STAT5 in hematopoietic stem cell biology and transplantation

Abstract

Signal transducer and activator of transcription 5 (STAT5) regulates normal lympho-myeloid development through activation downstream of early-acting cytokines, their receptors, and Janus kinases (JAKs). Despite a general understanding of the role of STAT5 in hematopoietic stem cell (HSC) proliferation, survival, and self-renewal, the transcriptional targets and mechanisms of gene regulation that control multi-lineage engraftment following transplantation for the most part remain to be understood. In this review, we focus on the role of STAT5 in HSC transplantation and recent developments toward identifying the relevant downstream target genes and their role as part of a pleiotropic STAT5 mediated signaling response.

STAT5 Activation in Hematopoietic Stem and Progenitor Cells

Hematopoiesis is a finely tuned process with intricate feedback mechanisms for regulating production of mature blood cells. Many of the lineage commitment and differentiation decisions of hematopoietic cells are dictated by the delicate balance of transcription factors. Most basal transcription factors are constitutively active, but some require activation in order to translocate to the nucleus and regulate gene expression. JAK-STAT pathway activation provides a rapid way for extracellular signals to rapidly transduce signals resulting in STAT activation within minutes and gene expression within hours of the initial cytokine exposure. Once tyrosine phosphorylated, transcriptionally activated STATs accumulate in the nucleus.

Signal transducer and activator of transcription-5 (STAT5) comprises two separate genes,¹ STAT5A and STAT5B, which collectively are major regulators of normal hematopoiesis with pleiotropic roles in hematopoietic stem cell (HSC),^1-7 hematopoietic progenitor cell (HPC),^8-10 and mature cell populations.^11-14 Although the differences in phenotype of mice lacking STAT5A vs. STAT5B^15,16 were primarily assumed to be due to differences in tissue specific gene expression, functional differences have been more recently discovered. For example, ERβ regulation appears to be mediated only by STAT5B,¹⁷ and STAT5B-deficient patients have been discovered that have reduced numbers of regulatory T cells and short stature.^18-20 These phenotypes are consistent with major non-redundant roles for STAT5B in regulation of target genes such as FoxP3 and IGF1. Knockdown studies of STAT5A and STAT5B in human CD4⁺ T cells confirmed this specificity for FoxP3²¹ and knockdown studies in IL-3 stimulated murine BaF3 cells identified overlapping and unique sets of STAT5 target genes by chromatin immunoprecipitation.²²

Interestingly, the ability for STAT5 binding to modulate gene expression appears to be under additional levels of regulation such as serine phosphorylation^23-25 and glycosylation²⁶ which may influence interactions with CREB-binding protein. Serine phosphorylation has been described for most STATs^27,28 although the positive or negative influence of this modification has been debated.²⁹ The normal role of STAT5 serine phosphorylation in hematopoiesis has not been tested and the effect may be very cell type and cell context specific. Serine phosphorylation could facilitate interaction of STAT5 with critical accessory proteins required for nuclear localization of tyrosine phosphorylated STAT5. Cooperative signals mediated by serine and tyrosine phosphorylation could also be lineage-specific and further studies of serine phosphorylation deficient STAT5 mutants are needed to fully understand this level of regulation in HSC and throughout hematopoiesis.

We have reported that STAT5 is required for HSC “fitness” with its deficiency resulting in greatly impaired long-term multilineage competitive repopulation capacity of fetal liver^4,10 and bone marrow^2-7 HSC in lethally-irradiated transplant recipients and a reciprocal permissiveness for wild-type donor HSC to engraft when transplanted into STAT5-deficient hosts in the absence of myeloablation.^2,7,10 In our study, using interferon-induced Cre-mediated deletion, STAT5-deficient HSC were found to be persistently more actively cycling and become apoptotic, resulting in a reduction of long-term HSC number⁷ (Fig. 1A). However in this model the interferon response could result in an initial stimulation of the HSC pool and contribute to the loss of quiescence phenotype. Although our analyses were done many months post interferon induction, the role of STAT5 in the complete absence of induced interferon requires additional HSC-specific deletion approaches. Figure 1B summarizes the current state-of-the-art of STAT5 conditional knockout in hematopoietic lineages.

