Molecular Inhibitor of QSOX1 Suppresses Tumor Growth in Vivo Amber L

Published OnlineFirst October 1, 2019; DOI: 10.1158/1535-7163.MCT-19-0233

MOLECULAR CANCER THERAPEUTICS | SMALL MOLECULE THERAPEUTICS

Molecular Inhibitor of QSOX1 Suppresses Tumor Growth In Vivo Amber L. Fiﬁeld1, Paul D. Hanavan2, Douglas O. Faigel3, Eduard Sergienko4, Andrey Bobkov4, Nathalie Meurice5, Joachim L. Petit5, Alysia Polito5, Thomas R. Caulﬁeld6,7,8,9,10, Erik P. Castle11, John A. Copland12, Debabrata Mukhopadhyay13, Krishnendu Pal13, Shamit K. Dutta13, Huijun Luo14, Thai H. Ho14, and Douglas F. Lake1

ABSTRACT ◥ Quiescin sulfhydryl oxidase 1 (QSOX1) is an enzyme over- inhibitors, known as “SBI-183,” suppresses tumor cell growth in a expressed by many different tumor types. QSOX1 catalyzes the Matrigel-based spheroid assay and inhibits invasion in a mod- formation of disulﬁde bonds in proteins. Because short hairpin iﬁed Boyden chamber, but does not affect viability of nonma- knockdowns (KD) of QSOX1 have been shown to suppress lignant cells. Oral administration of SBI-183 inhibits tumor tumor growth and invasion in vitro and in vivo, we hypothesized growth in 2 independent human xenograft mouse models of that chemical compounds inhibiting QSOX1 enzymatic activity renal cell carcinoma. We conclude that SBI-183 warrants further would also suppress tumor growth, invasion, and metastasis. exploration as a useful tool for understanding QSOX1 biology High throughput screening using a QSOX1-based enzymatic and as a potential novel anticancer agent in tumors that over- assay revealed multiple potential QSOX1 inhibitors. One of the express QSOX1.

Introduction largest cohort (N ¼ 126), to our knowledge, of matched patient primary and renal cell carcinoma (RCC) metastases, we identified the Cancer is a leading cause of death worldwide and distant metastases upregulation of ECM-related genes in metastases relative to primary are the major cause of patient mortality. Initially the primary tumor RCC tumors. Because these ECM genes are upregulated in metastases, grows in its microenvironment which consists of tumor cells and they may play an important role in the metastatic cascade across nonmalignant stroma, each secreting extracellular matrix (ECM; multiple solid tumors (5). ref. 1). The constituents of tumor ECM are a critical factor for cancer Because every protein in the ECM contains disulfide bonds, we invasion and metastasis (1) and many changes occur in the tumor hypothesized that QSOX1, an enzyme that generates disulfide bonds in microenvironment (TME) prior to physical migration of metastatic substrate proteins, is important for tumor cell growth, adherence, and cells away from the primary tumor. These changes include upregula- invasion. Overexpression of QSOX1 in cancer was discovered after a tion of matrix metalloproteinases (MMP; ref. 2), aberrant integrin C-terminal peptide was detected by mass spectrometry of plasma from signaling (3), and a loss of adherens junctions (3, 4). Each step of the patients with pancreatic cancer (6). Subsequently, QSOX1 overexpres- metastatic process is mediated by various ECM constituents (1). In the sion has been reported in many other cancers (7–12). Inhibition of QSOX1 activity with a mAb revealed that QSOX1 is active extracel- lularly in stromal fibroblasts and is required for proper incorporation 1School of Life Sciences, Arizona State University, Tempe, Arizona. 2RenBio, Inc., of laminin and fibronectin into the ECM (13, 14). Further, a small New York, New York. 3Division of Gastroenterology and Hepatology, molecule inhibitor of QSOX1, ebselen, reduced proliferation, and Department of Internal Medicine, Mayo Clinic, Phoenix, Arizona. 4Assay Devel- invasion of pancreatic and renal cancer cell lines in vitro, and reduced opment, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, tumor growth in vivo (15). California. 5Hematology/Oncology, Mayo Clinic, Scottsdale, Arizona. 6 7 Herein we demonstrate a novel small molecule derived from a high Department of Neuroscience, Mayo Clinic, Jacksonville, Florida. Mayo Grad- uate School, Neurobiology of Disease, Mayo Clinic, Jacksonville, Florida. throughput screen of 50,000 compounds, 3-methoxy-N-[4-(1-pyr- 8Department of Cancer Biology, Mayo Clinic, Jacksonville, Florida. 9Health rolidinyl)phenyl]benzamide (“SBI-183”), inhibits the enzymatic activ- Sciences Research, Division of Biomedical Statistics & Informatics, Mayo Clinic, ity of QSOX1, thereby suppressing the proliferative and invasive Jacksonville, Florida. 10Center for Individualized Medicine, Mayo Clinic, phenotype of 2 renal cancer cell lines (786-O and RCJ-41T2), a triple 11 Jacksonville, Florida. Department of Urology, Mayo Clinic, Phoenix, Arizona. negative breast cancer (TNBC) cell line (MDA-MB-231), a lung 12 13 Department of Cancer Biology, Mayo Clinic, Jacksonville, Florida. Department adenocarcinoma cell line (A549), and a pancreatic ductal adenocar- of Biochemistry and Molecular Biology, Mayo Clinic, Jacksonville, Florida. 14Division of Hematology/Oncology, Mayo Clinic, Phoenix, Arizona. cinoma (MIA PaCa2). Furthermore, we did not observe any compound-related toxicity of normal adherent fibroblasts or nonadherent Note: Supplementary data for this article are available at Molecular Cancer peripheral blood mononuclear cells (PBMC) supporting a role for Therapeutics Online (http://mct.aacrjournals.org/). QSOX1 in tumor-derived ECM. Corresponding Authors: Thai H. Ho, Mayo Clinic, 5777 East Mayo Boulevard, Phoenix, AZ 85054. Phone: 480-301-8335; Fax: 480-301-4675; E-mail: [email protected]; and Douglas F. Lake, School of Life Sciences, Arizona Materials and Methods State University, Tempe, AZ. E-mail: [email protected] Compounds Mol Cancer Ther 2019;XX:XX–XX SBI-183 (molecular weight 296.3723 g/mol) was purchased from doi: 10.1158/1535-7163.MCT-19-0233 ChemBridge Corp. Compounds were dissolved in tissue culture-grade 2019 American Association for Cancer Research. DMSO (Sigma-Aldrich) and kept at 80 C as 100 mmol/L stock

