AKR1C3 Inhibitor KV-37 Exhibits Antineoplastic Effects And

Published OnlineFirst June 11, 2018; DOI: 10.1158/1535-7163.MCT-17-1023

Small Molecule Therapeutics Molecular Cancer Therapeutics AKR1C3 Inhibitor KV-37 Exhibits Antineoplastic Effects and Potentiates Enzalutamide in Combination Therapy in Prostate Adenocarcinoma Cells Kshitij Verma1, Nehal Gupta1, Tianzhu Zang2, Phumvadee Wangtrakluldee2, Sanjay K. Srivastava1,3, Trevor M. Penning2, and Paul C. Trippier1,4

Abstract

Aldo-keto reductase 1C3 (AKR1C3), also known as type 5 sensitizes CRPC cell lines (22Rv1 and LNCaP1C3) toward the 17 b-hydroxysteroid dehydrogenase, is responsible for intra- antitumor effects of enzalutamide. Crucially, KV-37 does not tumoral androgen biosynthesis, contributing to the develop- induce toxicity in nonmalignant WPMY-1 prostate cells ment of castration-resistant prostate cancer (CRPC) and even- nor does it induce weight loss in mouse xenografts. Moreover, tual chemotherapeutic failure. Significant upregulation of KV-37 reduces androgen receptor (AR) transactivation and AKR1C3 is observed in CRPC patient samples and derived prostate-specific antigen expression levels in CRPC cell lines CRPC cell lines. As AKR1C3 is a downstream steroidogenic indicative of a therapeutic effect in prostate cancer. Combina- enzyme synthesizing intratumoral testosterone (T) and 5a- tion studies of KV-37 with enzalutamide reveal a very high dihydrotestosterone (DHT), the enzyme represents a promis- degree of synergistic drug interaction that induces significant ing therapeutic target to manage CRPC and combat the emer- reduction in prostate cancer cell viability via apoptosis, result- gence of resistance to clinically employed androgen depriva- ing in >200-fold potentiation of enzalutamide action in drug- tion therapy. Herein, we demonstrate the antineoplastic resistant 22Rv1 cells. These results demonstrate a promising activity of a potent, isoform-selective and hydrolytically therapeutic strategy for the treatment of drug-resistant CRPC stable AKR1C3 inhibitor (E)-3-(4-(3-methylbut-2-en-1-yl)-3- that invariably develops in prostate cancer patients following (3-phenylpropanamido)phenyl)acrylic acid (KV-37), which initial treatment with AR antagonists such as enzalutamide. reduces prostate cancer cell growth in vitro and in vivo and Mol Cancer Ther; 17(9); 1833–45. 2018 AACR.

Introduction such as gonadotropin-releasing hormone agonists [Leuprolide (5), goserelin (6), and buserelin (7)] and antiandrogens (bicalu- Prostate cancer is the third (1) leading cause of mortality among tamide; ref. 8) retard prostate cancer cell proliferation and lead to American men and has the highest incidence of all cancers remission. After an initial response to ADT that results in castrate reported in the United States (2). Surgical removal of the prostate levels of circulating androgens, the tumor adapts, giving rise to a by radical prostatectomy and radiotherapy to resect the localized more aggressive and fatal disease known as castration-resistant tumor are standard therapeutic interventions (3). The treatment prostate cancer (CRPC), which is characterized by molecular of advanced and metastatic forms of prostate cancer relies heavily changes that include an increase in the expression of androgen- on androgen deprivation therapy (ADT) that involves surgical synthesizing enzymes and reactivation of androgen signaling (orchiectomy) and/or chemical castration (4). Chemical agents (9, 10). Such adaptations can lead to relapse despite the presence of circulating castrate levels of androgens (11, 12). Abiraterone acetate (AA) was approved by the FDA for CRPC 1Department of Pharmaceutical Sciences, Texas Tech University Health Sciences Center, School of Pharmacy, Amarillo, Texas. 2Center of Excellence in Environ- treatment in 2011 and acts by inhibiting P450c17 (13), an mental Toxicology, Department of Systems Pharmacology and Translational upstream enzyme in the steroid biosynthetic pathway, and is Therapeutics, Perelman School of Medicine, University of Pennsylvania, Phila- effective in CRPC where tumor progression is dependent on delphia, Pennsylvania. 3Department of Immunotherapeutics and Biotechnology, intracrine androgen synthesis that consequently activates AR 4 Texas Tech University Health Sciences Center, Abilene, Texas. Center for signaling (14, 15). However, to prevent hypertensive crisis due Chemical Biology, Department of Chemistry and Biochemistry, Texas Tech to accumulation of desoxycorticosterone (DOC) in the adrenal University, Lubbock, Texas. gland, coadministration with prednisone is imperative (16). Note: Supplementary data for this article are available at Molecular Cancer Enzalutamide (ENZ) is another clinically used therapeutic that Therapeutics Online (http://mct.aacrjournals.org/). exerts potent androgen receptor (AR) antagonistic activity by K. Verma and N. Gupta contributed equally to this article. inhibiting AR nuclear translocation, coactivator recruitment, and Corresponding Author: Paul C. Trippier, Texas Tech University Health Sciences AR binding to androgen response elements (ARE; ref. 17). The Center, 1300 S. Coulter Street, Amarillo, TX 79106. Phone: 806-414-9245; E-mail: chemotherapeutic is effective up to stage 3 CRPC where prolifer- [email protected] ation is dependent on AR activation that occurs even in the doi: 10.1158/1535-7163.MCT-17-1023 absence of an androgen ligand (15). After an initial response to 2018 American Association for Cancer Research. AA or ENZ, resistance develops rapidly due to increases in

