Targeting mechanisms of immune evasion in chemotherapy-induced senescent cells

项目概要/摘要 乳腺肿瘤很少能通过化疗根除。残留疾病的存在有两个原因：肿瘤细胞 1) 避免因细胞凋亡、有丝分裂灾难和营养缺乏而死亡； 2）避免免疫清除。我们的 先前的研究已经证实，通过激活 p53 介导的停滞和衰老程序，肿瘤 细胞避免因有丝分裂灾难、细胞凋亡和营养缺乏而死亡。我们展示了化疗治疗 与 p53 突变型乳腺癌患者相比，p53 野生型乳腺癌患者的生存率较差 肿瘤。衰老细胞通过产生细胞因子和趋化因子（称为 衰老相关的分泌表型（SASP）。 先前研究的弱点是未能确定经过治疗的衰老肿瘤细胞如何避免 免疫清除。在本提案中，我们提供了令人信服的初步数据来解决这一弱点，并且 计划全面描述和研究旨在逃避细胞死亡的潜在治疗途径， 从而促进免疫清除并提高生存率。在初步数据中，我们发现了一个显着的 持续残留疾病中 PD-L1 的上调可能是通过与基质细胞的相互作用介导的， 并且在复发时仍保持表达，表明存在很强的选择压力。 该项目的目标是确定机制并制定专门针对免疫逃避的策略 在化疗后幸存的肿瘤细胞中进入衰老状态。 假设：基质干扰素 γ (IFNγ) 在化疗中的特定细胞亚群中诱导 PD-L1 治疗后的衰老肿瘤。可以使用一种新策略来靶向这些持续存在的衰老肿瘤细胞， 结合 senolytic 药物诱导免疫原性细胞死亡，然后使用免疫检查点抑制剂。 在目标 1 中，我们将使用小鼠乳腺肿瘤模型来确定表达 PD-L1 的肿瘤细胞是否具有更大的 与同一治疗肿瘤内不表达 PD-L1 的细胞相比，复发的可能性，以及每种细胞的反应如何 再治疗。我们将描述表达 PD-L1 的细胞与不表达 PD-L1 的细胞中的基因表达差异。 在目标 2 中，我们将确定衰老肿瘤细胞上调 PD-L1 所需的体内因素。 我们将把乳腺肿瘤移植到缺乏特定免疫成分（如 T 细胞）的同基因小鼠体内， 采用化疗治疗，并确定 PD-L1 表达和免疫环境的变化。 在目标 3 中，初步数据显示，只有三分之一的衰老小鼠乳腺肿瘤对抗 PD-L1 有反应 化疗后的治疗。我们将测试是否用 BH3 模拟药物诱导免疫原性细胞死亡 已证明具有 senolytic 活性，可以改善对免疫检查点抑制剂的反应。 在目标 4 中，我们将对人类乳腺癌标本前后进行多重免疫组织化学染色 化疗以确定衰老肿瘤中是否诱导 PD-L1 表达。

Project Summary/Abstract Breast tumors are rarely eradicated by chemotherapy. Residual disease exists for 2 reasons: the tumor cells 1) avoid death from apoptosis, mitotic catastrophe, and nutrient deprivation; 2) avoid immune clearance. Our previous research has established that by activating p53-mediated programs of arrest and senescence, tumor cells avoid death by mitotic catastrophe, apoptosis, and nutrient deprivation. We showed chemotherapy treated breast cancer patients with p53 wild type tumors have dismal survival compared to patients with p53 mutant tumors. Senescent cells promote relapse by their production of cytokines and chemokines (termed the senescence associated secretory phenotype, or SASP). The weakness of prior research has been a failure to identify how the treated, senescent tumor cells avoid immune clearance. In this proposal, we present compelling preliminary data addressing this weakness, and plans to fully characterize and investigate potential therapeutic avenues that target the evasion of cell death, thus facilitating immune clearance and improving survival. In preliminary data, we identify a remarkable upregulation of PD-L1 in the persisting residual disease likely mediated through interactions with stromal cells, and expression is maintained in the relapse, suggesting a strong selective pressure. The goal of this project is to identify mechanisms and develop strategies to target immune evasion specifically in tumor cells that have survived chemotherapy by entering senescence. Hypothesis: Stromal interferon gamma (IFNγ) induces PD-L1 in a specific subset of cells in a chemotherapy- treated, senescent tumor. These persisting, senescent tumor cells can be targeted using a novel strategy that combines senolytic drugs to induce immunogenic cell death followed by immune checkpoint inhibitors. In Aim 1 we will use mouse mammary tumor models to determine if PD-L1 expressing tumor cells have greater relapse potential than cells within the same treated tumor that do not express PD-L1, and how each responds to retreatment. We will characterize gene expression differences in cells that express PD-L1 vs. those that do not. In Aim 2 we will identify the factors in vivo that are required for PD-L1 upregulation on senescent tumor cells. We will transplant mammary tumors into syngeneic mice that lack specific immune components such as T cells, treat with chemotherapy, and determine changes in PD-L1 expression and immune contexture. In Aim 3, preliminary data show only one third of senescent mouse mammary tumors responded to anti-PD-L1 therapy following chemotherapy. We will test if inducing immunogenic cell death with BH3 mimetic drugs we have shown to have senolytic activity can improve the response to immune checkpoint inhibitors. In Aim 4, we will use multiplex immunohistochemistry staining of human breast cancer specimens pre and post chemotherapy to determine if PD-L1 expression is induced in senescent tumors.