Structure-Based Screen Identification of a Mammalian Ste20-Like Kinase 4 (MST4) Inhibitor with Therapeutic Potential for Pituitary Tumors

Published OnlineFirst December 31, 2015; DOI: 10.1158/1535-7163.MCT-15-0703

Small Molecule Therapeutics Molecular Cancer Therapeutics Structure-Based Screen Identiﬁcation of a Mammalian Ste20-like Kinase 4 (MST4) Inhibitor with Therapeutic Potential for Pituitary Tumors Weipeng Xiong1,3, Christopher J. Matheson2, Mei Xu1,3, Donald S. Backos2, Taylor S. Mills1, Smita Salian-Mehta1, Katja Kiseljak-Vassiliades1,3, Philip Reigan2, and Margaret E. Wierman1,3

Abstract

Pituitary tumors of the gonadotrope lineage are often large described as an Aurora kinase inhibitor, exhibited potent inhi- and invasive, resulting in hypopituitarism. No medical treat- bition of the MST4 kinase at nanomolar concentrations. The ments are currently available. Using a combined genetic and LbT2 gonadotrope pituitary cell hypoxic model was used to test genomic screen of individual human gonadotrope pituitary the ability of this inhibitor to antagonize MST4 actions. Under tumor samples, we recently identified the mammalian ster- short-term severe hypoxia (1% O2), MST4 protection from ile-20 like kinase 4 (MST4) as a protumorigenic effector, driving hypoxia-induced apoptosis was abrogated in the presence of increased pituitary cell proliferation and survival in response to hesperadin. Similarly, under chronic hypoxia (5%), hesperadin a hypoxic microenvironment. To identify novel inhibitors of blocked the proliferative and colony-forming actions of MST4 the MST4 kinase for potential future clinical use, computation- as well as the ability to activate specific downstream signaling al-based virtual library screening was used to dock the Sell- and hypoxia-inducible factor-1 effectors. Together, these data eckChem kinase inhibitor library into the ATP-binding site of identify hesperadin as the first potent, selective inhibitor of the the MST4 crystal structure. Several inhibitor candidates were MST4 kinase with the capacity to block pituitary tumor cell identified with the potential to bind with high affinity. Using a growth in a hypoxic microenvironment. Mol Cancer Ther; 15(3); TR-FRET in vitro recombinant kinase assay, hesperadin, initially 412–20. 2015 AACR.

Introduction increased risk of residual tumor regrowth in 30% after trans- sphenoidal surgical resection and need for additional surgery or Pituitary tumors are the most common type of brain tumor, radiation treatment (4). There are no widely accepted prognostic detected clinically in 1/10,000 persons, but present at autopsy in biomarkers and no medical therapies for gonadotrope pituitary up to 20% of the population (1, 2). These tumors are usually tumors. Thus, new treatment options are needed. derived from one of the five different pituitary cell types, including The underlying pathogenesis of pituitary tumors is poorly prolactin, growth hormone, gonadotrope, corticotrope, and understood due to a limited access to human tissue, lack of human thyrotrope. Gonadotrope, often called non-functioning or null cell lines, and/or optimal animal models (5, 6). Prior studies by our cell tumors, represent 35% of pituitary tumors, and are identified group and others have used gene expression microarray profiling of by expression of follicle-stimulating hormone, luteinizing hor- individual human pituitary tumor samples to identify and char- mone, and/or alpha-subunit mRNAs or protein by immunohis- acterize candidate genes involved in pituitary tumor promotion or tochemistry (3). These tumors present clinically more commonly maintenance (7–10). Using copy-number variation analysis and in men than in women, with hypogonadism due to low testos- DNA microarray profiling of individual human gonadotrope pitu- terone and tumor mass effects causing visual disturbances, and itary tumors and normal pituitaries, we recently identified the compromise to normal pituitary function, often resulting in mammalian Ste20-like kinase 4 (MST4) as a genetically and hypopituitarism (3). Local invasion into adjacent structures and genomically dysregulated gene (11). dura occurs in approximately 50% of patients, leading to Originally identified in yeast pheromone signaling, upstream of MAPK, Sterile 20 (Ste20) is a serine threonine kinase (12, 13). Mammalian Ste20-like kinases comprise a large family grouped 1 Division of Endocrinology, Department of Medicine, University of into two structurally distinct subfamilies: the p21-activated kinase Colorado School of Medicine, Aurora, Colorado. 2Department of Phar- maceutical Sciences, University of Colorado School of Pharmacy, family (PAK) and the germinal center kinase (GCK) family (12). Aurora, Colorado. 3Research Service,Veterans Affairs Medical Center, There are 8 subfamilies in the GCK group (GCKI to VIII; refs. 14– Denver, Colorado. 16). MST4, MST3, and SOK1 compromise the GCKIII subfamily, Corresponding Author: Margaret E. Wierman, 111H Endocrinology, Veteran which share approximately 90% of the amino acid sequence in the Affairs Medical Center, 1055 Clermont St. Denver, CO 80220. Phone: 303- kinase domain, but have less than 20% alignment in the C- 399-8020, ext. 3150; Fax: 303-393-5271; E-mail: terminal domain (12, 17). This GCK subfamily has shown a [email protected] broad array of cellular functions. SOK1 has a reported role in doi: 10.1158/1535-7163.MCT-15-0703 cell migration, and MST3 can play a proapoptotic role (18–20). 2015 American Association for Cancer Research. Depending on cell type, MST4 can play multiple roles in the cell.

