Abstract B05: Investigating the role of STK3/4 kinases in cancer

The serine/threonine-protein kinases 3 (STK3, MST2) and 4 (STK4, MST1) are the “core kinase cassette” of the Hippo signaling pathway, directing cell proliferation and differentiation through control of the downstream transcription factors, YAP and TAZ. In cancer, the ability of the Hippo pathway to regulate growth and differentiation provides a potential target for therapeutic intervention. Interestingly, it has been proposed that STK3/4 have a role in autophagy, a cellular, self-degradative process important for generating sources of energy in response to nutrient stress. These functions for STK3/4 are likely to be both cell type and context dependent. For example, we show that knocking down STK3 in prostate tumor cells in vitro decreases proliferation, suggesting that targeting STK3 with small-molecule inhibitors could be effective in the treatment of prostate cancer. However, current chemical probes for STK3/4 are suboptimal with respect to selectivity, potency, and drug-like properties. Better tools are required to obtain a detailed mechanistic understanding of the biologic roles of STK3/4. Thus, we propose to identify small-molecule modulators of STK3 and STK4 that could be used as chemical probes of the Hippo pathway or autophagy and ultimately lead to novel therapies for cancer. With this objective we established an in-house expression of both STK3 and STK4 protein and performed a pilot screen of >90 small molecules against purified STK3/4 using the ADP-Glo in vitro kinase assay. From this pilot screen, we identified nine STK3/4 inhibitors with low nanomolar potency and confirmed these hits as bona fide STK3/4 inhibitors using both protein-thermal shift and enzymatic kinetic assays. In addition, breast tumor cells treated with a selective STK3/4 inhibitor, PF-06447475, showed a concentration-dependent increase in microtubule-associated proteins 1A/1B light chain 3 (LC3) flux. In the future, we plan to further optimize our small-molecule hits and conduct a high-throughput screen to identify novel modulators of STK3/4. Citation Format: Nicole Bata, Allison Limpert, Lester J. Lambert, Apirat Chaikuad, Malene Hansen, Stefan Knapp, Nicholas D.P. Cosford. Investigating the role of STK3/4 kinases in cancer [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr B05.