Role of lipoxygenase-derived lipid mediators in the hematopoietic niche

PROJECT SUMMARY Hematopoietic stem and progenitor cells (HSPCs) closely interact with the niche microenvironment, allowing a dynamic regulation on proliferation, differentiation and self-renewal. Various niche cells have been identified, yet the secreted signaling factors in the hematopoietic niche are less well-defined, limiting the application of HSPC for ex vivo expansion and enhanced engraftment protocol for blood disease treatment. To identify novel niche factors that stimulate HSPC production, I performed an in vivo small molecule chemical screen and identified an antagonist of cysteinyl leukotriene receptor 1 (CysLTR1) that stimulates HSPC divisions that specifically occurs within a ‘pocket-like’ structure that is formed by a group of endothelial cells (ECs) in the niche. The HSPC is known to physically interact with these ‘pocket’ forming ECs and the event is termed ‘endothelial cuddling’. A HSPC division is often observed after endothelial cuddling, yet the mechanism is unknown. I performed single cell RNA-sequencing (scRNA-seq) analysis of zebrafish hematopoietic tissues and identified that CysLTR1 is specifically expressed in a subset of ECs with high expression of niche EC genes. I generated a stable cysltr1:mCherry reporter fish and found that cysltr1:mCherry+ ECs preferentially interact with HSPCs. I generated a stable cysltr1 knockout fish and it phenocopied the antagonist perturbations. Cysteinyl leukotrienes (CysLTs) are lipoxygenase (LOX)-derived lipid mediators. I further performed lipidomic analysis of zebrafish hematopoietic tissues and identified a series of LOX-derived lipid mediators. Interestingly, I performed scRNA- seq analysis and found that corresponding receptors of these lipid mediators were expressed in distinct cell types such as macrophages, neutrophils or stromal cells, indicating unique mechanisms. LOX-derived lipid mediators play key roles in inflammation. However, the role of LOX-derived lipid mediators in the hematopoietic niche remains unexplored. Given my preliminary data, I hypothesize that LOX-derived lipid mediators remodel hematopoietic niches by altering cellular interactions and secreted cytokines to regulate hematopoiesis. Under the supervision of Dr. Leonard Zon, the field-leading expert in hematopoiesis and zebrafish genetics, I will test my hypothesis using high resolution live cell imaging, chemical biology and CRISPR/Cas9 mutagenesis. I have put together a panel of scientific advisory committees who are field-leading experts in vascular biology, niche cell biology, and lipid mediator biology. Together with Dr. Zon, I will receive constant feedback on my research and career development. This K99/R00 award will enable me to gain trainings and acquire skills in scientific training and career development that prepare me to start my laboratory. My overarching goal is to investigate molecular mechanisms of niche-mediated regulations in hematopoiesis in my own laboratory. I aim to contribute my research discoveries to develop enhanced HSPC ex vivo expansion protocol through recapitulating the functional niche signaling and to improve the efficiency and safety of therapeutics through targeting/priming the hematopoietic niche.