Azd2014

AZD2014 was obtained from AstraZeneca (London, United Kingdom), dissolved in DMSO at a concentration of 10 mM, and stored at −20°C until further use. The stock solution was diluted to the appropriate concentration in culture medium containing serum just before addition to cell cultures. All antibodies used in this work were obtained from Cell Signaling Technology (Beverly, MA, USA).

Human cell lines included an NF2-null immortalized MN line Ben-Men-1, NF2 AC-CRISPR, and two independent human primary MN for which cell line establishment and growth conditions have been previously described (15 (link), 54 (link)). All primary cultures were collected following Massachusetts General Hospital Human Subjects protocols for tumor acquisition after informed consent. In addition, we generated an immortalized human MN line, MN1-LF from an independent NF2-null human primary MN cell line derived from a surgical resection. Immortalization methods and confirmation was carried out on a fee-for-service basis by Alstem, Inc. Briefly, 1 × 10⁵ cells were transduced with lentivirus encoding SV40 large T antigen and a puromycin resistance gene. Cells were infected at a multiplicity of infection of 2. Following selection with 1.5 μg/ml puromycin for 3 days, cells underwent an additional three passages. PCR amplification of SV40 was performed to confirm immortalization followed by expansion and freezing of cells.
Reagents included exogenous heregulin/NRG1 (Sigma); inhibitor drugs lapatinib, erlotinib, INK128/TAK-228, and BMS-754807 (Selleck Chemicals); rapamycin (EMD Millipore); AZD2014 (obtained from AstraZeneca); and MM-121 (generously provided by Merrimack Pharmaceuticals). Drug treatment concentrations and times are described in the figure legends.

Cell lines included immortalized NF2-deficient meningioma Ben-Men-1 [15 (link)], immortalized arachnoid AC007-hTERT derived from an NF2 patient expressing heterozygous level of NF2 [7 (link), 8 (link)], primary normal arachnoid AC028, and several primary NF2-deficient meningioma (MN) lines. Ben-Men-1 cells and AC007-hTERT were maintained under growth conditions described [7 (link), 15 (link)], and all primary lines were established and maintained as reported [8 (link)]. Low passage 293T cells for large-scale lentiviral packaging were obtained from TRC. Insulin was from Sigma (St. Louis, MO). Inhibitor reagents included rapamycin and AktVIII (EMD Millipore; Billerica, MA), AZD2014 (provided by AstraZeneca; Wilmington, DE), GSK650394 (Tocris; Minneapolis, MN), Torin1 (kindly provided by Dr. David Sabatini, Whitehead Institute/MIT, Cambridge, MA) and FRAX597 (generously given by Dr. Joseph Kissil, The Scripps Research Institute, Jupiter, FL). Inhibitor treatment times and concentrations are described in figure legends. All inhibitors were diluted as per manufacturer's recommendations.