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intratumoral androgen biosynthesis (18), AR overexpression, AR AKR1C3 were generated by stable transfection of AKR1C3 mutation that makes the receptor ligand promiscuous, or the plasmid as previously described (12). WPMY-1 cells were pur- appearance of AR splice variants that makes the AR constitutively chased from the ATCC (2017) and maintained in DMEM media active in the absence of the ligand-binding domain (15, 19, 20). supplemented with 5% FBS, 100 U/mL penicillin, and 0.1 mg/mL Due to tumor heterogeneity and the emergence of adaptive path- streptomycin. All cell lines were authenticated via short tandem ways of androgen biosynthesis (21), combined with frequent repeat analysis and tested for mycoplasma using the MycoAlert mutations in AR and androgen biosynthetic enzymes (10, 22), the mycoplasma detection kit as per the manufacturer's instructions prognosis for patients that have advanced to CRPC remains bleak. (Texas cancer cell repository) in May 2017, showing no contam- Thus, there is an urgent need for novel therapeutics that can delay ination. Cell line use was limited to passage nine or lower. Where or reverse the inevitable emergence of resistance. indicated, cells were also cultured in charcoal-stripped (CSS) Type 5 17b-hydroxysteroid dehydrogenase, also known as media prepared by supplementing RPMI 1640 without phenol aldo–keto reductase 1C3 (AKR1C3), acts downstream in the red with charcoal-stripped FBS. All cells were maintained at 37C steroidogenesis pathway and plays a pivotal role in the patho- in a humidified incubator with 5% carbon dioxide. ENZ or genesis and progression of CRPC by catalyzing the conversion of MDV3100 (catalog no. 50-101-3979) and INDO (catalog no. weak androgen precursors to the potent AR ligands: testosterone AAA1991006) were purchased from Fisher Scientific. Stock solu- (T) and 5a-dihydrotestosterone (5a-DHT; ref. 23). Also known as tions (200 mmol/L) of ENZ, INDO, baccharin, KV-32, and KV-37 prostaglandin (PG) F synthase, AKR1C3 catalyzes the conversion were prepared in DMSO and were serially diluted for cell culture of PGD2 to 11b-PGF2a and PGF2a prostanoids (24). An increase in treatments to a final concentration range of 1 to 200 mmol/L, PGF2a leads to activation of the FP receptor and consequently maintaining the final DMSO concentration at less than 1%. induces proliferation and radiation resistance in prostate cancer cells (25). Inhibition of testosterone production by AKR1C3 inhibitors Clinically, AKR1C3 has been shown to be the most upregulated Systems (200 mL) containing 100 mmol/L potassium steroidogenic enzyme in CRPC patients (26). Apart from inducing phosphate (pH 7.0), 0.9 mmol/L NADPH, 1.2 to 40 mmol/L a CRPC phenotype, AKR1C3 also mediates resistance to ENZ and 4-androstene-3,17-dione (D4-AD; containing 2.6 nmol/L [3H]- AA by providing a source of intratumoral androgens, and this 4-androstene-3,17-dione), KV-37 (0–10 mmol/L), and 0.3 to resistance can be surmounted somewhat by indomethacin 10 mmol/L inhibitor plus 1.65 mmol/L AKR1C3 were incubated (INDO), a known AKR1C3 inhibitor that also inhibits COX at 37C, and samples were prepared as previously reported (40). isozymes (27, 28). As the enzyme acts in the final steps of the Quantification of the individual steroid analytes was achieved by androgen synthesis pathway in the prostate (16), AKR1C3 inhi- integrating the radioactivity corresponding to each peak in the bitors would not cause the accumulation of DOC in the adrenal radiochromatogram and representing the radioactivity as a per- gland and would not have to be coadministered with prednisone. centage of the total corrected cpm for the organic fraction from These properties make AKR1C3 an attractive target for managing which it was derived. CRPC disease progression as well as countering therapeutic resistance. The related isoforms AKR1C1 and AKR1C2 share high Stability testing of AKR1C3 inhibitors homology with AKR1C3, function to eliminate DHT, and should Baccharin, KV-37, and a related ester analogue KV-32 (2 mmol/L not be inhibited (29). Numerous AKR1C3 inhibitors with diverse solution in DMSO) were incubated with male CD-1 murine liver scaffolds are known; however, none have made their way into S9 fractions containing a NADPH-generating system for 0 to 240 the clinic (30–32). Considerable interest has developed in the role minutes at 37C. Reactions were quenched with 0.5 mL (1:1) of of AKR1C3 in a variety of cancers of the breast (33), lung (34), and acetonitrile (ACN) containing 0.2% formic acid and 100 ng/mL colon (35). internal standard (IS; final conc. ¼ 50 ng/mL). Samples were Herein, we report on the actions of a novel, potent, isoform- vortexed for 15 seconds, incubated at room temperature for 10 selective and metabolically stable AKR1C3 inhibitor, KV-37 minutes, and spun for 5 minutes at 2,400 rpm. The supernatant (Fig. 1A; refs. 36–38), in androgen-sensitive prostate cancer cell (0.9 mL) was then transferred to an Eppendorf tube, clarified by lines and a xenograft model. We have previously reported centrifugation for 5 minutes at 13,200 rpm at 4C, and trans- AKR1C3 inhibitors that act synergistically with etoposide and ferred to an HPLC vial for analysis by Qtrap 4000 mass spec- daunorubicin as chemotherapeutic agents in a panel of leukemia trometry using the following parameters: Ion Source/Gas Para- cell lines (38). The AKR1C3 inhibitor KV-37 exerts high syner- meters: curtain gas (CUR) ¼ 35, collisionally activated disso- gistic drug interaction in combination with ENZ, which is superior ciation (CAD) ¼ medium, ionspray voltage (IS) ¼ 4,500, to that seen with INDO. Mechanistic studies reveal that KV-37 temperature (TEM) ¼ 600, nebulizing gas GS1 ¼ 70, drying causes apoptotic cell death in ENZ-resistant prostate cancer cell gas GS2 ¼ 70. Buffer A: dH2O þ 0.1% formic acid; Buffer B: lines with a consequent reduction in PSA levels, and in vivo studies ACN þ 0.1% formic acid; flow rate 1.5 mL/min; column Agilent demonstrate >50% reduction in tumor growth without observ- C18 XDB column, 5 mmpacking50 4.6 mm size; 0–1min able toxicity. 97% A, 1–2.5 min gradient to 99% B, 2.5–3.5 min 99% B, 4– 4.1 min gradient to 97% A, 4.1–4.5 min 97% A; IS: Warfarin (in MeOH, transition 307.3 to 160.9). Ion transitions followed Materials and Methods were 363.1 [M-H] to 186.9 for baccharin and KV-32, and 362.1 Cell culture and reagents [M-H] to 318.1 for KV-37. 22Rv1 and LNCaP cells were purchased from the American Type Culture Collection (ATCC in 2016) and cultured in RPMI Cell viability assays 1640 supplemented with 10% FBS, 100 U/mL penicillin, and Cells were seeded at a density of 10,000 cells/well in 96-well 0.1 mg/mL streptomycin (39). LNCaP1C3 cells overexpressing plates and were incubated in either normal media (24 hours) or

