Search

Tumor suppressor mutations in head and neck squamous cell carcinoma (HNSCC) dominate the genomic landscape, hindering the development of effective targeted therapies. Truncating and missense mutations in NOTCH1 are frequent in HNSCC, and inhibition of PI3K can selectively target NOTCH1 mutant (NOTCH1MUT) HNSCC cells. In this study, we identify several proteins that are differentially regulated in HNSCC cells after PI3K inhibition based on NOTCH1MUT status. Expression of Aurora kinase B (Aurora B), AKT, and PDK1 following PI3K inhibition was significantly lower in NOTCH1MUT cell lines than in wild-type NOTCH1 (NOTCH1WT) cells or NOTCH1MUT cells with acquired resistance to PI3K inhibition. Combined inhibition of PI3K and Aurora B was synergistic, enhancing apoptosis in vitro and leading to durable tumor regression in vivo. Overexpression of Aurora B in NOTCH1MUT HNSCC cells led to resistance to PI3K inhibition, while Aurora B knockdown increased sensitivity of NOTCH1WT cells. In addition, overexpression of Aurora B in NOTCH1MUT HNSCC cells increased total protein levels of AKT and PDK1. AKT depletion in NOTCH1WT cells and overexpression in NOTCH1MUT cells similarly altered sensitivity to PI3K inhibition, and manipulation of AKT levels affected PDK1 but not Aurora B levels. These data define a novel pathway in which Aurora B upregulates AKT that subsequently increases PDK1 selectively in NOTCH1MUT cells to mediate HNSCC survival in response to PI3K inhibition. These findings may lead to an effective therapeutic approach for HNSCC with NOTCH1MUT while sparing normal cells. Significance: Aurora B signaling facilitates resistance to PI3K inhibition in head and neck squamous cell carcinoma, suggesting that combined inhibition of PI3K and Aurora kinase is a rational therapeutic strategy to overcome resistance.

Targeted therapies are limited for head and neck squamous cell carcinoma (HNSCC), as it is driven by mutations in tumor suppressors, including NOTCH1. We previously identified loss of function NOTCH1 mutations in HNSCC to be sensitive to phosphoinositide-3 kinase (PI3K) inhibitors through sustained Aurora kinase B levels. However, therapy resistance and modest responses are the leading causes of failure for targeted therapies. To address this pressing clinical need, we sought to identify drugs that would enhance the efficacy of PI3K inhibitors. We tested 5768 drugs (0-1µM) with diverse targets in NOTCH1 mutant (NOTCH1MUT - HN31, UMSCC22A, PCI-15B) HNSCC cell lines and copanlisib acquired resistant (CAR) HN31 clones. We determined drug efficacy using two metrics-area over the curve lethal dose (AOC_LD>0, cytotoxic) and area under the growth curve (AUC_GRI2x), was evident when PI3K and PLK1 were concurrently inactivated as compared to single agent treatment in HNSCC models. Furthermore, HEK293 cells were unaffected at these doses. We then investigated the role of PLK1 following PI3K inhibition in HNSCC models and found PLK1 protein levels to be downregulated. As PLK1 is downstream of Aurora kinases, we further determined the phospho-PLK1 (p-PLK1) levels in HNSCC models. Interestingly, p-PLK1 levels remained unaltered in NOTCH1WT and CAR NOTCH1MUT cells despite total protein level depletion. However, p-PLK1 levels drastically decreased in NOTCH1MUT HNSCC cells. This finding could explain the importance of PLK1 inactivation in addition to PI3K inhibition as a requirement for increased cell death in HNSCC models. We will further validate this combination in vivo to determine the effect of combined PI3K and PLK1 inhibition on tumor growth and survival. These novel findings may lead to the development of a better therapeutic approach for NOTCH1MUT HNSCC and for patients who develop acquired resistance to targeted therapies. Citation Format: Pooja A. Shah, Tuhina Mazumdar, Reid T. Powell, Li Shen, Jing Wang, Clifford C. Stephen, Mitchell J. Frederick, Faye M. Johnson. Concurrent inactivation of PI3K and PLK1 is synergistic and overcomes acquired resistance to PI3K inhibitors in NOTCH1MUT HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2847.

Many lethal cancer types have inactivation of the retinoblastoma (Rb) tumor suppressor pathway including cancers caused by human papillomavirus (HPV), which causes >5% of all cancers worldwide. There are no therapies that uniquely target Rb-deficient cancers. Biomarker-selected, molecular targeted therapy for Rb-deficient cancers represents both an unmet need and a translational knowledge gap. To address this gap, we recently published data that support a model in which Rb-deficient cancers, having high levels of Mad2, survive owing to TRIP13-mediated inhibition of Mad2 and Aurora A-mediated mitotic progression. The combination of Aurora A inhibition (alisertib) plus TRIP13 depletion caused extensive apoptosis in Rb-deficient, but not in Rb-proficient, cancer cells. Both Aurora A and TRIP13 are involved in the cell’s response to DNA damage and apoptosis that can lead to immunogenic cell death (ICD), prompting us to investigate the mechanism underlying the synthetic lethality of TRIP13 and Aurora A in Rb-deficient cancer cells. We transfected two HPV+ cell lines with TRIP13 or control siRNA and incubated with 100nM alisertib. We also generated two stable HPV+ cell lines with TRIP13KO. In both these systems, TRIP13 KD or KO alone did not cause apoptosis. Incubation with alisertib resulted in significant cell death, that validated our previous findings. We then tested the effect of the combination on DNA damage. Alisertib caused robust increases in γH2AX, pTIF1β (KAP1, TRIM28), and DNA PKcs, measured using immunoblotting, in both TRIP13 KD and KO HPV+ cell lines. We also observed enhanced γH2AX staining using confocal microscopy. ICD leads to activation of immune cells by the dying cancer cells in the tumor microenvironment involving the release of antigenic fragments that are engulfed, processed and presented by antigen presenting cells for the induction of antigen-specific T-cell responses. Studies have shown DNA damage stimulates the cGAS/STING pathway leading to ICD. In our system, we saw that TRIP13KO and KD in HPV+ cells treated with alisertib, resulted in marked activation of the cGAS/STING pathway and release of cytochrome C and IL1α in the cell supernatant, which are markers of pyroptosis. Additionally, we also observed gasdermin-E cleavage. The combination further led to increased cancer cell secretion of GM-CSF, IL6, and IL18 that can activate macrophages, NK cells and T-cells. The selective effect of the combination in Rb-deficient cancer cells allowed us to significantly reduce the concentration of alisertib. By sparing normal cells that express Rb, this combination can potentially reduce treatment toxicity in patients with Rb-deficient cancers. The ICD induced by this combination may lead to host T-cell engagement, thereby further enhancing tumor elimination. This work can shape future clinical trials that can change current treatment regime. Citation Format: Soma Ghosh, Tuhina Mazumdar, Lacin Yapindi, Pragya Sinha, Jagannadha Sastry, Faye M. Johnson. Novel combination of TRIP13 and Aurora kinase A inhibition demonstrated extensive DNA damage and immunogenic cell death in RB-deficient cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1564.

