Targeting pyruvate kinase remains a challenge in cancer drug discovery. Despite compelling evidence highlighting the importance of the pyruvate kinase isoform M2 (PKM2) in cancer cell growth and proliferation, drug discovery efforts targeting the glycolytic activities of PKM2 have yet to yield a successful drug candidate for clinical use. Discovery efforts that are focused on developing chemical compounds that keep PKM2 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 PKM2 largely untouched. There is a critical need for drugs that can block both the glycolytic and non-glycolytic activities of PKM2. Recent evidence has shown that various heteronuclear RNA binding proteins (hnRNPs) and splicing factors are involved in formation of the PKM2 isoform in cancer cells. A novel approach to target cancer cells is to suppress PKM2 mRNA splicing in favor of splicing that leads to the non-oncogenic PKM1 isoform. Although this would prevent both glycolytic and non-glycolytic functions of PKM2, discovery efforts targeting PKM2 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 PKM2 mRNA levels in in vitro cell-based experiments. The splice sensor will produce fluorescence proportional to the amount of PKM2 mRNA in cells and not be affected by other interfering mRNA such as PKM1 or the PKM pre-mRNA. Prototype PKM1 and PKM2 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 PKM1 and PKM2 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 PKM2 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 PKM2 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: PUBLIC HEALTH RELEVANCE STATEMENT Cancer cells dependence on the embryonic form of pyruvate kinase (PKM2), which switches cellular glucose metabolism from oxidative phosphorylation to aerobic glycolysis to support increasing biosynthetic needs, makes PKM2 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 PKM2 largely untouched. We propose to develop an HTS-ready splice sensor assay that monitors the intracellular levels of PKM1/PKM2 mRNA and allows researchers to find drugs that target the PKM2 expression, thereby targeting both the glycolytic and non-glycolytic functions of PKM2.
Project Terms: aerobic glycolysis; Affect; angiogenesis; anti-cancer therapeutic; Antineoplastic Agents; Apoptosis; aptamer; assay development; base; Basic Cancer Research; Basic Science; Binding; Biological Assay; Biosensor; Buffers; cancer cell; Cancer Cell Growth; Cancer cell line; Cell Count; Cell Proliferation; Cells; Chemicals; Cisplatin; Clinical; Color; commercialization; Cultured Cells; Defect; Dependence; design; Development; Disease; docetaxel; drug candidate; drug discovery; Drug Industry; Drug Targeting; Embryo; Exhibits; Fluorescence; fluorophore; gemcitabine; glucose metabolism; Goals; Guidelines; high throughput screening; improved; In Vitro; inhibitor/antagonist; knock-down; Left; Ligands; Link; Marketing; Measures; Messenger RNA; Metabolic; Methods; Monitor; mRNA Expression; mRNA Precursor; Nature; neoplastic cell; non-oncogenic; novel; novel strategies; novel therapeutics; Oxidative Phosphorylation; paralogous gene; Performance; Pharmaceutical Preparations; Phase; Play; Preclinical Drug Evaluation; prevent; Process; Property; Protein Isoforms; Protein Kinase; Protein Splicing; prototype; public health relevance; Pyruvate Kinase; Reading; Research Personnel; research study; RNA; RNA Splicing; RNA-Binding Proteins; Role; Sampling; Scientist; Sensitivity and Specificity; sensor; Signal Transduction; small molecule; Sodium Chloride; Specificity; Spinach - dietary; System; Technology; Therapeutic Intervention; Time; Transcription Coactivator; tumor; tumorigenesis; tumorigenic; Validation; Warburg Effect; Work; Xenograft procedure
