Identifying the role of Ntrk1 in immunosuppression in Kras/p53 mutant lung cancer

项目摘要 肺癌是美国癌症相关死亡的主要原因，而肺癌患者的5年存活率 所有肺癌患者加起来只有18%左右。固体肿瘤免疫治疗的实施 肺癌等肿瘤显示出巨大的希望，并为改善患者的预后提供了可能性 一小部分患者。然而，大多数患者几乎没有反应或获得抵抗力。 在使用检查点抑制剂作为单一疗法提供的治疗期间。因此，识别抗性 机制和潜在的联合治疗方法是提高应答率的迫切需要 免疫检查点抑制剂与患者预后。为了解决这个问题，一种临床上相关的体内shRNA缺失 对FDA批准的药物靶点(FDAome)的编码基因进行筛查 间充质Kras/p53(KP)突变小鼠肺癌细胞。然后给小鼠注射同型或抗-HBs。 PD-1抗体。对条形码shRNAs的测序显示，NTRK1从 用PD-1阻滞剂挑战间充质瘤与同型治疗的肿瘤相比，表明它提供了 在免疫系统压力下，对这些肿瘤细胞来说是一种生存优势。初步数据已确认 PD-1抑制剂和来源的细胞系治疗的间叶性肿瘤中NTRK1转录水平上调 对人非小细胞肺癌细胞株的分析表明，NTRK1的mRNA水平与 更具侵袭性的间充质细胞表型。此外，NTRK1过表达细胞上调PD-L1 通过上调JAK信号与脾细胞共培养时的表达。稳定的NTRK1基因敲除 在间充质小鼠中，Kp突变的肺癌细胞体内肿瘤生长减少，并分析了肿瘤- 流式细胞仪检测显示CD8+T细胞耗竭明显减少， 而NTRK1过表达促进CD8+T细胞耗竭，从而降低效应器状态。这些 肿瘤的微环境也发生了改变，经典的免疫抑制细胞因子上调。 比如IL-10。在NTRK1高表达的人非小细胞肺癌细胞系中，PD-1蛋白水平也显著增加， 提供更多证据表明NTRK1可能是人类肺免疫系统功能的调节器 疾病。拟议工作的中心假设是NTRK1上调导致获得性抗药性 通过异常的JAK信号和下游CD8+T细胞功能障碍阻断PD-1，从而促进肿瘤的发生 细胞存活。将使用各种强大的工具来验证这一假设，包括对 T细胞-肿瘤细胞的动态相互作用、肺癌的基因工程和同基因临床前模型 分析免疫亚群与NTRK1表达的关系，以及人非小细胞肺癌组织的免疫组化分析 样本。拟议工作的目标是为NTRK1的合理药物组合提供强有力的证据 具有PD-1阻断的抑制剂将推广到临床以消除免疫检查点阻断 抵抗，并最终改善患者的预后。

Project Summary Lung cancer is the leading cause of cancer-related deaths in the United States, and the 5-year survival rate of all lung cancer patients combined is only about 18%. The implementation of cancer immunotherapeutics for solid tumors such as lung cancers has shown great promise and provided the possibility for improved outcome in a small percentage of patients. However, the majority of patients show little to no response or acquire resistance during treatment with checkpoint inhibitors delivered as a monotherapy. Therefore, identifying resistance mechanisms and potential combination therapy approaches is a critical need to improve response rates to immune checkpoint inhibitors and patient prognosis. To address this, a clinically relevant in vivo shRNA dropout screen focused on genes encoding for FDA-approved drug targets (FDAome) was performed in epithelial and mesenchymal Kras/p53 (KP) mutant murine lung cancer cells. Mice were then treated with either isotype or anti- PD-1 antibody. Sequencing for the barcoded shRNAs revealed that Ntrk1 was significantly depleted from mesenchymal tumors challenged with PD-1 blockade compared to isotype treated tumors, suggesting it provides a survival advantage to these tumor cells when under immune system pressure. Preliminary data confirmed Ntrk1 transcript levels are upregulated in mesenchymal tumors treated with PD-1 inhibitors and cell lines derived from resistant tumors, and analysis of human NSCLC cell lines revealed that Ntrk1 mRNA levels correlate with a more aggressive, mesenchymal cell phenotype. Additionally, Ntrk1 overexpressing cells upregulate PD-L1 expression when co-cultured with splenocytes through upregulation of JAK signaling. Stable knockdown of Ntrk1 in mesenchymal murine KP mutant lung cancer cells reduced tumor growth in vivo and analysis of tumor- infiltrating T cell populations via flow cytometry showed that CD8+ T cell exhaustion was significantly reduced, whereas overexpression of Ntrk1 promoted CD8+ T cell exhaustion, thus decreasing effector status. These tumors also have an altered microenvironment, with upregulation of classically immunosuppressive cytokines such as IL-10. PD-1 protein levels were also significantly increased in Ntrk1-high human NSCLC cell lines, providing additional evidence that Ntrk1 may be a modulator of immune system functionality in human lung disease. The central hypothesis of the proposed work is that Ntrk1 upregulation causes acquired resistance to PD-1 blockade via aberrant JAK signaling and downstream CD8+ T cell dysfunction, thereby promoting tumor cell survival. A variety of powerful tools will be utilized to test this hypothesis, including time-lapse imaging of dynamic T cell-tumor cell interactions, genetically-engineered and syngeneic preclinical models of lung cancer to analyze immune subpopulations as a function of Ntrk1 expression, and IHC analyses of human NSCLC tissue samples. The goal of the proposed work is to provide strong evidence for a rational drug combination of Ntrk1 inhibitors with PD-1 blockade to be carried forward into the clinic to abrogate immune checkpoint blockade resistance and ultimately improve patient outcomes.