Introduction

Granulocyte colony-stimulating factor (G-CSF) is a crucial regulator of granulopoiesis by stimulating the proliferation, survival and maturation of myeloid progenitor cells. The effects of G-CSF are mediated through a specific cell-surface receptor, the granulocyte colony-stimulating factor receptor (G-CSF-R).¹ The membrane-proximal region of the G-CSF-R contains two conserved sequences, called Box 1 and Box 2, as well as a more distal and less-conserved Box 3 region. In addition, the cytoplasmic domain of the human G-CSF-R contains four conserved tyrosine residues (Y704, Y729, Y744 and Y764) that serve as potential docking sites for Src homology 2 (SH2) domains of signaling proteins.^{2, 3} Pathways known to be stimulated by the G-CSF-R include the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway,^{4, 5, 6, 7, 8} the Ras/Raf/MEK/mitogen-activated protein kinase (MAPK) pathway^{9, 10, 11, 12, 13} and the phosphatidylinositol (PI) 3-kinase/Akt pathway.^{14, 15, 16}

We have previously identified a subset of severe congenital neutropenia (SCN) patients that harbor acquired mutations in the gene encoding the G-CSF-R, which serve to truncate the carboxyl-terminus of the receptor.^{17, 18, 19, 20} This cohort of patients also exhibits a strong predisposition to acute myeloid leukemia (AML).¹ Although the role of G-CSF-R truncations in the etiology of SCN remains controversial,^{21, 22, 23, 24, 25} there has been little argument about the contribution of such mutations to leukemogenesis. Truncated receptors show normal affinity for G-CSF,¹⁷ but transduce a strong hyperproliferative signal but a defective maturation signal when expressed in myeloid cells,¹⁹ a result confirmed in mice expressing truncated G-CSF-Rs.^{22, 25, 26}

We and others have shown that this hyperproliferation is due, at least in part, to defective internalization,^{27, 28, 29} as well as the loss of the receptor docking site for a string of negative regulators, including Src homology domain phosphatase-1, SH2 domain-containing 5'-phosphatase, cytokine-induced Src homology 2-containing protein and suppressor of cytokine signaling 3.^{29, 30, 31} Collectively, this leads to a decreased 'off-rate'^{26, 27, 32} and extended activation of various signaling pathways.^{15, 26, 27, 29, 32} However, those signaling pathways actually responsible for the hyperproliferation have remained unknown. Here, we utilize receptor mutants, dominant-negative STATs and pharmacologic inhibitors to show that sustained STAT5 activation, probably via JAK2, is the major mediator of the hyperproliferative responses, with a lesser contribution from the MAPK/extracellular signal-regulated kinase kinase (MEK) and PI 3-kinase pathways.

Materials and methods

Cell culture

A subline of the interleukin (IL)-3-dependent murine myeloid cell line 32Dcl3, called 32D.cl8.6,²⁷ and derivatives thereof were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS) and 10 ng/ml of murine IL-3, at 37°C and 5% (v/v) CO₂. Bone marrow cells were obtained from gcsfr-715/715 mice, as described.²² Where necessary, cells were deprived of serum and growth factors for 4 h at 37°C in RPMI 1640 medium at a density of 2–4 10⁶ per ml and then stimulated with 100 ng/ml human G-CSF.

