B4galt1 Regulates the WNT-β-Catenin Axis to Control Hematopoietic Stem and Progenitor Cells (HSPCs) Fitness

In achieving the optimal numbers of fully functional blood cells across different lineages, intrinsic developmental programs within the cells and external guidance mechanisms are crucial. Glycans on the cell surface and extracellular environment are pivotal in regulating cell maintenance, differentiation, and function. We recently discovered that the glycosyltransferase β-1,4-galactosyltransferase 1 (B4galt1) leads to dysplastic megakaryocytes, impaired thrombopoiesis, and increased hematopoietic stem cells (HSC).

To investigate the relationship between B4galt-dependent glycosylation and HSC function, we analyzed the glycan composition in control and B4galt1 null (B4^-/-) femurs by spatial mass spectrometry using matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI). In control femurs, we observed a distinct gradient of complex N-glycans, with higher expression at the distal ends and decreasing complexity towards the shaft, showcasing diverse glycan expression in the hematopoietic environment. However, this gradient was absent in B4^-/-femurs, where we detected an increase of immature N-glycans and a concurrent decrease in complex N-glycan structures (p<0.05). Additionally, we observed aberrant O-glycosylation in B4^-/- LT-HSCs, as determined using lectin arrays. The glycan composition in the absence of B4galt1-dependent glycosylation mirrors cancer-associated glycan patterns.

To further understand the role of B4galt1 in steady-state hematopoiesis, we next examined the bone marrow (BM) immunophenotypic composition of B4^-/- mice. Flow cytometry analysis revealed a significant expansion of immunophenotypically defined long term (LT)-HSCs (p=0.0061) and multipotent progenitors (MPPs) (p<0.05) in B4^-/-BM. Additionally, B4^-/- LT-HSCs showed higher expression of the platelet marker CD41 (p=0.0171), suggesting the presence of an expanded megakaryocyte (MK) biased LT-HSCs population (LT-HSC CD41+ cells). This expansion occurred without pro-inflammatory cues, as determined through cytokine arrays.

To investigate if the global transcriptional landscape in B4^-/- cells could explain the observed expansion phenotype, we conducted droplet-based single-cell RNA-seq. Transfer learning classification allowed us to identify an expansion of the transcriptional output associated with LT-HSC and MPP2 signatures in B4^-/- cells (p<0.05). Further transcriptional analysis revealed an increased number of B4^-/- cells expressing MKs specific markers (CD41, Pf4, GP9, Vwf, GP1ba), providing additional support for the expansion of B4^-/- MK-biased LT-HSCs. Gene Set Enrichment Analysis (GSEA) of B4^-/- LT-HSCs demonstrated enrichment in metabolism-associated pathways and cell cycle regulation (FDR<0.05). Conversely, critical differentiation processes at the stem cell level, such as cellular adhesion and lineage commitment, were downregulated (FDR<0.05).

To investigate the role of B4galt1 in HSC expansion, we transcriptionally classified LT-HSCs based on their cell cycle status (G1, G2M, and S). Analysis of LT-HSC CD41+ cell cycle distribution revealed an increased in S phase in B4^-/- LT-HSC CD41+ cells. Additionally, B4^-/- LT-HSC CD41+ cells exhibited enrichment in cell cycle regulation pathways, including DNA replication, DNA strand elongation, and G1 to S cell cycle control (FDR<0.05). Conversely, critical pathways associated with HSC homeostasis and quiescence were depleted in the absence of B4galt1 (FDR<0.05). GSEA analysis unveiled upregulation of Wnt and Myc pathways in B4^-/- LT-HSC CD41+ cells. Immunofluorescence and immunoblot analysis demonstrated elevated total β-catenin levels in B4^-/- LSK cells and enhanced nuclear translocation of non-phosphorylated Ser33/37/Thr41 β-catenin. To confirm the functional significance, we treated B4^-/- LSK cells with the Wnt inhibitor XAV939 which normalized cell numbers to control levels and cause a reduction in the observed expansion at the stem and progenitor compartments. These findings suggest that B4galt1 is vital in regulating HSC expansion, likely through the Wnt/β-catenin signaling pathway.

Our data provide compelling evidence that B4galt1 regulates steady-state hematopoiesis, through its influence on glycosylation in the bone marrow niche, highlighting the critical role of the B4galt1-Wnt-β-catenin axis in regulating LT-HSC fitness and expansion.