- Email: [email protected]
The Cancer Genome Atlas Data Suggest Only a Modest Role for TP53 in Head and Neck Squamous Cell Carcinoma Prognostication
Volume 94 Number 4 2016 either from postoperative complications or early secondary recurrence. The aim of the study was to identify clinical factors that might predict early failure and/or worse outcome after salvage laryngectomy in patients initially treated with definitive CRT for advanced laryngeal cancer. Materials/Methods: This was a retrospective chart review of salvage laryngectomies performed for advanced laryngeal squamous cell carcinoma at a single academic institution. An existing prospectively collected database of head and neck cancer patients was queried to identify patients who had undergone salvage laryngectomies. Additional pertinent information on the identified patients was gathered from manual chart review for analysis after institutional review board approval was obtained. Patients were separated into groups based upon how quickly they recurred after primary therapy with CRT: group 1 was defined as patients that relapsed in less than 1 year, and group 2 consisted of patients that recurred after the 1-year mark. Survival in each group was estimated using the Kaplan-Meier method, and differences between curves were assessed by the log-rank test. A multivariable Cox proportional hazards model was used to estimate the hazard ratio, adjusted for age and initial stage at diagnosis. Results: The 2 groups were homogeneous with respect to age, sex, race, and disease location/severity. The Kaplan-Meier analysis indicated that laryngeal cancer patients with a recurrence less than 1 year after definitive chemoradiation (group 1) fared poorer than those with later recurrences (PZ.06). After adjusting for patient age and stage at diagnosis, patients in group 1 still experienced poorer outcomes, although the results were not significant (hazard ratioZ2.30, 95% confidence interval: 0.79-6.73). Conclusion: Patients with larynx cancer who initially undergo CRT who relapse in less than a year have lower overall survival and are less likely to benefit from a salvage total laryngectomy. Author Disclosure: T.K. Hamilton: None. D. Bush: None. S. Langevin: None. K. Casper: None. J. Mark: None.
266 Novel Preclinical In Vitro and In Vivo Model Systems for Adenoid Cystic Carcinoma of Salivary Gland S. Agarwal, C. Chen, S. Choudhury, X. Liu, E. Glasgow, and R. Schlegel; Georgetown University Medical Center, Washington, DC Purpose/Objective(s): Adenoid cystic carcinoma (ACC) is one of the most malignant salivary gland cancers with a high incidence of both local and distant (lung and bone) metastases. In order to study this tumor type, a reliable model system is critical. Patient-derived mouse xenografts (PDX) have been shown to maintain the histology and gene expression profile of the primary tumor, making them a valid model system. However, PDX models suffer from high cost for generation and maintenance, low take rate (30%-50%), lack of manipulation, and high throughput capability. Lack of authenticated cell line has compromised studies of ACC basic biology and drug development. Materials/Methods: A new conditionally reprogrammed (CRC) method described previously combines the use of irradiated mouse fibroblasts and a ROCK inhibitor to induce the rapid and long-term growth of normal and tumor cells without any additional immortalization. We have used this technology to establish cell cultures using PDX tissue materials from 5 different individuals. We also established an in vivo zebrafish models system to study migratory and metastatic behavior of tumor cells using 2-day post fertilized embryos. We used transgenic zebrafish (flk:GFP) expressing green reef coral fluorescent protein in the vascular endothelial under the control of a VEGFR2 promoter, which makes it easy to follow the tumor cell trafficking in real time. The injected cells (tumor or normal) in the yolk sac were labeled with live dye (Mitotracker) allowed us to follow it in real time using live imaging system. Results: We have stable cultures from 5 individual PDX tumors using CRC technology. One ACC cell line, ACC11, has shown to maintain the MYB-NF1B translocation, mutations in FGFR2 and ATM genes and overexpression of Myb-NF1B fusion protein similar to the tumor of origin
Posters
935
(PDX tumor). We also established an in vivo zebrafish xenograft model system for ACCs, using ACC11 as a prototype, we showed that these cells are migratory and possess metastatic potential as a fraction of tumor cells moved from the yolk sac to the tail via vascular invasion within 3 days postinjection. Similar results were obtained when PDX tissue material for ACC11 was transplanted directly to the yolk sac. In contrast, injection of a non-ACC salivary gland cell line did not show any movement of cells even after 7 days of post injection. Conclusion: We have successfully established a toolkit consisting of an in vitro (cell line) and an in vivo (Zebrafish) model system for biological and translational research. Author Disclosure: S. Agarwal: Research Grant; Adenoid Cystic Carcinoma Research Foundation. C. Chen: None. S. Choudhury: None. X. Liu: None. E. Glasgow: None. R. Schlegel: Research Grant; National Institute of Health. Royalties from HPV vaccine; Georgetown University. Patent/License Fees/Copyright; Propagenix. Advise on scientific directions; Propagenix.
