|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Blood, 1 May 2007, Vol. 109, No. 9, pp. 3906-3914. Prepublished online as a Blood First Edition Paper on January 11, 2007; DOI 10.1182/blood-2006-07-036335.
NEOPLASIA Myeloproliferative disease induced by TEL-PDGFRB displays dynamic range sensitivity to Stat5 gene dosage1 Department of Internal Medicine, Division of Oncology, Washington University, Siteman Cancer Center, St Louis, MO; 2 Department of Pathology, Washington University School of Medicine, St Louis, MO; 3 Laboratory of Genetics and Physiology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD
Expression of the constitutively activated TEL/PDGFßR fusion protein is associated with the t(5;12)(q33;p13) chromosomal translocation found in a subset of patients with chronic myelomonocytic leukemia. TEL/PDGFßR activates multiple signal transduction pathways in cell-culture systems, and expression of the TEL-PDGFRB fusion gene induces myeloproliferative disease (MPD) in mice. We used gene-targeted mice to characterize the contribution of signal transducer and activator of transcription (Stat) and Src family genes to TEL-PDGFRB–mediated transformation in methylcellulose colony and murine bone marrow transduction/transplantation assays. Fetal liver hematopoietic stem and progenitor cells harboring targeted deletion of both Stat5a and Stat5b (Stat5abnull/null) genes were refractory to transformation by TEL-PDGFRB in methylcellulose colony assays. Notably, these cell populations were maintained in Stat5abnull/null fetal livers and succumbed to transformation by c-Myc. Surprisingly, targeted disruption of either Stat5a or Stat5b alone also impaired TEL-PDGFRB–mediated transformation. Survival of TPiGFP  Stat5a–/– and TPiGFPStat5a+/– mice was significantly prolonged, demonstrating significant sensitivity of TEL-PDGFRB–induced MPD to the dosage of Stat5a. TEL-PDGFRB–mediated MPD was incompletely penetrant in TPiGFPStat5b–/– mice. In contrast, Src family kinases Lyn, Hck, and Fgr and the Stat family member Stat1 were dispensable for TEL-PDGFRB disease. Together, these data demonstrate that Stat5a and Stat5b are dose-limiting mediators of TEL-PDGFRB–induced myeloproliferation.
Chronic myelomonocytic leukemia (CMML) is characterized by dysplastic monocytosis, hypercellular bone marrow, splenomegaly, variable bone marrow fibrosis, and progression to acute myelogenous leukemia (AML). The t(5;12) (q33;p13) chromosomal translocation occurs in a subset of CMML patients. The TEL/PDGFßR fusion protein retains the pointed (PNT) domain found in TEL and the split tyrosine kinase (intracellular) portion of platelet-derived growth factor receptor ß (PDGFßR).1 TEL/PDGFßR self-associates in the cytoplasm as oligomers via the PNT domain and becomes constitutively activated.2–4 This constitutive tyrosine kinase activity is required for recruitment of SH2-domain signaling intermediates including phosphatidylinositol 3-kinase (PI3K), phospholipase-C  (PLC), SHP2, as well as signal transducers and activators of transcription 1 and 5 (STAT1 and STAT5, respectively).2,5–7 A murine bone marrow transduction/transplantation model of TEL-PDGFRB is marked by a rapidly fatal myeloproliferative disorder (MPD) that recapitulates aspects of human CMML including leukocytosis, splenomegaly, and extramedullary hematopoiesis.8 A TEL/PDGFßR mutant with tyrosine to phenylalanine mutations in the juxtamembrane SH2-domain–binding tyrosine residues (TEL/PDGFßR-F2) retains tyrosine kinase activity but does not induce myeloproliferation in mice.8 TEL/PDGFßR-F2 fails to bind and activate Stat56 and, in the context of native PDGFßR, the F2 tyrosines mediate binding and activation of both Src and Stat proteins.9–11 Therefore Src and Stat are candidate signaling molecules, but their role in mediating disease induced by TEL-PDGFRB is unclear. We used gene-targeted mice to characterize the contribution of signal transducer and activator of transcription (Stat) and Src family genes to TEL-PDGFRB–mediated transformation in methylcellulose colony and murine bone marrow transduction/transplantation assays
