The Cell-Type-Specific Essentiality of the Tricarboxylic Acid (TCA) Cycle in the Hematopoietic System

Since its discovery by Krebs, the tricarboxylic acid (TCA) cycle has been regarded as the essential terminal catabolic pathway for energy production from all nutrients. However, we do not understand whether and how the metabolism of each hematopoietic cell type in vivo depends on a complete TCA cycle. This is an important problem because energy metabolism is one of the most fundamental activities in cells, and because the TCA cycle is dysregulated in many diseases including leukemia. Understanding hematopoietic cell type-specific metabolic dependencies can illuminate the link between metabolism and cell fate. Citrate synthase is the key reaction discovered by Krebs which regenerates citrate from oxaloacetate to maintain the cyclical nature of the TCA cycle. A complete turn of the cycle burns two carbons from nutrient-derived acetyl-CoA to CO2 coupled to the generation of reducing equivalents and energy. In the absence of CS, the catabolic engine loses its acetyl group fuel, but the conversion of citrate to oxaloacetate is not disrupted. Thus, deleting CS can block terminal nutrient catabolism by the TCA cycle without interrupting other reactions in the pathway that may have biosynthetic or signaling functions. However, a Cs conditional knockout mouse has not been previously generated. Therefore, it is not known if the catabolic TCA cycle is an essential metabolic pathway in any tissue. Here we have created a conditional (floxed) mouse for Cs and deleted Cs in adult hematopoiesis (Mx1Cre;CsΔ/Δ) for the first time to test if a complete TCA cycle turn is essential for hematopoietic stem cells (HSCs) and progenitor cells in vivo. Analysis of hematopoiesis showed that the development of some cell types but not others depended on CS. The development of T cells in the thymus, particularly double positive T cell progenitors required CS. Development of the erythroid lineage in the bone marrow was also highly dependent on CS, and Cs-deficient erythroid progenitors arrested at a CD71+Ter119mid stage. Development of the myeloid and B cell lineages did not require a complete TCA cycle. We developed rare cell metabolomics and stable isotope tracing methods to study the metabolism of HSCs and progenitor cells in vivo. We used them to test how hematopoietic cell metabolism responds to loss of TCA cycle fueling. Absolute quantification strategies assessed the effects of Cs deletion on oxygen consumption, ATP production, nutrient consumption, and metabolite excretion. With these strategies, we systematically defined the metabolic consequences of TCA cycle disruption and mapped their cell-type specificity in hematopoietic differentiation. We identified metabolic pathways that play critical roles in supporting the survival and function of hematopoietic cells in the absence of a complete TCA cycle. Bone marrow produces hundreds of billions of new cells daily, far more than any other tissue. Such massive turnover likely requires a specialized energy metabolism. Our work identifies the cell-type-specific necessity or dispensability of the TCA cycle in hematopoietic cells. Our work is the first to define the metabolic and functional roles of CS in any tissue in vivo. In addition to fundamental insights into the role of the TCA cycle, our work provides metabolic targets for regenerative therapies.