Immune Checkpoint Regulation by the Integrated Stress Response Pathway in Lung Ca

项目摘要 近年来，在识别和治疗靶向方面有了突破性的发现， PD-1/PD-L1免疫检查点轴。肺癌细胞表达高水平的程序性死亡配体 1（PD-L1），T细胞上PD-1的关键配体。PD-1/PD-L1相互作用允许肿瘤细胞直接 抑制抗肿瘤T细胞活性，导致免疫逃逸和肿瘤进展。尽管有这些 然而，随着研究的进展，患者PD-L1表达与治疗反应之间仍然存在脱节。这 强调迫切需要了解PD-L1上调的机制，确定 对PD-1/PD-L1治疗的耐药性，并确定其他免疫检查点或途径，以在临床上寻求 与这种疗法的结合。在肿瘤微环境应激反应中，如缺氧、血红素 剥夺和氨基酸饥饿，癌细胞激活整合应激反应（ISR）。ISR 激活允许癌细胞通过抑制整体蛋白质合成来逃避这些应激， 增加选择mRNA的翻译。ISR已被证明促进肿瘤发生，然而， ISR在免疫检查点蛋白的翻译控制中尚未得到充分研究。我们最近 证明ISR激活导致非小细胞肺癌（NSCLC）中PD-L1的强效诱导， 抑制抗肿瘤免疫在体外和体内，我们有新的证据表明，另一种免疫 在NSCLC细胞中，ISR激活后诱导检查点CD 155（分化簇155） 与PD-L1同时使用。我们的中心假设是ISR激活导致肿瘤细胞免疫逃逸 通过PD-L1和CD 155的翻译。在强有力的初步数据的指导下，我们将检验这一假设 通过追求三个具体目标：1）阐明ISR激活促进 CD 155的翻译; 2）确定ISR调节对免疫细胞应答的影响; 3）检查ISR的表达。 ISR抑制与PD-1阻断和/或TIGIT（CD 155免疫细胞 受体）阻断。我们将采用翻译研究，包括荧光素酶报告基因测定 和核糖体分析来剖析ISR介导的PD-L1和CD 155翻译控制的机制， NSCLC细胞。为了确定ISR激活对免疫细胞应答的影响，我们将测量免疫细胞的免疫应答。 共培养研究和免疫活性小鼠模型中ISR活化后的细胞应答。最后我们将 利用小鼠模型和ISR抑制剂来确定ISR抑制是否可以通过以下方式抑制肿瘤发生： 促进免疫应答，以及这是否可以与现有的免疫检查点疗法协同作用 (Fig 1，模型）。我们提出的研究是重要的，因为它将1）揭示新的监管电路， 控制免疫检查点蛋白表达，2）阐明癌细胞如何在免疫检查点中经历损伤， 肿瘤微环境调节免疫细胞的反应，3）我们的研究将提供证据， 肺癌的新的联合疗法的发展的概念。

PROJECT SUMMARY In recent years, there have been groundbreaking discoveries in the identification and therapeutic targeting of the PD-1/PD-L1 immune checkpoint axis. Lung cancer cells express high levels of Programmed Death Ligand 1 (PD-L1), a critical ligand for PD-1 on T cells. The PD-1/PD-L1 interaction allows tumor cells to directly suppress anti-tumor T cell activity, resulting in immune escape and tumor progression. Despite these advances, there remains a disconnect in patient expression of PD-L1 and treatment response. This underscores the critical need to understand mechanisms of PD-L1 upregulation, identify mechanisms of resistance to PD-1/PD-L1 therapy, and identify other immune checkpoints or pathways to pursue clinically in combination with this therapy. In response to tumor microenvironment stresses, such as hypoxia, heme deprivation, and amino acid starvation, cancer cells activate the integrated stress response (ISR). ISR activation allows cancer cells to escape these stresses through inhibition of global protein synthesis and increased translation of select mRNAs. The ISR has been shown to promote tumorigenesis, yet the role of the ISR in the translational control of immune checkpoint proteins has not been fully investigated. We recently demonstrated that ISR activation leads to potent induction of PD-L1 in non-small cell lung cancer (NSCLC) and suppression of anti-tumor immunity in vitro and in vivo, and we have new evidence that another immune checkpoint, CD155 (Cluster of differentiation 155), is induced upon ISR activation in NSCLC cells simultaneously with PD-L1. Our central hypothesis is that ISR activation causes tumor cell immune escape through translation of both PD-L1 and CD155. Guided by strong preliminary data, we will test this hypothesis by pursuing three specific aims: 1) Elucidate the mechanisms through which ISR activation promotes translation of CD155; 2) Determine the effect of ISR modulation on immune cell responses; 3) Examine the therapeutic efficacy of ISR inhibition in combination with PD-1 blockade and/or TIGIT (CD155’s immune cell receptor) blockade in mouse models. We will employ translational studies including luciferase reporter assays and ribosome profiling to dissect the mechanisms of ISR mediated PD-L1 and CD155 translational control in NSCLC cells. To determine the impact of ISR activation on immune cell responses, we will measure immune cell responses in co-culture studies and immunocompetent mouse models upon ISR activation. Finally, we will utilize mouse models and ISR inhibitors to determine whether ISR inhibition can suppress tumorigenesis by promoting an immune response and whether this can synergize with existing immune checkpoint therapies (Fig 1, model). Our proposed research is significant, because it will 1) uncover new regulatory circuits that govern immune checkpoint protein expression, 2) illuminate how insults experienced by cancer cells in the tumor microenvironment modulate the responses of immune cells, and 3) our studies will provide proof-of- concept for the development of new combination therapies for lung cancer.