Plasmid construction, transfections and infections

Cloning of wild-type (WT) GCSFR cDNA, the truncation mutant mM1 (d685) and the SCN patient-derived truncation mDA (d715) into the eucaryotic expression vector pLNCX has been described previously.⁸ These constructs were linearized by PvuI digestion and transfected into 32D.cl8.6 cells by electroporation. After 48 h incubation, cells were selected with G418 (Gibco-BRL, Invitrogen, Glen Waverley, Australia) at a concentration of 0.8 mg/ml with multiple clones expanded for further analysis. Equivalent receptor expression was determined for multiple independent clones using anti-G-CSF-R antibodies, as described.⁸ For retroviral infections, pMX clones were generated containing mSTAT5A(WT) and mSTAT5A750(DN)³³ cloned upstream of an IRES-enhanced green fluorescent protein (EGFP) and transfected into the Phoenix A amphotrophic packaging cell line (ClonTech, Palo Alto, CA, USA) to produce recombinant retrovirus, following standard protocols. Virus supernatants were used to infect 32D cells harboring the pLNCX.GCSFR(mDA) vector utilizing RetroNectin (Takara Biomedicals, Shiga, Japan), as described by the manufacturer. After 48 h, puromycin was added to 1 g/ml to select for stably transduced cells. EGFP expression levels were assessed by flow cytometry using a FACScan and sterile sorted using a fluorescence-activated cell sorter (FACS)-Calibur (Becton Dickinson, San Jose, CA, USA). For STAT3 experiments, stable 32D clones were generated by transfecting a pBabe clone of mutant mDA followed by puromycin selection and subsequent confirmation of expression, as described above. A positive clone was subsequently co-transfected with either pCAGGS-Neo or pCAGGS-Neo.HA-STAT3F³⁴ and clones selected using neomycin and screening for STAT3 expression using immunoprecipitation–Western blot analysis (see below).

Cell proliferation, cell cycle, apoptosis and morphological analysis

To quantify proliferation, cells were incubated at an initial density of 1–2 10⁵ cells/ml in 10% FBS/RPMI medium supplemented with 100 ng/ml of human G-CSF or 10 ng/ml of murine IL-3 and viable cells counted on the basis of Trypan blue exclusion. Where necessary, the medium was replenished every 1–2 days, and the cell densities were adjusted to 1–2 10⁵ cells/ml. Alternatively, cells were washed thoroughly before incubation in triplicate wells of a 96-well plate at 1 10⁶ cells/ml with appropriate growth factors for 30 h and pulsed with methyl-³H-thymidine for the last 24 h. In some studies, inhibitors of PI 3-kinase (LY294002), MEK (PD098059) or JAK2 (AG490), or vehicle (dimethyl sulphoxide (DMSO)), were added to these cultures. Cells were analyzed for DNA content with propidium iodide (PI)³⁵ and for apoptosis with PI/Annexin V,³⁶ using an Annexin V-FITC Apoptosis Detection Kit (Sigma-Aldrich, Castle Hill, Australia). To analyze the morphological features, cells were spun onto glass slides and examined after May–Grünwald–Giemsa staining.

Electrophoretic mobility shift assay

Nuclear extracts were prepared from appropriate cell populations as described.⁸ For electrophoretic mobility shift assay (EMSA) analysis, extracts from approximately 0.4–0.5 10⁶ cells were incubated for 20 min at room temperature with 0.2 ng of ³²P-labeled double-stranded oligonucleotide: m67 (5'-CATTTCCCGTAAATC), a high-affinity mutant of the sis-inducible element of the human c-fos gene,³⁷ which binds STAT1 and STAT3, or -cas (5'-AGATTTCTAGGAATTCAATCC), derived from the 5' region of the -casein gene,³³ which binds STAT5 and STAT1. The DNA complexes were separated by electrophoresis on 5% (w/v) polyacrylamide gels containing 5% (v/v) glycerol in 0.25 Tris-Borate-ethylenediaminetetraacetic acid (EDTA) as described.⁸ The gels were dried and subsequently analyzed by autoradiography.

