Adenylate Kinase 2 Links Energy Metabolism and Cell Fate in Hematopoietic Stem and Progenitor Cells

While hematopoietic stem and progenitor cells (HSPCs) were thought to rely mainly on glycolysis for energy supply, emerging evidence suggests that defects in mitochondrial functions can impact HSPC development with respect to self-renewal, differentiation and aging. The exact mechanisms underlying metabolic reprogramming and cell fate decisions during human hematopoiesis, however, remain elusive. Biallelic mutations in the mitochondrial enzyme adenylate kinase 2 (AK2), cause reticular dysgenesis (RD), one of the most profound forms of severe combined immunodeficiency (SCID). AK2 catalyzes the interconversion between adenine nucleotides and thereby controls the availability of ADP for oxidative phosphorylation. Clinically, RD patients not only present with profound lymphopenia, typical for classic SCID, but also suffer from severe congenital neutropenia. The developmental arrest across the T, NK and granulocytic lineages suggests that AK2 deficiency causes a metabolic defect with global impact on hematopoiesis. Our prior work in induced pluripotent stem cells (iPSCs) from RD patients has shown that maturation-arrested iPSC-derived HSPCs exhibit increased oxidative stress and an energy-depleted adenine nucleotide profile, suggesting that AK2-regulated mitochondrial bioenergetics play an integral role in HSPC differentiation. Therefore, RD serves as an excellent model to study the impact of mitochondrial metabolism during human HSPC development. Methods: Since iPSCs do not recapitulate definitive hematopoiesis, we developed an AK2 biallelic knock-out model in primary human HSPCs using CRISPR/Cas9 gene editing. Employing a homologous recombination-mediated dual color reporter strategy, we were able to select for HSPCs with biallelic AK2 knock-out. HSPCs edited at the safe harbor AAVS1 site were used as a control. FACS purified AK2-/- and AAVS1-/- HSPCs were in vitro differentiated along the granulocytic lineage, and cells at various differentiation stages were sorted for RNA-seq and metabolomics analysis. Results: We analyzed the myeloid differentiation potential of AK2-/- HSPCs in vitro. Compared to AAVS1-/- controls, AK2-/- HSPCs displayed a severely decreased colony forming potential of both myeloid and erythroid lineages. In addition, AK2-/- HSPCs showed a granulocytic maturation arrest at the HLA-DR-, CD117+ promyelocyte stage, consistent with the characteristic phenotype observed in RD patients. We then performed RNA-seq studies on in vitro differentiate