Original Article | Published:

Molecular Targets for Therapy

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

Leukemia volume 24, pages 13971405 (2010) | Download Citation

Abstract

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

Introduction

Signal transducer and activator of transcription-5 (STAT5) is a latent transcription factor that can be activated by phosphorylation by Janus kinases (JAKs) in the cytoplasm, leading to dimerization, DNA binding and retention within the nucleus.1 Tyrosine-phosphorylated STAT5 can be tracked by flow cytometry or immunostaining and is a biomarker associated with poor prognosis for juvenile myelomonocytic leukemia2 and acute myeloid leukemia.3 Recently, targeting transcription factor–cofactor complexes has become clinically plausible.4, 5 However, targeting of pSTAT5 or its aberrant signaling might be difficult and risky as complete inhibition of STAT5 may present significant side effects, for example, in hematopoietic cell types and liver function. Therefore, understanding aberrant STAT5 signaling in normal vs leukemic cells may allow for novel strategies for leukemia therapy.

Cooperative interactions and downstream targets of STAT5 responsible for its function in hematopoiesis are not well defined. Constitutive STAT5 activation with double-mutant STAT5aH299R/S711F causes myeloproliferative disease (MPD) in mice,6 and this disease development requires STAT5 expression in the hematopoietic stem cell (HSC).7 In studies using the single mutant STAT5aS711F, myeloid and lymphoid hyperplasias have been described.8, 9 To date most functions for STAT5 have been attributed to a growing list of well-characterized direct target gene products such as Osm, Cis, Socs, Pim1, Bcl-XL and c-Myc. We have recently shown that expression of bcl-2/bcl-XL mediated by STAT5 requires the N-domain and is critical for lethal MPD in mice.10

STAT5 and phosphatidylinositol-3′-kinase (PI3K) activation are required for prosurvival signaling,11 and cross talk between these pathways has been described downstream of interleukin (IL)-212 and thrombopoietin13 receptors. Enhanced sensitivity to inhibition of STAT5, SHP-2 and Grb2-associated binding protein (Gab2)14 was found in Bcr/Abl transformed cell lines. Cytoplasmic localization of phosphorylated STAT5 has recently been described, whereby STAT5 interacts with Gab28, 15 or with Shc, which in turn interacts with Grb2 and Gab2.16 In each case phosphorylated STAT5 promoted activation of Akt suggesting that Gab2/Akt might be a potential therapeutically relevant signaling node in hematologic malignancies. Gab2 is tyrosine phosphorylated by several early acting cytokine receptors such as Flt3, c-Kit, IL-3R and c-Mpl, and contains binding sites for SH2 and SH3 domains that promote binding to signaling molecules.17, 18, 19 Gab2 is involved in promoting the activation of the PI3K and the mitogen-activated protein kinase pathways and can regulate hematopoietic cell survival and migration functions.20 In BaF3 cells, Gab2 was found to associate indirectly with persistently active STAT5, p85 and Grb2, but not SHP-2, and to promote STAT5-mediated signaling through induction of PI3K and mitogen-activated protein kinase pathways.8, 15, 21, 22 This interaction required phosphorylation of STAT5. The STAT5–Gab2 complex was also observed in primary cells obtained from mice expressing STAT5aS711F where increased Akt activation was observed.8

In the studies reported here, we directly asked whether STAT5/Gab2 contribute to leukemic hematopoiesis in vivo by testing the genetic impact of Gab2 deficiency. We also tested the therapeutic efficacy of targeting the PI3K/Akt/mTOR pathway pharmacologically in STAT5-provoked MPD using rapamycin. The results indicated that this pathway can modulate cell growth but that targeting multiple STAT5-mediated survival signals including bcl-2/bcl-XL is required for efficient killing of myeloproliferative neoplasm cells.

Materials and methods

Cell lines, plasmids and antibodies

Murine stem cell virus (MSCV) vectors expressing green fluorescent protein (GFP) from an internal ribosomal entry sequence (IR) were generated for MSCV-STAT5a-IR-GFP and MSCV-STAT5aS711F-IR-GFP as described previously.9 All GP+E86-based retroviral producer cell lines were cultured in Hyclone Dulbecco's modified Eagle's medium containing 10% calf serum (Hyclone), 1% penicillin, 1% streptomycin and 1% amphotericin B (complete medium) at 37 °C in an atmosphere of 95% oxygen and 5% CO2. BaF3 and NB4 cells were maintained in RPMI 1640 medium and K562 cells were maintained in Iscove's modified Dulbecco's medium. All antibodies are described in Supplementary Materials and Methods.