Effective targeted therapies are lacking for head and neck squamous cell carcinoma (HNSCC) that is common and fatal. Developing targeted therapies for HNSCC is challenging as the genomic landscape is dominated by mutations in tumor suppressors, including NOTCH1. To address this need, we previously demonstrated that PI3K inhibition led to apoptosis in NOTCH1 mutant HNSCC cell lines. The underlying mechanism of this sensitivity is unknown and important to elucidate as modest responses and development of acquired resistance are the leading causes of failure for targeted therapies. To address this knowledge gap, we measured the levels of 304 proteins using reverse phase protein array in NOTCH1WT and NOTCH1MUT HNSCC cell lines upon PI3K inhibition and identified Aurora kinase B (AURKB) to be differentially regulated. We also identified AURKB levels to be maintained in the NOTCH1MUT cell lines with acquired resistance to PI3K inhibition. To determine if the maintenance of AURKB expression contributes to PI3K inhibitor resistance, we depleted AURKB in resistant NOTCH1WT HNSCC cells and overexpressed AURKB in sensitive NOTCH1MUT HNSCC cells. This manipulation of AURKB levels led to increased sensitivity and resistance to PI3K inhibition respectively. To use a pharmacologic approach, we combined the pan-Aurora kinase inhibitor danusertib (0-2µM) with PI3K/mTOR inhibitor omipalisib (0-200nM) in 56 HNSCC cell lines for 72h and observed a substantial decrease in cell viability in >80% of NOTCH1WT and >90% of NOTCH1MUT HNSCC lines. Additionally, concurrent Aurora kinase and PI3K inhibition in NOTCH1WT, NOTCH1MUT, and NOTCH1MUT acquired resistant HNSCC cells for 24h resulted in elevated cell death compared to single agents, as measured by the induction of cleaved PARP and cleaved Caspase 3; and Annexin V positive cells. Mice bearing NOTCH1MUT xenografts showed complete tumor regression when treated with a combination of pan-PI3K inhibitor Copanlisib and Aurora inhibitor Alisertib as compared to control groups. AURKB depletion in NOTCH1WT and overexpression in NOTCH1MUT HNSCC cells revealed significant changes in the total protein levels of AKT and PDK1. Similarly, AKT depletion and overexpression in NOTCH1WT and NOTCH1MUT HNSCC cells altered sensitivity to PI3K inhibition. However, manipulation of AKT levels affected PDK1 and not AURKB levels, demonstrating AURKB to be upstream to AKT and PDK1. Therefore, maintenance of AURKB levels mediates resistance to PI3K inhibition in HNSCC through AKT and PDK1. We identified AURKB as a central player governing the sensitivity to PI3K inhibitor-induced apoptosis in the context of NOTCH1 mutation status in HNSCC through its effects on AKT and PDK1. These novel findings may lead to the development of more robust therapeutic approach for NOTCH1 mutant squamous carcinoma as well as patients who develop acquired resistance to targeted therapies. Citation Format: Pooja A. Shah, Anne M. Fernandez, Vaishnavi Sambandam, Hongyun Zhao, Tuhina Mazumdar, Li Shen, Jing Wang, Faye M. Johnson. Aurora kinase B expression shields HNSCC from PI3K inhibition-induced apoptosis through downstream mediators AKT and PDK1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3251.

Human papilloma virus (HPV)-driven head and neck squamous cell carcinoma (HNSCC) is common in young patients ( Citation Format: Soma Ghosh, Tuhina Mazumdar, Wei Xu, Reid T. Powell, Clifford Stephan, Li Shen, Curtis R. Pickering, Jing Wang, Faye M. Johnson. Rb deficient HPV+ HNSCC experienced enhanced sensitivity to aurora kinase inhibitors by altering the balance of MAD2 and TRIP13 levels [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1858.

HPV-positive (HPV+) head and neck squamous cell carcinoma (HNSCC) tumors typically have p53 loss due to the activity of the human papillomavirus (HPV)-encoded E6 protein and the E6-associated protein (HPVE6-AP) which mediate the degradation of wild-type (WT) p53 (p53α). The loss of p53 is thought to be a major contributor to the pathogenesis of HPV+ HNSCC, which comprise approximately 35% of all HNSCC. Currently, standard care for HPV+HNSCC includes radiation and chemotherapy. However long-term toxicity related to these treatments is a concern, and there is a need for newer therapeutic strategies. Previously, we reported that two alternatively spliced, functional truncated isoforms of p53 (p53β and p53γ, comprising of exons 1 to 9β or 9γ, respectively) are degraded by nonsense-mediated decay (NMD), a regulator of aberrant mRNA stability. Here, using HPV+HNSCC cell line models, we show that NMD inhibition rescues p53β/γ isoforms and activates p53 pathway. Furthermore, we show that p53β/γ isoforms are more stable compared to p53α in these cells, with reduced vulnerabililty to HPVE6-AP- mediated degradation, and that p53β/γ isoforms contribute to increased expression of p53 transcriptional targets p21 and PUMA following NMD inhibition. Consistent with p53 pathway activation, NMD inhibition enhanced radiosensitivity of HNSCC cells. NMD inhibition attenuated colony forming ability and disrupted cell cycle progression. To evaluate the therapeutic implications of NMD inhibition, we assessed the in vivo growth of HPV+ UMSCC47 tumors. Nude mice were injected with UMSCC47 cells either subcutaneously or orthotopically in the tongue and randomized to receive vehicle or with an NMD inhibitor. In both tumor models, we observed a significant reduction in tumor volume with NMD inhibition as compared to the vehicle-treated animals. To investigate whether NMD inhibition induced the expression of p53β/γ isoforms and activated the p53 pathway in vivo, we collected tumor tissues from animals and evaluated expression of p53 isoforms and transcriptional targets by RT-PCR. We observed increased expression of p53γ, p21, GADD45A and PUMA mRNAs in NMD inhibitor treated UMSCC47 tumors, compared to their respective vehicle treated controls. These results identify NMD inhibition as a novel therapeutic strategy for restoration of p53 function in major subgroups of p53-deficient HPV+ HNSCC tumors. Citation Format: Jayanthi Gudikote, Tina Cascone, Alissa Poteete, Piyada Sitthideatphaiboon, Sonia Patel, Yan Yang, Fahao Zhang, Lerong Li, Li Shen, Monique Nilsson, Phillip Jones, Jing Wang, Jean-Christophe Bourdon, Faye M. Johnson, John V. Heymach. Targeting nonsense-mediated decay restores p53 function in HPV-associated head and neck cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5733.

Checkpoint kinase inhibitors (CHKi) exhibit striking single-agent activity in certain tumors, but the mechanisms accounting for hypersensitivity are poorly understood. We screened a panel of 49 established human head and neck squamous cell carcinoma (HNSCC) cell lines and report that nearly 20% are hypersensitive to CHKi monotherapy. Hypersensitive cells underwent early S-phase arrest at drug doses sufficient to inhibit greater than 90% of CHK1 activity. Reduced rate of DNA replication fork progression and chromosomal shattering were also observed, suggesting replication stress as a root causative factor in CHKi hypersensitivity. To explore genomic underpinnings of CHKi hypersensitivity, comparative genomic analysis was performed between hypersensitive cells and cells categorized as least sensitive because they showed drug IC50 value greater than the cell panel median and lacked early S-phase arrest. Novel association between CDKN2A/p16 copy number loss, CDK2 activation, replication stress, and hypersensitivity of HNSCC cells to CHKi monotherapy was found. Restoring p16 in cell lines harboring CDKN2A/p16 genomic deletions alleviated CDK2 activation and replication stress, attenuating CHKi hypersensitivity. Taken together, our results suggest a biomarker-driven strategy for selecting HNSCC patients who may benefit the most from CHKi therapy. Significance: These results suggest a biomarker-driven strategy for selecting HNSCC patients who may benefit the most from therapy with CHK inhibitors. Cancer Res; 78(3); 781–97. ©2017 AACR.

