Abstract P149: Novel compounds to probe Hippo kinase STK3 noncanonical function in prostate cancer

Abstract

Prostate cancer (PC) is the second cause of cancer related deaths in men. Most therapies target the Androgen Receptor (AR) which can prolong survival, but resistance emerges and patients eventually succumb to the disease. This highlights the need for new PC molecular targets. Our group observed in PC patient datasets, the Serine/threoninekinase3 (STK3) gene is frequently amplified and correlates with worse outcomes. This is against the established dogma that STK3 is a tumor suppressor in the Hippo Tumor suppressor pathway that canonically regulates oncogenic transcription factor YAP1. Counter to this role, we report that in PC, STK3 has a pro-tumorigenic role. Our work shows that genetic and chemical inhibition of STK3 in PC cells in vitro significantly slowed proliferation and protasphere growth. The goal of this study was to identify and validate novel STK3 kinase inhibitors with increased potency and specificity. Approach. We screened large assay panels of kinase inhibitors (ChEMBL) for STK3 active molecules, which identified two independent scaffolds. For assessment of STK3 inhibition, we utilized a battery of in cell and biochemical assays including western blot, STK3-NanoBRET assays, radiometric enzymatic assays and a broad KINOMEscan scanMax panels to assess leads and analogs. For phenotypic studies, we utilized Hi-myc ventral prostate (HMVP2) cells to test STK3 inhibition in 3D spheroid growth and spheroid invasion of matrigel by live cell imaging. Pharmacokinetics (PK) and pharmacodynamics (PD) were performed for oral (PO) and intraperitoneal (IP) routes of our top STK3 inhibitor. A subcutaneous HMVP2 allograft model was used to test in vivo efficacy of STK3 inhibition by daily IP injections at 10 mg/kg and 30 mg/kg doses vs. vehicle. Tumor volumes were tracked by caliper measurements and at endpoint, we measured tumor, liver, lung, heart and spleen weights. Results. The two lead scaffolds and a number of analogs showed significant inhibition of STK3 by western blot analysis, in cell STK3-NanoBRET assay and recombinant STK3 radiometric assays. Phenotypic studies in HMVP2 spheres treated with analog STK3 inhibitors showed dose dependent decrease in proliferation and matrigel invasion. From our assays, compound SOB-5-47 emerged as the most potent. PK and PD studies showed that IP injections of SOB-5-47 led to short time in plasma, but pro-longed half-life in the organs such as liver, heart and notably prostate with a terminal half-life clearance of 8.3hrs. Daily injections of SOB-5-47 at 10 and 30 mgs/kg significantly delayed HMVP2 tumor kinetics compared to vehicle control. Tumor weights in SOB-5-47 groups were significantly lower vs control. Importantly, no differences in organ weights or total body weights were observed between groups. Conclusions. Here we report a noncanical role of STK3 in PC and the development of novel STK3 kinase inhibitors that can be used as tools to further examine the role of STK3. Additionally, we show that STK3 can be targeted in vivo to slow PC growth without significant unwanted deleterious effects. Citation Format: Amelia U. Schirmer, Lucy M. Driver, Megan T. Zhao, Carrow I. Wells, Xuan Yang, David H. Drewry, Ivan Spasojevic, Everardo Macias. Novel compounds to probe Hippo kinase STK3 noncanonical function in prostate cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P149.