Oncogenic D816V-KIT signaling in mast cells causes persistent IL-6 production

Oncogenic D816V-KIT signaling in mast cells causes persistent IL-6 production by Araceli Tobío, Geethani Bandara, Denise A. Morris, Do-Kyun Kim, Michael P. O'Connell, Hirsh D. Komarow, Melody C. Carter, Daniel Smrz, Dean D. Metcalfe, and Ana Olivera Haematologica 2019 [Epub ahead of print] Citation: Araceli Tobío, Geethani Bandara, Denise A. Morris, Do-Kyun Kim, Michael P. O'Connell, Hirsh D. Komarow, Melody C. Carter, Daniel Smrz, Dean D. Metcalfe, and Ana Olivera. Oncogenic D816V-KIT signaling in mast cells causes persistent IL-6 production. Haematologica. 2019; 104:xxx doi:10.3324/haematol.2018.212126 Publisher's Disclaimer. E-publishing ahead of print is increasingly important for the rapid dissemination of science. Haematologica is, therefore, E-publishing PDF files of an early version of manuscripts that have completed a regular peer review and have been accepted for publication. E-publishing of this PDF file has been approved by the authors. After having E-published Ahead of Print, manuscripts will then undergo technical and English editing, typesetting, proof correction and be presented for the authors' final approval; the final version of the manuscript will then appear in print on a regular issue of the journal. All legal disclaimers that apply to the journal also pertain to this production process. Copyright 2019 Ferrata Storti Foundation. Published Ahead of Print on April 4, 2019, as doi:10.3324/haematol.2018.212126. Tobío et al., 1 Oncogenic D816V-KIT signaling in mast cells causes persistent IL-6 production Araceli Tobío1, Geethani Bandara1, Denise A. Morris1, Do-Kyun Kim1 , Michael P. O’Connell2, Hirsh D. Komarow1, Melody C. Carter1, Daniel Smrz1, Dean D. Metcalfe1 ¶, Ana Olivera1¶* 1Mast Cell Biology Section, Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA 2Genetics and Pathogenesis of Allergy Section, Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA ¶ DDM and AO are senior co-authors *Correspondence: Ana Olivera [email protected] Running title: STAT5 and IL-6 dysregulation in mastocytosis Word Count: Abstract: 250 Main text: 4,294 Number of Figures: 6 Contains a Supplementary Appendix file: Supplementary Methods, Supplementary Tables 1 and 2, Supplementary Figures S1-5 and References Acknowledgements We thank Robin Eisch in her role as protocol study coordinator, Linda Scott, the nurse practitioner on the study protocols, Pahul Hanjra and Irina Maric for providing information on patients and Daly Cantave for arranging for patient bone marrow samples. Tobío et al., 2 Abstract Persistent dysregulation of IL-6 production and signaling have been implicated in the pathology of various cancers. In systemic mastocytosis, increased serum levels of IL-6 associate with disease severity and progression, although the mechanisms involved are not well understood. Since systemic mastocytosis often associates with the presence in hematopoietic cells of a somatic gain-of-function variant in KIT, D816V-KIT, we examined its potential role in IL-6 upregulation. Bone marrow mononuclear cultures from patients with greater D816V allelic burden released increased amounts of IL-6 which correlated with the percentage of mast cells in the cultures. Intracellular IL-6 staining by flow cytometry and immunofluorescence was primarily associated with mast cells and suggested a higher percentage of IL-6 positive mast cells in patients with higher D816V allelic burden. Furthermore, mast cell lines expressing D816V-KIT, but not those expressing normal KIT or other KIT variants, produced constitutively high IL-6 amounts at the message and protein levels. We further demonstrate that aberrant KIT activity and signaling are critical for the induction of IL-6 and involve STAT5 and PI3K pathways but not STAT3 or STAT4. Activation of STAT5A and STATB downstream of D816V-KIT was mediated by JAK2 but also by MEK/ERK1/2, which not only promoted STAT5 phosphorylation but also its longterm transcription. Our study thus supports a role for mast cells and D816V-KIT activity in IL-6 dysregulation in mastocytosis and provides insights into the intracellular mechanisms. The findings contribute to a better understanding of the physiopathology of mastocytosis and suggest the importance of therapeutic targeting of these pathways. Tobío et al., 3 Introduction Mastocytosis defines a group of heterogeneous disorders characterized by the accumulation of neoplastic/clonal mast cells in the skin, bone marrow (BM) and other organs 1. Mastocytosis is clinically subdivided into systemic (SM) and cutaneous (CM) mastocytosis, both of which are comprised of several variants defined in accordance with histologic and clinical parameters and organ involvement 1. Somatic variants in the receptor for stem cell factor (SCF), KIT, that render it constitutively active often associate with SM, particularly p.