Cytotoxicity of Zardaverine in Embryonal Rhabdomyosarcoma from a Costello Syndrome Patient.

Donna M Cartledge,Katherine M Drake,Deborah L Stabley,Andrew D Napper,Katia Sol-Church,E Anders Kolb,Katherine M Robbins,Karen W Gripp,Rachel Sternberg

doi:10.3389/fonc.2017.00042

Abstract

Costello syndrome (CS) patients suffer from a very high 10% incidence of embryonal rhabdomyosarcoma (ERMS). As tools to discover targeted therapeutic leads, we used a CS patient-derived ERMS cell line (CS242 ERMS) harboring a homozygous p.G12A mutation in HRAS, and a control cell line derived from the same patient comprising non-malignant CS242 fibroblasts with a heterozygous p.G12A HRAS mutation. A library of 2,000 compounds with known pharmacological activities was screened for their effect on CS242 ERMS cell viability. Follow-up testing in a panel of cell lines revealed that various compounds originally developed for other indications were remarkably selective; notably, the phosphodiesterase (PDE) inhibitor zardaverine was at least 1,000-fold more potent in CS242 ERMS than in the patient-matched non-malignant CS242 fibroblasts, other ERMS, or normal fibroblasts. Chronic treatment with zardaverine led to the emergence of resistant cells, consistent with CS242 ERMS comprising a mixed population of cells. Many PDE inhibitors in addition to zardaverine were tested on CS242 ERMS, but almost all had no effect. Interestingly, zardaverine and analogs showed a similar cytotoxicity profile in CS242 ERMS and cervical carcinoma-derived HeLa cells, suggesting a mechanism of action common to both cell types that does not require the presence of an HRAS mutation (HeLa contains wild type HRAS). Two recent studies presented possible mechanistic explanations for the cytotoxicity of zardaverine in HeLa cells. One revealed that zardaverine inhibited a HeLa cell-based screen measuring glucocorticoid receptor (GR) activation; however, using engineered HeLa cells, we ruled out a specific effect of zardaverine on signaling through the GR. The second attributed zardaverine toxicity in HeLa cells to promotion of the interaction of phosphodiesterase 3A and the growth regulatory protein Schlafen 12. We speculate that this work may provide a possible mechanism for zardaverine action in CS242 ERMS, although we have not yet tested this hypothesis. In conclusion, we have identified zardaverine as a potent cytotoxic agent in a CS-derived ERMS cell line and in HeLa. Although we have ruled out some possibilities, the mechanism of action of zardaverine in CS242 ERMS remains to be determined.

Highlights

Embryonal rhabdomyosarcoma (ERMS) is the most common pediatric soft tissue sarcoma, and its incidence is significantly increased in children with Costello syndrome (CS), a rare genetic disorder resulting from heterozygous germline mutations in the proto-oncogene HRAS located on 11p15.5 [1,2,3,4,5]
A cell viability assay for CS242 embryonal rhabdomyosarcoma (ERMS) was developed in which the initial patient-derived CS242 culture was conditioned for high-throughput screening (HTS)
Cell growth over 4 days in the presence of a range of concentrations of Dimethyl sulfoxide (DMSO) showed that up to 0.5% DMSO had no effect on cell viability (Figure S1B in Supplementary Material), allowing for a screening protocol to be designed in which test compounds in 100% DMSO may be added to cells via a single intermediate dilution in media

Summary

Introduction

Embryonal rhabdomyosarcoma (ERMS) is the most common pediatric soft tissue sarcoma, and its incidence is significantly increased in children with Costello syndrome (CS), a rare genetic disorder resulting from heterozygous germline mutations in the proto-oncogene HRAS located on 11p15.5 [1,2,3,4,5]. Nemours has established a CS registry and tissue repository unparalleled in scope, allowing us to establish a CS patientderived ERMS cell line harboring a homozygous p.G12A mutation in HRAS (CS242 ERMS), as well as a control cell line from the same patient comprising non-malignant CS242 fibroblasts with a heterozygous p.G12A mutation. Using these cell lines, we endeavored to discover compounds selectively toxic to ERMS harboring a homozygous HRAS mutation. We chose to screen using a phenotypic assay of cell viability rather than an assay explicitly targeted to HRAS signaling to provide insights into disease biology and therapeutic opportunities by uncovering active compounds with unanticipated molecular target(s) and mechanism of action

Methods

Results

Conclusion