412 Mol Cancer Ther; 15(3) March 2016

Identifying a Novel MST4 Kinase Inhibitor

For example, MST4 is proapoptotic in breast cancer cells (21), but Molsoft ICM-Pro 3.8. The top 20 compounds that scored highest in embryonic kidney cells, cerebral cavernous modulator-3 in both sets of simulations were then docked again into MST4 (CCM3) shuttles MST4 from the Golgi to the plasma membrane using the flexible docking protocol in Discovery Studio (25). The to interact with Ezrin/Radixin/Moesin (ERM) proteins to promote final ranking of the docked poses was performed via consensus cell survival (17). In HeLa cells, MST4 may also be involved in scoring, combining the predicted binding energy with the Jain regulation of cell migration via its interaction with the Golgi (26), PLP2 (27), and Ludi3 (28) scoring functions. protein GM130 (22). Together, these data imply that MST4 serves a diversity of roles and cell-specific functions. Reagents The role of MST4 in tumor development or maintenance has The open reading frame of mMst4 was inserted into pcDNA3 not been extensively studied. Prior to our study, only one report vector as described (11). MST4 TR-FRET biochemical assay kit was showed that MST4 induces anchorage-independent growth purchased from Perkin Elmer. Hesperadin, PKI-587, and volaser- and increases in vitro proliferation as well as in vivo tumorigen- tib were purchased from SelleckChem. esis using prostate cancer cell lines (23), suggesting a role in prostate cancer progression. Our recent study demonstrated TR-FRET kinase assay that MST4 has the potential to promote pituitary tumorigenesis LANCE (Perkin Elmer) Europium TR-FRET kinase binding by modulation of cell proliferation and survival in response assays were performed in white 384-well plates (Perkin Elmer, to a hypoxic microenvironment (11). The functional effects of OptiPlate #6007299) using recombinant MST4 kinase (Carna MST4 in pituitary tumor cells were dependent on the MST4 Biosciences #07-119), ULight PKC substrate (Perkin Elmer kinase sequence and downstream signaling pathways (11). #TRF0108), ATP (Sigma Aldrich #A26209) and LANCE Eu- Additional data in our laboratory suggest the kinase is upre- anti-PKC (Ala25Ser) antibody (Perkin Elmer #TRF0207). Assays gulated in all pituitary tumor cell types, which support future were performed at 25 C in a reaction mixture consisting of 2 mL efforts to target this kinase as a potential new medical therapy serially diluted drug solution, 4 mL MST4 solution, 2 mL ULight for all types of human pituitary tumors and other tumors where PKC substrate solution, and 2 mL ATP solution. All reagents were MST4 is overexpressed. prepared as solutions in 1 kinase buffer (50 mmol/L HEPES, 1 In the present study, we utilized a computational-based and mmol/L EGTA, 10 mmol/L MgCl2, 1 mol/L DTT, 0.1% Tween, pH experimental screening approach to screen a database of small 7.5). Hesperadin solutions (0.02 nmol/L to 5 mmol/L final con- molecule compounds for potential MST4 inhibitors. Specifically, centration) were prepared from a DMSO stock solution through a virtual library screen was used to dock the commercially avail- serial dilution into kinase buffer such that final DMSO concen- able SelleckChem kinase inhibitor library into the ATP-binding trations did not exceed 0.5%, and this was shown to have no effect site of the MST4 crystal structure. This approach identified several on kinase activity. Required MST4 kinase and ATP concentrations, candidate MST4 kinase inhibitors with the potential to bind MST4 determined via titration, were 1.5 nmol/L and 17 mmol/L, respec- with high affinity. This docking analysis combined with an MST4 tively. ULight PKC substrate was used at a final concentration of TR-FRET in vitro kinase assay identified hesperadin as a candidate 50 nmol/L. All reagents were incubated together for 1 hour, before MST4 kinase inhibitor. Accordingly, functional studies using the reaction was halted through the addition of 5 mL 10 mmol/L hesperadin demonstrated nanomolar inhibition of the multiple EDTA solution in 1 LANCE detection buffer (Perkin Elmer functional roles of MST4, including blocking survival, growth, #CR97-100) followed by 5 mL LANCE Eu-anti-PKC (Ala25Ser) and tumorigenicity and activation of cell-specific downstream antibody solution to a final concentration of 2 nmol/L in 1 signaling pathways in a hypoxic LbT2 gonadotrope pituitary cell LANCE detection buffer. The plate was incubated in the dark for 1 model. Together, these data identify the first potent inhibitor of hour, before being read using a PerkinElmer Envision 2104 the MST4 kinase active at nanomolar concentrations with the Multilabel Reader enabled for TR-FRET (excitation ¼ 340 nm; capacity to selectively abrogate MST4 kinase effects under a tracer emission ¼ 665 nm; antibody emission ¼ 615 nm; delay ¼ hypoxic microenvironment and support future studies of its 100 ms; integration ¼ 200 ms). Emission ratios (665/615 nm) were ability to modulate pituitary tumor growth in rodent models determined for each inhibitor concentration (n ¼ 6), and the data and other cancers where the kinase is dysregulated. were analyzed using a nonlinear regression analysis of the log dose–response curve to afford an IC50 value.

Materials and Methods Cell culture Computational-based library screen LbT2 gonadotrope cells from P. Mellon (University of Califor- Computational modeling was performed using Accelrys Dis- nia, San Diego, San Diego, CA) were cultured as previously covery Studio 3.5 (Acclerys Inc.) and Molsoft ICM-Pro 3.8 (Mol- described (11). There are no human gonadotrope cell lines soft Inc.). Crystal structure coordinates for MST4 were down- available and these cells, immortalized with simian virus 40 T- loaded from the protein data bank (PDB ID: 3GGF, www.pdb. antigen, are the only functional gonadotrope tumor cell lines org). The protein structure was typed with the CHARMM force- available and used widely in the field. Cells were cultured in field (24) and energy minimized with the smart minimizer DMEM (Invitrogen) supplemented with 10% fetal bovine serum protocol within Discovery Studio using the Generalized-Born (FBS; Gemini), 100 U/mL penicillin, and 100 mg/mL streptomy- with simple switching implicit solvent model to a root mean cin at 37 C in humidified 5% CO2 incubation chamber. LbT2 square gradient (RMS) convergence <0.001 kcal/mol prior to use stable transfectants, including vector pcDNA3 and MST4 wild- in the docking studies. The commercially available (24) Sell- type, were generated using Lipofectamine 2000 (Invitrogen) eckChem kinase inhibitor library (SelleckChem, Inc.) was docked following the manufacturer's protocol. Selection of stably over- into the ATP-binding site of the MST4 crystal structure using the expressing pcDNA3 and MST4-WT cells were generated from the library docking tools in both Accelrys Discovery Studio 3.5 and population of clones under geneticin selection (600 mg/mL;

www.aacrjournals.org Mol Cancer Ther; 15(3) March 2016 413

Xiong et al.