Mice

The C57BL/6 (CD45.2) mice and the congenic B6.SJL-PtprcaPep3b/BoyJ (CD45.1) strain were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). All mutant mice were of the C57BL/6 background and housed in a specific pathogen-free environment. All mouse studies were approved by the institutional animal care and use committee at Case Western Reserve University.

Retroviral vector transduction and transplantation

Retroviral transduction of wild-type or Gab2−/− bone marrow (BM) was performed using concentrated retroviral supernatant and colony-forming unit in culture (CFU-C) assays were performed as described.23, 24 Five independent transduction experiments were performed with 0.8–4 million transduced BM cells transplanted per recipient mouse as described in the Results section. Typical donor/recipient ratios for these experiments were from 1.5:1 to 2:1. All adult recipient mice were conditioned with 950 rads of irradiation from a 137Cs source 3–6 h before cell injection.

Histology and flow cytometry of mouse tissues

Transplanted mice were killed when becoming moribund and the tissue specimens were prepared from freshly killed mice. Tissues were fixed in 10% neutral buffered formalin and embedded in paraffin. Paraffin sections (4 μm) were stained with hematoxylin and eosin. Histologic sections and stains were performed by the Case Comprehensive Cancer Center Histology core laboratory. Images were taken using an Olympus BX41 microscope equipped with PanAchromatic objectives ( × 20, × 40) and a mounted Spot In-Sight digital camera (Diagnostic Instruments Inc., Sterling Heights, MI, USA). The software for image acquisition was Spot Advanced. Cells from live mice were analyzed by flow cytometry as described in Supplementary Materials and Methods.

Drug treatments

Rapamycin (LC Laboratories, Woburn, MA, USA) was initially dissolved in 100% ethanol, stored at −20 °C, and further diluted in an aqueous solution of 5.2% Tween and 5.2% polyethylene glycol 400 (2% final ethanol concentration) immediately before use. Transplanted mice were treated with rapamycin (4 mg per kg, i.p.) every other day for 3–4 weeks and then discontinued. ABT-737 (Abbott Laboratories, Abbott Park, IL, USA) was dissolved in DMSO and stored in aliquots at −20 °C. BaF3 cells were cultured in the indicated concentration of rapamycin or ABT-737 and cell proliferation/viability was determined by either propidium iodide staining or Trypan blue exclusion assay. Both methods gave comparable percentages of cell death. Western blots were performed as previously described.21

Statistical analyses

To test for genetic interaction, we used two-way analysis of variance (ANOVA). We also used Wilcoxan–Mann–Whitney U-tests for wild-type vs mutant single comparisons. P-values of <0.05 were considered statistically significant and were calculated using SPSS 16.0 (SPSS Inc., Chicago, IL, USA). Student's two-tailed t-tests were performed using InStat 1.5 (University of Reading, UK). Kaplan–Meier comparisons were analyzed by log-rank test.

Results

Because our previous studies have shown evidence for STAT5-mediated activation of the PI3K pathway, we set out in these studies to interrogate the impact of PI3K signaling on a STAT5-provoked MPD in vivo. This is important to assess as a potential therapeutic approach as persistent STAT5 activation is the hallmark of myeloid hyperproliferation and myeloid cytokines and growth factors also activate STAT5. We, in our studies, used both genetic and pharmacologic modulation of the PI3K pathway to test the impact on MPD induced by transplantation of BM cells expressing STAT5aS711F into recipient mice (Supplementary Figure S1).

We examined whether there was a difference in the retroviral transduction efficiency between the wild-type and Gab2−/− BM cells. Similar transduction efficiencies were observed in both groups before transplantation within each experiment as determined by the percentage of GFP+ cells, which ranged from 10 to 40% for IR-GFP and from 10 to 30% for STAT5aS711F vector. Comparable levels of gene transfer (GFP+ cells) in vivo were also observed for the IR-GFP marking vector control in either wild-type or Gab2−/− BM recipient mice (data not shown), thus indicating that Gab2 deficiency did not impair transduction of cells capable of repopulating hematopoiesis. No defect in homing of c-Kit+ progenitors from wild-type or Gab2−/− BM cells was observed (data not shown) and mice engrafted with STAT5aS711F expressing donor BM cells showed marked expansion of the myeloid lineage but did not expand lymphoid or erythroid populations (data not shown).