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Figure 1. KV-37 induces potent antitumor activity in prostate cancer cell lines. A, Structure of KV-37. Percentage cell viability of 22Rv1 cells cultured in (B) normal media, (C) CSS media, and (D) normal media supplemented with 10 nmol/L D4-androstenedione, and (E) CSS media supplemented with 10 nmol/L D4-androstenedione. Percentage cell viability of LNCaP cells cultured in (F) normal media and (G)

CSS media. H, IC50 values of KV-37 determined in 22Rv1 and LNCaP cells. I, Comparative expression of AKR1C3 in prostate cancer cell lines and normal prostate cells (left) and overexpression of AKR1C3 in 22Rv1 cells when cultured in CSS media (right). Data shown as mean SD of at least three independent experiments, n ¼ 6. NM, normal media.

CSS media (48 hours). Treatments with ENZ, INDO, KV-37, or P/S. Cells were incubated with DMSO, or 10 or 30 mmol/L KV-37 combinations of ENZ and KV-37 were made with or without for 30 minutes at 37C. After the preincubation, 100 nmol/L 3H 10 nmol/L D4-AD (AKR1C3 substrate) and incubated at the D4-AD ﬁnal concentration was added to the wells and cells were indicated time points (24, 48, 72, and 96 hours). For pretreatment incubated for 24 hours at 37C. Cell media were transferred into experiments, cells were treated with KV-37 for 24 hours followed labeled borosilicate glass tubes after 48 hours and extracted with by the addition of ENZ and incubated for a further 72 hours. Cell 2 mL ethyl acetate. Samples were vortexed for 30 seconds and viability was determined by the MTS tetrazolium dye assay as placed in a –20C freezer for 1 hour to separate the phases. The described previously (38). No intrinsic absorbance was noted frozen samples were allowed to thaw at room temperature for 10 with either KV-37 or ENZ in the MTS assay. minutes and centrifuged for 20 minutes before the organic phase was extracted and the process repeated. The 4 mL organic layer Inhibition of testosterone production in LNCaP1C3 cells extract was then dried under vacuum for radiochromatography. LNCaP1C3 cells (1.5 106 cells per well) were placed in 2 mL The aqueous fractions were dried under vacuum for 30 minutes to of phenol red–free RPMI, 5% CD-FBS, 2 mmol/L L-glu, and 1% evaporate any leftover organic solvent residue. Note that 1%

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glacial acetic acid aqueous solution (25 mL) was added to the for Western blot analysis. Whole blood was collected in an samples to adjust the pH to 6.5, followed by addition of 18 mLof Eppendorf tube with ACD anti-coagulant for plasma separation 25 U/mL Escherichia coli b-glucuronidase. Samples were placed in a for pharmacokinetic evaluation of KV-37. water bath at 37C for 24 hours. Samples were then extracted as before. All samples were resuspended in 100 mL of ethyl acetate Pharmacokinetic evaluation of KV-37 and vortexed for 30 seconds to mix. Using 10 mL capillary tubes, For Standards, 98 mL of blank plasma was added to an Eppen- each sample was spotted onto a multi-channel UNIPLATE 20 dorf and spiked with 2 mL of IS (Warfarin). For QCs, 98.8 mL blank 20 cm TLC plate and analyzed as described before (32). The peak plasma was added to an Eppendorf and spiked with 1.2 mL of IS. of testosterone observed in LNCaP cells represented the basal Note that 100 mL of plasma was mixed with 200 mL of methanol formation of testosterone that was independent of AKR1C3, containing 0.15% formic acid and 37.5 ng/mL IS (IS ﬁnal conc. ¼ because LNCaP cells are AKR1C3 null. In the presence of 25 ng/mL). The samples were vortexed for 15 seconds, incubated 10 mmol/L AKR1C3 inhibitor, the amount of testosterone formed at room temperature for 10 minutes, and spun at 13,200 rpm in a in the LNCaP1C3 cells was identical to the basal level in LNCaP standard microcentrifuge. The supernatant was then analyzed by cells showing complete inhibition of AKR1C3-mediated testos- LC-MS/MS as described above to test the stability of the AKR1C3 terone production. inhibitors.

