Development of a single-cell fluorescence-based sensor to investigate Myc protein stability
The transcription factor Myc is expressed in cells in response to pro-growth signals and drives expression of a plethora of genes that promote proliferation. This pleiotropic activity renders loss of Myc regulation highly beneficial to tumour cells and, in fact, Myc is deregulated in most human cancers. While its contributions to cancer have made Myc a thoroughly studied protein and an extremely desirable drug target, over fifty years of research have failed to produce an inhibitor that has successfully reached the clinic. Drug design against Myc has been limited by its lack of a defined structure with exploitable druggable sites.
Recent strategies have been aimed at re-establishing post-translational regulation of Myc protein levels through inhibition of interacting partners that promote its accumulation in the cell. This exciting new therapeutic avenue is hindered by limited availability of high-throughput in vitro assays to measure changes in Myc stability in response to drug treatment. While many studies have been conducted to investigate regulation of Myc protein stability at the population level (for example, by immunoblotting for Myc), none have employed live imaging assays that are able to report on protein stability at the single cell level.
The work presented in this study has aimed to fill this gap in the molecular toolbox with the use of a tandem Fluorescent Protein Timer (tFT). tFTs are novel microscopy-based tools that act as sensors of protein age, and therefore stability, at a single-celllevel. They make use of differing maturation kinetics of fluorophores tagged in tandem to the protein of interest, where the ratio of the fluorescence signals of the fluorophores functions as a read-out for protein stability. This offers a high through-put assay employable in drug screening for compounds that affect Myc stability, but also allows for the investigation of single-cell regulation of Myc.
In this thesis, the generation and validation of Myc-tFT is described. Specifically, the system was challenged with known modulators of Myc stability, and the effect was assessed at the single-cell level. The functionality of Myc tagged with two fluorophores was also confirmed.
Once the ability of Myc-tFT to report on changes of Myc stability was confirmed, the assay’s screening window coefficient was evaluated and the Myc-tFT sensor assay was used in a medium-throughput screen of modifiers of Myc stability. This work was undertaken at industry partner AstraZeneca where their screening pipeline was employed. This work revealed variability in the data generated by the sensor that hindered the task of identifying significant hit compounds.
With the aim of making the assay more robust, the source of the variability in tFT sensor output was investigated and identified as a cell-autonomous oscillations in Myc stability and levels. The role of cell-cycle and circadian rhythms in regulation of Myc stability and levels was investigated using Myc-tFT, and confirmed in cancer cells expressing endogenous levels of Myc. This work has revealed previously undescribed post-translational regulation of Myc by the circadian rhythm. It highlights the need to investigate single-cell regulation of Myc and account for cell-autonomous post-translational regulation that could affect and bias identification of hits during drug screening. The findings also possess implications in the importance of circadian timing of treatment with modulators of Myc stability.