Splice sensors for cancer drug discovery

SUMMARY Targeting pyruvate kinase remains a challenge in cancer drug discovery Despite compelling evidence highlighting the importance of the pyruvate kinase isoform M PKM in cancer cell growth and proliferation drug discovery efforts targeting the glycolytic activities of PKM have yet to yield a successful drug candidate for clinical use Discovery efforts that are focused on developing chemical compounds that keep PKM arrested in an active activators or inactive state inhibitors are either mired in potency or specificity issues or have left the non glycolytic tumorigenic activity of PKM largely untouched There is a critical need for drugs that can block both the glycolytic and non glycolytic activities of PKM Recent evidence has shown that various heteronuclear RNA binding proteins hnRNPs and splicing factors are involved in formation of the PKM isoform in cancer cells A novel approach to target cancer cells is to suppress PKM mRNA splicing in favor of splicing that leads to the non oncogenic PKM isoform Although this would prevent both glycolytic and non glycolytic functions of PKM discovery efforts targeting PKM splicing are non existent This paucity is due to lack of good splice sensing platform technologies that are fast simple and HTS compatible The currently available methods are slow laborious or complicated Moreover they do not allow direct monitoring of endogenously spliced mRNA that forms in cells Here we propose a robust HTS compatible mix and read splice sensor assay that is based on a proven Spinach fluorescence biosensor technology This splice sensor would allow direct monitoring of endogenous PKM mRNA levels in in vitro cell based experiments The splice sensor will produce fluorescence proportional to the amount of PKM mRNA in cells and not be affected by other interfering mRNA such as PKM or the PKM pre mRNA Prototype PKM and PKM splice sensors developed by Lucerna scientists have demonstrated the feasibility of this concept established its specificity and confirmed that it is tunable In this project we will additionally construct PKM based sensors that allow simultaneous multiplexed monitoring of PKM and PKM RNA in the same sample We will optimize both the prototype sensors and the multiplexed sensors for HTS and develop assay conditions such that they exhibit high fluorescence sensitivity specificity and broad dynamic range while showing minimal background signal More importantly this new HTS compatible method will work directly on cell lysates and enable researchers for the first time to measure the endogenous PKM mRNA levels in a fast and reliable way At the end of this phase I project we will commercialize this splice sensor as assay kits In the next phase of the project we will validate the splice sensor assay for HTS based drug discovery by performing a pilot validation drug screen The HTS adaptation of the in vitro cell based splice sensor assay will enable the pharmaceutical industry to develop new drugs that block the PKM glycolytic and non glycolytic tumorigenic activities Lastly we will leverage the splice sensor platform to develop a suite of assays that would enable the pharmacologic targeting of aberrant splicing in other diseases PUBLIC HEALTH RELEVANCE STATEMENT Cancer cells dependence on the embryonic form of pyruvate kinase PKM which switches cellular glucose metabolism from oxidative phosphorylation to aerobic glycolysis to support increasing biosynthetic needs makes PKM an attractive target for drug discovery Current drug discovery efforts at targeting the glycolytic activity of pyruvate kinase leaves the other tumorigenic non glycolytic activity of PKM largely untouched We propose to develop an HTS ready splice sensor assay that monitors the intracellular levels of PKM PKM mRNA and allows researchers to find drugs that target the PKM expression thereby targeting both the glycolytic and non glycolytic functions of PKM