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Transcriptional control and signal transduction, cell cycle

Lineage-specific STAT5 target gene activation in hematopoietic progenitor cells predicts the FLT3+-mediated leukemic phenotype

Abstract

Mutations that activate FMS-like tyrosine kinase 3 (FLT3) are frequent occurrences in acute myeloid leukemia. Two distinct types of mutations have been described: internal duplication of the juxtamembranous domain (ITD) and point mutations of the tyrosine kinase domain (TKD). Although both mutations lead to constitutive FLT3 signaling, only FLT3-ITD strongly activates signal transducer and activator of transcription 5 (STAT5). In a murine transplantation model, FLT3-ITD induces a myeloproliferative neoplasm, whereas FLT3-TKD leads to a lymphoid malignancy with significantly longer latency. Here we report that the presence of STAT5 is critical for the development of a myeloproliferative disease by FLT3-ITD in mice. Deletion of Stat5 in FLT3-ITD-induced leukemogenesis leads not only to a significantly longer survival (82 vs 27 days) of the diseased mice, but also to an immunophenotype switch with expansion of the lymphoid cell compartment. Interestingly, we were able to show differential STAT5 activation in FLT3-ITD+ myeloid and lymphoid murine progenitors. STAT5 target genes such as Oncostatin M were highly expressed in FLT3-ITD+ myeloid but not in FLT3-ITD+ lymphoid progenitor cells. Strikingly, FLT3-TKD expression in combination with Oncostatin M is sufficient to reverse the phenotype to a myeloproliferative disease in FLT3-TKD mice. Thus, lineage-specific STAT5 activation in hematopoietic progenitor cells predicts the FLT3+-mediated leukemic phenotype in mice.

Introduction

Acute myeloid leukemia (AML) is a heterogeneous disease accounting for 10% of all hematologic malignancies.1 In recent years, the FMS-like tyrosine kinase receptor-3 (FLT3) has been identified as a main driver of AML transformation.2 FLT3 is physiologically expressed on early hematopoietic progenitor cells and plays an important role in proliferation and differentiation during hematopoiesis.3 It is a member of the receptor tyrosine kinase class III family that also includes c-kit receptor tyrosine kinase (c-KIT), colony-stimulating factor 1 receptor and platelet-derived growth factor receptor. This class of receptor proteins is characterized by an extracellular ligand-binding domain made up of five immunoglobulin-like domains, a regulatory juxtamembrane domain and an intracellular kinase domain split into two parts. Upon ligand binding the receptor dimerizes, followed by autophosphorylation in the intracellular kinase domains and subsequent phosphorylation of downstream signaling molecules.4

FLT3 is known to be overexpressed on the blasts of 75–100% of AML patients; mutations are detectable in 30% of cases overall, rendering it the most commonly mutated gene in AML.3 Internal tandem duplication (ITD) of the juxtamembraneous domain of FLT3 occurs in 20–27% of AML patients.5, 6 Point mutations in the tyrosine kinase domain (TKD) have been detected in 7% of patients with AML.7 Although both mutation types lead to constitutive activation of the receptor and confer factor-independent proliferation to murine myeloid and lymphoid cells, significant differences have been described: clinically, FLT3-ITD has been linked with elevated peripheral blood (PB) and bone marrow (BM) blast counts, as well as an increased chance of relapse and inferior overall survival.5, 6 No prognostic impact has been demonstrated for FLT3-TKD to date.6 Signal transducer and activator of transcription 5 (STAT5) is strongly activated by FLT3-ITD,8 but only to a much lesser extent by the FLT3 wild-type or TKD forms.9 FLT3-ITD induces myeloproliferative neoplasm (MPN) in a murine BM transplantation model, whereas FLT3-TKD leads to a lymphoid disease with significantly longer latency.10, 11 The signaling properties determining the different phenotypes and the impacts of these mutations have not been completely described to date.

There is evidence that STAT5 is constitutively activated in the majority of AML patient blasts,12 further underlining its relevance in the disease. Another study reported impaired growth of FLT3-ITD+ cells carrying a dominant-negative STAT5 in vitro;13 however, the role of STAT5 for in vivo transformation remained unclear. Unraveling the importance of STAT5 for FLT3-ITD-induced leukemogenesis could therefore reveal STAT5 as a potential target for therapeutic intervention.