Human papilloma virus (HPV) driven cancers are common and lethal. The standard of care chemo-radiation treatment for HPV+ head and neck squamous cell carcinoma (HNSCC) results in permanent, often life-altering, toxicities in a relatively young population (aged ~45-55). Additionally, those who recur have poor outcomes. Therefore, there is an urgent need for better treatment options. No biomarker-driven targeted therapies are available. To address the current unmet need, we hypothesized that a large-scale High Throughput Drug Screen (HTDS) comparing drug efficacy in HPV+ and HPV- cell lines would identify biomarker driven, effective and less toxic options for HPV+ cancers. We tested the drug- sensitivity of 864 unique drugs (0-3.1 µM) in 51 classes based on their targets in 16 HPV+ and 17 HPV- squamous cancer cell lines. Cells were treated for 72 hours and DAPI+ nuclei counted before and after drug treatment. We used the Area Over the Curve_Lethal Dose (AOC_LD) to measure drug sensitivity because it is independent of cell division. We defined effective drugs as those that led to cell death (i.e., less cells at the end of drug treatment than prior to treatment) in at least one cell line. About half of the drugs tested (439) were effective and certain classes of drugs were enriched in either the effective or ineffective categories (Pearson residual). We found that Aurora kinase inhibitors were the most effective class overall. Other effective drug classes included anthracyclines, vinca-alkaloids, and inhibitors of HDAC, proteasome, EGFR, CDC7, and BTK1. Only 30 drugs had a differential effect based on HPV status. All 16 Aurora kinase inhibitors were more effective in HPV+ than in HPV- cells and the difference in sensitivity (dichotomous variable) was statistically significant in 7 of them. To determine if factors other than HPV status predict response to Aurora kinase inhibitors, we compared the expression of 304 proteins (RPPA) and mutational status for the 50 most commonly mutated genes in HNSCC to drug efficacy. The expression of eight proteins were associated with sensitivity to multiple Aurora kinase inhibitors. We validated RPPA data by immunoblotting and observed that only Rb, pRB(S807/811) and p16 correlated with drug sensitivity. For gene mutations, only TP53 mutations correlated with drug resistance with 7 Aurora kinase inhibitors. Interestingly, these two biomarkers, Rb and TP53, corroborate well with HPV status. To validate the screening results, we treated HPV+ cells with two clinically relevant Aurora kinase inhibitors, alisertib and barasertib, and observed significantly more cell death in HPV+ cells in comparison with HPV- cells. Therefore, our present study portrays Aurora kinase inhibitors as an effective strategy for HPV+ cancer cells. Multiple Aurora kinase inhibitors are currently in clinical development making the clinical application of our data possible soon. Citation Format: Tuhina Mazumdar, Li Shen, Wei Xu, Soma Ghosh, Reid T. Powell, Clifford Stephan, Curtis R. Pickering, Jeffery N. Myers, Jing Wang, Faye M. Johnson. Aurora kinase inhibitors cause cell death in HPV+ cells: A plausible treatment option for HPV+ cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 368.

Head and neck squamous carcinoma (HNSCC) is dominantly driven by mutations in tumor suppressor genes making it challenging to devise biomarker-based targeted therapy. In order to meet this pressing clinical need, our group recently demonstrated that HNSCCs with loss of function NOTCH1 mutations were more sensitive to phosphoinositide-3 kinase (PI3K) pathway inhibition than their wild-type counterparts.Modest clinical responses and acquired resistance are leading causes of failure for molecular targeted therapies that are otherwise well tolerated. In order to address this challenge and identify drugs that could work in combination with PI3K inhibitors against NOTCH1-mutant HNSCC, we performed a high throughput screen of 5669 drugs (0-1µM) with diverse targets. We used a laser-based confocal imaging platform to determine actual cell numbers using DAPI staining before and after drug treatment of NOTCH1-mutant HNSCC cells (HN31, UMSCC22A). We used 2 metrics of efficacy to identify potential candidates to combine with PI3K inhibition: an arbitrary cut off value of ≤ 0.9 for the area under the curve, growth rate index and area over the curve lethal dose (AOC_LD). We calculate both metrics using the normalized growth rate inhibition curve in order to avoid the confounding effect of the rate of cell division. The AOC_LD is > 0 only when there are fewer cells after treatment than before (i.e., there was cell death). Of the resulting 340 candidates, we excluded chemotherapy, PI3K inhibitors, and non-specific drugs. When several candidates targeted the same pathway, we chose 2-3 of the most specific drugs to use in the combination screen. We combined the resulting 74 drugs with PI3K inhibitors, bimiralisib (0-1µM) or copanlisib (FDA approved, 0-100nM), for 72 h. Synergistic effects from these combinations were assessed using Bliss, HAS, Zip, and Loewe models. Trametinib (MEK inhibitor) and copanlisib were synergistic with the combination leading to 0.90 fraction of cells affected at concentrations of 30nM and 100nM respectively. These concentrations are target-specific and clinically achievable. Likewise, low concentrations of inhibitors of EGFR (10nM afatinib, 50nM AZ5104), HER2 (25nM sapitinib, 10nM poziotinib), and PLK1 (50nM BI2536, 50nM volasertib) were both effective and additive to synergistic with PI3K inhibitors. These combinations will further be validated in vitro using an independent approach to test for apoptosis (cleaved PARP and caspase-3 induction and TUNEL assay) and cell counts in multiple NOTCH1 mutant HNSCC cell lines. We will test the most promising combination in vivo. We have identified four drug classes that not only maximize the killing of NOTCH1-mutant HNSCC, but may also prevent resistance at clinically relevant concentrations. The identified pathways may give us insight into mechanisms of resistance. If validated, these combinations may lead to the first biomarker-specific, targeted therapy for HNSCC. Citation Format: Pooja A. Shah, Tuhina Mazumdar, Reid T. Powell, Li Shen, Jing Wang, Clifford C. Stephen, Mitchell J. Frederick, Faye M. Johnson. Identification of pathways that enhance cell death in NOTCH1-mutant HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 369.

Background Anaplastic thyroid cancer (ATC) is a rare, aggressive, and deadly disease. Robust pre-clinical models are needed to adequately develop and study novel therapeutic agents. Patient-derived xenograft (PDX) models are thought to closely resemble patient tumors by preserving the tumor microenvironment, making them excellent pre-clinical models for drug response evaluation. We used two distinct ATC PDX models and evaluated drug response following a high-throughput screening (HTS). Methods A HTS, using NCI's Approved Oncology Set V (n=112) and a custom collection of agents (n=145), was conducted on patient-derived thyroid cancer cell lines. To identify the most effective drugs, we selected individual agents with maximal growth inhibition at each dose level relative to wells examined on the day of treatment (top 25th percentile) and subsequently used non-parametric statistics to compare effect size with other drugs and controls. This allowed us to identify classes of systemic agents which demonstrated preferential effectiveness against ATC cell lines and certain mutations. Following our prior successful work on orthotopic xenograft models, we used two established ATC PDX models, HOSC68 and HOSC199, harboring distinct genetic profiles and expanded each of them into 50 athymic mice. HOSC68 has a BRAFV600E and a TP53 mutation, while HOSC199 is wild-type for both genes, but has an HRAS mutation. Equal pieces of 4 × 4mm of tumor were transplanted subcutaneously at the level of the right flank. Following tumor growth, the mice were separated into four treatment arms. All mice received their treatment intraperitoneally following standard drug administration schedules. Tumor volume was measured on the first day of treatment and every two to three days thereafter until trial completion (14 days). Drug response was analyzed by evaluating percent tumor growth inhibition (TGI). Mouse weight was recorded over time to evaluate treatment toxicity. Following treatment completion, tumors were surgically retrieved and evaluated morphologically and histologically. Results Microtubule inhibitors, antimetabolites, and HDAC inhibitors were some of the most effective drug classes identified against ATC cell lines. Specifically, in this study, mice were treated with control (CTR), Docetaxel (DOC)-microtubule inhibitor, Pralatrexate (PRA)-antimetabolite, and LBH-589 (LBH)-HDAC inhibitor. Forty-four HOSC68 and 43 HOSC199 mice successfully grew tumor and were included in the trial. Compared to CTR, HOSC68 mice treated with DOC showed a 37% TGI (p=0.04), 88% with PRA (p&lt0.001), and 83% with LBH (p&lt0.001), while HOSC199 mice had a 2% TGI with DOC (p=0.56), 76% with PRA (p=0.005), and 83% with LBH (p=0.002). PRA and LBH were significantly more toxic than DOC and CTR (p&lt0.001) in HOSC68 mice, while all three drugs were significantly more toxic than CTR in the HOSC199 mice (p&lt0.001). Conclusion We report the first large-scale evaluation of drugs identified through a HTS analysis on ATC PDX models. This trial demonstrates the feasibility of using this platform for in vivo drug testing, while providing an avenue for future drug testing and resistance evaluation, as well as personalized therapeutics development. Citation Format: Anastasios Maniakas, Abdallah S. Mohamed, Ying C. Henderson, Hu Hei, Shaohua Peng, Diana Bell, Michelle D. Williams, Steve Scherer, David A. Wheeler, Gary L. Clayman, Mark Zafereo, Jennifer R. Wang, Maria E. Cabanillas, Clifford Stephan, Faye M. Johnson, Stephen Y. Lai. In vivo drug response evaluation in anaplastic thyroid cancer patient-derived tumor xenografts following high-throughput screening [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1662.