Apoptosis assay Statistical analyses and quantification of degree of synergism Annexin V/FITC apoptosis assay was performed using a kit and Experiments were repeated at least thrice, and the statistical according to the manufacturer's instructions (BD Biosciences, significance was calculated using the Student t test. A P value catalog no. 556547). Cells were seeded at a density of 0.2 of <0.05 was considered statistically significant. IC50 values were 106 cells/well in 24-well plates and incubated in CSS media for calculated by GraphPad prism software. Combination treatments 48 hours followed by treatment with KV-37 for 48 and 72 hours. of KV-37 with ENZ were analyzed by CompuSyn software For pretreatment experiments, cells were treated with KV-37 for (Biosoft) based on the Chou–Talalay method. Combination 24 hours followed by the addition of ENZ and incubated for a index (CI) and dose reduction index (DRI) values were generated further 72 hours. After appropriate treatments, the samples were to evaluate the degree of synergistic drug interaction. analyzed by flow cytometery (Accuri C6). Results Western blotting Whole cell lysates were prepared using 4% (w/v) CHAPS in Inhibitors of AKR1C3 from various structural classes [flavones urea-tris buffer. Protein was quantified and electrophoresed as (30), jasmonates (31), and NSAIDS (23, 33)] have previously described previously (41). The membranes were probed with been described. We adopted a cinnamic acid derivative primary antibodies overnight against AR (Cell Signaling Technol- "baccharin" and conducted structure–activity relationship (SAR) ogy; #5153P, rabbit mAb), PSA (Cell Signaling Technology; studies to identify lead AKR1C3 inhibitors (37, 38). The cinnamic #5877S, rabbit mAb), AKR1C3 (Sigma; #A6229, mouse mAb), acid derivative KV-37 (Fig. 1A) was chosen for further studies as it ¼ C-PARP (Cell Signaling Technology; #5625S, Rabbit mAb), demonstrated potent AKR1C3 inhibition (IC50 66 nmol/L) and C-caspase3 (Cell Signaling Technology; #9661S, rabbit mAb), selectivity (109-fold over AKR1C2; ref. 38). In order to delineate and actin (Sigma; #A5441, mouse mb) followed by incubation the preliminary mechanism of action, we tested its mode of with anti-mouse (Sigma; #SAB4600224) or anti-rabbit AKR1C3 inhibition. We find that KV-37 displayed competitive (Perkin Elmer; #NEF812001EA) secondary antibody (1:2,000) enzyme inhibition versus AKR1C3 when D4-AD was used as horseradish peroxidase conjugate for 2 hours and detected by substrate, yielding a Ki value of 3.0 mmol/L for the E.NADPH. chemiluminescence (42). All the antibodies from Cell Signaling KV-37 complex. The difference between this Ki value and the IC50 Technology were diluted 1:1,000, AKR1C3 (1:500), and actin value seen in the preliminary inhibitor screen is consistent with þ (1:2,000). Bands were quantified by densitometry using ImageJ the lower Ki value for the E.NADP .inhibitor complex which is software. seen with other AKR1C3 inhibitors when they are screened in the oxidation direction (Supplementary Fig. S1; ref. 40). We also Tumor xenograft and pharmacokinetic study examined the mode of action of KV-37 in androgen-dependent All animal experiments were carried out according to approved 22Rv1 cells (high AKR1C3 expression) and LNCaP cells (low Institutional Animal Care and Use Committee protocols at the AKR1C3 expression). The 22Rv1 cell line shows increased expres- University of Texas Southwestern Medical Center (Dallas, TX). sion of AKR1C3 when grown in CSS media and possesses intrinsic Eighteen 6- to 8-week-old female NOD-SCID mice (low circulat- resistance to ENZ and AA (27). LNCaP cells stably overexpressing ing testosterone) were implanted with 4 106 22Rv1 cells in a AKR1C3 (LNCaP1C3) were used to further validate the effect of volume of 0.1 mL in the left flank. When tumor volume reached KV-37 on the target (12). Further, WPMY-1 cells, a nonmalignant approximately 100 mm3 (day 13), mice were randomly divided prostate stromal cell line, devoid of AKR1C3 expression (Fig. 1I), into two groups. One group was administered 20 mg/kg KV-37 were employed to evaluate the selective antineoplastic effects QD intraperitoneally, and the other group was given vehicle of KV-37. control (10% DMSO, 20% PEG400, 0.5% tween 80, 69.5% carbonate buffer, pH ¼ 9.2). Tumor volumes were measured KV-37 inhibits AKR1C3 activity in prostate cancer cell lines by twice a week with Vernier calipers, and tumor volume was inhibiting the conversion of D4-AD to testosterone calculated as (L W2) 3.14)/6 (41). On day 34, mice were KV-37 was used to inhibit the production of testosterone humanely euthanized, and tumors were collected, weighed, and in LNCaP1C3 cells following b-glucuronidase treatment of frozen in liquid nitrogen after taking pictures. Tumors were lysed the aqueous phase. At a concentration of 10 mmol/L, >80%