Gemini). In prior studies, we saw no effect of the MST4 kinase in with PBS and were fixed with 4% paraformaldehyde for 25 response to growth factors or in response to normoxia (11). The minutes at 4C. After being washed three times with PBS, the pituitary cell hypoxia model was created based upon the hypoxic cell membranes were permeabilized with 0.2% Triton X-100 in microenvironment that pituitary tumors grow within as described PBS for 5 minutes. TUNEL reaction mixture was made according (11). Briefly, short-term experiments were performed in 1% to the manufacturer's protocol (Promega), and 50 mL of the hypoxia. Long-term experiments for proliferation and colony TUNEL reaction mixture was added to the cells, then allowed to formation were performed under 5% hypoxia as no cells will incubate for 1 hour at 37C in a humidified chamber. After survive under more stringent conditions. washing with PBS 3 times, coverslips were mounted with DAPI ProLong Gold antifade reagent (Invitrogen). Using fluorescence Colony formation assay microscopy (Nikon Elipse E600), 500 cell nuclei were visualized Anchorage-dependent growth of tumor cells was investigated and the green fluorescence of apoptotic cells was counted. Counts by a standard colony formation assay. LbT2 transfectant cells were between replicate coverslips were averaged and reported as a 6 plated in triplicate in a 6-well plate (1.0 10 per well) and percentage of the 500 cells counted. incubated at 37 C under humidified 5% CO2. Twenty-four hours later, the transfectants were placed under hypoxia (5% O2)in Immunoblot analysis growth medium for 7 days in the absence or presence of hesper- Immunoblotting was performed as previously described (11). adin (20 nmol/L). Media were aspirated and replaced with fresh The bicinchoninic acid assay (BCA; Pierce) was performed to media containing hesperadin (20 nmol/L) or control media every quantify protein concentrations in cell lysates, and equal amounts 2 days during the incubation. Surviving colonies were stained of protein (25 mg) were separated by SDS-PAGE. Using the mini- with Crystal Violet for 10 minutes after methanol fixation for 5 transblotter system (Bio-Rad), the gels were blotted to polyvinyl minutes, and visible colonies (50 cells) were counted. difluoride membranes (PVDF). After blocking in a 3% BSA solution, membranes were incubated with primary antibodies at BrdUrd proliferation assay 4C overnight. Antibodies against mouse AKT, p38, ERK, phos- The proliferation assay was performed by plating 20,000 cells pho-AKT, phospho-p38, and phospho-ERK (Cell Signaling Tech- in a 24-well plate containing microscope coverslips in growth nology) were used at 1:1,000 dilutions. GAPDH (Millipore) was medium. Cells were grown under norrmoxic conditions for 24 used at a 1:2,000 dilution. The membranes were then washed with hours before adding hesperadin at various concentrations (0, 5, TBST and incubated for 1 hour with horseradish peroxidase– 10, 20, 40 nmol/L). After 3 hours of preincubation with hesper- conjugated secondary antibodies (Bio-Rad). Proteins were visu- adin, cells were placed in a hypoxic environment (5% O2) for 7 alized by enhanced chemiluminescence according to the manu- days. Due to the half-life of hesperidin, it was added every 2 days facturer's protocol (Pierce). in fresh hypoxic media. For BrdUrd staining, on day 6 the LbT2 transfectants were incubated with serum-free and antibiotic-free Statistical analysis (SFAF) DMEM containing BrdUrd (Sigma-Aldrich; 10 mmol/L) Data are presented as means SEM from three or more separate under 5% O2 for 24 hours. Cells were washed with PBS three times experiments. The P-value calculations were conducted using an and fixed in 4% paraformaldehyde for 20 minutes. To increase unpaired Student t test for two-group comparison or ANOVA permeability, cells were incubated in a buffer of 0.3% Triton X- (with Bonferroni posttest analysis for multiple comparisons). All 100 in PBS for 15 minutes. Cells were then blocked in a solution of data were analyzed and presented by using GraphPad Prism 10% bovine serum albumin (BSA) in PBS for 10 minutes. Anti- software (version 5.0; GraphPad Software Inc.). BrdUrd antibody (100 mL; BD Pharmingen) in 0.5% Tween-20 in PBS was added at a 1:100 concentration and incubated in a humidified chamber for 1 hour. After washing with PBS three Results times, cells were incubated for 30 minutes with rhodamine- Computational docking analysis identifies inhibitor candidates conjugated goat anti-mouse IgG, according to the supplier pro- In order to identify potential inhibitors of the MST4 kinase as a tocol (Life Technologies). Cells were washed with PBS three times, potential therapeutic target in pituitary tumors, we performed a then incubated with 0.5 mg/mL of diamidino-2-phenylindole virtual screen of the commercially available SelleckChem kinase (DAPI; Invitrogen) in methanol for 6 minutes. Prior to micro- inhibitor library consisting of 242 structurally diverse inhibitors scopic examination, cells were washed with H2O, and coverslips designed to target a variety of cellular kinases. Compounds were were applied using Flo-Texx mounting medium. A total of 500 docked into the ATP binding site of MST4 and an overall ranking cells per cover slip were counted and the percentage of BrdUrd- was generated according to predicted binding affinity as described positive cells for each treatment condition was recorded. Replicate in Materials and Methods (see Table 1). cover slips were averaged, and the experiment was repeated three times (N ¼ 3). Identification of MST4 protein expression levels in pituitary adenomas Apoptosis assay in response to a hypoxic microenvironment MST4 protein expression was detected using immunoblot To analyze apoptosis, terminal deoxynucleotidyl transferase analysis of normal pituitary and multiple types of pituitary dUTP nick end labeling (TUNEL) was performed. A total of adenomas [gonadotrope, as well as prolactin (PRL), adrenocor- 50,000 cells were plated in a 24-well plate containing microscope ticotropin (ACTH) and growth hormone; Fig. 1A]. MST4 was coverslips in growth medium and allowed to grow for 24 hours. consistently upregulated in all pituitary tumor samples analyzed. The cells were then starved overnight, treated with hesperadin (0, To create a model of gonadotrope tumorigenesis, MST4 was stably 5, 10, 20, 40 nmol/L) for 3 hours, then placed in a hypoxia overexpressed in LbT2 gonadotrope cells at levels equal to or less chamber (1% O2) for 17 hours. Cells were then washed 3 times than that seen in gonadotrope adenomas (Fig. 1B).

