Maintenance of hematopoietic stem cells by tyrosine-unphosphorylated STAT5 and JAK inhibition

• Tyrosine-unphosphorylated STAT5 maintains HSCs.

• JAK inhibition promotes unphosphorylated STAT5 activity and maintains mouse and human, normal and MPN HSPCs.

Adult haematopoietic stem cells (HSCs) are responsible for the lifelong production of blood and immune cells, a process regulated by extracellular cues including cytokines. Many cytokines signal through the conserved JAK/STAT pathway, in which tyrosine-phosphorylated STATs (pSTATs) function as transcription factors. STAT5 is a pivotal downstream mediator of several cytokines known to regulate haematopoiesis but its function in the HSC compartment remains poorly understood. Here, we show that STAT5-deficient HSCs exhibit an unusual phenotype: reduced multi-lineage repopulation and self-renewal, combined with reduced exit from quiescence and increased differentiation. This was driven not only by loss of canonical pSTAT5 signalling, but also by loss of distinct transcriptional functions mediated by STAT5 lacking canonical tyrosine phosphorylation (uSTAT5). Consistent with this concept, expression of an unphosphorylatable STAT5 mutant constrained wild-type HSC differentiation, promoted their maintenance and upregulated transcriptional programs associated with quiescence and stemness. The JAK1/2 inhibitor, ruxolitinib, which increased the uSTAT5:pSTAT5 ratio, had similar effects on murine HSC function: it constrained HSC differentiation and proliferation, promoted HSC maintenance and upregulated transcriptional programs associated with stemness. Ruxolitinib also enhanced serial replating of normal human HSPCs, CALR-mutant murine HSCs and HSPCs obtained from patients with myelofibrosis. Our results therefore reveal a previously unrecognized interplay between pSTAT5 and uSTAT5 in the control of HSC function and highlight JAK inhibition as a potential strategy for enhancing HSC function during ex vivo culture. Increased levels of uSTAT5 may also contribute to the failure of JAK inhibitors to eradicate myeloproliferative neoplasms.