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inhibition of testosterone production is blocked and the residual combination with ENZ. To test whether the sensitivity of 22Rv1 testosterone produced is that seen in untransfected LNCaP cells cells to ENZ could be restored with KV-37, 22Rv1 and LNCaP cells (Supplementary Fig. S2). were pretreated with KV-37 for 24 hours followed by exposure to ENZ. The combination demonstrated a remarkable synergistic KV-37 exhibits excellent plasma stability above that of the effect in 22Rv1 cells at both 48 and 72 hours after ENZ exposure in parent and other derivatives normal as well as CSS media (CI ¼ 0.19 and 0.07, respectively; Baccharin undergoes rapid hydrolysis to yield the inactive Fig. 2A–C, I). Concentrations as low as 1 mmol/L of KV-37 were phenol drupanin (AKR1C3 IC50 ¼ 15 mmol/L; ref. 37). In order capable of sensitizing 22Rv1 cells to only 1 mmol/L of ENZ and to compare the hydrolytic stability of lead compounds, baccharin reduced viability by more than 50%. Either agent employed alone and its derivatives were subjected to incubation with mouse S9 did not exert any antitumor effect. Among cultures supplemented fractions in the presence of phase I cofactors from 0 to 240 with 10 nmol/L D4-AD in normal and CSS media, the synergistic minutes. Aliquots were withdrawn and analyzed by LC-MS at drug interaction was maintained (CI ¼ 0.33 and 0.10, respective- various time points. As expected, baccharin and a related ester ly; Fig. 2D–F, I). Quantification of degree of synergism was made analogue (KV-32) were readily hydrolyzed (Supplementary using the Chou–Talalay method (45). Up to a 200-fold reduction Fig. S3). The amide-based inhibitor KV-37 displayed remarkable in ENZ dosing was achieved in 22Rv1 cells (Fig. 2I). LNCaP cells stability (t1/2 ¼ >240 minutes). however, due to their low expression of AKR1C3, displayed a lack of synergistic drug effect at the concentrations tested, in AKR1C3 inhibitor KV-37 exhibits greater reduction of prostate both normal and CSS media, and the interaction was merely cancer cell viability compared with baccharin additive (CI ¼ 1.12 and 1.14, respectively; Fig. 2G–I). On the Baccharin and its analogue KV-37 were screened for antineo- contrary, cotreatment experiments did not show any adjuvant plastic effects in a panel of human prostate cancer cell lines effect in either cell line cultured in normal or CSS media, with (LNCaP, 22Rv1, and LNCaP1C3). Baccharin consistently dis- or without D4-AD supplementation (Supplementary Fig. S6). played no toxicity among all the cell lines tested, up to concen- This observation suggests that the activity of AKR1C3 needs trations as high as 100 mmol/L (Supplementary Fig. S4). This is in to be inhibited prior to ENZ treatment in 22Rv1 cells for agreement with previous studies (38). However, treatment with therapeutic effect. KV-37 demonstrated a greater dose and time-dependent reduction in cell viability in androgen-dependent 22Rv1 and LNCaP AKR1C3 overexpression in LNCaP cells confers resistance to the cell lines. The antineoplastic effect was greater in 22Rv1 cells due antitumor effects of ENZ that is reversed upon KV-37 treatment to the higher expression of AKR1C3 as compared with the LNCaP The LNCaP1C3 cell line, stably overexpressing AKR1C3, was cell line (Fig. 1B–H). For 22Rv1 cells, a significant decline in IC50 used to evaluate the activity of KV-37 alone and in combination values (P ¼ 0.0015, <0.0001, <0.0001 at 24, 48, and 72 hours, with ENZ to further validate AKR1C3 as the target. KV-37 induced respectively) was observed after KV-37 treatment when the cells a dose- and time-dependent reduction in cell viability of were cultured in CSS media as compared with normal media LNCaP1C3 cells (Fig. 3A and table insert). The antitumor drug (Fig. 1B, C, H). This is attributable to the overexpression of effect increased further as compared with 22Rv1 and low AKR1C3 AKR1C3 when cells are grown in media devoid of androgens expressing LNCaP cells at all time points tested. These observa- (Fig. 1I). This drives the cellular phenotype to more closely tions demonstrate that the bioactivity of KV-37 is AKR1C3- resemble clinical castrate conditions, where the cancer cells are dependent and further corroborates AKR1C3 as the compound's dependent on AKR1C3 for survival and proliferation. Culture in target. Next, a dose–response curve of ENZ was evaluated in CSS media supplemented with an AKR1C3 substrate, 10 nmol/L LNCaP1C3 cells that revealed the generation of a resistant phe- 4 D -AD, led to a slight reduction in the antitumor effect and a notype as observed in previous findings (27). The 72-hour IC50 of significant increase in IC50 values (P ¼ <0.0001, <0.0009, <0.0001 ENZ increased to 150 mmol/L in LNCaP1C3 cells as compared at 24, 48, and 72 hours, respectively) as compared with those with 50 mmol/L in sensitive LNCaP cells (Fig. 3B; Supplementary observed in nonsupplemented CSS media (Fig. 1C, E, H). Addi- Fig. S5). To overcome this drug resistance, cells were treated with a tion of 10 nmol/L D4-AD to CSS culture mimics the serum level of combination of KV-37 and ENZ as cotreatments for 72 hours or as this androgen in CRPC patients (12, 43, 44). As D4-AD is a 24-hour pretreatments with KV-37 followed by ENZ exposure for substrate for AKR1C3, this observation suggests that the reduced a further 72 hours. Cotreatment experiments showed a moderate antineoplastic activity of KV-37 is due to the enzymatic formation degree of drug synergism (CI ¼ 0.54; Fig. 3C). Among the of T and DHT and corroborates AKR1C3 as the inhibitor target. pretreatment experiments, a very high degree of synergistic The antitumor activity of KV-37 was also evaluated in normal dug interaction was observed (CI ¼ 0.14), and concentrations media supplemented with 10 nmol/L D4-AD as a control exper- as low as 1 mmol/L of KV-37 were able to sensitize the cells to ENZ- iment to directly measure the difference in dose responses induced cytotoxicity. As a result, the effective ENZ concentration between normal and CSS media (Fig. 1D and H). to reduce prostate cancer cell viability was reduced by 25-fold and yielded an IC50 of 8.19 mmol/L for the drug combination (Fig. AKR1C3 inhibitor KV-37 sensitizes ENZ-resistant prostate 3D). These findings strongly suggest that KV-37, by virtue of its cancer cells to ENZ by exerting a synergistic drug effect AKR1C3-inhibiting properties, is able to resensitize ENZ-resistant The 22Rv1 cell line is intrinsically resistant to ENZ (27). The prostate cancer cells that are high expressers of AKR1C3 toward dose–response curve of ENZ in 22Rv1 cells displays increasing the antineoplastic effects of ENZ. resistance upon culture in CSS media (Supplementary Fig. S5), which are accompanied by an increase in AKR1C3 expression KV-37 induces cell death in prostate cancer cells via apoptosis (Fig. 1I). LNCaP cells that are sensitive to ENZ-induced cytotox- To elucidate the primary mechanism of cell death, 22Rv1 and icity were used as controls to compare the effect of KV-37 in LNCaP1C3 cells cultured in CSS media were treated with

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Figure 2. Pretreatment with KV-37 sensitizes prostate cancer cell lines to the antitumor effects of ENZ. Percentage cell viability of prostate cancer cells when pretreated with KV-37 for 24 hours followed by ENZ treatment at indicated concentrations and time points in (A)22Rv1cellsculturedinnormal media and (B and C) 22Rv1 cells cultured in CSS media, (D) 22Rv1 cells cultured in normal media supplemented with 10 nmol/L D4-androstenedione and (E and F) 22Rv1 cells cultured in CSS media supplemented with 10 nmol/L D4-androstenedione, and (G) LNCaP cells cultured in normal media and (H) LNCaP cells cultured in CSS media. I, Quantiﬁcation of the degree of synergism. CI and DRI values for KV-37 and ENZ combination treatments in prostate cancer cells. CI and DRI indices generated using CompuSyn software. Data shown as mean SD of at least three independent experiments, n ¼ 6.