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solutions. See Supplementary Fig. S1 for the chemical structure of SBI- 150 mmol/L NaCl pH 8.0 overnight at 4oC. The labeling ratio 183. was estimated using e ¼ 250,000 M 1cm 1 at 655 nm for DyLight 650 and e ¼ 93110 M 1cm 1 at 280 nm for QSOX1, and found Cell culture to be 1.1. RCC line 786-O was purchased from the ATCC and maintained in Microscale thermophoresis (MST) experiments were performed in RPMI1640 (Corning) containing 10% FBS (Atlanta Biologicals), 1% a Monolith NT.115 (Nanotemper). Sixteen serial dilutions of SBI-183 Penicillin-Streptomycin (Pen-Strep; Corning), and 1% Glutamax (from 250 to 0.0076 mmol/L) with 50 nmol/L Dylight 650-labeled (Gibco). A recently derived sarcomatoid RCC line from Mayo Clinic, QSOX1 in 1x PBS, pH 7.4, 5% DMSO, and 0.05% Tween 20 were RCJ-41T2 (16), was maintained in DMEM in 10% FBS, 1% Pen-Strep, loaded into standard MST capillaries and scanned at MST power of and 1% Glutamax. The TNBC adenocarcinoma cell line MDA-MB- 20% at 23 C. To obtain Kd, MST data were fitted using MO Affinity 231 (ATCC), lung adenocarcinoma cell line A549 (ATCC), and the Analysis software (Nanotemper). pancreatic ductal adenocarcinoma cell line MIA PaCa2 (ATCC) were also maintained 10% DMEM. MDA-MB-231-Luc (Cell Biolabs) was Small molecule docking maintained in 10% RPMI1640 without Pen-Strep. De-identified fibro- Docking for SBI-183 was performed using Glide (v.5.6) within the blasts derived from a 28-year-old Caucasian male with no overt disease Schrodinger€ software suite (Schrodinger,€ LLC; ref. 18). Our modeling were a kind gift from Dr. Clifford Folmes. PBMCs were obtained under techniques have been described (19–25). Briefly, we started with an IRB-approved protocol (#06010000548) from Arizona State Uni- conformation searches of the ligand via the method of Polak-Ribiere versity. The identity of all cell lines was confirmed by STR analysis. conjugate gradient (PRCG) energy minimization with the optimized Each cell line also tested negative for mycoplasma and mouse patho- potentials for liquid simulations (OPLS) 2005 force field (26) for 5,000 gens throughout the study, and were maintained at 37 Cin5%CO2. steps (or until the energy difference between subsequent structure was All cell lines were used immediately upon thawing throughout the less than 0.001 kJ/mol-Å; ref. 18). Our docking methodology has been study. described (19, 25, 27), and the scoring function utilized described elsewhere (28). Briefly, molecular refracting molecules were removed Stable lentiviral QSOX1 KD generation from the human QSOX1 crystal structure (PDB Codes: 3Q6O; ref. 29). Short hairpin (sh) lentiviral particles were purchased from Gene- Schrodinger's€ SiteFinder module focused the grid on the active site Copoeia containing either sh742 RNA as described (7) (catalog no. region for QSOX1 (Fig. 1C). Using this grid, initial placement for SBI- LPP-CS-HSH273J-LVRU6GP-100) or a shScramble (shScr) control 183 was docked using the Glide algorithm within the Schrodinger€ suite (catalog no. LPP- CSHCTR001-LVRU6GP-025). 786-O cells were as a virtual screening workflow (VSW). The docking proceeded from seeded at 2.5 104 cells/well in a 6-well plate in complete RPMI1640. lower precision through SP docking and Glide extra precision (XP; Adherent cells were transduced in triplicate with lentiviral particles Glide, v.5.6; Schrodinger,€ LLC; refs. 20, 30). The top poses were ranked following the manufacturer's instructions. After 72 hours, cells were for best score and unfavorable scoring poses were discarded. Multiple selected in puromycin and subcloned by limiting dilution. A mono- orientations were allowed in the site. Site hydroxyls were allowed to clonal population denoted as 786-O sh742.E11 was expanded. KD of move with rotational freedom. Full docking scores are given in QSOX1 was determined to be 90% by qRT-PCR as compared with the Supplementary Data File S1. This method provides the ideal confor- 786-O shScr cells (Supplementary Fig. S2). mation of ligand binding as utilized within Schrodinger€ suite, and the top docked pose represents the conformation of the ligand required to Enzymatic activity assay inhibit QSOX1. Hydrophobic patches were utilized within the VSW as PcDNA3.1 containing the short form of human QSOX1 (rQSOX1) an enhancement. XP descriptors were used to obtain atomic energy was used to transfect Freestyle 293F cells (Thermo Fisher Scientific). terms that result during the docking run (20, 30). Molecular modeling rQSOX1 was expressed by 293F cells, harvested from supernatants, for importing and refining the X-ray structure and generation of SBI- and purified on a nickel column via the C-terminal histidine tag. 183, as well as rendering of figure images were completed with Maestro Enzymatic activity of QSOX1 and inhibitory activity of SBI-183 was (Schrodinger,€ LLC). confirmed using a fluorogenic assay as previously reported (17). Briefly, a mixture of 150 mmol/L dithiothreitol (DTT) substrate Cellular viability assay (Sigma-Aldrich) was added to 150 nmol/L rQSOX1, 1.4 mmol/L horse Cells were plated at optimized densities (1,000 cells/well for 786-0 radish peroxidase (HRP; Thermo Fisher Scientific), and 1 mmol/L and RCJ-41T2; 750 cells/well for MDA-MB-231) in their respective homovanillic acid (HVA; Sigma-Aldrich) in PBS at ambient temper- media, and plated in Corning 3570 384-well white titerplates using a ature, pH 7.5. Assays were performed in a black plate in a total volume MultiFlo bulk dispenser (BioTek). The cells were allowed to adhere for of 150 mL in triplicate. Fluorescence was measured at 20 second 24 hours. Compound and assay controls diluted in 100% DMSO were l intervals over 15 minutes after the addition of DTT at ex 320 nm/ added to the cells using an ATS Gen4 acoustic transfer system (EDC l em 420 nm using a FlexStation spectrophotometer (Molecular Biosystems). A 1:1,000 compound:cell volume ratio was enforced to Devices). SBI-183 was preincubated with rQSOX1 for at least 10 avoid DMSO toxicity. After 72-hour compound incubation, 25 mL minutes at concentrations ranging from 6.25 to 50 mmol/L. CellTiter Glo reagent (Promega; G7573) diluted 1:4 in MilliQ water was added to the plates using the Multiflo dispenser and luminescent Microscale thermophoresis signal was read per standard assay protocol using a Molecular Devices rQSOX1 was labeled with DyLight 650 Amine-Reactive Dye Paradigm multimode reader (TUNE cartridge, luminescent mode). (Thermo Fisher Scientific). Briefly, Dylight-650 was dissolved at Prior to screening, assay optimization experiments were performed 10 mmol/L in dimethylformamide and added at 2:1 molar ratios for each cell line in the assay conditions described above. Cell densities m 3 to 86 mol/L QSOX1 in 50 mmol/L NaPO4 , 150 mmol/L NaCl, were titrated in control plates containing negative and positive controls pH 8.0. The mixture was incubated in the dark for 1e hour at (DMSO and 10 mmol/L staurosporine, 0.1% DMSO in assay wells) to room temperature on a rocker, and dialyzed to 50 mmol/L Tris, identify optimal seeding densities within linear ranges of luminescent