Plasmids TEL-PDGFRB ires green fluorescent protein (TPiGFP) was constructed in a murine stem cell virus (MSCV)2.2-ires-GFP vector backbone as previously described.8 TEL-PDGFRB-PGK-Neo (TPNeo) was generated by subcloning the TEL-PDGFRB cDNA into MSCV-Neo (Clontech, Mountain View, CA). MSCV-Myc was generated as described.12 TEL-PDGFRB ires c-Src (TPiSrc) was generated by inserting the c-Src cDNA (provided by Sara Courtneidge, Burnham Institute for Medical Research, La Jolla, CA) into MSCV2.2-ires-GFP, replacing GFP with c-Src. The TEL-PDGFRB cDNA was then excised from TPiGFP and placed into MSCV2.2-ires-cSrc. Stat51*6 ires GFP was created by placing the Stat51*6 cDNA (Toshio Kitamura, University of Tokyo, Japan) into MSCV2.2-ires-GFP. TEL-PDGFRB ires Stat5a (TPiStat5a) was created by placing the Stat5a cDNA into MSCV2.2-ires-GFP backbone to create MSCV2.2-ires-mStat5a, into which the TEL-PDGFRB cDNA was subcloned at the EcoRI site. TEL-PDGFRB ires Stat5b (TPiStat5b) was generated by inserting the mStat5b cDNA (courtesy of Lothar Hennighausen, NIDDK, Warren Leonard NHLBI, National Institutes of Health, Bethesda, MD) into MSCV2.2-ires-GFP. The TEL-PDGFRB cDNA was then subcloned into the EcoRI site of MSCV2.2-ires-mStat5b. Retrovirus production Retroviral supernatants were generated by transient transfection of Ecopac (Cell Genesys, Foster City, CA) with MSCV-based retroviral construct using Superfect transfection reagent (Qiagen, Chatsworth, CA) per the manufacturer's instructions. Retroviral supernatants were harvested 48 hours after transfection. To evaluate viral titer of constructs containing eGFP, Ba/F3 cells were transduced with retroviral supernatants and GFP expression was determined by flow cytometric detection of GFP positivity. To assess MSCV-Neo, TPiNeo, TPiStat5a, and TPiStat5b viral titer, 3T3 cells were transduced with retroviral supernatants. 3T3 DNA was isolated and quantitative polymerase chain reaction (PCR) amplication was used to detect psi retroviral sequences compared with control genomic sequences. Mouse strains Lyn, Hck, Fgr triply deficient mice13 were provided by Clifford Lowell (University of California, San Francisco). Stat1–/– mice14 were provided by Robert Schreiber (Washington University, St Louis). Lothar Hennighausen (NIDDK, National Institutes of Health) contributed Stat5a–/–15 and Stat5ab+/null mice.16 Stat5ab+/null mice were crossed with NIH Black Swiss female mice (Taconic Farms, Hudson, NY). The resulting outbred Stat5ab+/null male and female mice were used to generate Stat5abnull/null, Stat5abnull/null, and Stat5abnull/null fetuses. Stat5b–/– mice17 were acquired from Helen Davey (AgResearch, Hamilton, New Zealand). Assisted speed congenics18 were used to backcross the targeted Stat5a (N3 generation, approximately 94% balb/c) or Stat5b (N4 generation, approximately 99% balb/c) allele in singly deficient mice to balb/c (Taconic Farms). Methylcellulose colony formation Bone marrow transduction of whole bone marrow (Stat5a and Stat5b singly deficient mouse strains backcrossed to balb/c) was performed as previously described.8 Briefly, whole bone marrow was harvested from 150 mg/kg 5-fluorouracil–treated mice, and the red blood cells were removed by brief incubation in hypotonic lysis buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, pH 7.4). Unfractionated cells were prestimulated in media containing pen/strep, fetal bovine