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Figure 3. AKR1C3 overexpression in LNCaP cells (LNCaP1C3) increases the antineoplastic activity of KV-37 and induces resistance to ENZ that is overcome by pretreatment with KV-37. Percentage cell viability of LNCaP1C3 cultured in CSS media after (A) KV-37 treatment at indicated time points and concentrations IC50 values, (B) ENZ treatment at indicated time points and concentrations, (C) cotreatment with KV-37 and ENZ for 72 hours, and (D) pretreatment with KV-37 followed by ENZ exposure for 72 hours. Insert depicts the parameters for quantifying the degree of synergism. Data shown as mean SD of at least three independent experiments, n ¼ 6. increasing doses of KV-37. After 48- and 72-hour time periods were subjected to apoptosis analysis by Annexin V/PI costaining. after treatment, the cells were harvested and stained with Annexin In 22Rv1 cells, a combination of 10 mmol/L KV-37 with 25 mmol/L V/PI to measure apoptotic cell percentage. The percentage of the ENZ displayed a significant increase (P ¼ <0.05) in apoptotic cell apoptotic cell population increased with KV-37 concentration in a percentage as compared with 25 mmol/L ENZ treatment alone, dose- and time-dependent manner (Fig. 4A; Supplementary whereas a combination of 25 mmol/L KV-37 with 25 mmol/L ENZ Fig. S7). Consistent with the results from the cytotoxicity screen- displayed an even more significant (P ¼ <0.0001) increase in ing, the effect was significantly greater in the LNCaP1C3 cell line as apoptotic cell percentage as compared with either treatment alone compared with the 22Rv1 cells at both time points (P ¼ <0.05 at (Fig. 4B, left plot; Supplementary Fig. S7). Percent apoptosis was 25 mmol/L KV-37 and <0.003 at 50 mmol/L KV-37), underscoring also increased in LNCaP1C3 cells wherein both 10 and 25 mmol/L the significance of AKR1C3 overexpression in prostate cancer. In KV-37 plus 25 mmol/L ENZ demonstrated a significant (P ¼ <0.05 order to further corroborate these findings, Western blot analysis and <0.01, respectively) increase in percent apoptosis levels than was conducted after inhibitor treatment following an identical with either treatment alone (Fig. 4B, right plot; Supplementary dosing schedule. The results, in agreement with Annexin V/PI Fig. S7). These findings were further confirmed by Western assay, consistently show a dose-dependent increase in the levels of blot analysis showing a greater increase in C-caspase3 and C-PARP C-caspase3 and C-PARP with higher expression at 72 hours as levels in drug combination experiments performed with compared with 48-hour incubation time, among both 22Rv1 and 10 mmol/L KV-37 and 25 mmol/L ENZ in 22Rv1 (Fig. 4D, left LNCaP1C3 cell lines (Fig. 4C). Taken together, these data strongly plot) and LNCaP1C3 cells (Fig. 4D, right plot). These observations suggest apoptosis as the primary mechanism of antitumor action complement the cytotoxicity studies of the drug combinations for KV-37. and further strengthen the hypothesis of apoptotic cell death induced by KV-37 in prostate cancer cells. Combination of KV-37 with ENZ potentiates the degree of apoptosis in prostate cancer cells Molecular changes following KV-37 treatment in prostate To determine whether the effect of KV-37 to induce apoptosis cancer cells in 22Rv1 cells persists in the presence of ENZ, cells were pretreated Western blot analysis was conducted to measure the expression with KV-37 followed by ENZ incubation for 72 hours. The cells levels of AKR1C3, AR, and PSA. A slight decrease in the expression

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Figure 4. KV-37 induces apoptosis in prostate cancer cells alone and potentiates the degree of apoptosis when combined with ENZ. A, Dose- and time-dependent increase in apoptotic cell population after treatment with KV-37 in indicated cell lines as analyzed by Annexin V/PI costaining. B, Increase in apoptotic cell population after treatment with indicated concentrations of KV-37, ENZ, and a 24-hour pretreatment combination of KV-37 and ENZ in indicated cell lines analyzed 72 hours after ENZ exposure by Annexin V/PI costaining. C, Western blot showing an increase in C-caspase3 and C-PARP levels after treatment with KV-37 at indicated concentration and time points in prostate cancer cells. D, Western blot showing a greater increase in C-caspase3 and C-PARP levels with a 24-hour pretreatment combination of KV-37 and ENZ in prostate cancer cells analyzed 72 hours after ENZ exposure. Data shown as mean SD of at least three independent experiments, n ¼ 3. , P < 0.05; , P < 0.01; , P < 0.0001; and ns, nonsigniﬁcant.

level of AKR1C3 was noted in 22Rv1 cells at 48 hours, whereas (Fig. 5B). Because AKR1C3 serves as a coactivator for AR (46), it after 72-hour AKR1C3 expression increased, both changes were was prudent to measure AR levels after treatment. We observed observed at 25 and 50 mmol/L KV-37 concentrations (Fig. 5A). In that expression of AR complements AKR1C3 levels and was found LNCaP1C3 cells, a decrease in AKR1C3 expression was noted at to be decreased after 48 hours of treatment, whereas at 72 hours, only 50 mmol/L concentration at 48 hours. On the contrary, no no change in AR levels was observed in both 22Rv1 and change in AKR1C3 expression was observed in the LNCaP1C3 cell LNCaP1C3 cell lines. By contrast, a concentration-dependent line at all concentrations tested at 72 hours after KV-37 exposure decline in PSA levels was observed with KV-37 in both cell lines