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Figure 1. SBI-183 binds to and inhibits the enzymatic activity of QSOX1. A, Data were recorded in triplicate at time ¼ 15 minutes (steady state) after addition of DTT substrate. Error represents SEM. Signiﬁcance was determined by 2-way ANOVA and P < 0.05 and P < 0.0001. B, MST titrations of rQSOX1 with SBI-183. Red and blue data sets represent 2 independent titrations of 50 nmol/L Dylight650-labeled QSOX1 with increasing amounts of SBI-183 (0.0076 to 250 mmol/L). Fitting the data yielded

Kd ¼ 20 7 mmol/L. C, QSOX1 is shown with predicted binding sites 1 and 2 indicated by arrows. The boxed gray area for site 1 is where SBI-183 was shown to bind and is zoomed into for D. Atom colors are by atom type (C-gray, N-blue, O-red, S-yellow, H-white) and ribbons are colored by secondary structure (red-helix, cyan-sheet, gray-random coil/loop). D, SBI-183 docked with QSOX1 is given. Key interacting residues within 6Å cutoff are labeled and shown in licorice stick rendering. Dashed lines indicating hydrogen bonds, pi-cloud interactions, or electrostatics are shown. signal, minimizing CVs (<10%) and maximizing Z’ factors (>0.5), per Transwell invasion assay standard NIH assay guideline optimization criteria and methods. For A total of 1.0 105 786-O, RCJ-41T2, MDA-MB-231, and A549 or primary screens, 20-point 2-fold serial dilutions of SBI-183 in 100% 5.0 104 MIA PaCa2 cells were seeded in triplicate onto Matrigel- DMSO were prepared from a stock concentration of 40 mmol/L in coated 24-well invasion 8-mm pore-size inserts (Corning) in serum- acoustic-compatible Aurora microplates (Ref. ABA200100A); internal free media. Cells were allowed to adhere for 30 minutes prior to the plate controls for live cells (100% viability) and dead cells (0% viability) addition of DMSO or SBI-183 giving a final concentration in the well of were included in source plates. After 24-hour seeding time, compound 0.2% DMSO vehicle or 20 mmol/L SBI-183. Inserts were incubated for was added acoustically as described above. Cellular viabilities for each 4-5 hours (786-O, RCJ-41T2, MDA-MB-231, and A549) or overnight test well were derived from raw luminescent signal by normalization to (MIA PaCa2)at37 C. Noninvading cells were removed, membranes internal plate controls. Viability experiments were performed in were fixed in ice cold 100% methanol, and mounted on slides with triplicate, and normalized data points averaged per dose. Dose– DAPI (Vector Laboratories). Three unique fields were captured using response curves were calculated by logistic regression in TIBCO the 4 objective and then automatically counted on a Cytation 5 Spotfire (version 7.0.0). microscope (BioTek). Images were edited using ImageJ.

