Applying Small Molecule Signal Transducer and Activator of ... · P.T. Gunning and M.L. Fishel are...

Small Molecule Therapeutics

Applying Small Molecule Signal Transducer andActivator of Transcription-3 (STAT3) ProteinInhibitors as Pancreatic Cancer TherapeuticsCarolyn C. Arpin1, Stephen Mac1, Yanlin Jiang2, Huiwen Cheng2, Michelle Grimard2,Brent D.G. Page1, Malgorzata M. Kamocka3, Sina Haftchenary1, Han Su4, Daniel P. Ball1,David A. Rosa1, Ping-Shan Lai1, Rodolfo F. G�omez-Biagi1, Ahmed M. Ali1,5, Rahul Rana1,Helmut Hanenberg2,6,7, Kagan Kerman4, Kyle C. McElyea8, George E. Sandusky8,Patrick T. Gunning1, and Melissa L. Fishel2,9

Abstract

Constitutively activated STAT3 protein has been found to bea key regulator of pancreatic cancer and a target for moleculartherapeutic intervention. In this study, PG-S3-001, a smallmolecule derived from the SH-4-54 class of STAT3 inhibitors,was found to inhibit patient-derived pancreatic cancer cellproliferation in vitro and in vivo in the low micromolar range.PG-S3-001 binds the STAT3 protein potently, Kd ¼ 324 nmol/Lby surface plasmon resonance, and showed no effect in akinome screen (>100 cancer-relevant kinases). In vitro studiesdemonstrated potent cell killing as well as inhibition of STAT3activation in pancreatic cancer cells. To better model the tumorand its microenvironment, we utilized three-dimensional (3D)

cultures of patient-derived pancreatic cancer cells in the absenceand presence of cancer-associated fibroblasts (CAF). In thiscoculture model, inhibition of tumor growth is maintainedfollowing STAT3 inhibition in the presence of CAFs. Confocalmicroscopy was used to verify tumor cell death followingtreatment of 3D cocultures with PG-S3-001. The 3D modelwas predictive of in vivo efficacy as significant tumor growthinhibition was observed upon administration of PG-S3-001.These studies showed that the inhibition of STAT3 was ableto impact the survival of tumor cells in a relevant 3D model,as well as in a xenograft model using patient-derived cells.Mol Cancer Ther; 15(5); 794–805. �2016 AACR.

IntroductionApproximately 25% of all deaths per year in the United States

are caused by cancer where pancreatic cancer is one of the most

aggressive and lethal types (1). Patients face a one-year survivalrate of 25% and a 5-year survival rate of only 6% (2). A proposedfactor in the limited success of molecular therapies has been theheterogeneity found in pancreatic ductal adenocarcinoma(PDAC) samples pointing toward the need for strategies thattarget proteins that can affect multiple pathways (3–5). Lack ofclinical efficacy is due, in part, to the desmoplastic fibrosis thataccompanies pancreatic cancer (6, 7). The surrounding fibroticnetwork plays a principal role in supporting local tumor growthand distant metastasis (8–10). The challenge has been to identifythe molecular effectors that critically regulate the survival ofpancreatic cancer cells, to devise effective molecular-targetedstrategies that can prevent or minimize the selection of resistanttumor variants, and then to penetrate the fibrotic nature of thesetumors. Moreover, studies of stroma-specific depletion surpris-ingly found a decrease in survival rates of transgenic mousemodels of pancreatic cancer attributing the loss of activatedstroma to reduced survival (11, 12). As the contribution of thestroma is further investigated, future treatments for PDACmust beable to target the tumor in the presence of its associatedmicroenvironment.

These studies address the role of STAT3 protein as a criticalmolecular target in pancreatic cancer. STAT3 is a transcriptionfactor that regulates critical cell functions and has been implicatedin several cancers, including breast, prostate, liver, and pancreas(13–15). In pancreatic cancer, STAT3 has been associatedwith cellproliferation and viability as well as with angiogenesis andmetastases (16–19). In genetically engineered mouse (GEM)models of pancreatic cancer, STAT3 has been implicated in the

1Department of Chemistry, University of Toronto Mississauga, Missis-sauga, Ontario, Canada. 2Department of Pediatrics, Wells Center forPediatric Research, Indiana University School of Medicine, Indianapo-lis, Indiana. 3Department of Medicine, Division of Nephrology, IndianaCenter for Biological Microscopy, Indiana University School of Med-icine, Indianapolis, Indiana. 4Department of Physical and Environmen-tal Sciences, University of Toronto Scarborough, Toronto, Ontario,Canada. 5Department of Medicinal Chemistry, Faculty of Pharmacy,Assiut University, Assiut, Egypt. 6Department of Pediatrics III, Univer-sity Children's Hospital Essen, University of Duisburg-Essen, Essen,Germany. 7Department of Otorhinolaryngology and Head/Neck Sur-gery (ENT), Heinrich Heine University, Dusseldorf, Germany. 8Depart-ment of Pathology and Laboratory Medicine, Indiana UniversitySchool of Medicine, Indianapolis, Indiana. 9Department of Pharmacol-ogy and Toxicology, Indiana University School of Medicine, Indiana-polis, Indiana.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

P.T. Gunning and M.L. Fishel are co-last authors of this article.

Corresponding Authors: Melissa Fishel, Indiana University School of Medicine,1044 W. Walnut Street, R4-321, Indianapolis, IN 46202. Phone: 317-274-8810;Fax: 317-278-9298; E-mail: [email protected]; and Patrick T. Gunning, Departmentof Chemistry, University of Toronto Mississauga, Mississauga, ON L5L 1C6,Canada. Phone: 905-828-5354; Fax: 905-828-5425; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-15-0003

�2016 American Association for Cancer Research.