Figure 1. Conditional knockout strategies for STAT5 deletion in the hematopoietic system. (A) We have previously studied HSC with interferon-induced deletion of STAT5 using the Mx1-Cre system. This approach gives conditional deletion of STAT5 in adult mice. The limitation of the approach is the need to wait for the interferon response to subside before subsequent analysis. (B) Since 2006 various other groups have conditionally knocked out STAT5 floxed alleles of STAT5A and STAT5B that were generated by Lothar Hennighausen (NIDDK). These knockouts include expression of Cre recombinase in early hematopoietic stem cells (and vascular progenitors), as well as T cells, B cells, NK cells, and dendritic cell lineages.

STAT5 is activated by several early acting cytokines such as IL-3,^30-32 thrombopoietin (TPO),^33-37 granulocyte-colony stimulating factor (G-CSF),^38,39 and granulocyte-macrophage colony stimulating factor (GM-CSF).^38,40-42 Although initially all of these factors were believed to act on committee myeloid lineages, intracellular flow cytometry data from several groups using HSC/HPC fractions shows marked STAT5 activation.^38,43,44 In HSCs, Mpl synergizes with early acting cytokines^45-48 and regulates multilineage progenitors and HSC self-renewal.^49-51 Interestingly, Mpl also promotes HSC quiescence.⁵² Our prior studies have found close parallels between Mpl deficiency and STAT5 deficiency^2,3,7 (Fig. 2). These phenotypes include loss of multi-lineage engraftment ability, thrombocytopenia, decreased HSC self-renewal, loss of HSC quiescence, and decreased expression of Tie2 and p57. Although STAT5 may not be a major signaling partner utilized by TPO/Mpl in megakaryocytes and platelets, a direct functional link between Mpl/STAT5 is likely. Mpl promoter constructs driving STAT5A expression or generation of an inducible STAT5A expression vector crossed into a Mpl-deficient genetic background could address the role of STAT5 by testing rescue of function. Reciprocally, testing Mpl agonists in STAT5-deficient hosts might give important insight into whether STAT5 is essential for the hematopoiesis-promoting effects of these new clinically relevant agents.

Figure 2. Comparison between STAT5 and Mpl knockout phenotypes. While c-Mpl deficiency causes severe megakaryocyte and platelet production defects, most other lymphoid and myeloid lineages are unaffected. Interestingly, Mpl knockout also selectively impairs early HSC/HPC function as determined by analysis of peripheral blood chimerism following transplantation. STAT5 knockout mice share the common HSC/HPC level defects, do not have intrinsic megakaryocyte/platelet defects, but have severe combined immunodeficiency that is unique to the role of STAT5 downstream of the common gamma chain and JAK3.

Interestingly, in hematopoiesis, most of the classical bifurcation points in lineage development are regulated by at least two transcription factors that have antagonistic functions. Examples include Notch/Pax5 and C/EBPα/GATA-1 where progressive changes in the dosage of these factors results in a shift of lineage potential. Comparable antagonism may exist at the hematopoietic stem cell/progenitor cell commitment decision. Mpl appears to control myeloid commitment and its downregulation is associated with increased lymphoid priming in HSC.⁵³ It will be interesting to determine whether STAT5 similarly controls HSC fate decisions, perhaps downstream of Mpl. Recent studies of MPP heterogeneity, highlight downregulation of c-Mpl marking the transition to the lymphoid-primed multipotent progenitor (LMPP)⁵³ and we have previously linked c-Mpl to STAT5 in HSC maintenance and self-renewal^3,7 but have not examined lineage commitment. IL-7 is a major activator of STAT5 in the common lymphoid progenitor and the IL-7, common gamma chain, JAK3, STAT5 signaling axis is required for normal T and B lymphocyte development.⁵⁴ Expression of the IL-7 receptor marks the transition toward the common lymphoid progenitor (CLP). Additionally CD150^low HSC are functionally B-lymphoid-biased^55-57 and express flk2 and CD86.^55,58 Further, normal long-term repopulating HSC (LT-HSC) are CD150⁺48⁻ but become B-lymphoid biased when localized to the central bone marrow microenvironment.⁵⁹ Understanding the heterogeneity at all levels of hematopoiesis is currently a major area of study and new information is being obtained through techniques such as single cell PCR and lineage tracing with knock-in transgenic mouse models.

