Iacs 010759

OPM-1, KMS-11, BJAB, SUDHL10, U2932, HLY1, and HT cells were maintained in RPMI-1640 media containing 10% fetal bovine serum (FBS). HEK293T cells were maintained in Dulbecco’s modified Eagle’s media (DMEM) containing 10% FBS. HEK293T cells were purchased from ATCC. B-cell cancer cell lines were kindly provided by Dr. Laura Pasqualucci, Dr. Michele Pagano and Dr. Yibin Yang. Cell line authentication was not performed as cells were not listed in the commonly misidentified category. All cell lines were free of mycoplasma contamination (MycoAlert kit, Lonza). Cell lines were passaged for less than 6 months after receipt or resuscitation. The following drugs were used: MG132 (Peptides International, #IZL-3175-v; 10 μM final concentration), MLN4924 (EMD Millipore, #505477; 5 μM final concentration), cycloheximide (Sigma-Aldrich, #C7698; 20 mg/mL final concentration), Doxycycline hyclate (Sigma-Aldrich, #D9891; 1 ng/mL or 1 mg/mL final concentration), and IACS-010759 (Selleck Chemicals, #S8731), puromycin (Sigma-Aldrich, 0.5–1 μg/ml final concentration), hygromycin B (EMD Millipore, #400052, 100–200 μg/mL), zeocin (100–250 μg/mL), and blasticidin S HCl (Gibco, #R21001, 3–15 μg/mL). CRISPR-derived HEK293T FBXW7^–/– clones were selected using single cell cloning in 96-well plates.

HR-proficient OVCAR-8 (D. Scudiero, National Cancer Institute, Frederick, MD) and OVCAR-8-DR-GFP cells (14 (link)), the BRCA1-mutant COV362 cells (Robert van Waardenburg, University of Alabama at Birmingham, Birmingham, AL), prostate cancer PC3 (Haojie Huang, Mayo Clinic), and breast cancer MDA-MB-231 cells (Zhenkun Lou, Mayo Clinic) were cultured in RPMI-1640 media (Corning) supplemented with 8% fetal bovine serum (Millipore) and maintained in a humidified 5% CO₂ incubator at 37 °C. All cells were authenticated by autosomal STR profiling (University of Arizona Genetics Core) and were free of Mycoplasma contamination as determined by testing with a MycoAlert Mycoplasma Detection Kit (catalog no. LT07-118, Lonza) every 6 months. Cell lines reinitiated from cryopreserved stocks every 3 to 6 months.
Ceritinib used for cell culture experiments as well as olaparib, veliparib, IACS-010759, and linsitinib were obtained from Selleck Chemicals. The Ceritinib used for mouse studies was provided by Novartis. Cisplatin was obtained from Teva Pharmaceutical Industries. SL0101 was purchased from Millipore.

C6-ceramide (N-hexanoyl-D-erythro-sphingosine) was from Avanti Polar Lipids, Alabaster, AL. Ceramide nanoliposomes (CNL) were a generous gift from Keystone Nano, Inc. Control (ghost) formulations included all liposomal ingredients except C6- ceramide. Propidium iodide (PI) was obtained from Life Technologies/Thermo Fisher, Carlsbad, CA. SK1-i, a SPHK1 inhibitor, was from Enzo, Farmingdale, NY. Vincristine sulfate was purchased from LC Laboratories, Woburn, MA. Daunorubicin (DNR) and venetoclax (Vclax) were from R&D Systems, Minneapolis, MN. Metformin and phenformin were purchased from Sigma. IACS-010759 was purchased from SelleckChem. com (Cat # S8731). All chemicals for mitochondrial analysis were purchased from Sigma. D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) was purchased from Cayman, Ann Arbor, MI.

Xenograft experiments were approved by the Animal Care and Use Committee at the University of Pennsylvania. KELLY cells (3 × 10⁶) were injected in a 1:1 mixture of PBS and Matrigel (Corning; #356234) in a final volume of 200 μL into both flanks of female Balb/c nude mice (Charles River Laboratories: #194). Flank tumors were measured every other day by electronic calipers and tumor volumes were calculated as (π/6)(length × width²). Once the tumor reached approximately 100 mm³, mice were randomly assigned to two groups and treated with vehicle or IACS-010759 (Selleck-chem: #S8731). The vehicle (containing 0.5% Methyl Cellulose; viscosity: 4000cP; Sigma: no. M0512) or IACS-010759 (7.5 mg/kg per dose) was given to mice by oral gavage once daily on a 5-day on 2-day off regimen as described (24 (link)). At the endpoint of the experiments, animals were euthanized by CO₂ inhalation.