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Figure 5. Molecular changes in AR signaling following KV-37 treatment. Treatment with KV-37 alone at indicated concentration and time points in (A) 22Rv1 and (B) LNCaP1C3 cells cultured in CSS media. Treatment with indicated concentrations of KV-37, ENZ, and a 24-hour pretreatment combination of KV-37 and ENZ in (C) 22Rv1 cells and (D) LNCaP1C3 cells cultured in CSS media analyzed 72 hours after ENZ exposure. at 48- and 72-hour treatments. Such findings signify a beneficial tumor volume by greater than 50% and tumor weight by 35% therapeutic outcome in prostate cancer after treatment with KV-37 (P ¼ 0.0179; Fig. 6A–C) as compared with vehicle-treated (Fig. 5A and B). controls. No significant change in body weight was observed (Fig. 6D), indicative of a nontoxic effect. On day 34, mice were Combination of KV-37 with ENZ downregulates AR expression euthanized and blood collected at 10, 120, and 360 minutes after that increases after ENZ treatment KV-37 administration at 20 mg/kg and pharmacokinetic para- To determine the mechanism of synergistic interaction between meters analyzed. The peak plasma concentration (Cmax) was KV-37 and ENZ, Western blot analysis was performed on AR, determined to be 86.5 mg/mL, and Tmax, AUC, Vz/F, and CL/F AKR1C3, and PSA. In contrast to previous reports that indicate were calculated (Fig. 6E). Further, Western blot analyses of the AR degradation in 22Rv1 cells (47), treatment of 25 mmol/L tumor samples were conducted that showed no significant upre- ENZ alone increased AR expression in both 22Rv1 and LNCaP1C3 gulation of AKR1C3 expression in KV-37–treated tumors as cells signifying drug resistance. By contrast, a combination compared with nontreated controls (Fig. 6F). treatment of 25 mmol/L ENZ with 10 mmol/L KV-37 reduced the expression to less than control levels (Fig. 5C and D). Consistent INDO displays diminished cell viability reduction and a weak with inhibitor treatment alone, the combination produced no synergistic drug action compared with the more potent change in AKR1C3 expression levels. However, the combination inhibitor KV-37 of 10 mmol/L KV-37 with 25 mmol/L ENZ completely abrogates INDO (AKR1C3 Ki ¼ 8 mmol/L for the E.NADPH.Indometh- PSA expression in 22Rv1 cells and reduced PSA to control acin complex) has been shown to reverse ENZ resistance in levels in LNCaP1C3 cells. These data suggest apoptotic cell prostate cancer models (27). The dose–response curve of INDO death as a primary mechanism of synergistic antitumor effect showed a weak inhibition of cell viability at 96-hour incubation, between the two compounds leads to a decline in AR and IC50 ¼ 112.8 mmol/L (Supplementary Fig. S8A). Further, we also PSA expression. administered INDO as a 24-hour pretreatment to LNCaP1C3 cells followed by ENZ exposure for 72 hours. A moderate synergistic Antitumor effect and initial pharmacokinetics of KV-37 in a drug action was observed (CI ¼ 0.32; DRI ¼ 6.4; Supplementary 22Rv1 xenograft model Fig. S8B) as compared with the very high degree of synergism with Treatment with KV-37 at 20 mg/kg per day in female mice KV-37 under identical conditions (CI ¼ 0.14; DRI ¼ 24.8). These harboring 22Rv1 xenografts significantly (P ¼ 0.0003) inhibited findings further bolster the concept that AKR1C3 inhibition by

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Figure 6. KV-37 induces tumor growth inhibition in prostate cancer xenografts. Reduction in (A) prostate tumor volume and (B) prostate tumor weight in 22Rv1 murine xenografts after treatment with 20 mg/kg KV-37. C, Representative images showing a reduction in tumor size after treatment with 20 mg/kg KV-37. D, Mice body weight after KV-37 or vehicle treatment. E, Pharmacokinetic parameters determined in plasma after treatment with 20 mg/kg KV-37. F, Expression of AKR1C3 in tumor lysates (left plot), and each blot indicates tumor lysate from individual mouse. Bar graph representation of quantiﬁed blots (right plot).

KV-37 sensitizes ENZ-resistant prostate cancer cells to the che- observation that WPMY-1 cells are low expressers of AKR1C3, the motherapeutic, and is superior to INDO. reduction of cell viability induced by KV-37 was minimal with 96-hour IC50 ¼ 80 mmol/L. At concentrations of 1, 10, and KV-37 exhibits no toxicity alone, or in combination with ENZ, 25 mmol/L of KV-37, those used in our study of prostate cancer in WPMY-1 prostate cells cells, no reduction in WPMY-1 cell viability was observed, an The nonmalignant prostate stromal cell line WPMY-1 was observation consistent with no loss of body weight in mice chosen to evaluate the toxicity of KV-37. Consistent with the xenografts upon 20 mg/kg daily dosing. Further, pretreatment