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establishment of early PDAC lesions (15, 20) as well as associatedwith tumor and stromal cell proliferation and resistance togemcitabine therapy (21). STAT3 signaling is prevalent withinthepancreatic tumormicroenvironment as detailedherein. STAT3activation can be regulated by several mechanisms, includingphosphorylation and redox status (7, 22, 23). Several in vitro andin vivo studies using immortalized cells as well as tumor cell linesshowed that STAT3 blockade or inactivation (RNAi or pharma-cologic blockade) exerts inhibitory effects on the survival, prolif-eration, colony formation or invasiveness of human PDAC cells(24). Together, these results have identified STAT3 as an appealingtarget for therapeutic intervention (25–27).

STAT3 signaling (28) is initiated by extracellular cytokine/growth factor stimulation of a respective transmembrane protein,which results in the intracellular phosphorylation of key tyrosine(Y) residues. Cytokine binding to their cognate receptor leads toactivation of a JAK (Janus kinase) protein, which then phosphor-ylates and activates cytoplasmic STAT3 protein. These phospho-tyrosines (p-Y's) serve as docking sites for the Src Homology 2(SH2) domain of STAT3, and once bound, STAT3 is phosphor-ylated on Y705. Phosphorylated STAT3 then dissociates andforms active homodimers via reciprocal binding of the SH2domain of one protein and the p-Y705 of another. These tran-scriptionally active dimers then translocate to the nucleus, bind toDNA, and promote target gene expression.While STAT3 activity istransient in healthy cells, it is often aberrant and constitutivelyactive in cancer cells, including pancreatic cancer.

The STAT3 SH2 domain is integral to protein function. Thisshallow pocket binds transmembrane proteins, which enablesSTAT3 phosphorylation, activation, and formation of transcrip-tionally active STAT3 homodimers (29). Previous efforts havefocused on inhibiting STAT3 function by blocking the pY bindingsite within the SH2 domain (30) or the DNA binding domain. Ingeneral, STAT3 compounds have included peptidomimetics, (31,32) oligonucleotides (33–35), metal complexes (36, 37), andsmall molecule inhibitors (38–43). Inhibitors operating via DNAbinding domain blockade include inS3-54 (44) as well as Galeil-lalactone (45).Our efforts have focused on developing small-molecule inhibitors of the SH2 domain: BP-1-102 (46, 47),BP-5-87 (48), and SH-4-54 (49). Unfortunately, only a small numberof small molecule inhibitors for STAT3 protein have potenciesand selectivity profiles suitable for advanced preclinical evalua-tion (26, 50). The discovery of a clinically relevant direct-bindingSTAT3 inhibitor has yet to be achieved. In this study, we sought toidentify the most potent STAT3 inhibitors from a library of >100salicylic and benzoic acid–based inhibitors, known to have STATSH2 domain binding potential, and study the effects of these top-ranked analogs on PDAC tumor and stroma interactions.

Herein, we utilize state-of-the-art models, including 3D cul-tures of lowpassage patient-derived PDACcells in the absence andpresence of cancer-associated fibroblasts (CAF) to better modelthe pancreatic tumor and its tumor microenvironment (TME) invivo. Three-dimensional (3D) model systems more accuratelymimic the complexity of cancer biology, compared with mono-layer cell culture, and have the potential to provide relevantanswers related to cancer treatment and disease specific, pancre-atic cancer drug development (51, 52). Inhibition of tumorgrowth was observed following STAT3 inhibition as well as invivo tumor efficacy studies. Blockade of STAT3 activity using BP-1-102, SH-4-54, and PG-S3-001 lead to PDAC cell death in vitro andtumor regression both in 3D coculture systems and in vivo xeno-

graft models of PDAC. Moreover, these studies showed thatinhibition of STAT3 impacted the survival of tumor cells even inthe presence of CAFs from the tumor microenvironment. Thesestudies support our hypothesis that STAT3 is a significant molec-ular target in PDAC.

Materials and MethodsSynthesis of STAT3 small molecules

All of the details of the chemistry for the library preparation andsynthesis are contained in the Supplementary files. This filecontains a detailed description of the synthesis as well as thecharacterization of lead compounds PG-S3-001, PG-S3-002, andPG-S3-003.

Cell lines and patient-derived PDAC cellsPa03C, Panc10.05, Pa02C, and CAF19 were obtained fromDr.

AnirbanMaitra at The Johns Hopkins University (Baltimore, MD;ref. 3). Upon receipt of the cells, we used STR (short tandemrepeat) analysis (CellCheck with IDEXX BioResearch) to confirmthat we indeed received the aforementioned cells fromDr. Maitraand we rechecked them via the same method in June 2015.Normal lung fibroblasts, CCD-13Lu and MIA-PaCa-2 wereobtained from ATCC and were passaged for fewer than 6 monthsafter resuscitation. All cells were maintained at 37�C in 5% CO2,grown in DMEM (Invitrogen) with 10% serum (Hyclone), andmycoplasma-free. The CAF19 cells were transduced with a lenti-virus vector to make them stably express enhanced GFP (EGFP;ref. 53), and Pa03C cells were transduced with a lentivirus vector(pCL7TdTOMwo) tomake themstably express TdTomato. CAF19cellswere seeded 24hours before 150pfu/cell of the lentiviruswasadded to the media. One day later, the virus was removed andthen the cells were grown for an additional 2 days in regularmedia.

Survival and proliferation studiesThe proliferative capacity of PDAC and CAF19 cells as a

monolayer was assessed using MTS tetrazolium dye assay asdescribed previously (7). Using 96-well plates, we seeded eithertumor cells alone (2,000–3,000 cell/well), CAFs alone (4,000 cell/well), or tumor þ CAFs at a ratio of 2:1. STAT3 inhibitors wereadded 24 hours after the cells were seeded and MTS assay wasperformed 72 hours later.

