The E3 ubiquitin ligase Cul5 regulates hematopoietic stem cell function for steady-state hematopoiesis in mice

The balance of hematopoietic stem cell (HSC) self-renewal versus differentiation is essential to ensure long-term repopulation capacity while allowing response to events that require increased hematopoietic output. Proliferation and differentiation of HSCs and their progeny are controlled by the JAK/STAT pathway downstream of cytokine signaling. E3 ubiquitin ligases, like Cullin 5 (CUL5), can regulate JAK/STAT signaling by degrading signaling intermediates. Here we report that mice lacking CUL5 in hematopoietic cells (Cul5Vav-Cre) have increased numbers of hematopoietic stem and progenitor cells (HSPCs), splenomegaly, and extramedullary hematopoiesis. Differentiation in Cul5Vav-Cre mice is myeloid- and megakaryocyte-biased, resulting in leukocytosis, anemia, and thrombocytosis. Cul5Vav-Cre mice had increased HSC proliferation and circulation, associated with a decrease in CXCR4 surface expression. In bone marrow cells, we identified LRRC41 coimmunoprecipitated with CUL5, and vice versa, supporting that CRL5 forms a complex with LRRC41. We identified an accumulation of LRRC41 and STAT5 in Cul5Vav-Cre HSCs during IL-3 stimulation, supporting their regulation by CUL5. Whole-cell proteome analysis of HSPCs from Cul5Vav-Cre bone marrow identified upregulation of many STAT5 target genes and associated pathways. Finally, JAK1/2 inhibition with ruxolitinib normalized hematopoiesis in Cul5Vav-Cre mice. These studies demonstrate the function of CUL5 in HSC function, stem cell fate decisions, and regulation of IL-3 signaling.