Vaishnavi Sambandam

Genomic characterization of human papillomavirus-positive and -negative human squamous cell cancer cell lines

Human cancer cell lines are the most frequently used preclinical models in the study of cancer biology and the development of therapeutics. Although anatomically diverse, human papillomavirus (HPV)-driven cancers have a common etiology and similar mutations that overlap with but are distinct from those found in HPV-negative cancers. Building on prior studies that have characterized subsets of head and neck squamous cell carcinoma (HNSCC) and cervical squamous cell carcinoma (CESC) cell lines separately, we performed genomic, viral gene expression, and viral integration analyses on 74 cell lines that include all readily-available HPV-positive (9 HNSCC, 8 CESC) and CESC (8 HPV-positive, 2 HPV-negative) cell lines and 55 HPV-negative HNSCC cell lines. We used over 700 human tumors for comparison. Mutation patterns in the cell lines were similar to those of human tumors. We confirmed HPV viral protein and mRNA expression in the HPV-positive cell lines. We found HPV types in three CESC cell lines that are distinct from those previously reported. We found that cell lines and tumors had similar patterns of viral gene expression; there were few sites of recurrent HPV integration. As seen in tumors, HPV integration did appear to alter host gene expression in cell lines. The HPV-positive cell lines had higher levels of p16 and lower levels of Rb protein expression than did the HPV-negative lines. Although the number of HPV-positive cell lines is limited, our results suggest that these cell lines represent suitable models for studying HNSCC and CESC, both of which are common and lethal.

Abstract 2992: Identification of NOTCH1 inactivating mutation as a therapeutic vulnerability to PI3K/mTOR pathway inhibition in head and neck squamous cell carcinoma (HNSCC)

Background: Genomic alterations in the PI3K/mTOR pathway occur in 54% of HNSCC patients. However, clinical trials of PI3K/mTOR pathway inhibitors had limited success even in those tumors with pathway alterations, including PIK3CA mutations. To target genomic alterations in HNSCC, we tested the efficacy of 7 PI3K/mTOR pathway inhibitors in 59 HNSCC cell lines and determined the association between drug sensitivity and molecular characteristics in order to identify biomarkers of response. Methods: We systematically analyzed the association between drug sensitivity and genomic alterations in 59 HNSCC lines. Results: NOTCH1mut lines are significantly sensitive to PI3K/mTOR pathway inhibitors: GSK2126458 (13/16), BYL719 (6/16), PQR309 (13/16), BKM120 (14/16), BEZ235 (12/16), BAY806942 (14/16) and GDC0980 (13/16 lines). In contrast to PIK3CAmut cell lines, all 7 NOTCH1mut lines tested underwent apoptosis (14.3 fld; P Conclusion: In contrast to PIK3CAmut cells, NOTCH1mut HNSCC cells underwent apoptosis after PI3K/mTOR pathway inhibition in vitro and decreased tumor size in vivo. The ectopic activation of NOTCH1 rescued NOTCH1mut HNSCC cells from PI3K/mTOR inhibitor-mediated apoptosis. The underlying mechanism may involve differential effects on tumor metabolism and ROS production. This work is significant because inactivating NOTCH1 mutations, which occur in 18% of HNSCC patients and SCCs of the lung, esophagus, and other sites, may serve as a biomarker for response. Our future work may uncover previously unknown crosstalk between the PI3K/mTOR and NOTCH pathways in SCCs. Citation Format: Vaishnavi Sambandam, Li Shen, Pan Tong, Tuhina Mazumdar, Curtis Pickering, Jeffrey N. Myers, Jing Wang, Mitchell Frederick, Faye M. Johnson. Identification of NOTCH1 inactivating mutation as a therapeutic vulnerability to PI3K/mTOR pathway inhibition in head and neck squamous cell carcinoma (HNSCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2992. doi:10.1158/1538-7445.AM2017-2992

Abstract 393: NOTCH1 inactivating mutation mediates sensitivity to PI3K/mTOR inhibitors in head and neck squamous cell carcinoma

