Understanding heterogeneity of necroptotic cell death and mechanism(s) of necroptosis escape

Summary Receptor-interacting Protein Kinases 1 and 3 (RIPK1 and RIPK3) form necrosomes that, along with Mixed Lineage Kinase Domain-like (MLKL) pseudokinase protein, are well-established mediators of a regulated necrotic cell death process, known as necroptosis. Dysregulation of necroptosis has been implicated in many disease states, including inflammatory pathologies and cancer. Besides cell death, necrosome-driven pro- inflammatory transcriptional changes that are independent of cell death and the release of pro-tumorigenic DAMPs, cytokines, and chemokines by the dying cells can also drastically affect outcomes of necroptosis activation. Thus, how these diverse orthogonal processes intermingle in a heterogeneous population of cells undergoing necroptosis is crucial to understand. In addition, our new data show that upon activation of necroptosis in multiple necroptosis-sensitive human cancer cell lines, a subset of cells quickly develops an irreversible resistance to necroptotic stimuli and survives, which also needs to be accounted for when considering the outcomes of necroptosis activation. In our preliminary work, we used bulk transcriptomics of necroptosis-resistant clones to demonstrate systemic transcriptional shifts in cells upon acquisition of resistance to necroptosis. However, bulk approaches are generally not well suited for understanding cell death dynamics in a heterogeneous cell population. Instead, single-cell methods are needed to interrogate transcriptional signatures in cells undergoing necroptosis vs. developing resistance and surviving the insult. To address this gap, in Aim 1, we propose to validate and optimize a new assay format that we created, scAnnexinV-seq, allowing co-measurement of cell death status and short-term inflammatory gene expression changes with single- cell resolution in a cell population that is asynchronously undergoing necroptosis. These experiments will be done in human monocytic U937 cells. In addition, we will also examine, using scRNA-seq, gene expression patterns associated with a longer-term transition of these cells to a necroptosis-resistant state. Aim 2 will deploy a single-cell DAMP release assay to measure the dynamics of DAMP release and cell death in a necroptotic cell population. For this analysis, we will use cells with different degrees of acquired resistance to necroptosis from Aim 1 to examine whether a slower rate of cell death in cells transitioning toward resistance may lead to a prolonged pro-tumorigenic DAMP release. In addition, we will examine the roles that several specific factors involved in the regulation of necroptosis may have on the dynamics of DAMP release vs. cell death. The findings from this work will be impactful in developing new tools and elucidating population dynamics in necroptotic cells, thereby revealing new inflammatory mechanisms associated with necroptosis and cell trajectories toward necroptosis escape that may be highly relevant for inflammatory diseases and cancer. Project Number: 1R21AI185601-01A1 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: ALEXEI DEGTEREV (+2 co-PIs) | Institution: TUFTS UNIVERSITY BOSTON, BOSTON, MA | Award Amount: $450,764 | Activity Code: R21 | Study Section: Cellular Signaling and Regulatory Systems Study Section[CSRS] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R21AI18560101A1