A Metabolic Interplay Coordinated By Hlx Balances Hematopoietic Stem Cell Maintenance and Differentiation

Upregulation of the non-clustered homeobox transcription factor HLX (H2.0-like Homeobox) is frequently observed in patients with acute myeloid leukemia, but its mechanism of action remains obscure. Since developmental pathways are often reactivated in cancer, we asked whether hlx1 plays a role during hematopoietic development in zebrafish. Endothelial specific overexpression of human HLX led to aberrant production of hematopoietic stem and progenitor cells (HSPCs) and increased numbers of immature myeloid cells at 48 hpf (hours post fertilization), thereby mimicking a preleukemic phenotype. On the other hand, hlx1 morphants had diminished numbers of HSPCs, a defect that could be rescued by overexpressing human HLX under control of either an endothelial or HSPC specific promoter. To delve into the molecular mechanism of HLX function, we performed expression analysis of sorted endothelial cells from zebrafish that overexpress HLX and hlx1 morphants. Overexpression of HLX led to downregulation of mitochondrial electron chain enzymes (NDUFS4, SDHD, CYTB, COX7A2) and upregulation of the peroxisome proliferator activated receptor family member δ (PPARδ). Downregulation of hlx1 led to diminished expression of PPARδ. Analysis of open chromatin by ATAC-seq revealed altered chromatin accesibility around the vicinity of these genes in hlx1 morphants. To validate functionally these results we measured the mitochondrial membrane potential by Tetramethylrhodamine, Methyl Ester, Percholate (TMRM). TMRM was decreased upon HLX overexpression and increased in hlx1 morphants. These results suggested that HLX regulates the fate of hematopoietic cells by fine tuning mitochondrial membrane potential and PPARδ. Indeed PPARδ agonists could rescue HSPC generation in zebrafish morphants, while PPARδ antagonists relieved the differentiation block of myeloid cells. Direct regulation of mitochondrial genes and PPARδ by HLX was established by chromatin immunoprecipitation in human cell lines. To test whether there is a conserved, intrinsic role of HLX in primary hematopoietic progenitor cells, we used human progenitor CD34⁺ cells. In accordance with our results in zebrafish, overexpression of HLX led to increased numbers of colonies in colony forming unit assays and a myeloid differentiation block, whereas downregulation greatly compromised the ability of CD34⁺ cells to form colonies. HLX overexpression resulted in diminished oxidative phosphorylation and mitochondrial membrane potential whereas downregulation of HLX coincided with increased mitochondrial membrane potential. PPARδ agonists and antagonists could rescue TMRM staining and the colony forming capacity in HLX knockdown CD34+ cells and alleviated the myeloid differentiation block upon HLX overexpression, respectively. In summary, our findings show that HLX balances hematopoietic stem and progenitor cell maintenance and differentiation by modulating their metabolic status.