Three-dimensional growth assaysNinety-six well plates were coated with 1% noble agar (Difco,

214220) in 10% serum-containing media (50 mL/well) asdescribed previously (54). Pa03C cells were resuspended innormal growth media containing 3% Matrigel (BD Biosciences)at a cell density of 500 cells/well and plated on top of solidified1% noble agar. Cells were treated on days 4 and 8 followingplating with media containing 10% serum, 3% Matrigel, andSTAT3 inhibitors. CellTracker dye (25 mmol/L) or TdTomato vialentivirus was used to label these cells for confocal experiments topreserve the genetic characteristics of the low passage patient cells(3). The vehicle control was DMSO, was less than 0.01% of thevolume, and was equivalent in each well. On day 12, eitherAlamar blue reagent (Life Technologies) was added to each well(10 mL/well) and incubated for 4 hours or spheroids were ana-lyzed using Thermo ArrayScan high-content imaging system (55,56). For Alamar blue assays, fluorescence reading at 544, 590nmol/L was then taken and used to assess survival following

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STAT3 inhibition. Images of 3D structures were captured byArrayScan using a 2.5� objective for TdTomato and EGFP; thentwo-dimensional (2D) projections were processed to quantifydifferences in total intensity and total area of both CAFs andtumor. After summarizing 4–5 repeats in bothmonolayer and 3Dproliferation assays, we calculated effective dose-50 (ED50s) foreach compound using a line of best fit (i.e., linear regressionmodel) where the percent survival equaled 50%.

Western blot analysisCells were harvested, lysed in RIPA buffer (Santa Cruz Biotech-

nology), and protein was quantified and electrophoresed asdescribed previously (7). Immunoblotting was performed usingthe following antibodies: total STAT3, p-STAT3 (Y705), andSurvivin (Cell Signaling Technology) at a 1:1,000 dilution.

Imaging of 3D spheresTo visualize the tumor cells for imaging of the structure, we

labeled the Pa03C cells with CellTracker fluorescent probe (Invi-trogen, C34552).We incubated the cellswith 25mmol/L probe for45minutes in serum-freemedia, thenwe spundown the cells, andlet them recover in media containing serum for 30 minutes at37�C. CAFs utilized in these experiments stably express EGFP asdescribed above. We imaged the cells on days 5 and 11 to capturethe effect of STAT3 inhibition in the middle and end of the 3Dassay. On day 11, we added Alamar blue to the wells to confirmthe decrease in proliferation visualized by the confocalfluorescentimaging.

Confocal images of spheres were acquired with a confocal/two-photon Olympus Fluoview FV-1000 MPE system (OlympusAmerica) available at the Indiana Center for Biological Micros-copy (ICBM, Indianapolis, IN) facility, using an OlympusUMPLFL 10X W NA:0.30 air objective and XLUMPLFL 20X waterNA:0.95 lenses. Images were collected at 512 � 512 frame size, 8ms pixel dwell time, and in a sequential illumination mode using488- and 559-nm excitation laser lines. Emission light was col-lected with two spectral detectors set up at 500–545 nm and 570–670 nm filter ranges. Because of the large size and density of thecontrol and vehicle-treated spheres, accurate visualization of theentire sphere using confocal scanning mode was not possible onday 11. It was hypothesized that this was due to compromisedlight penetration through the tightly packed spheres. Therefore,transmitted light images were collected simultaneously withconfocal scans to evaluate overall size for the spheres. Axialscanning (Zstack) was performed, and optical consecutive andparallel slices were collected using optimal step size. Maximumintensity projection (MIP) images were created out of collectedimage data stacks using Olympus Fluoview Image Viewer v.3.0.3D reconstruction imageswere created out of collected image datastacks using Amira software (FEI Co.).

In vivo efficacy of STAT3 inhibitors in low passage patient-derived cells

All studies were carried out in accordance with, and approvalof, the Institutional Animal Care and Use Committee ofIndiana University Medical School, and the Guide for the Careand Use of Laboratory Animals. Male and female NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (common name NSG) 6–8 week oldmice were obtained from the In Vivo Therapeutics Core of theIndiana University Simon Cancer Center. Animals were housedunder pathogen-free conditions and maintained on Teklad Lab

Animal Diet (P/N TD 2014, Harlan Laboratories) with ad libitumaccess to sterile tap water under a 12-hour light/dark cycle at 22–24�C. Using low passage patient-derived cells, Pa03C, ectopicxenografts were grown in NSGmice. Pa03C cells (2.5� 106 cells/mouse) in 0.2 mL of DMEM:Matrigel (50:50) were implantedsubcutaneously into the rightflanks ofNSGmice. CompoundPG-S3-001 was dissolved in 4% CremophorEL:EtOH (1:1)/salinesolution. When tumor volumes were 71 � 4 mm3, compoundPG-S3-001 was administered once daily at 10 mg/kg i.p. for 15days. Mice were weighed twice weekly to assess toxicity. Tumorswere measured weekly during treatment. Tumor volumes weremonitored by caliper measurement [tumor volume ¼ length �(perpendicular width)2 � 0.5] and the average tumor volume inmm3 for each treatment group was plotted. Treatment started onday 8 postimplant. Average tumor volume� SE for the vehicle (n¼ 18) and PG-S3-001–treated (n ¼ 16) xenografts was analyzedby Student t test. On day 25, the mice were sacrificed and thetumors excised and weighed.

