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1.  Efficacious Intermittent Dosing of a Novel JAK2 Inhibitor in Mouse Models of Polycythemia Vera 
PLoS ONE  2012;7(5):e37207.
A high percentage of patients with the myeloproliferative disorder polycythemia vera (PV) harbor a Val617→Phe activating mutation in the Janus kinase 2 (JAK2) gene, and both cell culture and mouse models have established a functional role for this mutation in the development of this disease. We describe the properties of MRLB-11055, a highly potent inhibitor of both the WT and V617F forms of JAK2, that has therapeutic efficacy in erythropoietin (EPO)-driven and JAK2V617F-driven mouse models of PV. In cultured cells, MRLB-11055 blocked proliferation and induced apoptosis in a manner consistent with JAK2 pathway inhibition. MRLB-11055 effectively prevented EPO-induced STAT5 activation in the peripheral blood of acutely dosed mice, and could prevent EPO-induced splenomegaly and erythrocytosis in chronically dosed mice. In a bone marrow reconstituted JAK2V617F-luciferase murine PV model, MRLB-11055 rapidly reduced the burden of JAK2V617F-expressing cells from both the spleen and the bone marrow. Using real-time in vivo imaging, we examined the kinetics of disease regression and resurgence, enabling the development of an intermittent dosing schedule that achieved significant reductions in both erythroid and myeloid populations with minimal impact on lymphoid cells. Our studies provide a rationale for the use of non-continuous treatment to provide optimal therapy for PV patients.
doi:10.1371/journal.pone.0037207
PMCID: PMC3356383  PMID: 22623993
2.  STAT5 in hematopoietic stem cell biology and transplantation 
JAK-STAT  2013;2(4):e27159.
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.
doi:10.4161/jkst.27159
PMCID: PMC3906416  PMID: 24498540
cytokine signaling; JAK-STAT; hematopoiesis; engraftment; quiescence; self-renewal; transplantation; stem cell
3.  MIR29B regulates expression of MLLT11 (AF1Q), an MLL fusion partner, and low MIR29B expression associates with adverse cytogenetics and poor overall survival in AML 
British journal of haematology  2011;153(6):753-757.
Summary
MLLT11, an MLL fusion partner, is a poor prognostic biomarker for paediatric acute myeloid leukaemia (AML), adult normal cytogenetics AML, and adult myelodysplastic syndrome. MLLT11 is highly regulated during haematopoietic progenitor differentiation and development but its regulatory mechanisms have not been defined. In this study, we demonstrate by transfection experiments that MIR29B directly regulates MLLT11 expression in vitro. MIR29B expression level was also inversely related to MLLT11 expression in a cohort of 56 AML patients (P < 0·05). AML patients with low MIR29B/elevated MLLT11 expression had poor overall survival (P = 0·038). Therefore, MIR29B may be a potential prognostic biomarker for AML patients.
doi:10.1111/j.1365-2141.2011.08662.x
PMCID: PMC3896048  PMID: 21496004
AML prognostic marker; MLLT11; MLL fusion gene; MIR29; AML survival
4.  Identification of 6-Benzylthioinosine as a Myeloid Leukemia Differentiation–Inducing Compound 
Cancer research  2008;68(11):4369-4376.