Activated STAT5a and STAT5b molecules dimerize, translocate to the nucleus and activate transcription of various target genes. These include B-cell lymphoma 2 (Bcl-2), c-Myc14, 15 and the interleukin-6 (IL-6) family cytokine Oncostatin M (Osm),16 which has been shown to have both oncogenic and tumor-suppressing functions, depending on the cellular context.17, 18, 19, 20

In this study we used a murine conditional knockout model to investigate the necessity of STAT5 for inducing FLT3-ITD-mediated disease. Furthermore, we intend to prove that the different phenotypes induced by FLT3-ITD and TKD are due to the differential activation of STAT5 in myeloid and lymphoid progenitor cells.

We recently demonstrated that interaction of FLT3-ITD with SRC is necessary for STAT5 activation and proliferation, whereas FLT3-TKD is not dependent on SRC.21 These findings triggered our curiosity with regard to SRC expression levels in hematopoietic progenitor cells, as they might explain the differential activation of STAT5 by FLT3 mutants.

Materials and methods

Expression vectors

The retroviral vectors expressing FLT3-ITD and FLT3 D835Y have been previously described.10, 21 The high-expressing construct was used in all cases. The retroviral vector expressing DsRed Express 2 (pMiBerry) was kindly provided by Dr Tilman Brummer, Institute of Molecular Medicine and Cell Research, University of Freiburg (Freiburg, Germany).

Retrovirus preparation and helper virus assay

Transfection of PhoenixE cells and virus harvest was conducted as previously described.10

Transduction and transplantation of murine BM

Mice carrying a Stat5a and Stat5b locus flanked by loxP sites22 were crossed with mice containing the Cre recombinase under the interferon-dependent Mx1 promoter23 and subsequently backcrossed to a Balb/C background for at least 10 generations. For transplantation experiments, donor BM was collected, transduced and transplanted as previously described.10, 24 Recipient mice were female Balb/c wild type, 8 weeks of age. Induction of the Mx1 promoter by polyinosinic:polycytidylic acid (p(I:C; InvivoGen, San Diego, CA, USA) treatment was performed by (1) treatment of donor mice with p(I:C) for a total of three times within 6 days or (2) injection of p(I:C) on days 11, 14, 18 and 21 after BM transplantation. The animals were caged in a special caging system with autoclaved food and acidified water at the Technical University of Munich in accordance with national and institutional guidelines for animal care. The animal studies were not blinded or randomized.

Fetal liver cell culture

Fetal liver cells from Stat5 constitutive knockout and heterozygous mice22 were transduced by retroviral infection similar to the BM cells. For the methylcellulose assay, 1.25–1.5 × 104 enhanced green fluorescent protein (EGFP)-positive cells were seeded per well. Methylcellulose was purchased from Stem Cell Technologies (Cologne, Germany). After cultivation for 11 days, the colony numbers were documented.

Flow cytometric analysis and cell sorting

Flow cytometric analysis was performed using a XL cytometer (Beckman Coulter, Krefeld, Germany) or a FACSCantoII (BD Biosciences, Heidelberg, Germany). Cells were sorted on a Beckman Coulter MoFlo XDP. Antibodies used to stain cell surface markers were: anti-mouse CD11b (Mac-1, M1/70), CD45R/B220 (RA3-6B2), CD90.2 (Thy1.2, 30-H12), CD45 (30-F11), CD117 (c-kit, 2B8), CD127 (Il-7Ra, A7R34) and Sca-1 (D7), all obtained from BD Biosciences and eBiosciences (Frankfurt am Main, Germany). Lineage-positive cells were removed using the Lineage Cell Depletion Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) before sorting.

For intracellular staining, sorted progenitor cells were treated with Cytofix Buffer and Perm Buffer III (both from BD Biosciences) according to the manufacturer’s protocol. Cells were starved from cytokines for 4 h before fixation. As a positive control, starved multipotent myeloid progenitor (MMP) cells were treated with 10 ng/ml IL-3 for 15 min. Staining was performed using pSTAT5 antibody (pY694, clone 47, BD Biosciences) and FACSDIVA 6.1.2 (BD Biosciences) or FlowJo X 10.0.7 analysis software (FlowJo LLC, Ashland, OR, USA) was used to analyze flow cytometric data.

Quantitative reverse transcriptase-PCR (RT-PCR) analysis

Quantitative RT-PCR was performed using a StepOnePlus instrument (Thermo Fisher Scientific, Dreieich, Germany) and Platinum SYBR Green SuperMix-UDG Kit (Thermo Fisher Scientific). Primer sequences used for quantitative RT-PCR are available upon request. The levels of transcripts were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and TATA-box binding protein (TBBP) levels.

