A high-throughput Cyclin D-CDK4 NanoBiT assay facilitates identification and characterisation of antisense oligonucleotide-based CDK4 inhibitors

Disrupting the interaction between cyclin D and CDK4 is a potential approach to treating cyclin D-CDK4 related cancers, however this is hampered by a lack of high-throughput methodologies to measure their interaction. Here we report the development of NanoBiT luciferase assays for measuring each cyclin D-CDK4 interaction and use them to evaluate antisense oligonucleotides (AONs) targeting CDK4 as a potential cancer therapy. We demonstrate high specificity of the assays to measure each cyclin D-CDK4 interaction with cyclin D mutants with abolished CDK4 binding showing significantly decreased bioluminescence. Using the NanoBiT assays we identified two AONs targeting CDK4 that reduced cyclin D-CDK4 interactions by up to 72%. Viability assays revealed the best performing CDK4 AON caused a significant reduction in cell viability in MDA-MB-231 and MCF-7 cells, as well as a significant decrease in colony formation. Flow cytometry revealed an increase in late apoptotic cells, indicating cells entering apoptosis being the underlying cause of reduced cell number. This AON-based approach offers a potential alternative to current CDK4/6 inhibitors, particularly for resistant cancers. Furthermore, the NanoBiT assay provides a valuable tool for high-throughput screening of cyclin D-CDK4 interaction inhibitors.