As the pathophysiology of acute myelogenous leukemia (AML) involves a block of myeloid maturation, a desirable therapeutic strategy is to induce leukemic cell maturation to increase the efficacy and to avoid the side effects of traditional chemotherapeutics. Through a compound library screen, 6-benzylthioinosine (6BT) was identified as a promising differentiation-inducing agent. 6BT induces monocytic differentiation of myeloid leukemia cell lines such as HL-60 and OCI-AML3, as well as primary patient samples as evidenced by morphology, immunophenotyping, and nitroblue tetrazolium reduction. Not only can 6BT induce differentiation but a subset of AML cell lines such as MV4-11 and HNT34 instead undergo 6BT-mediated cell death. Despite inducing cell death in some leukemic cells, 6BT exhibits extremely low toxicity on several nonmalignant cells such as fibroblasts, normal bone marrow, and endothelial cells. This toxicity profile may relate to the function of 6BT as an inhibitor of the nucleoside transporter, ent1, which is thought to prevent it from entering many cell types. In contrast, 6BT likely enters at least some leukemic cell lines as shown by its requirement for phosphorylation for its differentiation activity. 6BT is also able to synergize with currently used myeloid differentiation agents such as ATRA and decitabine. Early studies indicate that the mechanism of action of this compound may involve ATP depletion that leads to growth inhibition and subsequent differentiation. Besides in vitro activity, 6BT also shows the ability to impair HL-60 and MV4-11 tumor growth in nude mice. 6BT is a promising new monocytic differentiation agent with apparent leukemic cell–specific activity.
doi:10.1158/0008-5472.CAN-07-6559
PMCID: PMC3896053  PMID: 18519698
5.  Molecular-Targeted Therapies for Hematologic Malignancies 
Advances in Hematology  2011;2012:606423.
doi:10.1155/2012/606423
PMCID: PMC3254001  PMID: 22242021
6.  Inhibition of PI3K/mTOR Overcomes Nilotinib Resistance in BCR-ABL1 Positive Leukemia Cells through Translational Down-Regulation of MDM2 
PLoS ONE  2013;8(12):e83510.
Chronic myeloid leukemia (CML) is a cytogenetic disorder resulting from formation of the Philadelphia chromosome (Ph), that is, the t(9;22) chromosomal translocation and the formation of the BCR-ABL1 fusion protein. Tyrosine kinase inhibitors (TKI), such as imatinib and nilotinib, have emerged as leading compounds with which to treat CML. t(9;22) is not restricted to CML, 20-30% of acute lymphoblastic leukemia (ALL) cases also carry the Ph. However, TKIs are not as effective in the treatment of Ph+ ALL as in CML. In this study, the Ph+ cell lines JURL-MK2 and SUP-B15 were used to investigate TKI resistance mechanisms and the sensitization of Ph+ tumor cells to TKI treatment. The annexin V/PI (propidium iodide) assay revealed that nilotinib induced apoptosis in JURL-MK2 cells, but not in SUP-B15 cells. Since there was no mutation in the tyrosine kinase domain of BCR-ABL1 in cell line SUP-B15, the cells were not generally unresponsive to TKI, as evidenced by dephosphorylation of the BCR-ABL1 downstream targets, Crk-like protein (CrkL) and Grb-associated binder-2 (GAB2). Resistance to apoptosis after nilotinib treatment was accompanied by the constitutive and nilotinib unresponsive activation of the phosphoinositide 3-kinase (PI3K) pathway. Treatment of SUP-B15 cells with the dual PI3K/mammalian target of rapamycin (mTOR) inhibitor BEZ235 alone induced apoptosis in a low percentage of cells, while combining nilotinib and BEZ235 led to a synergistic effect. The main role of PI3K/mTOR inhibitor BEZ235 and the reason for apoptosis in the nilotinib-resistant cells was the block of the translational machinery, leading to the rapid downregulation of the anti-apoptotic protein MDM2 (human homolog of the murine double minute-2). These findings highlight MDM2 as a potential therapeutic target to increase TKI-mediated apoptosis and imply that the combination of PI3K/mTOR inhibitor and TKI might form a novel strategy to combat TKI-resistant BCR-ABL1 positive leukemia.
doi:10.1371/journal.pone.0083510
PMCID: PMC3859659  PMID: 24349524
7.  Gab Adapter Proteins as Therapeutic Targets for Hematologic Disease 
Advances in Hematology  2011;2012:380635.
