Sirna Screen Of The Protein Kinome Identifies Therapeutic Targets In Neuroblastorna

AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO Background: High-risk neuroblastoma is a pediatric cancer that is often fatal despite intense multimodality therapy associated with significant late effects for those who survive. Therefore, future therapies will benefit from treatment strategies that rationally exploit signaling pathways for which a tumor cell is selectively dependent. In an effort to identify novel therapeutic targets, we performed a comprehensive siRNA knockdown screen of the protein kinome in neuroblastoma cell models. Methods: Using a validated siRNA library, which targets the human protein kinome (547 kinases), we transiently transfected individual kinase siRNAs into four neuroblastoma cell lines (KELLY, NLF, EBC1 and SKNAS) selected to represent clinically relevant aspects of neuroblastoma genomics (e.g. 2 with MYCN amplification). Each kinase was targeted by a pool of 4 siRNAs and each experiment included a non targeting control siRNA (or siGAPDH) and positive control siRNA. Using a real-time quantitative assay to evaluate inhibition of proliferation by siRNA knockdown, we compared the growth kinetic area under the curve (AUC) of the kinase to the AUC of the non-targeting control to obtain the primary outcome value of % relative growth. We then ranked the top 100 most potent kinase siRNAs per cell line and integrated the lists to identify kinases with inhibition in multiple lines. We further determined whether there was a correlation with inhibition by a kinase in a cell line and its gene expression and/or relative genomic copy number. Results: Of the 100 top kinases ranked per cell line, 73 inhibited two lines, 21 inhibited 3 lines and 4 kinases showed significant growth inhibition in all 4 cell lines tested. The kinases that inhibited 3 or more lines were enriched for genes involved in mitoses including CDK2, CDK4, AURKA, PLK1 and CHEK1 . Ten kinases were preferentially inhibited in the MYCN amplified cell lines (i.e. AURKB, ABL1, CHUK ) and 12 in MYCN single copy lines (i.e. ADORA2B, AKT2, CSNK2A2 ). When integrating kinase inhibition and genomic copy number status, several candidate kinases mapped to regions of genomic gain and for 2 kinases ( CSNK1D , 17q25 and CLK2 , 1q21) the presence of genomic gain mirrored growth inhibition. Conclusion: A functional siRNA screen designed to identify therapeutic targets in neuroblastoma has yielded several candidate oncogenes and/or activated pathways. The screen has internal validation as AURKA has been shown to be a molecular target (AACR Abstract 2297, 2008) and a Phase 1 study is ongoing, and several of the other kinases identified are oncogenic in other cancers. Current studies are focused on the preclinical evaluation of selected small molecular inhibitors of these candidate kinases for anti-neuroblastoma activity. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 5676.