414 Mol Cancer Ther; 15(3) March 2016 Molecular Cancer Therapeutics

Identifying a Novel MST4 Kinase Inhibitor

Table 1. Predicted MST4 inhibitors as determined by computational modeling a a Consensus ranking Name Structure Intended target Intended target IC50 1 PKI-587 PI3Ka, g, and mTOR 0.4, 5.4, and 1.6 nmol/L

2 Hesperadin Aurora kinase B 250 nmol/L

3 Apatinib VEGFR2 1 nmol/L

4 PHA-793887 CDK2, 5, and 7 8, 5, and 10 nmol/L

5 Volasertib PLK1 0.87 nmol/L

6 KU-60019 ATM 6.3 nmol/L

7 GSK1363089 HGFR and VEGFR2 0.4 and 0.9 nmol/L

8 Motesanib VEGFR1, 2, and 3 2, 3 and 6 nmol/L

9 SNS-314 Aurora kinase A, B, and C 9, 31, and 3 nmol/L

10 Tivozanib VEGFR1, 2, and 3 30, 6.5, and 15 nmol/L

a Intended target and relevant IC50 obtained from Selleck Chemicals inventory (http://www.selleckchem.com).

Comparison of the effects of selected inhibitor candidates in hesperadin and the Polo-like kinase 1 (PLK-1) inhibitor volaser- hypoxic cell model tib. The mTOR/PI3K pathway is a signal transduction cascade The computational modeling analysis revealed a list of 10 top- involved in cell growth and metabolism and has been previously ranked molecules that were predicted to bind the ATP-binding site shown to play a role in pituitary tumorigenesis (29). As expected, of MST4 (see Table 1). Based upon review of the literature and a PKI-587 abolished MST4 protected cell survival effects at less than goal of identifying novel inhibitors, 3 out of 10 inhibitor candi- 10 nmol/L (Fig. 1C), which was within the compounds IC50 range dates were selected and investigated in the pituitary cell model: against mTOR (30). Volasertib is a well-known small molecule the mTOR inhibitor PKI-587, the Aurora kinase B inhibitor inhibitor of PLK-1 (31). It did not show any effects on the ability

www.aacrjournals.org Mol Cancer Ther; 15(3) March 2016 415

Xiong et al.

A B Normal pituitary Gonadotrope PRL ACTH GH MST4 MST4

GAPDH b-Tubulin

CDE 60 60 60 pcDNA3 MST4 pcDNA3 MST4 pcDNA3 MST4 50 50 50 40 40 40 30 30 30 20 20 20 # * 10 ## # # # 10 *

10 cells nonviable % of * % of nonviable cells nonviable % of % of nonviable cells nonviable % of 0 0 0 10.10 10 1,000100 10.10 10 100 1,000 10.10 10 100 1,000 PKI-587 (nmol/L) Volasertib (nmol/L) Hesperadin (nmol/L)

Figure 1. Hypoxia model conﬁrms the effects of selected inhibitor candidates on cell survival under severe hypoxia. A, immunoblot analysis of MST4 protein levels in normal pituitary and pituitary adenomas [gonadotrope, prolactin (PRL), ACTH, and growth hormone]. GAPDH was used as a loading control. B, immunoblot analysis shows overexpression of MST4 in pcDNA3-MST4–stable transfectants. C, percentage of nonviable cells in vector control and MST4 cells in the presence of the mammalian target of rapamycin (mTOR) inhibitor PKI-587 (0, 0.1, 1, 10, 100, and 1,000 nmol/L). D, percentage of nonviable cells in vector controland MST4 cells in the presence of the Polo-like kinase 1 (PLK-1) inhibitor volasertib (0, 0.1, 1, 10, 100, and 1,000 nmol/L). E, percentage of nonviable cells in control and MST4 transfectants in the presence of hesperadin (0, 0.1, 1, 10, 100, and 1000 nmol/L). , P < 0.01; #, P < 0.0001, MST4 transfectants compared with pcDNA3 vector control cells.

of MST4 to protect cells from hypoxia-induced apoptosis (Fig. Determination of the in vitro inhibitory activity of hesperadin 1D). In contrast, incubation with hesperadin, an inhibitor ini- against MST4 tially described as an Aurora kinase B inhibitor, abolished MST4- Hesperadin is an indole-based compound (Fig. 2A) that is a induced cell survival (Fig. 1E). MST4 cells displayed decreased known ATP-competitive Aurora B kinase inhibitor. From our rates of hypoxia-induced apoptosis compared with vector con- computational screen, hesperadin displayed multiple binding trols (3.21-fold, P < 0.01). Incubation with hesperadin dimin- interactions in the ATP-binding domain of MST4 (Fig. 2B). The ished the antiapoptotic effects of MST4 in a dose-dependent inhibitory activity of hesperadin against recombinant human manner (2.6-fold at 0.1 nmol/L; 1.84-fold at 1 nmol/L; 1.49-fold MST4 was subsequently determined in an in vitro kinase time- at 10 nmol/L; 1.07-fold at 100 nmol/L and 1,000 nmol/L, resolved ﬂuorescence resonance energy transfer (TR-FRET) assay compared with vector controls , P < 0.05 by one-way ANOVA). (Fig. 2C). Potent inhibition of MST4 kinase activity was observed These data suggest that hesperadin may be a highly selective with hesperadin (IC50 ¼ 6.18 2.2 nmol/L, n ¼ 6). Importantly, inhibitor of the MST4 kinase. this IC50 is signiﬁcantly lower than the published IC50 value

O A HN H B N Figure 2. N Hesperadin is identiﬁed as a potent NH S inhibitor of MST4. A, the chemical O O Hesperadin structure of hesperadin. B, small C molecule docking depicting the Leu102 Glu100 predicted interaction of hesperadin (cyan) with the ATP-binding domain 0.5 Hesperadin of MST4. C, in vitro TR-FRET 0.4 PKI-587 recombinant kinase assay Volasertib Lys53 demonstrates direct inhibition of the 0.3 MST4 kinase by hesperadin at low 0.2 nanomolar concentrations. PKI-587 and volasertib were also screened in 0.1 the TR-FRET recombinant kinase Asp109 assay and show no direct inhibition 0.0 0.01 1 100 10,000 of MST4. Emission ratio (665/615 nm) Inhibitor (nmol/L)

