Zhengqi Wang

IL-3 Induces Basophil Expansion In Vivo by Directing Granulocyte-Monocyte Progenitors to Differentiate into Basophil Lineage-Restricted Progenitors in the Bone Marrow and by Increasing the Number of Basophil/Mast Cell Progenitors in the Spleen

Recent work has established important roles for basophils in regulating immune responses. To exert their biological functions, basophils need to be expanded to critical numbers. However, the mechanisms underlying basophil expansion remain unclear. In this study, we established that IL-3 played an important role in the rapid and specific expansion of basophils. We found that the IL-3 complex (IL-3 plus anti-IL-3 Ab) greatly facilitated the differentiation of GMPs into basophil lineage-restricted progenitors (BaPs) but not into eosinophil lineage-restricted progenitors or mast cells in the bone marrow. We also found that the IL-3 complex treatment resulted in ∼4-fold increase in the number of basophil/mast cell progenitors (BMCPs) in the spleen. IL-3-driven basophil expansion depended on STAT5 signaling. We showed that GMPs but not common myeloid progenitors expressed low levels of IL-3 receptor. IL-3 receptor expression was dramatically up-regulated in BaPs but not eosinophil lineage-restricted progenitors. Approximately 38% of BMCPs expressed the IL-3Rα-chain. The up-regulated IL-3 receptor expression was not affected by IL-3 or STAT5. Our findings demonstrate that IL-3 induced specific expansion of basophils by directing GMPs to differentiate into BaPs in the bone marrow and by increasing the number of BMCPs in the spleen.

HIF-1α deletion partially rescues defects of hematopoietic stem cell quiescence caused by Cited2 deficiency

Cited2 is a transcriptional modulator involved in various biologic processes including fetal liver hematopoiesis. In the present study, the function of Cited2 in adult hematopoiesis was investigated in conditional knockout mice. Deletion of Cited2 using Mx1-Cre resulted in increased hematopoietic stem cell (HSC) apoptosis, loss of quiescence, and increased cycling, leading to a severely impaired reconstitution capacity as assessed by 5-fluorouracil treatment and long-term transplantation. Transcriptional profiling revealed that multiple HSC quiescence- and hypoxia-related genes such as Egr1, p57, and Hes1 were affected in Cited2-deficient HSCs. Because Cited2 is a negative regulator of HIF-1, which is essential for maintaining HSC quiescence, and because we demonstrated previously that decreased HIF-1α gene dosage partially rescues both cardiac and lens defects caused by Cited2 deficiency, we generated Cited2 and HIF-1α double-knockout mice. Additional deletion of HIF-1α in Cited2-knockout BM partially rescued impaired HSC quiescence and reconstitution capacity. At the transcriptional level, deletion of HIF-1α restored expression of p57 and Hes1 but not Egr1 to normal levels. Our results suggest that Cited2 regulates HSC quiescence through both HIF-1-dependent and HIF-1-independent pathways.

Effective targeting of STAT5-mediated survival in myeloproliferative neoplasms using ABT-737 combined with rapamycin

Signal transducer and activator of transcription-5 (STAT5) is a critical transcription factor for normal hematopoiesis and its sustained activation is associated with hematologic malignancy. A persistently active mutant of STAT5 (STAT5aS711F) associates with Grb2-associated binding protein 2 (Gab2) in myeloid leukemias and promotes growth in vitro through AKT activation. Here we have retrovirally transduced wild-type or Gab2−/− mouse bone marrow cells expressing STAT5aS711F and transplanted into irradiated recipient mice to test an in vivo myeloproliferative disease model. To target Gab2-independent AKT/mTOR activation, we treated wild-type mice separately with rapamycin. In either case, mice lacking Gab2 or treated with rapamycin showed attenuated myeloid hyperplasia and modestly improved survival, but the effects were not cytotoxic and were reversible. To improve on this approach, we combined in vitro targeting of STAT5-mediated AKT/mTOR using rapamycin with inhibition of the STAT5 direct target genes bcl-2 and bcl-XL using ABT-737. Striking synergy with both drugs was observed in mouse BaF3 cells expressing STAT5aS711F, TEL-JAK2 or BCR-ABL and in the relatively single agent-resistant human BCR-ABL-positive K562 cell line. Therefore, targeting distinct STAT5-mediated survival signals, for example, bcl-2/bcl-XL and AKT/mTOR may be an effective therapeutic approach for human myeloproliferative neoplasms.

