Robust Mitochondrial Metabolism Provides a Fitness Advantage to Dnmt3a-Mutant Hematopoietic Stem Cells in the Context of Aging

Hematopoietic stem cells (HSCs) with mutations in the DNA methyltransferase DNMT3A gain a selective growth advantage leading to clonal hematopoiesis (CH), which increases all-cause mortality and the risk of developing blood cancers. Understanding the fundamental mechanisms by which DNMT3A-mutant HSCs outcompete wild-type HSCs is critical to develop therapeutic strategies to reduce their selective growth advantage and ultimately prevent progression to malignancy.

From our recent discovery that reduced local production of insulin-like growth factor 1 (IGF1) at middle age initiates functional decline of wild-type HSCs (Young et al., Cell Stem Cell 2021), we hypothesized DNMT3A-mutant HSCs would have a selective advantage in these conditions. We found that in vivo competitive transplant of Dnmt3a-mutant (Dnmt3a^R878H/+) HSCs into middle-aged recipient mice resulted in increased engraftment, Dnmt3a-mutant HSC expansion and myeloid lineage-biased Dnmt3a-mutant hematopoiesis. Similarly, we found that transplant of Dnmt3a-mutant HSCs into IGF1 conditional knockout recipient mice, as well as transplant of Dnmt3a-mutant HSCs into wild-type recipient mice followed by administration of an IGF1-mTOR inhibitor, replicated these phenotypes. As IGF1-mTOR signaling is well understood to regulate energy metabolism, we hypothesized that underlying metabolic differences in Dnmt3a-mutant HSCs enable their survival and selective growth advantage in a reduced IGF1/middle-aged context. RNA-seq analysis revealed strong enrichment of an oxidative phosphorylation gene signature in Dnmt3a-mutant vs. wild-type HSCs, suggesting differences in mitochondrial metabolism. Seahorse metabolic flux analyses demonstrated enhanced mitochondrial reserve capacity and mitochondrial activation potential in Dnmt3a-mutant vs. wild-type HSCs. Further, we found increased mitochondrial membrane potential, increased ATP production in the absence of glucose, and reduced mitochondrial and cellular reactive oxygen species in Dnmt3a-mutant HSCs, supporting that they have enhanced mitochondrial function. We then tested the hypothesis that enhanced mitochondrial function is a mechanism by which Dnmt3a-mutant HSCs outcompete wild-type HSCs in the middle-aged microenvironment. We performed a competitive transplant of Dnmt3a-mutant HSCs into middle-aged recipient mice and administered the mitochondrial-targeted antioxidant mitoquinol (MitoQ), which has been previously shown to enhance function of wild-type aged HSCs. We found that MitoQ enhanced hematopoiesis from wild-type HSCs and reduced the selective advantage and myeloid lineage-biased hematopoiesis of Dnmt3a-mutant HSCs. To evaluate direct effects of MitoQ on Dnmt3a-mutant hematopoiesis, we performed ex vivo assays. We found that addition of MitoQ reduced the serial colony-forming unit (CFU) replating capacity that is a hallmark of Dnmt3a-mutant HSCs. Further, Seahorse assays demonstrated that MitoQ depleted the mitochondrial reserve capacity of Dnmt3a-mutant HSCs, consistent with previous reports that MitoQ can cause mitochondrial depolarization and decreased ATP production in certain cellular contexts. Given that MitoQ improves mitochondrial function and hematopoiesis from aged wild-type HSCs, our work suggests that wild-type and Dnmt3a-mutant HSCs have fundamentally different responses to MitoQ treatment and that this difference may be capitalized upon as a therapeutic strategy to reduce CH burden.

Taken together, we report the discovery that mitochondrial metabolic regulation is a key mechanism by which DNMT3A-mutant HSCs gain a selective advantage over wild-type HSCs at middle age, when hematopoietic aging-associated phenotypes begin to be robustly observed. Targeting this mechanism may be used to maintain polyclonal hematopoiesis during aging and reduce the risk of CH-associated diseases.

Trowbridge:H3 Biomedicine: Consultancy, Research Funding.