Serine/threonine kinase 3 (STK3) is an essential member of the highly conserved Hippo Tumor suppressor pathway which regulates Yes 1 Associated protein (YAP1) and TAZ. STK3 and its paralog STK4 initiate a phosphorylation cascade that regulate YAP1/TAZ activation and degradation, which is important for regulated cell growth and organ size. Deregulation of this pathway leads to hyper-activation of YAP1 in various cancers. Counter to the canonical tumor suppression role of STK3, we report that in the context of prostate cancer (PC), STK3 has a pro-tumorigenic role. Our investigation started with the observation that STK3, but not STK4, is frequently amplified in PC. A high STK3 expression is associated with decreased overall survival and positively correlates with androgen receptor (AR) activity in metastatic castrate resistant PC. XMU-MP-1, an STK3/4 inhibitor, slowed cell proliferation, spheroid growth and matrigel invasion in multiple models. Genetic depletion of STK3 decreased proliferation in several PC cell lines. In a syngeneic allograft model, STK3 loss slowed tumor growth kinetics in vivo and biochemical analysis suggest a mitotic growth arrest phenotype. To further probe the role of STK3 in PC, we identified and validated a new set of selective STK3 inhibitors, with enhanced kinase selectivity relative XMU-MP-1, that inhibited tumor spheroid growth and invasion. Consistent with the canonical role, inhibition of STK3 induced cardiomyocyte growth and had chemo-protective effects. Our results contend that STK3 has a non-canonical role in PC progression and inhibition of STK3 may have therapeutic potential for PC that merits further investigation.
Current advancements in prostate cancer (PC) therapies have been successful in slowing PC progression and increasing life expectancy; however, there is still no curative treatment for advanced metastatic castration resistant PC (mCRPC). Most treatment options target the androgen receptor, to which many PCs eventually develop resistance. Thus, there is a dire need to identify and validate new molecular targets for treating PC. We found NUAK family kinase 2 (NUAK2) expression is elevated in PC and mCRPC versus normal tissue, and expression correlates with an increased risk of metastasis. Given this observation and because NUAK2, as a kinase, is actionable, we evaluated the potential of NUAK2 as a molecular target for PC.NUAK2 is a stress response kinase that also plays a role in activation of the YAP cotranscriptional oncogene. Combining pharmacological and genetic methods for modulating NUAK2, we found that targeting NUAK2 in vitro leads to reduction in proliferation, three-dimensional tumor spheroid growth, and matrigel invasion of PC cells. Differential gene expression analysis of PC cells treated NUAK2 small molecule inhibitor HTH-02-006 demonstrated that NUAK2 inhibition results in downregulation of E2F, EMT, and MYC hallmark gene sets after NUAK2 inhibition. In a syngeneic allograft model and in radical prostatectomy patient derived explants, NUAK2 inhibition slowed tumor growth and proliferation rates. Mechanistically, HTH-02-006 treatment led to inactivation of YAP and the downregulation of NUAK2 and MYC protein levels. Our results suggest that NUAK2 represents a novel actionable molecular target for PC that warrants further exploration.
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.
Obesity is a US and global epidemic that exacerbates various cancers including prostate cancer (PC). A recent meta-analysis of men with PC found a 20% increased risk of PC-specific mortality for each 5 kg/m2 increase in BMI. To identify ways to exploit this, we performed an in vivo screen to identify kinases that were causally involved in tumor/growth in obese mice. This resulted in the identification of the AMPK-related kinase SNARK a.k.a NUAK2, a YAP1 target gene and positive feedback regulator by inhibition LATS1 kinase. To identify obesity essential kinases LAPC-4 cells were inoculated with an shRNA library of ~5,000 lentivirus targeting 513 kinases. 5 × 106 cells were grafted to chronically obese mice. Tumors were established to ~200 mm3 and a portion collected for reference. Remaining mice were randomized to continue on ad lib WD or 25% CR diet. Genome-integrated shRNA inserts were amplified using nested barcoded primers and sequenced using Illumina Hi-Seq 2000 and quantified. NAUK2 shRNAs (3 of 10) were significantly depleted in LAPC4 xenograft tumors from mice on a Western diet (WD), but not tumors from mice on an isocaloric 25% calorie-restricted (CR) diet. Analysis of NUAK2 gene expression in human PCs shows NUAK2 expression is higher in PC tissue vs. normal benign. In addition, Cox regression analysis showed that NUAK2 is associated with a 3-fold increased chance of developing metastasis disease in high NUAK2 group. Proteomic data from metastatic PC rapid autopsy biopsies shows NUAK2 phospho-target LATS1 Ser464 is higher in metastatic PCs vs. benign localized prostatic tissues. Western blot analysis of PC cell lines shows NUAK2 is elevated in comparison to primary prostate epithelial cells. To test if NUAK2 is an actionable target in PC cells, we utilized two narrow-spectrum kinase inhibitors from the literature, WZ4003 and HTH-02-006. Pharmacologic inhibition of NUAK2 slows 2D PC cell growth and 3D PC spheroid growth of various PC cell lines with an IC50 range between 1-10 uM, with HTH-02-006 being the more potent. In addition, NUAK2 kinase inhibition with WZ4003 and HTH-02-006 significantly slowed migration and Matrigel invasion of PC3 and DU145 cells. To test if NUAK2 is associated with obesity-enhanced PC progression, we tested NUAK2 inhibition in combination with palmitic acid supplementation. Our preliminary studies show that pharmacologic inhibition of NUAK2 sensitized PC cells to fatty acid-induced cell death. In conclusion, our in vivo shRNA screen identified NUAK2 as an essential kinase for tumors grown in obese mice. NUAK2 expression correlates with worse outcomes in PC and NUAK2 kinase activity is elevated in metastatic vs. localized PCs. Our preliminary data show that pharmacologic inhibition of NUAK2 slows PC cell growth and migration/invasion. Lastly, our preliminary studies show that NUAK2 may lower the threshold for fatty acid-induced toxicity. Together, these preliminary data suggest NUAK2 is a relevant actionable target in PC and may synergize with obese host physiology to slow PC tumor growth. Citation Format: Weiwei Fu, Megan Zhao, Amelia Schirmer, Erick J. Maravilla, Junhee Yoon, Sungyong You, Stephen J. Freedland, Everardo Macias. NUAK2 inhibition for prostate 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 B07.
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