RIPK1 and RIPK3 are both kinases that belong to the subfamily of Ser/Thr Receptor-Interacting Protein Kinases (RIPK) within the tyrosine kinase-like family. These kinases are crucial mediators of inflammation, regulated cell death (necroptosis and apoptosis) and survival. Two aspects of RIPK1 will determine cellular fate: RIPK1 kinase activity initiates apoptosis or necroptosis upon activation, the latter occurring in caspase-8 deficient conditions, and RIPK1 also functions as a scaffold protein that is directly involved in inflammatory gene expression and cell survival. This scaffolding function is independent of its kinase activity. Necroptosis is a form of regulated necrotic cell death initiated by several receptors like TNFR1, toll-like receptors, IFNAR1 and is characterized by a signaling cascade with sequential phosphorylation of both RIPK1 and RIPK3 that will form a complex called necrosome. This necrosome will result in phosphorylation and activation of mixed lineage kinase-domain like protein (MLKL), a pseudokinase that was identified as a downstream substrate of RIPK3 and executioner of necroptosis. Necroptosis leads to the release of damage-associated molecular patterns (DAMPs) and cytokines, which cause an inflammatory response that results in various pathological conditions. As activation of RIPK1 and/or RIPK3 is important for the execution of necroptosis and these kinases are involved in many pathological conditions including ischemic insults, inflammatory and neurodegenerative diseases, they are promising therapeutic targets. As necrostatin-1, the first described RIPK1 inhibitor, has moderate potency, specificity and a narrow SAR profile, novel RIPK1 inhibitors are needed with higher therapeutic potential. The first part of this thesis focused on the identification and characterization of novel small molecule RIP kinase inhibitors with therapeutic potential to alleviate RIPK1/3-driven inflammatory disorders and tissue injury in mice. With high-content screenings, we identified Sorafenib tosylate (a multi-tyrosine kinase inhibitor that targets BRAF) as a potent inhibitor of necroptosis targeting RIPK1 and RIPK3. On the other hand, rational design of structural analogues of Tozasertib -an aurora kinase inhibitor shown to bind RIPK1- was used to develop novel RIPK1 inhibitors targeting the active kinase conformation. Aurora kinases regulate several phases during mitosis, including cytokinesis (splitting of one cell to two daughter cells) and inhibition of aurora kinases results in mitotic defects, deficient cytokinesis and eventually cell death. Here, we succeeded to develop novel Tozasertib analogues with increased specificity for RIPK1. Both Sorafenib tosylate as well as the Tozasertib analogues protected against a RIPK1/3-driven inflammatory disorder in vivo.
Necroptotic cell death is not only relevant in inflammatory and neurodegenerative disorders, but has also relevance in cancer. Cancers acquire several capabilities in order to survive during the multistep process in tumor development. Resisting cell death is one of them. Deregulation of RIPK1/RIPK3 has been reported in several cancer types, but little is known about the role of necroptosis-executor MLKL in tumorigenesis and tumor development. A few contradictory results on the possible association between MLKL expression and cancer prognosis have been reported. On the one hand, low MLKL expression is associated with decreased overall survival rates in colon cancer and pancreatic adenocarcinoma. On the other hand, higher levels of activated MLKL (phospho-MLKL) associated with lower survival rates in esophagus cancer and colon cancer. The contribution of MLKL regulation and activation during tumor development is therefore still obscure. Additionally, MLKL may have cell-death independent functions that influence tumor development. In the second part of this thesis, the role of MLKL in melanoma was explored using patient-derived melanoma cell lines and a genetic mouse melanoma model driven by BRAF mutation and PTEN deletion. This model mimics the mutational load that occurs in human melanoma and results in constitutive activation of the RAS/RAF/MEK/ERK MAPK signaling pathway that drives cell survival and proliferation in melanocytes. Upon increased mutational load, benign nevi will develop towards melanoma with radial expansion, followed by vertical growth into the dermis and metastasis. This metastatic process is facilitated by the high plasticity of melanoma cells including induction of ZEB1, a critical activator of epithelial-to-mesenchymal transition (EMT) that increases the invasiveness of melanoma cells. We found an intriguing positive correlation between MLKL and ZEB1 expression in melanoma, suggesting a possible functional link between MLKL and EMT-like processes. Also in patients, low MLKL RNA expression levels seem to result in higher survival rates, although this has no prognostic value to date. Although invasive melanoma cells express RIPK1, RIPK3 and MLKL, they are not sensitive to necroptosis. Understanding the necroptotic resistance mechanisms in melanoma will be crucial for the development of cell death-based therapies. Finally, MLKL has a modest but significant role in melanoma nevi growth.