Small Molecule Therapeutics

Potent Dual BET Bromodomain-KinaseInhibitors as Value-Added MultitargetedChemical Probes and Cancer TherapeuticsStuart W. Ember1, Que T. Lambert2, Norbert Berndt1, Steven Gunawan1,Muhammad Ayaz3, Marilena Tauro4, Jin-Yi Zhu1, Paula J. Cranfill1,Patricia Greninger4, Conor C. Lynch5, Cyril H. Benes4, Harshani R. Lawrence1,3,Gary W. Reuther2, Nicholas J. Lawrence1, and Ernst Sch€onbrunn1

Abstract

Synergistic action of kinase and BET bromodomain inhibitorsin cell killing has been reported for a variety of cancers. Using thechemical scaffold of the JAK2 inhibitor TG101348, we developedand characterized single agentswhich potently and simultaneous-ly inhibit BRD4 and a specific set of oncogenic tyrosine kinasesincluding JAK2, FLT3, RET, and ROS1. Lead compounds showedon-target inhibition in several blood cancer cell lines and werehighly efficacious at inhibiting the growth of hematopoieticprogenitor cells from patients with myeloproliferative neoplasm.

Screening across 931 cancer cell lines revealed differential growthinhibitory potential with highest activity against bone and bloodcancers and greatly enhanced activity over the single BET inhibitorJQ1. Gene drug sensitivity analyses and drug combination studiesindicate synergism of BRD4 and kinase inhibition as a plausiblereason for the superior potency in cell killing. Combined, ourfindings indicate promising potential of these agents as novelchemical probes and cancer therapeutics. Mol Cancer Ther; 16(6);1054–67. �2017 AACR.

IntroductionBromodomains (BRD) are about 110 amino acid domains that

bind to and "read" acetylated lysine (KAc) residues of histonestails in a process critical for chromatin organization and genetranscription (1). BRDs regulate transcription, chromatin remo-deling, gene splicing, protein scaffolding and signal transductionand, therefore, play fundamental roles in cell proliferation anddivision.Members of the bromodomain and extra terminal (BET)protein family (BRD2, BRD3, BRD4, and BRDT) have beenimplicated in a number of disease pathways and are thereforeconsidered promising drug targets (2). The BET protein BRD4facilitates transcriptional elongation via recruitment of the pos-itive transcription elongation factor (P-TEFb) anddisplacementofnegative regulators such as HEXIM1 and 7SK snRNA (3). BRD4 is

overexpressed in various cancers and can undergo translocationsthat are a hallmark of the lethal tumor NUT midline carcinoma(4). Chemical inhibition of BRD4 exerts a broad spectrum ofdesirable biologic effects such as anticancer and anti-inflamma-tory properties (5). All known BRD4 inhibitors target the KAcrecognition site, particularly through H-bonding interactionswith a conserved Asn residue. Importantly, BRD4 inhibitiondownregulates oncogenic MYC transcription factors in severalcancer cell lines. Intense efforts are underway to develop chem-ically diverse and highly potent BRD4 inhibitors as new cancertherapeutics (6). Currently, 8 different BRD4 inhibitors haveentered phase I trials for the treatment of liquid and solid tumors(www.clinicaltrials.gov).

We and others recently reported that diverse kinase inhibi-tors also inhibit the KAc-binding site of BRD4 (7–9). Two ofthe most potent compounds, BI2536 (primary target PLK1;IC50(BRD4) ¼ 25 nmol/L) and TG101209 (primary target JAK2,IC50(BRD4) ¼ 120 nmol/L) exerted cellular modes of actionconsistent with inhibition of BRD4, such as downregulation ofc-MYC in multiple myeloma and acute myeloid leukemia(AML) cells, accompanied by strong inhibition of cell prolif-eration. Combined, these findings provided compelling evi-dence that certain kinase inhibitor chemotypes could be furtherdeveloped to target specifically cancers that depend on BRD4functionality and aberrant kinase activity. The rationale todevelop dual BRD4-kinase inhibitors is relevant as single-activ-ity BRD4 and kinase inhibitors act synergistically in a variety ofcancers. JQ1 synergizes with rapamycin, an mTOR inhibitor, toinhibit proliferation and survival of human osteosarcoma cellsas well as to upregulate p21Cip1 and downregulate c-MYC(10). A quarter of AML cells undergo activating internal tandemduplication (ITD) mutations in FLT3. Combinations of JQ1

1Drug Discovery Department, Moffitt Cancer Center, Tampa, Florida. 2TumorBiology Department, Moffitt Cancer Center, Tampa, Florida. 3Chemical BiologyCore, Moffitt Cancer Center, Tampa, Florida. 4Massachusetts General HospitalCancer Center, Harvard Medical School, Boston, Massachusetts. 5Department ofMolecular Oncology, Moffitt Cancer Center, Tampa, Florida.

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

Current address for S.W. Ember: Reaction Biology Corp., Malvern, PA 19355,USA.

