ALKBH5 Modulates Hematopoietic Stem and Progenitor Cell Energy Metabolism through m ⁶a Modification-Mediated RNA Stability

During hematopoietic differentiation from hematopoietic stem cells (HSCs) to mature blood cells, cells undergo a metabolic shift from glycolysis to mitochondrial respiration. The mechanisms by which hematopoietic cells adjust their energy metabolism are still under investigation. N⁶-mehyladenosine (m ⁶A) mRNA modification has been reported to regulate numerous fundamental cellular processes through control of RNA stability or translational efficiency. The fat mass and obesity-associated protein (FTO), an m ⁶A _m and m ⁶A mRNA demethylase, has been reported to affect cellular metabolism in acute myeloid leukemia (AML). ALKBH5, the specific RNA m ⁶A demethylase, controls oncogene expression in AML. ALKBH5 becomes highly expressed in hematopoietic progenitors during hematopoietic development but the physiological role of RNA m ⁶A demethylase during hematopoiesis remains unknown.

To investigate the function of the RNA m ⁶A demethylase ALKBH5 in hematopoiesis, we generated Vav-iCre ⁺; Alkbh5^fl/fl (vcAlkbh5^-/-) mice, resulting in deletion of Alkbh5 specifically in the hematopoietic system. vcAlkbh5^-/-mice showed no hematopoietic defects at steady states up to 12 months of age. We applied TimeLapse-seq on lineage-depleted bone marrow cells of WT and vcAlkbh5^-/- mice to determine whether loss of ALKBH5 perturbed mRNA stability and/or RNA turnover. Ogdh mRNA was the most destabilized transcript resulting in significantly reduced OGDH protein levels.

OGDH is the rate-limiting enzyme in the tricarboxylic acid (TCA) cycle. Inhibition of OGDH subsequently induces production of L-2-hydroxyglutarate (L-2-HG), whose metabolism is closely coupled to energy metabolism through inhibition of oxygen consumption. L-2-HG, the enantiomer of D-2-HG, inhibits the function of a-ketoglutarate (a-KG)-dependent enzymes, including TET and KDM enzymes. We measured L- and D-2-HG in the plasma of WT and vcAlkbh5^-/- mice by chiral derivatization to distinguish the two enantiomers. Although D-2-HG levels were similar in the plasma of WT and vcAlkbh5^-/- mice, L-2-HG levels were significantly increased in the plasma of vcAlkbh5^-/- mice. We therefore determined the function of Jumonji C-domain lysine demethylases (JmjC-KDMs) by measuring histone methylation: H3K9me3, H3K27me3 and H3K36me3 modifications were all significantly increased in Alkbh5-deficient hematopoietic cells.

We next sought to understand whether reduction of OGDH expression and resulting increased L-2-HG levels production could impair energy metabolism via perturbation of the TCA cycle and oxidative phosphorylation (OXPHOS) in the mitochondria. We isolated lineage negative hematopoietic stem and progenitor cells (HSPCs) from WT and vcAlkbh5^-/- mice and subjected these to the Seahorse ATP Rate Assay. Comparing oxygen consumption rate (OCR) data and the kinetics of the Extracellular Acidification Rate (ECAR) of both groups, we found that less ATP was produced by mitochondria of the vcAlkbh5^-/- cells, while ATP produced by glycolysis showed no difference between the two groups. In the meantime, the ultrastructure of mitochondria in the Alkbh5-deficient cells remains normal.

We next determined whether the attenuated energy metabolism of Alkbh5-deficient HSPCs was functionally relevant by testing HSPC function in competitive transplantation assays. Interestingly, vcAlkbh5^-/- cells showed a significant competitive defect at all differentiation stages except in phenotypic long-term HSCs (LT-HSCs). This suggests that LT-HSCs, thought to preferentially rely on glycolysis as opposed to OXPHOS for their energy source, are protected from loss of ALKBH5 and OGDH.

In conclusion, our study demonstrates that ALKBH5 modulates energy metabolism by regulating mRNA stability of metabolic enzymes through its m ⁶A demethylation activity during hematopoiesis. This finding links Alkbh5 expression kinetics to the metabolic shift from glycolysis to mitochondrial OXPHOS during hematopoietic development.