Preparation of cell lysates, immunoprecipitation and Western blotting

Cells were washed with 10 volumes of ice-cold phosphate buffered saline (PBS) supplemented with 10 mol/l Na₃VO₄, and lyzed by incubation for 30 min at 4°C in lysis buffer (20 mmol/l Tris-HCl, pH 8.0, 137 mmol/l NaCl, 10 mmol/l EDTA, 100 mmol/l NaF, 1% Nonidet P-40, 10% glycerol, 2 mmol/l Na₃VO₄, 1 mmol/l Pefabloc SC, 50 g/ml aprotinin, 50 g/ml leupeptin, 50 g/ml bacitracin and 50 g/ml iodoacetamide). Insoluble materials were removed by high-speed centrifugation for 15 min at 4°C. Immunoprecipitation and Western blotting was performed as described.⁸ Antibodies used were: anti-STAT3C (Santa Cruz, Santa Cruz, CA, USA), anti-HA (12CA5) (Roche, Dee Why, Australia), anti-pERK (E4) (Santa Cruz); anti-ERK (K-23) (Santa Cruz), anti-pAkt (Thr 308) (New England Biolabs, Beverly, MA, USA), and anti-Akt (New England Biolabs).

Results

Analysis of G-CSF-R mutants

We have previously established the murine myeloid 32D cell line as a useful model for assessing the biological effects mediated by the G-CSF-R, including the identification of strong hyperproliferative responses induced by truncated G-CSF-Rs derived from patients with SCN/AML,^{3, 11, 19, 27} typified by mDA (Figure 1a). To further elucidate the regions of the receptor responsible for the hyperproliferative phenotype, an additional mutant mM1, which deletes all sequences beyond Box 2 (Figure 1a), was also analyzed using the 32D cell model. Clones expressing equivalent levels of receptor (Figure 1b) were analyzed for their responsiveness to G-CSF (Figure 1c and d). As described previously,³ the WT G-CSF-R mediates a transient proliferative response, followed by differentiation into morphologically identifiable neutrophilic granulocytes, whereas the SCN-derived mDA leads to sustained proliferation with no differentiation. In contrast, cells expressing the more severely truncated mutant mM1 showed no differentiation and limited proliferation, which ceased after 6–8 days on G-CSF. Therefore, the region spanning residues 684–715 appears essential for the sustained proliferation from truncated mDA receptors.

Figure 1.

Analysis of G-CSF-R mutants. (a) Schematic representation of G-CSF-R mutants. Cytoplasmic domains of WT and mutant receptors mDA (715) and mM1 (683) are shown. Boxes 1 and 2 denote subdomains conserved in members of the hematopoietin receptor superfamily, whereas Box 3 is conserved only with a limited number of family members. The numbers of key residues are indicated. (b) FACS analysis of G-CSF-R mutants. Representative 32D.c18.6 clones expressing WT, or mutant (mDA, mM1) G-CSF-Rs. Cells were either stained with biotinylated mouse anti-human G-CSF-R antibodies, followed by phycoerythrin (PE)-conjugated streptavidin, biotinylated anti-streptavidin and finally PE-conjugated streptavidin (unfilled), or without the anti-G-CSF-R step (filled). (c) Proliferation responses of G-CSF-R mutants. Cell proliferation of 32D.c18.6 clones expressing WT (empty square, thick line), mDA (cross, thin line) and mM1 (filled triangle, thin line) receptors. Data represent the growth of a representative clone of five examined for each receptor. (d) Differentiation responses of G-CSF-R mutants. Maturation of 32D.c18.6 cells expressing WT or mutant G-CSF-Rs, as detailed in panel c, expressed as the percentage of living cells showing signs of maturation at each time point. Data represent the growth of a representative clone of five examined for each receptor. Note that mDA and mM1 overlap at 0% neutrophils.

