Development and validation of an RNAi screen for ABT-737 sensitizers

Kenneth W Yip¹, Inki Kim¹, Jason Garrison¹, Apple Cortez¹, Nancy Cheung¹, Pedro Aza-Blanc¹, Marina Konopleva², Michael Andreeff³, John C Reed^1
1Sanford-Burnham Medical Research Institute, La Jolla, CA, USA; ²Department of Leukemia, ³Department of Stem Cell Transplantation and Cellular Therapy, MD Anderson Cancer Center, Houston, TX, USA

Abstract: The chemical compound ABT-737 is a nanomolar inhibitor of several antiapoptotic Bcl-2 family members with potential therapeutic efficacy for a variety of cancers. Herein, we describe the development of a complementation-based RNAi assay that can be used to identify mechanisms of ABT-737-resistance. HeLa cells, which were resistant to ABT-737, were optimized for reverse-transfection efficiency and tested for siRNA-mediated silencing. The developed assay utilized HeLa cell reverse-transfection with 10 nM siRNA, followed by 48 h incubation, ABT-737 or DMSO treatment for 24 h, and cell viability measurement using ATPlite (which measures ATP levels as an indicator of cell viability). As a validation, the kinase subset of the Ambion Silencer Human Druggable Genome siRNA Library V2, which consisted of 865 genes (three siRNA sequences per gene), was screened. Several assay-positive siRNAs were tested and confirmed to sensitize cells to ABT-737. Transfection of cells with siRNAs targeting Bcl-2 family member Mcl-1 also potently sensitized HeLa cells to ABT-737. The current assay thus represents a screen that can be utilized to identify ABT-737-sensitizing siRNAs and correspondingly, potential new targets for drug discovery.

Keywords: ABT-737, Bcl-2, apoptosis, RNAi screen, siRNA