Gab2 deficiency attenuates MPD and improves survival associated with activated STAT5

Because STAT5aS711F was incapable of conferring cytokine-independent growth to myeloid CFU-C, we tested the impact of Gab2 deficiency on murine MPLW506L-induced cytokine-independent CFU-C. Gab2 deficiency conferred a reduction in colony number (Supplementary Figure S2). To gain further insight into the contribution of Gab2 to STAT5aS711F-induced MPD in vivo, we transduced BM cells from wild-type or Gab2−/− mice with the IR-GFP control vector or STAT5aS711F expressing vector. The cells were then transplanted into lethally irradiated recipient mice. The engrafted mice were analyzed 4–6 weeks after transplantation. As expected, flow cytometry analyses showed that all wild-type mice expressing STAT5aS711F had an increased frequency of Gr-1+Mac-1+ cells compared with the empty vector control in the peripheral blood. Regardless of the myeloid frequencies, the white blood cell (WBC) counts from the mice transplanted with Gab2−/− background BM expressing STAT5aS711F were significantly lower than those receiving the wild-type counterpart (Figure 1a; P=0.001, Mann–Whitney U-test). The absolute number of Gr-1+Mac-1+ cells was accordingly reduced 3- to 4-fold relative to wild-type counterparts (Figure 1b; P=0.014, Mann–Whitney U-test). The genetic interaction between Gab2 and STAT5aS711F was advantageous for elevated WBC counts and myeloid cell expansion (WBC: P<0.001; two-way ANOVA; myeloid cells: P=0.022; two-way ANOVA), indicating that STAT5aS711F can cooperate with Gab2 to induce myeloid hyperplasia.

Figure 1
Figure 1

Deletion of Gab2 attenuates myeloid hyperplasia and improves survival of mice expressing persistently active STAT5. Wild-type or Gab2−/− mouse BM cells were retrovirally transduced with either IR-GFP control vector (IR) or STAT5aS711F, and transplanted into lethally irradiated recipients on three separate experiments. Mice were analyzed at the same time for all groups (26–32 days after transplant). (a) Total WBC counts are plotted for mice of various genotypes. Each dot represents an individual mouse that was analyzed. (b) The absolute number of Gr-1/Mac-1 double-positive cells in mice of various genotypes. Horizontal bars indicate the average for all mice analyzed per group. (c) The spleen weights were measured and plotted against each genotype analyzed. (d) The Kaplan–Meier survival curve of mice expressing persistently active STAT5a or IR-GFP control vector was also determined by following mice daily and recording deaths. The number of mice per group was as follows: WT-IR, N=6; Gab2−/− IR, N=6; Gab2−/− STAT5aS711F, N=6; WT STAT5aS711F, N=7. IR denotes IR-GFP vector and STAT5aS711F denotes STAT5aS711F-IR-GFP vector. The P-value in panel d (log-rank test) represents the comparison of survival by STAT5aS711F on wild-type vs Gab2−/− background.

At the time of death, tissues from mice were collected and analyzed to determine the degree of myeloid infiltration. Corresponding to the reduced peripheral myeloid expansion, spleen weights were reduced 2- to 3-fold for STAT5aS711F expressed in Gab2−/− background relative to STAT5aS711F expressed in wild-type background cells (Figure 1c; P=0.001, Mann–Whitney U-test). Genetic interaction between STAT5aS711F and Gab2 was observed (P<0.001; two-way ANOVA), consistent with our previous report of biochemical interaction between STAT5aS711F and Gab2.8 No mice died following transplant with control IR-GFP-expressing vector regardless of the genotype of the starting BM cells. The attenuated MPD was sufficient to improve survival, although most recipients of Gab2−/− background BM cells eventually died from MPD 68 days after transplant (Figure 1d).

