Sensitization to RIPK1-dependent death as a strategy to enhance response of renal cell carcinoma (RCC) to immunotherapy

对 RIPK1 依赖性死亡的敏感性作为增强肾细胞癌 (RCC) 对免疫治疗反应的策略

• 批准号: 10721156
• 负责人: ADRIAN T TING
• 金额: $ 41.5万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-19 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10721156
• 关键词: American Cancer Society; Apoptosis; Attention; Bacterial Infections; Behavior; CASP8 gene; CD8-Positive T-Lymphocytes; CTLA4 gene; Cause of Death; Cell Death; Cell Death Induction; Cell Line; Cell Survival; Cells; Cessation of life; Clinical; Coculture Techniques; Code; Complex; Conventional (Clear Cell) Renal Cell Carcinoma; Cytoplasmic Granules; Data; Dependence; Disease; Effectiveness; Engraftment; Exhibits; Failure; Family; Genetic Transcription; Goals; Granzyme; Human; Immune; Immune mediated destruction; Immune system; Immunotherapy; In Vitro; Inbred BALB C Mice; Individual; Knock-out; Lead; Life; Linear Regressions; Link; Lipopolysaccharides; Malignant Epithelial Cell; Malignant Neoplasms; Mediating; Methamphetamine; Modification; Mus; Nivolumab; PD-1/PD-L1; Patients; Peptides; Persons; Phosphorylation; Phosphotransferases; Predisposition; Proteins; RIPK1 gene; RIPK3 gene; Rag1 Mouse; Renal Cell Carcinoma; Renal carcinoma; Resistance; Serum; Signal Pathway; Signal Transduction; Signaling Molecule; T-Lymphocyte; TBK1 gene; TNF gene; TNFRSF1A gene; TRAF2 gene; Testing; The Cancer Genome Atlas; Toxin; Tumor Cell Line; Ubiquitin; Ubiquitination; Woman; adaptive immunity; antagonist; anti-PD-1; anti-PD-L1; antitumor agent; cancer immunotherapy; cancer therapy; cell killing; cytokine; cytotoxic; cytotoxic CD8 T cells; cytotoxicity; experimental study; immune checkpoint blockade; immune function; improved; in vivo; interest; ipilimumab; member; men; mouse model; neoplastic cell; novel strategies; perforin; pressure; prevent; response; success; targeted treatment; tumor; ubiquitin-protein ligase

Abstract The use of anti-CTLA, anti-PD1 and anti-PD-L1, collectively known as immune checkpoint blockade (ICB), has revolutionized the treatment of cancer. The effectiveness of ICB varies widely across different cancers and individuals. In kidney cancer, long term durable responses were observed in only a small percentage of patients treated with a combination of ipilimumab and nivolumab. Thus, new strategies to improve the effectiveness of ICB are needed. Whether a tumor is eradicated or not ultimately depends on whether the tumor cells are susceptible to ‘kill’ signals coming from immune cells. Therefore, we reason that understanding the mechanisms used by tumor cells to protect themselves against these ‘kill’ signals will allow us to target these mechanisms in immunotherapy. The cytokine tumor necrosis factor-alpha (TNF) was first identified as a factor that could induce tumor death in vitro and tumor regression in vivo by Carswell et al. However, TNF proved to be largely ineffective as an anti-tumor agent because other than the L929 and Meth A tumor lines initially used by Carswell et al, most murine and human tumor cells are resistant to TNF-induced cytotoxicity. Indeed, TNF confers the opposite effect on tumors by inducing a survival signal. We now know that TNF can induce both cell survival and death within the same cell, and tumor cells are resistant to TNF- induced death due to the suppression of the death-signaling machinery through both transcription-independent and -dependent mechanisms. Thus, tumor cells have disarmed a potential ‘kill’ signal, most likely due to selection pressure during immunoediting. Our lab discovered a proximal death suppression mechanism that is dependent on the non-degradative ubiquitination of the TNF signaling molecule RIPK1. When RIPK1 is modified by K63-linked and linear M1 ubiquitination, it functions as a survival-signaling molecule. These non- degradative ubiquitination modifications lead to the activation of multiple members of the IKK family, which can phosphorylate RIPK1 to inhibit its death-signaling function. Therefore, inhibiting ubiquitination of RIPK1, or the kinases that phosphorylate RIPK1, converts RIPK1 to a death-signaling molecule rendering cells sensitive to TNF-induced cell death. Essentially, RIPK1 acts as a toggle between life (when it is ubiquitinated) and death (when it is not ubiquitinated). Using the RAG cell line as a model for murine renal carcinoma cell (RCC), we generated knockouts defective in linear ubiquitination of RIPK1, which exhibit heightened sensitivity to RIPK1- mediated death following TNF stimulation. We propose experiments to demonstrate that RCC tumor lines RAG and RENCA that are deficient for ubiquitination or phosphorylation of RIPK1 are more susceptible to killing by T cells in a TNF-dependent manner, and therefore more sensitive to ICB. The goal of this study is to provide proof-of-concept evidence that disrupting RIPK1 ubiquitination or phosphorylation in tumor cells could enhance the effectiveness of ICB in immunotherapy of renal carcinoma, and potentially in other cancers as well.