267 WITHDRAWN
268 The Cancer Genome Atlas Data Suggest Only a Modest Role for TP53 in Head and Neck Squamous Cell Carcinoma Prognostication J.W. Rocco1 and E.A. Mroz2; 1The James Cancer Hospital and Solove Research Institute Wexner Medical Center at The Ohio State University, Department of Otolaryngology-Head and Neck Surgery, Columbus, OH, 2 The James Cancer Hospital and Solove Research Institute, The Ohio State University Wexner Medical Center, Columbus, OH Purpose/Objective(s): Identifying combinations of clinical variables and biomarkers best related to outcome is important for informing clinical decisions and for designing trials. Early studies could not distinguish human papillomavirus (HPV)-positive from HPV-negative head and neck squamous cell carcinoma (HNSCC), so it is now important to determine which combinations of prognostic variables are best related to outcome in these types of HNSCC. The Cancer Genome Atlas (TCGA) provides a rich set of clinical and molecular data on HNSCC; the mix of cases is representative of higher stage disease. Taking HPV status into consideration, we combined TCGA survival analysis with best-subset and LASSO variable selection to identify combinations of prognostic variables most closely associated with outcome. Materials/Methods: Ten prognostic variables potentially related to outcome had sufficient TCGA data for analysis (259 cases, 114 deaths): age, gender, smoking history, T and N classifications, TNM stage, tumor grade, TP53 mutation status, HPV status (32 HPV positivs including 1 TP53 mutant), and mutant-allele tumor heterogeneity (MATH). Bestsubset selection examined the relation of all combinations of variables to overall survival in Cox proportional hazards models. LASSO selection added individual variables sequentially as Cox model complexity was allowed to increase. To avoid overfitting, model complexity was penalized by the Akaike Information Criterion in best-subset analysis, and by the sum of the absolute values of regression coefficients in LASSO. Results: Surprisingly, best-subset selection omitted TP53 mutation status as a prognostic variable. Age (P<.001), smoking history (PZ.001), MATH (PZ.005), HPV status (PZ.008), and N classification (PZ.01) was the combination of variables best related to outcome. The pattern of sequential variable selection by LASSO provided an explanation for this omission. TP53 mutation was the first variable selected by LASSO for inclusion in the 259-case Cox model, followed by smoking history. As MATH, HPV status, N classification, and age entered the model, however, the
936
International Journal of Radiation Oncology Biology Physics
contribution of TP53 status diminished. Variable selection by LASSO was particularly striking in HPV-negative cases, as 5 other variables (smoking history, N classification, MATH, tumor grade, and age) were included in the model before TP53. Conclusion: Although TP53 mutation was identified long ago as a prognostic variable in HNSCC, these results suggest that its prognostic value might be modest outside of its acting as a surrogate for HPV status. These results should be interpreted cautiously, as they are based on a single data set and not from a predesigned clinical study. Nevertheless, despite its importance in the biology of HNSCC, the role of TP53 mutation as a prognostic variable in HPV-negative HNSCC may need to be reconsidered in future studies, particularly as novel biomarkers are evaluated. Author Disclosure: J.W. Rocco: Patent/License Fees/Copyright; Massachusetts General Hospital (MGH). Development and discussion of clinical trial ideas; NCI. comments and reviews on clinical trial ideas; NCI. E.A. Mroz: Patent/License Fees/Copyright; Massachusetts General Hospital (MGH).
primary tumors may antagonize HPV oncogenes to inhibit cell proliferation and tumor growth. Author Disclosure: M.T. Spiotto: None. J. Bechill: None. R. Zhong: None.
