Zach, R.; Annis, M.; Martin-Guerrero, S. M.; Alatawi, A.; Chia, K. H.; Meredith, M.; Osborn, K.; Peter, N.; Pearce, W.; Booth, J.; Rajasekaran, M.; Dias, S.; Coleman-Evans, L.; Foster, W. R.; Harper, J. A.; Herbert, A. D.; Tighe, C.; Reuillon, T.; West, R.; Busby, O.; Burdova, K.; Crepin, D.; Ortoll, S.; Vaeteewoottacharn, K.; Dejsuphong, D.; Spencer, J.; Patel, H.; Le Grand, D.; Hunt, T. A.; Andrews, D. M.; Yamano, H.; Cutillas, P. R.; Oliver, A. W.; Ward, S. E.; Hochegger, H.

The Greatwall kinase inhibits PP2A-B55 phosphatase activity during mitosis to stabilise critical Cdk1-driven mitotic phosphorylation. Although Greatwall represents a potential oncogene and prospective therapeutic target, our understanding of cellular and molecular consequences of chemical Greatwall inactivation remains limited. To address this, we introduce C-604, a highly selective Greatwall inhibitor, and characterise both immediate and long-term cellular responses to the chemical attenuation of Greatwall activity. We demonstrate that Greatwall inhibition causes systemic destabilisation of the mitotic phosphoproteome, premature mitotic exit and pleiotropic cellular pathologies. Importantly, we demonstrate that the cellular and molecular abnormalities linked to reduced Greatwall activity are specifically dependent on the B55 isoform rather than other B55 variants, underscoring PP2A-B55 phosphatases as key mediators of cytotoxic effects of Greatwall-targeting agents in human cells. Additionally, we show that sensitivity to Greatwall inhibition varies in different cell line models and that dependency on Greatwall activity reflects the balance between Greatwall and B55 expression levels. Our findings highlight Greatwall dependency as a cell-specific vulnerability and propose the B55-to-Greatwall expression ratio as a predictive biomarker of cellular responses to Greatwall-targeted therapeutics.