control). Tumors were resected at necropsy, and immunohistochemical and immunofluorescence staining were done.Results: In vitro, vandetanib caused dose-dependent inhibition of VEGFR-2 and EGFR phosphorylation in ACC cells. Vandetanib also inhibited the cell proliferation and induced their dose-dependent apoptosis. In vivo, mice in the vandetanib group had tumor volumes significantly lower than those in the control group (P < 0.01). In addition, immunohistochemical staining showed a decrease in microvessel density and an increase in apoptosis of both tumor cells and endothelial cells within the tumor xenografts.Conclusion: These results suggest that vandetanib inhibits the growth of ACC in vitro and in vivo, making it a promising novel agent for the treatment of ACC.

Purpose: We investigated the methylation status and protein expression of four tumor suppressor genes to determine their role in salivary gland tumorigenesis. Experimental Design: We performed methylation-specific PCR and protein analyses of 29 normal salivary glands, 23 benign, and 79 malignant salivary gland neoplasms to determine the pattern and potential diagnostic and/or biological role of the RASSF1, RARβ2, DAPK, and MGMT tumor suppressor gene methylation in these tumors. Results: No methylation was detected in the normal tissues. Methylation occurred in 9 of 23 (39.1%) benign tumors; 3 (25.0%) pleomorphic adenomas and 6 (66.7%) Warthin's tumors at the MGMT, DAPK, or RASSF1 genes. Methylation occurred in 33 of 79 (41.8%) malignant tumors; 8 (30.8%) adenoid cystic carcinomas, 6 (33.3%) mucoepidermoid carcinomas, 6 (42.9%) acinic cell carcinomas, and 13 (62.0%) salivary duct carcinomas. RASSF1 and RARβ2 represented 75.8% of methylation events occurring most frequently in salivary duct and acinic cell carcinomas. Overall, we found no significant correlation between protein expression and methylation status of individual genes, but observed low or absent protein expression in several methylated tumors. Significant correlations were found between methylation and aggressive malignant phenotypes (P = 0.0004) and age (P = 0.05). Conclusions: (a) Benign and malignant salivary tumors differed in the frequency and pattern of gene methylation; (b) high-grade carcinomas were significantly methylated compared with low-grade phenotypes; (c) RASSF1 and RARβ2 were highly methylated in malignant tumors and can be targeted for therapy; and (d) methylation pattern may serve as a diagnostic and biological marker in assessing these tumors.

Purpose: The insulin-like growth factor-I receptor (IGF-IR) and its ligands have been implicated in the pathogenesis and progression of various cancers, including those arising in the thyroid gland. We therefore evaluated whether the IGF-IR could serve as a potential target for therapy of anaplastic thyroid carcinoma (ATC). Experimental Design: The expression and activation of the IGF-IR and some of its downstream signaling pathway components were evaluated in both human thyroid cancer specimens and thyroid cancer cell lines. The therapeutic potential of a humanized monoclonal antibody (A12) directed against IGF-IR was assessed in vitro and in vivo in an orthotopic model of ATC. Tumor volume and overall survival time were analyzed to evaluate the efficacy of A12 in vivo. Results: IGF-IR was overexpressed in 94% of the thyroid cancers. Blockade of IGF-IR with A12 was effective in attenuating IGF-IR signaling both in vitro and in vivo. However, the inhibitory effects of A12 on cell proliferation were cell line dependent, as those ATC cell lines that had detectable levels of pIGF-IR were more sensitive to A12 treatment. A12 was equally effective in vivo, where it brought ∼57% (P = 0.041) inhibition in tumor volume. The concomitant use of A12 and irinotecan produced additive effects and resulted in a 93% (P < 0.001) reduction in tumor volume. Blocking IGF-IR blocked Akt phosphorylation and decreased proliferation and microvessel density but increased apoptosis within the tumor xenografts. Our results also highlighted a previously undefined IGF-IR-mediated antiangiogenic effect on tumor-associated endothelium in thyroid cancers. Conclusion: Blocking the IGF-IR with A12 seems to be a potential avenue for treating patients with ATC by its direct antitumor effects and its effects on the tumor vasculature.

Purpose: Patients with radioiodine-resistant follicular thyroid cancer (FTC) have a poor prognosis, if metastasized, with currently available treatment modalities. Epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) and their receptors (EGFR and VEGFR) have been reported to be overexpressed in FTC and have been implicated in FTC development. We hypothesized that inhibiting the phosphorylation of EGFR and VEGFR by treatment with NVP-AEE788 (AEE788), a novel dual specific EGFR and VEGFR inhibitor, either alone or in combination with paclitaxel, would inhibit the growth of FTC xenografts in an orthotopic nude mouse model. Experimental Design: To confirm previous reports, EGF and EGFR expression and vascularity were analyzed in human samples of FTC, Hürthle cell carcinoma, and normal thyroid tissues. EGFR expression in four FTC cell lines was measured using Western blotting. The antitumor effect of AEE788 on FTC cells in vitro was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays and Western blotting. The effect of AEE788, alone and in combination with paclitaxel, on FTC tumor growth in an orthotopic nude mouse model was also investigated. Immunohistochemical analysis of EGFR and VEGFR signaling status, cell proliferation, apoptosis, and microvessel density was done. Results: EGF, EGFR, and vascularity were increased in human thyroid tumor samples and EGFR was increased in FTC cells. AEE788 inhibited FTC cell growth in vitro and reduced the phosphorylation status of EGFR, VEGFR, and two downstream targets, AKT and mitogen-activated protein kinase, in FTC cells. AEE788 alone and, to a greater extent, AEE788 plus paclitaxel suppressed FTC tumor growth in the thyroids of nude mice. Conclusion: Dual inhibition of EGFR and VEGFR by AEE788 could represent a novel approach to the treatment of radioiodine-resistant FTC.

Purpose: We investigated whether concomitant blockade of the epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR) signaling pathways by AEE788, a dual inhibitor of EGFR and VEGFR tyrosine kinases, would inhibit the growth of cutaneous squamous cell carcinoma (SCC) cells and human cutaneous cancer xenografts in nude mice. Experimental Design: We examined the effects of AEE788 on the phosphorylation of EGFR and VEGFR-2 in cutaneous SCC cells expressing EGFR and VEGFR-2 and cutaneous SCC cell growth and apoptosis. We assessed the in vivo antitumor effects of AEE788 in a xenograft model in nude mice. AEE788 (50 mg/kg) was given orally thrice weekly to mice that had been s.c. injected with Colo16 tumor cells. Mechanisms of in vivo AEE788 activity were determined by immunohistochemical analysis. Results: Treatment of cutaneous SCC cells with AEE788 led to dose-dependent inhibition of EGFR and VEGFR-2 phosphorylation, growth inhibition, and induction of apoptosis. In mice treated with AEE788, tumor growth was inhibited by 54% at 21 days after the start of treatment compared with control mice (P < 0.01). Immunohistochemical analysis revealed that AEE788 inhibited phosphorylation of EGFR and VEGFR and induced apoptosis of tumor cells and tumor-associated endothelial cells. Conclusions: In addition to inhibiting cutaneous cancer cell growth by blocking EGFR and VEGFR signaling pathways in vitro, AEE788 inhibited in vivo tumor growth by inducing tumor and endothelial cell apoptosis.