Proliferation assay 3D spheroid invasion assay 786-O, RCJ-41T2, MDA-MB-231, A549, and MIA PaCa2 were The following protocol was performed as described in Vinci and seeded in triplicate at 2.5 103 cells/well (786-O, RCJ-41T2, A549, and colleagues (2015) with slight modiﬁcations as stated (31). 786-O and MIA PaCa2) or at 5.0 103 cells/well (MDA-MB-231) in phenol-red RCJ-41T2 were seeded in triplicate at 1.25 103 cells/well in 200 mL free 10% RPMI1640 (786-O) or 10% DMEM (RCJ-41T2, MDA-MB- 10% RPMI1640 or 10% DMEM respectively in ultra low attachment 231, A549, and MIA PaCa2) in 96-well plates. Adhered cells were (ULA) 96-well plates (Corning). MDA-MB-231, A549, and MIA incubated with 2-fold dilutions of SBI-183 starting at 20 mmol/L, or PaCa2 were seeded at 2.5 103 cells/well in 10% DMEM. Plates were vehicle (0.4% DMSO) for 5 days. Cell growth was determined at days 1, centrifuged at 1,000 g for 3 minutes then incubated and allowed to 3, and 5 in an MTT assay (Molecular Probes) following the manu- form spheroids for 3 days. Plates were chilled to 4C for 20 minutes and facturer's directions. all but 50 mL of media was removed. On ice, 50 mL of Matrigel Matrix

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(Corning) was added. Plates were centrifuged at 300 g for 3 minutes Statistical analysis at 4C, then incubated for 1 hour at 37C. Each well contained the Unless otherwise noted, all statistical analyses were performed using following final concentrations of SBI-183 in complete media: 20, 10, 5, GraphPad Prism version 7.04 for Windows, GraphPad Software, www. 2.5, or 0.4% DMSO vehicle. Cells were imaged on days 0, 2, 4, 6 (RCJ- graphpad.com. 41T2 and MDA-MB-231), and 8 (786-O). Invasion was quantified with ImageJ as total area of invaded cells. Rescue invasions were performed as stated above with the following Results modifications. To the 50 mL of media remaining in the wells, 50 mLof SBI-183 inhibits QSOX1 enzymatic activity in vitro media containing either PBS or rQSOX1, and SBI-183 or DMSO was SBI-183 emerged as a QSOX1 inhibitor from cell-free high added. Final concentrations in the well were 2.5 mmol/L SBI-183 and 5 throughput screening assays described previously (15). As shown mmol/L rQSOX1 or 0.025% DMSO (786-O), 5 mmol/L SBI-183, and 5 in Fig. 1A, SBI-183 inhibits rQSOX1 in a dose-dependent manner mmol/L rQSOX1 or 0.05% DMSO (RCJ-41T2), and 2.5 mmol/L SBI- in a fluorescence assay developed by Raje and colleagues (17). H2O2 183 and 2.5 mmol/L rQSOX1 or 0.025% DMSO (MDA-MB-231). produced by QSOX1 activity activates HRP to dimerize HVA Matrigel was added as above at a 1:1 dilution. resulting in fluorescence at 420 nm. The first bar in Fig. 1A demonstrates that SBI-183 does not inhibit HRP, nor does it appear fi Animal studies to scavenge H2O2,providingcon dence that the target of SBI-183 is 786-O: Fox1nu/nu mice were inoculated with 1.0 106 786-O QSOX1. cells in the right hind flank. Seven days post-implant (study day 0), mice were dosed daily by oral gavage with 400 mg/mouse/day SBI- SBI-183 binds to QSOX1 by MST 183 dissolved in 100% DMSO. Control mice received 100% Because SBI-183 appeared to inhibit the enzymatic activity of DMSO. Tumor length and width measurements were obtained QSOX1, we wanted to determine if it bound to QSOX1. MST was using Vernier calipers. performed in duplicate showing binding of SBI-183 to QSOX1 at a Kd RCJ-41T2: A part of RCJ-41T2 tumor was minced with a sterilized of 20 mmol/L (Fig. 1B). blade to slurry and mixed with equal volume of Matrigel. One hundred microliters of the resulting mixture was injected subcutaneously into Computer modeling predicting binding location of SBI-183 with 8- to 10-week-old male NSG mice using 1 mL syringes equipped with a QSOX1 in silico 16-gauge needle. When the tumor grew to approximately 1,500 Because crystal structures of QSOX1 have been generated (33) mm3, mice were sacrificed, the tumors were harvested and reimplanted and SBI-183 appeared to bind QSOX1, computer modeling was into 18, male NSG mice as described above. When the average size of performed predicting the binding location of SBI-183. From a the tumors was 100 mm3, mice were randomized into 2 groups: (i) SiteFinder search, 2 sites (Site 1 and Site 2) were identified as Vehicle: 20% DMSO þ 80% PEG-400, gavage daily, (ii) SBI-183, possible binding locations on QSOX1, however Site 1 was optimal 100 mg/kg dissolved in the vehicle, oral gavage daily. Treatment was for SBI-183 binding (Fig. 1C). Supplementary Table S1 displays the continued for 3 weeks. Then, mice were euthanized and tumors and results from our docking protocols. organs were harvested for further analysis. At Site 1, SBI-183 fits deep into a wedge-like crevice inside QSOX1 MDA-MB-231-Luc: Twenty-four female CB.17 SCID mice ages that includes the following residues within 6Å of SBI-183: C237, Y238, 8 weeks were obtained from Charles River. Mice were inoculated with L239, V251, L252, M253, F258, Y259, Y262, and L263. Interaction 0.1 mL of 50% Matrigel/50% Media containing 5 106 MDA-MB- pairs are formed between SBI-183 and QSOX1 with frequent ring-ring 231-Luc cells (Cell Biolabs) into the mammary fat pad. Seven days pi-clouds, H-bonds, and charge–charge interactions participating in post-implant (study day 1), daily oral administration of 100 mg/kg (n electrostatic interactions with the backbone carbonyls and hydroxyl ¼ 12) SBI-183 or vehicle control (n ¼ 12) began. Primary endpoint was residues, and transient pi-cloud interactions occurring with the phe- assessment of treatment effects on spontaneous distal lung metastases nyl-substituted tyrosine rings (Fig. 1D). determined by ex vivo bioluminescence imaging. SBI-183 was dissolved in DMA (10% total volume)/PEG400 (90% total volume). Stock SBI-183 suppresses tumor cell growth in vitro solution was made fresh weekly and stored at 20C. To determine if SBI-183 targets QSOX1 in tumor cells, All animal experiments were conducted in accordance with and we selected cell lines that were previously identified to express approved by an Institutional Animal Care and Use Committee QSOX1. The ability of SBI-183 to inhibit viability of tumor cells in a (IACUC). dose-dependent fashion was determined using the CellTiter Glo assay (Supplementary Fig. S3). Cells were treated with 2-fold Immunohistochemistry dilutions of SBI-183 between 40 mmol/L and 0.076 nmol/L (in RCJ-41T2 xenograft tumors from mice were mounted in paraffinon triplicate), incubated for 72 hours and analyzed. As shown in slides. Slides were deparaffinized, rehydrated, and then the antigen was Supplementary Fig. S3, inhibition of viability was observed for retrieved with citrate buffer (pH 6.0, 125 C for 1 minute). Slides were 786-O, RCJ-41T2, and MDA-MB-231 with IC50s of 4.6, 3.9, and m incubated in 3% H2O2 for 10 minutes at room temperature. Rabbit 2.4 mol/L, respectively. anti-laminin-a4 antibody (Novus Biologicals) was added at 1:300 and Because previous studies demonstrated reduced proliferation of incubated at 4C overnight. HRP conjugated anti-rabbit secondary tumor cells when QSOX1 was knocked down (KD) using antibody (Vector Laboratories) was added and incubated for 30 shRNA (7, 12, 15), we hypothesized that a compound which inhibits minutes at room temperature. 3,30-Diaminobenzidine (DAB) was QSOX1 would similarly decrease tumor growth in vitro.Totestthis, used as the chromogen with hematoxylin counter staining. tumor cells were cultured for 5 days in the presence of SBI-183 or Three unique images from each of 2 tumors per group were 0.4% DMSO vehicle control. An SBI-183 concentration-dependent obtained using the 10 objective on an Olympus BX51 microscope. reduction in cell growth was observed for each tumor cell line DAB intensity was measured using Fiji (32). (Fig. 2A–E). To determine if SBI-183 demonstrated selectivity for

