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Hippo Pathway Bioluminescent Biosensor

Novel Hippo pathway biosensors for drug screening and quantitative monitoring of Hippo pathway components

Hippo pathway illustration
Source: Novel Hippo pathway biosensors for drug screening and quantitative monitoring of Hippo pathway components

Background

The Hippo signaling pathway plays a critical role in tissue growth. It regulates numerous biological processes including organ development, tissue regeneration and stem cell differentiation/renewal. It has also been shown to promote tumorigenesis, metastasis and chemotherapy resistance. Dysregulation of the Hippo pathway is associated with various diseases, including heart disease and cancer, making it an attractive target for therapeutic intervention. Hippo signaling is activated by upstream regulators which result in the phosphorylation and activation of LATS kinase which subsequently phosphorylates downstream effectors YAP and its paralogue TAZ. Phosphorylating YAP and TAZ facilitates binding to protein 14-3-3 and prevents transcription of downstream gene targets. Although some regulatory factors have been identified, how the Hippo pathway is regulated by extracellular triggers is not completely understood. There are few quantitative, high-throughput and non-invasive tools to measure the dynamics and activity of the Hippo pathway.

Technology Overview

Queen鈥檚 researchers have developed and validated the first bioluminescence-based biosensor for studying the Hippo signaling pathway. The biosensor is a non-invasive tool for monitoring real time in vitro and in vivo activity of the Hippo core component LATS kinase. The biosensor screens for kinases that modulate LATS activity and has the potential to identify novel regulators of YAP/TAZ phosphorylation. Researchers have successfully used the technique to identify that VEGFR promotes angiogenesis through YAP/TAZ. The LATS biosensor can be used to provide preclinical data on effective combination treatment strategies that target VEGFR and Hippo.

Diagrams showing how the LATS biocsensor works
A. Mechanism of action for the LATS-Biosensor. At baseline, there is no interaction between YAP15 and 14-3-3; thus, the LATS-BS shows minimal bioluminescence activity. However, LATS-dependent phosphorylation of YAP15-S127 leads to 14-3-3 binding, luciferase complementation, and high biosensor signal. B. Validation of LATS-BS activity. LATS-BS was transfected alone or with LATS2 or/and MST2 into HEK293. Biosensor activity or NLuc-YAP15-S127 phosphorylation was determined 48 h after transfection. For lysophosphatidic acid (LPA) treatment, cells were stimulated with 10 渭M LPA for 1 h (n = 3). 

 

Diagram showing how LATS-BS responds to drug treatment
**A**. LATS-BS responds to drug treatments regulating Hippo signaling. HEK293 were transfected with LATS-BS and treated with the following: PI3K inhibitor 1 (GDC0941), 10 渭M for 4 h; PDK inhibitor (GSK2334470), 10 渭M for 4 h; EGF, 100 ng ml鈭1 for 1 h; Insulin, 10 渭gml鈭1 for 1 h; F/IBMX (Forskolin/IBMX), 10 渭M Forskolin/100 渭M IBMX for 1 h; PI3K inhibitor 2 (LY294002), 10 渭M for 4 h; LPA, 10 渭M for 1 h; Sphin gosine1-phosphophate (S1P), 1 渭M for 1 h; 12-O-tetradecanoylphorbol-13-acetate (TPA), 5 nM for 1 h; 2-deoxy glucose, 25mM for 1 **B**. Biosensor activity was determined by luciferase assay or BLI of live cells (n = 3). h LATS-BS can be used to determine LATS activity in vivo. HEK293 were transfected with LATSBS. Cells were injected into the mammary fat pad of immunocompromised mice. After 48 h, BLI was performed. Data are represented as mean 卤 SD

 

Further Details

Benefits

The biosensor is a non-invasive, high throughput quantitative tool that can be used for

  • High throughput drug screening for inhibitors of LATS, YAP and TAZ
  • Enables In Vivo evaluation of LATS, YAP, and TAZ and inhibitor effects
  • Identifying kinases that modulate LATS activity 
  • Identifying novel regulators of YAP/TAZ phosphorylation
  • Validating drug-target interaction 
  • Study Hippo signaling in different metastasis models

Applications

Drug Screening for inhibitors of LATS, YAP, AND TAZ

Enable in vivo imaging of hippo pathway and its modulation

Providing data on effective combination treatment strategies that target VEGFR and Hippo.

Opportunity

 Out-licensing or research collaboration

Patents

  • US, US16/177691 鈥淗ippo Pathway Bioluminescent Biosensor鈥
  • Canadian, Serial No. 3,022,981 鈥淗ippo Pathway Bioluminescent Biosensor鈥

IP Status

  • Patented
  • Patent application submitted

Seeking

  • Development partner
  • Commercial partner
  • Licensing
  • Seeking investment

Posted

November 16, 2022