IHC of tumor tissueTissues were fixed overnight at room temperature in 10% NBF

after which they were transferred through graded concentrationsof alcohol to xylene inside a Leica Automated Vacuum TissueProcessor. Tissues were embedded in paraffin before being cutinto 5-mm thick sections, mounted onto positively charged slides,and baked at 60�C. The slides were then deparaffinized in xyleneand rehydrated through graded alcohols to water. Antigen retriev-al was performed by immersing the slides in a Target RetrievalSolution (Dako) for 20minutes at 90�C (in awater bath), coolingat room temperature for 10 minutes, washing in water, and thenproceeding with immunostaining. Slides were blocked with pro-tein blocking solution (Dako) for 30 minutes. All subsequentstaining stepswere performedusing theDako FLEXSYSTEMonanautomated Immunostainer; incubations were done at room tem-perature and Tris-buffered saline plus 0.05% Tween 20, pH 7.4(TBS - Dako Corp) was used for all washes and diluents. Theprimary antibodies were anti-mouse phospho-Histone H3(1:500, Dako) and p-STAT3 (1:25, Cell Signaling Technology).Control sections were treated with an isotype control using thesame concentration as primary antibodies to verify the stainingspecificity. For whole slide digital imaging, the Aperio ScanScopeCS system was used. The system imaged all slides at 20�. Slideswere reviewed by two pathologists, and tissue was recorded as%tumor, %necrosis, %inflammation, and %stroma. The pathol-ogy hand count was only evaluated in an area of viable tumorcells. An average of mitotic figures (at �20) was hand-countedfrom four hotspot areas on each tissue. The control and treatmentgroups were then evaluated for statistical differences.

Determination of inhibitory constants for small moleculesusing fluorescence polarization assays

Competitive binding fluorescence polarization (FP) assayswere performed for the lead inhibitors PG-S3-001, PG-S3-002,and PG-S3-003 against STAT3 protein. The assays were performedin flat black 384-well plates (Corning #3573) and FP measure-ments were taken with the Infinite M1000 machine (Tecan).The buffer conditions for all assays were 10 mmol/L HEPES,50 mmol/L NaCl, 1 mmol/L EDTA, 2 mmol/L TCEP, pH 7.5,and the final DMSO concentration in the wells was kept constantat 10%. Calibration curves for the wild-type STAT3 proteins werederived by incubating a 10 nmol/L final concentration of a

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Mol Cancer Ther; 15(5) May 2016 Molecular Cancer Therapeutics796




fluoresceinated-phosphopeptide (5-FAM-GpYLPQTV for STAT3),with increasing concentrations of protein. The point atwhich 80%of the fluoresceinated-phosphopeptide was bound was used asthe optimal protein concentration required for the competitive FPassays. For the STAT3 FP assays, the 5-FAM-GpYLPQTV-NH2

peptide and STAT3 protein were incubated for 30 minutes atroom temperature. Inhibitors were titrated at concentrationsranging from 1 nmol/L to 500 mmol/L and incubated for a further15 minutes at which point FP measurements were taken intriplicate. The final well concentrations of the fluoresceinated-phosphopeptide and STAT3 protein were 10 nmol/L and 250nmol/L, respectively. FP measurements were normalized andplotted against inhibitor concentration on a logarithmic scale.The raw data were fitted with a standard dose-normalizedresponse inhibition curve using GraphPad Prism 6 software. Allinhibitory constants and Ki values are summarized in Fig. 2 (57).

Surface plasmon resonance spectroscopySurface plasmon resonance (SPR) binding experiments were

performed using a ProteOn XPR36 (Bio-Rad Laboratories Ltd).ProteOn HTE sensor chips (#176-5033) that had an elevated tris-NTA surface density optimal for protein–small molecule interac-tions were used in connection with ProteOn HTE capturing kit.The horizontal and vertical spots of the sensor chips were con-ditioned using consecutive injections of 0.5% SDS, 50 mmol/LNaOH, 100 mmol/L HCl followed by 300 mmol/L EDTA for 1minute each at a flow rate of 30 mL/minute. Then, the sensor chipswere activated using 10 mmol/L NiSO4 for 2 minutes in thehorizontal orientation at a flow rate of 30 mL/minute. Immedi-ately after the activation step, His-tagged STAT3 (0.5–25 mg/mL,SignalChem) protein was immobilized until the desired ligandimmobilization level of approximately 12 kRU was obtained at aflow rate of 30 mL/minutes. Small molecules were injected atvarious concentrations both in vertical (control spots with noprotein) and horizontal (protein-immobilized spots) orienta-tions at a flow rate of 5 mL/minute. To determine the full kineticprofile, small molecule–protein-binding spectrograms were eval-uated using the ProteOn Manager software.

Docking of inhibitors to STAT3 and visualization of thecomputed receptor–ligand complex

Ligand structures weremodeled and the geometry optimized inMaestro 9.9 (Schr€odinger Suite). The STAT3 crystal structure incomplex with DNA (PDB ID:1Y1U) was imported in Maestrowhere water molecules and the DNA oligonucleotides wereremoved. Hydrogen atoms were added followed by assigningGasteiger charges to the receptor. Molecular docking simulationswere performed with Glide 6.5 using a receptor grid localizedwithin the STAT3 SH2 domain. Simulation results were analyzedusing Pose Viewer and imported to MacPyMol v.1.7.6. Moleculardocking protocols are outlined in the supporting information(57–59).

qPCR off-target screening for kinome activityA competitive qPCR screening was employed to identify off-

target activity of PG-S3-001 against a comprehensive DiscoveRxKINOMEscan library of 132 human kinases. In this assay, kinaseslabeled with DNA were treated with PG-S3-001 (5 mmol/L singleconcentration) and incubated with an immobilized liganddesigned to capture its target kinase. Ultra-sensitive qPCR wasemployed tomeasure levels of immobilized kinases when treated

with PG-S3-001 and relative kinase levels compared with controlsamples. In this screen, hits are classified as compounds wherecaptured kinase levels fall below a 30% threshold. Images weregenerated using TREEspot software tool and reprinted with per-mission fromKINOMEscan, adivision ofDiscoveRxCorporation.Results pertaining to the interaction of PG-S3-001 with relevantkinases are included in the Supplementary files (SupplementaryTable S1).

