Abstract 3030: De novo engineering of chromosomal amplifications in human cells

Abstract

Abstract Chromosomal amplifications are among the most common genetic alterations found in human cancers. However, experimental systems to study the processes that lead to recurrent amplification events are lacking. Moreover, some common amplification events, such as that at 8p11-12 in breast cancer, harbor multiple candidate driver oncogenes, which are poorly modeled by conventional overexpression approaches. We sought to develop an experimental system to model recurrent chromosomal amplification events in human cell lines. Our general strategy is to use homologous-recombination-mediated gene targeting to deliver a dominantly selectable, amplifiable marker to a specified chromosomal location. We chose regions recurrently amplified at high frequency in breast cancer, including 8p11-12, 17q12, and 11q13-14. After confirmation of successful gene targeting, the targeted cells are subjected to selective pressure directed at the amplifiable marker. Spontaneous DNA breakage events lead to varying degrees of co-amplification of linked loci near the targeting site. We used adeno-associated virus vectors to target human MCF-7 breast cancer cells at the ZNF703 locus, in the recurrent 8p11-12 amplicon, using either the E. coli inosine monophosphate dehydrogenase enzyme IMPDH or a mutant dihydrofolate reductase gene as a marker. We applied selective pressure using IMPDH inhibitors. Surviving clones were found to have increased copy number of ZNF703 (average 2.5-fold increase) by droplet digital PCR. Genome-wide array comparative genomic hybridization confirmed that amplification had occurred on the short arm of chromosome 8, without changes on 8q or other chromosomes. Patterns of amplification were variable and similar to those seen in primary human breast cancers, including “sawtooth” patterns, distal copy number loss suggesting a breakage-fusion-bridge mechanism, and large continuous regions of copy number gain. Amplification was confirmed by FISH. RT-PCR and immunoblotting demonstrated overexpression of selected amplified transcripts and proteins. In summary, we have used genome engineering techniques to create focal chromosomal amplification events de novo in human cell lines. These amplifications share many of the features of amplifications observed in primary human cancers. This system will allow study of the cis- and trans-acting factors that are permissive for chromosomal amplification and provide a system to model oncogene cooperativity in amplifications harboring multiple candidate driver genes. Citation Format: Josh Lauring. De novo engineering of chromosomal amplifications in human cells. [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 3030. doi:10.1158/1538-7445.AM2015-3030