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HNSCC is 6th most common malignancy in the world. Despite advances in diagnosis and treatment, the survival rates remain low due in large part to metastatic disease. The underlying biology associated with metastatic disease and poor outcome in HNSCC remains unclear. Importantly, metastatic cells acquire new properties that permit them to invade surrounding tissues and seed metastasis at distant sites. While these acquired properties contribute to aggressiveness of metastatic cancer, they can be exploited to target metastatic cells selectively, sparing toxicity in normal tissues. We used functional genomic technologies to identify new therapeutic targets for advanced disease in HNSCC. These targets were identified by conducting whole genome shRNA screens in matched sets of cell lines derived from primary HNSCC tumors and their respective metastatic sites/recurrences. Since hypoxia is an important attribute of aggressive and therapy resistant subpopulations of HNSCC tumor cells, we also aimed to identify genes that became essential when cells are exposed to hypoxia. To complement the data from the functional screens and to further characterize our cell line collection, we utilized several high through put approaches including mutational analysis, proteomics and gene expression profiling. Moreover, we performed chemical screens using libraries of over 4000 FDA approved drugs with the aim to combine our functional genomic data with the results from the drug screens to discover drug/gene “hit” combinations that would provide a basis for development of novel anti-cancer therapies.While HNSCC derived cell lines represent a valuable tool to study this disease, their biology might not always accurately represent the biology of the tumors they were derived from. Therefore, we looked at the expression of the components of several key pathways that we discovered using the in vitro characterization in a patient material from over HNSCC surgical samples constructed into a TMA. Five of the hits belonged to the Notch signalling pathway and 4 others came from the proteomic analysis as they showed higher levels of surface expression in metastatic cells and/or under hypoxic conditions. Interestingly, higher proportion of patients with late stages of HNSCC belonged to the high Jag2 and Hey1 populations as compared to patients with low stages of HNSCC that mostly expressed low or intermediate levels of these proteins. This data is in line with our observations that Jag2 and Hey1 expression is elevated in cells derived from metastatic sites and is low in cells derived from primary tumors. Proportion of cells with high CD66 and TRAIL proteins was also higher among the late stage tumors suggesting that these biomarkers are of interest for further investigation. We discovered several molecular pathways that are important in metastatic/hypoxic HNSCC. We are investigating our ability to target these pathways using FDA approved drugs in order to achieve durable cures. Citation Format: Maria Kondratyev, Aleksandra Pesic, Anna Dvorkin-Sheva, Troy Ketela, Natalie Stickle, Laurie Aiiles, Reidar Grenman, Marianne Koritzinsky, Brad Wouters. Identification of therapeutic targets in 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 2311.

HNSCC is 6th most common malignancy in the world. Despite advances in diagnosis and treatment, the survival rates remain low due in large part to metastatic disease. The underlying biology associated with metastatic disease and poor outcome in HNSCC remains unclear. Importantly, metastatic cells acquire new properties that permit them to invade surrounding tissues and seed metastasis at distant sites. While these acquired properties contribute to aggressiveness of metastatic cancer and interfere with success of therapies, they can also potentially be exploited to target metastatic cells selectively, sparing toxicity in normal tissues. We used functional genomic technologies to identify new potential therapeutic targets for advanced disease in HNSCC. These targets were identified by conducting whole genome shRNA screens in matched sets of cell lines derived from primary HNSCC tumors and their respective metastatic sites or recurrences. While extensive efforts are being made by both industry and academia to develop novel anti-cancer drugs, this process is still very slow and expensive. Drug repurposing is becoming increasingly popular due to a reduced risk to patients and lower costs of drug development compared to “de novo” discovery. We utilized 2 large chemical libraries (Selleck and Prestwick) that together contain about 4000 drugs approved by FDA and similar agencies worldwide. The libraries were screened against the HNSCC lines described above in order to discover new drugs targeting head and neck cancer including drugs that target selectively metastatic cells compared to their primary tumor counterparts. We accomplished screening of 30 HNSCC lines for all of which we have also obtained the functional genomic, mutational and gene expression data. We aim to combine our functional genomic data with the results from the drug screens to discover drug/gene “hit” combinations that would provide a basis for development of novel anti-cancer therapies. For this purpose, we are looking at correlations between the drugs and the shRNAs that have similar pattern of effects across the 30 cell lines. To complement the functional screens data, we performed targeted sequencing of the most commonly altered genes in HNSCC as reported by the TCGA. This data will help understand which pathways are affected by the drugs we select to investigate. Out of the 30 lines, we have 13 sets of “matched” lines that belong to the same patients, coming from primary tumors, metastatic sites or recurrences. As a first step of the analysis, we looked at drugs that affected cells derived from metastases but not those from the respective primary tumors. Interestingly, many of the metastasis-specific drugs were antibiotics. We selected 10 of the drugs for further validation; dose response-based viability assays and growth curves experiments confirmed that 5 of the drugs were selectively affecting survival and proliferation of the metastatic cell lines; the mechanism of this effect are currently being investigated. Citation Format: Maria Kondratyev, Aleksandra Pesic, Anna Dvorkin-Sheva, Troy Ketela, Jason Moffat, Marianne Koritzinsky, Brad Wouters. Repurposing of FDA approved drugs for treatment of metastatic HNSCC [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 507.