ResultsScreening of novel STAT3 inhibitors in patient-derivedpancreatic cancer cell lines

To identify compounds best suited for targeting p-STAT3 inPDAC cell lines, we conducted a screen of 52 salicylic and benzoicacid containing compounds prepared in our laboratory withdemonstrated anticancer activity on p-STAT3–containing tumorcell lines. In total, 52 compounds (including a range of previouslypublished inhibitors) were screened using a cell viability assayemploying lowpassage patient-derived cells, Panc10.05. Previouswork demonstrated that both these cells and the additionalpatient derived cells (Pa03C) used here, express p-STAT3 and canbe killed following inhibition of p-STAT3 (7). This initial screenrevealed that three of the newly synthesized derivatives PG-S3-001, PG-S3-002, and PG-S3-003 (Fig. 1A) were more potent thanBP-1-102. For our proliferation assays, BP-1-102 and SH-4-54were utilized as comparator parent compounds (Fig. 1B and C).Specifically, PG-S3-001 and PG-S3-002 displayed enhanced effi-cacy against PDAC cells, with respective ED50 values of 2.4 � 0.2mmol/L and 3.0 � 0.1 mmol/L (Fig. 1C). PG-S3-003 was lesseffective than PG-S3-001, PG-S3-002, and SH-4-54 at blockingproliferation of Panc10.05 cells, with an ED50 value of 18.7� 1.1mmol/L for PG-S3-003 and 8.7 � 1.9 mmol/L for SH-4-54 (Fig.1C). In the case of PG-S3-003, the valine linker afforded amodestincrease in potency over parent compound, BP-1-102. A similartrend was observed with SH-4-540s congener, PG-S3-001 (Fig.1B), where an approximate 3-fold enhancement in activity wasobserved. The increase in potency could be attributed to valineacting as a conformational lock; restricting the number of acces-sible conformations anddecreasing the entropic cost of binding toSTAT3.

Predictive computational modeling of PG-S3-001 to the SH2domain of STAT3: analysis of ligand–receptor interaction

The initial lead STAT3 inhibitors, BP-1-102 and SH-4-54both possess a relatively flexible glycine core. The conforma-tional effect of the bulkier isopropyl alkyl appendage of PG-S3-001 was investigated in silico using the molecular dockingsoftware, Glide 6.5 (Schr€odinger Suite). As previouslyreported, BP-1-102 and SH-4-54, which differ only by a phe-nolic hydroxide, have essentially identical structural orienta-tion and energetic contacts with STAT3 (49). Comparativedocking within the SH2 domain of PG-S3-001 is shownin Fig. 2A. It revealed analogous electrostatic interactionsbetween the carboxylate group of the inhibitor with the pos-itively charged and hydrophilic pY-recognition pocket consist-ing of Arg609, Ser611, Ser613, and Lys591. The cyclohexyl-benzyl appendage of PG-S3-001 projects towards Glu638,whereas the pentafluorobenzene of PG-S3-001 was shown toadopt a different conformation relative to both SH-4-54 andBP-1-102. As seen from Fig. 2A and a 2D interaction map






(Fig. 2B), the pentafluorobenzne group forms dipolar inter-action with the backbone carbonyl oxygen of Pro715 andfavorable hydrophobic contact with Phe716. Finally, it wasobserved that PG-S3-001 adopts a conformation such that theisopropyl appendage interacts with the alkyl side chain ofIle634.

Measuring inhibitor binding to STAT3 proteinWith three lead compounds in hand, two biophysical techni-

ques were used to assess inhibitor binding to the STAT3 protein.First, a FP assay was employed to measure the disruption of thenative p-STAT3 SH2 domain complex in the presence of inhibitoras an initial gauge of binding affinity. The lead compounds, PG-S3-001, PG-S3-002, and PG-S3-003 demonstrated modest

Figure 2.A, computational modeling of PG-S3-001 to the SH2 domain of STAT3: analysis of ligand–receptor interaction. B, two-dimensional interaction map highlightingsmall molecule–receptor interaction of lead compound PG-S3-001.

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PG-S3-001 PG-S3-002 PG-S3-003 Figure 1.A, structure of lead STAT3 inhibitors:PG-S3-001, PG-S3-002, and PG-S3-003.B, top compounds were screened andcompared with the parent compoundsBP-1-102 and SH4-54; average � SE, n ¼4–5. C, bar graph of ED50s for comparisonwith new STAT3 inhibitors; average� SE,n ¼ 4–5.

Arpin et al.





binding affinities to STAT3 protein (Ki ¼ 72 � 6 mmol/L, 45 � 7mmol/L, and 39 � 3 mmol/L, respectively; Fig. 3A).

Second, SPR spectroscopy was used to determine the Kd (kon/koff) values for PG-S3-001 and PG-S3-002 against STAT3. Calcu-latedKds for PG-S3-001andPG-S3-002were 324� 37nmol/L and301 � 32 nmol/L, respectively (Fig. 3B), which were comparablewith that of parent compound SH-4-54 (300� 27 nmol/L). It canbe seen from the SPR traces that both compounds appear to haveeither slow dissociative off-rates (koff), suggesting longer target–drug residence times, or are irreversible inhibitors. For example, it ispossible that PG-S3-001 is alkylating the STAT3 protein throughcysteine-mediated nucleophilic aromatic substitution at the para-arylfluoride position of the pentafluorobenzene. The difference inactivity relative to the FP data is likely explained by PG-S3-001binding to a region of STAT3 other than the SH2 domain. Thispostulated mechanism of action will be investigated in futurestudies. However, given the Panc10.05 cell viability and biophys-ical data, PG-S3-001 was selected as the lead compound.

