3146 – INVESTIGATING THE UBIQUITIN E3 LIGASE, FBXO21, IN REGULATION OF HEMATOPOIETIC STEM CELLS

Hematopoietic stem cells allow for the formation of all cell types in the blood and maintain these populations throughout a person’s life. Hematopoietic cell fate decisions such as self‐renewal and differentiation are highly regulated through multiple molecular pathways and dysregulation can lead to the initiation of malignant hematopoietic disorders like acute myeloid leukemia (AML), immune deficiencies, and anemia. One pathway that regulates hematopoietic cell fate decisions is the ubiquitin proteasome system (UPS). The UPS controls protein levels by tagging them with polyubiquitin chains and promoting their degradation through the proteasome. The substrate recognition component, the protein that decides which substrate will be chosen for degradation, of the UPS is the ubiquitin E3 ligase. Through investigating a specific family of ubiquitin E3 ligases, the Fbox family of proteins, we discovered that Fbxo21was highly expressed in the hematopoietic stem and progenitor (HSPC) population, and was more highly expressed in this population when compared to other Fbox genes suggesting a possible role in maintaining the HSPC population. Likewise, Western blot confirmed high levels of FBXO21 in HSPCs and revealed low to no protein in mature myeloid populations. To determine the role of FBXO21 on HSPC maintenance, self‐renewal, and differentiation, we generated shRNAs against both Fbxo21 and FBXO21. We found that silencing Fbxo21/FBXO21 in healthy HSPCs and in human AML cell lines lead to a loss in colony formation, a decrease in proliferation, and an increase in differentiation towards the mature myeloid lineage. While overexpression of FBXO21 lead to an increase in colony formation and proliferation. This leads us to hypothesize that the ubiquitin E3 ligase, FBXO21, maintains the hematopoietic stem cell population while loss of Fbxo21/FBXO21 leads to a promotion in myeloid differentiation.We then generated the first conditional Fbxo21 knockout (KO) mouse model to delete Fbxo21 in hematopoietic cells. Our Fbxo21 KO mice exhibited no significant difference in primary sites of hematopoietic development, but display changes in hematopoietic cell populations in both young and aged mice. To further identify the substrates and pathway of FBXO21, we performed TMT Mass Spectrometry to analyze changes in protein levels upon knockdown of Fbxo21/FBXO21 in healthy HSPC and human AML cell lines. We found that both populations displayed an increase in proteins involved in the inflammatory response, the immune response primarily maintained by the myeloid cells through cytokine signaling. Which among both our Fbxo21 KO mice and human AML cell lines with FBXO21 knocked down, we see alterations in the cell’s response to certain cytokines, including IL‐6 and G‐CSF, upon Fbxo21/FBXO21 being knocked down. Therefore, deciphering the role of FBXO21 could expand the current known molecular mechanisms that regulate hematopoietic lineage specification and stem cell maintenance.

MYC Promotes Bone Marrow Stem Cell Dysfunction in Fanconi Anemia.

Hematopoietic stem cell-specific GFP-expressing transgenic mice generated by genetic excision of a pan-hematopoietic reporter gene.

The role of chemokine activation of Rac GTPases in hematopoietic stem cell marrow homing, retention, and peripheral mobilization

On the symmetry of siblings: automated single-cell tracking to quantify the behavior of hematopoietic stem cells in a biomimetic setup

Transcriptome Analysis Identifies Regulators of Hematopoietic Stem and Progenitor Cells

Cellular and Molecular Analysis of Hematopoietic Stem and Progenitor Cells in Shwachman-Diamond Syndrome

Roles for Heme Synthesis in the Maintenance of Hematopoietic Stem and Progenitor Cells

Bnip3lb Regulated Mitophagy Maintains the Embryonic Pool of Hematopoietic Stem Cells By Protecting Them from ROS Induced Apoptosis

3226 – THE ROLE OF UBIQUITIN E3 LIGASE FBXO21 IN REGULATING HEMATOPOIETIC STEM AND PROGENITOR CELLS (HSPC) THROUGH CYTOKINE MEDIATED PATHWAYS

Mechanisms of hematopoietic stem cell mobilization: When innate immunity assails the cells that make blood and bone

Ubiquitin E3 ligase FBXO21 regulates cytokine-mediated signaling pathways, but is dispensable for steady-state hematopoiesis

Endothelial cells (ECs) line blood vessels and serve as a niche for hematopoietic stem and progenitor cells (HSPCs). Recent data point to tissue-specific EC specialization as well as heterogeneity; however, it remains unclear how ECs acquire these properties. Here, by combining live-imaging-based lineage-tracing and single-cell transcriptomics in zebrafish embryos, we identify an unexpected origin for part of the vascular HSPC niche. We find that islet1 (isl1)-expressing cells are the progenitors of the venous ECs that constitute the majority of the HSPC niche. These isl1-expressing cells surprisingly originate from the endoderm and differentiate into ECs in a process dependent on Bmp-Smad signaling and subsequently requiring npas4l (cloche) function. Single-cell RNA sequencing analyses show that isl1-derived ECs express a set of genes that reflect their distinct origin. This study demonstrates that endothelial specialization in the HSPC niche is determined at least in part by the origin of the ECs.

Differential effects of mixed lymphocyte reaction supernatant on human mesenchymal stromal cells

Blood Cell Replenishment and Bone Marrow Stem Cell Pool Renewal Are Regulated By Different Circadian Peaks Via Norepinephrine and TNFα/S1P Signaling