Background: DYRK1B, one of the kinases specified in this RFA, is the key regulator of steady state turnover of the gatekeeper prostate cancer suppressor protein NKX3.1. NKX3.1 loss occurs in the majority of prostate cancers and the NKX3.1 is the most frequently deleted gene in prostate cancer. NKX3.1 is a haploinsufficient protein and reduction of cellular protein levels by as little as 1/3 results in a neoplastic phenotype of prostate epithelial cells. However, some NKX3.1 expression is retained even in advanced prostate cancer cells so that the residual protein expression is exploited by pathologists as a tissue specific marker for prostate cancer. Moreover, NKX3.1 is a potent growth suppressor and differentiation factor. It follows that increasing NKX3.1 levels is a logical therapeutic strategy to reverse the neoplastic phenotype of prostate cancer. To prove the validity of this approach, we showed that whereas Nkx3.1+/- mice developed prostate hyperplasia and dysplasia within 6 months of age, loss of the single Nkx3.1 Dyrk1b phosphorylation site at serine 186 essentially reversed this phenotype in monoallelic Nkx3.1S186A/- mice. This remarkable finding provides preclinical justification to identify DYRK1B inhibitors for the treatment of prostate cancer. We have also demonstrated that short-term administration of a small molecule DYRK1B inhibitor to Nkx3.1+/- mice increased Nkx3.1 levels in prostate epithelial cells. Hypothesis: DYRK1B inhibition will increase intracellular Nkx3.1, resulting in retarded or reversed prostate carcinogenesis, epithelial cell differentiation, and prostate cancer growth inhibition. Experiments: In a collaboration with Chris Hulme, PhD, Director of the University of Arizona BIO5 Translational Drug Discovery Center, we will test a large panel of DYRK inhibitors from his lab. In the one year of this project, we will identify the most potent and highest affinity inhibitors of DYRK1B by in vitro assay in LNCaP cells by assessing inhibition of NKX3.1 degradation. NKX3.1 half-life is ~30 minutes. Therefore, screening drug candidates can be done by treating cycloheximide-exposed cells for up to 3 hours and assaying for NKX3.1 levels. In Aim 2 we will chose up to five of the agents most effective in vitro for administration over one week to Nkx3.1+/- mice at different doses to determine the potency of each to increase Nkx3.1 expression levels in vivo. To assure drug availability in vivo we will conduct, full KinomeScans™ and PK studies of each inhibitor to assure in vivo effectiveness. In addition, we will conduct RNAseq analysis of Nkx3.1+/- prostate tissue after one week of exposure to the most potent DYRK inhibitors to define their target pathways. As controls, the inhibitors will be administered over one week to Nkx3.1+/+ and Nkx3.1-/- mice to carry out RNAseq to identify on-target, off-Nkx3.1 gene expression effects and off-target effects. Comparison will also be made with prostate gene expression from untreated Nkx3.1...