Study of the Roles of SDF1 and CXCR4 in Hematopoiesis

Division Of Basic Sciences - Nci

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Abstract

Previous studies from many groups, including our group, have identified a critical role of the chemokine CXCL12/SDF1 and its receptor CXCR4 in the retention and mobilization of myeloid cells from the bone marrow. CXCL12 is mostly produced by the non-hematopoietic cells of bone marrow, particular in the CAR (CXCL12 abundant reticular) and in other mesenchymal cells in the bone marrow. The bone marrow hematopoietic cells generally express CXCR4, and the signaling interaction between CXCL12/CXCR4 serves to retain hematopoietic cells in the bone marrow. G-CSF is a critical physiological regulator of granulopoiesis: mice carrying homozygous deletions of colony-stimulating factor (G-CSF) or its receptor are severely neutropenic, and dominant-negative mutations of G-CSFR have been linked to severe defects of granulopoiesis. Administration of G-CSF induces an expansion of myeloid lineage cells in the bone marrow, and promotes the release of mature myeloid cells and hematopoietic progenitor cells from the bone marrow to the peripheral blood. Thus, G-CSF at pharmacologic concentrations is widely used to induce granulopoiesis and to mobilize hematopoietic progenitors to the peripheral blood. Functionally, G-CSF reduces CXCR4 and CXCL12 levels in the bone marrow through a variety of mechanisms. As a consequence, the glue that retains hematopoietic cells in the bone marrow is loosened and massive exit of mature and immature hematopoietic cells is achieved. A CXCR4 competitive inhibitor, AMD3100/Plerixafluor, has been approved by FDA as a mobilizing agent for hematopoietic precursors in conjunction with G-CSF. Recently, our studies on HSPC mobilization have detected an important role of the receptor/ligand pair EphrinB2/EphB4. We unveiled the mutually exclusive bone marrow distribution of EphB4 receptors in the sinusoids and EphrinB2 ligands in hematopoietic cells, and discovered an EphB4/EphrinB2-dependent pathway that controls HSPCs mobilization. Blockade of EphB4/EphrinB2 reduced HSPCs and other myeloid cells mobilization to the circulation. In murine cancer models, in which hematopoietic cells derived from the bone marrow promote tumor growth, EphB4/EphrinB2 blockade reduced tumor infiltration with HSPCs and tumor progression. These results identify EphB4/EphrinB2 signaling as critical to hematopoietic cells mobilization from bone marrow and provide a new strategy for reducing cancer progression by targeting the bone marrow. Other ongoing studies stem from previous observations in the neuronal system, particularly in the cerebellum, showing a biochemical link between EphrinB2 and CXCR4 that regulates cerebellar cell motility. More specifically, these studies showed that PDZ-dependent signaling by EphrinB2 recruits the PDZ-containing RGS3 protein, which in turn can bind and shut down CXCR4 signaling. In so doing, active EphrinB2 regulates CXCL12-induced motility of CXCR4-positive neuronal cells. We hypothesized that this interaction may also occur in the context of the bone marrow where CXCL12 (produced by non-hematopoietic cells), CXCR4 and EphrinB2 (expressed by hematopoietic cells) are abundant. In proof-of-principle experiments we have documented the proximal co-localization of CXCR4 and EphrinB2 in mouse hematopoietic cells. Initial mapping of sites where these molecules associate in the bone marrow showed a broad pattern, with some degree of signal concentration at sites more proximal to the spongiform bone. This mapping will be continued by bone marrow imaging of WT and genetically-modified mouse lines. In vitro experiments have clarified how EphrinB2 regulates hematopoietic cell responses to CXCL12. In brief, phosphorylated EphrinB2 reduced hematopoietic cell migration in response to CXCL12. This effect is more prominent in subsets of immature hematopoietic cells. Current studies are focused on identifying the specific bone marrow niche that includes CXCL12-producing cells, CXCR4 and EphrinB2 co-expressing cells, and EphB expressing cells. Based on our experiments in vitro, we have hypothesized that such niche would serve as a "motility unit" in the bone marrow, promoting the movement of specific hematopoietic a stem or progenitor cells within the bone marrow. Other ongoing studies have identified a novel population of endothelial cells in the mouse bone marrow that bears markers of typical endothelial cells and also bears some features of mesenchymal cells. This identification is based on analysis of single-cell transcriptomic analysis of hematopoietic-cell depleted bone marrow cell populations. We have now been able to mark this population by generating a mouse line in which Col1a2 (protein Collagen Type1 Alpha 2 chain)-expressing cells are tracked by a fluorescent reporter after Tamoxifen-induced Cre recombination. We have isolated an endothelial (VE-Cadherin+) bone marrow cell population that expressed Col1a2 and have begun to analyze its function in the adult bone marrow. Since individual cell-components of this population share the markers of endothelial and mesenchymal cells, current efforts aim at the purification and characterization of these cells genetically, epigenetically, phenotypically, and functionally. These efforts have highlighted the importance of single-cell technologies. Current technological limitations have prevented effective multiplex analysis of the epigenome at a single-cell level. They have also preventer single-cell analysis of the transcriptome and epigenome in individual cells. To overcome this limitation, we have developed a new technology that relies on the establishment of "re-usable" single-cells and proximity ligation-based mapping of epigenetic marks to the genome. This technology has successfully been applied to single-cell analysis of a model hematopoietic cell line (K562 cells). More recent developments of this technology have envisioned and provided proof-of-principle that single-cell analysis of transcriptome and epigenome is possible. An invention report and patent application have been filed. Importantly, we have applied this development to a fundamental question in hematopoiesis, i.e. the relationship between epigenome and transcriptome in regulation of stem and progenitor hematopoietic cells. Currently, the results of RNA sequencing from single-cells is being analyzed.