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Figure 2. SBI-183 inhibits proliferation of tumor cells, but does not kill ﬁbroblasts or rapidly proliferating PBMC. Inhibition of proliferation of 786-O (A), RCJ-41T2 (B),

MDA-MB-231 (C), A549 (D), and MIA PaCa2 (E) with SBI-183 exhibits a dose response. This phenotype is similar to that seen in the QSOX1 stable KD cell line 786-O sh742.E11 (F). Significance was determined by 2-way ANOVA. No significant toxicity was observed when fibroblasts (G) or PHA-stimulated PBMC (H) were incubated with SBI-183 for 5 days. Significance was determined by 1-way ANOVA, Kruskal–Wallis Test. Experiments were performed in triplicate and error represents SEM. Cells incubated with DMSO vehicle alone were used to calculate percentage growth with the following equation: [(Cells þ SBI-183)/ (Cells þ 0.4% DMSO)] 100. Percentage growth of QSOX1 sh742 KD was calculated against shScr. P < 0.05; P < 0.01; P < 0.001; P < 0.0001.

tumor cells, non-malignant adherent human ﬁbroblasts and non- SBI-183 inhibits tumor invasion in both 2D and 3D models adherent PHA-stimulated PBMC from healthy human donors were We previously reported that silencing QSOX1 expression with incubated with SBI-183 for 5 days under the same conditions. No shRNA and inhibiting QSOX1 with the small molecule ebselen signiﬁcant inhibition of cell growth was observed compared with reduced the invasiveness of cancer cell lines in vitro (7, 12, 15). Sim- vehicle control (Fig. 2G and H). Supplementary Table S2 lists ilarly, we hypothesized that another small molecule inhibitor of percent growth for Fig. 2A–D. QSOX1 would also suppress invasion. It is well known that 3D culture

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Figure 3.