Microscope image acquisition

Slides were viewed with a Axioplan 2 microscope (40 × /0.75NA Plan-Neofluar air objective, Zeiss, Jena, Germany). Images were acquired using a Zeiss Axiocam MRc 5 camera and processed with Axiovision Rel 4.6 scanning software.

Statistical analysis

Statistical analysis was performed using Prism (GraphPad, La Jolla, CA, USA)software. P-values were determined by two-sided Student’s t-test or logrank test where appropriate. Center values are given as mean, and error bars as s.d. Fluorescence intensity center values are given as median. Sample sizes were estimated in cooperation with the Center for Medical Biometry and Medical Informatics at the University of Freiburg. Variances were similar between groups that were compared.

Results

STAT5 is necessary for FLT3-ITD-mediated proliferation in vitro

Earlier studies8, 10, 25, 26 have accumulated evidence that STAT5 may be necessary for FLT3-ITD-mediated proliferation in hematopoietic progenitor cells. To test this hypothesis we infected fetal liver cells from Stat5−/− mice, heterozygous Stat5+/− mice and Stat5+/+ control mice with FLT3-ITD, FLT3-TKD (FLT3 D835Y) or empty MiG vector. The term ‘Stat5’ refers to both Stat5a and Stat5b alleles. After seeding in methylcellulose colony formation was greatly reduced in FLT3-ITD-positive Stat5−/− cells in comparison with Stat5+/− or Stat5+/+ cells infected with FLT3-ITD (Figure 1a). Stat5 deletion in FLT3 D835Y-expressing cells also led to a significant reduction of colony formation, but with a much smaller effect than it was the case for FLT3-ITD. Our findings thereby suggest an important role of STAT5 in FLT3-ITD-mediated proliferation in murine hematopoietic progenitor cells.

Figure 1
figure1

STAT5 is crucial for FLT3-ITD-mediated proliferation in vitro and in a murine BM transplantation model. (a) Statistical analysis of colony formation reveals strongly decreased colony numbers of FLT3-ITD+ Stat5−/− cells and slightly decreased colony numbers of FLT3 D835Y+ Stat5−/− cells. Three independent experiments were analyzed. Each Stat5+/− or Stat5−/− colony number was normalized to its corresponding Stat5+/+ control colony number. A mean of the resulting values was calculated and presented as percentage of Stat5+/+ colony number. **P<0.01; ***P<0.001. (b) Kaplan–Meier plots for survival of recipients of 20 000 Stat5fl/flBM cells transduced with FLT3-ITD or FLT3 D835Y. A total of 18 mice are presented that received FLT3-ITD+ Stat5fl/flMx1Cre BM. Seven mice received FLT3-ITD+ BM without Mx1Cre; 10 mice received FLT3 D835Y+ BM in the presence and 8 in the absence of Mx1Cre, respectively. (c) CD45+ BM cells from moribund mice were stained for CD11b, B220 or Thy1.2 and analyzed by flow cytometry. Numbers indicate percentage of cells in the respective quadrant gate. Mice transplanted with FLT3-ITD+ Stat5fl/flMx1Cre BM cells display a phenotypic shift to a lymphoid disease with high numbers of EGFP+/ Thy1.2+ cells. Mice transplanted with FLT3-ITD+ Stat5fl/fl BM cells develop a myeloid phenotype as indicated by the increased percentage of CD11b+/EGFP+ cells. FLT3 D835Y+ mice develop a lymphoid disorder regardless of Stat5 status.

STAT5 is necessary for FLT3-ITD-mediated proliferation in a murine model

To study the in vivo role of STAT5 for FLT3-ITD-mediated leukemogenesis, we combined a conditional knockout model with an inducible Mx1Cre system, in which the floxed Stat5 allele is deleted upon p(I:C) injection. Stat5 was deleted after transplanting the oncogene (FLT3-ITD vs FLT3 D835Y) infected BM into recipient animals.

