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Endogenous thymic regeneration is a crucial process that allows for the renewal of immune competence following stress, infection or cytoreductive conditioning. Fully understanding the molecular mechanisms driving regeneration will uncover therapeutic targets to enhance regeneration. We previously demonstrated that high levels of homeostatic apoptosis suppress regeneration and that a reduction in the presence of damage-induced apoptotic thymocytes facilitates regeneration. Here we identified that cell-specific metabolic remodeling after ionizing radiation steers thymocytes towards mitochondrial-driven pyroptotic cell death. We further identified that a key damage-associated molecular pattern (DAMP), ATP, stimulates the cell surface purinergic receptor P2Y2 on cortical thymic epithelial cells (cTECs) acutely after damage, enhancing expression ofFoxn1, the critical thymic transcription factor. Targeting the P2Y2 receptor with the agonist UTPγS promotes rapid regeneration of the thymusin vivofollowing acute damage. Together these data demonstrate that intrinsic metabolic regulation of pyruvate processing is a critical process driving thymus repair and identifies the P2Y2 receptor as a novel molecular therapeutic target to enhance thymus regeneration.SUMMARYThymocytes rapidly and transiently undergo pyroptosis after acute thymic damage and promote regeneration.Damage-induced redirection of pyruvate acutely enhances mitochondrial OXPHOS in thymocytes.Elevated mitochondrial ROS promotes pyroptosis in thymocytes after acute insult by driving caspase 1 cleavage.Extracellular ATP release promotesFoxn1expression in cTECs via activation of P2Y2Therapeutic targeting of the P2Y2 receptor promotes thymic regeneration.

The oxidative tricarboxylic acid (TCA) cycle is a central mitochondrial pathway integrating catabolic conversions of NAD+ to NADH and anabolic production of aspartate, a key amino acid for cell proliferation. Several TCA cycle components are implicated in tumorigenesis, including loss of function mutations in subunits of succinate dehydrogenase (SDH), also known as complex II of the electron transport chain (ETC). Mechanistic understanding of how proliferating cells tolerate the metabolic defects of SDH loss is still lacking. Here, we identify that SDH supports cell proliferation through aspartate synthesis but, unlike other ETC impairments, is not restored by electron acceptor supplementation. Interestingly, we find aspartate production and cell proliferation are restored to SDH impaired cells by concomitant inhibition of ETC complex I (CI). We determine that the benefits of CI inhibition in this context are dependent on decreasing mitochondrial NAD+/NADH, which drives SDH-independent aspartate production. We also find that genetic loss or restoration of SDH selects for cells with concordant CI activity, establishing distinct modalities of mitochondrial metabolism for maintaining aspartate synthesis. Collectively, these data identify a metabolically beneficial mechanism for CI loss in proliferating cells and reveal that compartmentalized redox changes can impact cellular fitness.

It is not well understood how physiological environmental conditions and nutrient availability influence cancer cell proliferation. Production of oxidized biomass, which requires regeneration of the cofactor NAD+, can limit cancer cell proliferation1-5. However, it is currently unclear which specific metabolic processes are constrained by electron acceptor availability, and how they affect cell proliferation. Here, we use computational and experimental approaches to demonstrate that de novo lipid biosynthesis can impose an increased demand for NAD+ in proliferating cancer cells. While some cancer cells and tumors synthesize a substantial fraction of their lipids de novo6, we find that environmental lipids are crucial for proliferation in hypoxia or when the mitochondrial electron transport chain is inhibited. Surprisingly, we also find that even the reductive glutamine carboxylation pathway to produce fatty acids is impaired when cancer cells are limited for NAD+. Furthermore, gene expression analysis of 34 heterogeneous tumor types shows that lipid biosynthesis is strongly and consistently negatively correlated with hypoxia, whereas expression of genes involved in lipid uptake is positively correlated with hypoxia. These results demonstrate that electron acceptor availability and access to environmental lipids can play an important role in determining whether cancer cells engage in de novo lipogenesis to support proliferation.