Abstract 4547: High-throughput screen to identify compounds targeted to embryonal rhabdomyosarcoma harboring mutant HRAS using cells derived from a pediatric Costello syndrome patient.

Abstract

Abstract Costello syndrome (CS) is a disorder caused by HRAS gene mutations. Substitutions in glycine residues at positions 12 or 13 of the HRAS gene lead to aberrant activation of the guanine-nucleotide binding protein, HRAS. Homozygous expression of mutant HRAS predisposes pediatric patients to early onset of cancer, especially embryonal rhabdomyosarcoma (ERMS), while heterozygosity at this locus results in non-malignant abnormalities. Since the mid 1970’s there has been only a modest increase in the 5-year survival rate for ERMS in the United States to no more than 70%, so new treatment strategies are needed. To address this need, we have acquired, propagated, and banked patient-derived ERMS cells harboring a homozygous p.G12A substitution and have used these cells to develop a high-throughput screening (HTS) assay to discover compounds that selectively kill cells bearing homozygous HRAS mutation. The assay employs luminescent detection of ATP using CellTiter-Glo to quantify cell viability. Cell number, growth conditions, and compound delivery were optimized to obtain a robust 72-hr cell growth assay. The potent cytotoxic agent dactinomycin was used to ensure reproducible sensitivity of the assay. The assay was validated for HTS by pilot screening of a library of 2,000 known drugs and bioactive natural products, and hits were confirmed by retesting in dose-response. Confirmed active compounds were tested for selectivity for ERMS harboring homozygous mutant HRAS in a panel of patient-derived and purchased cell lines: RD, an ERMS line not driven by HRAS mutation; RH36, a mutant HRAS (p.Q61K) ERMS line that is not derived from CS; fibroblasts derived from CS patients with heterozygous HRAS mutations (CS242, p.G12A and CS066, p.G12S), and normal fibroblasts. In future work, compounds identified by HTS as displaying selectivity for homozygous mutant HRAS-expressing ERMS cells will be evaluated in pairwise combinations and also in combination with currently used chemotherapeutics. Targeting mutant HRAS-activated cell growth could direct the design of a novel therapeutic strategy for treatment of children with ERMS. Citation Format: Donna M. Cartledge, Katherine M. Drake, Karen W. Gripp, Katia Sol-Church, E. Anders Kolb, Andrew D. Napper. High-throughput screen to identify compounds targeted to embryonal rhabdomyosarcoma harboring mutant HRAS using cells derived from a pediatric Costello syndrome patient. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4547. doi:10.1158/1538-7445.AM2013-4547