Corresponding Authors: Ernst Sch€onbrunn, Moffitt Cancer Center, 12902 Mag-nolia Drive, Tampa, FL 33612. Phone: 813-745-4703; Fax: 813-745-6748; E-mail:ernst.schonbrunn@moffitt.org; and Nicholas J. Lawrence,nicholas.lawrence@moffitt.org

doi: 10.1158/1535-7163.MCT-16-0568-T

�2017 American Association for Cancer Research.

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and the FLT3 inhibitors quizartinib and ponatinib are syner-gistically lethal in AML cells driven by FLT3-ITD (11). Inmetastatic breast cancer, mutations of c-MYC and members ofthe PI3K signaling pathway are often concurrent. In such cells,inhibition of either BET proteins or PI3K is ineffective. How-ever, when combined, MS417 (an analogue of JQ1) and thePI3K inhibitor GDC-0941 induce cell death and tumor regres-sion (12). BET inhibition in mantle cell lymphoma cellsdecreases CDK4/6 and Bruton tyrosine kinase (BTK) levels,and co-treatment with JQ1 and the BTK inhibitor ibrutinib orthe CDK4/6 inhibitor palbociclib synergistically induces apo-ptosis (13). Combinatorial treatment of osteosarcoma cellswith JQ1 and CDK inhibitors induces potent synergistic activity(14). Significant molecular synergism between BET inhibitorsand the TK inhibitor lapatinib occurs in ERBB2þ breast cancercells, indicating that targeting broad-acting epigenetic regula-tors is needed to suppress the induction of gene expressionfollowing adaptive kinome response (15). In acute T-cell lym-phoblastic leukemias, BRD2 inhibition by JQ1 showed strongsynergy with tyrosine kinase inhibitors in inducing apoptosis(16).

Here, we describe the development of potent dual BET-kinaseinhibitors using the dianilinopyrimidine scaffold of the JAK2/FLT3 inhibitor TG101348. Lead compounds selectively inhibitBET bromodomains and a set of tyrosine kinases including thecancer targets JAK2, FLT3, RET, and ROS1. Screening across 931cancer cell lines revealed potent growth-inhibitory potential overJQ1 and TG101348. Dual BRD4-kinase inhibitors were highlyeffective against cell lines and patient samples of JAK2-drivenmyeloproliferative neoplasm (MPN), and drug combinationstudies indicated synergistic lethality in cell lines with highsensitivity for these compounds. Combined, our findings indicatepromising potential of these first-in-class dual BRD4-kinase inhi-bitors as cancer therapeutics.

Materials and MethodsReagents and compounds for biochemical and crystallographic

experiments were purchased from Sigma-Aldrich, Selleck Chemi-cals, and Hampton Research unless otherwise indicated. BRD4-1was purified and crystallized as described previously (7). Thesynthesis of compounds 1–6 is described elsewhere (17). HumanMM1.S, MV-4-11, and SAOS2 cells were purchased from ATCCandwere passaged in the laboratory for fewer than 6months afterreceipt or resuscitation. Human myeloproliferative syndrome–derived UKE-1 cells, which express the constitutively active JAK2-V617F mutant enzyme, were kindly provided by Dr. Ross Levine(Memorial Sloan Kettering Cancer Center, New York, NY).Human HCC-78 cells were kindly provided by Dr. Uwe Rix(Moffitt Cancer Center, Tampa, FL). UKE-1 and HCC-78 cellswere authenticated by short tandem repeat (STR) DNA typing intheMoffitt Genomics core.MM1.S,UKE-1, andHCC-78 cellsweremaintained in RPMI-1640 medium (Life Technologies), andMV4-11 cells were maintained in IMDMmedia (Lonza) contain-ing 10% FBS (Atlanta Biologicals). SAOS2 cells were maintainedin McCoy 5A containing 15% FBS. All cells were grown andmaintained at 37�C in a humidified atmosphere containing5% CO2. The following antibodies were purchased from CellSignaling: phospho-STAT3 (Y705) (#9145), STAT3 (#9139), phos-pho-FLT3 (Y591) (#3466), FLT3 (#3462), cMYC (#5605),p21Cip1 (#2946), cleaved PARP (#5625), and cleaved caspase-

3 (#9661). Vinculin antibody was purchased from Sigma-Aldrich(#V9131). Peroxidase-conjugated secondary antibodies were pur-chased from Jackson ImmunoResearch.

Differential scanning fluorimetryThe binding potential of compounds against BRD4-1 was

assessed by differential scanning fluorimetry (DSF) using a Ste-pOnePlus Real-Time PCR system (Applied Biosystems). PurifiedBRD4-1 [4 mmol/L final concentration; 10 mmol/L HEPES(pH7.5), 100 mmol/L NaCl, and 1 mmol/L DTT] was assayed,in quadruplicates, in a 96-well plate. Inhibitors were added to afinal concentration of 100 mmol/L and 2% DMSO. Protein Ther-mal Shift Dye (1:8000; Applied Biosystems) was used as thefluorescent probe, and fluorescence was measured using the ROXReporter channel (620 nm). Protein stability was investigated byprograming the thermocycler to increase the temperature from25�C to 99�C using 0.2�C increments and 10-second incubationsper increment. The inflectionpoint of the transition curve/meltingtemperature (Tm) was calculated using the Boltzmann equationwithin the Protein Thermal Shift Software (v.1.1) (Applied Bio-systems). (þ)-JQ1 (18) and dinaciclib (8) were used as controlsfor strong andweak binders of BRD4-1, respectively. The DTmwascalculated by using DMSO control wells as reference.