3D and 2D invasion. Inhibition of invasion in 3D of 786-O (i), RCJ-41T2 (ii), MDA-MB-231 (iii), A549 (iv), and MIA PaCa2 (v) exhibits a dose–response relationship. This phenotype is similar to that seen in the QSOX1 stable KD cell line 786-O sh742.E11 (vi). Representative images of 3D invasion on day 0 (C, F, I, L, O) and day 4 or day 6 (cell line dependent) with no compound (0.4% DMSO vehicle only; A, D, G, J, M), or 20 mmol/L SBI-183 (B, E, H, K, N). Images of 786-O cells transduced with GFP- expressing shRNA scramble (shScr) and QSOX1 KD (sh742.E11) on day 0 (P, R)orday6(Q, S). Data are representative of 3 experiments performed in triplicate. Scale bar, 300 mm. 786-O sh742.E11 forms smaller spheroids than 786-O shScr. To account for this, 786-O shScr spheroids at all time points were normalized against 786-O sh742.E11 as follows: (calculated area shScr) (average area day 0 shScr – average area day 0 sh742.E11). Invasion of all cell lines through a Matrigel-coated membrane (2D) was signiﬁcantly inhibited (vii, viii, ix, x, xi and T, U, V, W, X, Y, Z, Ai, Aii, Aiii). Experiments were performed in triplicate. Error represents SEM. Signiﬁcance was determined by 2-way ANOVA and P < 0.05; P < 0.01; P < 0.001; P < 0.0001.

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systems more closely recapitulate in vivo tumor phenotypes than 2D volume ¼ 0.5 a b2,wherea and b are the longest and shortest cultures. As seen in vivo, compounds may have difficulty diffusing diameters, respectively. Over the course of the experiment, treat- to the center of a spheroid, or may be inhibited by hypoxia, leading ment with SBI-183 resulted in an average 51% tumor volume to decreased efficacy, increased cellular survival, and reduced reduction compared with control (Fig. 5). No differences were compound sensitivity (34–36). Therefore, in order to more closely observed in the overall body weight. These data suggest that SBI- mimic how naturally occurring tumors would be affected by SBI- 183 inhibits the growth of a highly aggressive sarcomatoid RCC 183, we utilized a 3D invasion model. 786-O, RCJ-41T2, MDA-MB- in vivo. 231, A549, and MIA PaCa2 were grown as spheroids. After the addition of Matrigel, spheroids were imaged on the indicated days SBI-183 reduces laminin-a4 deposition in RCJ-41T2 mouse (Fig. 3i, ii, iii, iv, and v). 786-O, RCJ-41T2, and A549 initially xenografts formed dense spheroids (Fig. 3C, F, and L), which expanded over Because QSOX1 has previously been shown to be involved in the thecourseoftheexperiment(Fig. 3A, D, and J). RCJ-41T2 formed deposition of laminin-a4 in the ECM (13), we hypothesized that wandering tendrils as it invaded the surrounding matrix (Fig. 3D). laminin-a4 deposition would be reduced in xenograft tumors from MDA-MB-231 and MIA PaCa2 initially formed loose, grape clus- mice treated with SBI-183. DAB staining intensity due to laminin-a4 ter-like spheroids (Fig. 3I and O). At the end of the experiment deposition was shown to be significantly reduced in SBI-183–treated these clusters were greatly enlarged with projections from the main mice compared with vehicle control (Fig. 6). body, and single cells migrating from the spheroid (Fig. 3G and M). In each cell line tested, incubation with SBI-183 reduced invasion through Matrigel (Fig. 3i, ii, iii, iv, and vB,E,H,K,N), similar to Discussion the reduction observed in QSOX1 stable KD cell line 786-O sh742. Despite systemic therapy, distant metastases are the major E11 (Fig. 3viS). cause of cancer mortality. QSOX1 secreted from tumor and stromal To ensure the observed decrease in invasion was not simply due to a cells is involved in ECM formation including laminin and fibro- decrease in proliferation due to the length of the 3D experiment, nectin deposition (13, 14), and posttranslational activation of modified Boyden chamber invasion assays were also performed MMPs (12). Taken together, QSOX1 plays an important role in (Fig. 3vii–xi). These invasion assays confirmed the 3D spheroid ECM-mediated invasive processes. Because tumor-stroma-derived results. ECM is crucial for metastasis, targeting a potential master regulator of the ECM such as QSOX1 may affect multiple ECM proteins Exogenous addition of rQSOX1 partially rescues invasion involved in invasion and metastasis. There are several lines of induced by SBI-183 evidence supporting QSOX1 as a potential therapeutic target. First, – QSOX1 is overexpressed by tumor cells, localizes to the ER and QSOX1 is overexpressed in several malignancies (7 12, 37) and is Golgi, and is secreted. Because SBI-183 inhibits the activity of QSOX1 an indicator of poor relapse free and overall survival in luminal B resulting in a decrease in invasion, we added exogenous rQSOX1 to breast cancer (7, 8, 38). Second, enzymatic inhibition of QSOX1 rescue the invasive phenotype (7, 15). Addition of a 2-fold molar excess usingeithersmallmoleculesormAbsinterfereswithECMdepo- – of rQSOX1:SBI183 to 786-O cells partially rescued invasion (Fig. 4A). sition and reduces tumor invasion (13 15). Because shRNA silenc- Addition of equimolar concentration of rQSOX1 partially rescued the ing of QSOX1 previously demonstrated suppression of tumor invasive phenotype of both RCJ-41T2 cells (Fig. 4B) and MDA-MB- growth and invasive phenotype (7, 15), we embarked on a screening 231 cells (Fig. 4C). strategy to identify chemical probes to examine the effects of QSOX1 inhibition in vitro and in vivo. We demonstrate that the small molecule, SBI-183, (i) inhibits QSOX1 enzymatic activity SBI-183 inhibits tumor growth of 786-O in vivo in vitro, (ii) binds to QSOX1, (iii) inhibits tumor cell growth and Because SBI-183 inhibits invasion in vitro, we tested the activity of invasion in vitro, and (iv) reduces tumor size in 2 independent SBI-183 in 2 independent RCC mouse xenografts. Tumor measure- mouse models. ments were obtained at the intervals indicated in Fig. 5. One mouse We used an enzymatic assay developed by Colin Thorpe's group (17) from the test group was terminated according to IACUC protocol on to screen for QSOX1 inhibitors in a library of 50,000 compounds. day 21. At the end of the experiment (day 41), SBI-183-treated 786-O SBI-183 was identified as a lead compound for the inhibition of xenografts had average tumor volumes that were 86% smaller than QSOX1 enzymatic activity (Fig. 1A). We previously reported that vehicle-treated mice. These results indicate that SBI-183 inhibits the ebselen bound covalently to QSOX1 by LC/MS-MS analysis (15), but growth of a RCC tumor cell line in vivo. SBI-183 does not appear to bind covalently to QSOX1. Another measure of binding is MST which measures the motion of proteins SBI-183 inhibits tumor growth of RCJ-41T2 in vivo along microscopic temperature gradients and is affected by changes in Sarcomatoid RCC is associated with an aggressive, mesenchymal protein hydration, charge, and size originating from ligand binding. phenotype, and is intrinsically resistant to antiangiogenic therapy. Incubation of serial dilutions of SBI-183 with QSOX1 demonstrated a To extend our findings to a sarcomatoid RCC line recently derived temperature shift indicative of binding (Fig. 1B). This physical from a patient, 18 NSG mice were inoculated with minced RCJ- interaction between QSOX1 and SBI-183 supports computer models 41T2 tumors obtained from patient-derived xenografts in 50% showing SBI-183 fitting into a crevice in QSOX1 at the C-terminal end Matrigel and tumors were established for 10 days prior to dosage of the second thioredoxin domain. The strong docking score via the with 100 mg/kg SBI-183 or vehicle control. Data are from 9 control SBI-183 benzyl-moiety and the tyrosine ring at Y262, along with the mice and 6 experimental mice (3 mice were lost in the experimental SBI-183 carbonyl oxygen electrostatic interactions at the nearby group due to an oral gavage problem, not due to the compound). tyrosines (Y259, Y262) and various van der Waals interactions on Tumor volume was measured every 7 days with calipers the hydrophobic residues (V251, M253, L252, F258) with the alkane and volume was calculated using the following formula: Tumor atoms of SBI-183 creates a solid anchored position for SBI-183 on

