Sphingolipids Regulate Myeloid-Erythroid Fate Determination in Human Hematopoiesis
Authors: Xie Stephanie Zhi-Juan , Kaufmann Kerstin , Gan Olga I , Zandi Sasan , Takayama Naoya , Dick John E. 
Published: November 2019

Abstract
The established model of hematopoiesis posits mature blood lineages are derived through successive stages of progenitors that become increasingly lineage-restricted. Whereas the master transcriptional regulators of myeloid-erythroid fate specification such a in Pu.1, Gata1 and CEBPalpha are well studied, the signaling networks that link extrinsic niche signals to lineage commitment is ill-defined. Sphingosine-1-phosphate (S1P) is a bioactive lipid produced from sphingolipid metabolism that in mice has been implicated in HSC egress, lymphocyte trafficking and lymphocyte lineage determination, mainly through the receptor S1PR1. However, the role of sphingolipid biology in human lineage specification is unknown.
Gene expression profiling of 11 highly resolved populations of human stem, progenitor and lineage commited cells was undertaken to gain insight into the transcriptional signatures that define each cell population and the changes that occur during lineage commitment. We previously established that sphingolipid metabolism is transcriptionally distinct between HSC and progenitors (Xie et al In prep). Unbiased clustering of RNA-seq data of 46 sphingolipid genes was sufficient to segregate mature human erythroid, lymphoid, and myeloid cells suggesting that tight regulation of S1P signaling is required for lineage commitment. Myeloid cells have the highest transcriptional expression of S1P receptors among mature lineages whereas erythroid cells have little to no expression predicting that S1P signaling may be important in governing myeloid-erythroid fate. We found that manipulating S1P transport via overexpression of the S1P transporter SPNS2 in cord blood was sufficient to limit erythropoiesis as assayed by single cell in vitro assays and in vivo xenotransplantation. S1P signals through a family of 5 G-protein coupled S1P receptors, with S1PR3 expression being myeloid-specific. S1PR3 protein expression is restricted in the primitive human hematopoietic hierarchy to only a subset of granulocyte-macrophage progenitors. Enforced expression of S1PR3 in HSC, MPP (multipotent progenitors) and CMP (common myeloid progenitors) is sufficient to inhibit erythropoiesis and upregulate myelopoiesis in single cell stromal-based assays suggesting S1PR3 has a unique role in myeloid-erythroid fate specification. Flow cytometry analysis shows S1PR3 protein is highly overexpressed in primary AML relative to normal blood cells, suggesting S1P biology is dysregulated in AML. Collectively, our studies provide the first direct evidence that the S1P pathway governs fate determination along myeloid-erythroid lineage commitment. Future studies will need to be undertaken to determine how this new mechanism of lineage commitment is linked to the transcription factor network. Our studies also suggest that this pathway may play a role in AML biology raising the possibility of a new therapeutic direction for AML.

Disclosures
No relevant conflicts of interest to declare.