Mice transplanted with 20 000 Stat5fl/fl Mx1Cre FLT3-ITD-positive BM cells were treated with p(I:C) on days 11, 14, 18 and 21 after transplantation to delete floxed Stat5 alleles. Deletion of Stat5 by p(I:C) injection was efficient, as shown by immunohistochemistry in Supplementary Figure 1B. Recipients showed significantly prolonged survival after transplantation (median 82 days; P<0.01; Figure 1b) and lower median spleen weight of 75±10 mg compared with 255±36 mg of control mice with intact Stat5 (Supplementary Figures 2B and C). Strikingly, we observed a massive expansion of lymphoid EGFP-positive T cells in the BM (Figure 1c) as well as PB, spleen and thymus (Supplementary Figure 3A), and only a small fraction of EGFP-positive CD11b cells (Figure 1c). These results strongly indicate that STAT5 is required to induce MPN by FLT3-ITD.

Control mice were transplanted with donor BM cells possessing a floxed Stat5 allele but lacking Cre recombinase; nevertheless, the recipient mice were also treated with p(I:C). These animals displayed a rapid onset of disease, with a median survival of 27 days after transplantation (Figure 1b). Examination of the mice revealed splenomegaly (Supplementary Figures 2B and C). Using flow cytometric analysis, an increase in the fraction of CD11b-positive cells (Figure 1c and Supplementary Figure 3B) was detected, resembling the phenotype described in earlier studies.10

Next, we sought to clarify whether STAT5 is required for the induction of FLT3-TKD-mediated lymphoid disease. The median survival of mice transplanted with FLT3 D835Y-positive Stat5fl/fl Mx1Cre (n=10) BM cells was slightly higher compared with recipients of FLT3 D835Y-transduced control BM cells (155 vs 109 days) (Figure 1b); however, the difference did not reach significance. Phenotypic analysis revealed splenomegaly and enlarged thymi (Supplementary Figures 2B and C). All recipient mice demonstrated lymphoid expansion of mainly T cells in BM, spleen and thymus (Figure 1c and Supplementary Figures 1C and 3C) by both flow cytometry and immunohistochemistry analysis.

Control mice transplanted with FLT3 D835Y-positive BM cells lacking Mx1Cre also presented with enlarged spleens and thymi (Supplementary Figures 2B and C). All mice showed a lymphoid phenotype; four of the six analyzed mice had an expansion of T cells (Figure 1c and Supplementary Figures 1C and 3B), whereas two of six mice had increased numbers of B cells. Phenotypes are summarized in Table 1.

Table 1 Analysis of mice that received a transplant of Stat5fl/fl and FLT3-mutant-transduced bone marrow

These results were confirmed in a second experiment where donor Stat5fl/flMx1Cre mice were treated with p(I:C) before BM cell harvest, retroviral infection and transplantation to recipient mice. Mice that received a transplant of 100 000 FLT3-ITD-positive Stat5del/del BM cells pretreated with p(I:C) showed a significant delay in the onset of disease (median survival 96 days; Supplementary Figure 4A). Interestingly, these mice did not develop a myeloproliferative disease; they rather suffered from a B-lymphoid malignancy marked by the expansion of EGFP+/B220+ cells in the BM, spleen and lymph nodes (Supplementary Figure 4B). In contrast, control mice transplanted with 100 000 FLT3-ITD-positive wild-type BM cells rapidly succumbed to a fatal MPN, with a median survival of 10 days after transplantation (Supplementary Figure 4A). These mice showed a phenotype similar to that described for FLT3-ITD-induced disease,10, 11 most notably splenomegaly and expansion of CD11b-positive cells in BM, spleen and PB (Supplementary Figure 4C).

Taken together, our results reveal a remarkable similarity in the lymphoid phenotype of mice transplanted with FLT3-ITD-positive Stat5del/del BM compared with FLT3 D835Y-positive mice, where the Stat5 deletion has no gross impact on disease latency and phenotype. STAT5 is therefore strictly required to induce MPN by FLT3-ITD, but might be dispensable for induction of FLT3 D835Y-mediated lymphoid malignancy.

Differential STAT5 target gene activation in myeloid progenitor cells compared with lymphoid progenitor cells transduced with FLT3-ITD

FLT3-ITD leads to a myeloproliferative disease in a murine mouse model exclusively in the presence of active STAT5. Stat5 deletion leads to a phenotype shift toward a lymphoid phenotype. We therefore hypothesized that activation of STAT5 by FLT3-ITD may be critical in myeloid but not lymphoid progenitor cells. We thus sorted murine MMP cells and common lymphoid progenitor (CLP) cells27, 28 (Figure 2a) and subsequently infected the sorted progenitors with FLT3-ITD and empty control vector (MiG) (Figures 2b and c). We then analyzed STAT5 activation by intracellular staining of phospho-STAT5 (pSTAT5) levels and flow cytometry. We were able to observe higher STAT5 activation in myeloid compared with lymphoid progenitors cells expressing high levels of FLT3-ITD (Figure 2d). As a positive control, MMP cells were stimulated with IL-3 (Figure 2e). The 5% highest FLT3-ITD-expressing cells display a significantly (1.8-fold) increased pSTAT5 signal (Figures 2f and g). This finding suggests differential STAT5 activation in hematopoietic progenitor cells by FLT3-ITD.