The Grb-2 associated binder (Gab) family of scaffolding/adaptor/docking proteins is a group of three molecules with significant roles in cytokine receptor signaling. Gabs possess structural motifs for phosphorylation-dependent receptor recruitment, Grb2 binding, and activation of downstream signaling pathways through p85 and SHP-2. In addition, Gabs participate in hematopoiesis and regulation of immune response which can be aberrantly activated in cancer and inflammation. The multifunctionality of Gab adapters might suggest that they would be too difficult to consider as candidates for “targeted” therapy. However, the one drug/one target approach is giving way to the concept of one drug/multiple target approach since few cancers are addicted to a single signaling molecule for survival and combination drug therapies can be problematic. In this paper, we cover recent findings on Gab multi-functionality, binding partners, and their role in hematological malignancy and examine the concept of Gab-targeted therapy.
doi:10.1155/2012/380635
PMCID: PMC3246295  PMID: 22216034
8.  Grb2-associated binding (Gab) proteins in hematopoietic and immune cell biology 
Grb2-associated binding (Gab) scaffolding/adapter proteins are a family of three members including mammalian Gab1, Gab2, and Gab3 that are highly conserved. Since the discovery of these proteins, there has been an extensive amount of work done to better understand Gab functional roles in multiple signaling pathways, typically acting as a downstream effectors of receptor-tyrosine kinase (RTK)-triggered signal transduction. In addition to their participation in hematopoiesis, Gabs play important roles in regulation of immune response and in also in cancer cell signaling. Gabs may play complex roles and thus a complete understanding of their interactions and how they modulate hematopoietic and immune cell biology remains to be determined. This review will cover the most recent findings including the involvement of Gabs in disease development and signaling which will be important for design of future therapeutic interventions.
PMCID: PMC3232456  PMID: 22163099
Adapter protein; cytokine signaling; Grb2-associated binding protein; Gab; receptor tyrosine kinase; cancer signaling
9.  Molecular Targets for the Treatment of Juvenile Myelomonocytic Leukemia 
Advances in Hematology  2011;2012:308252.
Significant advances in our understanding of the genetic defects and the pathogenesis of juvenile myelomonocytic leukemia (JMML) have been achieved in the last several years. The information gathered tremendously helps us in designing molecular targeted therapies for this otherwise fatal disease. Various approaches are being investigated to target defective pathways/molecules in this disease. However, effective therapy is still lacking. Development of specific target-based drugs for JMML remains a big challenge and represents a promising direction in this field.
doi:10.1155/2012/308252
PMCID: PMC3226315  PMID: 22162691
10.  Psoriasis-associated variant Act1 D10N with impaired regulation by Hsp90 
Nature immunology  2012;14(1):72-81.
Act1 is an essential adaptor molecule in IL-17-mediated signaling and is recruited to the IL-17 receptor upon IL-17 stimulation. Here, we report that Act1 is a client protein of the molecular chaperone, Hsp90. The Act1 variant (D10N) linked to psoriasis susceptibility is defective in its interaction with Hsp90, resulting in a global loss of Act1 function. Act1-/- mice modeled the mechanistic link between Act1 loss of function and psoriasis susceptibility. Although Act1 is necessary for IL-17-mediated inflammation, Act1-/- mice exhibited a hyper TH17 response and developed spontaneous IL-22-dependent skin inflammation. In the absence of IL-17-signaling, IL-22 is the main contributor to skin inflammation, providing a molecular mechanism for the association of Act1 (D10N) with psoriasis susceptibility.
doi:10.1038/ni.2479
PMCID: PMC3522792  PMID: 23202271
11.  Grb2-associated binding (Gab) proteins in hematopoietic and immune cell biology 
Grb2-associated binding (Gab) scaffolding/adapter proteins are a family of three members including mammalian Gab1, Gab2, and Gab3 that are highly conserved. Since the discovery of these proteins, there has been an extensive amount of work done to better understand Gab functional roles in multiple signaling pathways, typically acting as a downstream effectors of receptor-tyrosine kinase (RTK)-triggered signal transduction. In addition to their participation in hematopoiesis, Gabs play important roles in regulation of immune response and in also in cancer cell signaling. Gabs may play complex roles and thus a complete understanding of their interactions and how they modulate hematopoietic and immune cell biology remains to be determined. This review will cover the most recent findings including the involvement of Gabs in disease development and signaling which will be important for design of future therapeutic interventions.