269 miR-203 Inhibits Human Papillomavirus Oral Tumor Growth by Suppressing Proliferation in Differentiated Tumor Cells M.T. Spiotto,1 J. Bechill,2 and R. Zhong2; 1University of Chicago, Chicago, IL, 2The University of Chicago, Chicago, IL Purpose/Objective(s): In order to facilitate viral replication, the human papillomavirus (HPV) oncogene E7 stimulates differentiated keratinocytes in the suprabasal epithelial layers to initiate DNA synthesis. Here, we studied the extent to which HPV oncogenes caused proliferation in differentiated tumor cells and the pathways governing this process. Materials/Methods: Tamoxifen treatment of triple transgenic KHR mice induced expression of the HPV oncogenes E6 and E7 and a mutant KrasG12D oncogene to cause oral tumor development. As a control, we used HPV-negative KR mice that developed oral tumors in which proliferation was limited to the basal stem cell compartment. To study how a regulator of epithelial differentiation, miR-203, impacted primary tumor growth, we generated a dual inducible mouse model, KHR-203, where doxycycline (DOX) induced miR-203 expression independent of HPV and KrasG12D oncogenes. Gene expression was assessed using Affymetrix Mouse Gene 1.0 ST arrays, quantitative RT-PCR, and in situ hybridization (ISH). The proliferation markers Mcm7, Pcna, and BrdU as well as the keratinocyte differentiation marker CK10 were assessed by immunohistochemistry or immunofluorescence. Results: Compared to HPV-negative KR tumors, HPV-positive KHR tumors grew faster (tumor volume at d24: 387.174.3 mm3 for KHR tumors vs 140.166.9 mm3 for KR tumors; P<.001). KHR oral tumors had more cells expressing proliferation markers Mcm7 (81.52.2% of KHR vs 23.80.7% of KR tumors; P<.001) and Pcna (76.81.4% of KHR vs 31.91.4% of KR tumors; P<.001). Furthermore, KHR tumors, but not KR tumors, had partially differentiated CK10+ cells incorporating BrdU indicating that proliferation occurred in partially differentiated cells. Expression profiling demonstrated that 112 genes and 1 miRNA, miR-203, were differentially expressed between HPV-positive and HPV-negative tumors. qRT-PCR confirmed that miR-203 was downregulated 1.9-fold in HPV-positive tumors (P<.01), and ISH demonstrated that loss of miR-203 occurred primarily in the suprabasal layers. In KHR-203 oral tumors, DOX treatment induced miR-203 expression and suppressed HPV-positive tumor growth (tumor volume at d24: 147.318.9 mm3 for DOX-treated vs 426.125.3 mm3 for vehicle-treated tumors; P<.0001). The decrease growth of DOX-treated tumors was associated with decreased proliferation in differentiated tumor cells as measured by percentages of Mcm7-positive cells (P<.001) and Pcna-positive cells (P<.001). Conclusion: HPV oncogenes accelerated oral tumor growth by inducing proliferation of partially differentiated tumor cells that was regulated by miR-203. Our results suggest that restoring the miR-203 pathway in
270 A Regimen Combining the Wee1 Inhibitor AZD1775 With Histone Deacetylase Inhibitor Vorinostat is Highly Active Against Head and Neck Squamous Cell Carcinoma Harboring High-risk TP53 Mutations A.A. Osman, N. Tanaka, N.L. Silver, N.N. Kalu, A.A. Patel, J. Wang, L. Tang, M.J. Frederick, F.M. Johnson, S. Fu, and J.N. Myers; The University of Texas MD Anderson Cancer Center, Houston, TX Purpose/Objective(s): The cure rate for patients with advanced head and neck squamous cell carcinoma (HNSCC) remains poor due to resistance to standard therapy primarily consisting of chemoradiation. Since mutation of TP53 in HNSCC occurs in 60% to 80% of nonhumanpapillomavirus (HPV)-associated cases and is in turn associated with resistance to these treatments, novel therapeutic approaches are needed to overcome drug resistance and improve survival outcomes in patients with advanced HNSCC. Wee-1 is a kinase that has been linked to DNA damage-induced G2/M arrest, owing to its ability to inactivate cyclin-dependent kinase 1 (CDK1) through phosphorylation of the Tyr15 residue. Our laboratory has shown that the Wee-1 kinase inhibitor AZD1775 sensitizes HNSCC cells harboring high-risk p53 mutations to cytotoxic therapies both in vitro and in vivo. Vorinostat is a small molecule inhibitor of histone deacetylase (HDAC) that has been shown in vitro and in vivo to have promising anticancer activity. Treatment with vorinostat alone shows preferential cytotoxicity for mutant p53 HNSCC cells. This finding supports the rationale to use vorinostat-based regimens to achieve