Figure 2
figure2

FLT3-ITD activates STAT5 in myeloid but not in lymphoid progenitor cells. (a) Cell sorting of common myeloid (MMP) and lymphoid progenitor (CLP) cells. (b) Flow cytometric analysis of MMP and CLP cells infected with empty (MiG) vector. (c) Flow cytometric analysis of MMP and CLP cells infected with MiG-FLT3-ITD vector. (d) Histogram analysis of pSTAT5 signal in EGFP+ MMP and CLP cells infected with MiG-FLT3-ITD vector or empty vector. (e) Histogram analysis of pSTAT5 signal in MMP cells stimulated with IL-3 or left untreated. (f) Gating strategy to identify the 5% highest FLT3-ITD-expressing MMP or CLP cells. (g) Statistical analysis of median pSTAT5 fluorescence intensity of the 5% highest FLT3-ITD-expressing cells from total FLT3-ITD+ MMP or CLP cells. pSTAT5 signal is significantly higher in MMP cells compared with CLP cells. Three independent experiments using 10 mice each were analyzed. Median fluorescence intensity was normalized to CLP cell sample values.

We thus analyzed activation of STAT5 target genes like c-Myc, Bcl-2 and Osm14 by quantitative RT-PCR in sorted myeloid and lymphoid progenitor cells infected with FLT3 mutants. Interestingly, Osm expression displayed significant upregulation exclusively in FLT3-ITD-positive MMP cells, but not in lymphoid progenitor cells (Figure 3a). Similarly, upregulation of other STAT5 targets genes (c-Myc, Bcl-2) by FLT3-ITD was restricted to the myeloid compartment and could not be shown in lymphoid progenitor cells (Figures 3b and c). c-Jun, a gene not regulated by STAT5, served as internal control (Figure 3d).

Figure 3
figure3

FLT3-ITD activates STAT5 via SRC in myeloid but not in lymphoid progenitor cells. (ad) Quantitative RT-PCR analysis of STAT5 target genes Oncostatin M, Bcl-2 and c-Myc in MMP and CLP cells transduced with FLT3-ITD or FLT3 D835Y. Expression levels of an unrelated control gene (c-Jun) were also determined. PCR analysis was performed in duplicate. A representative of two independent experiments is shown. Osm mean upregulation was 3.7-fold in FLT3-ITD+ MMP cells vs MiG-infected MMP cells in both independent experiments. All data were normalized to empty vector (MiG)-infected MMP cells and to GAPDH expression values. (e) Quantitative RT-PCR of Src expression in MMP cells and CLP cells after cultivation for 2 days together with viral supernatant. MMP cells show a significant upregulation of Src levels compared with CLP cells, irrespective of the virus supernatant used for stimulation. PCR analysis was performed in triplicate. A representative of two independent experiments is shown. Data were normalized to MMP cells w/o culture and to GAPDH and TBBP expression values. (f) Quantitative RT-PCR analysis of Osm expression in BM cells of representative mice transplanted with FLT3 mutants with or without deletion of Stat5. Data were normalized to wild-type (WT) sample and to GAPDH expression values. ND, no detectable transcripts of target gene above background level. *P<0.05; **P<0.01; ***P<0.001.

We recently demonstrated that STAT5 activation by FLT3-ITD is dependent on the interaction of FLT3-ITD and SRC tyrosine kinase.21 We thus examined whether the predominant activation of STAT5 in myeloid progenitor cells correlates with the level of Src expression in myeloid and lymphoid progenitor cells. We therefore determined Src expression levels in sorted progenitor cells by quantitative RT-PCR and detected significantly higher Src levels in MMP cells compared with CLP cells (Figure 3e). It is of note that the differences of Src expression levels between the myeloid and lymphoid progenitor cells are much higher than what one would suspect from the differences in STAT activation and might hint to signaling differences of these cells mainly downstream of STAT5.