HNSCC is the 6th most common malignancy in the world. The prognosis is favorable during early stages; however, the disease is rarely diagnosed early due to the lack of symptoms. Most HNSCC patients present with metastatic disease for which the survival rates remain low. Hypoxia serves as a bad prognostic factor in HNSCC correlating with worse survival and resistance to radiotherapy. We aimed to discover novel targets in metastatic HNSCC utilizing a unique collection of matched sets of cell lines derived from primary tumors and their respective metastatic sites. We carried out expression profiling across the lines to reveal potential common changes in gene expression between the cells derived from primary and metastatic sites in each patient as well as between normoxia and hypoxia. This analysis revealed significant changes in gene expression between the described conditions. Interestingly, beta defensin 2 was one of the genes that came up as overexpressed in metastasis as well as in cells cultured in hypoxia. Beta defensins are small cationic peptides that belong to the innate immune system and exhibit antimicrobial and antiviral activities. Few studies reported their abnormal expression patterns in various cancers including HNSCC. While various novel anticancer therapies are currently being developed, early diagnosis remains one of the biggest challenges in cancer research. A discovery of soluble serum factors that can reliably detect the presence of primary or metastatic disease would serve as an extremely valuable tool in defining diagnosis and prognosis, predicting the response to treatment, and monitoring disease progression. We hypothesized the beta defensin 2 can serve as a serum biomarker of hypoxia and metastasis in HNSCC patients. Utilizing a commercial ELISA kit developed to detect and quantify levels of beta-defensin in human sera, we demonstrated that media collected from cell lines derived from metastases contained higher levels of beta-defensin compared to the cell lines derived from the primary tumors. Moreover, sera from HNSCC patients showed higher levels of beta defensin compared to the normal controls. As a next step, we tested sera samples from 40 HNSCC patients, of whom 20 had lymph node metastasis and 20 did not. While these data are still under analysis, preliminary results suggest that higher concentrations of beta-defensin correlate with the presence of lymph node metastasis in HNSCC patients. Utilizing two large chemical libraries that together contain about 4,000 FDA-approved drugs, we performed high-throughput screening of the HNSCC lines described above in order to discover new drugs targeting head and neck cancer, including drugs that target selectively metastatic cells compared to their primary tumor counterparts. Interestingly, many of the metastasis-specific drugs were antibiotics. Together with the findings described above, these data clearly suggest a connection between patient microbiome and the metastatic process in HNSCC. Citation Format: Maria Kondratyev, Aleksandra Pesic, Carl Virtanen, Marianne Koritzinsky, Brad Wouters. Antibacterial defense and metastatic progression—lessons from head and neck cancer [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr B10.