Protein crystallographyCrystals of BRD4-1 were grown in the presence of 1 mmol/L

ligand and 10% (v/v) DMSO from vapor diffusion hanging dropsusing reservoir as described previously (7), harvested in cryopro-tectant [reservoir containing 25% (v/v) ethylene glycol and 0.5mmol/L ligand], and flash-frozen in a stream of nitrogen gas. X-ray diffraction data were recorded at�180�C at the beamline 22-BM, SER-CAT, Advanced Photon Source, Argonne National Lab-oratories. Data were reduced and scaled with XDS (19); PHENIX(20) was employed for phasing and refinement, and modelbuilding was performed using Coot (21). All structures weresolved bymolecular replacement and the monomer of PDB entry4O7A (7) as the search model. Initial models for the small-molecule ligands were generated using MarvinSketch (Che-mAxon) with ligand restraints from eLBOW of the PHENIX suite.Figures were prepared using PyMOL (Schr€odinger, LLC).

Cell viability assaysHuman cells were seeded in 96-well plates at approximately

3,000 adherent or 20,000 suspension cells per well (0.1 mL).Adherent cells were allowed to attach overnight, and suspensioncells were incubated for 1 hour before dosing. Cells were incu-batedwith the compounds indicated in the figure legends rangingfrom 1 nmol/L to 10 mmol/L in the presence of vehicle (0.1%DMSO) for 72 hours, with 6 replicates per concentration. Afterdrug treatment, 15 mL of CellTiter Blue reagent (Promega) wasadded to each well, followed by vigorous orbital shaking for 5minutes and incubation for 3 hours at 37�C. Plates were placed ina Wallac EnVision 2103 Multilabel Reader (PerkinElmer), andfluorescence was determined using excitation and emission filtersof 570 and 615 nm, respectively. Growth inhibition data wereanalyzed with the Prism6 software (GraphPad).

Development of drug resistance was assessed by culturingUKE-1 cells (1 � 105/mL) in DMSO (0.1%) and inhibitors atconcentrations around the respective IC20 values. Cell cultureswere counted every 2 to 3 days at which point they were dilutedback to the starting cell density. If drug-treated cells grew at a rate

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of 90% or more of DMSO control, the drug concentration wasdoubled, and cells growing at a rate less than 90% of DMSOcontrol were maintained at their current drug concentration.

Dose–effect analysis of drug combinations was performedessentially as described (22). Growth inhibition data upon 72-hour drug treatment with single drugs and constant 1:1 ratios ofJQ1 and ruxolitinib, quizartinib, or TG101209 were determinedin parallel for each cell line and the data were fit to the Hillequation (Eq. A) to account for the differences inmaximum effectlevels (Max) reached for each inhibitor. Rearrangement of Eq. Ayields the dose (D) as a function of the fraction affected (fa; Eq. B).The combination index (CI) at a given fa value was calculatedusing Eq. C where (Dc)1 and (Dc)2 are the concentrations of eachdrug in the combination treatment and (Ds)1 and (Ds)2 are thoseof the single drugs.

fa ¼ Max

1þ ð DEC50

Þn ðAÞ

D ¼ Maxfa

� 1� �1

n

� EC50 ðBÞ

CI ¼ Dcð Þ1Dsð Þ1

þ Dcð Þ2Dsð Þ2

ðCÞ

A different dose–effect analysis was performed for a separatestudy inwhichUKE-1 cells treatedwithnon-constant ratios of JQ1and ruxolitinib were counted over a period of 6 days. Growth rateconstants (kobs) were determined for each drug treatmentusing Eq. D, where y0 is the number of cells at t ¼ 0 (2 � 105

cells/mL plated). Cell numbers were determined at days 2, 3, 4,and 6 by trypan blue exclusion from 2 separate experiments.

y ¼ y0 � ekobs�t ðDÞ

Fa values were calculated from ekobs values for each drug/combination relative to DMSO control. Dose–effect analysiswas performed using CompuSyn (http://www.combosyn.com/index.html).

MPN patient samplesPeripheral blood was obtained from JAK2-V617F–positive

MPN patients, consented through theMoffitt Cancer Center TotalCancer Care protocol (MCC14690/Liberty IRB #12.11.0023) andapproved by the Moffitt Cancer Center Scientific Review commit-tee. Blood was treated with HetaSep (STEMCELL Technologies,Inc.) to remove the majority of red blood cells. Peripheral bloodmononuclear cells (PBMC) were isolated by ficoll separation.PBMCs (1 � 105 to 4 � 105) were then plated in 1 mL ofmethylcellulose medium containing rhSCF, rhIL-3, and rhGM-CSF (MethoCult #H4534; STEMCELL Technologies, Inc.). Alldrug-treated samples contained 0.1% DMSO as the final concen-tration. Erythroid colonies were counted after 14 days.

