Single-Cell STAT5 Signal Transduction Profiling in Normal and Leukemic Stem and Progenitor Cell Populations Reveals Highly Distinct Cytokine Responses.

Abstract 2510

Poster Board II-487

Signal Transducer and Activator of Transcription 5 (STAT5) plays critical roles in normal and leukemic hematopoiesis. We have observed that introduction of activated STAT5A in human stem/progenitor cells enhances long-term stem cell self-renewal, while lentiviral downmodulation of STAT5 expression in both normal as well as primary leukemic CD34+ cells impairs long-term growth and self-renewal. Many cytokines that act on the immature human stem cell compartment are known to be able to activate STAT5, such as TPO and SCF. Yet, little is known about the specificity and kinetics of STAT5 signaling in response to early-acting and lineage-restricted cytokines in specifically defined stem cell and progenitor compartments, particularly in the case of acute myeloid leukemia. We optimized a multiparametric flow cytometry protocol to analyze STAT5 phosphorylation upon cytokine stimulation in stem and progenitor cell compartments at the single-cell level. In normal cord blood (CB) cells, STAT5 phosphorylation was efficiently induced by TPO, IL-3 and GM-CSF within CD34⁺CD38⁻ hematopoietic stem cells (HSCs). EPO and SCF-induced STAT5 phosphorylation was largely restricted to the megakaryocyte-erythroid progenitor (MEP) compartment, while G-CSF, as well IL-3 and GM-CSF were most efficient in inducing STAT5 phosphorylation in the myeloid progenitor compartments. Strikingly, mobilized adult peripheral blood (PB) CD34⁺ cells responded much less efficiently to cytokine-induced STAT5 activation, with the exception of TPO. In leukemic stem and progenitor cells, highly distinct cytokine responses were observed, differing significantly from their normal counterparts. A number of different types of responses were observed, being a) a strong STAT5 activation induced by TPO only; b) a strong STAT5 activation induced by IL3 and GM-CSF, but not TPO; c) STAT5 activation induced by various cytokines; and d) constitutive STAT5 phosphorylation that could not significantly be further induced by cytokines. These responses could not be predicted by the expression level of cytokine receptors. Also, heterogeneity existed in cytokine requirements for long-term expansion of AML CD34⁺ cells on stroma. In most examined cases multiple cytokines acted in an additive fashion in inducing long-term growth of primary AML CD34+ cells in bone marrow stromal cocultures, and in only 1 out of 10 cases cytokine-independent growth was observed. In none of the cases only IL3 or TPO were sufficient to induced long-term expansion of primary AML CD34+ cells. In conclusion, our study demonstrates variable cytokine responses in STAT5 phosphorylation in both normal and leukemic stem/progenitor cells.