(D816V), a missense in the tyrosine kinase domain of KIT. D816V-KIT may be accompanied by variants in other genes that further contribute to the oncogenic expansion of mast cells 2-4. Interleukin-6 (IL-6) is a pleiotropic cytokine produced by several cell types including stromal, hematopoietic and tumor cells. In addition to its involvement in normal inflammatory processes and host immune defense mechanisms, IL-6 may contribute to malignancy in a range of cancers including multiple myeloma, B cell and non-B cell leukemias and lymphomas 5, 6, by modulating cellular development, growth, apoptosis, metastasis and/or cellular resistance to chemotherapy 6. As elevated IL-6 levels in the serum of patients with such malignancies have been associated with poor clinical outcomes, blocking IL-6 or its synthesis 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 mastocytosis 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 significantly correlate with disease progression and lower progression-free survival, suggesting that blockade of IL-6 synthesis or function may be beneficial in cases with aberrant 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 accumulation 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 constitutively elevated IL-6 levels in mastocytosis are not known. In this study, we test the hypothesis that cells expressing 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 mechanisms independent of autocrine feed-forward loops involving IL-6 and signal transducer and activator of transcription 3 (STAT3) described in other malignant cells, but dependent on oncogenic KIT-derived signals. These signals include phosphatidylinositide 3-kinase (PI3K) pathways and oncogenic STAT5 activation by both janus kinase 2 (JAK2) and, unexpectedly, by the mitogen-activated protein kinase Tobío et al., 4 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 Patients of study BM samples were obtained when clinically indicated from patients with SM classified according to the WHO guidelines 1, 3, 14 (Online Supplementary Table 1). 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 08I-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 PCR from patient blood 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 plated in 6 well plates and incubated with or without the indicated inhibitors for 2 h in serum-free media. Cells were lysed as 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 µM PMA plus 1 µM 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 to 4 days. IL-6 released into the media was determined by ELISA. Alternatively, IL-6 expression in single cells was determined by flow cytometry using a LSRII flow cytometer. BM cells were incubated with Brefeldin A for 4 h and stained with an antibody cocktail containing anti-CD3-QDOT605, anti-CD34-APC, antiKIT-BV605 and anti-Fc ε RI-FITC, for 30 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+/Fc ε RI+) was analyzed using FlowJo software. Quantitative real-time PCR Tobío et al., 5 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 extracted 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) as described 17. STAT5 mRNA was silenced by a small interference-RNA (si-RNA) “ON-TARGET” pool from Dharmacon (Lafayette, CO), introduced into cells by electroporation. Statistical analysis Data were expressed as mean ±SEM. Values were from at least 3 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 and ****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 serum 9 as well as the allelic frequency 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 mononuclear cells isolated from patients with SM showed varied ability to release IL-6 into the culture media after 2 to 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 (Figure 1B), suggesting a contributory role for mast cells in IL-6 production. Additionally, we analyzed intracellular IL-6 staining in single BM mast cells by flow cytometry from three separate patients described in the Online