PMCID: PMC3232456  PMID: 22163099
Adapter protein; cytokine signaling; Grb2-associated binding protein; Gab; receptor tyrosine kinase; cancer signaling
12.  Direct and Differential Suppression of Myeloid-derived Suppressor Cell Subsets by Sunitinib is Compartmentally Constrained 
Cancer research  2010;70(9):3526-3536.
The anti-angiogenic drug sunitinib is a receptor tyrosine-kinase inhibitor with significant, yet not curative, therapeutic impacts in metastatic renal cell carcinoma (mRCC). Sunitinib is also an immunomodulator, potently reversing myeloid-derived suppressor cell (MDSC) accumulation and T-cell inhibition in the blood even of non-responder RCC patients. We observed that sunitinib similarly prevented MDSC accumulation and restored normal T-cell function to spleens of tumor-bearing mice, independent of sunitinib's capacity to inhibit tumor progression (RENCA>CT26>4T1). Both monocytic and neutrophilic splenic MDSC were highly repressible by sunitinib. In contrast, MDSC within the microenvironment of 4T1 tumors or human RCC tumors proved highly resistant to sunitinib, and ambient T-cell function remained suppressed. Proteomic analyses comparing tumor to peripheral compartments demonstrated that GM-CSF predicted sunitinib resistance, and recombinant GM-CSF conferred sunitinib resistance to MDSC in vivo and in vitro. MDSC conditioning with GM-CSF uniquely inhibited STAT3 and promoted STAT5 activation, and STAT5ab(null/null) MDSC were rendered sensitive to sunitinib in the presence of GM-CSF in vitro. We conclude that compartment-dependent GM-CSF exposure in resistant tumors may account for sunitinib's regionalized impact upon host MDSC modulation, and hypothesize that ancillary strategies to decrease such regionalization will enhance sunitinib's potency as an immunomodulator and a cancer therapy.
doi:10.1158/0008-5472.CAN-09-3278
PMCID: PMC3426924  PMID: 20406969
RCC; myeloid derived suppressor cells (MDSC); sunitinib; STAT; GM-CSF
13.  Mutation of STAT1/3 binding sites in gp130FXXQ knock-in mice does not alter hematopoietic stem cell repopulation or self-renewal potential 
American journal of stem cells  2012;1(2):146-153.
Interleukin (IL)-6 family cytokine signaling through gp130 and signal transducer and activator of transcription (STAT) activation is believed important for early hematopoiesis. To determine whether gp130/STAT1/3 physical interaction is required, we compared hematopoietic repopulating activities of embryonic day (E)14.5 fetal liver cells from gp130FXXQ/FXXQ knock-in mice, which have four mutated STAT1/3 binding sites. In hematopoietic cells, failure to tyrosine phosphorylate STAT3 by gp130 did not cause any significant effects on myeloid progenitor colony forming units (CFU) in vitro and or on competitive multilineage hematopoietic reconstitution. Serial transplantation of fetal liver (FL) cells was unaffected throughout primary, secondary, and tertiary transplants indicating normal self-renewal capacity. Even gp130FXXQ/FXXQ on the background of STAT5 deficiency, with known hematopoietic stem cell (HSC) repopulating dysfunction, did not further impair HSCs beyond that of STAT5 alone. Overall, the defective gp130-mediated STAT1/3 signaling is surprisingly dispensable for HSC function. However, since these mice lack both STAT1/3 binding sites there are several possible explanations for this result and these are discussed.