We also analyzed STAT5 target gene expression in the BM of diseased mice and found Osm mRNA to be increased up to 4.1-fold in FLT3-ITD-positive Stat5fl/fl mice compared with the wild-type mice, whereas we detected only low levels of Osm transcripts in FLT3-ITD-positive Stat5fl/fl Mx1Cre mice (Figure 3f). These findings suggest an important role of the STAT5/Oncostatin axis for transformation of myeloid cells by FLT3-ITD.

To further underline this myeloid bias of STAT5 activation in the human disease, we have analyzed microarray expression databases for STAT5 target gene expression. Interestingly, the STAT5 target genes Osm (1.14-fold, P<0.001), c-Myc (1.02-fold, P<0.001), Pim-1 (1.06-fold, P<0.001) and Pim-2 (1.03-fold, P<0.001) were found to be significantly upregulated in a panel of AML patients compared with acute lymphoblastic leukemia patients.29 Pim-1 was also found 1.16-fold upregulated in FLT3-ITD+ AMLs compared with FLT3-TKD+ AMLs (P=0.0079).30

Ectopic Oncostatin M expression is sufficient to switch the immunphenotype in FLT3-TKD+ mice to a myeloproliferative disease

To further examine the importance of STAT5 activation for induction of myeloid neoplasms we hypothesized that ectopic expression of STAT5 target genes in a lymphoid mouse model may lead to a phenotype reversion with development of myeloid disease. We thus expressed the STAT5 target gene Oncostatin M in a FLT3 D835Y lymphoid mouse model, where STAT5 activation is not necessary for disease induction.

Strikingly, after transplantation of double-infected FLT3 D835Y- and Oncostatin M-expressing BM cells, transplanted mice succumbed to MPN with expansion of Gr-1+ cells in the BM (Figure 4b) and PB. Approximately 80% of the FLT3-D835Y/EGFP-positive cells were Gr-1+ in BM and PB (Figures 4c and d). Notably, <1% of total BM cells were detected as Oncostatin+/Berry cells (Figure 4a), suggesting that only few Oncostatin-releasing cells are required to drive FLT3 D835Y+ myeloid cell expansion. Furthermore, the combined expression of FLT3 D835Y and Oncostatin has an additive effect on the disease itself, as these mice also display a significantly reduced survival compared with mice transplanted with a single oncogene (Figure 4e).

Figure 4
figure4

Oncostatin M expression is sufficient to induce a myeloproliferative disease in FLT3 D835Y-transplanted mice. Mice were transplanted with BM cells co-transfected with MiG-FLT3 D835Y (EGFP) and pMiBerry-Oncostatin M (DsRedExpress2) constructs (n=6). Control mice received BM cells transfected with empty vector and a single oncogene (n=4 for each group). (a) Flow cytometric analysis of cell surface markers in the BM from representative mice transplanted with 20 000 FLT3 D835Y+/ 10 000 Oncostatin M+ cells (left panel), 20 000 FLT3 D835Y+/ 10 000 pMiBerry cells (middle panel) and 20 000 MiG/10 000 Oncostatin M+ cells (right panel). Numbers indicate percentage of cells in their respective quadrant gate. (b) Histogram analysis of Gr-1+ cells in the EGFP+ fraction of the BM of representative mice. (c) Statistical analysis of the percentage of Gr-1+ in the BM EGFP+ fraction. In the presence of Oncostatin M, the original lymphoid phenotype of FLT3 D835Y+ cells is switched to the myeloid (Gr-1+) lineage. (d) Statistical analysis of the percentage of Gr-1+ in the PB EGFP+ compartment. **P<0.01; ***P<0.001. (e) Kaplan–Meier plots for survival of recipients of 20 000 OSM+/FLT3 D835Y+ BM cells or bone cells transfected with single oncogenes. (f) Flow cytometric analysis of Thy1.2 surface expression on BM cells from representative mice.

Mice transplanted with BM cells expressing FLT3 D835Y together with an empty vector displayed low numbers of Gr-1+ cells in the BM (Figure 4b), as it has been described previously.10 The fraction of Gr-1+ cells in the FLT3-D835Y/EGFP-positive compartment was <10% in BM (Figures 4c and d) and <20% in PB (Figure 4d). Instead, these mice show expansion of EGFP+/Thy1.2+ cells (Figure 4f).