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with KV-37 for 24 hours followed by ENZ exposure for 72 hours concentration of KV-37 (10 mmol/L), which exerts no more than resulted in no loss of cell viability (Supplementary Fig. S8C 20% reduction in prostate cancer cell viability, was chosen for the and S8D). drug combination treatments. In combination drug treatments, Annexin V/PI staining demonstrated a significant increase in the Discussion percentage of apoptotic cells compared with either treatment alone, which was confirmed by an increase in C-caspase3 and Prostate tumors resistant to AA and ENZ are characterized by C-PARP levels. These data indicate that apoptotic cell death is a overexpression of steroidogenic enzymes where AKR1C3 is primary mechanism of the synergistic drug effect. Androgen- among the most overexpressed (26, 48, 49). Thus, in situ or dependent gene expression was analyzed after KV-37 treatment, intratumoral androgen biosynthesis is a predominant factor of and a dose-dependent reduction in PSA levels was observed. therapeutic failure with second-line ADT (13, 48). Overexpres- Although the activity of AKR1C3 was inhibited by KV-37, the sion of AKR1C3 by stable transfection in prostate cancer cells protein expression levels either remained unchanged or slightly imparted ENZ resistance which was overcome by AKR1C3 increased in 22Rv1 cells, after treatment, indicating a feedback knockdown by shRNA. INDO, a weak AKR1C3 inhibitor, was increase in AKR1C3 expression. Interestingly, we observed an also able to rescue xenografts from ENZ and AA resistance increase in the AR expression levels after treatment with ENZ (27, 28). Continuing our efforts to identify potent and selective alone in both 22Rv1 and LNCaP1C3 cell lines, which is one of the AKR1C3 inhibitors, we synthesized and characterized KV-37,a signatures of drug resistance. A combination of ENZ with KV-37 nanomolar inhibitor of AKR1C3 with >100-fold isoform selec- reduced AR expression to control levels or less. Further, treatment tivity and metabolic stability (38). We demonstrate that the with KV-37 alone in 22Rv1 murine xenografts reduced tumor synergistic effects of KV-37 with ENZ in reducing prostate volume by more than 50%, whereas no change in mouse body cancer cell viability are much greater than that seen with INDO weight was observed. In order to compare the degree of synergistic (Supplementary Fig. S9) and that the effects of KV-37 are drug interaction exhibited by the weak AKR1C3 inhibitor INDO mediated by AKR1C3 inhibition. (Ki ¼ 8 mmol/L) with KV-37 (Ki ¼ 3 mmol/L), pretreatment We demonstrate that KV-37 inhibits the activity of AKR1C3 in experiments with INDO were conducted in LNCaP1C3 cells prostate cancer cells by inhibiting the conversion of D4-AD to followed by ENZ treatment that revealed a moderate degree of testosterone. Crucially, KV-37 exerts a preferential antineoplastic drug synergism, much lower than that achieved with KV-37. This effect in AKR1C3-overexpressing 22Rv1 and LNCaP1C3 cells as study serves as proof of concept that AKR1C3 inhibition has the compared with the low expressing LNCaP cell line and nonma- potential to overcome ENZ resistance and corroborates previous lignant WPMY-1 cells. Furthermore, culture of 22Rv1 cells in CSS findings (27). Further, we also analyzed the activity of KV-37 media, which are devoid of androgens, upregulates AKR1C3 alone and in combination with ENZ in the nonmalignant human expression further and results in greater susceptibility to prostate stromal cell line WPMY-1. No reduction in cell viability KV-37--induced antineoplastic effects. Because the physiologic up to 25 mmol/L was observed, whereas no cell viability reduction androgen level in clinical castrate conditions is of the order in combination experiments was noted, indicating selectivity to of 1 to 10 nmol/L, CSS was supplemented with 10 nmol/L cells overexpressing AKR1C3. D4-androstenedione to further mimic the CRPC phenotype In conclusion, we highlight the potential of KV-37 as a (44). KV-37 displayed an equally robust inhibition of cell combination therapy with ENZ for CRPC because it retards viability in such cultures. The 22Rv1 cell line is inherently ENZ-resistant prostate cancer cell growth and induces apopto- resistant to ENZ, whereas LNCaP cells became resistant to ENZ sis. Furthermore, it is conceivable from the results of this study when AKR1C3 was stably expressed in this cell line. Pretreatment that the structurally novel AKR1C3 inhibitor KV-37 scaffold can with KV-37 restored the sensitivity of 22Rv1 cells to ENZ and be developed as a stand-alone therapeutic for prostate cancer provided a strong synergistic effect resulting in >200-fold reduc- management. tion in chemotherapeutic dose under conditions that mimic the CRPC disease phenotype. The strong synergistic effect was main- Disclosure of Potential Conflicts of Interest tained in LNCaP1C3 cells upon pretreatment with KV-37.A K. Verma has ownership interest (including stock, patents, etc.) in a moderate synergistic effect was also observed in these cells if provisional patent application describing KV-37.T.M.Penningreports KV-37 was coadministered with ENZ. By contrast, as LNCaP cells receiving commercial research grant from Forendo and other commercial are low expressers of AKR1C3, only an additive effect was research support from Constellation, and is a consultant/advisory board observed, an observation that may be ascribed to the general member for Research Institute for Fragrance Materials, Markey Cancer Center, Mailman School of Public Health, Columbia U, and EOSHI, Rutgers toxicity of KV-37 toward cancer cells as evidenced previously in University. P.C. Trippier has ownership interest (including stock, patents, leukemic cell lines (38). etc.) in a provisional patent application describing KV-37. No potential Insights into the mechanism of apoptosis mediated by KV-37 conflicts of interest were disclosed by the other authors. were obtained by measuring an increase in the apoptotic markers C-caspase3 and C-PARP, as well as a dose-dependent increase in Authors' Contributions Annexin V/PI after KV-37 treatment. The observation that percent Conception and design: K. Verma, N. Gupta, T.M. Penning, P.C. Trippier apoptotic cells were greater in LNCaP1C3 cells at any given Development of methodology: K. Verma, N. Gupta, T. Zang, P. Wangtrakluldee, concentration, compared with 22Rv1 cells, corroborates AKR1C3 S.K. Srivastava, T.M. Penning, P.C. Trippier as the target of KV-37. In combination drug treatments, 25 mmol/L Acquisition of data (provided animals, acquired and managed patients, ENZ was employed, as this corresponds to the minimum steady- provided facilities, etc.): K. Verma, N. Gupta, T. Zang, P. Wangtrakluldee, T.M. Penning state plasma concentration of 12 mg/mL of the drug, the physi- Analysis and interpretation of data (e.g., statistical analysis, biostatistics, ological concentration achieved clinically in prostate cancer computational analysis): K. Verma, N. Gupta, T. Zang, P. Wangtrakluldee, patients at 150 mg/day dosing regimen (17). A subtherapeutic S.K. Srivastava, T.M. Penning, P.C. Trippier

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Writing, review, and/or revision of the manuscript: K. Verma, T. Zang, Financial support for this work was provided by Texas Tech University Health P. Wangtrakluldee, S.K. Srivastava, T.M. Penning, P.C. Trippier Sciences Center (to P.C. Trippier). Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): T. Zang The costs of publication of this article were defrayed in part by the Study supervision: K. Verma, S.K. Srivastava, T.M. Penning, P.C. Trippier payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate Acknowledgments this fact. We are indebted to Drs. Noelle Williams and Sukesh Voruganti at the University of Texas Southwestern Medical Center Preclinical Pharmacology Received October 30, 2017; revised March 1, 2018; accepted June 4, 2018; Core for conducting in vivo pharmacokinetic studies and tumor measurements. published ﬁrst June 11, 2018.

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AKR1C3 Inhibitor KV-37 Exhibits Antineoplastic Effects and Potentiates Enzalutamide in Combination Therapy in Prostate Adenocarcinoma Cells

Kshitij Verma, Nehal Gupta, Tianzhu Zang, et al.

Mol Cancer Ther 2018;17:1833-1845. Published OnlineFirst June 11, 2018.

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