serum, SCF, IL3, FLT3, and Tpo for 48 hours. Cells were transduced with retroviral supernatants of equivalent titer by 2 rounds of centrifugation at 2500g for 90 minutes in the presence of 8 µg/mL polybrene (American Bioanalytical, Natick, MA). Twenty-four hours following the second transduction, cells were washed thrice with cold PBS and resuspended in methylcellulose with cytokines (2 x 104 cells/mL in M3434 methylcellulose; Stem Cell Technologies, Vancouver, BC) or without cytokines (105 cells/mL in M3234; Stem Cell Technologies) in the presence or absence of 1 mg/mL G418 (Gibco/Invitrogen, Grand Island, NY; from a 20-mg/mL stock solution in 0.1 M Hepes [Cambrex, Walkersville, MD]) and plated in triplicate. Colonies containing at least 30 cells were counted 10 days after plating. Transduction efficiency for each genotype and experimental condition was determined by plating cells in the presence of cytokines and dividing the average number of G418-resistant colonies per plate by the average number of colonies per plate in a duplicate set of plates not containing G418. Murine 14.5-dpc fetuses were harvested from Stat5ab+/null x Stat5ab+/null timed pregnancies and were washed in sterile PBS (Gibco/Invitrogen). Fetal livers were placed into RPMI containing penn/strep and 10% fetal bovine serum and brought to a single-cell suspension by passing through a 27-gauge needle. Red blood cells were lysed as in the paragraph above, and the remaining cells from each fetal liver were resuspended into fetal liver transplant media (RPMI containing pen/strep, 10% fetal bovine serum, 10 ng/mL IL6, 100 ng/mL SCF, 6 ng/mL IL3, 50 ng/mL FLT3, and 10 ng/mL Tpo) and placed at 37°C in 5% CO2. Fetal liver cell DNA was isolated using Puregene DNA purification kit (Gentra Systems, Minneapolis, MN) and genotyped as described.16 Cells of each genotype were pooled and replated in fetal liver transplant media and incubated at 37°C in 5% CO2. Beginning 36 hours after harvest, cells were transduced every 6 hours with retroviral supernatants of equivalent titer by centrifugation at 2500g for 10 minutes in the presence of 8 µg/mL polybrene (American Bioanalytical) for a total of 4 transductions. Twenty-four hours following the final transduction, cells were washed and plated in methylcellulose as described in the preceding paragraph. Colonies containing at least 30 cells were counted 10 days after plating. Transduction efficiency was determined as described above for TPNeo and MSCV-Neo experiments, while detection of GFP-positive cells by flow cytometry immediately prior to plating cells in methylcellulose was used to evaluate MSCV-Myc transduction efficiency. Retroviral transduction and transplantation Transduction and transplantation were performed as previously described.8 Syngeneic recipient mice were lethally irradiated and were injected with 106 bone marrow cells in 750 µL Hanks buffered saline solution via lateral tail-vein injection. Doses of irradiation were as follows: Lyn–/–Hck–/–Fgr–/–: 450 cGy C57/Black6 (Taconic Farms); Stat1–/–: 1000 cGy C57BL/6 x 129S6/SvEv F1 (B6129; Taconic Farms); outbred Stat5a–/–: 1000 cGy B6129 (Taconic Farms); balb/c backcrossed Stat5a–/–: 900 cGy wild-type littermate control; outbred Stat5b–/–: 1000cGy B6129 (Taconic Farms); balb/c backcrossed Stat5b–/–: 900 cGy wild-type littermate control. Mouse analysis Peripheral blood was obtained from the saphenous vein of moribund mice and blood counts were analyzed (Hemavet; CDC Technologies, Oxford, CT). Moribund mice were killed and spleen weight was recorded. Heart, lungs, tibia, spleen, kidney, and liver were fixed using 10% neutral buffered formalin (Sigma-Aldrich, St Louis, MO). Slides of peripheral blood and formalin-fixed tissue were hematoxylin and eosin (H&E) stained and imaged using an Olympus BX40 F4 microscope with an oil-immersion 50x/0.90 or 100x/1.30 objective lens (Olympus Optical, Tokyo, Japan) using an Olympus DP70 digital camera using DPController software (Olympus Optical). Statistical analyses were generated with StatView (SAS Institute, Cary, NC). Kaplan-Meier plots were generated using Excel (Microsoft, Redmond, WA). Flow cytometry Bone marrow cells and splenocytes were brought to a single-cell suspension in RPMI-1640 (Cambrex) and the red blood cells were removed by brief incubation in hypotonic lysis buffer. Cells were washed once with flow buffer (PBS, 0.5% BSA, 0.1 mM EDTA), and 106 cells were resuspended in 100 µL flow buffer and incubated with 1 µL each of the antibodies recognizing murine Gr-1 and Mac-1 conjugated to PE and PECy7, respectively (eBiosciences, San Diego, CA) for 1 hour on ice. Cells were then washed twice with flow buffer and resuspended in 250 µL flow buffer. Data were collected using either MoFlo (Dako, Carpinteria, CA) or Cytomics FC 500 (Beckman Coulter, Fullerton, CA). Figures were prepared using FloJo software (Tree Star, San Carlos, CA). Progenitor analysis methods As described under "Methylcellulose colony formation." 14.5-dpc fetal liver cells were harvested, brought to a single-cell suspension in PBS (Gibco/Invitrogen), and stored at 4°C during DNA isolation (REDExtract; Sigma-Aldrich) and genotyping (as described16). Unfractionated fetal liver cells of each genotype were pooled into Stat5ab–/– (n = 8 fetal livers), Stat5ab+/– (n = 6), and Stat5ab+/+ (n = 7) cohorts, filtered through a 50-µM filter (Partec, Münster, Germany), and stained for flow cytometric analysis of progenitor populations as described.19 Antibody staining for lineage markers was performed using FITC-conjugated CD3e (145-2C11), CD4 (RM4-5), CD8a (53-6.7), CD19 (1D3), CD45R (RA3-6B2), and GR1 (RB6-8C5) acquired from BD PharMingen (San Diego, CA) and TER119 (TER-119) and CD127 (A7R34) from eBiosciences. Cells were also stained using biotin-CD34 (RAM34), PE-streptavidin, APC-Sca-1 (D7), PE-Cy7-CD16/32 (93), and APC-Alexa750-CD117 (2B8) antibodies obtained from eBiosciences. Data were collected from 5 x 106 cells per genotype using MoFlo flow cytometer (Dako), and figures were prepared using FloJo software (Tree Star).
Stat5 is required in primary murine hematopoietic cells for TEL-PDGFRB–mediated growth Mice harboring targeted deletions of both Stat5a and Stat5b genes (Stat5abnull/null) rarely survive past weaning.16,20 Therefore, we evaluated TEL-PDGFRB transformation in methylcellulose colony formation assays using fetal liver hematopoietic stem cells (HSCs) and progenitor cells. We transduced unfractionated Stat5ab+/+, Stat5ab+/null, and Stat5abnull/null16 fetal liver cells with retroviral supernatants encoding either TEL-PDGFRB with a neomycin resistance cassette (TPNeo) or Neo alone (MSCV-Neo) and plated them in methylcellulose in the absence of cytokines. Transduction efficiency was equivalent among Stat5ab+/+, Stat5ab+/null, and Stat5abnull/null cells. TEL-PDGFRB induced G418-resistant colony formation in Stat5ab+/+ and Stat5ab+/null cells. In contrast, Stat5abnull/null cells were completely resistant to TEL-PDGFRB–mediated transformation (Figure 1A). MSCV-Neo did not cause cytokine independent colony formation in Stat5ab+/+, Stat5ab+/null, or Stat5abnull/null cells (data not shown). As a positive control for the transduction of hematopoietic stem and progenitor cells from these mice, we transduced Stat5ab+/+ and Stat5abnull/null cells with c-Myc. c-Myc readily transformed both Stat5ab+/+ and Stat5abnull/null cells to generate cytokine-independent colonies (Figure 1A), confirming efficient retroviral transduction of Stat5-deficient cells.