Supplementary Table 1. Patient 1 had idiopathic anaphylaxis and did not meet criteria for SM and thus was used as a control. This patient had no detectable D816V-KIT, 0.098% of BM cells were CD3-/CD34-/KIT+/Fc ε RI+ (mast cells) and a minor percentage of these were IL-6 positive (0.063%) (Figure 1C, left panel). However, CD3- /CD34-/KIT+/Fc ε RI+ cells from patients 2 and 3, with BM D816V frequencies of 2.7% and 5.5%, were ∼77% and 99% positive for IL-6, respectively (Figure 1C, middle and right panels). In this ex-vivo experiment, where BM cells were cultured up to 4 d in the presence of Tobío et al., 6 SCF, cell lineages other than mast cells (KIT+/Fc ε RI-, KIT-/Fc ε RI+ and KIT-/Fc ε RI-) also showed positive intracellular IL-6 staining (Online Supplementary Table 2). However, the highest IL-6 mean fluorescence intensity (MFI) was associated with KIT+ cells. As SCF in the media may induce IL-6 directly or through autocrine/paracrine signals in clonal and nonclonal cells in these cultures, we examined the expression of IL-6 in BM biopsies by immunofluorescence (IF). IF images of BM of patients with SM indicated that IL-6 intracellular content was mostly associated with mast cells (Online Supplementary Figure S1). Thus, the combination of ex-vivo and in situ experiments suggests mast cells as the predominant producers of IL-6, with variable participation of other cell lineages. Data are also consistent with an association between increased IL-6 expression by BM mast cells and D816V-KIT frequency. Expression of D816V-KIT causes IL-6 upregulation and secretion Given these associations and that mast cells and their progenitors often carry somatic D816V-KIT variants in SM, we investigated the hypothesis that D816V-KIT expression intrinsically promotes IL-6 production. Thus, we analyzed the production of IL-6 at the protein and message levels in various cell lines expressing or not D816V-KIT. In agreement with our previous observations, the HMC-1.2 mastocytosis mast cell line which harbors KIT with D816V plus another missense variant in the juxta membrane domain of KIT (V560G), showed markedly higher IL-6 mRNA synthesis13 (Figure 2A, left panel) and IL-6 release than HMC-1.1 cells, which express only the monoallelic V560G-KIT variant (Figure 2A, right panel). The mastocytoma mouse mast cell line P815 carrying the homolog to the D816V-KIT variant (D814Y-KIT), also released significantly more IL-6 than primary mouse BMMCs (Figure 2B). Furthermore, expression of human D816V-KIT in an immortalized mouse mast cell line that lacks KIT 13, 19, unlike cells expressing normal KIT or vector alone, released significant amounts of IL-6 into the media (Figure 2C). In aggregate, the results using the various mast cell lines demonstrate an association between expression of D816V-KIT in mast cells and persistent transcription and release of IL-6. This may not be restricted to mast cells, since introduction of D816V-KIT by CRISPR in the colorectal carcinoma cell line HCT116 also promoted IL-6 transcription and release (Figure 2D left and right panels, respectively) when cells were stimulated with PMA and ionomycin, indicating that in these cells D816V-KIT primes HCT116 cells for more robust IL-6 production. KIT tyrosine kinase activity is required for IL-6 production in mast cells Ligand-activated KIT signaling induces IL-6 production in mast cells and enhances IgEreceptor-driven IL-6 production 20. Indeed, stimulation of KIT by SCF in the LAD2 cell line, which expresses normal KIT, induced IL-6 release, albeit the amounts were quantitatively limited (Figure 3A). HMC-1.1 showed higher production of IL-6 than LAD2 cells, particularly when stimulated with SCF (Figure 3A). However, not only was IL-6 production ~100 fold higher in unstimulated HMC-1.2 than in LAD2 or HMC-1.1 cells, but ligand- Tobío et al., 7 induced stimulation of KIT did not cause any further IL-6 secretion by HMC-1.2 cells (Figure 3A). These data are consistent with the conclusion that ligand-independent signals induced by oncogenic KIT activity, particularly those from D816V-KIT, are more effective in inducing IL-6 secretion than ligand-activated KIT signals. We next blocked D816V-KIT activity in HMC-1.2 cells with dasatinib, a tyrosine kinase inhibitor that effectively suppresses the activity of the active, open conformation of D816VKIT. Dasatinib markedly reduced IL-6 mRNA levels (Figure 3B) and IL-6 secretion (Figure 3C), while