Cancer cell line screeningCompounds 2, 3, and 6 were screened across 931 cell lines

using curve fitting and IC50 estimation essentially as described(23). All cell lines were sourced from commercial vendors. Cellswere grown in RPMI or DMEM/F12 medium supplemented with5% FBS and penicillin/streptavidin and maintained at 37�C in ahumidified atmosphere at 5% CO2. Cell lines were propagated in

these 2 media to minimize the potential effect of varying themedia on sensitivity to therapeutic compounds in our assay andto facilitate high-throughput screening. To exclude cross-contam-inated or synonymous lines, a panel of 92 SNPs was profiled foreach cell line (Sequenom) and a pairwise comparison scorecalculated. In addition, STR analysis (AmpFlSTR Identifiler,Applied Biosystems) was conducted and was matched to anexisting STR profile. More information on the cell lines screened,including their SNPandSTRprofiles, is available on theGenomicsofDrug Sensitivity in Cancer project website (www.cancerRxgene.org). Cells were seeded at variable density (�10%–25% conflu-ence) to ensure optimal proliferation during the assay. For adher-ent cell lines, after overnight incubation, cells were treated with 9concentrations of each compound (3-fold dilutions series) usingliquid handling robotics and then returned to the incubator forassay after 96 hours. For suspension cell lines, cells were treatedwith compound immediately following plating and were alsoreturned to the incubator for 96 hours. Cells were then stainedwith 55 mg/mL resazurin (Sigma) prepared in glutathione-freemedia for 4hours.Quantitationoffluorescent signal intensitywasperformed using a fluorescent plate reader at excitation andemission wavelengths of 535/595 nm. All screening plates weresubjected to stringent quality control measures (including coef-ficient of variations of untreated control wells <20%). Effects oncell viability were measured and a curve-fitting algorithm wasapplied to this raw dataset to derive a multiparameter descriptionof drug response, including the half maximal inhibitory concen-tration (IC50). Data for JQ1 (733 cell lines overlapping withcompound 3) and TG101348 (885 cell lines overlapping withcompound 3) were collected in separate experiments using thesame experimental conditions and procedures with the exceptionthat cells were exposed to drug for 72 hours. Gene mutation andexpression status of drug targets and biomarkers for the screenedcell lines were obtained from online databases of the GDSC(www.cancerrxgene.org), Cosmic (cancer.sanger.ac.uk/cosmic),and cBioPortal (www.cbioportal.org).

ImmunoblottingCells were seeded in 6-well plates (2 � 105 adherent or

1 � 106 suspension cells per well) and incubated for 6 hourswith increasing inhibitor concentrations. Cells were harvestedby centrifugation at 300 � g for 5 minutes and resuspended inCelLytic M Cell Lysis Reagent (Sigma-Aldrich) containing HaltProtease Inhibitor Cocktail and Halt Phosphatase InhibitorCocktail (Thermo Scientific) and 5 mmol/L EDTA at 4�C.Protein concentrations were determined with Bio-Rad ProteinAssay Reagent, and samples were diluted with 1:3 volume 4�SDS sample buffer and heated at 95�C for 5 minutes. Sampleswere subjected to 10% or 12.5% SDS-PAGE and transferredto polyvinylidene difuoride (PVDF) or nitrocellulose mem-branes. Western blot analyses were developed with the appro-priate pairs of primary and secondary antibodies, and signalswere visualized using HyGLO Chemiluminescent reagent(Denville Scientific).

Flow cytometryMM1.S cells were treated with 0.5 mmol/L compound or 0.1%

vehicle (DMSO) for 24 hours. Cells were harvested and spundownat 4�C,washedwith ice-cold PBS, andfixedon ice for at least30 minutes with 70% ethanol. Cells were washed again with ice-cold PBS, filtered with a cell strainer to achieve a single-cell

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suspension, and stained with 1 mg/mL DAPI (BD Biosciences#564907) at a cell density of 1 � 106 to 2 � 106 cells/mL for 1 to2 hours. Sample analysis was performed on a FACSCanto II (BDBiosciences) with DIVA 8 software, and histograms were gener-ated using FlowJo v9 cytometry analysis software (Tree Star, Inc.).