Lead STAT3 inhibitor effectively disrupts PDAC and CAF cellsurvival and proliferation individually and in coculture

In addition to the tumor cell proliferation studies in Fig. 1,additional validating studies in low passage patient-derivedcell line, Pa03C, were performed. The cytotoxicity of PG-S3-001 was evaluated in the MTS assay in these cells (Fig. 4A), CAFs(CAF19, Fig. 4B) and in a 2:1 coculture (Fig. 4C) to determine itscellular efficacy. In all three cultures, PG-S3-001 (IC50¼ 3.7� 0.4mmol/L, tumor cells; 4.0 � 0.7 mmol/L, CAFs; 4.7 � 0.1 mmol/L,coculture) exceeded the performance of parent compounds BP-1-102 (IC50 ¼ 19.1 � 0.9 mmol/L, tumor cells; 15.2 � 2.7 mmol/LCAFs; 16.4�0.6mmol/L coculture) andSH-4-54 (IC50¼9.2�1.7

mmol/L tumor cells; 7.6 � 0.9 mmol/L CAFs; 11.8 � 1.1 mmol/Lcoculture) at concentrations below their half maximal inhibitionconcentration (IC50s). Furthermore, cell proliferation was mon-itored using normal human lung fibroblasts, CCD-13Lu follow-ing STAT3 inhibition. It has been previously shown that STAT3inhibition is more cytotoxic to cancer cells as compared withnormal cells; therefore, primary, normal fibroblasts, CCD-13Luwere included to demonstrate that the cytotoxicity observedfollowing inhibition of STAT3 was preferential to tumor cellsand activated fibroblasts (Fig. 4D). After incubation for 72 hours,PG-S3-001 severely impaired the growth of Pa03C and CAF19cells, whereas the primary CCD-13Lu cell line retained 84% of itsrelative growth. Compared with parent compounds, PG-S3-001exhibited superior efficacy (�10-fold) for mitigating growth ofthe patient derived adenocarcinoma Pa03C cells. When compar-ing the three compounds inCAF19 cells, the activity of PG-S3-001is, within error, comparable with BP-1-102 and SH-4-54.

STAT3 inhibitors decrease STAT3 phosphorylationTreatment with BP-1-102, SH-4-54, and PG-S3-001 inhibited

phosphorylation of STAT3 at Y705 (Fig. 4E) as well as cellularproliferation in PDAC cells, CAFs, and cocultures (Fig. 4A–C).Inhibition of STAT3 activity correlated well to the amount of cellkilling by our lead compounds. In Fig. 4E, concentration of parentcompounds BP-1-102 and SH-4-54 was 10 mmol/L and PG-S3-001 was 5 mmol/L. The amount of inhibition of STAT3 activity vialevels of p-STAT3 correlatedwith the amount of cell killing of eachof these compounds: PG-S3-001 > SH-4-54 > BP-1-102. PG-S3-001 can significantly inhibit dose-dependent activation of STAT3at Y705 in two additional PDAC cell lines, established cell lineMIA-PaCa-2 and low passage patient-derived line Pa02C [Fig. 4F;

Figure 3.A, FP competition assay curves for PG-S3-001, PG-S3-002, PG-S3-003. B, SPR curves displaying the binding affinity for PG-S3-001 and PG-S3-002.






PaCa-2, P < 0.05, comparing IL6 control with 5 mmol/L and P <0.01 with 10 mmol/L PG-S3-001 and Pa02C, P < 0.01 (IL6 controlvs. 5 mmol/L and P < 0.001 for IL6 control vs. 10 mmol/L)]. Theactivation of p-STAT3 is also dramatically blocked in the CAF cellsas shown in Fig. 4G. This blockade of STAT3 in the CAFs led to>60% downregulation of STAT3-regulated gene, SURVIVIN, inthe CAF cells as well (P < 0.01).

Lead compound PG-S3-001 does not significantly inhibitupstream kinases as assessed by a kinome screen

To probe for off-target effects, PG-S3-001 (5 mmol/L concen-tration) was submitted to a kinome screen against 132 differentkinases. PG-S3-001 exhibitedminimal affinity for kinases directlyinvolved in STAT3 phosphorylation. Only proto-oncogene SRCkinase was moderately inhibited (36 % inhibition at 5 mmol/L,Supplementary Information, Table S1). These data further vali-dated PG-S3-001 as a candidate for the 3D tumor model and invivo efficacy studies.

Inhibitionofp-STAT3 inhibits cell growth in3Dculture systemsState-of-the-art three-dimensional (3D) culture systems (54)

in the presence and absence of CAFs were utilized to furtherscreen our lead compound for efficacy at killing PDAC cells.Three-dimensional tumor–stromal cell–associated spheroidmodels (Fig. 5) include selective pressures akin to the TMEwhich has several advantages over traditional monolayer cellculture (51, 52, 60, 61). CAF cells isolated from a pancreaticcancer patient (62) were included to recapitulate a more accu-rate picture of cell–cell interactions in vivo (Fig. 5). Because ofthe importance of the stroma in this disease (51, 52), CAFs andtumor cells were labeled to assess which cell type in the 3Dcoculture was most affected by STAT3 inhibition. As expected,coculture of CAFs with tumor cells increased the growth oftumor spheres (Fig. 5). Inhibition of p-STAT3 via BP-1-102, SH-4-54, and PG-S3-001 resulted in inhibition of growth in 3D aswas also observed with monolayer cell cultures. Inhibition ofSTAT3 was found to effectively slow the growth rate of cells in a