Tissue histology of mice receiving wild-type or Gab2−/− background transduced BM cells was compared at the time of killing. In the liver of wild-type mice expressing STAT5aS711F, the hepatic lobular architecture was markedly distorted by dense infiltration of mostly mature myeloid cells but including rare early precursors in the hepatic lobules or portal triads (Figure 2a). However, in the mice transplanted with Gab2−/− background BM cells, the hepatic architecture was largely intact with significantly less infiltrate in the hepatic lobules or periportal areas. In the spleen, the wild-type mice expressing STAT5aS711F showed pronounced splenomegaly (approximately fivefold) with markedly distorted splenic architecture (Figure 2b). The red and white pulps were diffusely effaced by extramedullary hematopoiesis and myelomonocytic cells at mostly immature stages of differentiation. However, the splenic architecture for STAT5aS711F on the Gab2−/− background was largely intact and associated with approximately twofold increased spleen weights. Spleen and liver of wild-type mice expressing STAT5aS711F showed increased percentages of Gr-1+Mac-1+ myeloid lineage cells. In contrast, there was markedly less myeloid involvement in spleen and liver of mice receiving Gab2−/− BM cells expressing STAT5aS711F. In the absence of Gab2, about half of the mice expressing STAT5aS711F died early and had higher percentages of myeloid cells than those that survived longer (Supplementary Figure S3A). Notably, significant expansion of non-GFP+ cells was also observed (Supplementary Figure S3B).

Figure 2
Figure 2

Deletion of Gab2 attenuates liver and spleen pathology resulting from expression of persistently active STAT5. Tissues from transplanted mice expressing either STAT5aS711F or IR-GFP control vector were collected, formalin fixed and histologic sections were stained with hematoxylin and eosin. Representative sections of liver and spleen are shown. The liver (a) or spleen (c), at × 20 magnification was analyzed for wild-type and Gab2−/− BM-transplanted mice expressing the IR-GFP control vector. The liver (b) or spleen (d) was also analyzed at × 20 and × 40 magnification for wild-type and Gab2−/− groups expressing STAT5aS711F.

Persistently active STAT5 induces Akt activation in myelomonocytic infiltrates

Although Gab2 deficiency attenuated the MPD by STAT5aS711F in vivo, it did not fully block the MPD progression. Previous reports indicated that STAT5aS711F can induce Akt activation in vitro and we showed that TAT-Gab2 decoy molecules can significantly block this Akt activation.8 We therefore next examined the pAkt level in the spleen of mice transplanted with wild-type or Gab2−/− BM cells expressing either empty vector or STAT5aS711F. A similar basal level of Akt activation was observed in the mice transplanted with IR-GFP vector expressing BM cells of either genotype (Supplementary Figure S4A). However, there was a dramatic increase in the number of positive cells for phosphorylated species of Akt staining in mice transplanted with wild-type or Gab2−/− BM cells expressing STAT5aS711F (Supplementary Figure S4B). The pAkt-positive cells correlated with the expanding myeloid population in the tissues, consistent with the finding that more myeloid infiltration occurs in the liver of mice transplanted with wild-type BM cells expressing STAT5aS711F.

Rapamycin treatment is cytostatic and delays MPD induced by persistently active STAT5

To determine whether targeting downstream of Gab2-mediated PI3K/Akt signaling would be effective, we used rapamycin to inhibit mammalian target of rapamycin (mTOR). Rapamycin was very effective at reducing the number of CFU-C driven by either STAT5aS711F cultured in 2 ng/ml IL-3 (Figure 3a) or MPLW506L (Figure 3b) in the absence of cytokines. In the presence of IL-3, IL-6 and SCF, a trend toward reduced CFU-C was observed but it was not significant (Figure 3b). The observation that STAT5 hyperactivation induced Akt activation in vivo provided a potential therapeutic target. mTOR, a serine/threonine kinase downstream of Akt, is involved in regulation of the cell cycle, apoptosis and angiogenesis. Tumors harboring dysregulated activation of the PI3K/Akt pathway are sensitive to mTOR inhibition.25 We next tested whether inhibition of mTOR activity could attenuate MPD promoted by STAT5aS711F. Owing to the rapid onset of MPD as early as 10 days after transplantation, we decided to perform our drug treatments at the early stage of MPD. We delayed the disease by transplanting 0.8–1 million STAT5aS711F expressing BM cells into wild-type mice compared with 1.5–4 million for the Gab2−/− experiments. At 1 month after transplantation, when all mice were still healthy from appearance but had a significant fraction of GFP+ cells in the peripheral blood, we administered rapamycin every other day (4 mg/kg, i.p. injection or vehicle). The average percent GFP+ cells pretreatment for all control (21±10%, N=12) or rapamcyin (17±5%, N=13) groups was not significantly different (P=0.21, t-test). The average WBC counts before treatment for all control (9.2 × 106±7.9 × 106 per ml; N=12) or rapamycin (7.8 × 106±4.0 × 106 per ml; N=13) groups were not significantly different (P=0.58, t-test). Rapamycin treatment for 3–4 weeks followed by discontinued treatment resulted in a slight drop in WBC counts compared to vehicle treatment and relative to mice transplanted with BM cells transduced with the IR-GFP control retroviral vector (Figures 4a and b). In contrast, mice transplanted with BM cells expressing STAT5aS711F showed a significant reduction in WBC count in a time span of 10 days after initiation of rapamycin treatment (Mann–Whitney U-test, P=0.002; Figures 4c and d). Control vehicle treatment had no effect on STAT5aS711Fmediated MPD progression. Consistent with inhibition of STAT5aS711F-induced myeloid outgrowth, the mice expressing STAT5aS711F had improved survival during the 3 weeks of treatment of rapamycin and lived approximately 1 month longer than the vehicle-treated control group (Figure 4e; P=0.03). All control mice died within 2 months of transplantation. Overall, rapamycin therapy reduced the outgrowth of the myeloid expansion by STAT5aS711F and attenuated progression of disease.