Human papilloma virus (HPV) is an oncogenic driver for a subset of head and neck squamous cell carcinomas (HNSCC), primarily from the oropharyngeal tissue subsite (OPSCC). These tumors are increasing in incidence and have recently surpassed cervical cancer as the most common HPV-driven malignancy in the United States. Fortunately, these tumors generally respond well to radiation-based therapy (XRT), and long-term (5 yr) survival is around 85%. However, the XRT treatment can generate significant morbidity, including problems with speech and swallowing. There is a clinical effort to reduce the treatment-related morbidity without compromising survival outcomes, through de-escalation treatment protocols. However, there is a subset of HPV+ OPSCC patients who do not respond to the current therapies and should not be given less intense treatment. This has generated the need to stratify patients based on their risk of recurrence or death, but currently no molecular biomarkers are available for risk assessment in OPSCC. By analyzing genomic data from The Cancer Genome Atlas (TCGA) we have identified a gene expression signature associated with expression of HPV genes. This signature identified 2 groups within the HPV+ tumors that demonstrate different levels of HPV function. One group seems to have reduced HPV function and present with intermediate phenotypes between HPV+ and HPV- tumors. Importantly, this signature is also highly prognostic in HPV+ OPSCC (p Citation Format: Frederico O. Gleber-Netto, Meng Gao, Xiayu Rao, Christopher P. Vellano, Joseph R. Marszalek, Jing Wang, Faye M. Johnson, Curtis R. Pickering. Variations in HPV function are associated with patient outcome and identify new candidate therapeutic approaches [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4942.

Head and neck squamous cell carcinoma (HNSCC) is primarily driven by the loss of tumor suppressor genes. Although approaches to target driver oncogenes have been clinically successful, targeting tumor suppressors has been challenging. To address this translational gap, we previously demonstrated that HNSCC cell lines with loss of function (LOF) NOTCH1 mutations (NOTCH1MUT n=14) were more sensitive to 6 PI3K pathway inhibitors (Omipalisib, Alpelisib, Bimiralisib, Dactolisib, Copanlisib, Apitolisib) than NOTCH1WT cells (n=45). In contrast to PIK3CAMUT cell lines, NOTCH1MUT lines underwent significant apoptosis after PI3K/mTOR pathway inhibition. NOTCH1MUT lines also showed significantly reduced clonogenic growth and significant tumor growth inhibition in both oral orthotopic and subcutaneous xenograft models. We employed CRISPR-Cas9 gene editing technology to knock out NOTCH1 gene in two NOTCH1WT lines, PJ34 and UMSCC49. After Omipalisib treatment, NOTCH1-KO lines showed decreased cell viability compared to parental lines. The NOTCH1 knock out lines showed significantly increased apoptosis after Omipalisib treatment compared to parental lines (PJ34 KO: 1.62-fld, P Citation Format: Vaishnavi Sambandam, Li Shen, Shaohua Peng, Tuhina Mazumdar, Curtis Pickering, Jeffrey Myers, Jing Wang, Mitchell Frederick, Faye Johnson. PDK1 drives susceptibility of NOTCH1 mutant head and neck squamous carcinoma to PI3K/mTOR pathway inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3913.

Genomic alterations in the PI3K/mTOR pathway occur in 54% of HNSCC patients. To identify novel biomarkers of response to PI3K/mTOR pathway inhibitors 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 genomic alterations. We identified that NOTCH1mut lines were significantly more sensitive to PI3K/mTOR pathway inhibitors than NOTCHWT lines: GSK2126458 (12/14 NOTCH1Mut lines), BYL719 (6/14), PQR309 (12/14), BKM120 (14/16), BEZ235 (12/16), BAY806942 (13/14) and GDC0980 (5/14 lines). In contrast to PIK3CAmut cell lines that experienced cell cycle arrest, after PI3K/mTOR pathway inhibition, NOTCH1mut lines underwent significant apoptosis in addition to G1/S cell cycle arrest. NOTCH1mut lines also showed reduced clonogenic growth in vitro and tumor growth inhibition in vivo in both oral orthotopic and subcutaneous xenograft mouse models. NOTCH1 knock out (KO) by CRISPR-Cas9 system in a NOTCH1WT line (PJ34) rendered it more sensitive to PI3K/mTOR inhibition. After PI3K/mTOR inhibition, PJ34-NOTCH1 KO showed significant reduction in clonogenic growth (1.57-fold; P As no canonical pathways account for the underlying mechanism of sensitivity, we measured the level of 301 proteins by reverse phase protein array (RPPA) in 3 NOTCH1mut and 3 NOTCH1WT lines after GSK2126458 treatment. Several proteins related to cell cycle were differentially regulated in NOTCH1mutcells compared to wild type lines. Notably, both mRNA and protein levels of Aurora B were significantly decreased in NOTCH1mutcells but not in NOTCHwt cells following PI3K/mTOR inhibition. Aurora B is an important cell cycle regulator and deregulation of Aurora kinases leads to defective chromosomal segregation and mitotic catastrophe in numerous cancers. Aurora kinase inhibitors as single agent are highly effective in a panel of NOTCHwt cell lines as demonstrated by decreased colony formation ability and proliferation as well as G2/M arrest and apoptosis. Inhibition of Aurora kinases in combination with PI3K inhibitors displayed synergy (Combination Index 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 present work may uncover potential combination therapies for HNSCC. Citation Format: Vaishnavi Sambandam, Li Shen, Pan Tong, Shaohua Peng, Tuhina Mazumdar, Ratnakar Singh, Curtis R. Pickering, Jeffrey N. Myers, Jing Wang, Mitchell Frederick, Faye M. Johnson. PI3K/mTOR pathway inhibition induces Aurora B mediated cell death in NOTCH1 mutant head and neck squamous (HNSCC) cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2977.

Determining which HPV+ oropharyngeal squamous cell carcinoma (OPSCC) patients will respond to therapy is of utmost important for implementation of treatment de-escalation to reduce morbidity or testing of novel therapeutic approaches. Due to the lack of reliable indicators of treatment response, we explored the gene expression profile of OPSCC in order to better understand the biology of these tumors and identify novel biomarkers. In this study, we investigated human genes associated with HPV transcription in 80 OPSCC samples from The Cancer Genome Atlas (TCGA). This exploratory analysis provided a list of 582 human genes associated with HPV biology. Hierarchical clustering analysis using the 582 human genes was used to separate HPV+ OPSCC in two groups with significantly distinct survival (log-rank test p < 0.001) and differential expression of HPV genes. A refinement of this analysis generated a prognostic gene expression signature for HPV+ OPSCC containing 41 human genes. These results were validated in two independent cohorts of HPV+ OPSCC (n=83 and n=47) and one independent cohort of cervical cancer (n=83). In all three validation cohorts our 41 gene expression signature was able to identify a group of HPV+ OPSCC patients with worse survival. Further refinement and validation of the signature is ongoing. In order to understand the biology related to poor outcome in HPV+ OPSCC, a whole transcriptome analysis between the two initial HPV+ OPSCC TCGA groups was performed. Pathway analysis identified cell metabolism, cell stress, and DNA damage related genes were highly associated with poor outcome. Similar patterns of gene expression were found in vitro in a panel of 10 HPV+ cell lines, and markers of the poor outcome were found to be associated with reduced radiation sensitivity in vitro. Our study has identified prognostic biomarkers in HPV+ OPSCC with potential clinical importance. These biomarkers may be useful for selection of low risk patients for treatment de-escalation or selection of high risk patients for novel therapeutic approaches. These biomarkers are also functionally linked to radiation sensitivity and may include new therapeutic targets. Citation Format: Frederico O. Gleber-Netto, Xiayu Rao, Kelly Erikson, Keiko Akagi, Faye M. Johnson, Jing Wang, Joseph Califano, Maura L. Gillison, Jeffrey N. Myers, Curtis R. Pickering. Risk stratification and biomarker discovery in HPV-positive oropharynx squamous cell carcinoma determined by HPV and human gene expression profile associations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4621.

