Abstract Pituitary tumors of the gonadotrope lineage are often large and invasive, resulting in hypopituitarism. There are no medical treatments currently available. Using a combined genetic and genomic screen of human pituitary tumor samples, we identified the Mammalian sterile-20 like kinase 4 (MST4) as a pro-tumorigenic effector, driving increased pituitary cell proliferation and survival in response to a hypoxic microenvironment. MST4 is a member of the germinal center kinase III subfamily of Ste-20 kinases with limited data available concerning its role in tumorigenesis. To identify selective inhibitors of the MST4 kinase, computational-based virtual library screening was employed to dock the commercially available SelleckChem kinase inhibitor library into the ATP binding site of the MST4 crystal structure. Several candidate MST4 kinase inhibitors were identified with the potential to bind with high affinity. Using a newly developed TRFRET in vitro kinase assay, one of the small molecule compounds exhibited a potent inhibition of the MST4 kinase (IC50 = 6.18 nM +/- 2.2 nM). To determine the ability of this inhibitor to antagonize MST4 actions, a hypoxic LβT2 gonadotrope pituitary cell model was used. Under chronic hypoxia (5% O2), cells stably overexpressing MST4 showed an increased rate of proliferation (at 7 days, 3.16-fold, p = 0.006) compared to vector control cells as assessed by BrdU staining. These effects were completely blocked by the inhibitor at 50nM (-2.61-fold, p = 0.01, NS from controls). Similarly, MST4 protection from hypoxia induced apoptosis under 1% O2 for 17h was also inhibited at the same dose as assessed by TUNEL staining (-2.72-fold, p<0.001, NS from controls). Analysis of transfectants expressing wildtype and Mst4-mutants (K53E-kinase dead, T178A-loss of autophosphorylation, and C-terminus deletion) confirmed the inhibitory effect was dependent on the kinase sequence. Together, these data identify a potent inhibitor of the MST4 kinase with the capacity to selectively block pituitary cell growth under a hypoxic microenvironment and support future studies of its ability to modulate pituitary tumor growth in rodent models. Citation Format: Weipeng Xiong, Chris Matheson, Donald S. Backos, Mei Xu, Katja Kiseljak-Vassiliades, Smita Salian-Mehta, Philip Reigan, Margaret E. Wierman. Identification of a mammalian sterile-20 like kinase 4 (MST4) inhibitor to target pituitary tumorigenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-303. doi:10.1158/1538-7445.AM2015-LB-303