Abstract 487: Whole genome screen to identify genes targeting MYCN-driven embryonal tumors

Abstract

Abstract MYCN is a driver of neuroblastoma (NB) tumorigenesis and is over-expressed in a number of tumors of embryonal origin, including rhabdomyosarcoma, medulloblastoma and diffuse intrinsic pontine gliomas. We sought to identify regulators of MYCN transcription by performing a whole genome screen (WGS) for regulators of MYCN promoter activity using a NB cell model. A plasmid containing the MYCN promoter (1.3kb upstream of MYCN TSS) fused to luciferase and stably integrated into the genome of NGP NB cells was the readout system. NGP-MYCNpluc, was selected based on MYCN luciferase activity inhibition by ATRA and HDAC inhibitors to a similar extent as endogenous MYCN mRNA levels. After assay optimization, siRNAs targeting 11,000 genes using 3 siRNAs/gene were evaluated. The siRNA library encompassed the druggable genome and the majority of human transcription factors. Using a 384-well format, siRNAs were reverse transfected in duplicate into NGP-MYCNpluc cells, cultured for 3 days at 37oC and analyzed for luciferase activity and cell viability using a ONE-Glo and Cell Titer-Glo assays (Promega), respectively. A robust statistical measure of median absolute deviation (MAD) was used to standardize siRNA activities in the screen. This identified 36 “high-confidence” genes in which all 3 siRNAs significantly decreased NGP-MYCNpluc luciferase activity, and 49 genes which significantly decrease cell viability. Most drugs associated with the essential viability genes have shown activity in NB cells (bortizamab, gemcitabine/paclitaxel, erlotinib/gemcitabine, UCN-01 and BI 2536). A number of MYCN reporter hits also decreased a CMV luciferase promoter in HEK293 cells, suggesting these hits did not specifically regulate the MYCN promoter. However, several hits did not affect CMV reporter activity, suggesting specificity to the MYCN reporter system. Low-throughput secondary screens are being utilized for assay confirmation and mechanistic evaluation. A 23,000 unique gene set is currently under evaluation and more elegant informatics tools are being used to further illuminate actives within the data Citation Format: Carol J. Thiele, Zhihui Liu, Veronica Veschi, Eugene Buehler, Scott Martin. Whole genome screen to identify genes targeting MYCN-driven embryonal tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 487. doi:10.1158/1538-7445.AM2015-487