concentrations of the tyrosine kinase inhibitor imatinib that are ineffective in blocking D816V-KIT, did not alter IL-6 mRNA levels (Figure 3B), further suggesting an involvement of D816V-KIT signaling. Inhibition by gefitinib of the epidermal growth factor receptor (EGFR), a tyrosine kinase receptor that can drive IL-6 production in transformed cells 21, had no significant effects on IL-6 mRNA levels in HMC-1.2 cells (Figure 3B). Similar to HMC-1.2 cells, dasatinib effectively inhibited IL-6 release in P815 murine mastocytoma cells (Figure 3D). In contrast, persistent IL-6 production in HMC-1.2 cells did not appear to be mediated via feed-forward loops of activation by other receptors as those reported for the IL-6R 22, sphingosine-1-phosphate receptors 23 or the TGF β receptor 24 in other neoplastic cells, since it was not altered by specific blockage of these receptors (Online Supplementary Figure S2 A-C). Overall, these data suggest that signals from D816V-KIT can promote ligand-independent IL-6 production without involving some of the most common autocrine feed-forward loops described in malignant cells. The constitutive release of IL-6 by unstimulated HMC-1.2 was enhanced by stimuli such as complement component 5a (C5a), IL-1 β, 10% FBS and PMA/ionomycin (Online Supplementary Figure S3A, left panel) suggesting that the production and secretion of IL-6 due to D816V-KIT can synergize with other complementary signals or environmental cues. Of interest, the intracellular content of IL-6 protein was only ~10% of the total IL-6 released (Online Supplementary Figure S3A), and although dasatinib did not affect this percentage, it also reduced the total intracellular content. Thus, dasatinib inhibited the transcription, intracellular content and release of IL-6, (Figure 3B-C and Online Supplementary Figure S3A, right panel), consistent with the conclusion that D816V-KIT signals cause constitutive de novo production and release of IL-6 without regulating storage. In addition, we demonstrate that IL-6 protein released by HMC-1.2 cells is biologically active since conditioned media of these cells caused IL-6 receptor (IL-6R)-mediated STAT3 phosphorylation in LAD2 cells which express and respond to IL-6R activation 12 (Online Supplementary Figure S3B). D816V-KIT-induced IL-6 production is dependent on JAK2, ERK and PI3K pathways MAPKs (ERK1/2 and p38) and PI3K pathways are part of the oncogenic signals derived from D816V-KIT activity 2, 25. Since these pathways may affect IL-6 expression 26-28, we investigated their potential roles in D816V-KIT-induced IL-6 production. While inhibition of p38 with SB203580 had minor effects on IL-6 synthesis, inhibition of the ERK1/2 pathway using a MEK1/2 inhibitor (U0126) (Figure 4A) or inhibition of the PI3K pathway by the Tobío et al., 8 PI3Kα/δ/β inhibitor LY294002 (Figure 4B), caused a 50 to 60% reduction, respectively, in IL-6 production at the message (left panels) and protein levels (right panels). The JAK/STAT axis is also known to be prominently upregulated by D816V-KIT activity 29, 30. JAK2 is activated by SCF 31 leading to STAT phosphorylation and translocation into the nucleus, where it exerts its transcriptional activity 32. Inhibition of JAK2 by the JAK2 selective inhibitor fedratinib (TG101348) markedly blocked IL-6 constitutive transcription and cytokine release (Figure 4C, left and right panels, respectively). Ruxolitinib which inhibits JAK1 in addition to JAK2, similarly inhibited IL-6 expression, although at higher concentrations than fedratinib (Figure 4D). However, tofacitinib, a pan-JAK inhibitor preferential for JAK3 and to a lesser extent JAK1, was less effective (Figure 4D). Similar to HMC-1.2 cells, in mouse P815 cells inhibition of MEK/ERK1/2, PI3K or JAK2 pathways markedly reduced IL-6 release in mouse P815 cells (Figure 4E). The data thus implicate JAK2 in D816V-KIT induced IL-6 production. Since JAK2 activation has also been reported in certain cells to activate PI3K or ERK 32, 33, we further investigated the potential inter-relationships between JAK2 and the ERK and PI3K pathways. While inhibition of KIT by dasatinib, as expected, blocked the phosphorylation of JAK2, AKT and ERK1/2 signaling pathways downstream of the receptor by 40 to 80% (Figure 4F), inhibitors of JAK2, PI3K/AKT or MEK1/2/ERK1/2 reduced phosphorylation of their respective targets, but did not show significant effects on any of the others, suggesting that