Sialylated Glycans Regulate MUC13 and the Proto-Oncogenes Pim-1 and Myc to Control Hematopoietic Stem and Progenitor Cell Numbers

The enzyme β-1-4 galactosyltransferase 1 (β4GalT1) plays a critical role in thrombopoiesis by modulating sialo-glycan (sialyl N-acetyl-lactosamine or LacNAc) content and function of the β1 integrin on megakaryocytes (MKs) (Nat. Commun. 2020;11(1):356). Recent data, however, point to a more complex role for β4GalT1 in hematopoiesis, as the promoter region of its conserved gene, B4galt1, is rich in enhancer sequences for transcription factors associated with cell identity and pro-oncogenic regulatory programs.

Here, we investigated the homeostasis of hematopoietic stem and progenitor cells (HSPCs) in B4galt1^-/- mice. We demonstrate that lack of Sialylated LacNAc synthesis perturbs HSPCs beyond the homing deficiency associated with lack of homing sialo-glycan motifs. Flow cytometry analysis showed that B4galt1^-/- mouse bone marrows have increased numbers of Long-Term HSCs (LT-HSCs, defined as Lineage^Neg/Sca-1^Pos/c-Kit^Pos/CD150^Pos/CD48^Neg). The increase in HSC numbers led us to investigate their phenotypic and functional features further. While quiescence markers in B4galt1^-/- LT-HSCs were indistinguishable relative to controls, LT-HSCs expressed more of the platelet marker CD41 on their surface, supporting a highly expanded CD41⁺ subset of LT-HSCs. Platelet-bias of LT-HSCs has been associated with inflammation and aging. However, our data do not support an increased cytokine inflammatory profile in the bone marrow.

Instead, single-cell RNA sequencing (scRNA seq) of sorted β4galt1^-/- Lineage^Neg/Sca-1^Pos/c-Kit^Pos (LSK) cells showed a significantly increased expression of the proto-oncogene Pim-1, its target, Myc, and the heavily O-glycosylated transmembrane receptor mucin 13 (MUC13), compared to control cells. Analysis of LT-HSC glycan expression using lectin microarray showed the expected decrease in N-glycosylation associated with B4galt1 deficiency, but also an increase in O-glycans, consistent with overexpression of MUC13, expression of which was enriched compared to other surface mucins. The data show that B4galt1 deletion leads to overexpression of the proto-oncogenes Pim-1, Myc, and MUC13 in HSPCs. The data suggest that MUC13-associated O-glycans and glyco-synthetic genes are potential therapeutic targets for hematologic malignancies since mucins have anti-inflammatory functions and alterations in mucin expression are with inflammation and cancer.