416 Mol Cancer Ther; 15(3) March 2016 Molecular Cancer Therapeutics

Identifying a Novel MST4 Kinase Inhibitor

A Hypoxia (1% O2, 17 h) B Figure 3. pcDNA3 MST4 Hesperadin blocks the effects of MST4 pcDNA3 MST4 on cell survival under acute hypoxia (1% O2). A, representative 40 immunocytochemistry of apoptotic cells as assessed by TUNEL in the 0 nmol/L presence or absence of hesperadin 30 (40 nmol/L) under hypoxia for 17 hours. B, rates of apoptosis were 20 expressed as a percentage of TUNEL- positive cells to total cells in the absence or presence of various Hesperadin 10 concentrations of hesperadin (5, 10,

20, and 40 nmol/L). , P ¼ 0.01 (at 10 cells TUNEL-positive % of 0 nmol/L); #,P ¼ 0.04 (at 20 nmol/L); 40 nmol/L , P < 0.001, MST4 transfectants 0 5 10 20 40 compared with pcDNA3 control cells. Hesperadin (nmol/L)

(250 nmol/L) of hesperadin to inhibit Aurora kinase B (32). PKI- increase the rates of proliferation in a dose-dependent fashion, 587 and volasertib were also tested in the TR-FRET assay and compared with controls (Fig. 4A and B; 2.60-fold at 5 nmol/L, P < neither compound inhibited MST4 within the range of the assay 0.001; 2.41-fold at 10 nmol/L, P ¼ 0.002; 1.38-fold at 20 nmol/L, (up to 5 mmol/L; Fig. 2B), confirming that the effects PKI-587 on P ¼ 0.12; 2.16-fold at 40 nmol/L, P ¼ 0.91). To examine if hypoxia-induced apoptosis of MST4 transfectants was not via hesperadin would modulate the rates of colony formation, actions through MST4. The TR-FRET assay results confirmed the LbT2-MST4 or vector controls were grown under chronic hypoxic data derived from the computational-based screen and the effects conditions (5% O2) for 14 days (Fig. 4C and D). Under hypoxia, of hesperadin in the hypoxic cell model. Collectively, these data an increased number of colonies were observed in MST4 trans- support hesperadin as the first selective nanomolar inhibitor of fectants (7,837 401 colonies per well in MST4 cells compared MST4 and supported further analysis of hesperadin and its inhib- with 3,917 135 colonies/well in vector control cells, a 2.0-fold itory effects on MST4 in the hypoxic pituitary tumor cell model. increase, P < 0.001; Fig. 4C). In contrast, incubation with hesperadin (20 nmol/L) completely blocked the promotion of colony Hesperadin abolishes MST4-protected cell survival in response formation induced by MST4 overexpression (2,941 253 colo- to hypoxia nies per well in MST4 transfectants compared with 3,031 622 The computational modeling data and in vitro kinase assay colonies per well in vector cells, 0.97-fold, P ¼ 0.91; Fig. 4D). revealed hesperadin as an MST4 kinase inhibitor. To further These results confirm that the hesperadin blocks MST4-induced evaluate this compound, hesperadin was investigated for its ability cell proliferation and colony formation of gonadotrope pituitary to abrogate the protective effect of MST4 in response to hypoxic tumor cells in a hypoxic microenvironment. stress. To mimic a hypoxic milieu, control and stable MST4 LbT2 transfectants were exposed to severe hypoxia (1% O2) for 17 hours MST4-regulated signaling is blocked by hesperadin in the absence or presence of hesperadin in various doses, and cell We have previously demonstrated that the MST4 kinase death was assessed by TUNEL staining (Fig. 3). Decreased rates of regulates pituitary cell proliferation and survival via p38 MAPK apoptosis were detected in MST4 cells in response to hypoxia and AKT signaling cascades (see the model in Fig. 5A; ref. 11). compared with those observed in vector controls (2.67-fold at To investigate the inhibitory effect of hesperadin on MST4 17 hours; P < 0.001). Incubation with hesperadin abolished the signaling, LbT2 transfectants were incubated in the absence or MST4-protective effects on cell survival in a dose-dependent fash- presence of hesperadin for 3 hours (20 or 40 nmol/L), and ion with an EC50 of 10 nmol/L. Treatment with hesperadin (40 lysates were used for immunoblots to analyze the expression nmol/L) completely blocked MST4 protection against hypoxia- and activation of p38 MAPK and AKT pathways (Fig. 5B). In the induced apoptosis with rates of cell death similar to that of vector absence of hesperadin, activated p-p38 MAPK and p-AKT were controls (0.93-fold, P ¼ 0.69). These data confirm that hesperadin detected in MST4 transfectant compared with vector controls. inhibits the ability of MST4 to promote pituitary tumor cell survival In contrast, incubation with hesperadin attenuated this MST4- in a hypoxic microenvironment. increased activation of p38 MAPK and AKT. These data support the assertion that hesperadin blocks pathways downstream of Hesperadin blocks MST4 promotion of proliferation and the MST4 kinase. colony formation in pituitary tumor cells under long-term hypoxic stress Hesperadin blocks MST4 activation of the hypoxia-inducible To further explore the ability of hesperadin to modulate MST4 factor (HIF-1) pathway effects, we examined its impact on gonadotrope cell proliferation Our prior studies demonstrated that in addition to activation of and tumorigenicity. Cell proliferation was assessed by BrdUrd the p38 MAPK and PI3K/AKT signaling pathways, MST4 activates incorporation under chronic hypoxia (5% O2) for 7 days in the HIF-1 and its downstream effectors (11). To confirm that hesper- absence or presence of hesperadin (Fig. 4A and B). Incubation adin may also interrupt this pathway, control and MST4 cells were with hesperadin inhibited the ability of MST4 transfectants to exposed to hypoxia (1% O2) for 17 hours in the absence or

www.aacrjournals.org Mol Cancer Ther; 15(3) March 2016 417

Xiong et al.

