Therapeutics , Targets , and Chemical Biology Targeting the EWSR 1-FLI 1 Oncogene-Induced Protein Kinase PKC-b Abolishes Ewing Sarcoma Growth

Ewing sarcoma is a rare but aggressive diseasemost common in young adults. This cancer is driven by a unique chimeric fusion oncogene but targeted strategies have been elusive. Here we report the identification of the protein kinase PKC-ß (PRKCB) as a disease-specific druggable target for treatment of Ewing sarcoma. We found that transcriptional activation of PRKCBwas directly regulated by the chimeric fusion oncogene EWSR1-FLI1 that drives this cancer. PRKCB phosphorylated histone H3T6 to permit global maintenance of H3K4 trimethylation at a variety of gene promoters. PRKCB loss induced apoptosis in vitro and prevented tumor growth in vivo. Gene expression profiling revealed a strong overlap between genesmodulated by EWSR1-FLI1 andPRKCB in regulating crucial signaling pathways. Taken together, our findings offer a preclinical proof-of-concept for PRKCB as a promising therapeutic target in Ewing sarcoma. Cancer Res; 72(17); 1–10. 2012 AACR. Introduction Ewing sarcoma is the second most frequent childhood bone tumor. Clinically, Ewing sarcoma is a highly metastatic tumor with around 25% of patients presenting metastasis at the time of diagnosis. Although great advances have been made in the treatment of local disease, therapies used for advanced stages of the disease are still disappointing, the 5-year overall survival still being critically low. Thus the discovery of novel druggable targets allowing targeted therapies is mandatory. Since the discovery and characterization of the causal translocation event (1, 2), researches were driven by the quest for targets of the chimeric EWSR1-FLI1 transcription factor, themain oncogenic event in more than 85% of Ewing sarcoma. Most of the studies aiming at identifying important molecules involved in Ewing sarcoma oncogenesis were based either on EWSR1-FLI1 inhibition in Ewing sarcoma cells or EWSR1-FLI1 expression in non-Ewing cell lines. Thus, many different studies, including ours, identified genes involved in EWSR1-FLI1– dependent cell survival, such as IGF-1R (3) and IGFBP (4, 5), PPP1R1A (6), MK-STYX/STYXL1 (7), NR0B1 (8, 9), NKX2-2 (10), GSTM4 (11), and SOX2 molecules (12). Proposed as a promising targeted therapy, IGF signaling inhibition has been extensively investigated in the context of Ewing sarcoma, and several clinical trials have been conducted. Although well tolerated, several reports of phase II studies suggested that only about one patient of 4 would benefit from IGF-1R monoclonal antibodies as single therapy (13). On the other hand, some studies analyzed primary tumors to find specific gene signature of Ewing sarcoma (6, 14–16). Aiming at finding genes that are specifically found in primary Ewing tumors compared with other tumors types and modulated by EWSR1-FLI1, we identified the protein kinase C b (PRKCB). In the context of Ewing sarcoma, we show here that PRKCB is crucial for cell survival in vitro and tumor development in vivo. We show that inhibition of PRKCB induces apoptosis and stimulates TNF and NF-kB signaling and that concomitant inhibition of both PRKCA and PRKCB, by pharmacologic inhibition or RNA silencing, is required to decrease histone H3 lysine 4 methylation levels. Materials and Methods Microarrays and statistical analysis Ewing tumors samples (GSE34620), together with samples of other pediatric or bone tumors including 32 small round cell desmoplastic tumors, 52 medulloblastomas (GSE12992), 64 neuroblastomas (GSE12460), 122 rhabdomyosarcoma (17), 27 osteosarcomas (GSE14827), and 34 synovial sarcomas (GSE20196), were used for this analysis. All microarray data Authors' Affiliations: Institut Curie; INSERM U830, Unité de Génétique et Biologie des Cancers; Institut Curie, Unité de génétique somatique, Paris; LUNAM Universités, Université de Nantes, EA3822: Laboratoire de Physiopathologie de la Résorption Osseuse et Thérapie des Tumeurs Osseuses Primitives; INSERM, UMR957, Nantes; Département de Biopathologie, Centre LéonBérard, Lyon; Institut GustaveRoussy, Université Paris-Sud XI, Villejuif; Centre National de Recherche Scientifique (CNRS), Laboratoire de Vectorologie et Thérapeutiques; Anticancéreuses, UMR 8203,Orsay; and Service d'Anatomie Pathologique, CHUdeTours, Tours, France Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: Franck Tirode, Unit e deG en etique et Biologie des Cancers, INSERM U830, Section de Recherche, Institut Curie, 26 rue d'Ulm, 75248 Paris Cedex 05, France. Phone: 33-1-56-24-66-83; Fax: 33-1-56-24-66-30; E-mail: franck.tirode@curie.fr doi: 10.1158/0008-5472.CAN-12-0371 2012 American Association for Cancer Research. Cancer Research www.aacrjournals.org OF1 Research. on May 29, 2017. © 2012 American Association for Cancer cancerres.aacrjournals.org Downloaded from Published OnlineFirst August 28, 2012; DOI: 10.1158/0008-5472.CAN-12-0371