these signals are activated independently from each other. D816V-KIT-induced IL-6 production is dependent on STAT5 JAK phosphorylates STATs, and STAT family members such as STAT3 34, 35, STAT4 36 and STAT5 37 have been implicated in the regulation of IL-6 transcription in various cells and conditions. As the levels of expression or phosphorylation of STAT3, STAT4 and STAT5 (Online Supplementary Figures S4 A-C) are increased in HMC-1.2 and other cells with D816V-KIT 30; and STAT4 and STAT5 are upregulated in BM mast cells from patients with SM 29, 38, 39, we investigated their possible involvement in the induction of IL-6 transcription by silencing STAT3-5 expression using sh-RNA or si-RNA. Sh-RNA-mediated STAT3 silencing resulted in >75% reduction in STAT3 at the messenger and protein levels (Figure 5A) but did not affect IL-6 production by HMC-1.2 (Figure 5A, red bar). Similarly, a selective small inhibitor molecule for STAT3, C188-9, at concentrations that caused >80% reduction in STAT3 phosphorylation (Online Supplementary Figure S5A, upper panel) did not alter constitutive IL-6 production by these cells (Online Supplementary Figure S5A, lower panel). Neutralizing antibodies for the IL6R, which signals through STAT3, were also ineffective on IL-6 production (Online Supplementary Figure S2A). Reduction of STAT4 message and protein by >50% using shRNA knockdown was also inconsequential for IL-6 persistent production by these cells (Figure 5B). Tobío et al., 15 D816V mutation burden in adults with indolent systemic mastocytosis. Eur J Haematol. 2013;91(2):106-111. 19. Smrz D, Bandara G, Zhang S, et al. A novel KIT-deficient mouse mast cell model for the examination of human KIT-mediated activation responses. J Immunol Methods. 2013;390(1-2):52-62. 20. Ito T, Smrz D, Jung MY, et al. Stem cell factor programs the mast cell ac tivation phenotype. J Immunol. 2012;188(11):5428-5437. 21. Gao SP, Mark KG, Leslie K, et al. Mutations in the EGFR kinase domain mediate STAT3 activation via IL-6 production in human lung adenocarcinomas. J Clin Invest. 2007;117(12):3846-3856. 22. Grivennikov S, Karin M. 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Stat5a serine 725 and 779 phosphorylation is a prerequisite for hematopoietic transformation. Blood. 2010;116(9):1548-1558. 50. Pircher TJ, Petersen H, Gustafsson JA, Haldosen LA. Extracellular signal-regulated kinase (ERK) interacts with signal transducer and activator of transcription (STAT) 5a. Mol Endocrinol. 1999;13(4):555-565. Tobío et al., 17 Figure legends Figure 1. Production of IL-6 by bone marrow cells and mast cells is enhanced in patients with SM in association with the D816V-KIT allelic burden. (A) IL-6 release from BM mononuclear cells isolated from five patients with SM with D816V-KIT bone marrow allelic frequencies of <5 (patients 4 and 5 in Supplementary Table 1) or >5 (patients 6, 7, and 10). BM aspirates were cultured for 2-4 days and IL-6 released into the culture media was measured by ELISA. Data are the mean ± SEM. (B) Correlation between IL-6 released into the media by BM cells and the percentage of mast cells in those cultures which was determined by flow cytometry. (C) Flow cytometry histograms showing intracellular IL6 staining in BM mast cells. Mast cells were gated as CD3-/CD34-/KIT+/Fc ε RI+ within the BM cells of three patients (patients 1-3 in Supplementary Table 1), with D816V-KIT allelic frequencies in the bone marrow also indicated in the figure. Patient 1had idiopathic anaphylaxis and did not meet criteria for SM but was used as a control. The percentage of IL6 positive cells within the mast cell population is indicated in the histograms. Figure 2. Cells with D816V-KIT constitutively express and release IL-6. (A) IL-6 mRNA expression (left) and IL-6 released into the media (right) by the mastocytosis cell lines HMC1.1 (with V560G) and HMC-1.2 (with V560G and D816V) after 2 h in serum free media. (BC) Comparison of IL-6 released into the media by the mouse P815 mastocytoma mast cell line (with D814Y-KIT) compared to normal murine BMMCs (B), and by the murine mast cell line MCBS-1 (which lacks c-Kit) transfected with human KIT or D816V-KIT compared to MCBS-1 transfected with vector alone (C). (D) Comparison of IL-6 mRNA expression (left) and IL-6 released into the media (right) by the human colorectal carcinoma cell line HCT116 expressing or not D816V-KIT. HCT116 cells were stimulated with PMA and ionomycin