摘要 使用抗CTLA、抗PD 1和抗PD-L1，统称为免疫检查点阻断 (ICB)彻底改变了癌症的治疗方法。国际竞争性商业银行的有效性在不同的 癌症和个人。在肾癌中，仅在一小部分患者中观察到长期持久的反应。 用伊匹单抗和纳武单抗的组合治疗的患者的百分比。因此，新的战略 需要提高国际竞争力。肿瘤是否被根除最终取决于 肿瘤细胞是否容易受到来自免疫细胞的“杀死”信号的影响。因此，我们推断， 了解肿瘤细胞用来保护自己免受这些“杀死”信号的机制将使我们能够 我们可以在免疫治疗中靶向这些机制。细胞因子肿瘤坏死因子-α（TNF）首先是 Carswell等鉴定为可诱导体外肿瘤死亡和体内肿瘤消退的因子。 然而，TNF被证明作为抗肿瘤剂在很大程度上是无效的，因为除了L929和MethA之外， Carswell等人最初使用的肿瘤细胞系，大多数鼠和人肿瘤细胞对TNF诱导的 细胞毒事实上，TNF通过诱导存活信号对肿瘤赋予相反的作用。我们现在知道 TNF可以在同一细胞内诱导细胞存活和死亡，并且肿瘤细胞对TNF具有抗性。 由于通过转录非依赖性的死亡信号机制受到抑制而诱导死亡 和依赖机制。因此，肿瘤细胞已经解除了潜在的“杀死”信号，这很可能是由于 免疫编辑期间的选择压力。我们的实验室发现了一种近端死亡抑制机制， 依赖于TNF信号分子RIPK 1的非降解性泛素化。当RIPK 1 通过K63连接和线性M1泛素化修饰，它作为生存信号分子发挥作用。这些非- 降解性泛素化修饰导致IKK家族多个成员的激活， 磷酸化RIPK 1以抑制其死亡信号功能。因此，抑制RIPK 1的泛素化， 磷酸化RIPK 1的激酶，将RIPK 1转化为死亡信号分子，使细胞对 TNF诱导的细胞死亡。从本质上讲，RIPK 1在生命（当它被泛素化时）和死亡之间起着切换的作用 (when它不是泛素化的）。以RAG细胞系作为小鼠肾癌细胞（RCC）的模型， 产生RIPK 1线性泛素化缺陷的敲除，其表现出对RIPK 1的高度敏感性。 介导的死亡。我们提出实验来证明RCC肿瘤细胞系RAG RIPK 1的泛素化或磷酸化缺陷的RENCA和RENCA更容易被 T细胞以TNF依赖的方式存在，因此对ICB更敏感。本研究的目的是提供 概念验证证据表明，破坏肿瘤细胞中的RIPK 1泛素化或磷酸化可以增强 ICB在肾癌免疫治疗中的有效性，以及在其他癌症中的潜在性。