STAT5 is potently activated by IL-3/IL-5/GM-CSF all of which utilize the common β chain in the myeloid lineage to produce mast cells, basophils, eosinophils, granulocytes, and monocytes, including the basophil/mast cell progenitor.^14,60 It is clear that STAT5 deletion causes significant development defects in both lymphoid and myeloid lineages. Interestingly, when STAT5 is deleted in fetal liver HSC, we have observed increased myelopoiesis and decreased lymphopoiesis following transplant into lethally-irradiated adult hosts.⁶ However, STAT5 is required at later stages for normal granulopoiesis.⁴² Therefore, STAT5 acts at many levels of hematopoiesis and the overall output in the peripheral blood lineages is a composite of the effects on HSC, primitive HPC, and the lineage committed progenitors. EPO is also a major activator of STAT5 in the erythroid lineage^61-63 and it can synergize with SCF to activate STAT5,⁶⁴ whereas activation of STAT5 by SCF alone in HSC/HPC still remains unclear.^13,38,65,66 In addition to the canonical receptor/JAK interactions, cytokine mediated functional activation of STAT5 may depend on additional factors such as lipid rafts, Src family kinase activation, or recruitment by Grb2-associated binding proteins (Gabs). Prospective isolation of committed progenitor pools has not yet been tested, so it is difficult to separate the role of STAT5 in hematopoietic stem cells from the various progenitor cells and the frequency and output of progenitor populations must be considered when interpreting the outcome of long-term multilineage competitive repopulation assays.

STAT5 Regulation of Hematopoietic Stem Cell Transplantation

Recent advances in understanding HSC heterogeneity^67,68 have defined a gradient of high CD150 expression regulating self-renewal potential, quiescence in long-term repopulating HSC (LT-HSC), and myeloid lineage bias and low CD150 expression associated with loss of self-renewal potential, loss of quiescence, and lymphoid-biased differentiation in short-term repopulating HSC (ST-HSC).^55-57,69,70 While intermediate term HSC are beginning to be identified, their genetic control is not well characterized. Regulation of quiescence by STAT5 may be secondary to other defects in hematopoietic stem cell potential. Greater understanding of the impact of STAT5 on HSC heterogeneity and commitment may lead to improved methods for mobilization and stem cell engraftment in the absence of genotoxic myeloablative conditioning. It still remains to be determined whether enforced quiescence alone would be sufficient to correct function. Recently a method for induced quiescence was described that was based on pharmacologic stabilization of Hif-1α causing increased quiescence of HSC and accelerated recovery from myelosuppression.⁷¹

HSC niche occupancy is an essential ingredient for successful stem cell transplantation. In mice lacking STAT5, it is unclear why some niches are open and available to be filled by a donor graft. Interestingly, a percentage of the filled niches remain occupied by host cells and donor grafts do not eventually take over. Instead there is equilibrium between donor and host that is maintained long-term in the KLS compartment, although several peripheral blood lineages are dominated by wild-type grafts which selectively out-compete the STAT5-deficient host cells (Fig. 3). HSC could leave but not be able to compete for the niche once a specific set of HSC niches are filled. There are likely unique HSC and HPC niches and only the HPC niches may be fully open and able to accept wild-type donor HSC. HSC lacking STAT5 may also never leave the niche and instead act as a “dominant negative” by blocking niche space from the wild-type cells and continuing to produce blood cells, albeit at a reduced rate and without the production of mature T/B lymphocytes.

Figure 3. Possible hematopoietic stem/progenitor fates in the absence of STAT5. We have observed high levels of HSC/HPC engraftment in mice lacking STAT5. However, due to the strong multilineage selective growth advantage of the wild-type donor cells vs. the STAT5 knockout cells, it has been difficult to assess the impact in HSC vs. HPC compartments of the bone marrow. It is possible that only a subset of niches are vacated in the absence of STAT5 and that competition for these niches is not selectively impaired (homing, engraftment). STAT5 deficient HSC may not leave the niches as part of normal trafficking but rather prevent donor HSC from occupying niches. STAT5 deficient HPC may be vacated from putative HPC niches resulting in greater multilineage engraftment from multipotent progenitors.

Mechanistic studies have provided insight into how STAT5 modulates HSC engraftment and self-renewal. STAT5 transcriptional targets differ between human HSC and progenitor compartments.⁷² We observed that STAT5 promotes HSC quiescence and self-renewal but promotes proliferation in more differentiated cell types. More detailed molecular understanding of this dichotomy of function remains to be discovered. Roles for phosphorylated STAT5 in HSC vs. HPC might relate to the levels of phosphorylation. This is important because changes in phosphorylation might directly affect gene expression of distinct genes.⁷³ STAT5 transcriptional function in cultured human cells has been described to involve Gata-dependent and independent pathways in control of megakaryocyte–erythrocyte development.⁷⁴ STAT5 also controls key target genes such as Hif-2α to regulate HSC expansion in vitro.⁷² While constitutively active STAT5 cannot confer self-renewal on progenitors,⁷⁵ STAT5 targets differ between HSC and progenitors in human cells.⁷² STAT5 is required for efficient lympho-myeloid repopulation and HSC quiescence⁷ and the hypoxia inducible gene Cited2 (STAT5 target gene) plays similar roles in fetal⁷⁶ and adult HSC.⁷⁷

