Poster Presentation Hunter Cell Biology Meeting 2022

Metabolic- and Malignancy-mediated Akt signaling revealed by Akt-FRET biosensor mouse (#75)

James RW Conway 1 2 , Sean C Warren 2 , Young-Kyung Lee 2 , Andrew T McCulloch 2 , Astrid Magenau 2 , Victoria Lee 2 , Xanthe L Metcalf 2 , Janett Stoehr 2 , Lea Abdulkhalek 2 , Cristian S Guaman 2 , Clare Zhang 2 , Kendelle J Murphy 2 , Brooke A Pereira 2 , Pauline Mélénec 2 , Sharissa L Latham 2 , Helen Lenthall 2 , Elissa K Deenick 2 , Yuanqing Ma 2 , Tri Phan 2 , Stacey Walters 2 , Shane T Grey 2 , Yan-Chuan Shi 2 , Lei Zhang 2 , Herbert Herzog 2 , David R Croucher 2 , Andy Philp 2 , David Herrmann 2 , Owen J Sansom 3 4 , Jennifer P Morton 3 4 , Antonella Papa 5 , Jody J Haigh 6 , Paul Timpson 2 , Max Nobis 2
  1. Turku Bioscience Centre, University of Turku and Åbo Akademi University, Turku, Finland
  2. Garvan Institute of Medical Research, The Kinghorn Cancer Centre, St Vincent's Clinical School, Faculty of Medicine, Sydney, NSW, Australia
  3. Cancer Research UK Beatson Institute, Glasgow, Lanarkshire, UK
  4. Institute of Cancer Sciences, Wolfson Wohl Cancer Research Centre, Institute of Cancer Sciences, University of Glasgow, Glasgow, UK
  5. Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Mebourne, VIC, Australia
  6. Research Institute in Oncology and Hematology, Department of Pharmacology and Therapeutics, University of Manitoba, Manitoba, Canada

Akt (protein kinase B) is a key regulator in a variety of cellular processes such as glucose metabolism, cell survival, proliferation and cell migration. Its activity is aberrantly upregulated in a plethora of cancers, metabolic and immune disorders, including but not limited to breast, pancreatic and prostate cancer. More specific, time-resolved monitoring of key drivers of metabolism and proliferation in tissue specific contexts can be achieved in an in vivo setting with the use of FRET-biosensor mice to track protein activity and the effect of therapeutic intervention.

Here, we describe the generation and characterization of a FRET-biosensor mouse to examine Akt [1.] activity in an in vivo setting in a variety of tissues and cancers by the application of optical windows [2.]. Elevated levels of Akt activity were observed in the pancreatic cancer models driven by mutant KRasG12D/+ and KRasG12D/+;p53R172H/+, including PTEN loss driven PDAC as reported previously [3.] and Akt activity mapped over the course of disease progression. Whole body PTENG129E/+ mutation or loss (PTENfloxed/+) mice were also crossed to the Akt-FRET biosensor mouse and Akt activity measured in several cancers such as lymphomas, adrenal, mammary and prostate cancer. Cell lines and organoid cultures established from these tumours retained the AKT-FRET reporter expression and inhibition of Akt was mapped in vitro over time in 2D and 3D contexts. Akt activity was also effectively inhibited by administration of a Pi3K inhibitor and pharmacodynamics mapped live in vivo in a breast cancer setting. Akt activity was further successfully quantified in a variety of tissues by specific Cre driver lines such as in the brain via neuropeptide Y receptor-Cre (NPY-Cre), in pancreatic beta islets (RIP-Cre) or in the entire pancreas (Pdx1-Cre). Metabolic challenge in mice bearing optical windows over the pancreas (RIP-Cre), white fat or brown fat was moreover successfully imaged live in vivo in Akt-FRET mice following ip administration of glucose or insulin.

In conclusion, the described Akt-FRET biosensor mouse can be applied to a wide range of metabolic, immune and cancer settings and used successfully in characterizing disease etiology and monitoring treatment outcomes.

  1. Komatsu, N. et al. (2011) Development of an optimized backbone of FRET biosensors for kinases and GTPases. Mol. Biol. Cell 22, 4647–56
  2. Ritsma, L. et al. (2013) Surgical implantation of an abdominal imaging window for intravital microscopy. Nature Protocols. 8, 583–594
  3. Conway, JRW. et al. (2018) Intravital Imaging to Monitor Therapeutic Response in Moving Hypoxic Regions Resistant to PI3K Pathway Targeting in Pancreatic Cancer. Cell Rep. 23, 3312–3326