Full figure and legend (83K)

STAT activation by G-CSF-R mutants

As STATs have been implicated in the control of differentiation and proliferation from the WT G-CSF-R,^{3, 7, 8, 38, 39, 40} we analyzed STAT activation from these various receptor forms. Whereas both WT and mDA receptors strongly activated STAT3 in both short-term (Figure 2a) and long-term (Figure 2b) assays, the mM1 form produced negligible activation in either assay, consistent with a role for Y704 in facilitating STAT3 docking.⁸ In contrast, the control of STAT5 activation was more complex. Short-term activation of STAT5 from WT receptors was strong but transient, whereas from SCN-derived mDA receptors it was strong and sustained, as previously reported.²⁷ In contrast, mM1 activated moderate levels of STAT5, indicating that both the membrane proximal region and residues 684–715 contribute to STAT5 activation. However, like mDA, the kinetics of activation from mM1 was sustained relative to the WT receptor in these short-term assays (Figure 2a). This is consistent with the notion that receptor truncation leads to loss of a di-leucine internalization motif^{27, 28} and binding sites for negative regulators that collectively lead to sustained signaling.^{29, 30, 31} However, long-term activation of STAT5 from mM1 was similar to the WT receptor, being considerably lower than the robust activation from mDA, suggesting other mechanisms might control STAT5 activation over more extended periods.

Figure 2.

STAT activation from G-CSF-R mutants. 32D.c18.6 cells expressing WT or mutant G-CSF-Rs were assessed for their ability to activate STAT proteins using the probes m67 (upper panels) and -cas (lower panels). The positions of the various dimers of STAT1 (S1), STAT3 (S3) and STAT5 (S5), as determined by supershift experiments, are indicated. (a) Short-term STAT activation. Cells were starved for 4 h then stimulated with G-CSF for the times indicated and assayed for STAT activation. Results are from a representative of at least three independent clones examined. (b) Long-term STAT activation. Cells were either grown in IL-3 (I) or switched to G-CSF (G) for 24 h and assayed for STAT activation. Results are from a representative of at least three independent clones examined.

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Role of STAT3 and STAT5 in hyperproliferation responses from truncated receptors

The collective results described above suggested that robust activation of STAT5, rather than STAT3, might contribute to the hyperproliferative responses of truncated receptors. To directly address this question, we separately introduced dominant-negative forms of these two STAT proteins into 32D[mDA] cells to assess their effects. Clones containing empty vector (mDA[vec]) or expressing HA-tagged dominant-negative STAT3 (mDA[S3F]) (Figure 3a) were compared for their responses to growth factors. No difference was observed in IL-3 responses between the clones (data not shown). However, clones expressing the dominant-negative STAT3 showed a slight, but statistically significant, increase in proliferation in response to G-CSF (Figure 3b). None of the clones produced appreciable differentiation (data not shown).

Figure 3.

STAT3 exerts a negative effect on hyperproliferative responses from truncated G-CSF-Rs. (a) Expression of dominant-negative STAT3. Western blot analysis of anti-STAT3 immunoprecipitation of cell lysates prepared from 32D[mDA] cells co-transfected with either empty vector (mDA[vec]) or one encoding a HA-tagged dominant-negative STAT3 (mDA[S3F]). The Western blot was probed with anti-HA antibodies (-HA: upper panel) to detect exogenous STAT3 before stripping and re-probing with anti-STAT3 antibodies (-STAT3: lower panel) to quantitate total STAT3 levels. A representative independent clone is shown out of four analyzed. (b) Proliferation responses of STAT3 clones. Cytokine-mediated cell proliferation of mDA[vec] (+IL-3: cross; +G-CSF: open square) and mDA[S3F] (+IL-3: plus; +G-CSF: closed square). Data are derived from a representative of three experiments performed on four clones. ^**Calculated doubling times were significantly different: mDA[vec]+G=22.5 h (95% confidence interval (CI): 19.6–26.4 h); mDA[S3F]+G=16.6 h (95% CI: 15.7–17.7 h).