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dose-dependent manner (Fig. 5A–C), with IC50's ranging fromapproximately 15.2–27.0 mmol/L in 3D culture and approxi-mately 23.5–42.5 mmol/L for 3D culture containing tumor andCAFs. As expected, PDAC cells grown in 3D as well as theaddition of CAFs to the 3D cultures resulted in an increase inresistance to STAT3 inhibition. However, in this assay, PG-S3-001 (IC50 ¼ 15.2 � 2.0 mmol/L, tumor and 23.5 � 4.4 mmol/L,coculture) was more potent than the parent compounds, evenin the presence of stromal CAFs (Fig. 5C). The data in Fig. 5A–Cwere generated using a proliferation-based Alamar blue assaywhich provided reliable information about the effects of thecompounds on the proliferation of both cell types but does notdelineate which cell type was affected by inhibition of STAT3activity. Therefore, we used two additional methods to quan-titate and visualize the effects of PG-S3-001 on PDAC cellsalone and in coculture.

Two-dimensional projections of 3D images capturedbyArrayS-can high-content screening system were used to quantitate thearea and relative intensity (RLU) of the red and green channels.

Both the tumor cells alone and in coculture are sensitive to theSTAT3 inhibitor, PG-S3-001, as evidenced by a dose-dependentdecrease in area and intensity (Fig. 5DandE). As shown in Fig. 5D,the area of the tumor spheroids is more dramatically affected bythe PG-S3-001 compound than the area of the CAFs (P < 0.01comparing the area of the tumor to the area of the CAFs). Thefluorescent intensity of both tumor cells and CAFs was decreasedfollowing treatment (Fig. 5E). The CAFs provide significant pro-tection to the tumor cells as there are significant differencesbetween the tumor alone and the tumor in coculture both inarea and intensity (Fig. 5D and E).

Confocal microscopy further demonstrates the decrease inviability of PDAC cells following p-STAT3 inhibition

Confocal microscopy confirms the results from the high con-tent imaging system demonstrating enhanced effects of thePG-S3-001 STAT3 inhibitor on tumor proliferation (Fig. 5F andG). At days 5 and 11 after treatment, confocal images wereacquired to confirm the presence of both cell types in the spheres

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Figure 5.Inhibition of STAT3 by PG-S3-001 inhibits cell growth in 3D culture systems using proliferation-based assays and confocal microscopy. A–C, proliferation assays forlead inhibitor (C) in comparison with parent compounds (A and B) following 12 days in 3D culture. Low passage patient-derived spheres, Pa03C (gray bars)or the coculture spheres (black bars, 1:4 tumor:CAFs)were quantitated usingAlamar blue assay, average�SE, n¼ 3–4; �� ,P<0.01; #,P<0.001 comparedwithDMSOvehicle control (Veh). Quantitation of the images acquired by ArrayScan automated imaging system for the area (D) and the intensity (E) of the red channeland the green channel corresponding to the tumor and the CAFs, respectively. Fold change refers to the comparison of each data point to the fluorescence(RLU)/area of the untreated tumor alone culture. Average � SE, n ¼ 4; � , P < 0.05; �� , P < 0.01. Three-dimensional reconstruction of imagesacquired on day 5 (F) and day 11 (G) following treatment on day 3 and 7 of 3D cocultures with PG-S3-001.






as well as the effects of PG-S3-001 on both cell types (Fig. 5F andG). At day 11, transmitted light images were acquired simulta-neously with confocal images. Both CAFs (green signal) andtumor cells (red signal) decreased following treatment withPG-S3-001, but tumor cell intensity was more drastically reducedat day 5. Again, the confocal microscopy data confirmed that thetumor cells appeared to be more sensitive to the inhibition ofSTAT3 than theCAFs as EGFPfluorescence persists.On thebasis ofrecent data, which suggests that depletion of the stroma in PDACis not beneficial to the patient and can accelerate the disease (11,12), it is important to understand the effects of p-STAT3 inhibi-tion in both cell types when they are cocultured and interacting.When analyzed in 3D coculture, encouragingly, based on EGFPfluorescent readout, the CAF cells are still present followingtreatment with STAT3 inhibitors (Fig. 5D–G), suggesting a prom-ising selectivity profile. As PG-S3-001 effectively inhibited p-STAT3 activity and blocked proliferation in traditional cell culture

models, as well as in 3D models, PG-S3-001 was next assessed inan in vivo model.

3D coculture assay predicts the in vivo efficacy of a potent STAT3inhibitor on pancreatic cancer xenografts

A patient-derived xenograft (PDX) model was utilized to dem-onstrate that STAT3 inhibitor, PG-S3-001 was effective as a singleagent. After tumors from patient-derived cells, Pa03C reached anaverage of 71 � 4 mm3, treatment with PG-S3-001 was initiated(10 mg/kg i.p.). PG-S3-001 was not overly toxic to the animals asmeasured by weight loss (Fig. 6A). In contrast to the vehicle-control tumors, patient-derived xenografts demonstrated a sig-nificant tumor growth delay (54%–62 %) after treatment withPG-S3-001 for 15 days (Fig. 6B). To further confirm the mecha-nismof actionof PG-S3-001, IHCwas conducted to quantitate thelevels of activated STAT3 (p-Y705) and found a 45% reduction inthe levels of activated STAT3 following treatment with PG-S3-001

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Figure 6.Significant growth delay on pancreatic cancer xenografts with lead STAT3 inhibitor. NSG mice were implanted with patient-derived cells Pa03C and treatedwith 10 mg/kg PG-S3-001 for 15 days. There was significant growth delay in the tumors by volume (B) and weight (D) with acceptable body weight loss (A);� , P < 0.05; �� , P < 0.01; #, P < 0.001, n ¼ 18 for vehicle control (VEH)-treated tumors and n ¼ 16 for PG-S3-001. Levels of p-STAT3 (Y705, C) and p-Histone H3(E, P < 0.05) were also reduced in the tumors following treatment with STAT3 inhibitor.