Recently published whole exome sequencing studies in head and neck squamous cell carcinoma (HNSCC) tumors revealed that few had therapeutically targetable alterations using current strategies. This finding defines translational gap between genomics and HNSCC treatment. One potential targetable alteration is PIK3CA mutations. However, clinical trials testing PI3K/mTOR pathway inhibitors have had limited success and these inhibitors only lead to cell cycle arrest in PIK3CA mutant HNSCC cell lines. Thus, there is a critical need to identify therapeutic vulnerabilities for common mutation groups, including tumor suppressors, in HNSCC. One of these molecular subgroups is NOTCH1 which is the second most frequently mutated gene in HNSCC, with a 10-15% prevalence of inactivating mutations. Although there are several studies underscoring the importance of NOTCH1 as a tumor suppressor in HNSCC, none has identified a therapy that targets NOTCH1 mutant (mut) HNSCC. Our objective was to identify predictive biomarkers of sensitivity to PI3K/mTOR inhibitors by integrating drug and multiple-omics data. Cell viability with six PI3K/mTOR inhibitors in 68 HNSCC lines was measured by the CellTiter Glo assay. The peak plasma concentration of each drug was used as the cut-off to determine sensitivity. We observed a striking correlation between NOTCH1mut and sensitivity to PI3K/mTOR pathway inhibitors. When fisher9s exact test was performed, NOTCH1mut lines were more sensitive to GSK2126458 (P NOTCH1mut lines underwent more apoptosis after GSK2126458 treatment compared to NOTCH1wt lines (PCI15B- 48.1 fold; P Our data suggests that loss of active NOTCH1 signaling confers sensitivity to PI3K/mTOR inhibition. If the combination of NOTCH1 and PI3K/mTOR inhibition leads to apoptosis, this combination could be translated into the clinic. Citation Format: Faye M. Johnson, Vaishnavi Sambandam, Li Shen, Ming Zhang, Rishi Saigal, Pan Tong, Tuhina Mazumdar, Lauren A. Byers, Curtis Pickering, Jeffrey N. Myers, Jing Wang, Mitchell Frederick. NOTCH1 inactivating mutation mediates sensitivity to PI3K/mTOR inhibitors in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 393.

Abstract 4757: Identification of biomarkers that predict response of head and neck squamous cell carcinoma to mitotic inhibitors

Objectives: It is urgent to explore novel biomarkers and therapeutic targets for that are relevant to head and neck squamous cell carcinoma (HNSCC), which is the 6th most common cancer worldwide. Based on a prior drug screen, we identified 3 mitotic inhibitors (AZD7762, AZD1775, volasertib) as effective therapies for HNSCC. Our objective with this study is to identify mechanisms of response and potential biomarkers of response and Methods: Cell viability assays were performed by the CellTiter-Glo Luminescent method in a panel of 68 fingerprinted HNSCC cell lines using the 3 drugs at concentrations of 0.018 to 9.613 μM. Cell cycle, apoptosis and altered pathway protein expression after cells treated by the polo-like kinase 1 (PLK1) inhibitor volasertib were investigated by FACS, TUNEL and western blots respectively. An orthotopic mouse model of HNSCC was used to confirm the antitumor effects of PLK1 inhibition in vivo. To determine the mechanisms of drug sensitivity, we analyzed the correlation between gene expression, protein expression, gene mutation and drug sensitivity using modified two-sample t-tests were performed. The beta-uniform mixture (BUM) model was used to control false discovery rate (FDR). For correlations between drug sensitivity and gene mutations, we performed Fisher9s exact test. Results: Using the IC80 values with the peak plasma concentration of each drug as the cut-off to determine sensitivity, 34, 44 and 20 HNSCC cell lines were sensitive AZD1775 (Wee inhibitor), AZD7762 (CHK1/2 inhibitor) and volasertib (PLK1 inhibitor) respectively. HNSCC harboring AJUBA mutations were more sensitive to these 3 inhibitors and those with RAS mutations more resistant. PLK1 inhibition led to G2/M arrest, but only sensitive cell lines underwent substantial apoptosis following PLK1 inhibition. Decreases of the levels of phosphorylated TCTP were observed following treatment with volasertib confirming PLK1 inhibition. There was a significant decrease of tumor volumes and prolongation of survival in the mice bearing orthotopic HNSCC tumors treated with volasertib in vivo. Conclusions: PLK1 inhibition was an effective therapy in vitro and in vivo models of HNSCC. We identified the AJUBA and RAS mutations as potential candidate biomarkers of response to these mitotic inhibitors in HNSCC. This study identified the therapeutic potential of PLK1 as a novel therapeutic target for HNSCC. Citation Format: Ming Zhang, Shaohua Peng, Tuhina Mazumdar, Vaishnavi Sambandam, Li Shen, Pan Tong, Lerong Li, Lauren Byers, Curtis Pickering, Mitchell Frederick, Jeffrey N. Myers, Jing Wang, Faye M. Johnson. Identification of biomarkers that predict response of head and neck squamous cell carcinoma to mitotic inhibitors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4757.