overnight. IL-6 mRNA expression was determined by q-RT-PCR and relative expression was calculated in relationship to the expression of GAPDH using the ΔCt method and expressed as fold change compared to HMC-1.1 (A, left panel), or the HCT116 parental cell line (D, left panel). All data (A-D) are the mean ± SEM of three independent experiments done in triplicates. Figure 3. D816V-KIT oncogenic activity drives ligand-independent IL-6 induction. (A) IL-6 released to the extracellular media was measured in LAD2, HMC-1.1 and HMC-1.2 cells incubated for 48 h (37C, 5%CO2) in serum free medium in the presence or absence of 100 ng/mL SCF. (B) IL-6 mRNA levels were measured in HMC-1.2 after 2 h incubation in serum free medium in the presence or absence of the KIT inhibitors, dasatinib and imatinib, or the EGFR inhibitor gefitinib at the indicated concentrations. Relative expression of IL-6 mRNA was obtained by normalizing to the expression of GAPDH using the ΔCt method and the results are expressed as fold change compared to untreated cells. The effect of the KIT inhibitor dasatinib (0.5 µM) on the secretion of IL-6 by HMC-1.2 (C) or by P815 mast cells (D) was determined after 6 h incubation in serum free medium. All data are the mean ± SEM of three independent experiments done in triplicates. Tobío et al., 18 Figure 4. MEK/ERK-, PI3Kand JAK2-mediated pathways are independently activated by D816V-KIT and contribute to ligand-independent IL-6 induction. (A-B) Effect of inhibition of MEK/ERK1/2 (U0126), p38 (SB203580) (A) or PI3K pathways (LY294002) (B) on the expression of IL-6 mRNA (left panels) after treatment for 2 h and the release of IL-6 into the media (right panels) by HMC-1.2 cells for 16 h. (C) Effect of the JAK2 inhibitor fedratinib at the indicated concentrations on the expression of IL-6 mRNA (left) and the release of IL-6 into the media (right) by HMC-1.2 cells. (D) Effect of various concentrations of inhibitors for JAK1/2 (ruxolitinib) and JAK3 (tofacitinib) on the expression of IL-6 mRNA by HMC-1.2 cells. E) Effect of inhibition of MEK/ERK1/2, PI3K or JAK2 on the production of IL-6 by mouse P815 cells after 6 h of incubation. (F) Effect of inhibitors for KIT (KIT-I: dasatinib; 0.5 µM), JAK2 (JAK2-I: fedratinib; 1 µM), PI3K (PI3K-I: LY294002; 10 µM) and MEK/ERK1/2 (ERK-I: U0126; 10 µM) on the phosphorylation of KIT, JAK2, AKT and ERK1/2 in the indicated amino acid residues. In F, inhibitors were incubated for 2 h in serum free medium. Numbers under each phosphorylated band are mean ± SEM of at least three experiments and represent fold changes in the relative band fluorescence (normalized to the corresponding total expression) compared to untreated cells. Relative expression of IL-6 mRNA was obtained by comparing to the expression of GAPDH using the ΔCt method and the results were expressed as fold change compared to untreated cells. Data represent the mean ± SEM and are from three independent experiments. Figure 5. STAT5A and B are key in mediating D816V-KIT-induced persistent IL-6 production. Effect of STAT3 (A) and STAT4 (B) knockdown by lentiviral sh-RNA on their respective targets and on the expression of IL-6 mRNA by HMC-1.2 cells. For sh-STAT4, the effect of two different constructs are shown (#1 and #3). Western blot gels underneath the bar graphs represent the effect of sh-STAT3 (A) or sh-STAT4 (B) constructs on the protein levels of STAT3 and STAT4, respectively. Lysates from duplicate samples are shown. The numbers under each pair are mean ± SEM of at least 3 separate experiments and represent fold change in the relative band fluorescence compared to the sh-RNA non-target (control). Actin content was used as loading control. (C, D) Effect of silencing STAT5A, STAT5B or the combination of both STAT5A&B by si-RNA on the expression of STAT5A and STAT5B mRNA (C) and of IL-6 mRNA (D) in HMC-1.2. The blots under the bar graph in C show the effect of STAT5 silencing in the protein levels of STAT5A/B and the numbers under the blot, the fold change as in A and B. Of note, the antibody used recognizes both STAT5A and B. Figure 6. MEK/ERK signaling pathway cooperates with JAK2 in the regulation of STAT5 to induce IL-6 while PI3K regulates IL-6 independently of STAT5. (A) Effect of inhibitors for KIT (KIT-I: dasatinib; 0.5 µM), JAK2 (JAK2-I: fedratinib; 1 µM), PI3K (PI3K-I: LY294002; 10 µM) MEK/ERK1/2 (ERK-I: U0126; 10 µM), STAT5 (STAT5-I: CAS285986-31-4; 50 µM), or their