Figure 3
Figure 3

Inhibition of myeloid colony formation resulting from STAT5aS711F or MPLW506L expression by treatment with rapamycin. (a) Wild-type BM cells were retrovirally transduced with MSCV-STAT5aS711F-IR-GFP, sorted for GFP+ cells and plated in methylcellulose medium. The average number of colonies (from duplicate plating) was determined in the absence or presence of recombinant IL-3 (2 ng/ml) from three independent assays that yielded comparable results. (b) Wild-type BM cells were retrovirally transduced with MSCV-MPLW506L-IR-YFP, sorted for YFP+ cells and plated in the presence or absence of IL-3 (20 ng/ml), IL-6 (50 ng/ml) and SCF (50 ng/ml).

Figure 4
Figure 4

Rapamycin inhibits myeloid expansion in the peripheral blood and prolongs the survival of mice expressing STAT5aS711F. BM cells were transduced with retroviral vectors expressing either IR-GFP or STAT5aS711F. Equal donor equivalents of GFP+ cells were subsequently transplanted into irradiated recipients. At 1 month after transplantation, mice were treated with either rapamycin (4 mg/kg every other day) or vehicle control by i.p. administration for 3–4 weeks and then discontinued. Peripheral white blood cell counts were determined at the indicated times after treatment. IR-GFP control mice treated with either vehicle control (a), or rapamycin (b) and STAT5aS711F-expressing mice treated with either vehicle (c) or rapamycin (d). Each line represents an individual mouse. (e) The four groups of mice were monitored daily and mouse deaths were recorded. Mouse survival was plotted on Kaplan–Meier curve. IR-GFP vehicle control, N=3; IR-GFP rapamycin, N=3; STAT5aS711F vehicle control, N=9; STAT5aS711F rapamycin, N=10. The P-value in panel e (log-rank test) represents the comparison of survival in mice expressing STAT5aS711F treated with rapamycin vs vehicle control.

Rapamycin synergizes with ABT-737 to inhibit cytokine-independent myeloid cell survival

We next tested whether the mTOR inhibitor rapamycin would be effective at suppressing growth of a broader set of hematopoietic cells expressing mutations relevant to human disease. BaF3 cells expressing STAT5aS711F were compared with BaF3 cells expressing BCR-ABL or TEL-JAK2. All three cell lines were cytokine independent and were sensitive to rapamycin-induced growth suppression. Cell lines expressing only the IR-GFP empty vector still required IL-3 for growth stimulation. The BaF3 cells were cultured in a low dose of 0.01 ng/ml IL-3 and this concentration was sufficient to keep the control cells alive during 48 h without significant loss of viability. Overexpression of STAT5aS711F enhanced Akt activation and downstream phosphorylation of p70S6 kinase (p70S6K) and AKT relative to the IR-GFP control (Figure 5a). Treatment with rapamycin for 24 h at the concentration of 1 nM effectively blocked STAT5aS711F-mediated growth and suppressed p70S6K without having any direct impact on STAT5 tyrosine or Akt serine phosphorylation. It is noteworthy that although rapamycin significantly inhibited proliferation (Figure 5b), it did not induce significant loss of cell viability in any of the BaF3 cell lines.