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QSOX1. Additionally, the area of interaction includes C237 within 6Å invasion observed in the trans-well invasion assay (Fig. 3vii-xi). of SBI-183 binding (Fig. 1D). C237 is one of 2 cysteines that covalently All invasion results are consistent with decreased invasion observed bound ebselen in our previous study (15). It is thought that C237 is not in cells stably expressing shRNA specific for QSOX1 (7, 15), involved in QSOX1 enzymatic activity (39), however, it is interesting and could be at least partially due to a malformed ECM lacking that 2 compounds which inhibit QSOX1 interact with it in this laminin, fibronectin (13, 14), and reduced MMP-2 and MMP-9 location. These data suggest that this region may be important for activity (12). Furthermore, addition of exogenous rQSOX1 partially QSOX1 activity. rescued SBI-183–induced invasion suppression observed in 786-O, In addition to metastatic processes, the ECM is involved in RCJ-41T2, and MDA-MB-231 (Fig. 4; A549 and MIA PaCa2 were not signaling. SBI-183 suppressed growth in each tumor cell line tested tested). Rescue experiments are difficult to perform with small mole- in a concentration-dependent manner (Fig. 2A–E), but no signif- cules because the small molecule can enter the cell while the target icant reduction in growth of fibroblasts or PHA-stimulated PBMC protein remains extracellular. A review of polypharmacology discusses was observed (Fig. 2G–H). This finding agrees with previously that most drugs interact with 5 or more targets (44). In line with this, published IHC results in a tumor tissue biopsy that show no QSOX1 some VEGF tyrosine kinase inhibitors such as sunitinib, are known to protein expression in nonmalignant tissue or infiltrating lympho- also interact with other kinases (45). Similarly, although SBI-183 is cytes (6, 40). Furthermore, Supplementary Table S3 demonstrates active against QSOX1 (Fig. 1A) it likely has various other targets in that the maximal tolerated dose of SBI-183 in healthy nude tumors, explaining why exogenous addition of rQSOX1 does not mice is over 200 mg/kg and was limited by solubility of the completely rescue the invasive phenotype. However, if QSOX1 is a compound, not toxicity. We do note, however, that SBI-183 has master regulator of multiple disulfide-bonded proteins, even partial ahighIC50 in the tested cell lines (Supplementary Fig. S3). As such, inhibition of QSOX1 may disrupt folding or proper association of it is possible that there are other targets of SBI-183 in vivo that proteins in the ECM. suppress tumor growth. To examine the in vivo effects of SBI-183 on tumor growth To further examine the cancer phenotype, we utilized a well- and metastasis, we inoculated mice with 786-O, RCJ-41T2, and accepted model of 3D invasion of spheroids into Matrigel MDA-MB-231 (Supplementary Fig. S4). Mice bearing 786-O or (31, 41–43). Incubation with SBI-183 significantly reduced invasion RCJ-41T2 tumors that were treated with SBI-183 exhibited a statis- in all 5 cell lines (Fig. 3i-v). Our 3D results are consistent with reduced tically significant reduction in tumor volume compared with controls

Figure 4. Partial rescue of invasive phenotype by addition of exogenous rQSOX1. Addition of 5 mmol/ L rQSOX1 increased invasion of 786-O (A)by 10% by day 6. By day 4, 5 mmol/L rQSOX1 increased invasion of RCJ-41T2 (B) by 17% and increased invasion of MDA-MB-231 (C) by 20%. Experiment was performed in triplicate. Error represents SEM. Signiﬁcance was determined by 2-way ANOVA and P < 0.01; P < 0.001; P < 0.0001.