Perspective on Targeting STAT5 in Normal and Leukemic Hematopoiesis

Recycling of STATs between nucleus and cytoplasm and retention in the nucleus only occurring following activation has been described.^78,79 It is now clear that molecules such as Rac-GAP are important for STAT5 nuclear import.⁸⁰ The negative regulation of this pathway is also critically important and much is known about the inducible negative regulators Cis and Socs. Hyper-activation of STAT5 is a validated leukemia driver and is currently a high value target for therapy. In chronic myeloid leukemia, unique roles for STAT5A and STAT5B in drug resistance downstream of BCR-ABL have been described.⁸¹ Activating mutations of STAT5 (STAT5B Y665F and N642H) in leukemia are also found in large granular lymphocyte (LGL) leukemia.⁸² With STAT5, it is clear that phosphorylation by JAKs is the major mechanism of activation; however, other mechanisms appear to play important roles in non-canonical activation of STAT5. These mechanisms include activation by Src family kinases downstream of Flt3 mutations, although conflicting results have been reported.^83,84 Roles for integrin signaling and the spleen tyrosine kinase (Syk) in activation of STAT5 in AML blasts, including MLL-AF9 positive AML have also been reported.^85,86

Interestingly, retention of phosphorylated STAT5 in the cytoplasm has been observed in leukemias and the cytoplasmic functions for STAT5 will be important to define. STAT5 nuclear accumulation occurs predominately in Flt3-ITD⁺ AML⁸⁷ where other mutations and pathways could provide complimentary signals. Constitutively active STAT5 via Socs1 is capable of p53 activation and induction of an oncogene-induced cell senescence response.^88,89 Therefore, negative regulatory mechanisms may be overcome in AML through alternative pathway activation. In contrast, BCR-ABL driven leukemogenesis in CML and ALL may require engagement of mechanisms to restrain STAT5 activation because of the principal requirement of STAT5 for leukemic progression and the absence of other compensating pathways. Notably, CML is associated with pSTAT5 localization in the cytoplasm at an unusually high level compared with normal cells.⁹⁰ Since nuclear pSTAT5 might not be well tolerated in CML, understanding mechanisms controlling nuclear and cytoplasmic distribution of STAT5 has emerged as an important area.

It has been estimated that about half of STAT5 binding sites are in introns and half are in the promoter region.^91,92 Nelson et al. propose about 200 highly conserved tandem STAT5 binding sites.⁹¹ Perhaps providing clues to the activity of STAT5 in HSC, transcriptional activation or repression determined by gene expression array has been completed in mouse embryonic fibroblasts lacking STAT5.⁹³ Although STAT5 targets in various tissues such as liver⁹⁴ may have some likely conservation with hematopoietic cells, the microenvironment of these diverse cell types is different and the intrinsic genetic programs and co-activators are different. Importantly, it has recently been shown that STAT binding patterns are cell type specific.⁹⁵ Perhaps better understanding the HSC and HPC niches will elucidate novel mechanisms of gene regulation that are niche-dependent. Furthermore, better understanding of how STAT5 switches from activation to repression and on what sites in HSC that STAT5 is active, will be important for moving the field forward. Recent work has suggested that in lymphocyte development STAT5 tetramers play an important role in normal cytokine responses and normal immune function.⁹⁶

Therapeutics for improving the transplantation into non-ablated hosts is the “Holy Grail” for many biological disorders. Also, the same principles may apply for leukemic HSC and their targeted therapy. STAT5 targeted small molecules have been recently described but more sophisticated efforts may also help such as targeting protein:protein interactions with the N-domain or by targeting STAT5 activation signatures. STAT5 serine phosphorylation also represents a potential new target.²⁴ Identification of the serine kinase(s) could yield druggable targets capable of eradicating pSTAT5 in leukemias. Due to the prime role of STAT5 at the intersection of normal and leukemic hematopoiesis, innovative approaches for modulating STAT5 activation hold much promise for future therapeutic applications.

10.4161/jkst.27159