The receptor tyrosine kinase-like orphan receptor 1 (ROR1) has been identified as an attractive target for ovarian and endometrial cancer therapy. Using the pipeline established from a large collaborative drug repurposing project, we identified Ivacaftor, an FDA-approved cystic fibrosis medication, as a candidate predicted to interact with ROR1. This study aimed to provide preclinical evidence supporting the potential repurposing of Ivacaftor for HGSOC and endometrial cancer treatment using 2D preclinical models as well as 3D patient-derived organoid models in vitro.

Dose response analysis was undertaken in ROR1 expressing HGSOC cell lines OVCAR4, KURAMOCHI, COV362 and COV318 as well as endometrial cancer cell lines Ishikawa and ARK1 using a serial dilution of eight concentrations of Ivacaftor (3.9 µM to 100 µM). Cell viability was assessed at 72 hours post drug treatment using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay. Cells were treated with 15uM ivacaftor and 30uM carboplatin for 24, 48, and 72 hrs for flow cytometry analysis using Apoptosis, DNA Damage and Cell Proliferation Kit (BD Biosciences) which stained for fluorophores against cleaved PARP, H2Ax, and BrdU. Additionally, ROR1-expressing HGSOC and molecularly classified endometrial cancer patient-derived organoids (n=3 for HGSOC and n=6 for endometrial cancer) underwent dose-response analysis, with CellTiter Glo-based viability measurements determined at 72 hours post-treatment. Finally, the mechanisms associated with Ivacaftor treatment were explored in the cell lines and organoids through Western blotting, flow cytometry and real-time Annexin V assays.

The IC50 for Ivacaftor ranged from 6.5 to 13.2 µM in 2D HGSOC cell lines cultured in 2D and 0.004 to 1.5 µM in endometrial cancer cell lines. In organoid models, IC50 values ranged from 11.6 to 15.1 µM for HGSOC and from 0.4 to 18.6 µM for endometrial cancer. Notably, mismatch repair deficient (MMRd) endometrial cancer models exhibited the highest sensitivity, with IC50 values lower than 1µM, in contrast to p53 wildtype and p53 abnormal models. Ivacaftor treatment induces cell apoptosis similar to carboplatin treatment, but does not increase DNA damage in the cells, as opposed to carboplatin treatment. Ivacaftor treatment does not cause an increase in cells stalling in S phase. In addition, Ivacaftor treatment suppressed tumour stemness and modulated ROR1 signaling associated oncogenic pathways including the PI3K/AKT pathway and epithelial to mesenchymal transition (EMT).

In conclusion, Ivacaftor demonstrated significant single agent anti-tumour potential in preclinical HGSOC and endometrial cancer models, supporting its further investigation as a repurposed therapy for ROR1-expressing ovarian and endometrial cancers.