Our laboratorys accomplishments this year begin with full characterization of the tumor microenvironment of two different models of murine oral cavity (MOC) cancer. Both models are carcinogen-induced, fully syngeneic models of oral cancer that are transplantable into immunocompetent C57BL/6 mice. MOC1 generated tumors are slow growing, do not metastasize and demonstrate a T-cell inflamed phenotype with high baseline CD8 T-lymphocyte and NK cell infiltration. MOC1 tumors have a high mutational burden and are predicted to have dozens of neoantigens with high affinity binding to MHC class I complexes. MOC2 tumors are very aggressive, metastasize early, and demonstrate a non-T-cell inflamed microenvironment with low CD8 T-lymphocyte and NK infiltrate but robust infiltration of myeloid derived suppressor cells (MDSCs). MOC2 tumors have low mutation burden and are predicted to have very few neoantigens with high MHC class I binding affinity. These two tumors, which are both carcinogen-induced oral cavity squamous cell carcinomas, represent powerful models that can be used to study highly (MOC1) and poorly (MOC2) immunogenic tumors in immune competent mice. We further characterized the expression of programmed death ligand 1 (PD-L1) in both models, and demonstrated high PD-L1 expression in MOC1 tumors and very low PD-L1 expression in MOC2 tumors, consistent with their baseline phenotype. Using small molecule inhibitors to target oncogenic pathways important for the growth and survival of MOC cells, we revealed that this PD-L1 expression in MOC1 was not associated with oncogenic signaling, but rather associated with baseline and changes in interferon levels within the tumor microenvironment (Head and Neck, In Press). This work further established the MOC1 and 2 models as useful tools to study treatment-induced changes in the tumor microenvironment. Our first series of therapeutic experiments were designed to understand how small molecule inhibitors that target the phosphoinositide 3-kinase/mammalian target of rapamycin (PI3K/mTOR) and mitogen-activated protein kinase (MAPK) pathways, both shown to be commonly co-activated in head and neck cancers, affect tumorigenesis in MOC1 and MOC2 tumors. While the growth of both primary tumors was significantly suppressed while on treatment, we demonstrated that mTOR inhibition resulted in durable anti-tumor responses following cessation of treatment in immunogenic MOC1 but not poorly immunogenic MOC2 mice. This was a CD8 but not NK dependent mechanism as CD8 depletion completely abolished the anti-tumor effect of rapamycin in immunogenic MOC1 tumors. Conversely, inhibition of the MAPK pathway was found to be very immunosuppressive in immunogenic MOC1 tumors, blocking tumor infiltration and expansion of antigen-specific CD8 T-lymphocytes and NK cells and prevent durable treatment responses (Oncotarget, In Press). This data challenges existing paradigms as rapamycin, more so than MAPK inhibition, is generally felt to be immunosuppressive in the context of preventing solid organ transplant rejection. This data, combined with existing clinical data demonstrating objective tumor responses to single agent rapamycin in a subset of patients with treatable oral cavity cancer, suggested that rapamycin could synergize with immunotherapies to improve anti-tumor immune responses. We next combined rapamycin and a MEK inhibitor with anti-PD-L1 antibody immunotherapy. We demonstrated that while no significant responses were observed in MOC2 tumors following PD-L1 inhibition, transient anti-tumor responses with PD-L1 blockade and durable anti-tumor responses in a subset of tumors following rapamycin were significantly enhanced following combination therapy. No synergy was observed between MEK and PD-L1 inhibition; rather, MEK inhibition appeared to reverse the favorable CD8 and NK responses observed with PD-L1 blockade alone. Mechanistically combination rapamycin and PD-L1 blockade enhanced IFN production from antigen-specific T-cells both in the tumor and in the periphery, and was again a CD8 and not NK dependent process (Manuscript Submitted). We have continued to work to understand the exact mechanism of how rapamycin in enhancing immunotherapeutic responses, with the ultimate goal of gathering enough pre-clinical evidence to justify combining rapamycin with checkpoint inhibitor therapy in the clinic. Given this demonstration of how targeted therapies can critically alter the tumor microenvironment to either enhance or limit immunotherapeutics, we have expanded our study to how various targeted and adjuvant therapies effect T-lymphocyte and NK cell function both in vitro and in vivo with the goal of optimizing different treatment combinations to maximize anti-tumor activity. We will continue to use the MOC model system as a means of pre-clinically evaluating different combinations, with correlative and mechanistic studies validating which combinations carry the most clinical promise.

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2015
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Deafness & Other Communication Disorders
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Moore, Ellen C; Sun, Lillian; Clavijo, Paul E et al. (2018) Nanocomplex-based TP53 gene therapy promotes anti-tumor immunity through TP53- and STING-dependent mechanisms. Oncoimmunology 7:e1404216
Ahn, Julie; Bishop, Justin A; Roden, Richard B S et al. (2018) The PD-1 and PD-L1 pathway in recurrent respiratory papillomatosis. Laryngoscope 128:E27-E32
Friedman, Jay; Morisada, Megan; Sun, Lillian et al. (2018) Inhibition of WEE1 kinase and cell cycle checkpoint activation sensitizes head and neck cancers to natural killer cell therapies. J Immunother Cancer 6:59
Zolkind, Paul; Przybylski, Dariusz; Marjanovic, Nemanja et al. (2018) Cancer immunogenomic approach to neoantigen discovery in a checkpoint blockade responsive murine model of oral cavity squamous cell carcinoma. Oncotarget 9:4109-4119
Morisada, Megan; Chamberlin, Michael; Allen, Clint (2018) Exploring the rationale for combining ionizing radiation and immune checkpoint blockade in head and neck cancer. Head Neck 40:1321-1334
Morisada, Megan; Clavijo, Paul E; Moore, Ellen et al. (2018) PD-1 blockade reverses adaptive immune resistance induced by high-dose hypofractionated but not low-dose daily fractionated radiation. Oncoimmunology 7:e1395996
Gadkaree, Shekhar K; Pandian, Vinciya; Best, Simon et al. (2017) Laryngotracheal Stenosis: Risk Factors for Tracheostomy Dependence and Dilation Interval Otolaryngol Head Neck Surg 156:321-328
Tatar, Emel Çadalli; Kupfer, Robbi A; Barry, Jonnae Y et al. (2017) Office-Based vs Traditional Operating Room Management of Recurrent Respiratory Papillomatosis: Impact of Patient Characteristics and Disease Severity. JAMA Otolaryngol Head Neck Surg 143:55-59
Kuo, Chia-Wei; Allen, Clint Tanner; Huang, Chu-Chun et al. (2017) Murray secretion scale and fiberoptic endoscopic evaluation of swallowing in predicting aspiration in dysphagic patients. Eur Arch Otorhinolaryngol 274:2513-2519
Tran, Linda; Allen, Clint T; Xiao, Roy et al. (2017) Cisplatin Alters Antitumor Immunity and Synergizes with PD-1/PD-L1 Inhibition in Head and Neck Squamous Cell Carcinoma. Cancer Immunol Res 5:1141-1151

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