The majority of neuroblastoma patients have tumors that initially respond to chemotherapy, but a large proportion of patients will experience therapy-resistant relapses. The molecular basis of this aggressive phenotype is unknown. Whole genome sequencing of 23 paired diagnostic and relapsed neuroblastomas showed clonal evolution from the diagnostic tumor with a median of 29 somatic mutations unique to the relapse sample. Eighteen of the 23 relapse tumors (78%) showed mutations predicted to activate the RAS-MAPK signaling pathway. Seven events were detected only in the relapse tumor while the others showed clonal enrichment. In neuroblastoma cell lines we also detected a high frequency of activating mutations in the RAS-MAPK pathway (11/18, 61%) and these lesions predicted for sensitivity to MEK inhibition in vitro and in vivo. Our findings provide the rationale for genetic characterization of relapse neuroblastoma and show that RAS-MAPK pathway mutations may function as a biomarker for new therapeutic approaches to refractory disease.

Purpose Neuroblastoma is a pediatric cancer that continues to exact significant morbidity and mortality. 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, a highly specific CDK4/6 inhibitor. Results Treatment with LEE011 significantly reduced proliferation in 12 of 17 human neuroblastoma-derived cell lines by inducing cytostasis at nanomolar concentrations (mean IC50 = 307 ± 68 nM in sensitive lines). LEE011 caused cell cycle arrest and cellular senescence that was attributed to dose-dependent 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. While our data indicate that neuroblastomas sensitive to LEE011 were more likely to contain genomic amplification of MYCN (p = 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.