maximum synthetic lethality in HNSCC with p53 mutations. In this study, we evaluated the efficacy of a regimen combining vorinostat and AZD1775 in HNSCC cells with a variety of p53 mutations. Materials/Methods: Clonogenic survival assays were performed to examine the in vitro sensitivity of several TP53 mutant HNSCC cell lines following treatment with vorinostat and AZD1775. Cell cycle and western blotting analyses were performed to investigate cellular mechanisms. An orthotopic mouse model of oral cancer and HNSCC patient-derived tumor xenografts (PDX) were used to evaluate in vivo efficacy of the drugs. Results: Vorinostat synergized with AZD1775 in vitro and reduced cell survival of mutant p53 HNSCC cells. Interestingly, addition of vorinostat had no effect on AZD1775 responses in the wild-type p53 HNSCC cells. It appears that the reduction in cell survival with vorinostat or combination treatment is mediated through apoptosis. Treatment of HNSCC cells with vorinostat and AZD1775 increased p21 induction and promoter activity independent of p53 expression. Interestingly, shRNA knock-down of p21 did not attenuate the lethal effect of combined treatment, suggesting that p21 might be necessary but not sufficient for vorinostat-mediated cell death in these cells. Finally, coadministration of vorinostat with AZD1775 resulted in significant tumor growth inhibition and prolonged animal survival in an orthotopic mouse model of oral cancer and HNSCC patientderived xenografts. Conclusion: A regimen combining AZD-1775 with vorinostat is highly active in preclinical models of p53 mutant head and neck cancer. Author Disclosure: A.A. Osman: None. N. Tanaka: None. N.L. Silver: None. N.N. Kalu: None. A.A. Patel: None. J. Wang: None. L. Tang: None. M.J. Frederick: None. F.M. Johnson: None. S. Fu: None. J.N. Myers: None.
271 E2F1 Mediates Human Papillomavirus (HPV) Oncogene Toxicity and Suppresses HPV Oral Tumor Growth M.T. Spiotto,1 R. Zhong,2 J.M. Melotek,1 and J. Bechill2; 1University of Chicago, Chicago, IL, 2The University of Chicago, Chicago, IL
266 Novel Preclinical In Vitro and In Vivo Model Systems for Adenoid Cystic Carcinoma of Salivary Gland S. Agarwal, C. Chen, S. Choudhury, X. Liu, E. Glasgow, and R. Schlegel; Georgetown University Medical Center, Washington, DC Purpose/Objective(s): Adenoid cystic carcinoma (ACC) is one of the most malignant salivary gland cancers with a high incidence of both local and distant (lung and bone) metastases. In order to study this tumor type, a reliable model system is critical. Patient-derived mouse xenografts (PDX) have been shown to maintain the histology and gene expression profile of the primary tumor, making them a valid model system. However, PDX models suffer from high cost for generation and maintenance, low take rate (30%-50%), lack of manipulation, and high throughput capability. Lack of authenticated cell line has compromised studies of ACC basic biology and drug development. Materials/Methods: A new conditionally reprogrammed (CRC) method described previously combines the use of irradiated mouse fibroblasts and a ROCK inhibitor to induce the rapid and long-term growth of normal and tumor cells without any additional immortalization. We have used this technology to establish cell cultures using PDX tissue materials from 5 different individuals. We also established an in vivo zebrafish models system to study migratory and metastatic behavior of tumor cells using 2-day post fertilized embryos. We used transgenic zebrafish (flk:GFP) expressing green reef coral fluorescent protein in the vascular endothelial under the control of a VEGFR2 promoter, which makes it easy to follow the tumor cell trafficking in real time. The injected cells (tumor or normal) in the yolk sac were labeled with live dye (Mitotracker) allowed us to follow it in real time using live imaging system. Results: We have stable cultures from 5 individual PDX tumors using CRC technology. One ACC cell line, ACC11, has shown to maintain the MYB-NF1B translocation, mutations in FGFR2 and ATM genes and overexpression of Myb-NF1B fusion protein similar to the tumor of origin
Posters
935