Mice that received BM transfected with Oncostatin M and empty vector succumbed to MPN, revealing expansion of Gr-1+ cells in BM and PB (Figures 4b–d), confirming results from earlier studies.31

Taken together, ectopic expression of FLT3-TKD and a major STAT5 target gene—Oncostatin M—can mimic the FLT3-ITD-induced myeloid phenotype, highlighting the complete dependency of FLT3-induced myeloid malignancies on precise STAT5 activation.

Discussion

The characterization of signaling pathways aberrantly activated in malignant transformation, along with the discovery of novel targets for therapy, are both major challenges in modern leukemia research. In the present study we demonstrate for the first time that STAT5 is a critical player in inducing a myeloid disease by FLT3-ITD in vivo, whereas it seems not necessary for a FLT3 TKD-induced lymphoid disorder in mice. Moreover, we show that STAT5 is activated by FLT3-ITD in myeloid hematopoietic progenitor cells, and only to a lower degree in lymphoid hematopoietic progenitor cells. Interestingly, murine MMP cells also express significantly higher levels of Src compared with CLP cells. This finding may explain the exclusive activation of STAT5 by FLT3-ITD in these cells, as we recently demonstrated that interaction between SRC and FLT3-ITD is essential for proliferative signaling.21

Within our study we used a conditional Stat5 knockout model that was also used in other studies proving the necessity of STAT5 for oncogenes such as Bcr/abl32 or JAK2 V617F.33, 34 In those studies, Stat5 deletion led to longer disease latency, but no shift in the hematologic phenotype could be found. However, the data from the described conditional knockout model may not be directly transferred to the situation in humans, as FLT3-ITD occurs there mainly in AML cases and can only rarely be found in human MPNs. Moreover, in humans it is also frequently associated with different other mutations, such as cytoplasmic NPM1 or mixed-lineage leukemia–partial tandem duplication.35, 36 Here, we demonstrate that in FLT3-ITD-induced disease STAT5 is necessary not only for latency but also for the induction of the myeloproliferative phenotype in mice.

To gain insights into FLT3-ITD signaling properties in early hematopoietic progenitor cells, we analyzed STAT5 activation at the CLP and MMP cell stage and showed that FLT3-ITD induces significant, but not very prominent, STAT5 activation in murine myeloid progenitors compared with lymphoid progenitor cells. However, STAT5 target genes were highly and significant activated by FLT3-ITD in MMP cells and we were thus able to describe a novel type of cell-specific signaling by oncogenes that has also been observed in different cellular contexts.37, 38

The precise stage of these progenitor cells driven by FLT3-ITD toward a myeloid rather than a lymphoid lineage remains controversial. As long-term and short-term hematopoietic stem cells both lack FLT3 expression,39, 40 the multipotent progenitor cells MPP cells contain the most primitive progenitor cell population expressing the FLT3 receptor.41 A fraction of FLT3-positive, lymphoid-primed multipotent progenitor cells within the multipotent progenitor cell population has been described;42 these also possess the ability to differentiate into granulocytic and monocytic lineages, but not into megakaryocytic or erythroid lineages. In the present study we demonstrate that FLT3-ITD signaling is myeloid biased in early hematopoietic progenitor cells. Our results indicate that FLT3-ITD does not block lymphoid expansion, as CLP cells also activate STAT5 but at lower levels. However, MMP cells show increased STAT5 activation, leading to a marked upregulation of STAT5 target genes. They encode for anti-apoptotic proteins (for example, Bcl-243) as well as for transcription factors enhancing proliferation (for example, c-Myc44), and as a consequence, the myeloid cells simply overgrow the lymphoid cells. Consistent with this observation, there is recent evidence that FLT3-ITD leads to an upregulation of myeloid program genes in lymphoid-primed multipotent progenitor cells, whereas the expression of lymphoid-affiliated genes was markedly reduced.45 These findings might explain the myeloproliferative phenotype detected in the murine model, as well as the frequent occurrence of FLT3-ITD in human AML.