Full figure and legend (75K)

To examine the role of STAT5, a similar strategy was employed to that described for STAT3. However, despite several rounds of transfection, no clones showed reduced STAT5 were produced (data not shown). To overcome this problem, 32D[mDA] cells were infected with retroviruses expressing either WT or dominant-negative STAT5 cloned as a transcriptional fusion with EGFP. The EGFP⁺ population was sorted (Figure 4a) and expanded in IL-3 for further analysis from two independent batches of infected cells. In each case, the EGFP⁺ population derived from cells infected with dominant-negative STAT5 (mDA[S5dn]) showed reduced STAT activation compared to those derived from cells infected with WT STAT5 (mDA[S5wt]), in both short-term (Figure 4b) and long-term (Figure 4c) assays. STAT3 activation was also reduced, but to a lesser extent. Cells infected with the dominant-negative form of STAT5 also showed approximately 50% of the proliferative responses of the WT controls in response to G-CSF (Figure 4d), as well as even greater reduction in IL-3 responses, both of which were statistically significant. The experiment was also repeated and EGFP⁺-expressing cells plated out by limiting dilution to obtain EGFP⁺ clones. Such clones produced a similar profile of G-CSF responses (data not shown).

Figure 4.

STAT5 contributes to the hyperproliferation from truncated G-CSF-Rs. (a) Transduction with WT and dominant-negative STAT5. Flow cytometric analysis of EGFP expression of 32D[mDA] cells infected in bulk with either WT STAT5-IRES-EGFP (mDA[S5wt]) or dominant-negative STAT5-IRES-EGFP (mDA[S5dn]), with the sorted EGFP⁺ population used for further experiments indicated with the box. This is a representative of two independent transductions; in each case the percentage of EGFP⁺ cells was 2.5–3.5%. (b) Effect of dominant-negative STAT5 on short-term STAT activation. EMSA of nuclear extracts prepared from populations sorted from two independent transductions, as shown in panel a. Cells were starved for 4 h, and then incubated for 10 min in absence (-) or presence (+) of G-CSF, and nuclear extracts analyzed using m67 and -cas probes. (c) Effect of dominant-negative STAT5 on long-term STAT activation. EMSA of nuclear extracts prepared from sorted populations shown in panel a. Cells were either grown in IL-3 (I) or switched to G-CSF (G) for 24 h and nuclear extracts analyzed using m67 and -cas probes. (d) Effect of dominant-negative STAT5 on proliferation. Proliferation of mDA[S5wt] or mDA[S5dn] cells in response to IL-3 or G-CSF, measured via ³H-thymidine incorporation, with that of mDA[S5wt] stimulated with IL-3 set to 100% in each case. Results are from a representative experiment with the s.d. indicated. Significance was determined using an unpaired t-test assuming unequal variance: ^***P<0.005.

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Role of other pathways in hyperproliferative responses

The results described above suggested a positive role for STAT5 in mediating the hyperproliferative responses of truncated G-CSF-Rs. However, previous studies have shown that truncated G-CSF-Rs also produce sustained activation of both extracellular signal-regulated kinase (ERK) and Akt,^{15, 16} a result confirmed in our 32D[mDA] clones (data not shown). To assess the relative effects of these pathways on the hyperproliferative responses of truncated receptors, we used specific pharmacological agents, LY294002, PD098059 and AG490, targeting the kinases PI 3-kinase (upstream of Akt), MEK (upstream of ERK) and JAK2 (upstream of STAT5), respectively. The JAK2 inhibitor was the most potent inhibitor of proliferation, causing a 85–95% reduction when used at 20 M (Figure 5a and b). In contrast, the MEK inhibitor resulted in a reduction of approximately 40–50% at 10–40 M, whereas the PI 3-kinase inhibitor inhibited proliferation by 30–40% at 12.5–50 M. Each was effective in 32D[mDA] cells at these concentrations, as judged by inhibition of activation of the respective downstream signaling molecule (Figure 5c–e). The JAK2 inhibitor produced similar levels of inhibition of 32D[WT] cells (data not shown). Analysis of the cell cycle profile revealed no gross changes in the presence of the inhibitors, with only a small increase in the S- and G2/M-phase content observed with AG490 (Figure 5f). Again, only AG490 caused any appreciable increase in apoptosis (Figure 5g), although this was still small, and insufficient to explain the inhibition of cell growth as judged by thymidine incorporation and Trypan blue exclusion. Interestingly, in mDA[S5dn] cells, the relative potency of the MEK and PI 3-kinase inhibitors increased (Figure 5h), suggesting that these pathways act synergistically with the STAT5 pathway. Application of the inhibitors to bone marrow cells of gcsfr-715/715 mice, which carry a knock-in mutation equivalent to mDA,²² largely recapitulated the inhibitor results seen in 32D[mDA] cells (Figure 5i), confirming their in vivo relevance.