PMCID: PMC3385990  PMID: 22754757
Signal transducer and activator of transcription; hematopoietic stem cell; cytokine signaling; fetal liver
14.  Mutation of STAT1/3 binding sites in gp130FXXQ knock-inmice does not alter hematopoietic stem cell repopulation or self-renewal potential 
American Journal of Stem Cells  2012;1(2):146-153.
Interleukin (IL)-6 family cytokine signaling through gp130 and signal transducer and activator of transcription (STAT) activation is believed important for early hematopoiesis. To determine whether gp130/STAT1/3 physical interaction is required, we compared hematopoietic repopulating activities of embryonic day (E)14.5 fetal liver cells from gp130FXXQ/FXXQ knock-in mice, which have four mutated STAT1/3 binding sites. In hematopoietic cells, failure to tyrosine phosphorylate STAT3 by gp130 did not cause any significant effects on myeloid progenitor colony forming units (CFU) in vitro and or on competitive multilineage hematopoietic reconstitution. Serial transplantation of fetal liver (FL) cells was unaffected throughout primary, secondary, and tertiary transplants indicating normal self-renewal capacity. Even gp130FXXQ/FXXQ on the background of STAT5 deficiency, with known hematopoietic stem cell (HSC) repopulating dysfunction, did not further impair HSCs beyond that of STAT5 alone. Overall, the defective gp130-mediated STAT1/3 signaling is surprisingly dispensable for HSC function. However, since these mice lack both STAT1/3 binding sites there are several possible explanations for this result and these are discussed.
PMCID: PMC3385990  PMID: 22754757
Signal transducer and activator of transcription; hematopoietic stem cell; cytokine signaling; fetal liver
15.  Normal Hematopoietic Stem Cell Function in Mice with Enforced Expression of the Hippo Signaling Effector YAP1 
PLoS ONE  2012;7(2):e32013.
The Hippo pathway has recently been implicated in the regulation of organ size and stem cells in multiple tissues. The transcriptional cofactor yes-associated protein 1 (Yap1) is the most downstream effector of Hippo signaling and is functionally repressed by the upstream components of the pathway. Overexpression of YAP1 stimulates proliferation of stem and progenitor cells in many tissues, consistent with inhibition of Hippo signaling. To study the role of Hippo signaling in hematopoietic stem cells (HSCs), we created a transgenic model with inducible YAP1 expression exclusively within the hematopoietic system. Following 3 months induction, examination of blood and bone marrow in the induced mice revealed no changes in the distribution of the hematopoietic lineages compared to control mice. Moreover, the progenitor cell compartment was unaltered as determined by colony forming assays and immunophenotyping. To address whether YAP1 affects the quantity and function of HSCs we performed competitive transplantation experiments. We show that ectopic YAP1 expression does not influence HSC function neither during steady state nor in situations of hematopoietic stress. This is in sharp contrast to effects seen on stem- and progenitor cells in other organs and suggests highly tissue specific functions of the Hippo pathway in regulation of stem cells.
doi:10.1371/journal.pone.0032013
PMCID: PMC3283704  PMID: 22363786
18.  Ubiquitous Expression of MAKORIN-2 in Normal and Malignant Hematopoietic Cells and Its Growth Promoting Activity 
PLoS ONE  2014;9(3):e92706.
Makorin-2 (MKRN2) is a highly conserved protein and yet its functions are largely unknown. We investigated the expression levels of MKRN2 and RAF1 in normal and malignant hematopoietic cells, and leukemia cell lines. We also attempted to delineate the role of MKRN2 in umbilical cord blood CD34+ stem/progenitor cells and K562 cell line by over-expression and inhibition of MKRN2 through lentivirus transduction and shRNA nucleofection, respectively. Our results provided the first evidence on the ubiquitous expression of MKRN2 in normal hematopoietic cells, embryonic stem cell lines, primary leukemia and leukemic cell lines of myeloid, lymphoid, erythroid and megakaryocytic lineages. The expression levels of MKRN2 were generally higher in primary leukemia samples compared with those in age-matched normal BM cells. In all leukemia subtypes, there was no significant correlation between expression levels of MKRN2 and RAF1. sh-MKRN2-silenced CD34+ cells had a significantly lower proliferation capacity and decreased levels of the early stem/progenitor subpopulation (CFU-GEMM) compared with control cultures. Over-expression of MKRN2 in K562 cells increased cell proliferation. Our results indicated possible roles of MKRN2 in normal and malignant hematopoiesis.
