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

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.