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Figure 5. Treatment with SBI-183 suppresses 786-O and RCJ-41T2 growth in mice. A, 786-O cells were subcutaneously injected into 4 nude mice per group, and tumors were established before initiation of daily oral gavage of 400 mg/mouse/day SBI-183 or DMSO vehicle. Percentage of decrease was calculated with the following formula: 100 [(average SBI-183)/(average vehicle)] 100. B, Daily treatment with SBI-183 suppresses RCJ- 41T2 growth in NSG mice. Data are from 9 control mice and 6 experimental mice. Percentage decrease was calculated as above. Error bars represent SEM. Signiﬁ- cance was determined by 2-way ANOVA and P < 0.01; P < 0.0001.

(Fig. 5A ad B). Mice bearing the TNBC cell line MDA-MB-231-luc and modiﬁed Boyden chamber models which are in vitro surrogates for interestingly did not exhibit a reduction in primary tumor volume metastasis. It should be noted that the effect of SBI-183 observed (Supplementary Table S4) but rather exhibited a suppression of depends on the cell line tested, suggesting that cells depend differently metastasis as evidenced by a reduction in mean lung radiance of on QSOX1 activity. 76% when compared with controls (Supplementary Fig. S4). This Our data show that both in vitro and in vivo, SBI-183 suppresses reduction, while striking, did not reach statistical signiﬁcance, likely QSOX1 enzymatic activity which results in inhibition of tumor due to the death of 2 control mice. Our MDA-MB-231 in vivo data growth, invasion, and possibly metastasis in vivo. Further, our data differs from our in vitro data in that SBI-183 slows tumor growth suggest that SBI-183 may be a useful tool to increase our understanding in vitro, but did not slow primary tumor growth in mice. However, of the role of QSOX1 activity in the ECM of cancer and stromal cells MDA-MB-231 cells were inhibited from invading in the 3D spheroid during invasion and metastasis. Because metastasis is the main cause of

Figure 6. Treatment with SBI-183 reduced laminin-a4 deposition in RCJ-41T2 mouse xenografts. DAB staining intensity (log OD) due to laminin-a4 deposition was 0.115 0.022 for vehicle-treated mice and 0.088 0.008 for SBI-183– treated mice (P ¼ 0.0101). Optical density (OD) was estimated from 3 unique images from each of 2 tumors per group with the following formula: OD ¼ Log(max intensity/mean intensity), where max intensity ¼ 255 (46). Error represents SEM and was calculated in Microsoft Excel. Scale bar, 50 mm. Signiﬁcance was determined using Welch t test.

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death from cancer, even partial inhibition of this process may prolong Administrative, technical, or material support (i.e., reporting or organizing data, fi patient survival. Finally, our study provides further evidence of QSOX1 constructing databases): A.L. Fi eld, D.O. Faigel, J.L. Petit, T.H. Ho, D.F. Lake as an anti-neoplastic target. Study supervision: T.H. Ho, D.F. Lake Other (Assay development and HTS that identified the studied molecule): E. Sergienko Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed. Acknowledgments ’ This work was supported in part by the Gloria A. and Thomas J. Dutson Jr. Authors Contributions Kidney Research Endowment. T.H. Ho is supported by NCI (R01 CA224917) fi Conception and design: A.L. Fi eld, P.D. Hanavan, D.O. Faigel, J.L. Petit, T.H. Ho, and the Department of Defense (W81XWH-17-1-0546). Opinions, interpreta- D.F. Lake tions, conclusions, and recommendations are those of the author and are not fi Development of methodology: A.L. Fi eld, P.D. Hanavan, E. Sergienko, A. Bobkov, necessarily endorsed by the Department of Defense. The funding agencies had fi T.R. Caul eld, J.A. Copland, D. Mukhopadhyay, T.H. Ho, D.F. Lake no role in the study design. This work was also partially supported by grants Acquisition of data (provided animals, acquired and managed patients, provided to D.O. Faigel, E. Sergienko, and D.F. Lake (R01 CA201226) and to fi facilities, etc.): A.L. Fi eld, P.D. Hanavan, E. Sergienko, A. Bobkov, N. Meurice, D. Mukhopadhyay (R01 CA78383-20). J.L. Petit, A. Polito, E.P. Castle, D. Mukhopadhyay, K. Pal, S.K. Dutta, H. Luo, T.H. Ho, D.F. Lake Analysis and interpretation of data (e.g., statistical analysis, biostatistics, The costs of publication of this article were defrayed in part by the payment of computational analysis): A.L. Fifield, P.D. Hanavan, D.O. Faigel, A. Bobkov, page charges. This article must therefore be hereby marked advertisement in N. Meurice, J.L. Petit, A. Polito, T.R. Caulfield, D. Mukhopadhyay, K. Pal, accordance with 18 U.S.C. Section 1734 solely to indicate this fact. T.H. Ho, D.F. Lake Writing, review, and/or revision of the manuscript: A.L. Fifield, D.O. Faigel, A. Bobkov, N. Meurice, J.L. Petit, T.R. Caulfield, E.P. Castle, J.A. Copland, Received March 12, 2019; revised July 1, 2019; accepted September 24, 2019; D. Mukhopadhyay, K. Pal, T.H. Ho, D.F. Lake published first October 1, 2019.

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Molecular Inhibitor of QSOX1 Suppresses Tumor Growth In Vivo

Amber L. Fifield, Paul D. Hanavan, Douglas O. Faigel, et al.

Mol Cancer Ther Published OnlineFirst October 1, 2019.

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