doi:10.1371/journal.pone.0092706
PMCID: PMC3968021  PMID: 24675897
19.  STAT5 requires the N-domain for suppression of miR15/16, induction of bcl-2, and survival signaling in myeloproliferative disease 
Blood  2009;115(7):1416-1424.
Phosphorylated signal transducer and activator of transcription 5 (STAT5) is a biomarker and potential molecular target for hematologic malignancies. We have shown previously that lethal myeloproliferative disease (MPD) in mice mediated by persistently activated STAT5 (STAT5aS711F) requires the N-domain, but the mechanism was not defined. We now demonstrate by retrovirally complementing STAT5abnull/null primary mast cells that relative to wild-type STAT5a, STAT5a lacking the N-domain (STAT5aΔN) ineffectively protected against cytokine withdrawal-induced cell death. Both STAT5a and STAT5aΔN bound to a site in the bcl-2 gene and both bound near the microRNA 15b/16 cluster. However, only STAT5a could effectively induce bcl-2 mRNA and reciprocally suppress miR15b/16 leading to maintained bcl-2 protein levels. After retroviral complementation of STAT5abnull/null fetal liver cells and transplantation, persistently active STAT5aS711F lacking the N-domain (STAT5aΔNS711F) was insufficient to protect c-Kit+Lin−Sca-1+ (KLS) cells from apoptosis and unable to induce bcl-2 expression, whereas STAT5aS711F caused robust KLS cell expansion, induction of bcl-2, and lethal MPD. Severe attenuation of MPD by STAT5aΔNS711F was reversed by H2k/bcl-2 transgenic expression. Overall, these studies define N-domain–dependent survival signaling as an Achilles heel of persistent STAT5 activation and highlight the potential therapeutic importance of targeting STAT5 N-domain–mediated regulation of bcl-2 family members.
doi:10.1182/blood-2009-07-234963
PMCID: PMC2826763  PMID: 20008792
20.  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 (MPD) model. To target Gab2-independent AKT/mTOR activation, wild-type mice were treated separately with rapamycin. In either case, mice lacking Gab2 or treated with rapamycin displayed attenuated myeloid hyperplasia and modestly improved survival, but the effects were not cytotoxic and were reversible. To improve upon this approach, in vitro targeting of STAT5-mediated AKT/mTOR using rapamycin was combined 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, e.g. bcl-2/bcl-XL and AKT/mTOR may be an effective therapeutic approach for human myeloproliferative neoplasms.
doi:10.1038/leu.2010.131
PMCID: PMC2921023  PMID: 20535152
cytokine signaling; JAK/STAT; hematopoiesis; leukemogenesis; flow cytometry; hematopoietic stem cell
21.  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 1 
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 + anti-IL-3 antibody) greatly facilitated the differentiation of granulocyte/monocyte progenitor (GMPs) into basophil lineage-restricted progenitors (BaPs) but not into eosinophil lineage-restricted progenitors (EoPs) or mast cells in the bone marrow. We also found that the IL-3 complex treatment resulted in about 4-fold increase in the number of basophil/mast cell progenitors (BMCPs) in the spleen. IL-3-driven basophil expansion depended on signal transducer and activator of transcription 5 (STAT5) signaling. We showed that GMPs but not common myeloid progenitors (CMPs) expressed low levels of IL-3 receptor. IL-3 receptor expression was dramatically upregulated in BaPs but not EoPs. About 38% of BMCPs expressed the IL-3Rα chain. The upregulated 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.
doi:10.4049/jimmunol.0802870
PMCID: PMC2756103  PMID: 19234178
22.  Gab2 Promotes Hematopoietic Stem Cell Maintenance and Self-Renewal Synergistically with STAT5 
PLoS ONE  2010;5(2):e9152.