Purpose. To address the unmet need for biomarker-driven, effective, targeted therapy for human papillomavirus (HPV)-associated head and neck squamous cell carcinoma (HNSCC) and cervical epithelial squamous cell carcinoma (CESC), we conducted a high-throughput drug screen (HTDS) using 1122 compounds in all readily available HPV-positive HNSCC and CESC cell lines and an equal number of matched HPV-negative lines. Methods. Cells were incubated in drug concentrations ranging from 0.01 μM to 3.16 μM for 72 h, fixed and stained with DAPI, and counted. Of the 1122 analyzable compounds, 865 unique drugs were tested because of overlap. All drugs were assigned to one of 36 classes based on their primary targets. Drug concentrations resulting in a 50% reduction in cell proliferation (GI50) and the area under the dose response curve were calculated. Two biological replicates were performed for all cell lines on separate days and at least 1 week apart. Results. The HTDS was conducted using 24 cell lines. We identified 493 highly effective compounds, which we defined as those with GI50 values less than 0.5 μM in 2 or more of the cell lines screened. The most effective drug classes were inhibitors of polo-like kinase, proteasomes, histone deacetylase, and Aurora kinases. Of the 19 Aurora kinase inhibitors tested, 18 were highly effective. We confirmed the efficacy of 3 Aurora kinase inhibitors using colony formation assays in 15 cell lines. Treatment with a dual Aurora A/B inhibitor, danusertib, led to G2M arrest and apoptosis in all 6 tested cell lines. Additionally, danusertib treatment decreased tumor size compared to controls in patient-derived xenograft mouse models of HNSCC. To identify biomarkers predicting response to Aurora kinase inhibitors, we tested for associations between mutations in the cell lines and sensitivity to the Aurora kinase inhibitors using whole exome mutation data for the 50 most common driver mutations in HNSCC. To validate our findings in an independent dataset, we queried the Genomics of Drug Sensitivity in Cancer database. In both data sets, cancer cell lines with KMT2D (MLL2) mutations were more sensitive to Aurora kinase inhibitors than cells without mutations. KMT2D mutations are inactivating; experiments to knock down KMT2D in wild-type cell lines and assess sensitivity to Aurora kinase inhibitors are ongoing. Conclusions. We identified Aurora kinase inhibitors as effective and understudied drugs in HNSCC and CESC. These drugs cause apoptosis and cell cycle arrest in vitro and decrease tumor size in vivo. This is the first published study to demonstrate that mutations in KMT2D (MLL2), which are common in many cancers (16% HNSCC, 12% CESC), correlate with drug sensitivity in 2 independent data sets. Citation Format: Tuhina Mazumdar, Nene N. Kalu, Shaohua Peng, Pan Tong, Li Shen, Jing Wang, Jeffrey N. Myers, Curtis R. Pickering, David Brunell, Clifford C. Stephan, Faye M. Johnson. Pharmacogenomic screen identifies KMT2D mutations as a biomarker of sensitivity to Aurora kinase inhibition in head and neck and cervical squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4646.

Introduction: Plk1 is a serine-threonine protein kinase that is overexpressed in cancer cells, and plays a major role in regulating tumor growth. Plk1 inhibitors are well tolerated, but only a few unselected patients with non-small cell lung cancer (NSCLC) respond to single-agent therapy. Our lab discovered that mesenchymal NSCLC cell lines are more sensitive to Plk1 inhibitors than epithelial cell lines in vitro and in vivo. However, mechanisms of resistance to Plk1 inhibitors have not been elucidated and this unknown is a major gap in knowledge. Experimental procedure: To study the mechanisms of Plk1 inhibitor-induced apoptosis we used 3 pairs of isogenic epithelial NSCLC cell lines induced to a mesenchymal phenotype with TGF-β. These isogenic pairs were treated with the Plk1 inhibitor volasertib for 24 h and levels of 301 proteins and phosphoproteins were simultaneously measured using reverse phase protein array (RPPA). Volasertib acquired resistance (VAR) cell lines were generated by exposing cells to increasing doses of volasertib. Results: The induction of a mesenchymal phenotype using TGF-β increased Plk1 inhibition-induced apoptosis in all 3 cell lines. To further elucidate mechanisms of resistance, we compared protein expression in these isogenic cell lines, 24 h after Plk1 inhibition. There were 33 proteins differentially regulated following Plk1 inhibition in parental vs TGF-β induced isogenic cells (p-value < 0.05). Notably, phosphorylated c-Met (Y1234/1235), FAK (Y397) and Src (Y416) were consistently inhibited following Plk1 inhibition in the mesenchymal lines. These changes were confirmed by Western blotting. Total c-Met, FAK and Src protein levels were not affected, implicating a post-translational changes. Likewise, VAR cell lines exhibited an epithelial phenotype and c-Met phosphorylation was persistent even after Plk1 inhibition. Simultaneous c-Met and Plk1 inhibition or silencing increased apoptosis in NSCLC cell lines tested compared to single agent inhibition or silencing. Combination of Plk1 and c-Met inhibitors decreased tumor volume and increased mouse survival in vivo in patient derived and cell line xenograft models. Similarly VAR cells also showed more apoptosis when treated with combination of Plk1 and c-Met inhibitors. Levels of the c-Met ligand HGF were unchanged after Plk1 inhibition and further mechanistic studies are on-going. Conclusion: NSCLC cell lines have diverse sensitivities to Plk1 inhibition, which is consistent with the results of clinical trials of Plk1 inhibitors in solid tumors. This study reveals a novel mechanism of non-canonical c-Met activation in resistant epithelial NSCLC after Plk1 inhibition. We demonstrate a profound effect of combination Plk1 and c-Met inhibition in vivo in multiple mouse models that could be a novel therapy for NSCLC patients. Citation Format: Ratnakar Singh, Pavitra Viswanath, Shaohua Peng, Vaishnavi Sambandam, Li Shen, Lerong Li, Jing Wang, Bingliang Fang, Faye M. Johnson. Noncanonical c-Met activation mediates de novo and acquired resistance to polo-like kinase 1 inhibitor-induced apoptosis in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 895.

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

The genomic alterations identified in head and neck squamous cell carcinoma (HNSCC) tumors have not resulted in any changes in clinical care, making the development of biomarker-driven targeted therapy for HNSCC a major translational gap in knowledge. To fill this gap, we used 59 molecularly characterized HNSCC cell lines and found that mutations of AJUBA,SMAD4 and RAS predicted sensitivity and resistance to treatment with inhibitors of polo-like kinase 1 (PLK1), checkpoint kinases 1 and 2, and WEE1. Inhibition or knockdown of PLK1 led to cell-cycle arrest at the G2/M transition and apoptosis in sensitive cell lines and decreased tumor growth in an orthotopic AJUBA-mutant HNSCC mouse model. AJUBA protein expression was undetectable in most AJUBA-mutant HNSCC cell lines, and total PLK1 protein expression was increased in cell lines wild-type for AJUBA. Exogenous expression of wild-type AJUBA in an AJUBA-mutant cell line partially rescued the phenotype of PLK1 inhibitor-induced apoptosis and decreased PLK1 substrate inhibition, suggesting a threshold effect in which higher drug doses are required to affect PLK1 substrate inhibition. PLK1 inhibition was an effective therapy for HNSCC in vitro and in vivo. However, biomarkers to guide such therapy are lacking. We identified AJUBA, SMAD4 and RAS mutations as potential candidate biomarkers of response of HNSCC to treatment with these mitotic inhibitors. Citation Format: Ming Zhang, Singh Ratnakar, Shaohua Peng, Mazumdar Tuhina, Shen Li, Pan Tong, Curtis Pickering, Jeffrey N. Myers, Jing Wang, Faye M. Johnson. Mutations of the lim protein ajuba mediate sensitivity of head and neck squamous cell carcinoma to treatment with cell cycle inhibitors [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 3816. doi:10.1158/1538-7445.AM2017-3816

