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

我们实验室今年的成就开始与两种不同的小鼠口腔（MOC）癌症模型的肿瘤微环境的充分表征。这两种模型都是致癌物诱导的，完全同源的口腔癌模型，可移植到免疫活性的C57 BL/6小鼠中。MOC 1产生的肿瘤生长缓慢，不转移，并表现出具有高基线CD 8 T淋巴细胞和NK细胞浸润的T细胞发炎表型。MOC 1肿瘤具有高突变负荷，并且预测具有数十种与MHC I类复合物具有高亲和力结合的新抗原。MOC 2肿瘤具有很强的侵袭性，早期转移，并且表现出非T细胞发炎的微环境，具有低的CD 8 T淋巴细胞和NK浸润，但髓源性抑制细胞（MDSC）的强烈浸润。MOC 2肿瘤具有低突变负荷，并且预测具有非常少的具有高MHC I类结合亲和力的新抗原。这两种肿瘤都是致癌物诱导的口腔鳞状细胞癌，代表了可用于研究免疫活性小鼠中高（MOC 1）和低（MOC 2）免疫原性肿瘤的强大模型。我们进一步表征了两种模型中程序性死亡配体1（PD-L1）的表达，并证明MOC 1肿瘤中PD-L1表达较高，MOC 2肿瘤中PD-L1表达极低，与其基线表型一致。使用小分子抑制剂靶向对MOC细胞生长和存活重要的致癌途径，我们发现MOC 1中的PD-L1表达与致癌信号传导无关，而是与肿瘤微环境中干扰素水平的基线和变化相关（Head and Neck，In Press）。这项工作进一步建立了MOC 1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。 我们的第一系列治疗实验旨在了解靶向磷酸肌醇3-激酶/哺乳动物雷帕霉素靶点（PI 3 K/mTOR）和丝裂原活化蛋白激酶（MAPK）途径的小分子抑制剂如何影响MOC 1和MOC 2肿瘤的肿瘤发生，这两种途径通常在头颈癌中被共激活。虽然两种原发性肿瘤的生长在治疗时均被显著抑制，但我们证明了mTOR抑制在免疫原性MOC 1小鼠而非免疫原性较差的MOC 2小鼠中停止治疗后产生持久的抗肿瘤应答。这是一种CD 8依赖性机制，而不是NK依赖性机制，因为CD 8消耗完全消除了雷帕霉素在免疫原性MOC 1肿瘤中的抗肿瘤作用。相反，发现MAPK途径的抑制在免疫原性MOC 1肿瘤中具有非常强的免疫抑制性，阻断肿瘤浸润和抗原特异性CD 8 T淋巴细胞和NK细胞的扩增，并阻止持久的治疗应答（Oncotarget，In Press）。这一数据挑战了现有的范例，因为雷帕霉素在预防实体器官移植排斥的背景下通常被认为是免疫抑制的，而不是MAPK抑制。该数据与现有的临床数据相结合，证明了在可治疗的口腔癌患者亚组中对单药雷帕霉素的客观肿瘤应答，表明雷帕霉素可以与免疫疗法协同作用以改善抗肿瘤免疫应答。 我们接下来将雷帕霉素和MEK抑制剂与抗PD-L1抗体免疫疗法组合。我们证明，虽然在PD-L1抑制后在MOC 2肿瘤中未观察到显著反应，但在联合治疗后，PD-L1阻断的瞬时抗肿瘤反应和雷帕霉素治疗后肿瘤亚组中的持久抗肿瘤反应显著增强。在MEK和PD-L1抑制之间未观察到协同作用;相反，MEK抑制似乎逆转了单独PD-L1阻断时观察到的有利的CD 8和NK应答。雷帕霉素和PD-L1阻断的机械组合增强了肿瘤和外周中抗原特异性T细胞的IFN产生，并且再次是CD 8而非NK依赖性过程（Mandelipt Submitted）。我们继续努力了解雷帕霉素如何增强免疫应答的确切机制，最终目标是收集足够的临床前证据，以证明雷帕霉素与检查点抑制剂治疗相结合的合理性。 鉴于靶向治疗如何严重改变肿瘤微环境以增强或限制免疫治疗的这一证明，我们将我们的研究扩展到各种靶向和辅助治疗如何在体外和体内影响T淋巴细胞和NK细胞功能，目的是优化不同的治疗组合以最大化抗肿瘤活性。我们将继续使用MOC模型系统作为临床前评估不同组合的手段，相关和机制研究验证哪些组合具有最大的临床前景。

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.