Background
Grb2-associated binding (Gab) adapter proteins play major roles in coordinating signaling downstream of hematopoietic cytokine receptors. In hematopoietic cells, Gab2 can modulate phosphatidylinositol–3 kinase and mitogen associated protein kinase activities and regulate the long-term multilineage competitive repopulating activity of hematopoietic stem cells (HSCs). Gab2 may also act in a linear pathway upstream or downstream of signal transducer and activator of transcription-5 (STAT5), a major positive regulator of HSC function. Therefore, we aimed to determine whether Gab2 and STAT5 function in hematopoiesis in a redundant or non-redundant manner.
Methodology/Principal Findings
To do this we generated Gab2 mutant mice with heterozygous and homozygous deletions of STAT5. In heterozygous STAT5 mutant mice, deficiencies in HSC/multipotent progenitors were reflected by decreased long-term repopulating activity. This reduction in repopulation function was mirrored in the reduced growth response to early-acting cytokines from sorted double mutant c-Kit+Lin−Sca-1+ (KLS) cells. Importantly, in non-ablated newborn mice, the host steady-state engraftment ability was impaired by loss of Gab2 in heterozygous STAT5 mutant background. Fetal liver cells isolated from homozygous STAT5 mutant mice lacking Gab2 showed significant reduction in HSC number (KLS CD150+CD48−), reduced HSC survival, and dramatic loss of self-renewal potential as measured by serial transplantation.
Conclusions/Significance
These data demonstrate new functions for Gab2 in hematopoiesis in a manner that is non-redundant with STAT5. Furthermore, important synergy between STAT5 and Gab2 was observed in HSC self-renewal, which might be exploited to optimize stem cell-based therapeutics.
doi:10.1371/journal.pone.0009152
PMCID: PMC2818849  PMID: 20161778
23.  An ENU-induced Recessive Mutation in Mpl Leads to Thrombocytopenia with Overdominance 
Experimental hematology  2008;37(2):276-284.
Objective
The aim of this study was to identify and characterize the causative mutation in the thrombocytopenic mouse strain HLB219 that was generated at the Jackson Laboratory as part of a large scale ENU-mutagenesis screen.
Methods
The HLB219 mutation was identified by interval mapping of F2 mice generated from intercross breeding of HLB219 to both BALB/cByJ (BALB) and 129/SvImJ (129/Sv). Mpl was identified as a candidate gene and sequenced. The mutation was characterized in vivo in mouse hematopoietic stem/progenitor cell assays and in cell culture by expression in Ba/F3 cells.
Results
A novel mutation in the thrombopoietin (TPO) receptor Mpl in HLB219 mice caused a Cys→Arg substitution at codon 40 in the extracellular region of the receptor. Mice homozygous for the Mplhlb219 mutation had an 80% decrease in the number of platelets in comparison to the wild type C57BL/6J strain, low numbers of bone marrow megakaryocytes, high TPO levels, and decreased competitive repopulating ability, consistent with a loss-of-function mutation in the receptor. Mice heterozygous for Mplhlb219 however, showed an overdominance effect with a significant increase in platelet number. Functional analysis in vitro demonstrated that Ba/F3 cells expressing the mutant MPLhlb219 protein failed to activate ERK and STAT5, but proliferated in the absence of TPO and required constitutive phosphorylation of AKT for cytokine-independent growth.
