Development of novel small molecule inhibitors targeting the stress kinase LKB1 as a therapeutic target in EOC metastasis
Charles B Trelford1,4, Ahmed Abdelhameed5, Yudith Ramos Valdes1, Sarah Mcarthur1,4, Corey Fan1,4, Jason Say5, Babu Joseph5, Laurent Hoffer5, Richard Marcellus5, Methvin Isaac5, Radek Laufer5, David Uehling5, Rima Al-awar5, Trevor G Shepherd1,2,3,4.
1The Mary & John Knight Translational Ovarian Cancer Research Unit, Verspeeten Family Cancer Centre, London, ON, Canada; 2Dept. of Obstetrics & Gynaecology, Western University, London, ON, Canada; 3Dept. of Oncology, Western University, London, ON, Canada; 4Dept. of Anatomy & Cell Biology, Western University, London, ON, Canada; 5Drug Discovery Program, Ontario Institute for Cancer Research, Toronto, ON, Canada
Stress adaptation is used by epithelial ovarian cancer (EOC) cells to survive during metastasis. Liver kinase B1 (LKB1) stress signaling is crucial for EOC spheroid cell viability, invasion, and metastatic potential. We hypothesize LKB1 is a novel therapeutic target for advanced EOC. Herein, we present results from our screen and synthesis of small-molecule LKB1 inhibitors. A library of 720 kinase inhibitors was screened for LKB1 inhibitory activity. ASC-069 (IRE1i) and dinaciclib (CDKi) were selected for medicinal chemistry approaches to improve LKB1 selectivity. Iterative in vitro kinase and cell-based NanoBRET assays were used to define a short list of LKB1-specific inhibitors. Cellular thermal shift assays (CETSA) were used to measure compound binding to the LKB1 complex in HeyA8 and OVCAR8 cells. Spheroid cell viability and reattachment in response to drug treatment were assessed. ASC-069 (70nM IC50) was identified as the top hit in our screen, and dinaciclib (500nM IC50) was fifth. Given predicted docking to LKB1 and utility for medicinal chemistry, over 200 derivatives were generated for in vitro kinase assays. Several dinaciclib series compounds had improved specificity for LKB1 vs CDK2 by several orders of magnitude, yet this selectivity was not met for ASC-069. Conversely, LKB1 NanoBRET assays yielded potent inhibition using ASC-069 derivatives, but little activity was seen with the dinaciclib series. CETSA results demonstrated the dinaciclib series increased thermal stability of LKB1 and its partner STRADα in EOC cells meaning an alternative binding mode may exist. LKB1 substrates AMPK and NUAK1 had reduced phosphorylation in drug-treated EOC cell lines. Lastly, several compounds representing both series reduced EOC spheroid cell viability and reattachment. Our work is the first report of small-molecule LKB1 inhibitors. Importantly, we now have compounds with distinct MOAs to test as therapeutics for advanced EOC using preclinical models of metastasis.
OvCAN/CRS-OCC Strategic Partnership Grant.