A Hypoxia (5% O2, 7d) B pcDNA3 MST4 pcDNA3 MST4 20 ** ** 15 *

0 nmol/L Figure 4. Hesperadin blocks the effects of MST4 on 10 proliferation and colony formation under chronic hypoxic stress (5% O2). A, representative immunocytochemistry of the 5 rates of BrdUrd incorporation in pcDNA3 Hesperadin control and MST4 transfectants in the absence

% of BrdUrd-positive cells 0 and presence of hesperadin (40 nmol/L) under 40 nmol/L 0 5 10 20 40 chronic hypoxia (5% O2) for 7 days. B, Hesperadin (nmol/L) hesperadin abolishes MST4 increased cell proliferation. In the absence or presence of various doses of hesperadin (0, 5, 10, 20, and 40 nmol/L), cell proliferation was measured by Hypoxia (5% O2, 7d) D C BrdUrd after exposure to chronic hypoxia (5% pcDNA3 MST4 pcDNA3 O2) for 7 days. C, photomicrograph of colony 10,000 formation in vector and MST4 transfectants ** MST4 incubated with DMSO or hesperadin 8,000 (20 nmol/L). D, hesperadin decreases the ability of MST4 to promote increased colony formation. Numbers of pcDNA3 and MST4 6,000 transfectant colonies were counted after DMSO exposure to chronic hypoxia (5% O2) for 7 days. # , P ¼ 0.002; , P < 0.001, MST4 transfectants 4,000 compared with pcDNA3 vector cells; #, P < 0.01, MST4 transfectants with 2,000 hesperadin treatment (20 nmol/L) Mean of colony of numbersMean

(20 nmol/L) compared with the cells treated with DMSO.

0 DMSO Hesperadin (20 nmol/L) Hesperadin

presence of hesperadin (20 or 40 nmol/L) and activation of HIF-1 with hesperadin (IC50 ¼ 6.18 2.2 nmol/L). This is in compar- was assessed by a HIF-1 reporter construct (Fig. 5C, inset). Control ison to the reported cell-free inhibitory activity of the compound cells exposed to DMSO showed a nonsignificant activation of the against Aurora B at 250 nmol/L (32). These data support that HRE-luciferase construct (1.95-fold, P ¼ 0.2) in the presence of hesperadin is a potent inhibitor of MST4, demonstrate greater hypoxia. In contrast, the MST4 transfectants hyper-responded to selectivity for MST4 compared with Aurora B kinase, and suggest hypoxia activating the HIF-1 reporter, with the HRE-luciferase that hesperadin might be more appropriately termed an MST4 activity increasing by 29.8-fold (P < 0.001). Coincubation with inhibitor. hesperadin decreased this activity in a dose-dependent fashion These studies strengthened the rationale to analyze the ability (0.8-fold at 20 nmol/L, P ¼ 0.22; 0.49-fold at 40 nmol/L, P ¼ of hesperadin to block the proliferative, tumorigenic, and pro- 0.002), compared with untreated MST4 cells. These results again survival effects of MST4 in the hypoxic pituitary cell model. confirm the potent ability of hesperadin to limit the effects of Pituitary tumors enlarge within a restricted space and blood MST4 to drive the hyper-responsiveness of downstream targets supply and are exposed to a hypoxic microenvironment (33, containing a HIF-1 response element. 34). We developed both a short-term severe hypoxia model and a longer term pituitary tumor cell hypoxia model (11) to analyze Discussion the function of MST4 and to test the ability of hesperadin to antagonize MST4 actions. In a chronic hypoxic microenviron- A computational-based virtual screen of the Selleckchem kinase ment, hesperadin blocked the ability of MST4 to promote colony inhibitor library against the MST4 crystal structure identified formation in soft agar, and inhibited MST4 increased cell prolif- several potential inhibitors of MST4. Following the computation- eration. Under short-term severe hypoxia, MST4 pituitary cell al screen, several inhibitors were identified with optimal docking transfectants incubated with hesperadin lost their survival advan- scores to bind to the ATP binding site of the MST4 crystal structure. tage. We then demonstrated that the activation of the downstream We examined the mTOR inhibitor PKI-587, the Aurora kinase effectors of MST4, including p-p38, p-AKT, and HIF-1, was abro- inhibitor hesperadin, and the Polo kinase inhibitor volasertib in gated in the presence of hesperadin. Together, these data support our hypoxic cell model, and hesperadin diminished the antia- efforts to further investigate hesperadin as a potential MST4 poptotic effects of MST4 in a dose-dependent manner. We then inhibitor in vivo, toward a future goal to utilize the compound examined inhibition of MST4 kinase activity using a TR-FRET in as a potential novel medical therapy for patients with pituitary vitro kinase assay and found potent low nanomolar inhibition tumors.

418 Mol Cancer Ther; 15(3) March 2016 Molecular Cancer Therapeutics

Identifying a Novel MST4 Kinase Inhibitor

A Hypoxic stress

MST4 O HN H N N S NH O O p38 Hesperadin AKT HIF-1

Figure 5. A, illustration of MST4 signaling pathways in response to hypoxic stress. B, hesperadin blocks Proliferation MST4 downstream signaling effectors. Survival HIF-1 Tumorigenicity Phosphorylation of AKT, p38 MAPK, and ERK were determined by immunoblot in the presence HRE or absence of hesperadin (0, 20, and 40 nmol/L). C, MST4-induced HIF-1 activity is blocked by hesperadin. Control or MST4 cells were transfected with HRE-luciferase reporter constructs. After 24 hours of transfection, cells B C were subjected to normoxia or hypoxia (1% O2) Hesperadin 0 20 nmol/L 40 nmol/L Luciferase for 17 hours. HRE-luciferase values were detected HRE HRE HRE and normalized to Renilla control luciferase values. , P < 0.001, MST4 transfectants 25 compared with pcDNA3 vector cells; p-p38 pcDNA3 * #, P ¼ 0.002, MST4 transfectants with 20 hesperadin treatment compared with MST4 p38 * DMSO-treated controls. 15 ,# p-AKT * 10 AKT Fold change

(HRE-LUC/Renilla) 5 p-ERK 0 ERK Hesperadin 0204002040 GAPDH (nmol/L) Normoxia 1% Hypoxia