We were impressed by the dramatic reduction of TEL-PDGFRB–induced cytokine-independent colonies in the absence of Stat5 and were concerned that our data might be explained by reduced hematopoietic stem and progenitor cells in these mutant mice. To further assess the presence and quantity of hematopoietic stem and progenitor cells in Stat5abnull/null fetal livers, we performed multiparameter immunophenotypic analysis using high-speed flow cytometry.19 Less mature, lineage-negative cells were twice as prevalent among Stat5abnull/null fetal liver cells compared with Stat5ab+/+ cells (data not shown). Among lineage-negative cells, there were equivalent numbers of HSC (KLS) cells present in Stat5abnull/null and Stat5ab+/+ fetal livers (Figure 2A). Together, these data indicate that HSCs are twice as prevalent among Stat5abnull/null fetal liver cells, consistent with the findings of other investigators.21 The incidence of common myeloid progenitor (CMP) was the same among Stat5abnull/null, Stat5ab+/null, and Stat5ab+/+ Kit+Lin–Sca– fetal liver cells (Figure 2A). Megakaryocyte-erythrocyte progenitor (MEP) populations were moderately increased in Stat5abnull/null fetal liver cells (Figure 2A). Notably, granulocyte-monocyte progenitors (GMPs) were markedly decreased (Figure 2A). When evaluated as subsets of all fetal liver cells, immunophenotypically defined hematopoietic stem cell–enriched and progenitor populations were moderately increased in Stat5abnull/null fetal liver cells compared with wild-type littermate controls (summarized in Figure 2B).
We plated transduced fetal liver cells in methylcellulose containing SCF, IL3, IL6, and EPO to determine if Stat5abnull/null progenitor cells form colonies in methylcellulose. MSCV-Neo–transduced Stat5abnull/null cells generated half the number of colonies found on Stat5ab+/+ plates (Figure 2C). Expression of TEL-PDGFRB rescued the colony formation defect in Stat5abnull/null fetal liver cells, suggesting that TEL-PDGFßR activated Stat5-independent signaling pathways to restore colony formation capacity to that of Stat5ab+/+ cells (data not shown). Taken together, these data suggest that the inability of TEL-PDGFRB to transform Stat5abnull/null fetal liver cells was not due to the absence of HSC or progenitor-cell populations. Stat5a and Stat5b share 96% similarity on an amino acid level22 and serve largely redundant functions in hematopoiesis.15–16,17,23 Therefore, we hypothesized that TEL-PDGFRB–mediated transformation would not be affected by deficiency of either Stat5a or Stat5b alone (ie, either Stat5a–/–Stat5b+/+ or Stat5a+/+Stat5b–/–). Stat5a–/– and Stat5b–/– mice have no defects in basal hematopoiesis15,17 (and data not shown), however Stat5a and Stat5b possess distinct DNA-binding specificities24–25 and have unique roles in response to high doses of Flt3 ligand.26 We tested the role of individual Stat5 genes in TEL-PDGFRB transformation using methylcellulose colony assays and bone marrow transduction/transplantation assays. We transduced whole bone marrow cells harboring targeted disruption of either Stat5a or Stat5b with either TPNeo or MSCV-Neo retroviral supernatants and plated them in the absence of cytokines. TEL-PDGFRB transformed wild-type cells to cytokine-independent growth (Figure 1B-C). Remarkably, there was a downward trend of TEL-PDGFRB–transformed colonies when either Stat5a+/– singly targeted or Stat5b+/– singly targeted bone marrow cells were used. Finally, deficiency in either Stat5a or Stat5b alone caused a significant reduction in the incidence of TEL-PDGFRB–transformed colonies (Figure 1B-C), indicating that both Stat5a and Stat5b contribute to transformation. Transduction efficiency was equivalent in all cohorts. Taken together, these data demonstrate that Stat5 is critical to TEL-PDGFRB–induced transformation. Stat5a gene dosage mediates the latency of TEL-PDGFRB–induced myeloproliferation
