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

摘要 使用抗CTLA、抗PD1和抗PD-L1，统称为免疫检查点阻断 (ICB)，使癌症的治疗发生了革命性的变化。ICB的有效性在不同的地区差异很大 巨蟹座和个人。在肾癌中，只有一小部分患者观察到长期持久反应。 联合使用ipilimumab和nivolumab的患者百分比。因此，新的战略将 需要提高ICB的有效性。肿瘤能否被根除最终取决于 肿瘤细胞是否对来自免疫细胞的“杀伤”信号敏感。因此，我们推断 了解肿瘤细胞用来保护自己免受这些“杀伤”信号伤害的机制将使 我们希望在免疫治疗中针对这些机制。细胞因子肿瘤坏死因子-α(TNF)排在第一位 被Carswell等人确定为体外诱导肿瘤死亡和体内肿瘤消退的因素。 然而，事实证明，除了L929和Meth A外，肿瘤坏死因子作为抗肿瘤药物在很大程度上是无效的 Carswell等人最初使用的肿瘤细胞系，大多数小鼠和人类肿瘤细胞对肿瘤坏死因子诱导的肿瘤细胞具有耐药性 细胞毒性。事实上，肿瘤坏死因子通过诱导生存信号对肿瘤产生相反的作用。我们现在知道 肿瘤坏死因子可以在同一细胞内诱导细胞存活和死亡，并且肿瘤细胞对肿瘤坏死因子具有抵抗力。 通过转录非依赖性抑制死亡信号机制导致的死亡 和依赖的机制。因此，肿瘤细胞解除了潜在的“杀死”信号，这很可能是由于 免疫编辑过程中的选择压力。我们的实验室发现了一种近端的死亡抑制机制 依赖于肿瘤坏死因子信号分子RIPK1的非降解性泛素化。当RIPK1为 经K63连接和线性M1泛素化修饰后，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家族的多个成员被激活，这可以 使RIPK1磷酸化以抑制其死亡信号功能。因此，抑制RIPK1的泛素化，或 磷酸化RIPK1的激酶，将RIPK1转化为死亡信号分子，使细胞对 肿瘤坏死因子诱导的细胞死亡。从本质上讲，RIPK1充当了生命(当它被无处不在)和死亡之间的切换 (当它不是无所不在的时候)。用RAG细胞系作为小鼠肾癌细胞(RCC)的模型，我们 在RIPK1的线性泛素化中存在缺陷的产生的敲除，对RIPK1-表现出高度的敏感性 肿瘤坏死因子刺激后介导性死亡。我们提出的实验证明肾细胞癌肿瘤株RAG 和缺乏RIPK1泛素化或磷酸化的RencA更容易被 T细胞以肿瘤坏死因子依赖的方式，因此对ICB更敏感。这项研究的目标是提供 概念验证证据表明，干扰肿瘤细胞中RIPK1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疗效。

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.