BRD inhibition/binding assays and profilingThe half maximal inhibitory concentration (IC50) of each

compound against BETs was determined by Reaction BiologyCorp. using a chemiluminescent alpha screen binding assay.Briefly, donor beads coatedwith streptavidin were incubatedwithbiotinylated histone H4 peptide (residues 1–21) containing KAc(K5/8/12/16Ac). In the absence of inhibitor, His-tagged BRDbinds to KAc-histone H4 peptide, thereby recruiting acceptorbeads coated with a nickel chelator. Binding potential is assessedby detecting light emission (520 to 620 nm) from acceptor beadsfollowing laser excitation (680 nm) of a photosensitizer withinthe donor beads which converts ambient oxygen to singlet oxy-gen. Binding potential for BRD4-1 and profiling across 32 humanbromodomains was performed by Discoverx Corp. The amountof BRD captured on an immobilized ligand in the presence orabsence of compound was measured using a quantitative real-time PCR (qPCR) method that detects the associated DNA labeltagged to the bromodomain. The results are reported as:

% of control¼ inhibitor signal�positive control signalnegative control signal DMSOð Þ� positive control signal

Profiling of compounds 3 and 5 was performed at a singleconcentration of 2 mmol/L.

Kinase activity assays and profilingInhibitory activity of compounds against JAK2, FLT3, RET,

ROS1, and other kinases was determined in dose response byReaction Biology Corp using a 33P-ATP radiolabeled assay(10 doses from 0.5 nmol/L to 10 mmol/L). ATP concentrationwas 10 mmol/L and staurosporine served as a positive control.Residual enzymatic activity (in % of DMSO controls) was deter-mined in duplicate. Profiling of compounds 3 and 5 against apanel of 365 kinases was performed by Reaction Biology at asingle concentration of 0.1 mmol/L in duplicate.

Accession codesAtomic coordinates and structure factors for complexes of

BRD4-1 with compounds 1–5 have been deposited in the ProteinData Bank (PDB) under accession codes 5F5Z, 5F60, 5F61, 5F62,and 5F63.

ResultsDesign and structure–activity relationship studies of dualBET-kinase inhibitors

BRDs and kinases are functionally and structurally unrela-ted, and the respective KAc- and ATP-binding sites are uniquelydifferent in architecture. TG101209, a close analogue ofTG101348 (fedratinib), inhibits JAK2 and the first bromo-domain of BRD4 (BRD4-1) with IC50 values of 0.5 and 130nmol/L, respectively (Table 1). The functional groups requiredfor binding to the hinge region of the ATP site in JAK2 (Fig. 1A)directly interact with the side chain of Asn140 in the KAc site ofBRD4-1 (7), a conserved residue among bromodomains whichis critical for the binding of acetylated proteins (Fig. 1B). Toincrease binding potential for BRD4 while maintaining highactivity against JAK2 and FLT3, we designed analogues ofTG101209 to explore the KAc site in several regions, of whichthe `WPF shelf' (W81, P82, F83), the `KL flank' (K91, L92), andthe `ZA-channel' (structurally conserved water molecules) areprominent features (Fig. 1C). For structure–activity relation-ship (SAR) studies, the binding potential of new compoundswas initially assessed by DSF using a standard curve of knownBRD4 inhibitors covering a range of IC50 values between 0.025and 25 mmol/L (7). Inhibitory activity was determined byalpha screen assay for BRD4 and other BETs, and a 33P-labeledassay was employed to determine activity against kinases.High-resolution co-crystal structures of compounds withhuman BRD4-1 were determined throughout to guide thedesign of high-affinity inhibitors (Supplementary Fig. S1 andSupplementary Table S1). The chemical synthesis of this seriesof compounds is described elsewhere (17). Here, we charac-terized a select set of compounds that emerged as promisingleads for the development of dual BET-kinase inhibitors ascancer drugs.

Table 1. Properties of dual BRD4-kinase inhibitors in comparison with single activity inhibitors

BRD4-1 binding/inhibition Kinase inhibition Cell growth inhibitionCompound DTm,a �C IC50,

b nmol/L Kd,c nmol/L IC50,

d nmol/L IC50,e mmol/L

ID DSF Alpha screen qPCR JAK2 FLT3 MM1.S UKE-1 SAOS2 HCC78

1 7.5 105 86 2.7 0.9 0.38 0.13 — —

2 9.6 27 43 11 10 0.16 0.35 — —

3 11.0 34 35 1.1 1.1 0.15 0.08 0.40 0.664 12.6 14 6.8 3.4 11 0.07 0.16 — —

5 12.5 21 12 12 32 0.08 0.16 0.34 0.796 5.1 260 — 31,000 1,700 1.5 1.8 — —

TG101209 6.8 130 — 0.51 1.5 1.4 0.32 1.5 1.9(þ)-JQ1 8.8 21 9.1 n.a. n.a. 0.10 0.21 1.5 2.6Ruxolitinib <1 n.a. — 2.8f — >10 0.17 >10 >10Quizartinib <1 n.a. — — 1.3g >10 9.5 >10 >10Abbreviation: n.a., no activity (>90% of DMSO control at 10 mmol/L concentration).aMean value of 3 experiments in quadruplicate.bPerformed by Reaction Biology (mean value of 2 to 3 experiments).cPerformed by DiscoveRx (mean value of single experiment in duplicate).dPerformed by Reaction Biology using 33P-labeled assay (single experiment).eCellTiter Blue assay (mean value of 2 experiments in hexaplicate) for cell lines studied in detail.fReference 50.gReference 26.

