Nutlin-3a Is a Potential Therapeutic for Ewing Sarcoma

Abstract

Purpose: Although mutations in the TP53 gene occur in half of all cancers, approximately 90% of Ewing sarcomas retain a functional wild-type p53. The low frequency of TP53 alterations in Ewing sarcoma makes this tumor type an ideal candidate for p53-targeted therapies. In this study, we have examined the molecular and cellular responses of cultured Ewing sarcoma cell lines following exposure to Nutlin-3a, a recently developed MDM2 antagonist.Experimental Design: The ability of Nutlin-3a to impart apoptosis or cell cycle arrest in a p53-dependent manner was determined in a comprehensive panel of Ewing sarcoma cell lines. The capacity of Nutlin-3a to augment the antitumor activity of MDM4 antagonists and cytotoxic agents currently used in the clinical treatment of Ewing sarcoma was also investigated.Results: Apoptosis was the primary response of wild-type p53 expressing Ewing sarcoma cell lines. The cytotoxicity of Nultin-3a was also synergistic with the chemotherapeutic agents, vincristine, actinomycin D, doxorubicin, and etoposide in a concentration-dependent manner. Significant MDM4 protein overexpression was observed in Ewing sarcoma cell lines of wild-type p53 status, providing a mechanism through which Ewing sarcomas can develop in the absence of TP53 alterations. This study provides the first evidence of synergism between targeted inhibition of MDM2 and MDM4.Conclusion: Our findings suggest that p53-dependent apoptosis is the primary cellular response of Ewing sarcoma cell lines following exposure to Nutlin-3a. Furthermore, Nutlin-3a can synergize with the current Ewing sarcoma chemotherapy protocols, suggesting p53 activation as a novel systemic therapeutic approach for this disease.

Mechanism-specific signatures for small-molecule p53 activators

“…Such compounds should selectively induce p53-dependent G 1 and/or G 2 cell cycle arrest in normal tissues while leaving cancer cells lacking functional p53 vulnerable to subsequent treatment with S-or M-phase poisons. 30,31 Initial studies with nutlin-3 7,32,50,51 or LDactD 9,52 have yielded interesting and, in some cases, promising results.…”

Mutant p53 drives invasion in breast tumors through up-regulation of miR-155

“…Briefly, total RNA was extracted from cells using RNeasy mini kit (Qiagen), with quantitative realtime PCR performed as previously described (37). Specific primers for real-time PCR are listed in Supplementary Table 3.…”

Nutlin-3a Efficacy in Sarcoma Predicted by Transcriptomic and Epigenetic Profiling

“…gDNA was extracted using DNeasy mini kit (Qiagen), with copy number variation (CNV) determined using a previously described method (7) and normalized using the a-albumin (AFM) housekeeping gene. Realtime PCR reactions were processed and normalized as previously described (17). Primer sequences are listed in Supplementary Table S1.…”

“…Ewing sarcoma cell lines were maintained as previously described (17) and supplied by G. Hamilton (University of Vienna, Vienna, Austria; TC252), F. van Valen (Westf€ alischeWilhelms-University, Germany; WE-68, VH-64), J. Sonnemann (University Children's Hospital Jena, Germany; CADO-ES1), and P. Ambros (St. Anna Children's Hospital, Austria; STA-ET1). Cell lines were not authenticated by our laboratory.…”