HNSCC is 6th most common malignancy in the world. Despite advances in diagnosis and treatment, the survival rates remain low due in large part to metastatic disease. The underlying biology associated with metastatic disease and poor outcome in HNSCC remains unclear. Importantly, metastatic cells acquire new properties that permit them to invade surrounding tissues and seed metastasis at distant sites. While these acquired properties contribute to aggressiveness of metastatic cancer and interfere with success of therapies, they can also potentially be exploited to target metastatic cells selectively, sparing toxicity in normal tissues. We used functional genomic technologies to identify new potential therapeutic targets for advanced disease in HNSCC. These targets were identified by conducting whole genome shRNA screens in matched sets of cell lines derived from primary tumors and their respective metastatic sites, with the goal of identifying genes that become essential for cell survival only following metastasis. Since hypoxia is an important attribute of aggressive and therapy resistant subpopulations of HNSCC tumor cells, we also aimed to identify genes that became essential when cells are exposed to hypoxia. We are particularly interested in the identification of contextual synthetic lethal oncogenes expressed on the cell surface, as those are easily targetable by therapeutic antibodies. To identify these targets, we performed high-throughput flow cytometry screening that enables evaluation of 370 validated cell surface antibodies. Cell surface targets differentially expressed in metastatic lines included CECAM and CCR6 that were previously reported to be implicated in metastasis and tumor progression as well as Thy1, a known marker of stem cells involved in regulation of cell adhesion. Cell surface targets induced under hypoxic conditions across the cell lines included CA9, an enzyme that is known to regulate pH in hypoxic cells and be associated with tumor progression, as well as CD338, CD264 and CD312, that were previously associated with stemness in a few models of cancer. Interestingly, the described proteins were also found to be differentially essential in the shRNA screens, highlighting their functional importance in tumor progression and hypoxia survival. We are currently investigating the role of these proteins in HNSCC metastasis utilizing our unique collection of matched pairs of HNSCC lines from multiple patients. Moreover, we are utilizing our pipeline of patient derived HNSCC xenografts to test the effect of knocking down the described genes in patient tumors. We are also testing the expression of selected hits in histological sections of patient tumorsthe 400-patient TMA by immunohistochemistry looking for correlation with tumor grade, aggressiveness, levels of hypoxia as well as presence/absence of metastasis in the patient. Citation Format: Maria Kondratyev, Aleksandra Pesic, Azin Sayad, Troy Ketela, Natalie Stickle, Carl Virtanen, Jason Moffat, Laurie Ailles, Marianne Koritzinsky, Brad Wouters. Cell surface targets in head and neck cancer [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 3054.

Head and neck carcinoma (HNSCC) is 6th most common malignancy in the world. Despite advances in diagnosis and treatment, the survival rates remain low due in large part to metastatic disease. The underlying biology associated with metastatic disease and poor outcome in HNSCC remains unclear. Importantly, metastatic cells acquire new properties that permit them to invade surrounding tissues and seed metastasis at distant sites. While these acquired properties contribute to aggressiveness of metastatic cancer and interfere with success of therapies, they can also potentially be exploited to target metastatic cells selectively, sparing toxicity in normal tissues. The idea behind this selective targeting is based on discovering molecular pathways that became essential in metastatic cells, and then exploiting this vulnerability through targeted agents. We used functional genomic technologies to identify new potential therapeutic targets for advanced disease in HNSCC. These targets were identified by conducting whole genome shRNA screens in matched sets of cell lines derived from primary tumors and their respective metastatic sites, with the goal of identifying genes that become essential for cell survival only following metastasis. Since hypoxia is an important attribute of aggressive and therapy resistant subpopulations of HNSCC tumor cells, we also aimed to identify genes that became essential when cells are exposed to hypoxia. To complement the functional screens data, we performed targeted sequencing of the most commonly altered genes in HNSCC as reported by the TCGA profiled gene expression in the HNSCC cell lines cultured under normoxia and hypoxia using Illumina microarrays. These analyses led to the discovery of genes that are differentially essential in metastatic cells as well as in cells exposed to hypoxic conditions. Utilizing CRISPR technology, we assembled a library of guide RNAs targeting the discovered hits and are currently validating the top identified genes using both in vitro and in vivo systems. To validate hits that are essential in metastatic cells in vivo, we engineered selected “matched” pairs of HNSCC cell lines to express CAS9 protein upon induction with doxycycline. The cells are then transduced with the library of guides and injected subcutaneously into mice; tumors from control and doxycycline treated animals are compared. The difference in genes that are essential for growth of tumors seeded by primary tumor derived and metastasis derived HNSCC cell lines is then assessed in a quantitative manner. Citation Format: Maria Kondratyev, Aleksandra Pesic, Troy Ketela, Azin Sayad, Stephano Marastoni, Jason Mofat, Carl Virtanen, Natalie Stickle, Reider Grenman, Brad Wouters. Novel targets in metastatic HNSCC [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 407.

HNSCC is 6th most common malignancy in the world. Despite advances in diagnosis and treatment, the survival rates remain low due to frequent recurrences the biology of which remains unclear. Using functional genomic technologies, we identified new therapeutic targets for metastatic disease in HNSCC. Whole genome shRNA screens were conducted in matched sets of cell lines derived from primary tumors and respective metastatic sites, identifying genes essential for cell survival only following metastasis. To test if knockdown of selected targets inhibits metastasis in a therapeutic setting, we established orthotropic model of HNSCC that metastasize to lymph nodes in the mouse. Components of Notch pathway were identified as essential for survival of cells derived from metastatic sites. Whole exome sequencing identified a novel mutation in one of the EGF domains of Notch3 that was acquired only in the metastatic line. Utilizing CRISPR methodology, we established that “fixing” the mutation results in reversal of metastatic phenotype of the cells, making them Notch independent similar to their primary tumor counterparts. Mutations in EGF domains have been reported to influence interaction with specific ligands, dictating which ligand can activate Notch signaling. Our data indicate that a distinct set of target genes is induced upon interaction between Notch3 and Jag2 ligand. Furthermore, our results indicate that suppression of Notch3 improves survival in mice bearing orthotropic tumors derived from the metastatic HNSCC lines. Overall, our data demonstrate that metastatic cells from head and neck tumors acquire dependency on Notch3 signaling. Novel treatments targeting components of this pathway may prove effective in targeting metastatic cells alone or in combination with conventional therapies. Citation Format: Maria Kondratyev, Aleksandra Pesic, Troy Ketela, Azin Sayad, Stephano Marastoni, Carl Virtanen, Laurie Ailles, Soroush Samadian, Mikhail Bashkurov, Marianne Koritzinsky, Brad Wouters. Novel therapeutic targets in head and neck 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 3020. doi:10.1158/1538-7445.AM2017-3020