Given the surprising attenuation of TEL-PDGFRB–mediated transformation in the absence of Stat5a, we sought to determine the impact of Stat5a deficiency upon the myeloproliferative disease induced by TEL-PDGFRB in vivo. Unlike Stat5abnull/null mice, Stat5a-deficient mice have abnormal lactogenesis, but no defects in steady-state myelopoiesis,15 and can be used as donors for bone marrow transplant assays. We assessed the relative contribution of the Stat5a gene to TEL-PDGFRB–mediated transformation in bone marrow transduction/transplantation experiments using Stat5a singly deficient (Stat5a–/–), heterozygous (Stat5a+/–), and wild-type (Stat5a+/+) donor mice with retroviral supernatants encoding TEL-PDGFRB with eGFP (TPiGFP). Survival of TPiGFP
Despite significant prolongation of survival, all mice eventually succumbed to disease. We characterized TEL-PDGFRB disease in each Stat5a genotype cohort at time of death. Histopathologic analysis of TPiGFP Stat5a+/+ mice revealed severe extramedullary hematopoiesis with accumulation of large numbers of mature myeloid cells in the liver sinusoids and perivascular regions of the liver and profoundly disrupted splenic architecture (data not shown). Blood vessels and capillaries were filled with granulocytes with multifocal congestion and hemorrhage in the lungs of TPiGFPStat5a+/+ mice. Consistent with the prolonged survival of both TPiGFPStat5a–/– and TPiGFPStat5a+/– mice, only mild extramedullary hematopoiesis was detected in both TPiGFPStat5a–/– and TPiGFPStat5a+/– mice (data not shown), although all cohorts had splenomegaly. There were slight to moderate accumulations of granulocytes in the livers and diffuse expansion of granulocytes in the spleens of both TPiGFPStat5a–/– and TPiGFPStat5a+/– cohorts at time of death due to disease (data not shown). Well-differentiated granulocytes infiltrated the lungs of TPiGFPStat5a–/– and TPiGFPStat5a+/– recipient mice, with mild to moderate multifocal congestion in the lungs. TPiGFPStat5a+/+ mice displayed severe leukocytosis, while leukocytosis in TPiGFPStat5a+/– and TPiGFPStat5a–/– mice was significantly reduced (median WBC, 29 x 109/L [29 000/µL] and 28 x 109/L [28 000/µL], respectively; Figure 3B). Taken together, these data show that TEL-PDGFRB–induced MPD was attenuated in both disease latency and severity in TPiGFPStat5a–/– and TPiGFPStat5a+/– mice, suggesting that TEL-PDGFRB–induced MPD was significantly reduced by absence or haploinsufficiency of Stat5a.
While transduction efficiency was equivalent among Stat5a–/–, Stat5a+/–, and Stat5a+/+ cells in a methylcellulose assay for TEL-PDGFRB–mediated transformation, we sought to confirm that we had efficiently transduced and transplanted Stat5a–/– and Stat5a+/+ cells in our in vivo model. To address the possibility that pretreatment with 5-FU reduces the Stat5a–/– transduction/transplantation target population relative to that in Stat5a+/+ cells, we transduced Stat5a–/– donor cells with retroviral supernatants encoding a constitutively activated Stat5a, Stat51*6.27. As expected, Stat51*6
We were struck by the dramatic reduction in disease severity afforded by loss of a single Stat5a allele in TPiGFP TEL-PDGFRB–mediated MPD is incompletely penetrant in the absence of Stat5b
TPiGFP