Limitations of the studies include the lack of human pituitary of cancer tissues suggests that 4% to 5% of prostate cancers in their cell lines and that currently available mouse gonadotrope cell dataset have a genetic amplification of the MST4 locus as a lines are SV40 immortalized. However, the effects of immor- mechanism for overexpression (36, 37). These data suggest that talization would be expected to be similar in both vector prostate cancer is a potential area of interest. MST4 is also control and MST4 transfectants and not the major mediator amplified in ovarian, stomach, and esophageal cancers and some of the differential effects observed. In addition to Aurora kinase head and neck cancers (36, 37). Further studies are needed to B, hesperadin has been shown to block several other kinases, determine if MST4 is a biomarker for disease activity or plays a including AMPK, Lck, MKK1, MAPKAP-K1, CHK1, and PHK tumorigenic role in these cancers, and if hesperadin as a single (32); however, these were observed at doses of 1 mmol/L, far in agent or in combination therapy may be a novel therapeutic excess of the low nanomolar doses used in our studies. Future approach for the treatment of pituitary tumors and in additional studies will be needed to examine if there are any off-target cancer types. effects of hesperadin on other kinases in the low nanomolar range. Disclosure of Potential Conflicts of Interest What about dysregulation of the MST4 kinase in other cancers? No potential conflicts of interest were disclosed. To date, only one report suggested that MST4 is overexpressed in prostate cancer samples and cell lines, and that ectopic expression Authors' Contributions of wild-type, but not kinase dead, MST4 induced cell proliferation Conception and design: W. Xiong, C.J. Matheson, M. Xu, M.E. Wierman and in vivo tumorigenesis in nude mice (28). In a recent evaluation Development of methodology: W. Xiong, C.J. Matheson, M. Xu, D.S. Backos of 110 prostate biopsy cores, MST4 was not expressed in benign Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): W. Xiong, C.J. Matheson, D.S. Backos, M.E. Wierman prostatic hypertrophy samples, but was detected at high levels by Analysis and interpretation of data (e.g., statistical analysis, biostatistics, immunohistochemistry in 39% of prostate cancer samples (35). computational analysis): W. Xiong, C.J. Matheson, M. Xu, D.S. Backos, Using the Bioportal website, analysis of The Cancer Genome Atlas S. Salian-Mehta, K. Kiseljak-Vassiliades, P. Reigan, M.E. Wierman

www.aacrjournals.org Mol Cancer Ther; 15(3) March 2016 419

Xiong et al.

Writing, review, and/or revision of the manuscript: W. Xiong, C.J. Matheson, Cancer Center (P30-CA046934). Molecular modeling studies were conducted at M. Xu, D.S. Backos, T.S. Mills, S. Salian-Mehta, K. Kiseljak-Vassiliades, P. Reigan, the University of Colorado Computational Chemistry and Biology Core Facility, M.E. Wierman which is supported in part by NIH/NCATS Colorado CTSA Grant Number UL1 Administrative, technical, or material support (i.e., reporting or organizing TR001082. data, constructing databases): W. Xiong, M. Xu, M.E. Wierman The costs of publication of this article were defrayed in part by the payment of Study supervision: P. Reigan, M.E. Wierman page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Acknowledgments This work was supported by VA Merit Review 001 (to M.E. Wierman), NIH Received August 25, 2015; revised October 27, 2015; accepted November 27, K12CA086913-12 (to K. Kiseljak-Vassiliades), and the University of Colorado 2015; published OnlineFirst December 31, 2015.