(PDX tumor). We also established an in vivo zebrafish xenograft model system for ACCs, using ACC11 as a prototype, we showed that these cells are migratory and possess metastatic potential as a fraction of tumor cells moved from the yolk sac to the tail via vascular invasion within 3 days postinjection. Similar results were obtained when PDX tissue material for ACC11 was transplanted directly to the yolk sac. In contrast, injection of a non-ACC salivary gland cell line did not show any movement of cells even after 7 days of post injection. Conclusion: We have successfully established a toolkit consisting of an in vitro (cell line) and an in vivo (Zebrafish) model system for biological and translational research. Author Disclosure: S. Agarwal: Research Grant; Adenoid Cystic Carcinoma Research Foundation. C. Chen: None. S. Choudhury: None. X. Liu: None. E. Glasgow: None. R. Schlegel: Research Grant; National Institute of Health. Royalties from HPV vaccine; Georgetown University. Patent/License Fees/Copyright; Propagenix. Advise on scientific directions; Propagenix.
267 WITHDRAWN
268 The Cancer Genome Atlas Data Suggest Only a Modest Role for TP53 in Head and Neck Squamous Cell Carcinoma Prognostication J.W. Rocco1 and E.A. Mroz2; 1The James Cancer Hospital and Solove Research Institute Wexner Medical Center at The Ohio State University, Department of Otolaryngology-Head and Neck Surgery, Columbus, OH, 2 The James Cancer Hospital and Solove Research Institute, The Ohio State University Wexner Medical Center, Columbus, OH Purpose/Objective(s): Identifying combinations of clinical variables and biomarkers best related to outcome is important for informing clinical decisions and for designing trials. Early studies could not distinguish human papillomavirus (HPV)-positive from HPV-negative head and neck squamous cell carcinoma (HNSCC), so it is now important to determine which combinations of prognostic variables are best related to outcome in these types of HNSCC. The Cancer Genome Atlas (TCGA) provides a rich set of clinical and molecular data on HNSCC; the mix of cases is representative of higher stage disease. Taking HPV status into consideration, we combined TCGA survival analysis with best-subset and LASSO variable selection to identify combinations of prognostic variables most closely associated with outcome. Materials/Methods: Ten prognostic variables potentially related to outcome had sufficient TCGA data for analysis (259 cases, 114 deaths): age, gender, smoking history, T and N classifications, TNM stage, tumor grade, TP53 mutation status, HPV status (32 HPV positivs including 1 TP53 mutant), and mutant-allele tumor heterogeneity (MATH). Bestsubset selection examined the relation of all combinations of variables to overall survival in Cox proportional hazards models. LASSO selection added individual variables sequentially as Cox model complexity was allowed to increase. To avoid overfitting, model complexity was penalized by the Akaike Information Criterion in best-subset analysis, and by the sum of the absolute values of regression coefficients in LASSO. Results: Surprisingly, best-subset selection omitted TP53 mutation status as a prognostic variable. Age (P<.001), smoking history (PZ.001), MATH (PZ.005), HPV status (PZ.008), and N classification (PZ.01) was the combination of variables best related to outcome. The pattern of sequential variable selection by LASSO provided an explanation for this omission. TP53 mutation was the first variable selected by LASSO for inclusion in the 259-case Cox model, followed by smoking history. As MATH, HPV status, N classification, and age entered the model, however, the
936
International Journal of Radiation Oncology Biology Physics
contribution of TP53 status diminished. Variable selection by LASSO was particularly striking in HPV-negative cases, as 5 other variables (smoking history, N classification, MATH, tumor grade, and age) were included in the model before TP53. Conclusion: Although TP53 mutation was identified long ago as a prognostic variable in HNSCC, these results suggest that its prognostic value might be modest outside of its acting as a surrogate for HPV status. These results should be interpreted cautiously, as they are based on a single data set and not from a predesigned clinical study. Nevertheless, despite its importance in the biology of HNSCC, the role of TP53 mutation as a prognostic variable in HPV-negative HNSCC may need to be reconsidered in future studies, particularly as novel biomarkers are evaluated. Author Disclosure: J.W. Rocco: Patent/License Fees/Copyright; Massachusetts General Hospital (MGH). Development and discussion of clinical trial ideas; NCI. comments and reviews on clinical trial ideas; NCI. E.A. Mroz: Patent/License Fees/Copyright; Massachusetts General Hospital (MGH).
