Midostaurin

Two BL cell lines, Raji and Ramos, were purchased from American Type Culture Collection (Manassas, VA) and were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (GIBCO BRL, Grand Island, NY) and 1% penicillin/streptomycin (Ambion, Austin, TX).
As a complement resource, normal human serum (NHS) was pooled from 10 healthy persons, aliquoted and stored at −80 °C until use. The phosphoinositide 3-kinase (PI3K) inhibitor IPI-145^{31 (link)} and the protein kinase C (PKC) inhibitor midostaurin were purchased from Selleck Chemicals (Houston, TX), and dissolved in dimethyl sulfoxide (DMSO) in use. Consequently, same volume DMSO was used as control.

Antibodies against phospho-AKT(Ser473), ERK, phospho-ERK(Thr202/Tyr204), STAT5, and phospho-STAT5 were obtained from Cell Signaling Technology (Danvers, MA); anti-KIT from R&D Systems (Minneapolis, MN) and Santa Cruz Biotechnology (Santa Cruz, CA); anti-phospho-KIT(Tyr823) from Invitrogen (Carlsbad, CA); anti-AKT from BD Biosciences (San Diego, CA); and anti-β actin from Sigma Aldrich (St. Louis, MO); and anti-rabbit IgG 800CW and anti-mouse IgG 680 RD from Licor Biosciences (Lincoln, NE). Human TruStain FcX and APC-conjugated anti-human c-KIT (clone 104D2), unlabeled anti-human KIT antibody (clone 104D2) and Zombie Aqua fixable viability dye were obtained from Biolegend (San Diego, CA); KIT antibody for IHC from Dako-Agilent (Santa Clara, CA); human SCF from R & D Systems; avapritinib, imatinib, dasatinib, midostaurin, fedratinib, STAT5-IN-1, SH4-54, venetoclax, ripretinib (DCC-2618), ruxolitinib, tofacitinib, and C188-9 from SelleckChem (Houston, TX); LY294002 and U0126 from Tocris (Minneapolis, MN); Pierce BCA assay and CyQuant cell proliferation assay from Thermo Fisher Scientific (Waltham, MA); and ViaStain propidium iodide (PI) staining solution from Nexelom Biosciences (Lawrence, MA).

All cell lines were kept at 37° C and 5% CO₂. 293T cells and JIMT-1 cells were grown in DMEM (Corning) supplemented with 10% fetal bovine serum (FBS, Gibco). SMF cells were grown in DMEM (Corning) supplemented with 10% FBS and 5 mg/ml insulin (Gemini Bio-Products). All media contained 2 mM glutamine (Corning) and penicillin/streptomycin (Corning), unless stated otherwise. Lapatinib was purchased from Santa Cruz Biotech. Axitinib, Dovitinib, KW-2449, staurosporine, UCN-01, midostaurin, lestaurtinib, PHA-665752, SU-14913, and sunitinib were purchased from Selleck Chemicals.

Quizartinib, midostaurin, and gilteritinib were purchased from Selleck Chemicals (Houston, TX). 17-AAG, 17-DMAG, retaspimycin hydrochloride, and propidium iodide (PI) were obtained from Alomone Labs (Jerusalem, Israel), APExBIO (Houston, TX), and Wako (Tokyo, Japan), respectively. Daunorubicin (DNR) was from Sigma-Aldrich (St. Louis, MO). All other inhibitors were obtained from their original developers.

Midostaurin and crenolanib were purchased from Selleckchem (Houston, TX). DUB inhibitors HBX19818 and P22077 were purchased from Medchem Express. HBX19818 analogues, including C673-0105, C598-0556, C598-0563, C598-0466, C598-0515 and C598-0468, were purchased from ChemDiv. UPLC-MS analysis of all compounds was consistent with reported purity and molecular weight. All inhibitors were dissolved in DMSO to obtain a 10 mM stock solution. Serial dilutions were then made, to obtain final dilutions for cellular assays with a final concentration of DMSO between 0.2 and 0.5%.

Venetoclax, AZD-4320, Navitoclax, ABT-737, AZD-5991, 5-Azacytidine (5-Aza), Etoposide and MK-571 were purchased from MedChemExpress (USA). Midostaurin, Gilteritinib, Ivosidenib and Enasidenib were acquired from Selleck Chemicals (USA). Reversan was obtained from Merck (Germany). Ethacrynic acid (EA) was purchased from Sigma-Aldrich (USA). All compounds were diluted in DMSO. Buthionine sulfoximine (BSO) was purchased from Merck (Germany) and diluted in ddH₂O. For in vivo experiments, AZD-4320 was dissolved in 30% 2-Hydroxypropyl-β-cyclodextrin (HP-β-CD, Merck (Germany) solution in ddH₂O (3 mM stock).

Dimethyl sulfoxide (DMSO), Roswell Park Memorial Institute (RPMI) 1640, characterized and dialyzed fetal bovine serum (FBS), MSC-qualified FBS, glutamine, phosphate-buffered saline (PBS), 96-well plates, and heat-sealing foil were bought from Thermo Fisher Scientific (Waltham, MA). CellTiter-Glo 2.0 Assay kit was purchased from Promega (Madison, WI). 384-well plates (white and flat bottom, opaque) and Dulbecco’s Modified Eagle’s Medium (DMEM) were bought from Fisher Scientific (Waltham, MA). The drug library, midostaurin, FF-10101, and gilteritinib were purchased from Selleck Chemicals (Houston, TX), and tandutinib was from LC Laboratories (Woburn, MA), while the other drugs were from Cayman Chemical (Ann Arbor, MI). Filter plates (96-well, 0.45 µm PTFE) were bought from Pall Corporation (Port Washington, NY). Isotope tracers were purchased from Cambridge Isotope Laboratories (Tewksbury, MA). PCR plates (96 LoBind) were purchased from Eppendorf (Enfield, CT). APC Annexin-V (Cat. 550475) was bought from BD Biosciences (San Jose, CA). CountBright counting beads (Cat. C36950) was bought from Invitrogen (Carlsbad, CA). All solvents and chemicals for mass spectrometry were LC/MS grade from Thermo Fisher Scientific (Waltham, MA).