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(Fig. 6C). The tumor growth delay also resulted in a significantlysmaller tumor weight. Tumor weight was 36% lower in PG-S3-001–treated mice compared with vehicle-treated mice whenweighed at the conclusion of the study (Fig. 6D). The antitumoreffect is at least partially due to a reduction in cell proliferation aswe observed a significant decrease in phospho-histone H3, amarker of mitotic cells (63) in tumors treated with PG-S3-001.The preclinical results suggested that targeting STAT3 in PDACmight have clinical efficacy and further confirmed that the 3Dcoculture model is predictive of in vivo efficacy.

DiscussionNovel models were used to characterize a new class of STAT3

inhibitors, including low passage patient-derived cells and 3Dcoculture systems to address the role of the TME. Three-dimen-sional tumor–stromal cell–associated spheroid models in Fig. 5include both tumor and CAFs to better model PDAC in vitro. Thishas several advantages over traditionalmonolayer cell culture (51,52, 60, 61). Monolayers are grown on stiff polystyrene plastic,while 3D culture systems are grown as floating tumor spheroidsallowing for diffusion of nutrients similar to human tumors (54).In addition, spheroids provide a more accurate portrayal oftumors in vivo due to the altered proliferation and morphologyin 3D and the increased length of time for culturing whencompared with the traditional monolayer techniques. CAF cellsfrom the TME were included to recapitulate a more accuratepicture of cell–cell interactions in vivo (Fig. 5). The sensitivity ofCAF cells to STAT3 inhibition was similar to tumor cells inmonolayer; however, in 3D coculture, we observed promisingselectivity for inhibition of tumor cell proliferation that againspeaks to the relevance of the in vitro 3D model.

In summary, we have reported the use of a low micromolar p-STAT3 inhibitor to selectively target patient-derived pancreaticcancer cells in the presence of CAFs, and provided preliminarypreclinical evidence to suggest that p-STAT3 inhibition mightafford an effective therapy for pancreatic cancer patients. Whilemore potent and selective p-STAT3 inhibitors are required torealize this goal, the described 3D PDAC tumor model providesamore realisticmodel of the disease for screening potential STAT3inhibitors. For example, in coculture with CAFs, compoundactivity, relative to that observed inmonolayer PDAC cell culture,drops significantly. Thus, inhibitors that can selectively target 3DPDAC tumor cells in stroma will likely have better efficacy inpreclinical models. As demonstrated, this 3D model approachwill serve as a most useful screening tool as we seek to discovernew and more effective regimens for treating pancreatic cancer.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: C.C. Arpin, S. Mac, B.D.G. Page, S. Haftchenary,H. Su, P.T. Gunning, M.L. FishelDevelopment of methodology: C.C. Arpin, S. Mac, Y. Jiang, S. Haftchenary,H. Su, G.E. Sandusky, P.T. Gunning, M.L. FishelAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): C.C. Arpin, H. Cheng, M. Grimard, M.M. Kamocka,H. Su, R.F. Gomez-Biagi, M.L. FishelAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): C.C. Arpin, Y. Jiang, H. Cheng, M. Grimard, H. Su,D.P. Ball, D.A. Rosa, R.F. Gomez-Biagi, A.M. Ali, K.C. McElyea, G.E. Sandusky,P.T. Gunning, M.L. FishelWriting, review, and/or revision of the manuscript: C.C. Arpin, H. Su,D.P. Ball, D.A. Rosa, P.-S. Lai, R.F. Gomez-Biagi, A.M. Ali, R. Rana,G.E. Sandusky, P.T. Gunning, M.L. FishelAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): C.C. Arpin, S. Mac, Y. Jiang, H. Su, D.A. Rosa,R.F. Gomez-Biagi, A.M. Ali, M.L. FishelStudy supervision: C.C. Arpin, P.T. Gunning, M.L. FishelOther (chemical synthesis): B.D.G. PageOther (contributed toward synthesis of intermediates, designed molecularmodeling protocol, carried out computational studies and analysis; writingsections of the manuscript involving the computational studies): R. RanaOther (provided unique novel research material and technical advice):H. HanenbergOther (SPR-based data of the inhibitors): K. Kerman

AcknowledgmentsThe authors thankDrs.Murray Korc and JesseGore,Department ofMedicine,

for help with establishing the 3D culture assay.

Grant SupportThis work was supported by grants from the NIH, NCI CA167291 (to M.L.

Fishel) with additional support from the IUSCC ITRAC basic science pilotfunding mechanism, Indiana Clinical Translational Science Institute, ProjectDevelopment Teamwithin the ICTSI NIH/NCRRGrant Number UL1TR001108(to M.L. Fishel). M.L. Fishel supported in part by Jeff Gordon Children'sFoundation. P.T. Gunning was supported by CIHR, Leukemia and LymphomaSociety, Canada Research Chair Program, Alberta Innovates, and CanadianFoundation for Innovation. A MITACS post-doctoral fellowship supported R.F.Gomez-Biagi.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received January 7, 2015; revised January 4, 2016; accepted January 29, 2016;published OnlineFirst February 12, 2016.

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2016;15:794-805. Published OnlineFirst February 12, 2016.Mol Cancer Ther Carolyn C. Arpin, Stephen Mac, Yanlin Jiang, et al. TherapeuticsTranscription-3 (STAT3) Protein Inhibitors as Pancreatic Cancer Applying Small Molecule Signal Transducer and Activator of

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