Abstract A29: The NF1 and NF2 pharmacome project, a course in high-throughput screening to identify targets and profile the sensitivity of MPNST cells to candidate drugs

Abstract

There are several large-scale efforts to compare the sensitivities of tumor cells to chemotherapeutic drugs and small-molecule pathway probes. However, to date, none of these profiling efforts systematically probed malignant peripheral nerve sheet tumors (MPNST). We initiated a database of drug sensitivities of MPNST as part of a course in high-throughput screening. We first developed a panel of 130 drugs highly relevant to neurofibromatosis (NF1 and NF2) that included a comprehensive set of MEK, RAF, RAS, FTI, PAK, ERK inhibitors, a representative set of drugs against many other cancer pathways including Wnt, Hedgehog, p53, EGF, HDAC, as well as classical cytotoxic agents such as doxorubicin and taxol. Many of the drugs in our panel are in clinical trials themselves, or closely related to drugs in trials for NF1. The drugs were tested against cells in 384-well plates at eight concentrations ranging from .004 μM to 10 μM. Cells were allowed to attach overnight, incubated for 72 hours with drugs, and then analyzed for ATP content using ATPlite. To date, we have profiled nine MPNST cell lines (ST88-14, ST88-3, 90-8, SNF02.2, STS26, T265, S462TY, SNF96.2, SNF94.3) and one NF2 Schwannoma cell line (HEI193). We also tested several cell lines that were tested in other screens so that we can compare our results to the other databases. The IC50 was calculated as a common measure of how effective each drug is using GraphPad Prism software. NF1 cells were distinguished from NF2 cells and STS26 cells (a spontaneous MPNST cell line derived from a patient who did not have NF1) by their strong sensitivity to MEK and Bromodomain inhibitors. Some drugs, including cytotoxic agents, Pak inhibitors, and HDAC inhibitors, were broadly toxic, inhibiting growth regardless of NF1 and NF2 status. None of the drugs in our panel exclusively inhibited HEI193 cells. With this study we initiated a database to archive the drug sensitivities of NF1 and NF2 cells that will be expanded in future versions of our course. Sponsored by Children's Tumor Foundation. Citation Format: Jianman Guo, Michael Grovola, Grace Coggins, Patrick Duggan, Jiale Huang, Claire Song, Gabriela Witek, Danny Lin, Hong Xie, David Schultz, Simon Berritt, Jeffrey Field. The NF1 and NF2 pharmacome project, a course in high-throughput screening to identify targets and profile the sensitivity of MPNST cells to candidate drugs [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr A29.

p53 modulates kinase inhibitor resistance and lineage plasticity in NF1-related MPNSTs

“…Cell lines were verified to be free of Mycoplasma contamination every 6 months by PCR (ATCC). Human-derived MPNST cell lines were cultured as described previously [ 85 ]. Capmatinib (Novartis), trametinib (Novartis), everolimus (Selleckchem), and afuresertib (Selleckchem) solutions were prepared in DMSO.…”

“…Capmatinib (Novartis), trametinib (Novartis), everolimus (Selleckchem), and afuresertib (Selleckchem) solutions were prepared in DMSO. For human cell line [ 86 ] IC 50 experiments, drugs were purchased as 10 mM stock solutions (Selleckchem) and handled as previously described [ 85 ]. All cell culture experiments were performed 3 independent times, unless otherwise noted.…”

“…Emmeans and ggplot2 were also used for comparison of doubling times. For human MPNST cell lines, the IC 50 was determined as previously described [ 85 ]. Briefly, cells were allowed to adhere and then treated with DMSO or the indicated serially diluted drug (4.6 nM–10 μM) for 72 h, cell viability was measured by ATPlite Luminescence Assay (PerkinElmer), and IC 50 was calculated using GraphPad Prism 7.…”

p53 modulates kinase inhibitor resistance and lineage plasticity in NF1-related MPNSTs

“…Cell lines were veri ed to be free of Mycoplasma contamination every 6 months by PCR (ATCC). Humanderived MPNST cell lines were cultured as described previously (34). Capmatinib (Novartis), trametinib (Novartis), everolimus (Selleckchem), and afuresertib (Selleckchem) solutions were prepared in DMSO.…”

“…Capmatinib (Novartis), trametinib (Novartis), everolimus (Selleckchem), and afuresertib (Selleckchem) solutions were prepared in DMSO. For human cell line (35) IC 50 experiments, drugs were purchased as 10 mM stock solutions (Selleckchem) and handled as previously described (34).…”