Figure 5
Figure 5

Rapamycin combined with ABT-737 synergistically kills transduced BaF3 cells expressing STAT5aS711F, BCR-ABL or TEL-JAK2. (a) BaF3 cells were transduced with the IR-GFP, STAT5aS711F, BCR-ABL or TEL-JAK2 vector. The cells were treated with 1 nM rapamycin overnight. Recombinant IL-3 at a minimal concentration of 0.01 ng/ml was added to IR-GFP cells to maintain their viability. Cell extracts were resolved by SDS–PAGE and immunoblot was performed with the indicated antibody. (b) Cells were cultured in serial concentrations of rapamycin for 48 h and cell expansion was measured by manual scoring using a hemacytometer. (c) Cells were cultured with the indicated concentration of ABT-737 for 48 h and cell viability was measured by Trypan blue exclusion. (d) Cells were cultured in 0.2 ng/ml IL-3 or without IL-3 as indicated. For each group, the cells were treated with either rapamycin (1 nM) or ABT-737 (5 μm) alone or in combination. After 48 h, cell death was determined by Trypan blue exclusion assay. P-values shown are for comparison of ABT-737 alone vs ABT-737/rapamycin combination groups (*indicates P<0.05).

Because rapamycin alone was not effective at killing the BaF3 cells, we combined it combined with ABT-737, an inhibitor of bcl-2/bcl-XL. ABT-737 was toxic in a dose-dependent manner up to 10 μM to all BaF3 cell lines (Figure 5c). However, when 5 μM ABT-737 was combined with a concentration of 1 nM rapamycin, a striking synergy was observed in cell lines expressing TEL-JAK2, BCR-ABL and STAT5aS711F increasing from 20 to >80% killing (Figure 5d). To extend this observation further, we assayed the effects of rapamycin or ABT-737 alone in human BCR-ABL-positive K562 cells. Human myeloproliferative neoplasms are more complex genetically than the primary BM cell or BaF3 model cells. Despite inhibition of phosphorylation of p70S6K at concentrations above 10 nM (Figure 6a), rapamycin alone had minimal effects on these cells (Figure 6b). K562 cells were then exposed to ABT-737, which showed very low toxicity at concentrations 5 μM and up to 30% death at 10 μM (Figure 6c). However, the combination of 10 μM ABT-737 and a nontoxic concentration of 1 nM rapamycin gave a synergistic increase in the percentage of cell killing (Figure 6d). In contrast, NB4 cells were more sensitive to rapamycin alone but showed no synergy when combined with ABT-737 (Figures 6e–h) indicating that the effect is not generalizable to all types of leukemia cells. Various doses and timing were tested for NB4 cells and no evidence of synergy was observed (data not shown). Similar results were also obtained in HL-60 cells (data not shown).

Figure 6
Figure 6

Rapamycin combined with ABT-737 synergistically kills human K562 cells. (a, e) K562 cells and NB4 cells were cultured at the indicated concentration of rapamycin overnight. The rapamycin activity was determined by phosphorylation of the p70S6K protein. Total p70S6K was used a control. (b, f) K562 and NB4 cells were cultured in the indicated concentration of rapamycin for 48 h and the fold proliferation of the cells was determined by manual cell count. (c, g) K562 cells and NB4 cells were cultured in the presence of ABT-737 for 48 h. The cell viability was determined by both PI staining and Trypan blue exclusion. Both methods gave similar results. (d, h) K562 and NB4 cells were cultured in rapamycin alone (10 nM), ABT-737 alone (5 μM) or the combination of both for 48 h. Cell viability was determined as described in Materials and methods.

Discussion

Activation of STAT5 has been frequently observed in human myeloid leukemias and myeloproliferative disorders. Persistent activation of STAT5 in a mouse model mimics the effect of upstream activating tyrosine kinases that activate tyrosine phosphorylate STAT5 and drive mouse MPD (Figure 7). Our transplant model thus has relevance for leukemia and MPDs in patients. Important roles for STAT5 were reported in the propagation of leukemias induced by BCR-ABL,26 Flt3-ITD27 and TEL-PDGFR28 in mouse models. The advantage of this model for study of downstream signaling from STAT5 is that the aberrant signaling is initiated by STAT5 and not through receptors that are capable of activating multiple signaling pathways.