Figure 5.

Involvement of other pathways in hyperproliferative responses from truncated G-CSF-Rs. (a and b) Effect of inhibitors on G-CSF-mediated proliferation. Proliferation of 32D[mDA] cells in the presence of 100 ng/ml G-CSF as well as specific inhibitors for PI 3-kinase (LY294002, LY – with M concentration shown), MEK (PD098059, PD) and JAK2 (AG490, AG). Proliferation was measured on triplicate samples via viable cell number enumeration (a) or ³H-thymidine incorporation (b), with DMSO-treated samples used as controls. The results show mean and s.d. from a representative of three experiments. (c and d) Effectiveness of inhibitors on downstream signaling molecules. 32D[mDA] cells were starved for 4 h before stimulation for 10 min with (+) or without (-) G-CSF (100 ng/ml), in the presence of specific inhibitors of PI 3-kinase (c), MEK (d) and JAK2 (e). Total cell lysates subjected to Western blot analysis with activation-specific antibodies for the downstream signaling molecules Akt (PI 3-kinase – c) and ERK (MEK – d). Membranes were subsequently stripped and reprobed with a corresponding antibody to detect total Akt and total ERK, respectively. Alternatively, nuclear extracts were analyzed for activation of STAT5 and STAT3 using the probes -cas (e – upper panel) and m67 (e – lower panel). This was a representative of three experiments. (f and g) Analysis of cell cycle profile and apoptosis in the presence of inhibitors. 32D[mDA] cells were treated as described in panel a, and subjected to cell cycle analysis using PI-staining (f) and apoptosis analysis using PI/Annexin V (g). (h and i) Effect of inhibitors on other cells. Proliferation of mDA[S5dn] cells (h) or gcsfr-715/715-derived bone marrow cells (i) in the presence of specific inhibitors for PI 3-kinase (LY), MEK (PD) and JAK (AG) pathways measured via ³H-thymidine incorporation, as described above. The results presented are a representative of three experiments.

Full figure and legend (198K)

Discussion

The results presented here show unequivocally in the 32D cell model that sustained STAT5 activation, most probably via JAK2, is a key mediator of the hyperproliferative responses triggered by truncated G-CSF-Rs. In addition, signaling via other pathways, including those involving both PI 3-kinase and MEK, also appears to make a contribution to the enhanced mitogenic response. In contrast, STAT3 appears to exert a negative influence, in agreement with recent studies showing that STAT3 inhibits proliferative responses in the context of the full-length G-CSF-R.^{40, 41} Therefore, it is likely that the consequences of receptor truncation are a result of the net effect of a complex combination of signals, at least in 32D and gcsfr-715/715 cells, although it remains to be confirmed in primary cells from appropriate SCN/AML patients.

The PI 3-kinase/Akt pathway has been shown to be an important component of oncogenic signaling generally. For example, its activation is required for Ros-induced cell transformation,⁴² and it also contributes to the mitogenic signaling from the oncogenic Xmrk.^{43, 44} Studies in chicken B cells suggest that it has a role in proliferation as well as survival signaling from the full-length G-CSF-R,⁴⁵ whereas others have shown that PI 3-kinase is activated by G-CSF and correlates with inhibition of apoptosis.¹⁴ Furthermore, one of the key signaling molecules downstream of PI 3-kinase, Akt, has been shown to be activated in a sustained manner by truncated G-CSF-Rs.^{15, 16} Our results show that this prolonged activation of the PI 3-kinase/Akt pathway probably also contributes to proliferative signaling in the context of the truncated G-CSF-R.