Different groups have generated murine knock-in models of FLT3-ITD.46, 47 Small and colleagues48 have also investigated differences in early hematopoietic progenitor cells in their FLT3-ITD knock-in model, noting an expansion of the MMP cell population, supporting our data that FLT3-ITD expression increases STAT5 activation in myeloid progenitor cells and thereby leads to myeloproliferation. On the other hand, we demonstrate that CLP cells transduced with FLT3-ITD show minor STAT5 activation, whereas FLT3-ITD knock-in mice display an increase in the percentage of CLP cells.48 One possible explanation could be that CLP cells give rise to Pre-Pro B cells that also expand in the FLT3-ITD knock-in mouse model and are known to have myeloid rather than B-cell potential.45 Furthermore, there is evidence that FLT3-ITD leads to a myeloid tendency in lymphomyeloid multipotent progenitors that lie upstream of the CLP cell population.45

Whereas numerous studies have focused on FLT3-ITD, the signaling properties of FLT3 tyrosine kinase domain mutations have remained largely unknown. In marked contrast to FLT3-ITD, Stat5 deletion in FLT3 D835Y mice leads to no gross differences in disease onset and progression, suggesting a somehow more dispensable role for STAT5 in FLT3 D835Y signaling. Interestingly, the immunophenotype of FLT3 D835Y-positive mice is remarkably similar to that of FLT3-ITD-positive Stat5del/del mice, suggesting that STAT5 activation in FLT3-ITD cells is the exclusive target that induces the differences in the immunophenotypes of FLT3 D835Y vs FLT3-ITD. Moreover, solely Stat5 deletion is not sufficient to impede disease onset in FLT3-ITD and FLT3 D835Y mice.

It has been shown previously that STAT5 activation by FLT3-ITD is dependent on SRC,21 but independent of JAK (Janus kinase) or SOCS (suppressor of cytokine signalling) signaling,25 and that tyrosine residues 589 and 591 are critical for signal transduction.26 Our results show that high Src expression levels in hematopoietic progenitor cells attend effective STAT5 target gene activation, adding further evidence that SRC is a critical mediator in FLT3-ITD signaling,

Oncostatin M involvement in malignant transformation has been discussed controversially. Whereas initial reports described Oncostatin M as a tumor suppressor that inhibits the proliferation of a melanoma cell line,17 other groups demonstrated growth-stimulating potential of the protein in different cellular contexts.18, 19, 20 Interestingly, murine BM reconstitution with Oncostatin M-infected BM cells results in a phenotype remarkably similar to FLT3-ITD transduction with splenomegaly and expansion of Gr-1/Mac-1-positive cells.31 Here, we were able to show the impact of Oncostatin M as an important STAT5 target in FLT3-ITD leukemogenesis in mice. Furthermore, we prove the influence of previously published STAT5 targets such as c-Myc49 on FLT3-ITD-mediated transformation. Interestingly, co-expression of Oncostatin M can reverse the original lymphoid phenotype of FLT3 D835Y-induced disease to a myeloproliferative disease in mice, suggesting that ectopic expression of the STAT5 target gene Oncostatin M is sufficient to induce a myeloid lineage commitment in FLT3 D835Y cells.

Taken together, we propose a novel concept of aberrant cell-specific signaling in murine hematopoietic progenitor cells. Our findings indicate that internal tandem duplication of FLT3 results in differing activation of downstream signaling pathways depending on the precise cell type. In case of a lymphoid progenitor cell, the absence of SRC leads to diminished STAT5 signaling, and thus less proliferation. However, when FLT3-ITD is expressed in a myeloid progenitor cell, STAT5 target gene activation leads to subsequent increased proliferation and cell survival. In case of Stat5 deletion, minor proliferative signals can be transmitted in both myeloid and lymphoid progenitor cells and will eventually trigger lymphoid expansion. The involved signaling pathway might be identical to FLT3 D835Y signaling, as the resulting disease phenotype is remarkably similar.

Therefore, our study might refer to STAT5 and its activated genes as possible targets in the treatment of FLT3-ITD-positive AML, and identify an important role of Oncostatin M in the induction of disease.

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Acknowledgements

We thank M Follo for help with FACS sorting and G Schäfer for technical assistance. This work was supported by a DFG grant (FOR 2033 to JD and TAM). LH was supported by the Intramural Research Program (IRP) of the National Institutes of Diabetes, Digestive and Kidney Disease, NIH (Bethesda, MD, USA). ALI was supported by a Research grant from University Clinic Freiburg and from the government Baden-Württemberg (BSL).

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Müller, T., Grundler, R., Istvanffy, R. et al. Lineage-specific STAT5 target gene activation in hematopoietic progenitor cells predicts the FLT3+-mediated leukemic phenotype. Leukemia 30, 1725–1733 (2016). https://doi.org/10.1038/leu.2016.72

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