Resistance to therapy, including conventional chemotherapy, targeted therapy and immunotherapy, continues to plague cancer treatment. Moreover, mechanisms governing resistance are poorly characterized leading to a dearth of rational combinatorial and sequential treatment strategies. While drug response data is abundant across myriad tumor types and drug classes, there exists no high-throughput method to probe such data with a query as simple as “If tumors are resistant to drug X, to what drug(s) are they sensitive?”- a seemingly trivial problem beset by immense data sets and imprecise definitions of sensitivity and resistance. Here, we present DISARM, a novel approach designed specifically to screen for drugs that are active in spite of resistance to a reference drug. DISARM selects candidates based on the proportion of samples that are resistant to a reference drug but sensitive to a candidate drug with simultaneous consideration to relatively lower IC50 values for candidate drugs and higher IC50 values for reference drugs. As candidates may work in only a subset of resistant models and precise delineation between sensitivity and resistance may vary between experimental settings, DISARM permits flexibility in dichotomizing drug data and uses grid search to optimize specifications. To illustrate, we analyzed publically available cell line data (IC50 data) from several cancer types for which platinum-based therapy is a standard of care, identifying multiple drugs that demonstrate activity in cisplatin-resistant models across tumor types such as the BCL-2 inhibitor obatoclax in small cell lung cancer, lung adenocarcinoma, gastric adenocarcinoma and bladder cancer, and the farnesyltransferase inhibitor tipifarnib in small cell lung cancer, bladder cancer, esophageal cancer, colon adenocarcinoma and head and neck squamous cell carcinoma. Frequently, multiple drugs from the same class were selected by DISARM for a single tumor type and, in these cases, we found statistically significant similarity between sensitive cell lines suggesting a subset of cisplatin-resistant cell lines that are repeatedly sensitive to a drug class. While translating preclinical observations into approved clinical use is often thwarted by an inability to identify predictive biomarkers, DISARM also allows us to select cell lines that are especially sensitive to candidate drugs or drug classes on which to perform biomarker analysis. To demonstrate this approach, we chose drugs with activity in multiple cancer types and compared mRNA and protein expression data to highlight potentially novel common and tumor-specific biomarkers for concomitant candidate drug sensitivity and cisplatin resistance. Thus, DISARM offers a simple yet effective approach for both drug and biomarker discovery within a specified clinical niche. Citation Format: Carl M. Gay, Pan Tong, Robert J. Cardnell, Xiao Su, Nene N. Kalu, Upasana Banerjee, Rasha O. Bara, Faye M. Johnson, John V. Heymach, Jing Wang, Lauren A. Byers. Differential sensitivity analysis for resistant malignancies (DISARM), a novel approach for drug screen analysis, identifies common candidate drugs across platinum-resistant cancer types [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 1560. doi:10.1158/1538-7445.AM2017-1560

Background: Inhibiting polo-like kinase 1 PLK1 may be an effective treatment for non-small cell lung cancer (NSCLC). PLK1 is a key regulator of mitosis and DNA damage checkpoints. PLK1 inhibitors are well tolerated, but only a few unselected patients with NSCLC respond to single-agent therapy. However, predictive biomarkers have not been used to select patients who are likely to experience a response to PLK1 inhibitors, and the mechanisms of resistance to PLK1 inhibitors have not been elucidated, making these unknowns a major gap in knowledge. To address this gap, we compared basal gene and protein expression in 63 NSCLC cell lines and discovered that mesenchymal NSCLC cell lines were more sensitive to PLK1 inhibitors than epithelial cell lines in vitro and in vivo. The induction of apoptosis in some NSCLC cell lines at very low drug concentrations and the need to find better therapy for mesenchymal NSCLC motivated us to further study PLK1 inhibition. Methods: To identify the pathways involved in PLK1 inhibitor-induced apoptosis, we used 3 pairs of isogenic NSCLC cell lines in which we had induced a mesenchymal phenotype using TGF-β. These isogenic lines were treated with the PLK1 inhibitor (volasertib) for 24 hours and levels of 301 proteins and phosphoproteins were simultaneously measured before and after treatment with volasertib using reverse phase protein array (RPPA). Results: The induction of a mesenchymal phenotype using TGF-β increased PLK1 inhibition-induced DNA damage and apoptosis in 3 NSCLC cell lines. To further elucidate mechanisms of resistance to PLK1 inhibition, we compared gene and protein expression in these isogenic cell lines, before and after PLK1 inhibition. There were 35, 12 and 43 proteins differentially regulated following PLK1 inhibition in epithelial vs. mesenchymal lines in HCC366, H1975, and HCC4006 cell lines, respectively at False Discovery rate 0.1. Phosphorylated FAK (Y397) and c-Met (Y1234/1235) were consistently inhibited following PLK1 inhibition in the mesenchymal lines but activated in the epithelial lines. These changes were confirmed by Western blotting. Total FAK and c-Met protein and mRNA levels were not affected, demonstrating post-translational changes. The inhibition of c-Met using EMD 1214063 led to FAK inhibition but FAK inhibition did not affect c-Met activation. The combination of c-Met inhibitor and volasertib increases sensitivity in NSCLC cell lines tested. The combinations led to more apoptosis than the single-agent inhibitors. Conclusions: NSCLC cell lines have diverse sensitivities to PLK1 inhibition, which is consistent with the results of clinical trials of PLK1 inhibitors in solid tumors, but no studies to date explain these diverse responses to PLK1 inhibition. We have identified c-Met activation as a previously unknown pathway of resistance to PLK1 inhibition in epithelial NSCLC. Citation Format: Ratnakar Singh, Li Shen, Pan Tong, Jing Wang, Faye M. Johnson. c-Met activation mediates resistance to polo-like kinase 1 inhibitor-induced apoptosis in non-small cell lung cancer [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 4095. doi:10.1158/1538-7445.AM2017-4095

High-risk human papillomavirus (HPV) is an oncogenic virus associated with 90% of cervical cancers, over 60% of oropharyngeal carcinoma cases and over 90% of anogenital cancers. Although HPV-positive cancers are molecularly, clinically and epidemiologically distinct from HPV-negative cancers, there are no specific or targeted therapies for HPV-positive cancers. In order to identify small molecule inhibitors that target HPV-positive cancers, we performed a high throughput drug screen on 24 cervical cancer and head and neck squamous cell carcinoma (HNSCC) cell lines (12 HPV-positive and 12 HPV-negative) to determine if these cell lines display differential sensitivity based on HPV status. HPV-positive cell lines with doubling times of less than 72 hours were selected for the screen. Unsupervised clustering of HNSCC cell lines based on protein expression levels obtained from reverse phase protein array (RPPA) was used to select matched HPV-negative cell lines by Spearman's rank-order correlation. Cytotoxic chemotherapeutics and agents targeting a broad range of processes including cell cycle control and DNA damage response were obtained from commercial vendors and academic collaborators. Drug sensitivity was measured by using nuclear staining to monitor cell death and proliferation after 72h of treatment. The screen of 1062 unique compounds at 6 different concentrations (0-3μM) in 24 cell lines represents 25,448 cell line – drug interactions. To ensure the robustness and reproducibility of the screen, the Z-factor and standard deviation for biological replicates were calculated. A Z-factor greater 0.5 was considered acceptable and the mean standard deviation across all drugs for each cell line was 0.06. Drug sensitivity was determined by calculating IC50 and area under the curve (AUC) values. Overall, HPV-positive HNSCC cell lines showed greater sensitivity to 13 drugs from different classes. Particularly, HPV-positive HNSCC cells were more sensitive to p38 MAPK and B-Raf inhibitors including LY2228820 (p < 0.01), regorafenib (p < 0.01), sorafenib (p < 0.05) and SB590885 (p < 0.05). On the other hand, HPV-negative HNSCC lines showed increased sensitivity to 30 drugs among these, palbociclib (p < 0.01), a CDK 4/6 inhibitor, and ryuvidine (p Citation Format: Nene N. Kalu, Tuhina Mazumdar, Pan Tong, Li Shen, Jing Wang, Lauren Averett Byers, Faye M. Johnson. Cell-based, high-throughput screen for small molecule inducers of cell death in HPV-associated head and neck cancers. [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 3793.

