Abstract 4517: A CRISPR approach to monitoring hypoxia-inducible proteins in real-time

Abstract

Abstract Hypoxia-inducible factor 1A (HIF1A) regulates expression of genes implicated in various aspects of oncogenesis, including angiogenesis, cell survival, metastasis, and glucose metabolism. Overexpression or hypoxia-induced stabilization of HIF1A has been associated with poor prognosis in cancer patients, making HIF1A and its associated pathway a high-profile target for anticancer therapies. We sought to develop a live-cell assay to monitor abundance of endogenous HIF1A and HIF1A-inducible proteins that could be used to identify potent and specific inhibitors of the hypoxia signaling pathway. To accomplish this goal, mammalian cell lines were edited by CRISPR using a Cas9:crRNA ribonucleoprotein complex with a single-stranded oligonucleotide donor DNA to introduce the HiBiT tag at the C-terminus of HIF1A and a number of known hypoxia-inducible proteins, including BNIP3, ANKRD37, HILDPA and KLF10. The 11 amino acid HiBiT peptide and its complementing 18 kDa polypeptide, known as LgBiT, spontaneously reconstitute into an active luciferase derived from the NanoLuc enzyme. Co-expression of LgBiT in edited cells, followed by addition of the cell-permeable luciferase substrate, leads to generation of a bright, steady luminescent signal that directly correlates with abundance of the HiBiT fusion. The edited cells were treated with several known modulators of the HIF1A signaling pathway, and changes in the abundance of the protein fusions were followed in real-time by monitoring luminescence. The HiBiT tag was also used to validate size and subcellular localization of the fusion proteins using bioluminescence imaging and antibody-free blotting. As expected, all tested compounds induced HIF1A accumulation. However, the downstream targets of HIF1A generated differing response to the chemical modulators, warranting further investigation into the modes by which these compounds act. By coupling the speed and efficiency of CRISPR-mediated editing with the small size and brightness of HiBiT, it was possible to generate a live-cell assay to monitor abundance of proteins along the HIF1A pathway. This assay could easily be adapted to screen for compound-induced effects on protein levels of HIF1A, as well as HIF1A- induced changes in expression patterns. Citation Format: Marie K. Schwinn, Thomas Machleidt, Brock F. Binkowski, Christopher T. Eggers, Keith V. Wood. A CRISPR approach to monitoring hypoxia-inducible proteins in real-time [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4517. doi:10.1158/1538-7445.AM2017-4517