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Early into the SAR studies, we realized that reversal of thesulfonamide functionality in the 30-position of the A-ring(�SO2NH-R to �NHSO2-R) along with the introduction ofhalogen substituents in either aniline ring significantly increasedbinding potential for BRD4 (Table 1). Compound 1, an isomer ofTG101209, assumes an extended conformation in the KAc site,stabilized by H-bonding interactions of the sulfonamide groupwith Lys91 and Asp88 below the KL flank (Fig. 1D and E). Thebinding potential of compound 1 toward BRD4 and kinases JAK2and FLT3 was similar to TG101209. Introducing a fluorine in the30-position of the B-ring (compound 3) increased binding activityfor BRD4 4-fold (IC50¼ 34 nmol/L). As the binding partners andthe conformations of compounds 1 and 3 in the KAc site areidentical, the substantial increase in binding activity of compound3 is likely a result of the fluorine acting as an electron sink,rendering the �NH groups flanking the pyrimidine core moreacidic for H-bonding with Asn140 and Pro82. Similarly, intro-ducing a chlorine in the 40-position of the A-ring (compound 2)also resulted in a 4-fold increase in activity for BRD4 but renderedthe compound10-fold less active against JAK2 andFLT3 (Table 1).It appears that chlorine in 40-position of the A-ring favors adifferent inhibitor conformation in the KAc site, the tert-butylgroup now neatly positioned in a hydrophobic subsite above theWPF shelf (Fig. 1E). Notably, the chlorophenyl group of JQ1occupies the same subsite in BRD4, and superimposition of the

respective co-crystal structures revealed that compound 2 adopts aconformation mimicking the binding mode of JQ1 (Supplemen-tary Fig. S2). To further diversify the parent compound, severalother modifications were introduced, of which compounds 4 and5were among themost potent BRD4 inhibitors described to date,with IC50 values of 14 and 21 nmol/L, respectively, similar to JQ1(IC50¼ 21 nmol/L). As expected, the 40 Cl containing compounds4 and 5 were less active against kinases and assumed conforma-tions in the KAc site identical to compound 2 (Fig. 1E). Therefore,compounds2,4, and 5 can be considered equipotent inhibitors ofBRD4and JAK2/FLT3 (IC50 10–30nmol/L),whereas compound3is about 10-fold more potent against these kinases. Notably,compounds 2–5 were equally active against the first and secondbromodomains of BRD4 and showed only slightly reduced activ-ity against BRDT-1, whereas JQ1 was 7-fold less active againstBRDT-1 (Supplementary Table S2). Compound 6,which carries acyclohexyl moiety fused to the pyrimidine ring of compound 1(Supplementary Fig. S3), was about 10-fold less active againstBRD4 and greatly reduced kinase activity; it served as a negativecontrol in cellular assays.

Dual BRD-kinase inhibitors are selective for BETs and a definedset of kinases

To assess inhibitory potential against BRDs outside the BETfamily, compounds 3 and 5 were profiled against a panel of 32

Figure 1.

Structure-based design of dianilinopyrimidines yields highly potent BRD4 inhibitors. A, Diaminopyrimidine core of the parent compound TG101209 bindsto JAK2 through direct H-bonds with the main chain of Leu932 which is part of the hinge region (PDB: 4JI9). The hinge region is shown in orange, the gatekeeperresidue in red, other residues in pale green, and water molecules in cyan. B, In the KAc site of BRD4-1, TG101209 binds directly to the side chain of Asn140(magenta) and the main chain of Pro82 (PDB: 4O76). C, KAc site is a U-shaped cavity characterized by Asn140, 2 largely hydrophobic flanking regions andthe so-called ZA-channel composed of structurally conserved water molecules. D, H-bonding (black dotted lines) and van-der Waals (VDW) interactions (greendotted lines) of compound 3 in BRD4-1. E, Comparison of the binding modes of compounds 1–5 and TG101209 in BRD4-1. The orientation of the KAc site is thesame as in (C) and (D). The crystallographic data and refinement statistics are shown in Supplementary Table S1 and stereo presentations of the bindinginteractions and electron density maps in Supplementary Fig. S1.

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human BRDs (Fig. 2A). Both compounds showed high selectivityfor BETs (Family II). Outside the BET family, only compound 5showed weak binding potential for TAF1 (Family VII) and EP300and CREBBP (both Family III). Profiling against a panel of 365kinases confirmed that compound3displayed significantly higheractivity against the kinome than compound 5 (Fig. 2B). Theprimary targetswere tyrosine kinases (TK) particularly JAK2, FLT3,and RET with IC50 values between 0.9 and 1.1 nmol/L. Other TKssignificantly inhibited by compound 3 and causally implicated incancer (ref. 24; cancer.sanger.ac.uk/census)wereNTRK3 (IC50¼ 5nmol/L), ROS1 (IC50¼11nmol/L), PDGFRb(IC50¼16nmol/L),and FGFR1 (IC50 ¼ 43 nmol/L). The most significant differencesin inhibitory potential were noted for ULK3, ULK1, and ERN2/IRE2,whichwerepotently inhibitedby compound3but appearedto be insensitive towards compound 5. Statistical evaluation ofkinase selectivity using the Gini coefficient (25) yielded selectivityscores for compounds 3 and 5 similar to those of ruxolitinib,quizartinib, and fedratinib using published kinome profiling data(ref. 26; Supplementary Fig. S4).