combination on the protein and phosphorylation levels of STAT5 in HMC-1.2 cells. (B) Effect of inhibitors of JAK2 and ERK1/2 alone or in combination on STAT5 phosphorylation in HMC-1.2 cells. In A and B, lysates were obtained after 2 h incubation with the indicated inhibitors in serum free medium. Data under the blots are the mean ± SEM of three independent experiments done in duplicates and represent fold Tobío et al., 19 change in the relative band fluorescence compared to the untreated. STAT5 content was used to normalize the data. (C) Inhibition of MEK/ERK1/2 reduces STAT5A and STAT5B expression (upper and lower panels, respectively) after 16 h incubation. Relative expression of STAT5 mRNA was obtained by comparing to the expression of GAPDH using the ΔCt method and the results are expressed as fold change compared to untreated cells at each time (2 h or 16 h). (D, E) Inhibitors for STAT5, PI3K and MEK/ERK1/2 additively prevent IL-6 mRNA induction (D, left panel) and IL-6 release in HMC-1.2 cells (D, right panel) and P815 cells (E). The release of IL-6 into the media was determined after 6 h. (F) Inhibitors for JAK2, PI3K and MEK/ERK1/2 additively prevent IL-6 mRNA induction in HMC-1.2 cells. All data represent mean ± SEM of at least three independent experiments. Tobío et al., 6 RNA “ON-TARGET” pool for human STAT5A (L-005169-00-0005), STAT5B (L010539-00-0005), or a non-targeting si-RNA pool (D-001810-10-05) from Dharmacon (Lafayette, CO). Transfected cells were kept in culture at 37oC in 5% CO2 for 72 h. Cells were then washed, incubated in serum free culture media for 2 h and their cellular protein or RNA contents extracted. Tobío et al., 7 Supplementary Table 1D816V-KIT allelic frequency and tryptase levels in the patients used in this study Patient number Diagnosis Sex BM D816V-KIT Frequency (%) Serum Tryptase (ng/mL) 1 IA* F Negative 4 2 ISM M 2.74 35 3 ISM M 5.50 182 4 ASM F 1.41 479 5 ISM F 0.35 105 6 ISM F 31.08 293 7 ISM M 13.44 291 8 ISM M 0.06 14 9 ISM M 0.03 10 10 SSM M 44.66 590 *BM aspirates were obtained from this patient with idiopathic anaphylaxis (IA) during an evaluation for possible SM. SM was not diagnosed in this patient and thus findings were used as a control in this study. ISM: indolent systemic mastocytosis; SSM: smouldering systemic mastocytosis. Patients were classified according to the WHO criteria 8-11 Tobío et al., 8 Supplementary Table 2Intracellular IL-6 staining of bone marrow cells in patients with SM Patient / D816V-KIT Frequency (%) KIT+/Fc e RI+ KIT+/Fc e RIKIT-/Fc e RI+ KIT-/Fc e RIIL-6 MFI % IL6+cells IL-6 MFI % IL6+cells IL-6 MFI % IL6+cells IL-6 MFI % IL6+cells 1/ (0.0%) - 21 - 17 - 20 - 17 2/ (2.7%) 336 74 167 56 254 61 117 47 3/ (5.5%) 3371 98 768 88 524 86 522 73 BM cells were cultured for 3-4 d and intracellular IL-6 staining in Brefeldin Atreated cells was analyzed by FACS. CD3-/CD34cells were gated and the mean fluorescence intensity (MFI) of intracellular IL-6 and the percentage of cells with IL-6+ staining determined within each population (KIT+/FcεRI+, KIT+/FcεRI-, KIT- /FcεRI+ and KIT-/FcεRI-). Patients 1-3 are the same as shown in Figure 1C and as defined in Supplementary Table 1. Patient 1 had idiopathic anaphylaxis and did not meet criteria for SM but was used as a control. Tobío et al., 9 Supplementary Figure S1. Intracellular IL-6 in bone marrow specimens from patients with SM is mainly associated with mast cells. Bone marrow biopsies from patients # 7 (A) and #10 (C) defined in Supplementary Table 1 were prepared for immunofluorescence staining with anti-mast cell tryptase (red) and anti-IL-6 (green). Nuclei were identified by DAPI staining. In the overlay, proximal co-localization is shown in yellow or white depending on the intensity of the individual anti-tryptase and anti-IL-6 staining. Shown are areas with disperse mast cell infiltration for better visualization. Scale bars are 50 µM (left panels) and 20 µM (right panels). Right panels are the magnified areas indicated by white squares in the left panels. Anti-IL-6 Anti-tryptase Overlay+DAPI Patient#7 Patient#10 A B 20 µM 20 µM Tobío et al., 10 Supplementary Figure S2Autocrine activation of IL-6, S1P and TGF-β receptors does not mediate the constitutive expression of IL-6 in HMC-1.2 cells. Effect of antiIL-6 receptor (IL-6R) antibody (tocilizumab; 100 µg/mL) (A, left panel), anti-gp130 antibody (1 µg/mL) (A, right panel), S1PR1,3, S1PR2 and S1PR4 antagonists (VPC 