HNSCC is 6th most common malignancy in the world. Despite advances in diagnosis and treatment, the survival rates remain low due to frequent recurrences the biology of which remains unclear. Using functional genomic technologies we identified new therapeutic targets for metastatic disease in HNSCC. Whole genome shRNA screens were conducted in matched sets of cell lines derived from primary tumors and respective metastatic sites, identifying genes essential for cell survival only following metastasis. To test if knockdown of selected targets inhibits metastasis in a therapeutic setting, we established orthotropic model of HNSCC that metastasize to lymph nodes in the mouse. Components of Notch pathway were identified as essential for survival of cells derived from metastatic sites. Whole exome sequencing identified a novel mutation in one of the EGF domains of Notch3 that was acquired only in the metastatic line. Mutations in EGF domains have been reported to influence interaction with specific ligands, dictating which ligand can activate Notch signaling. Our data indicate that metastatic, but not primary tumor cells, undergo apoptosis upon knockdown of Notch3 and that a distinct set of target genes is induced upon interaction between Notch3 and Jag2 ligand. Furthermore, our results indicate that suppression of Notch3 improves survival in mice bearing orthotropic tumors derived from the metastatic HNSCC lines. Our data demonstrate that metastatic cells from head and neck tumors acquire dependency on Notch3 signaling. Novel treatments targeting components of this pathway may prove effective in targeting metastatic cells alone or in combination with conventional therapies. Citation Format: Maria Kondratyev, Aleksandra Pesic, Stephano Marastoni, Troy Ketela, Jason Moffat, Carl Virtanen, Azin Sayad, Mikhail Bashkurov, Alessandro Dati, Laurie Ailles, Reidar Grenman, Marianne Koritzinsky, Brad Wouters. Novel therapeutic targets in head and neck 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 3794.

Introduction: Enzalutamide is a recently approved potent androgen receptor antagonist for prostate cancer both in the pre- and post-docetaxel settings. Its action relies upon 3 defined mechanisms of action: 1) blocking testosterone binding to the androgen receptor (AR), 2) preventing nuclear translocation of AR, and 3) inhibiting AR-DNA binding. However, despite its utility in prolonged overall survival in both these settings, disease relapse is inevitable, suggesting that resistance to the drug is somehow being acquired and is of vital concern. Methods: Using a combination of high-throughput functional genomic (kinome-shRNA) and chemical genomic (pharmacological inhibitor) screens, along with various in vitro / in vivo assays (proliferation, apoptosis, necrosis, soft agar growth, etc.) we have identified the IkB kinase (IKK) / IkB / NFkB signaling axis as important for acquisition of, and continued resistance to, Enzalutamide. Results: Using a panel of Enzalutamide-resistant and -sensitive cell lines, we show that targeting this signaling axis will selectively kill Enzalutamide-resistant cells while not affecting the proliferation of sensitive cells. Moreover, our data shows that the pharmacological inhibitors and shRNAs are effective even without Enzalutamide administration, negating any potential drug-drug interaction issues. Finally, we have found that our cells are resistant to Enzalutamide in an AR-V7-independent manner, suggesting that NFkB signaling is acting in a novel manner to bring about the resistance phenotype. Conclusions: Through in vitro and in vivo experiments, we provide evidence that targeting activators of NFκB signalling can be a strategy to help patients with Enzalutamide-resistant prostate cancer. Note: This abstract was not presented at the meeting. Citation Format: Sujeeve Jeganathan, Amina Zoubeidi, Martin Gleave, Brad G. Wouters, Anthony M. Joshua. Using functional and chemical genomics to identify mechanisms of Enzalutamide resistance in prostate cancer. [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 732. doi:10.1158/1538-7445.AM2015-732