Dual BRD4-kinase inhibitors exhibit on-target inhibition incell lines of hematologic malignancies

On-target inhibition of dual BRD4-kinase inhibitors wasassessed in the multiple myeloma and AML cell lines MM1.S andMV-4-11, both of which are highly sensitive to JQ1 accompaniedby downregulation of c-MYC levels (9, 27). Growth of MM1.S

cells was potently inhibited by compounds 1–5 with IC50 valuesbetween 0.07 and 0.38 mmol/L (Table 1, Fig. 3A). Growth-inhib-itory activity of the most potent BRD4 inhibitors 4 and 5 wassimilar to that of JQ1 (IC50 ¼ 0.1 mmol/L) and greatly improvedover JAK2 inhibitor ruxolitinib (IC50 > 10 mmol/L) or FLT3inhibitor quizartinib (IC50 > 10 mmol/L), both of which lackbinding potential for BRD4. Inhibition of BRD4 by JQ1 has beenreported to induce G1 arrest in MM1.S cells (27). Flow cytometricanalysis of cells treatedwith dual BRD4-kinase inhibitors revealeda substantial increase of cells in G1 arrest similar to JQ1 (Fig. 3B;Supplementary Fig. S5). Single kinase inhibitors ruxolitinib andquizartinib only slightly affected MM1.S cell cycle, whereas theparent compound TG101209 showed amoderate increase of cellsin G1. It appears that the growth inhibition and the ability toinduceG1 arrest are directly correlated. Compounds1–5 showedadose-dependent reduction of c-MYC and p-STAT3 levels, reflect-ing the concomitant inhibition of BRD4 and JAK2, respectively(Fig. 3C). Notably, p-STAT3 levels were detectable only uponstimulation with human IL6, which did not alter STAT3 or c-MYClevels (Supplementary Fig. S6). The pSTAT3 and c-MYC reductionpotential of compounds was in general agreement with theinhibitory activities against JAK2 and BRD4, the weaker kinaseinhibitors 2 and 5 being less effective at reducing pSTAT3 levelsthan compounds 1 and 3. MYC levels were affected strongest bycompounds 3 and 5, slightly improved over JQ1, compound 3being most active in attenuating both c-MYC and p-STAT3 levels.

Figure 2.

Lead compounds 3 and 5 are highlyselective for BET BRDs and a subset ofkinases. A, Compounds were screenedagainst 32 human BRDs at a singleconcentration of 2 mmol/L. Bindingactivity is expressed as a percentage ofthe positive control, with small valuesindicating higher binding affinity (largercircles). Shown is an artisticrepresentation of the human BRDphylogenetic tree highlighting thepotency and selectivity against BETBRDs (Family II). Other BRDs weaklyinhibited by compound 5 were TAF1-2(Family VII) and CREBBP and EP300(Family III). B, Compounds werescreened against 365 kinases at a singleconcentration of 100 nmol/L, andinhibitory activity is shown among thekinase family tree from blue (lowactivity) to red (high activity).Experimental values for BRD and kinaseinhibition are listed in SupplementaryTables S3 and S4.

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As expected, control compound 6 showed only moderate c-MYCreduction potential without affecting p-STAT3 levels, and ruxo-litinib and JQ1 exclusively reduced p-STAT3 or c-MYC levels,respectively. Similar results were obtained for the FLT3-driven cellline MV-4-11 (28) which upon treatment with compounds 1–5showed dose-dependent reduction of p-FLT3 and c-MYC levels(Supplementary Fig. S7). Compounds were also probed for theirability to affect levels of p21Cip1, previously reported to increasein certain cell lines upon treatment with JQ1 (10). In MM1.S,upregulation of p21Cip1 and downregulation of c-MYC levelscaused byBRD4 inhibitionwere time-dependent (SupplementaryFig. S8). Increase in p21Cip1 levels was paralleled by a decrease ofc-MYC levels for compounds 1–6, compounds 3 and 5 showingactivities similar to JQ1 (Fig. 3C). Onset of apoptosis, assessed byincreased levels of c-PARP, was pronounced for dual BRD4-kinaseinhibitors, whereas the single activity inhibitors JQ1, ruxolitiniband compound 6, were ineffective.