References 1. Ezzat S, Asa SL, Couldwell WT, Barr CE, Dodge WE, Vance ML, et al. The 20. Chen XD, Cho CY. Downregulation of SOK1 promotes the migration of prevalence of pituitary adenomas: a systematic review. Cancer 2004;101: MCF-7 cells. Biochem Biophys Res Commun 2011;407:389–92. 613–9. 21. Dan I, Ong SE, Watanabe NM, Blagoev B, Nielsen MM, Kajikawa E, et al. 2. Melmed S. Update in pituitary disease. J Clin Endocrinol Metab 2008;93: Cloning of MASK, a novel member of the mammalian germinal center 331–8. kinase III subfamily, with apoptosis-inducing properties. J Biol Chem 3. Molitch ME. Nonfunctioning pituitary tumors. Handb Clin Neurol 2014; 2002;277:5929–39. 124:167–84. 22. Preisinger C, Short B, De Corte V, Bruyneel E, Haas A, Kopajtich R, et al. 4. Freda PU, Beckers AM, Katznelson L, Molitch ME, Montori VM, Post KD, YSK1 is activated by the Golgi matrix protein GM130 and plays a role in et al. Pituitary incidentaloma: an endocrine society clinical practice guide- cell migration through its substrate 14-3-3zeta. J Cell Biol 2004;164: line. J Clin Endocrinol Metab 2011;96:894–904. 1009–20. 5. Herman V, Fagin J, Gonsky R, Kovacs K, Melmed S. Clonal origin of 23. Sung V, Luo W, Qian D, Lee I, Jallal B, Gishizky M. The Ste20 kinase MST4 pituitary adenomas. J Clin Endocrinol Metab 1990;71:1427–33. plays a role in prostate cancer progression. Cancer Res 2003;63:3356–63. 6. Alexander JM, Biller BM, Bikkal H, Zervas NT, Arnold A, Klibanski A. 24. Brooks BR, Brooks CL 3rd, Mackerell AD Jr, Nilsson L, Petrella RJ, Roux B, Clinically nonfunctioning pituitary tumors are monoclonal in origin. J Clin et al. CHARMM: the biomolecular simulation program. J Comput Chem Invest 1990;86:336–40. 2009;30:1545–614. 7. Shorts-Cary L, Xu M, Ertel J, Kleinschmidt-Demasters BK, Lillehei K, 25. Koska J, Spassov VZ, Maynard AJ, Yan L, Austin N, Flook PK, et al. Fully Matsuoka I, et al. Bone morphogenetic protein and retinoic acid-inducible automated molecular mechanics based induced fit protein-ligand docking neural specific protein-3 is expressed in gonadotrope cell pituitary adeno- method. J Chem Inf Model 2008;48:1965–73. mas and induces proliferation, migration, and invasion. Endocrinology 26. Jain AN. Scoring noncovalent protein–ligand interactions: a continuous 2007;148:967–75. differentiable function tuned to compute binding affinities. J Comput 8. Xu M, Shorts-Cary L, Knox AJ, Kleinsmidt-DeMasters B, Lillehei K, Wierman Aided Mol Des 1996;10:427–40. ME. Epidermal growth factor receptor pathway substrate 8 is overexpressed 27. Gehlhaar DKB, Bouzida D, Rejto P. Rational drug design: novel method- in human pituitary tumors: role in proliferation and survival. Endocrinol- ology and practical applications.Parrill L, Reddy MR, editors. Washington, ogy 2009;150:2064–71. DC 1999. 9. Michaelis KA, Knox AJ, Xu M, Kiseljak-Vassiliades K, Edwards MG, Geraci 28. Bohm HJ. On the use of LUDI to search the Fine Chemicals Directory for M, et al. Identification of growth arrest and DNA-damage-inducible gene ligands of proteins of known three-dimensional structure. J Comput Aided beta (GADD45beta) as a novel tumor suppressor in pituitary gonadotrope Mol Des 1994;8:623–32. tumors. Endocrinology 2011;152:3603–13. 29. Monsalves E, Juraschka K, Tateno T, Agnihotri S, Asa SL, Ezzat S, et al. The 10. Xu M, Knox AJ, Michaelis KA, Kiseljak-Vassiliades K, Kleinschmidt-DeMas- PI3K/AKT/mTOR pathway in the pathophysiology and treatment of pitu- ters BK, Lillehei KO, et al. Reprimo (RPRM) is a novel tumor suppressor in itary adenomas. Endocr Relat Cancer 2014;21:R331–44. pituitary tumors and regulates survival, proliferation, and tumorigenicity. 30. Venkatesan AM, Dehnhardt CM, Delos Santos E, Chen Z, Dos Santos O, Endocrinology 2012;153:2963–73. Ayral-Kaloustian S, et al. Bis(morpholino-1,3,5-triazine) derivatives: 11. Xiong W, Knox AJ, Xu M, Kiseljak-Vassiliades K, Colgan SP, Brodsky KS, potent adenosine 50-triphosphate competitive phosphatidylinositol-3- et al. Mammalian Ste20-like kinase 4 promotes pituitary cell proliferation kinase/mammalian target of rapamycin inhibitors: discovery of com- and survival under hypoxia. Mol Endocrinol 2015;29:460–72. pound 26 (PKI-587), a highly efficacious dual inhibitor. J Med Chem 2010; 12. Ling P, Lu TJ, Yuan CJ, Lai MD. Biosignaling of mammalian Ste20-related 53:2636–45. kinases. Cell Signal 2008;20:1237–47. 31. Gjertsen BT, Schoffski P. Discovery and development of the Polo-like 13. Leberer E, Dignard D, Harcus D, Thomas DY, Whiteway M. The protein kinase inhibitor volasertib in cancer therapy. Leukemia 2015;29:11–9. kinase homologue Ste20p is required to link the yeast pheromone response 32. Hauf S, Cole RW, LaTerra S, Zimmer C, Schnapp G, Walter R, et al. The small G-protein beta gamma subunits to downstream signalling components. molecule hesperadin reveals a role for Aurora B in correcting kinetochore- EMBO J 1992;11:4815–24. microtubule attachment and in maintaining the spindle assembly check- 14. Dan I, Watanabe NM, Kusumi A. The Ste20 group kinases as regulators of point. J Cell Biol 2003;161:281–94. MAP kinase cascades. Trends Cell Biol 2001;11:220–30. 33. Vidal S, Scheithauer BW, Kovacs K. Vascularity in nontumorous human 15. Pombo CM, Force T, Kyriakis J, Nogueira E, Fidalgo M, Zalvide J. The GCK II pituitaries and incidental microadenomas: a morphometric study. Endocr and III subfamilies of the STE20 group kinases. Front Biosci 2007;12: Pathol 2000;11:215–27. 850–9. 34. Kristof RA, Aliashkevich AF, Hans V, Haun D, Meyer B, Thees C, et al. The 16. Strange K, Denton J, Nehrke K. Ste20-type kinases: evolutionarily con- regional oxygen saturation of pituitary adenomas is lower than that of the served regulators of ion transport and cell volume. Physiology (Bethesda) pituitary gland: microspectrophotometric study with potential clinical 2006;21:61–8. implications. Neurosurgery 2003;53:880–5; discussion 5–6. 17. Sugden PH, McGuffin LJ, Clerk A. SOcK, MiSTs, MASK and STicKs: the 35. Zhang H, Ma X, Peng S, Nan X, Zhao H. Differential expression of MST4, GCKIII (germinal centre kinase III) kinases and their heterologous protein- STK25 and PDCD10 between benign prostatic hyperplasia and prostate protein interactions. Biochem J 2013;454:13–30. cancer. Int J Clin Exp Pathol 2014;7:8105–11. 18. Lin CY, Wu HY, Wang PL, Yuan CJ. Mammalian Ste20-like protein kinase 3 36. Gao J, Aksoy BA, Dogrusoz U, Dresdner G, Gross B, Sumer SO, et al. induces a caspase-independent apoptotic pathway. Int J Biochem Cell Biol Integrative analysis of complex cancer genomics and clinical profiles using 2010;42:98–105. the cBioPortal. Sci Signal 2013;6:pl1. 19. Wu HY, Lin CY, Chen TC, Pan ST, Yuan CJ. Mammalian Ste20-like protein 37. Cerami E, Gao J, Dogrusoz U, Gross BE, Sumer SO, Aksoy BA, et al. The cBio kinase 3 plays a role in hypoxia-induced apoptosis of trophoblast cell line cancer genomics portal: an open platform for exploring multidimensional 3A-sub-E. Int J Biochem Cell Biol 2011;43:742–50. cancer genomics data. Cancer Discov 2012;2:401–4.

420 Mol Cancer Ther; 15(3) March 2016 Molecular Cancer Therapeutics

Structure-Based Screen Identification of a Mammalian Ste20-like Kinase 4 (MST4) Inhibitor with Therapeutic Potential for Pituitary Tumors

Weipeng Xiong, Christopher J. Matheson, Mei Xu, et al.

Mol Cancer Ther 2016;15:412-420. Published OnlineFirst December 31, 2015.

Updated version Access the most recent version of this article at: doi:10.1158/1535-7163.MCT-15-0703

Cited articles This article cites 36 articles, 8 of which you can access for free at: http://mct.aacrjournals.org/content/15/3/412.full#ref-list-1

Citing articles This article has been cited by 2 HighWire-hosted articles. Access the articles at: http://mct.aacrjournals.org/content/15/3/412.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at Subscriptions [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://mct.aacrjournals.org/content/15/3/412. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.