Novel Mechanism for FcϵRI-mediated Signal Transducer and Activator of Transcription 5 (STAT5) Tyrosine Phosphorylation and the Selective Influence of STAT5B over Mast Cell Cytokine Production

Background: STAT5 is a transcription factor that is vital for mast cell function. Results: Loss of Fyn kinase prevents, whereas loss of Lyn, Gab2, or SHP-1 enhances, FcϵRI-mediated STAT5 tyrosine phosphorylation. Conclusion: IgE-mediated STAT5 activation in mast cells requires Fyn kinase. Significance: Elucidating the mechanisms of mast cell activity is essential to understanding and treating allergic pathologies. Previous studies indicate that STAT5 expression is required for mast cell development, survival, and IgE-mediated function. STAT5 tyrosine phosphorylation is swiftly and transiently induced by activation of the high affinity IgE receptor, FcϵRI. However, the mechanism for this mode of activation remains unknown. In this study we observed that STAT5 co-localizes with FcϵRI in antigen-stimulated mast cells. This localization was supported by cholesterol depletion of membranes, which ablated STAT5 tyrosine phosphorylation. Through the use of various pharmacological inhibitors and murine knock-out models, we found that IgE-mediated STAT5 activation is dependent upon Fyn kinase, independent of Syk, PI3K, Akt, Brutons tyrosine kinase, and JAK2, and enhanced in the context of Lyn kinase deficiency. STAT5 immunoprecipitation revealed that unphosphorylated protein preassociates with Fyn and that this association diminishes significantly during mast cell activation. SHP-1 tyrosine phosphatase deficiency modestly enhanced STAT5 phosphorylation. This effect was more apparent in the absence of Gab2, a scaffolding protein that docks with multiple negative regulators, including SHP-1, SHP-2, and Lyn. Targeting of STAT5A or B with specific siRNA pools revealed that IgE-mediated mast cell cytokine production is selectively dependent upon the STAT5B isoform. Altogether, these data implicate Fyn as the major positive mediator of STAT5 after FcϵRI engagement and demonstrate importantly distinct roles for STAT5A and STAT5B in mast cell function.

Multiple apoptotic defects in hematopoietic cells from mice lacking lipocalin 24P3

The lipocalin mouse 24p3 has been implicated in diverse physiological processes, including apoptosis, iron trafficking, development and innate immunity. Studies from our laboratory as well as others demonstrated the proapoptotic activity of 24p3 in a variety of cultured models. However, a general role for the lipocalin 24p3 in the hematopoietic system has not been tested in vivo. To study the role of 24p3, we derived 24p3 null mice and back-crossed them onto C57BL/6 and 129/SVE backgrounds. Homozygous 24p3−/− mice developed a progressive accumulation of lymphoid, myeloid, and erythroid cells, which was not due to enhanced hematopoiesis because competitive repopulation and recovery from myelosuppression were the same as for wild type. Instead, apoptotic defects were unique to many mature hematopoietic cell types, including neutrophils, cytokine-dependent mast cells, thymocytes, and erythroid cells. Thymocytes isolated from 24p3 null mice also displayed resistance to apoptosis-induced by dexamethasone. Bim response to various apoptotic stimuli was attenuated in 24p3−/− cells, thus explaining their resistance to the ensuing cell death. The results of these studies, in conjunction with those of previous studies, reveal 24p3 as a regulator of the hematopoietic compartment with important roles in normal physiology and disease progression. Interestingly, these functions are limited to relatively mature blood cell compartments.

STAT5 in hematopoietic stem cell biology and transplantation

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-deficient hematopoiesis is permissive for Myc-induced B-cell leukemogenesis

Despite being an attractive molecular target for both lymphoid and myeloid leukemias characterized by activated tyrosine kinases, the molecular and physiological consequences of reduced signal transducer and activator of transcription-5 (Stat5) during leukemogenesis are not well known. Stat5 is a critical regulator of mouse hematopoietic stem cell (HSC) self-renewal and is essential for normal lymphocyte development. We report that pan-hematopoietic deletion in viable adult Vav1-Cre conditional knockout mice as well as Stat5abnull/null fetal liver transplant chimeras generated HSCs with reduced expression of quiescence regulating genes (Tie2, Mpl, Slamf1, Spi1, Cited2) and increased expression of B-cell development genes (Satb1, Dntt, Btla, Flk2). Using a classical murine B-cell acute lymphoblastic leukemia (B-ALL) model, we demonstrate that these HSCs were also poised to produce a burst of B-cell precursors upon expression of Bcl-2 combined with oncogenic Myc. This strong selective advantage for leukemic transformation in the background of Stat5 deficient hematopoiesis was permissive for faster initiation of Myc-induced transformation to B-ALL. However, once established, the B-ALL progression in secondary transplant recipients was Stat5-independent. Overall, these studies suggest that Stat5 can play multiple important roles that not only preserve the HSC compartment but can limit accumulation of potential pre-leukemic lymphoid populations.

