STAT5 Promotes Hematopoietic Stem Cell Maintenance by Increasing Quiescence-Associated Gene Expression and Suppressing Lymphoid Commitment

Abstract 851

Signal transducer and activator of transcription 5 (STAT5) is a critical regulator of lympho-myeloid hematopoiesis through activation downstream of early-acting cytokines, their receptors (e.g. c-Mpl), and janus kinases (JAKs). We have previously reported that hematopoietic stem cells (HSC) in pI:pC treated Mx1-Cre conditional STAT5 knockout mice, like c-Mpl deficient HSC, are persistently more actively cycling and become apoptotic, reducing long-term HSC number. Since down-regulation of c-Mpl is associated with HSC heterogeneity defined by differential CD150/Flk2 expression and skewing toward lymphoid-primed multipotential progenitors (LMPP), we set to investigate whether STAT5-deficient cycling KLS cells are similarly lymphoid-primed. First, we generated novel Vav1-Cre/+STAT5abfl/fl hematopoietic-specific conditional knockout mice with 98% deletion efficiency in sorted KLS cells as determined by real-time PCR (N=3 mice). The majority of Vav1-Cre/+STAT5abfl/fl mice were viable with significantly reduced white blood cell counts (1.84±1.01 x10³/μl, N=17 vs 8.46±2.26 x10³/μl, N=27), red blood cell counts (7.53±0.78 x10⁶/μl vs 10.19±0.80 x10⁶/μl) and hematocrits (33.2±4.3% vs 47.1±3.2%) relative to wild-type. Competitive repopulation was 16-fold reduced CD45.2 donor chimerism with Vav-Cre/+STAT5abfl/fl (3.3±0.7%; N=5) compared to wild-type (55±2%; N=5) in a 1:1 mix with wild-type Boy J bone marrow cells. In non-ablative bone marrow transplantation, injection of a single dose of 5 million donor bone marrow cells gave rise to high levels of engraftment (84±8%, N=5) in the peripheral blood of congenic adult Vav-Cre/+STAT5abfl/fl recipients compared to no detectable engraftment controls. The KLS fraction was analyzed using CD34 and FLK2 markers. Despite severe reduction in the long-term repopulating HSC CD34-FLK2-KLS (N=3, p<0.001) and short-term repopulating HSC CD34+FLK2-KLS (p=0.03) fractions, the CD34+FLK2+KLS fraction enriched for LMPP was relatively maintained. We obtained similar results using CD150/CD48 in both Vav-Cre/+STAT5abfl/fl and STAT5ab null/null fetal liver transplanted mice. Using Affymetrix mouse gene chips to compare global gene expression between CD45.2+ KLS cells from STAT5ab null/null vs. wild-type fetal liver transplanted mice, several HSC-related genes were among those decreased whereas the majority of increased genes were associated with B-lineage commitment and immunoglobulin production. Furthermore, single cell gene expression analysis on the Fluidigm BioMark 48.48 chip (20 individual cells each genotype) was consistent with the microarray profiling. Half of wild-type KLS cells expressed high levels of HSC transcripts such as c-Mpl and Gfi1b and low levels of lymphoid transcripts whereas half were the opposite. In contrast, all STAT5 deficient KLS cells were lymphoid-primed and deficient in HSC transcripts. Due to the STAT5-dependent block at the pre-pro-B-cell stage, we are unable to functionally readout the donor B-lymphoid cells in the peripheral blood of transplanted mice. Since the combination of bcl-2 and myc induces aggressive B-cell acute lymphoblastic leukemia, we set out to use both bcl-2 and myc transgenic mouse models to test whether lymphoid-primed KLS cells lacking STAT5 would promote B cell development through a non-canonical pathway. Transgenic myc driven from an immunoglobulin H chain enhancer (Eμ-Myc) combined with H2K-bcl-2 induced faster B-ALL in STAT5ab null/null vs. wild type control fetal liver transplanted mice. The latency was significantly reduced from a median survival of 91 days in the wild type control to a median survival of 44 days in lethally-irradiated recipients (P<0.001; N range from 8–14 mice/group). Similar results were obtained in sub-lethally irradiated recipients (P=0.007; N range 10–20 mice/group). Loss of STAT5 promoted the accumulation of B-lineage committed progenitor cells in combination with pre-pro-B blockage. We also found a recently described Sca1+c-Kit-Lin- alternative lymphoid progenitor population to be expanded only in the STAT5ab null/null genotype expressing myc and bcl-2. Whether this population possesses the leukemia-initiating potential is still under investigation. Overall, we have found that STAT5 plays an unexpected role in hematopoietic stem cell differentiation by limiting early lymphoid lineage commitment.