The p21^ras/MEK/MAPK pathway is also a key component of mitogenic responses in a wide variety of cell systems.⁴⁶ Moreover, several studies have also implicated components of the p21^ras/MEK/MAPK pathway in proliferative responses from the full-length G-CSF-R, including the upstream signaling molecules Shc, Grb2 and SHP-2,^{2, 3, 47}, p21^ras itself,^{10, 11, 48, 49} MEK^{50, 51} and ERK.^{13, 51} In addition, expression of dominant-negative p21^ras is able to inhibit G-CSF-mediated proliferation without affecting differentiation.⁴⁸ In the context of the full-length receptor, the p21^ras/MEK/MAPK pathway is abrogated by removal of the C-terminal tyrosine, Y764, which also blocks proliferation but not differentiation.^{11, 13} The truncated mDA receptor, on the other hand, is able to activate this pathway despite the absence of this tyrosine.³² Interestingly, unlike other pathways, activation of ERK by mDA is similar in extent and length to that seen from the WT receptor,³² perhaps providing some explanation for why inhibitors of this pathway only exert a reduced effect on proliferation.

Perturbed activation of JAK-STAT pathway components has been associated with several hematopoietic disorders.⁵² From these studies, it is clear that hyperactivation of JAK2 results in myeloproliferative disorders⁵³ and constitutive STAT5 activation is consistently observed in leukemia (compared to STAT3 in lymphoma).⁵² In addition, STAT5 appears to be essential for the myelo- and lymphoproliferative disease induced by TEL–JAK2 fusions,⁵⁴ and is also required for cell cycle progression, survival and leukemogenesis induced by BCR-ABL.^{55, 56} Potential roles in other cancers have also been identified, including melanoma⁴⁴ and mammary cancer.⁵⁷ By corollary, expression of constitutively active forms of STAT5 has been shown to result in enhanced proliferation of myeloid cell lines³⁸ and of hematopoietic cells in vivo,⁵⁸ which we have recently confirmed.⁵⁹ The results presented here showing sustained STAT5 activation downstream of JAK2 as a crucial mediator of the hyperproliferative responses from truncated G-CSF-Rs associated with AML further emphasizes the importance of the JAK2-STAT5 module in malignancy – a role that has apparently been conserved throughout evolution.^{59, 60} This is perhaps not surprising given the important cell cycle regulators known to be stimulated by STAT5, including cyclin D1⁶¹ and c-myc,⁶² not to mention its positive effects on cell survival mediators, such as bcl-XL and bcl-2.^{56, 63} Together, this suggests that suppression of STAT5 function may represent an important general strategy in the treatment of leukemias and other cancers.

However, our results also point to a conspiracy of pathways being required to mediate full proliferative potency from truncated G-CSF-Rs, which appears to be a common theme in oncogenesis. For example, the full mitogenic signaling potency from the oncogenic Xmrk kinase is apparently also mediated by PI 3-kinase, MAPK and STAT5 pathways.^{43, 44} The challenge remains to delineate the exact contribution of each of these pathways to the transformation process at the molecular level, which is finally become a reality using microarray and other technologies.

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Acknowledgements

We thank Cristal Peck for expert help with paper preparation, Dora McPhee, Louise Wangerek and Michelle Hookham for technical assistance, as well as Mirjiam Hermans, Jim Johnston, Karim Dib and Massimo Gadina for reagents. This work was supported by grants from the KWF Kankerbestrijding, the NWO and the Deakin University Central Research Grants Scheme. ACW was variously supported by an EMBO Long Term Fellowship, a Sylvia and Charles Viertel Senior Medical Research Fellowship and a Deakin University International Study Program Grant.