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6 protocols using ruxolitinib

1

Evaluating JAK1/2 and IGF1R Inhibitors

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Patients were seen at the Oregon Health & Science University Center for Hematologic Malignancies (patients #1 to #3, #5 and #6) or at the Hematology and Hemotherapy Center of the University of Campinas (patients #4 and #7). Following informed consent, peripheral blood was collected and mononuclear cells were isolated by Ficoll-gradient centrifugation. Cells were resuspended in RPMI 1640 medium supplemented with 10% FBS, L-glutamine, and penicillin/streptomycin and plated in 384-well plates (9000 cells/well) in triplicate in the presence of the JAK1/2 inhibitor ruxolitinib (0-4000nM), the IRS/IGF1R inhibitor NT157 (0-4000nM; Axon MedChem, USA), or both inhibitors in combination at the indicated concentrations. Plates were cultured at 37°C for 72h, and absorbance was measured by a methanethiosulfonate (MTS)-based assay, according to the manufacturer's protocol (CellTiter 96 AQueous One; Promega) for patients #1 to #3, #5 and #6, or by methylthiazole tetrazolium (MTT) assay for patients #4 and #7. Data were normalized to the mean percent of the untreated control wells.
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2

CHOP Cytotoxicity in Farage Cells

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CHOP consisted of four drugs (cyclophosphamide, vincristine, adriamycin, and prednisone) in a ratio of 80/5.5/0.16/11.1, respectively [45 (link)]. All four drugs were purchased from Selleckchem (Houston, TX, USA). Farage cells were treated with various concentrations of CHOP for 2 days or 3 days. Other drugs and their sources were as follows: Velcade (Selleckchem), ruxolitinib (Axon Medchem, Groningen, Netherlands), lenalidomide (Axon Medchem), SP 100030 (Tocris Bioscience, Bristol, United Kingdom), pimozide (Calbiochem, Darmstadt, Germany), AS 1517499 (Axon Medchem) and SH-4-54 (Selleckchem). The determination of IC50 and generation of the graphs were accomplished using GraphPad software.
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3

Inhibitor Screening for Epigenetic Targets

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Chidamide was provided by Shenzhen Chipscreen Biosciences Ltd. SAHA was obtained from Sigma–Aldrich. Trichostatin A (TSA), belinostat and tofacitinib were purchased from Selleckchem. Romidepsin was obtained from MedChemExpress. Ruxolitinib and Stattic were purchased from Axon Medchem and Millipore, respectively.
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4

Cytokine Signaling Inhibitor Screening

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TGFβ1 was purchased from Peprotech (#100-21, Peprotech, Rocky Hill, NJ) and was used at 2 ng ml−1; recombinant human LIF was purchased from Millipore (#LIF1005,Millipore, Billerica, MA), and was used at a concentration of 2 ng ml−1. LIF neutralizing antibody (AB-250-NA, R&D, Minneapolis, MN) was used at 10 μg ml−1. Neutralizing antibodies were incubated for 1 h with CM before experiments. The following inhibitors were used in this study: Pyridone 6 (#42009, Calbiochem, Los Angeles, CA) was used at 5 μM, Ruxolitinib (#1598, Axon medchem, Groningen, The Netherlands) at 10 μM, 5′-Aza (#A2385, Sigma, Saint Louis, USA) was used daily at 2.5 μM, PTP inh 1 (#540200, Millipore, Billerica, MA) at 10 μM, Sodium Orthovanadate (#567540, Millipore, Billerica, MA) at 1 mM, C646 (#SML002, Sigma, Saint Louis, USA) and CTPB (#sc- 202558, Santa Cruz, Biotechnology, Santa Cruz, CA) at 25 μM. A list of the concentrations used for the inhibitors/activators in Fig. 1d,e is provided in Supplementary Table 1.
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5

Evaluating MPN Cell Responses to Stromal Interactions

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HS-5 and KM-102 cell lines were cultured to 70% confluence and the MPN cells added to the stromal layer of HS-5 (+ HS-5) or KM-102 (+ KM-102) at 0.1x106 cells/ml in the appropriate culture medium, either directly (for cell to cell contact) or indirectly (separated by a 0.4-mm-thick micropore membranes +HS-5 TW). In addition, MPN cells were incubated without any stromal support (no stroma) or with the HS-5 conditioned media (+ CM), diluted 50% in the respective culture media. Vorinostat (Selleckchem), Ruxolitinib (Axon Medchem), SP600125 (JNK inhibitor) (Selleckchem) and LY294002 (PI3K inhibitor) (Cayman Chemicals) were added to the co-cultures once the MPN cells adhered to the BM stroma. At the indicated time points the cells were harvested and assessed as described below for viability, gene expression and immunoblotting.
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6

Cytokine Signaling Pathway Modulation

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TGFβ1 was purchased from Peprotech (#100-21, Peprotech, Rocky Hill, NJ) and was used at 2 ng/ml; recombinant human GCSF (#300-23) and IL-6 (#200-06) were purchased from Peprotech and were used at 10ng/mL, recombinant human LIF was purchased from Millipore (#LIF1005, Millipore, Billerica, MA), and was used at a concentration of 2 ng/ml. Recombinant TNF alpha was produced in E Coli and purified under native conditions using an N-terminal 6-his tag. ICAM-1 neutralizing antibody (#BBA3, R&D, Minneapolis, MN) was used at 10 μg/ml. The following inhibitors were used in this study: Ruxolitinib (#1598, Axon medchem, Groningen, The netherlands) at 10μM, CYT387 (#S2219, Selleckchem, Huston, TX) at 10μM Y27632 (#1254, Tocris bioscience, Ellisville, MO) at 10μM, Blebbistatin (#B0560, Sigma, Saint Louis, MO) at 10μM, SU6656 (#572635, CalbioChem, Los Angeles, CA) at 10μM and Verteprofin (#SML0534, Sigma, Saint Louis, MO) at 4μg/mL.
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