Decrypting the USP22-homologous DUB network to sensitize pancreatic cancer for STING-mediated necroptosis

坏死性凋亡是程序性细胞死亡（PCD）的一种裂解形式，涉及感染、炎症和肿瘤形成。胰腺癌（PC）的预后非常差，但干扰素（IFN）和STING诱导的坏死性凋亡可能成为靶向化疗和化疗耐药PC的有吸引力的治疗选择。坏死性凋亡的中心决定因素是MLKL的RIPK 3依赖性磷酸化，导致MLKL寡聚化、膜积累和透化，最终触发细胞死亡。RIPK 3和MLKL的泛素化可微调坏死性凋亡，但其潜在机制、E3连接酶和去泛素化酶仍在很大程度上未被探索。我们已发表的和初步的数据确定USP 22是通过RIPK 3和MLKL泛素化控制坏死性凋亡的新型DUB，并揭示USP 22是紧张性IFN信号传导、STING泛素化和STING诱导的坏死性凋亡的负调节因子。目前的理论认为USP 22嵌入佐贺复合物中，通过调节组蛋白H2 B单泛素化来控制基因转录，但我们的研究结果表明USP 22具有新的SAGA独立作用。值得注意的是，两种高度USP 22同源的DUB USP 51和USP 27 X与USP 22一起发挥作用，但USP 22-USP 51-USP 27 X网络对（STING介导的）坏死性凋亡的机制、调节和功能相关性仍不清楚。因此，该提案旨在了解USP 22同源DUB网络在控制坏死性凋亡中的细胞和生物学相关性，以及如何靶向该网络以使PC对STING介导的坏死性凋亡敏感。为此，我们将使用多学科最先进的成像系统，包括3D光片荧光显微镜和激光扫描荧光显微镜，以及先进的近距离相互作用组质谱法和对健康和致瘤性原发性人类胰腺类器官中坏死性凋亡的深入分析。由于RIPK 3-MLKL信号传导的物种特异性差异，分析坏死性凋亡中的USP 22-USP 51-USP 27 X网络不仅与PCD、基因转录和类器官的基础研究领域极其相关，而且通过使抗凋亡PC对STING介导的坏死性凋亡敏感而具有翻译相关性。因此，这种竞争性研究方法提供了高度创新和信息丰富的方法，以揭示同源USP 22-USP 51-USP 27 X三联体的新型隔室和蛋白质复合物特异性作用。与此同时，这些知识将用于翻译环境中，通过在临床相关和接近生理的多细胞人类类器官中重新编程泛素化，STING功能和PCD敏感性来对抗PC。

Necroptosis is a lytic form of programmed cell death (PCD) involved in infection, inflammation and tumor formation. Pancreatic cancer (PC) has an extremely dismal prognosis, but interferon (IFN)- and STING-induced necroptosis potentially serves as attractive therapeutic option to target chemotherapy- and apoptosis-resistant PC. A central determinant of necroptosis is the RIPK3-dependent phosphorylation of MLKL, leading to MLKL oligomerization, membrane accumulation and permeabilization, ultimately triggering cell death. Ubiquitination of RIPK3 and MLKL fine-tunes necroptosis, but the underlying mechanisms, E3 ligases and deubiquitinating enzymes remain largely unexplored. Our published and preliminary data identify USP22 as novel DUB that controls necroptosis via RIPK3 and MLKL ubiquitination and reveal USP22 as negative regulator of tonic IFN signaling, STING ubiquitination and STING-induced necroptosis. The current dogma states that USP22 is embedded in the SAGA complex to control gene transcription via regulating histone H2B mono-ubiquitination, but our findings suggest novel, SAGA-independent roles of USP22. Of note, the two highly USP22-homologous DUBs USP51 and USP27X function together with USP22, but the mechanisms, regulation and functional relevance of the USP22-USP51-USP27X network for (STING-mediated) necroptosis remain unclear. This proposal therefore aims to understand the cellular and biological relevance of the USP22-homologous DUB network in controlling necroptosis and how this network can be targeted to sensitize PC for STING-mediated necroptosis. To this end, we will use multidisciplinary state-of-the-art imaging systems, including 3D light-sheet fluorescence microscopy and laser scanning fluorescence microscopy, with advanced proximity interactome mass-spectrometry and in-depth analysis of necroptosis in healthy and tumorigenic primary human pancreatic organoids. Due to species-specific differences in RIPK3-MLKL signaling, analyzing the USP22-USP51-USP27X network in necroptosis will not only be extremely relevant for the fundamental research fields of PCD, gene transcription and organoids, but also has translational relevance by sensitizing apoptosis-resistant PC for STING-mediated necroptosis. This competitive research approach therefore offers highly innovative and informative approaches to unravel novel compartment- and protein complex-specific roles of the homologous USP22-USP51-USP27X triad. At the same time, this knowledge will be used in translational settings to combat PC by reprogramming ubiquitination, STING function and PCD sensitivities in clinically relevant and near-physiological, multicellular human organoids.