For the purpose of improving recombinant protein production from mammalian cells, an unbiased, high-throughput whole-genome RNA interference screen was conducted using human embryonic kidney 293 (HEK 293) cells expressing firefly luciferase. 21,585 human genes were individually silenced with three different siRNAs for each gene. The screen identified 56 genes that led to the greatest improvement in luciferase expression. These genes were found to be included in several pathways involved in spliceosome formation and mRNA processing, transcription, metabolic processes, transport and protein folding. The 10 genes that most enhanced protein expression when down regulated, were further confirmed by measuring the effect of their silencing on the expression of three additional recombinant proteins. Among the confirmed genes, OAZ1- the gene encoding the ornithine decarboxylase antizyme1- was selected for detailed investigation, since its silencing improved the reporter protein production without affecting cell viability. Silencing OAZ1 caused an increase of the ornithine decarboxylase enzyme and the cellular levels of putrescine and spermidine; an indication that increased cellular polyamines enhances luciferase expression without affecting its transcription. The study shows that OAZ1 is a novel target for improving expression of recombinant proteins. The genome-scale screening performed in this work can establish the foundation for targeted design of an efficient mammalian cell platform for various biotechnological applications.

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