Figure 7
Figure 7

Proposed model for targeting synergistic signaling nodes mediated by distinct STAT5 survival targets in myeloproliferative disease. On the basis of our earlier studies, it was important to assess the impact of the Gab2 interaction and PI3K/Akt pathway activation in the pathophysiology of MPD induced by persistently activated STAT5. In the STAT5aS711F murine retroviral transduction model, we were able to initiate MPD at the level of STAT5 without activating first the upstream receptor or nonreceptor tyrosine kinases that would simultaneously activate PI3K/Akt at the receptor level. Using genetic deletion of Gab2 or pharmacologic inhibition of mTOR with rapamycin, we were able to interrogate this signaling axis and determine its importance in the development and progression of MPD. Note that this diagram is overly simplified regarding PLCγ and MAPK pathway activation to focus on STAT5 and PI3K/Akt activation.

We have recently shown that persistently active STAT5a can interact physically with Gab2 to promote Akt activation in BaF3 cells and in primary BM cultures.8 However, the in vitro effects of TAT-Gab proteins on cell growth may not have recapitulated the complex intrinsic and extrinsic disease in vivo. Therefore, it was necessary to test the impact of PI3K pathway activation in oncogenic STAT5a-mediated MPD to establish the definitive role in a disease model. Although Gab2 was not essential for STAT5-induced leukemic expansion in vivo, Gab2 did have an important supportive role in several aspects of MPD induced by STAT5aS711F. In the absence of Gab2, myeloid cells were reduced in peripheral blood and tissues, such as the liver, spleen weights were normalized and overall survival was improved. These effects were also not related to transduction efficiency of the Gab2−/− host as levels of GFP+ cells in the BM before transplant were comparable. Moreover, we have previously reported that the numbers of hematopoietic stem cell and early progenitors are normal in the absence of Gab2.24 In addition, the percentage of GFP+ cells obtained using IR-GFP control were the same regardless of Gab2 genotype and we found that homing of Gab2−/− BM c-Kit+ cells was normal.

Unlike previous studies, we did not observe evidence of lymphoid hyperplasia induced by STAT5aS711F. This is perhaps related to a very different transplant protocol that differs in 5-fluorouracil treatment that increased retroviral transduction efficiency (percentage of GFP+ cells) and the use of pure C57BL/6 mouse strains for donor/host in these experiments compared with previous studies that used C57BL/6 × 129/Sv F1 mice (which achieved 5% GFP+ cells8, 9). Overall, we did not observe expansion of a myeloblastic c-Kit+ population in the transplant protocol. Thus, we refer to the disease as MPD instead of a myeloid leukemia. Of note, we observed expansion of nontransduced donor BM cells (GFP negative), indicating that STAT5-induced MPD may also involve cell extrinsic promoting factors. Oncostatin M is a myeloid cytokine and target gene of STAT5, which might partially explain this response. Similar cell extrinsic effects have been observed in retroviral models expressing TEL-JAK26 or JAK2V617F vectors,29 which activate both STAT5 proteins.

The presence of substantial phosphorylated Akt in the absence of Gab2 could be due to a number of possible alternative activation routes and will be the focus of future studies. Activation of cytokine receptor signaling through secreted growth factors such as IL-3 or granulocyte-monocyte colony stimulating factor is possible. Other Gab family members such as Gab1 or Gab3 could compensate for Gab2 in this disease model. All three Gabs are highly homologous and may have a redundant role in multiple aspects of hematopoietic development. Alternatively, STAT5 activation in the absence of Gab2 protein could lead to genetic compensation. Nevertheless, the phosphorylated Akt represented a key protein downstream of STAT5aS711F/Gab2/PI3K and this led us to question whether efficient targeting by the inhibitor of mTOR would be effective in this model.

We used rapamycin to test whether it would have a similar efficacy in the STAT5aS711F MPD model as was observed in the Gab2−/− genetic background. Strikingly, treatment with rapamycin at the early stage of MPD was very effective at preventing further development and expansion of myeloid cells. This effect was cytostatic but did not prevent the subsequent recurrence of MPD once the treatment was stopped. Compared with the permanent deletion of Gab2, rapamycin treatment gave a similar response regarding Gr-1+/Mac-1+ cell expansion and prolonged survival. Treatment with rapamycin in the transplant model is a very stringent system, as it was necessary to wait 4 weeks until hematopoietic reconstitution before initiation of therapy, to prevent graft failure. To slow down disease progression, we injected fewer donor cells, which allowed for a balance between donor engraftment and early disease development. In either the Gab2 genetic model or the rapamycin pharmacologic model, the survival was improved. Preliminary data show that the combination of rapamycin and Gab2 targeting may be effective but this finding needs to be further tested in vivo and will be more challenging to translate to the clinic. Although there is a complex interplay between Akt and the mTOR complexes and a negative feedback loop mediated by p70S6K inhibition of IRS controls serine 473 phosphorylation of Akt,30 we did not observe increased p70S6K in our BaF3 studies with our short 24 h rapamycin treatment. However, with this in mind, we may not have achieved the maximal attenuation of mTOR signaling in vivo, which may have limited our efficacy in controlling myeloid expansion and survival.

