(335b) Engineering a Bioluminescence-Based Protein Kinase Reporter for in vivo, Longitudinal Studies

A hallmark of many cancers is the aberrant activation of protein kinases that promote tumorigenic phenotypes. To measure the activity of these kinases, cancerous tissues must typically be manipulated ex vivo and then processed using immunological techniques such as western blotting or immunofluorescence microscopy. Newer techniques such as fluorescence resonance energy transfer (FRET) and fluorescence-lifetime imaging microscopy (FLIM) have been developed to monitor the activity of kinases in live cells, but these methods are often challenging to utilize for live-animal studies. These challenges stem from inherent limitations of utilizing light microscopy for in vivo tumor studies, mainly low light penetrance and high tissue autofluorescence. As a result, most in vivo studies assess the activity of a given kinase at the end-point of an experiment without any knowledge of signal dynamics. Here, we outline our work adapting an extracellular signal-regulated kinase (ERK) activity reporter for use in longitudinal animal experiments. This reporter expands upon a previously developed construct that exploits ERK’s ability to phosphorylate and stabilize a protein domain in the transcription factor fos-related antigen 1 (FRA1)¹. First, we validated that a fluorescent version of the FRA1-based Integrative Reporter (FIRE) faithfully reported ERK activity in a cancer cell line in vitro. We then used cloning techniques to modify the FIRE reporter plasmid, replacing fluorescent proteins with click beetle luciferase enzymes. This cloned vector was used to engineer a glioblastoma multiforme cell line to express the bioluminescence-based FIRE reporter. Finally, we used this cell line for subcutaneous xenograft mouse studies to evaluate the ability to measure ERK activity quantitatively in vivo. Ultimately, the ability to monitor the activity of oncogenic kinases in real-time during longitudinal, in vivo tumor studies can considerably reduce the number of animals needed for experiments that require activity measurements throughout an experimental timeline. Furthermore, this method eliminates concerns regarding how ex vivo manipulation and processing might perturb kinase activity. This work serves as a proof of concept in the development of future reporters for other oncogenic kinases, demonstrating significant technical improvements for kinase activity measurement in vivo.

1 - Albeck, J. et al. Mol Cell, 49: 249-61, 2013.