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 fisher's 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.

Introduction: Activation of epidermal growth factor receptor (EGFR) leads to the development and progression of human epithelial cancers, including non-small cell lung cancer (NSCLC). EGFR tyrosine kinase inhibitors (EGFR-TKI) are effective therapy for NSCLC with activating EGFR mutations. However, acquired resistance to EGFR-TKIs is unavoidable and occurs through various molecular mechanisms including the development of T790M secondary EGFR mutations, MET amplification, epithelial-mesenchymal transition (EMT) and other mechanisms. One of potential strategy to overcome EGFR-TKIs is through inhibition of polo-like kinase 1 (PLK1). PLK1 is one of the key regulators of mitotic progression and a DNA damage recovery checkpoint. PLK1 inhibitors are at various stages of clinical development. Our previous studies showed that mesenchymal NSCLC cell lines are more sensitive to PLK1 inhibitor than epithelial ones. This study examines the efficacy of PLK1 inhibition in NSCLC cell lines with acquired resistance to the EGFR-TKI erlotinib. Methods: Erlotinib resistant cell lines were developed in EGFR mutant, erlotinib sensitive cell lines PC9, HCC4006 and HCC827 by chronic exposure to stepwise increased concentrations of erlotinib. The effects of the PLK1 inhibitor volasertib alone or combined with erlotinib on proliferation, apoptosis, cell cycle, EGFR-dependent signaling, DNA damage and DNA damage response signaling were evaluated using CellTiter-Glo assay, TUNEL assay, BrDU incorporation assay, comet assay, western blot and immunofluorescence. Sensitivity of volasertib alone or in combination with erlotinib was also detected in the human tumor xenograft model. The combination index (CI) was calculated by Calcusyn software. Results: Acquired erlotinib resistant NSCLC cell lines with endogenous EGFR mutations (PC9, HCC4006 and HCC827) showed diverse resistance mechanisms: T790M EGFR mutation, MET amplification and EMT. Four of the 6 ER clones that underwent EMT became more sensitive to volasertib. Volasertib and erlotinib demonstrated synergistic effects only in cells bearing T790M EGFR mutations where we observed more pronounced G2M arrest and increased apoptosis. Volasertib alone or in combination showed enhanced DNA damage, activation of CHK1/ATR and CHK2/ATM pathways and an increase in γH2AX foci but only a modest effect on canonical signaling downstream of EGFR. The combination treatment exhibited a pronounced reduction in the size of tumor compared to single agent treatment in subcutaneous xenograft model generated with T790M mutant EGFR TKI-resistant PC9 cell line. Conclusions: These data demonstrate that PLK1 inhibition alone induces apoptosis and DNA damage in EGFR-TKI resistant cell lines with EMT. The combination of volasertib and erlotinib overcomes T790M-mediated drug resistance in vitro and in vivo. Citation Format: Ratnakar Singh, Yuehong Wang, Liguang Wang, Monique Nilsson, Ruchitha Goonatilake, Pan Tong, Lerong Li, Uma Giri, Jing Wang, John V. Heymach, Faye M. Johnson. The combination of polo-like kinase 1 inhibition and erlotinib overcomes T790M-mediated drug resistance in vitro and in vivo in non-small cell lung cancer. [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 4665.

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 Fisher's 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.

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 Ward's 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

Background:Head and neck squamous cell carcinoma (HNSCC) is the 6th most frequently diagnosed non-skin cancer in the world. Risk factors associated with HNSCC include smoking, alcohol use and human papillomavirus (HPV) infection. With the decline in tobacco use, there has been an increase in the incidence of HPV-associated head and neck cancers. Our increased understanding of the mutational landscape demonstrates that HPV-positive and HPV-negative HNSCC are molecularly distinct. Additionally, patients with HPV-positive tumors respond better to therapy and have better prognosis overall. However, there are no targeted therapies for HPV+ HNSCC. Human cell lines can serve as preclinical models for studying cancer progression and identifying possible therapeutic targets. Our aim was to determine if HNSCC tumors and cell lines derived from head and neck tumors display similar proteomic profiles. Methods: Reverse phase protein array (RPPA) analysis was performed on 66 HNSCC cell lines. The HPV-status of these cell lines was determined by quantitative and reverse transcriptase polymerase chain reaction (PCR). Results: Real-time PCR analysis confirmed that 9 of the 66 cell lines were HPV-positive. Differential expression of 156 proteins was analyzed and proteomic pathway scores were determined by t-test in HPV-positive and HPV-negative HNSCC cell lines. We identified significant proteomic differences between HPV-positive and negative HNSCC cell lines. As observed in HPV-positive head and neck tumors, the clinical biomarker p16 was overexpressed in HPV-positive cell lines (p-value Citation Format: Nene N. Kalu, Tuhina Mazumdar, Lixia Diao, Patrick Kwok Shing Ng, Jing Wang, Jeffery Myers, Faye M. Johnson, Lauren Averett Byers. Characterization of HPV-positive head and neck cancer cell lines as preclinical models for targeted therapy. [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 1826. doi:10.1158/1538-7445.AM2015-1826

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 IC50 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 DrugDrug's targetMedian IC50 (μM)Range of IC50(μM)Cmax (μM)ErlotinibEGFR5.970.12- >9.614LEE011CDK4/69.610.01- >9.61NABKM120PI3K0.640.01- >1.434MEK162MEK9.610.01- >9.611BosutinibSrc2.320.19- >9.610.8SeliciclibCDK2/59.610.01- >9.619.02VolasertibPLK1.120.01- >9.611.2AZD7762CHK1/20.120.02- >3.02NADocetaxelChemotherapy9.610.01- >9.610.08CisplatinChemotherapy8.620.01- >9.617.33RuxolitinibJAK9.610.01- >9.611.48AZD1775Wee10.250.03- >7.521.65BMN673PARP9.610.20- >9.610.07 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

Head and neck Squamous cell carcinoma (HNSCC) is a lethal, disabling and disfiguring cancer with no biomarker-directed therapeutic options. We are in a desperate need for novel targeted therapies. PI3K/AKT/mTOR is one of the most often altered pathways in HNSCC. This pathway is important for cancer cell signaling and several pharmacological inhibitors are already in clinical trials for other cancer types. Our lab previously found that HNSCC cell lines with PIK3CA mutations were more sensitive to pathway inhibitors compared to other PI3K pathway alterations such as PIK3CA amplification or PTEN loss and that activation of ERK is one mechanism responsible for PI3K inhibitor resistance. In this report we extended our study to further investigate the predictive biomarkers, biological effects of PI3K inhibition, the potential pathways of resistance and the use of rational drug combination in a diverse panel of HNSCC cell lines. We tested 64 HNSCC cell lines with the PI3K inhibitor GDC0941 and correlated drug sensitivity with basal PI3K pathway activation, PI3K copy number or PIK3CA mutation. We confirmed that cell lines with PIK3CA mutations were more sensitive to GDC0941 (P= 6.35X 10 -13) and those with amplification were not. We tested another four PI3K pathway inhibitors, including one PI3K inhibitor GSK1059615, one dual PI3K/mTOR inhibitor GDC0980, one AKT inhibitor GDC690693 and one mTOR inhibitor AZD8055 in a panel of 18 cell lines and found that the trend of drug sensitivity was similar (P < 0.05) in two PI3K inhibitors and one dual inhibitor where as AKT and mTOR inhibitors had a distinct trend. We evaluated the basal activation of PI3K/AKT/mTOR pathway using reverse phase protein array (RPPA) with two proteomic scores: PI3K/AKT and TSC/mTOR. Neither score predicted sensitivity to GDC0941. We also analyzed the PI3K/AKT and TSC/mTOR scores in 200 HNSCC tumors from the TCGA . In the same way PIK3CA amplification did not correlate with the scores whereas the PI3K/AKT score was higher in the PIK3CA mutants (P < 0.039) compared to WT PIK3CA. The RPPA did identify several biomarkers, including VEGFR2, pCRAF and PCNA, whose expression correlated with GDC0941 sensitivity. Treatment with GDC0941 caused cell cycle arrest in sensitive cell lines, but not in resistant cell lines. To identify potential resistance pathways following GDC0941 treatment in resistant lines, we tested 39 phosphoproteins using phosphoscan analysis and found that EGFR, IGF-1R, RET, FGFR3, and M-CSFR were activated in resistant cell lines after incubation with GDC0941. Combination therapy with GDC0941 and the EGFR inhibitor erlotinib was synergistic in resistant cell lines. Our data will be useful to conduct biomarker-based clinical trials in HNSCC in the near future. Citation Format: Tuhina Mazumdar, Lauren A. Byers, Patrick Ng, Gordon B. Mills, Shaohua Peng, Lixia Diao, Youhong Fan, Katherine Stemke-Hale, John V. Heymach, Jeffrey N. Myers, Bonnie S. Glisson, Faye M. Johnson. Evaluation of predictive biomarkers and resistance mechanisms of PI3K pathway inhibition in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 903. doi:10.1158/1538-7445.AM2014-903

