Characterization and Enhancement of Anti-Tumor Immune Responses in Head and Neck Cancer

我们的实验室今年的成就始于对两种不同小鼠口腔（MOC）癌模型的肿瘤微环境的全面表征。这两种模型都是致癌物诱导的、完全同基因的口腔癌模型，可移植到免疫活性的C57BL/6小鼠体内。MOC1产生的肿瘤生长缓慢，不转移，表现为t细胞炎症表型，伴有高基线CD8 t淋巴细胞和NK细胞浸润。MOC1肿瘤具有很高的突变负担，预计具有数十种与MHC I类复合物高亲和力结合的新抗原。mo2c肿瘤具有很强的侵袭性，早期转移，并表现出非t细胞炎症微环境，CD8 t淋巴细胞和NK细胞浸润低，但骨髓源性抑制细胞（MDSCs）浸润强烈。MOC2肿瘤具有较低的突变负担，预计具有高MHC I类结合亲和力的新抗原很少。这两种肿瘤都是致癌物诱导的口腔鳞状细胞癌，它们代表了强大的模型，可用于研究免疫功能正常小鼠中高（MOC1）和低（MOC2）免疫原性肿瘤。我们进一步表征了程序性死亡配体1 （PD-L1）在两种模型中的表达，并证明了MOC1肿瘤中PD-L1的高表达和MOC2肿瘤中PD-L1的极低表达，与它们的基线表型一致。使用小分子抑制剂靶向对MOC细胞生长和存活至关重要的致癌途径，我们发现MOC细胞中PD-L1的表达与致癌信号无关，而是与肿瘤微环境中干扰素水平的基线和变化有关（Head and Neck, in Press）。这项工作进一步建立了MOC1和2模型，作为研究治疗诱导的肿瘤微环境变化的有用工具。

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.