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， 所有肺癌患者加起来只有大约18%。癌症免疫治疗的实施对于固体 肿瘤如肺癌已经显示出很大的希望，并提供了改善预后的可能性， 小部分患者。然而，大多数患者几乎没有反应或获得耐药性 在作为单一疗法递送的检查点抑制剂治疗期间。因此，鉴定耐药性 机制和潜在的联合治疗方法是一个关键的需要，以提高反应率， 免疫检查点抑制剂和患者预后。为了解决这个问题，临床相关的体内shRNA缺失 在上皮细胞中进行集中于编码FDA批准的药物靶点（FDAome）的基因的筛选， 间充质Kras/p53（KP）突变小鼠肺癌细胞。然后用同种型或抗- PD-1抗体。对条形码化的shRNA进行测序显示，Ntrk 1从 与同种型治疗的肿瘤相比，用PD-1阻断剂攻击的间充质肿瘤，表明它提供了 在免疫系统压力下，这些肿瘤细胞的生存优势。初步数据证实 在用PD-1抑制剂处理的间充质肿瘤中Ntrk 1转录水平上调， 对人NSCLC细胞系的分析显示，Ntrk 1 mRNA水平与 更具侵略性的间充质细胞表型此外，Ntrk 1过表达细胞上调PD-L1 当与脾细胞共培养时，通过上调JAK信号传导表达。Ntrk 1的稳定敲除 在间充质鼠KP突变肺癌细胞中减少体内肿瘤生长和分析肿瘤- 通过流式细胞术的浸润T细胞群显示CD 8 + T细胞耗竭显著降低， 而Ntrk 1的过表达促进CD 8 + T细胞耗竭，从而降低效应子状态。这些 肿瘤还具有改变的微环境，具有经典免疫抑制细胞因子的上调 例如IL-10。PD-1蛋白水平在Ntrk 1高人NSCLC细胞系中也显著增加， 提供了Ntrk 1可能是人肺中免疫系统功能的调节剂的额外证据 疾病这项工作的中心假设是Ntrk 1的上调导致了对大肠杆菌的获得性抗性。 通过异常JAK信号传导和下游CD 8 + T细胞功能障碍阻断PD-1，从而促进肿瘤 细胞存活各种强大的工具将被用来测试这一假设，包括时间推移成像的 动态T细胞-肿瘤细胞相互作用、肺癌的基因工程和同基因临床前模型 分析作为Ntrk 1表达函数的免疫亚群，以及人NSCLC组织的IHC分析 样品这项工作的目的是为Ntrk 1的合理药物组合提供强有力的证据。 将PD-1阻断剂用于临床，以消除免疫检查点阻断 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.