Our data demonstrated that Stat5b was not absolutely required for TEL-PDGFRB–mediated MPD. However, we noted that 5 of 13 TPiGFP Stat5b–/– mice appeared to escape rapidly fatal MPD induced by TEL-PDGFRB and were longer lived (Figure 4A). We confirmed equivalent GFP expression in TPiGFPStat5b+/+ and TPiGFPStat5b–/– recipient bone marrow cells and splenocytes (data not shown), showing that transduced and transplanted cells persist in surviving TPiGFPStat5b–/– mice. We evaluated myeloproliferation in the longer-lived subset of TPiGFPStat5b–/– mice. We assessed these mice by gross and microscopic pathology, peripheral blood cell count, and flow cytometric analysis of spleen and bone marrow. However, we did not detect significant myeloproliferation in long-lived (> 100 days after transplantation) TPiGFPStat5b–/– mice by histopathology and detected only normal to mildly elevated peripheral blood cell counts in 2 of 2 mice analyzed (mean WBC, 11 x 109/L [11 000/µL]). However, we noted that leukocytosis at time of death due to disease was somewhat reduced in TPiGFPStat5b–/– mice (median WBC, 80 x 109/L [80 000/µL]) compared with TPiGFPStat5b+/+ littermate controls (median WBC, 213 x 109/L [213 000/µL]). While dispensable for TEL-PDGFRB–induced myeloproliferation, Stat5b was required for complete penetrance of rapidly fatal disease. Stat5b can restore MPD when coexpressed with TEL-PDGFRB in Stat5a-deficient bone marrow cells
We found significant differences in disease latencies between TPiGFP
Stat1 and Src family kinases are dispensable for TEL-PDGFRB–mediated myeloproliferative disease
We evaluated the contribution of the remaining candidate signaling molecules, the Src family kinases, and Stat1 to TEL-PDGFRB–mediated MPD using mice either triply deficient in myelomonocytic Src family kinases Lyn, Hck, and Fgr (Lyn–/–Hck–/–Fgr–/–) or deficient for Stat1 (Stat1–/–). We transduced unfractionated bone marrow mononuclear cells from Lyn–/–Hck–/–Fgr–/– or strain-matched wild-type mice with TPiGFP retroviral supernatants and transplanted them into lethally irradiated syngeneic recipient mice. We analyzed mice from each cohort for survival and myeloproliferative disease burden at time of death by peripheral blood cell count (WBC), as well as gross and microscopic pathology. All recipient mice (100%) that received TEL-PDGFRB–transduced Lyn–/–Hck–/–Fgr–/– bone marrow cells and either GFP (TPiGFP
To determine the role of Stat1 in the development of TEL-PDGFRB–induced MPD, we expressed TEL-PDGFRB in whole bone marrow harvested from Stat1-deficient (TPiGFP Stat1–/–) or wild-type control (TPiGFPStat1+/+) mice. Again, all mice (100%) in both groups developed rapidly fatal MPD characterized by leukocytosis, extramedullary hematopoiesis, and splenomegaly (data not shown). TPiGFPStat1–/– mice developed MPD with a shorter latency than their TPiGFPStat1+/+ counterparts (45 versus 53 days, P = .013), consistent with the role of Stat1 as a tumor suppressor.30 The significantly shortened survival of TPiGFPStat1–/– mice indicates that Stat1 is not required for TEL-PDGFRB disease development. Taken together, these data demonstrate that, although TEL/PDGFßR induces both Stat5-dependent and -independent signaling, Stat5 is critical for TEL-PDGFRB–mediated transformation of primary hematopoietic cells.
Using a genetic approach, we found that the signaling pathways activated by TEL/PDGFßR in cell culture do not contribute equally to transformation. The combined loss of both Stat5a and Stat5b (Stat5abnull/null) eliminated TEL-PDGFRB–mediated transformation as measured by growth of cytokine-independent colony formation in methylcellulose (Figure 1A). Confirming the presence of hematopoietic stem and progenitor populations among Stat5abnull/null fetal liver cells, we found that they formed colonies in the presence of cytokines and that immunophenotypically-defined stem and progenitor cells were moderately increased among Stat5abnull/null over Stat5ab+/+ fetal liver cells. Future comprehensive analysis of the repopulating capacity of Stat5abnull/null hematopoietic stem and progenitor cells will clarify the contribution of Stat5 to normal hematopoiesis. Although Stat5a a | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Top
Abstract
Introduction
) developed moderate or severe MPD (spleen weight median, 770 ± 300 mg; range, 350-1120 mg; n = 7). TPiGFP
). TPiGFP
) or c-Src (