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death. Therapies that target specific genetic aberrations are very effective in a subset on NSCLC. An effective approach for discovering novel therapeutic targets is to characterize responsive tumors. In this regard, we discovered a novel, kinase-inactivating BRAF mutation in the tumor of a patient with a marked and durable response to the multi-targeted kinase inhibitor dasatinib. Likewise, dasatinib induces senescence in NSCLC cells with endogenous kinase-inactivating BRAF mutations (KIBRAF). To identify the mechanism underlying this effect, we used reverse phase protein array, Western blotting and gene expression analysis on NSCLC cell lines with KIBRAF or WTBRAF incubated with dasatinib. We found that ATM, ATR, Rad 51, pH2AX, CDC6, CHEK1 (Chk1), pLATS and TAZ, were differentially regulated in mutant vs. WT cell lines. Quantitative real-time PCR analysis revealed that CHEK1, TAZ and TAZ's downstream targets were decreased following incubation with dasatinib in KIBRAF cells. Overexpression of CHEK1 or TAZ in KIBRAF NSCLC cells resulted in dasatinib resistance and decreased dasatinib-induced senescence. Given the differential effect on CHEK1 and pH2AX, we studied the effect of dasatinib on DNA damage using the COMET assay and found that dasatinib induces a significant increase in COMET-positive KIBRAF NSCLC cells. These data suggest that the inhibition of Chk1 along with inhibition of Hippo pathway results in the dasatinib sensitivity in NSCLC with KIBRAF. Citation Format: Banibrata Sen, Shaohua Peng, Tuhina Mazumdar, Lauren A. Byers, Humam Kadara, Faye M. Johnson. The multitargeted kinase inhibitor dasatinib induces DNA damage, Hippo pathway engagement and senescence in lung cancer cell lines that possess kinase-inactivating BRAF mutations. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3707. doi:10.1158/1538-7445.AM2014-3707

HNSCC is a common, deadly, and disfiguring disease. While combinations of radiotherapy, surgery, and chemotherapy are highly effective in HNSCC, there is significant morbidity associated with the disease and recurrence is common. There is great interest in identifying molecular events and pathways which drive HNSCC progression in order that targeted therapies may be developed. The loss of the tumor suppressor function such as p53 and NOTCH1, as well as activation of the STAT3 and STAT5 pathways, has been implicated in HNSCC progression. We have identified interactions between STAT3, STAT5, and the novel tumor suppressor SOCS2 to be important in HNSCC. To extend these findings to humans, we performed experiments to identify prognostic markers in HNSCC and to investigate the expression of STAT3, STAT5, and SOCS2 in HNSCC tissue. Immunohistochemistry was performed on tissue microarrays from resected tumor specimens of 123 stage I-IVB oral cavity SCC patients (treated with surgery +/- adjuvant radiation therapy) with annotated clinical outcome information from a median follow up of 76 months collected at the UT/MD Anderson Cancer Center. The array was screened with antibodies against STAT5, STAT3, and SOCS2, and scored in a blinded fashion by a pathologist. SOCS2 expression (present vs. absent) correlated significantly with recurrence-free survival in both the univariate (hazard ratio 0.24, p=0.0003; Cox proportional hazards model) and multivariate (hazard ratio 0.24, p=0.0004) analyses. Notably all patients who lacked SOCS2 tumor expression recurred within 45 months. There was a trend towards a correlation of SOCS2 expression with overall survival (HR 0.50, p=0.11) and disease-specific survival (HR 0.52, p=0.28). SOCS2 expression did not significantly correlate with tumor stage and extracapsular extension emphasizing that its absence is an independent marker of poor prognosis. Our prior published work demonstrates that STAT5 drives SOCS2 expression. The expression of SOCS2 in these patient samples positively correlated with total and phosphoSTAT5 (r=0.29 and 0.21 respectively; p This work was supported The University of Texas SPORE in Head and Neck Cancer (P50 CA097007), The Cancer Center Support Grant, and the ASCO Cancer Foundation Young Investigator Award (WW). Citation Format: Courtney Nicholas, Maria I. Nunez, Nusrat Harun, J. Jack Lee, Jeffrey Myers, Ignacio I. Wistuba, Banibrata Sen, Adel K. El-Naggar, Stephen Y. Lai, Faye M. Johnson, William N. William. SOCS2 (suppressor of cytokine signaling protein 2) is a prognostic indicator of progression-free survival in head and neck squamous cell carcinoma (HNSCC) patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5578. doi:10.1158/1538-7445.AM2013-5578

Head and neck squamous cell carcinoma (HNSCC) is a deadly and disfiguring disease for which better systemic therapy is desperately needed. The development of new therapies for HNSCC and the understanding of its biology both depend upon clinically relevant animal models. An increasingly promising xenograft model, the heterotransplant model, is developed by surgically implanting tumor tissue directly from a patient into an immunocompromised mouse. We have transplanted 30 HNSCC primary tumors from untreated patients directly into mice that included 27 tongue tumors, one maxillary gingival tumor, and two from the floor of the mouth. Five of 30 (17%) transplanted tumors could be serially passaged and used for therapeutic and mechanistic studies. One cell line has been established from a tongue primary. Histopathologic characterization of our panel showed a concordance between xenografts (up to the tenth generation) and the corresponding patients’ tumors in terms of tumor differentiation, cancer cell appearance, necrosis, and keratin production. To further characterize our model, we conducted gene expression analysis with Affymetrix U133A-microarrays on 20 patient tumors; 5 third generation and one tenth generation tumor in mice; one tumor-derived cell line; and 2 established cell lines (OSC19, Tu167). Although unsupervised clustering clearly separates patient tumors, xenografts, and cell lines into distinct groups, the early and late xenografts that were derived from the same patient clustered together. From the >54,000 probes tested, there were 1417 probes that were distinct between the tumors growing in mice vs. the corresponding human tumors from which they were derived (p,0.01, fold change 1.4). There were 992 probes that were distinct between the human tumors that subsequently grew in mice vs. those that did not - suggesting that this model enriches for cancers with distinct biological features (p,0.01, fold change 1.4). We have successfully used one heterotransplant model for multiple studies of cancer drug efficacy. Our results demonstrate the feasibility of a heterotransplant model of HNSCC. Although the gene expression patterns are distinct, the histology of the tumors in mice is similar to that of the same tumor in humans. Additionally, gene expression patterns and histology are relatively stable over multiple generations. Biologic gene pathways analysis for similarities and differences in this model will further refine the promise of heterotransplants as an in vivo model to test the efficacy of anti-cancer drugs. This work was supported by R01-CA143369-01 Citation Format: Shaohua Peng, Chad Creighton, Yiqun Zhang, Banibrata Sen, Tuhina Mazumdar, Jeffrey Myers, Diana Bell, Adel El-Naggar, Michelle Williams, Faye Johnson. Molecular and histologic characterization a patient-derived heterotransplant mouse model of head and neck squamous carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 333. doi:10.1158/1538-7445.AM2013-333