Investigating synergistic combinations of MRT68921 and afatinib as a therapeutic strategy in epithelial ovarian cancer
Tiffany Johnston1,2, Yudith Ramos Valdes1, Matthew Borrelli1,2, Emily Tomas1,2, Bart Kolendowski1, Gabriel E DiMattia1,4,5, Trevor G Shepherd1,2,3,4.
1The Mary & John Knight Translational Ovarian Cancer Research Unit, Verspeeten Family Cancer Centre, London, ON, Canada; 2Anatomy and Cell Biology, Western University, London, ON, Canada; 3Obstetrics and Gynaecology, Western University, London, ON, Canada; 4Oncology, Western University, London, ON, Canada; 5Biochemistry, Western University, London, ON, Canada
Introduction: Epithelial ovarian cancer (EOC) is often diagnosed at advanced stages with a unique metastatic pattern occurring by direct dissemination of cells via spheroids within the peritoneal cavity. We have shown that autophagy activation in spheroids contributes to enhanced cell survival and may contribute to chemoresistance. Here, we investigated the inhibition of unc51-like kinase 1 (ULK1), a crucial regulator of autophagy, using MRT68921 (MRT) and pursued its combination with the tyrosine kinase inhibitor afatinib to investigate drug synergism.
Methods: Cell lines representing high-grade serous and clear cell carcinoma were used in adherent and spheroid cultures. Immunoblotting was performed to verify on-target inhibitor effects of MRT and afatinib as well as markers of autophagy and apoptosis. mCherry-eGFP-LC3 cell lines were used to monitor autophagic flux. Drug combination matrices were used to assess spheroid cell viability followed by Synergy Finder analysis to determine drug synergy. Synergistic MRT and afatinib combinations were then tested in a larger panel of cell line spheroids as well as ascites- and patient-derived organoids.
Results: MRT inhibited ULK1 activity rapidly, leading to effective autophagy inhibition at low micromolar concentrations. Afatinib treatment induced autophagy likely via AKT blockade. Indeed, MRT blocked autophagic flux while afatinib increased it in EOC spheroids; and, in combination MRT completely blocked autophagy. Spheroid reattachment and cell viability assays demonstrated robust effects of combination treatment driven by MRT, and early results in ascites-derived organoids support this finding.
Conclusions: We are the first to assess the potential drug synergism of MRT and afatinib in EOC. These results may offer a novel therapeutic strategy against chemo-resistant disease and warrant further evaluation of combined autophagy and tyrosine kinase inhibition in EOC.
Mary and John Knight Translational Ovarian Cancer Research Unit. Lawson Health Research Institute . London Health Sciences Centre. Obstetrics and Gynecology, Western University. Cancer Research Society. London run for ovarian cancer.