Abstract 2427: The difference of drug sensitivity between HPV-positive and HPV-negative head and neck squamous cell carcinoma cell lines

Objectives: Infection with the human papillomavirus (HPV) is an important risk factor for development of head and neck squamous cell carcinoma (HNSCC). Strikingly, HPV-positive HNSCCs carry a more favorable prognosis and are a biologically distinct subgroup when compared with their HPV-negative counterparts. We aimed to discover novel therapeutic targets by conducting a high-throughput drug screening platform in vitro with both FDA approved and investigational drugs. Based on our prior proteomic analysis we tested inhibitors of pathways activated in HPV+ HNSCC tumors. Methods: Cell viability assays were performed by the Cell Titer Glo method in a panel of 66 fingerprinted HNSCC cell lines using 13 drugs at concentrations of 0.011 to 9.613 μM. This panel includes 9 HPV+ lines and 57 HPV- lines (5 oropharynx, 15 oral cavity, and 37 others). The IC 50 values were calculated. Western Blot assay was used to confirm that the drugs inhibited their targets. Results: We observed a wide range of sensitivities to all 13 drugs with the exception of BKM120 which was effective in all tested lines (Table 1). In contrast, nearly all cell lines were resistant to BMN673, ruxolitinib, LEE011, and selicilib. The HPV+ cell lines were more sensitive to MEK162 (p Conclusions: For most agents tested, HNSCC cell lines displayed similar drug sensitivity regardless of the tumor site. However, HPV+ lines were more sensitive to MEK162 and more resistant to Bosutinib. Future analysis will include comparing drug sensitivity to mutation, gene and protein expression in these lines. Taken together, the results may provide a rationale for the clinical evaluation of MEK inhibitors as a molecular targeted approach for the treatment of HPV+ HNSCC. Table 1. IC50 values in 66 HNSCC cell lines Citation Format: Ming Zhang, Tuhina Mazumdar, Shaohua Peng, Pan Tong, Vaishnavi Sambandam, Lauren A. Byers, Jeffrey N. Myers, Jing Wang, Faye M. Johnson. The difference of drug sensitivity between HPV-positive and HPV-negative head and neck squamous cell carcinoma cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2427. doi:10.1158/1538-7445.AM2015-2427

Abstract 417: Integrative drug sensitivity analysis of PI3K /mTOR pathway inhibitors in Head and Neck Squamous Cell Carcinoma (HNSCC)

Background: HNSCC is the sixth most common cancer worldwide. To date, Cetuximab is the only approved targeted therapy for HNSCC treatment. Thus,there is an immediate need to discover effective targets. Two pharmacogenomic HTS studies, Cancer Genome Project (CGP) and Cancer Cell Line Encyclopedia (CCLE) provide a large repository of drug sensitivity data. The PI3K/mTOR pathway is one of the frequently activated signaling cascades in HNSCC. However, it is unclear which class of PI3K/mTOR inhibitors is most promising and which biomarkers may be used to predict sensitivity. The rationale for this study is to identify novel biomarkers to targets such as PI3K/mTOR pathway by data mining these public databases. The potential biomarkers will be characterized in vitro in 68 HNSCC cell lines.Methods:The landscape of drug sensitivity profiles in 23 HNSCC cell lines (CGP) was analyzed by boxplot illustrations. Drugs that induce growth inhibition at low doses (median≤10 μM) were considered “effective”. Chemotherapy drugs, drugs with missing values and unknown targets were excluded from analyses. Hierarchical clustering of cell lines was performed based on drug sensitivity using GOWER distance metric and Ward9s linkage after normalization. Clustering of 140 drugs based on their sensitivity profiles was also done. In vitro, drug response to PI3K pathway inhibitors in 28 HNSCC cell lines was assessed by ATP based cell viability assay (CellTiter-Glo). Results: In the CGP datasets, we identified a set of effective drugs with median IC75 Citation Format: Vaishnavi Sambandam, Li Shen, Ming Zhang, Rishi Saigal, Lauren A. Byers, Curtis Pickering, Jeffrey N. Myers, Jing Wang, Faye M. Johnson. Integrative drug sensitivity analysis of PI3K /mTOR pathway inhibitors in Head and Neck Squamous Cell Carcinoma (HNSCC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 417. doi:10.1158/1538-7445.AM2015-417