Splice Sensors for Cancer Drug Discovery
Award last edited on: 3/2/2021

Sponsored Program
Awarding Agency
Total Award Amount
Award Phase
Solicitation Topic Code

Principal Investigator
Balajee Somalinga

Company Information

Lucerna Inc

PO Box 1342
New York, NY 10021
Location: Single
Congr. District: 12
County: New York

Phase I

Contract Number: 1R43CA206804-01A1
Start Date: 7/5/2016    Completed: 6/30/2017
Phase I year
Phase I Amount
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

Phase II

Contract Number: 2R44CA206804-02
Start Date: 7/5/2016    Completed: 9/30/2020
Phase II year
(last award dollars: 2020)
Phase II Amount

RNA splicing plays a central role in the generation of proteome diversity and in gene regulation. Splicing impacts vital cellular processes, such as cell-fate and differentiation, acquisition of tissue-identity, and organ development. Thus, defects in splicing has been linked to many diseases, including spinal muscular atrophy, Duchenne muscular dystrophy, Parkinson?s disease, and several types of cancer. RNA splicing is even more relevant in cancer due to the higher incidence of mis-splicing events than in normal cells. Thus, it is not surprising that aberrant splicing has been linked to important hallmarks of cancer such as proliferation, proliferation, apoptosis evasion, metastasis, and angiogenesis, and in the development of cellular resistance against cancer therapeutics. Despite the significance of splicing in cancer and other diseases, drug discovery efforts targeting them are far and few. A critical bottleneck for such efforts is the lack of robust high-throughput assay tools to monitor endogenous spliced RNA in the cell. Even though there are excellent tools such as RT-qPCR and RNA- seq to study RNA splicing, they are not readily adaptable for high-throughput screening (HTS) due to their complex and time-consuming methodology. While splice-mini-gene method offers the advantage of higher- throughput, it lacks in the ability to monitor the endogenous target RNA due its artificial design. Thus, there is an unmet need for simple and robust HTS-ready assay tools to monitor RNA splicing. Addressing this need, we proposed the development of an easy-to-use, HTS-ready splice sensor platform that can specifically detect a spliced RNA isoform of interest. In the Phase I, we used pyruvate kinase isoforms M1 (PKM1) and M2 (PKM2) as model targets and developed prototype sensors that emit fluorescence signal when they bind to their respective target RNA isoforms. PKM isoform switching is one of the ways cancer cells reprogram their glycolytic pathways to meet cellular demands of energy and biosynthetic intermediates for growth and proliferation. With the goals of commercializing the splice sensor platform for drug discovery, in the Phase II, we will perform a pilot HTS for chemical modulators of PKM splicing and validate the splice sensor as a HTS-ready platform. To demonstrate the versatility of the assay platform to target any RNA isoform of interest, we will port the modular splice sensor platform to detect two new RNA isoforms linked to cancer. To operationalize the splice sensor development process in the Aim 3, we will streamline standard operating procedures and create benchmarks for the development, production, and kitting of the splice sensor assay. Lastly, we will create a next-generation splice sensor-origami (SSO) system that will comprise of a Spinach reader and a user-customizable companion probe set to target any spliced RNA isoform of interest. The SSO will rely on toe-hold mediated strand displacement to achieve specificity and activation of Spinach fluorescence. Thus, at the end of Phase II, we will commercialize the splice sensors developed as assay kits for direct sales and work with biopharma companies to create custom splice sensors for internal drug discovery needs.

Thesaurus Terms:
Active Sites; Address; Angiogenesis; Antineoplastic Agents; Apoptosis; Aptamer; Area; Assay Development; Automation; Base; Benchmarking; Binding; Biochemical; Biological Assay; Cancer Cell; Cancer Type; Cell Growth; Cell Physiology; Cell Proliferation; Cells; Cellular Development; Chemicals; Commercialization; Companions; Complex; Custom; Defect; Design; Detection; Development; Disease; Drug Candidate; Drug Discovery; Drug Screening; Drug Targeting; Duchenne Muscular Dystrophy; Event; Experimental Study; Fda Approved; Fluorescence; Fluorescence Resonance Energy Transfer; Gene Expression Regulation; Generations; Genes; Glycolysis; Goals; Growth; High Throughput Screening; Incidence; Interest; Libraries; Link; Malignant Neoplasms; Manuals; Mediating; Methodology; Methods; Modeling; Monitor; Neoplasm Metastasis; New Therapeutic Target; Next Generation; Normal Cell; Oncogenic; Organ Growth; Parkinson Disease; Pathway Interactions; Performance; Pharmaceutical Preparations; Phase; Phosphotransferases; Play; Portability; Procedures; Process; Product Development; Production; Protein Isoforms; Proteins; Proteome; Prototype; Pyruvate Kinase; Quality Assurance; Quality Control; Reader; Readiness; Reporter; Research Personnel; Resistance; Rna; Rna Splicing; Role; Sales; Sampling; Sensor; Signal Transduction; Specificity; Spinach - Dietary; Spinal Muscular Atrophy; Success; Supporting Cell; System; Therapeutic; Therapeutic Target; Time; Tissues; Toes; Tool; Transcriptome Sequencing; Translations; Validation; Warburg Effect; Work;