Rapamycin is an effective inhibitor of mTORC1 and has been previously shown to synergize with protein tyrosine kinase inhibitors.31, 32 Rapamycin also targets mcl-1 in glucocorticoid-resistant acute lymphoblastic leukemia33 and is synergistic with the BH3 mimetic, and bcl-2/bcl-XL inhibitor ABT-737 due to its contribution to disabling resistance to the intrinsic apoptotic pathway. For example, in lung tumor xenografts, ABT-737 synergized with rapamycin34 and the homologue ABT-263 synergized with rapamycin on lymphoma cells.35 We recently reported that induced expression of bcl-2 by STAT5 is critical for the development of lethal MPD.10 Therefore, we hypothesized that additional STAT5 direct target genes promoting cell survival might also need to be targeted. Indeed, we observed striking synergy in killing cells expressing BCR-ABL, TEL-JAK2 or mutant STAT5 when rapamycin was combined with ABT-737. Importantly, in the ABT-263 resistant cell line K562,36 which we show is relatively resistant to rapamycin and ABT-737 alone, was much more sensitive to the combination of rapamycin and ABT-737. In contrast, the classic APL subtype cell line NB4 that lacks constitutive STAT5 activation was not synergistically sensitive to the combined treatment. It is possible that STAT5 regulates mcl-1 or bcl-2A1 expression through both direct and indirect mechanisms to promote cell survival in MPD, comparable to recent reports.37, 38 However, in this study we focused on the therapeutic end points and did not profile expression of all bcl-2 family members. Further analysis of additional STAT5 target genes may be important for optimization of the approach outlined in Figure 7.

Overall, the similarity in response suggests that the in vivo STAT5aS711F model may be a useful tool for further testing drug combinations in vivo for their impact upon MPD progression and lethality. Targeting using specific Akt and PI3K inhibitors or combination mTORC1/2 inhibitors39 in our model may show even greater translational potential. Overall, our studies validate that the Gab2/PI3K/Akt/mTOR signaling axis is a therapeutic target capable of attenuating hematologic disease provoked by persistently active STAT5, which may find clinical use as an adjuvant in combination with drugs directed toward STAT5 target genes such as bcl-2 and bcl-XL.

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Acknowledgements

We thank Henry Koon for critical review of the article and Fang Xu for biostatistical support. We also thank Jalpa Patel and Emma Arriola at Abbott Laboratories for supplying ABT-737. This work was supported by NIH R01DK059380 (KDB), The Center for Stem Cell and Regenerative Medicine, SFB-F28 (RM) and the Flow Cytometry, Radiation Resources, and Histology Core Facilities of the Case Comprehensive Cancer Center (P30CA43703).

Author information

Author notes

    • Z Wang
    •  & K D Bunting

    Current address: Department of Pediatrics, Aflac Cancer Center and Blood Disorders Service, Emory University, Atlanta, GA, USA.

    • W Tse

    Current address: Department of Medicine, Mary Babb Randolph Cancer Center, West Virginia University, Morgantown, WV, USA.

    • W Tse
    •  & K D Bunting

    Current address: Case Comprehensive Cancer Center, Cleveland, OH, USA.

Affiliations

  1. Division of Hematology-Oncology, Department of Medicine, Case Western Reserve University, Cleveland, OH, USA

    • G Li
    • , K L Miskimen
    • , Z Wang
    • , W Tse
    •  & K D Bunting
  2. Department of Pathology, Summa Health Barberton Hospital, Barberton, OH, USA

    • X Y Xie
  3. INSERM, Department of Medicine, Universite de Picardie J. Verne, Amiens, France

    • F Gouilleux
  4. Ludwig Boltzmann Institute for Cancer Research, Vienna, Austria

    • R Moriggl

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Competing interests

The material is original research, has not been previously published and has not been submitted for publication elsewhere while under consideration. The authors also have no conflicts of interest to declare regarding the studies performed.

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Correspondence to K D Bunting.

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https://doi.org/10.1038/leu.2010.131

Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)

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