Cancer Therapy : Preclinical Dual CDK 4 / CDK 6 Inhibition Induces Cell-Cycle Arrest and Senescence in Neuroblastoma

Purpose:Neuroblastoma is a pediatric cancer that continues to exact significantmorbidity andmortality. Recently, a number of cell-cycle proteins, particularly those within the Cyclin D/CDK4/CDK6/RB network, have been shown to exert oncogenic roles in neuroblastoma, suggesting that their therapeutic exploitation might improve patient outcomes. Experimental Procedures: We evaluated the effect of dual CDK4/CDK6 inhibition on neuroblastoma viability using LEE011 (Novartis Oncology), a highly specific CDK4/6 inhibitor. Results: Treatment with LEE011 significantly reduced proliferation in 12 of 17 human neuroblastomaderived cell lines by inducing cytostasis at nanomolar concentrations (mean IC50 1⁄4 307 68 nmol/L in sensitive lines). LEE011 caused cell-cycle arrest and cellular senescence that was attributed to dosedependent decreases in phosphorylated RB and FOXM1, respectively. In addition, responsiveness of neuroblastoma xenografts to LEE011 translated to the in vivo setting in that there was a direct correlation of in vitro IC50 values with degree of subcutaneous xenograft growth delay. Although our data indicate that neuroblastomas sensitive to LEE011 were more likely to contain genomic amplification of MYCN (P 1⁄4 0.01), the identification of additional clinically accessible biomarkers is of high importance. Conclusions: Taken together, our data show that LEE011 is active in a large subset of neuroblastoma cell line and xenograft models, and supports the clinical development of this CDK4/6 inhibitor as a therapy for patients with this disease. Clin Cancer Res; 1–10. 2013 AACR. Introduction Neuroblastoma is a pediatric cancer that originates in tissues of the developing sympathetic nervous system. It is typically diagnosed in very young children and has a highly variable clinical presentation, with tumors displaying significant genomic and biological heterogeneity (1–3). Although patients with favorable clinical and biological features (low-risk disease) can often be cured by surgery alone, patients with high-risk disease, particularly those harboring amplification of the MYCN oncogene, have survival rates of less than 40% despite intensive multimodal therapy (1, 4). Suchhighmortality thusmandates a need for the development of novel therapies that will significantly improve high-risk patient survival. The identification of molecular abnormalities driving neuroblastoma tumorigenesis and disease progression followed by their targeted treatment is a pragmatic strategy for meeting this need. Currently, converging evidence points to members of the Cyclin D/CDK4/CDK6/RB cell-cycle regulatory pathway as potential candidates for such therapeutic exploitation. Both CDK4 and CDK6 (CDK4/6) encode cyclin-dependent serine–threonine kinases that, in response tomitogenic or pro-proliferative stimuli, complex with D-type cyclins to phosphorylate the RB tumor suppressor protein. This phosphorylation induces the release of RB from E2F transcription factors, and thus enables E2F to transcribe the genes that are required for G1–S phase cellcycle progression and ultimately cellular proliferation (5– 7). In addition to cell-cycle regulation, CDK4/6 signaling has also been linked to senescence suppression via regulation of the FOXM1 transcription factor (8). Given its demonstrated ability to override suppressive cues in favor of cellular proliferation, it is not surprising that deregulation of theCyclinD/CDK4/CDK6/RBpathway is associatedwith unrestricted growth and is a hallmark of nearly every tumor histotype. Authors' Affiliations: Division of Oncology and Center for Childhood Cancer Research; Division of Pathology, Children's Hospital of Philadelphia; Department of Pediatrics; Abramson Family Cancer Research Institute, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania; and Novartis Institutes for Biomedical Research, Cambridge, Massachusetts Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Corresponding Author: John M. Maris, Children's Hospital of Philadelphia, Colket Translational Research Building, Room 3060, 3501 Civic Center Blvd., Philadelphia, PA 19104. Phone: 215-590-5244; Fax: 267426-0685; E-mail: maris@email.chop. edu doi: 10.1158/1078-0432.CCR-13-1675 2013 American Association for Cancer Research. Clinical Cancer Research www.aacrjournals. org OF1 Research. on January 7, 2018. © 2013 American Association for Cancer clincancerres.aacrjournals. org Downloaded from Published OnlineFirst September 17, 2013; DOI: 10.1158/1078-0432.CCR-13-1675