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STAT5 and IL-6 dysregulation in mastocytosis
ated with poor clinical outcomes, blocking IL-6 or its syn- thesis in these patients is viewed as a potential therapeutic avenue.7,8
In SM, the levels of serum IL-6 are higher in patients with aggressive versus indolent variants of SM and have been associated with adverse clinical features of mastocy- tosis such as accumulation of mast cells in the BM, organomegaly, elevated tryptase levels,9,10 osteoporosis and/or bone pain.11 Although progression into more aggressive disease within patients with indolent SM (ISM) occurs only in a subset of patients, IL-6 plasma levels sig- nificantly correlate with disease progression and lower progression-free survival, suggesting that blockade of IL-6 synthesis or function may be beneficial in cases with aber- rant IL-6 pathways.10 Other studies have shown that IL-6 promotes the differentiation, growth and degranulation of normal mast cells,12 and induces the production of reactive oxygen species by malignant mast cells and their accumu- lation in tissues in a model of mastocytosis.13 Despite the potential implications for disease pathology, the cell types and the mechanisms that may contribute to the constitu- tively elevated IL-6 levels in mastocytosis are not known.
In this study, we test the hypothesis that cells express- ing gain of function variants of KIT, particularly D816V- KIT, confer the ability to constitutively produce IL-6. As will be shown, ex vivo BM mast cells from patients with SM release IL-6 in correlation with the allelic frequency of D816V-KIT. We further demonstrate that expression of D816V-KIT causes persistent IL-6 induction by mecha- nisms independent of autocrine feed-forward loops involving IL-6 and signal transducer and activator of tran- scription 3 (STAT3) described in other malignant cells, but dependent on oncogenic KIT-derived signals. These sig- nals include phosphatidylinositide 3-kinase (PI3K) path- ways and oncogenic STAT5 activation by both janus kinase 2 (JAK2) and, unexpectedly, by the mitogen-acti- vated protein kinase MEK/ERK1/2 pathways. These data expand our understanding of the potential mechanisms initiating enhanced IL-6 production in mastocytosis and emphasize targets for therapeutic intervention in cases of high IL-6 profiles and suspected disease progression.
Methods
A detailed description of the methods used in this study can be found in Online Supplementary Appendix.
Study patients
Bone marrow samples were obtained when clinically indicated from patients with SM classified according to the World Health Organization (WHO) guidelines1,3,14 (Online Supplementary Table S1). All human samples were obtained after informed consent, on clinical protocols approved by the Institutional Review Board of the National Institute of Allergy and Infectious Diseases (02-I-0277 and 08-I-0184) in agreement with the Declaration of Helsinki. D816V-KIT mutation analysis and its allele burden in the BM (D816V-KIT frequency) were determined by allele-specific poly- merase chain reaction (PCR) from patient blood and BM genomic DNA.15
Cell lysates
Cell lines were cultured as described in the Online Supplementary Appendix. To obtain lysates for western blots, 3x106 cells were plat- ed in 6-well plates and incubated with or without the indicated
inhibitors for 2 hours (h) in serum-free media. Cells were lysed as previously described.16
IL-6 measurements
Cells (3x106) were plated in 6-well plates for 2 h to overnight in 6 mL of serum-free media to exclude the possibility that any extrinsic stimulant present in the serum would influence the results. IL-6 released into the media was measured by ELISA (R&D Systems). Human colorectal carcinoma HCT116 cells were stimulated with 20 ng/mL PMA plus 1 mM ionomycin overnight and the supernatants then collected for IL-6 measurements.
Mononuclear cells in BM aspirates from patients were cultured in StemPro-34 medium with human recombinant SCF (100 ng/mL) for 2-4 days. IL-6 released into the media was determined by ELISA. Alternatively, IL-6 expression in single cells was deter- mined by flow cytometry using a LSRII flow cytometer. BM cells were incubated with Brefeldin A for 4 h and stained with an anti- body cocktail containing anti-CD3-QDOT605, anti-CD34-APC, anti-KIT-BV605 and anti-FcεRI-FITC, for 30 minutes (min). Cells were fixed, permeabilized and stained with anti-IL-6-PE for 30 min. Expression of IL-6 in mast cells (CD3–/CD34–/KIT+/FcεRI+) was analyzed using FlowJo software.
Quantitative real-time polymerase chain reaction
HMC-1.2 cells (3x106) were plated in 6-well plates in 6 mL and incubated for 2 h in serum-free media. Cellular RNA was extract- ed and reverse-transcribed into cDNA. cDNA was then amplified using TaqMan® Gene Expression Master Mix and Taqman® Gene Expression Assays for IL-6, STAT3, STAT4, STAT5A, STAT5B or GAPDH as described in the Online Supplementary Appendix.
Knockdown of STAT transcription factors
Knockdown of STAT3 and STAT4 was performed by lentiviral- mediated transduction of small hairpin RNA (sh-RNA) (Sigma- Aldrich, St. Louis, MO, USA) as previously described.17 STAT5 mRNA was silenced by a small interference-RNA (si-RNA) “ON- TARGET” pool from Dharmacon (Lafayette, CO, USA), intro- duced into cells by electroporation.
Statistical analysis
Data were expressed as mean±Standard Error of Mean (SEM). Values were from at least three independent experiments, each performed at least in duplicate. Statistically significant differences were calculated by using the Student t-test (unpaired). Statistical significance was indicated as follows: *P<0.01; **P<0.01; ***P<0.001; ****P<0.0001.
Results
Release of IL-6 from patient’s bone marrow cells and its association with D816V-KIT and mast cell frequencies
The levels of IL-6 in serum9 as well as the allelic frequen- cy of D816V-KIT18 correlate with the levels of tryptase, a surrogate marker of mast cell burden. As mast cells often accumulate in the BM in SM, we tested the ability of BM cells to produce IL-6 in short-term cultures. BM mononu- clear cells isolated from patients with SM showed varied ability to release IL-6 into the culture media after 2-4 days, with more release observed in cells from patients with a higher BM D816V-KIT allelic frequency (Figure 1A). Although cells other than mature mast cells may express D816V-KIT,18 the release of IL-6 into the media correlated with the percentage of mast cells within BM live cells
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