primary tumors may antagonize HPV oncogenes to inhibit cell proliferation and tumor growth. Author Disclosure: M.T. Spiotto: None. J. Bechill: None. R. Zhong: None.
269 miR-203 Inhibits Human Papillomavirus Oral Tumor Growth by Suppressing Proliferation in Differentiated Tumor Cells M.T. Spiotto,1 J. Bechill,2 and R. Zhong2; 1University of Chicago, Chicago, IL, 2The University of Chicago, Chicago, IL Purpose/Objective(s): In order to facilitate viral replication, the human papillomavirus (HPV) oncogene E7 stimulates differentiated keratinocytes in the suprabasal epithelial layers to initiate DNA synthesis. Here, we studied the extent to which HPV oncogenes caused proliferation in differentiated tumor cells and the pathways governing this process. Materials/Methods: Tamoxifen treatment of triple transgenic KHR mice induced expression of the HPV oncogenes E6 and E7 and a mutant KrasG12D oncogene to cause oral tumor development. As a control, we used HPV-negative KR mice that developed oral tumors in which proliferation was limited to the basal stem cell compartment. To study how a regulator of epithelial differentiation, miR-203, impacted primary tumor growth, we generated a dual inducible mouse model, KHR-203, where doxycycline (DOX) induced miR-203 expression independent of HPV and KrasG12D oncogenes. Gene expression was assessed using Affymetrix Mouse Gene 1.0 ST arrays, quantitative RT-PCR, and in situ hybridization (ISH). The proliferation markers Mcm7, Pcna, and BrdU as well as the keratinocyte differentiation marker CK10 were assessed by immunohistochemistry or immunofluorescence. Results: Compared to HPV-negative KR tumors, HPV-positive KHR tumors grew faster (tumor volume at d24: 387.174.3 mm3 for KHR tumors vs 140.166.9 mm3 for KR tumors; P<.001). KHR oral tumors had more cells expressing proliferation markers Mcm7 (81.52.2% of KHR vs 23.80.7% of KR tumors; P<.001) and Pcna (76.81.4% of KHR vs 31.91.4% of KR tumors; P<.001). Furthermore, KHR tumors, but not KR tumors, had partially differentiated CK10+ cells incorporating BrdU indicating that proliferation occurred in partially differentiated cells. Expression profiling demonstrated that 112 genes and 1 miRNA, miR-203, were differentially expressed between HPV-positive and HPV-negative tumors. qRT-PCR confirmed that miR-203 was downregulated 1.9-fold in HPV-positive tumors (P<.01), and ISH demonstrated that loss of miR-203 occurred primarily in the suprabasal layers. In KHR-203 oral tumors, DOX treatment induced miR-203 expression and suppressed HPV-positive tumor growth (tumor volume at d24: 147.318.9 mm3 for DOX-treated vs 426.125.3 mm3 for vehicle-treated tumors; P<.0001). The decrease growth of DOX-treated tumors was associated with decreased proliferation in differentiated tumor cells as measured by percentages of Mcm7-positive cells (P<.001) and Pcna-positive cells (P<.001). Conclusion: HPV oncogenes accelerated oral tumor growth by inducing proliferation of partially differentiated tumor cells that was regulated by miR-203. Our results suggest that restoring the miR-203 pathway in