Imipramine blue sensitively and selectively targets FLT3-ITD positive acute myeloid leukemia cells

Aberrant cytokine signaling initiated from mutant receptor tyrosine kinases (RTKs) provides critical growth and survival signals in high risk acute myeloid leukemia (AML). Inhibitors to FLT3 have already been tested in clinical trials, however, drug resistance limits clinical efficacy. Mutant receptor tyrosine kinases are mislocalized in the endoplasmic reticulum (ER) of AML and play an important role in the non-canonical activation of signal transducer and activator of transcription 5 (STAT5). Here, we have tested a potent new drug called imipramine blue (IB), which is a chimeric molecule with a dual mechanism of action. At 200–300 nM concentrations, IB is a potent inhibitor of STAT5 through liberation of endogenous phosphatase activity following NADPH oxidase (NOX) inhibition. However, at 75–150 nM concentrations, IB was highly effective at killing mutant FLT3-driven AML cells through a similar mechanism as thapsigargin (TG), involving increased cytosolic calcium. IB also potently inhibited survival of primary human FLT3/ITD+ AML cells compared to FLT3/ITDneg cells and spared normal umbilical cord blood cells. Therefore, IB functions through a mechanism involving vulnerability to dysregulated calcium metabolism and the combination of fusing a lipophilic amine to a NOX inhibiting dye shows promise for further pre-clinical development for targeting high risk AML.

STAT5 activation in B-cell acute lymphoblastic leukemia: Damned if you do, damned if you don't

A significant role of the microenvironment in leukemogenesis is beginning to emerge. The leukemia cell microenvironment consists of not only the stromal and endothelial cell components but also the normal hematopoietic cells. Signal transducer and activator of transcription 5 (STAT5) is a latent transcription factor that is normally transiently activated by phosphorylation in response to microenvironmental signals. In hematopoietic cells, persistently activated STAT5 via aberrant receptor signaling, Janus kinases (JAKs), or intracellular tyrosine kinases is a bona fide driver of leukemogenesis. However, active IL-7/STAT5 signaling also protects the early B-cell genome by suppressing error-prone recombination and vulnerability to transformation. Along these lines, we have reported that lymphocyte development from transplanted STAT5-deficient fetal liver cells was blocked at the pre-pro-B-cell stage but when combined with transgenic Myc and Bcl-2 promoted faster initiation of B-ALL. Furthermore, inflammatory responses may also be involved in leukemia initiation in both pediatric and adult patients which are associated with decreased phosphorylation of STAT5. Likewise, additional targeted agents continue to be developed for precision medicine that prominently suppress signaling pathways. A common theme of all of these perturbations is potential risk for dysregulating hematopoiesis through general transcriptional modulation. Here we discuss the potential for STAT5 inhibition as a double edged sword in certain hematologic disorders, such as early B-cell lymphoblastic leukemias. Considering the rapid pace of understanding of the pre-leukemic decrease in poly-clonality that precedes leukemia, the functional changes associated with microenvironmental influences are thus of potential clinical significance.

The Cooperative Relationship between STAT5 and Reactive Oxygen Species in Leukemia: Mechanism and Therapeutic Potential

Reactive oxygen species (ROS) are now recognized as important second messengers with roles in many aspects of signaling during leukemogenesis. They serve as critical cell signaling molecules that regulate the activity of various enzymes including tyrosine phosphatases. ROS can induce inactivation of tyrosine phosphatases, which counteract the effects of tyrosine kinases. ROS increase phosphorylation of many proteins including signal transducer and activator of transcription-5 (STAT5) via Janus kinases (JAKs). STAT5 is aberrantly activated through phosphorylation in many types of cancer and this constitutive activation is associated with cell survival, proliferation, and self-renewal. Such leukemic activation of STAT5 is rarely caused by mutation of the STAT5 gene itself but instead by overactive mutant receptors with tyrosine kinase activity as well as JAK, SRC family protein tyrosine kinases (SFKs), and Abelson murine leukemia viral oncogene homolog (ABL) kinases. Interestingly, STAT5 suppresses transcription of several genes encoding antioxidant enzymes while simultaneously enhancing transcription of NADPH oxidase. By doing so, STAT5 activation promotes an overall elevation of ROS level, which acts as a feed-forward loop, especially in high risk Fms-related tyrosine kinase 3 (FLT3) mutant leukemia. Therefore, efforts have been made recently to target ROS in cancer cells. Drugs that are able to either quench ROS production or inversely augment ROS-related signaling pathways both have potential as cancer therapies and may afford some selectivity by activating feedback inhibition of the ROS-STAT5 kinome. This review summarizes the cooperative relationship between ROS and STAT5 and explores the pros and cons of emerging ROS-targeting therapies that are selective for leukemia characterized by persistent STAT5 phosphorylation.