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

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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.