270 A Regimen Combining the Wee1 Inhibitor AZD1775 With Histone Deacetylase Inhibitor Vorinostat is Highly Active Against Head and Neck Squamous Cell Carcinoma Harboring High-risk TP53 Mutations A.A. Osman, N. Tanaka, N.L. Silver, N.N. Kalu, A.A. Patel, J. Wang, L. Tang, M.J. Frederick, F.M. Johnson, S. Fu, and J.N. Myers; The University of Texas MD Anderson Cancer Center, Houston, TX Purpose/Objective(s): The cure rate for patients with advanced head and neck squamous cell carcinoma (HNSCC) remains poor due to resistance to standard therapy primarily consisting of chemoradiation. Since mutation of TP53 in HNSCC occurs in 60% to 80% of nonhumanpapillomavirus (HPV)-associated cases and is in turn associated with resistance to these treatments, novel therapeutic approaches are needed to overcome drug resistance and improve survival outcomes in patients with advanced HNSCC. Wee-1 is a kinase that has been linked to DNA damage-induced G2/M arrest, owing to its ability to inactivate cyclin-dependent kinase 1 (CDK1) through phosphorylation of the Tyr15 residue. Our laboratory has shown that the Wee-1 kinase inhibitor AZD1775 sensitizes HNSCC cells harboring high-risk p53 mutations to cytotoxic therapies both in vitro and in vivo. Vorinostat is a small molecule inhibitor of histone deacetylase (HDAC) that has been shown in vitro and in vivo to have promising anticancer activity. Treatment with vorinostat alone shows preferential cytotoxicity for mutant p53 HNSCC cells. This finding supports the rationale to use vorinostat-based regimens to achieve maximum synthetic lethality in HNSCC with p53 mutations. In this study, we evaluated the efficacy of a regimen combining vorinostat and AZD1775 in HNSCC cells with a variety of p53 mutations. Materials/Methods: Clonogenic survival assays were performed to examine the in vitro sensitivity of several TP53 mutant HNSCC cell lines following treatment with vorinostat and AZD1775. Cell cycle and western blotting analyses were performed to investigate cellular mechanisms. An orthotopic mouse model of oral cancer and HNSCC patient-derived tumor xenografts (PDX) were used to evaluate in vivo efficacy of the drugs. Results: Vorinostat synergized with AZD1775 in vitro and reduced cell survival of mutant p53 HNSCC cells. Interestingly, addition of vorinostat had no effect on AZD1775 responses in the wild-type p53 HNSCC cells. It appears that the reduction in cell survival with vorinostat or combination treatment is mediated through apoptosis. Treatment of HNSCC cells with vorinostat and AZD1775 increased p21 induction and promoter activity independent of p53 expression. Interestingly, shRNA knock-down of p21 did not attenuate the lethal effect of combined treatment, suggesting that p21 might be necessary but not sufficient for vorinostat-mediated cell death in these cells. Finally, coadministration of vorinostat with AZD1775 resulted in significant tumor growth inhibition and prolonged animal survival in an orthotopic mouse model of oral cancer and HNSCC patientderived xenografts. Conclusion: A regimen combining AZD-1775 with vorinostat is highly active in preclinical models of p53 mutant head and neck cancer. Author Disclosure: A.A. Osman: None. N. Tanaka: None. N.L. Silver: None. N.N. Kalu: None. A.A. Patel: None. J. Wang: None. L. Tang: None. M.J. Frederick: None. F.M. Johnson: None. S. Fu: None. J.N. Myers: None.
271 E2F1 Mediates Human Papillomavirus (HPV) Oncogene Toxicity and Suppresses HPV Oral Tumor Growth M.T. Spiotto,1 R. Zhong,2 J.M. Melotek,1 and J. Bechill2; 1University of Chicago, Chicago, IL, 2The University of Chicago, Chicago, IL