23019; 1 µM, JTE 013; 1 µM and CYM 50358; 1 µM) (B) and a TGFβ-R antagonist (SD 208; 5 µM) (C), on IL-6 secretion by HMC-1.2 cells. The antibodies and antagonists were incubated for 24 h in serum free media. The data represent mean ± SEM of two experiments performed in duplicate. Tobío et al., 11 Supplementary Figure S3. Constitutive secretion of IL-6 by HMC-1.2 is enhanced by various stimuli; and secreted IL-6 is biologically active. (A) Total IL-6 released into the media by HMC-1.2 in comparison with total intracellular IL-6. Cells were incubated at 37oC overnight in serum free media with or without C48/80 (500 ng/mL), C5a (500 ng/mL), PMA (20 ng/mL) and Ionomycin (Io) (1 µM), LPS (10 µg/mL), IL-1b (100 ng/mL), or full media containing 10% FBS. In the right panel, the effects of the KIT inhibitor dasatinib (0.5 µM) on released IL-6 and intracellular IL-6 are shown. The percentages of intracellular compared to released IL-6 were 10% with or without dasatinib, or in the presence of C48/80; 6% after stimulation with C5a, LPS and PMA/Io; and 2% after incubation with IL-1b or 10% FBS. (B) IL-6 released by HMC-1.2 induces STAT3 phosphorylation in LAD2 cells. LAD2 cultures were treated for 30 min with conditioned media from HMC-1.2 (HMC-1.2-C.M.) in the presence or absence of the neutralizing IL6R antibody tocilizumab (100 µg/mL) or incubated with serum-free media with or without 50 ng/mL recombinant IL-6. Cell lysates were obtained and phosphorylation of STAT3 determined by Western blotting. Tobío et al., 12 Supplementary Figure S4. Baseline expression levels and phosphorylation state of STAT family members in HMC-1.2 compared to HMC-1.1 cells. (A) Expression levels of STAT3 mRNA (upper panel) and protein (lower panel) in HMC-1.1 and HMC-1.2 cells. (B) Expression levels of STAT4 mRNA (upper panel) and protein (lower panel) in HMC1.1 and HMC-1.2 cells. (C) Expression levels of STAT5 mRNA (left panel, STAT5A; middle panel, STAT5B) and protein (right panel) in HMC-1.1 and HMC-1.2 cells. Both phosphorylated and total STAT family members are shown. Relative expression of STAT mRNA was obtained by comparing to the expression of GAPDH using the DCt method and the results were expressed as fold change compared to HMC-1.1 cells. Cells were incubated for 2 h in serum-free media before obtaining the cell lysates or RNA. The data represent mean ± SEM of three experiments performed in duplicate. Tobío et al., 13 Supplementary Figure S5. Inhibition of STAT5 but not STAT3 reduces constitutive expression of IL-6 by HMC-1.2 cells. (A) Effect of the small STAT3 inhibitor, C188-9, at the indicated concentrations, on STAT3 phosphorylation (upper panel) and the expression of IL-6 mRNA (lower panel). (B-C) Effect of the STAT5 inhibitor CAS28598631-4, at the indicated concentrations, on STAT5 phosphorylation (B, upper panel), the expression of IL-6 mRNA (B, lower panel) and IL-6 protein (C) in HMC-1.2 cells. The expression of IL-6 protein in C was evaluated by intracellular staining and FACS analysis as described in Methods. (D) Effect of the STAT5 inhibitor on IL-6 secretion by P815 cells. In A-D, cells were incubated in serum-free media with the corresponding inhibitors for 2 h (IL-6 mRNA expression) or 6 h (IL-6 release). (E) Combined effect of knocking down both STAT5A and B by si-RNA and incubation with the STAT5 inhibitor for 2 h, at the indicated concentrations, on the constitutive expression of IL-6 in HMC-1.2. Relative expression of IL-6 mRNA was obtained by comparing to the expression of GAPDH using the DCt method and the results were expressed as fold change compared to untreated or cell treated with si-non-target control. The data represent mean ± SEM of at least two individual experiments. 0.0 0.5 1.0 1.5 Relative IL-6 mRNA 010 15 STAT3-I (µM) (C188-9) 020 50 0.0 0.5 1.0 1.5 Relative IL-6 mRNA **** STAT5-I (µM) (CAS 285986-31-4) 0 50 100 150 IL-6 release (pg/mL) *** 050 STAT5-I (µM) (CAS 285986-31-4) P815 cells A B 0.0 0.5 1.0 1.5 Relative IL-6 mRNA STAT5A&B siRNA STAT5-I (µM) (CAS 285986-31-4) si-Control 0 20 50 HMC-1.2 C D E HMC-1.2 MFI Unstained 74 Vehicle 666 STAT5-I 442 10.3±0.1 HMC-1.2 0 10 15 (µM) pSTAT3 (Y705) STAT3 STAT3-I : KDa 86 86 0.1±0.1 β Actin46 pSTAT5 (Y694) STAT5 β Actin 1 0.6±0.04 HMC-1.2 0.5±0.1 46 90 90 0 10 15 (µM) STAT5-I : KDa Tobío et al., 14 Supplementary References 1. Butterfield JH, Weiler D, Dewald G, Gleich GJ. 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