Conclusion
Thrombocytopenia in HLB219 mice is caused by a recessive mutation in Mpl that abrogates MAPK-ERK and JAK-STAT signaling.
doi:10.1016/j.exphem.2008.10.005
PMCID: PMC2656350  PMID: 19059699
Receptors; Thrombopoietin; Thrombopoiesis; Models; Genetic
24.  Hematopoiesis in mice is extremely resilient to wide variation in TIMP/MMP balance 
Blood cells, molecules & diseases  2008;41(2):179-187.
Tissue inhibitor of matrix metalloproteinases (TIMPs) are natural inhibitors of matrix metalloproteinases (MMPs) and are associated with normal and pathologic extracellular matrix turnover. Because the microenvironment is critical for normal hematopoietic stem/progenitor cell function, we aimed to determine whether alterations in the TIMP/MMP balance impact upon normal hematopoiesis in mice. We have used both overexpression and knockout mouse models to determine whether early hematopoiesis is susceptible to potentially pathologic changes in TIMP/MMP level. These studies used TIMP-1−/− mice and retroviral vectors co-expressing human TIMP-1 or TIMP-2 linked with the green fluorescent protein (GFP) transduced into bone marrow (BM) cells and transplanted into lethally irradiated recipient mice. Loss of TIMP-1 in knockout mice or retroviral overexpression of TIMP-1 or TIMP-2 did not alter hematopoietic stem/progenitor function during steady-state hematopoiesis. Surprisingly, even when applying hematopoietic stress through mobilization, chemotaxis, or myelosuppression, murine hematopoiesis was not adversely affected by TIMP-1 or TIMP-2 level. We conclude that TIMP/MMP balance alone does not exert significant influence on blood cell development and homeostasis. An important corollary of these studies is that specific modulation using MMP inhibitors for cancer or immunologic therapy is unlikely to have adverse hematopoietic side effects.
doi:10.1016/j.bcmd.2008.03.005
PMCID: PMC2600540  PMID: 18487063
25.  STAT5 requires the N-domain to maintain hematopoietic stem cell repopulating function and appropriate lymphoid-myeloid lineage output 
Experimental hematology  2007;35(11):1684-1694.
Objective
STAT5 is a critical regulator of hematopoietic development and its impaired activation is associated with hematopoietic and immune cell defects. However, much of this information has been learned from knockout mice that still retain the potential for expression of STAT5 proteins that are N-terminally truncated due to alternative internal translation initiation codons. The goal of these studies was to use transplantation based assays to analyze the degree of STAT5ΔN activity in HSC and throughout lymphomyeloid development.
Methods
We have directly compared E14.5 fetal liver cells from mice with potential to express STAT5abΔN (STAT5abΔN/ΔN) with mice completely lacking STAT5a and STAT5b (STAT5abnull/null). We have also utilized retroviral complementation of STAT5abnull/null fetal liver hematopoietic stem cells (HSC) to enforce expression of full-length STAT5a or STAT5a lacking the first 136 amino acids (STAT5aΔ N).
Results
We report that STAT5 is required for HSC, lymphocyte, and erythrocyte development. We demonstrate that restored expression of STAT5a in STAT5abnull/null HSC provides a strong selective advantage, correcting T/B lymphocyte and erythrocyte development. Interestingly, Gr-1+ blood cells were inversely correlated with B-lymphocytes and both were normalized by STAT5a expression. In contrast, transduction of STAT5aΔN only provided partial B-lymphocyte development.
Conclusions
These studies define the role of STAT5 in maintaining normal lymphoid vs. myeloid balance during hematopoiesis and highlight a major role for the N/domain in HSC function. The platform of retroviral complementation described here will be particularly useful for future studies to sub-define the N-domain regions that are critical for hematopoiesis.
doi:10.1016/j.exphem.2007.08.026
PMCID: PMC2134320  PMID: 17976521
cytokine signaling; JAK/STAT; hematopoiesis; hematopoietic stem cell; retroviral transduction

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