Dual BET-kinase inhibitors are efficacious against JAK2-drivencell lines and the neoplastic growth of hematopoieticprogenitor cells from MPN patients

We investigated the effect of compounds on the JAK2-drivenUKE-1 cell line which is a model for MPNs (29). Aberrant JAKactivation is a fundamental feature of many hematologic malig-nancies, including pediatric and Down syndrome–associated

precursor B-cell acute lymphoblastic leukemia (B-ALL; ref. 30),Hodgkin lymphoma (31), and Philadelphia chromosome–neg-ative MPNs, including polycythemia vera, essential thrombocy-tosis, and primary myelofibrosis (32). Chromosomal transloca-tions or somatic alteration (e.g., V617F) result in overactivation ofthe STAT proteins and other JAK2 effector pathways and is thereason behind the high transforming potential associated withconstitutively active JAK2 (33). UKE-1 cells were sensitive forruxolitinib (IC50 ¼ 0.17 mmol/L) and JQ1 (IC50 ¼ 0.21 mmol/L),but insensitive to quizartinib (IC50 ¼ 9.5 mmol/L; Fig. 4A).Parent compound TG101209 showed good activity (IC50 ¼0.32 mmol/L), whereas most dual activity inhibitors showedincreased activity, compound 3 being the most active (IC50 ¼0.08 mmol/L). Growth of the erythroleukemia cell line HEL and aJAK2-V617F–transformed BaF3 cell line was inhibited by com-pound 3 with IC50 values of 0.37 and 0.04 mmol/L, respectively,essentially identical to ruxolitinib (Supplementary Fig. S9). DualBRD4-kinase inhibitors reduced phospho-STAT3 and c-MYClevels similar to those observed in MM1.S cells, whereas JQ1 andruxolitinib exclusively reduced c-MYC and pSTAT3 levels, respec-tively (Fig. 4B).

To assess the ability of MPN cells to develop resistance towardsingle and dual activity inhibitors, UKE-1 cells were exposed todrug over a period of 2months, starting with drug concentrationsequaling the respective IC20 values (seeMaterials andMethods for

pSTAT3 (Y705)

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Dual BRD4-kinase inhibitors are efficacious at inhibiting the growth of MM1.S cells consistent with the simultaneous inhibition of BRD4 and kinases. A, Cellviability after 72-hour drug exposure. B, Cell-cycle distribution determined by flow cytometry after 24-hour exposure to 0.5 mmol/L drug. Nocodazole (50 ng/mL)was used as a positive control for G2–M arrest (original data shown in Supplementary Fig. S5). Averages of diploid and tetraploid cells were calculated.C, Cells were exposed to drug for 6 hours, and lysates were subjected to immunoblotting for biomarkers of BRD4 inhibition (c-MYC and p21Cip1), JAK2inhibition (pSTAT3), and onset of apoptosis (cPARP). Vinculin served as a loading control.

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Figure 4.

Dual BET-kinase inhibitors are efficacious at inhibiting the growth of JAK2-driven cell lines and erythroid colonies of MPN patient samples. A, UKE-1 cell viabilityupon treatment with drugs for 72 hours determined by CellTiter Blue assay. B, Dose-dependent reduction of phospho-STAT3 and c-MYC levels in UKE-1 cellsas a result of JAK2- and BRD4 inhibition upon 6-hour drug exposure detected by immunoblotting. C, Upon prolonged drug exposure, UKE-1 cells developedresistance for TG101209 and ruxolitinib but not for compound 3. Cells were cultured at inhibitor concentrations around the respective IC20 values andinhibitory activity is expressed as fold increase of IC20 (see Materials and Methods for details). D, UKE1-R cells, which are resistant to ruxolitinib, were culturedin 1 mmol/L of the indicated drugs alone or in combination. Cell counts were determined by trypan blue exclusion. E, Growth rate constants (kobs) weredetermined for UKE-1 cells treated with ruxolitinib, JQ1 and non-constant combinations thereof over a period of 6 days (n ¼ 2). Insert, Isobologram from thecorresponding drug–effect analysis using CompuSyn. Points below the diagonal indicate synergistic action of the 2 drugs. Original data and correspondingdata reduction are shown in Supplementary Fig. S10. F, Inhibition of erythroid colony formation of cells from JAK2-V617F–positive MPN patients. PMCs wereplated in methylcellulose, containing cytokines but no erythropoietin, with the indicated concentrations of drug.

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details). While cells readily developed resistance for TG101209and ruxolitinib, they remained highly sensitive to compound 3(Fig. 4C), suggesting that the concomitant inhibition of BRD4andJAK2 could not be escaped. A similar observation was previouslyreported for JAK2 inhibitor–resistantHEL cells that didnot readilydevelop resistance to BRD4 inhibitors (34). Selection of JAK2-V617F–driven cells in escalating doses of JAK2 inhibitors leads tocells that are cross-resistant to type I JAK2 kinase inhibitors (35).Such cells do not contain JAK2 mutations that induce permanentdrug resistance (a phenomenon that mimics clinical observa-tions), as cells can become resensitized to the drug after culturingin the absence of drug (35). Remarkably, UKE-1 cells growingpersistently in 1 mmol/L ruxolitinib (UKE-R cells) and that arecross-resistant toother JAK2 inhibitors (35, 36) continued to growin the presence of single inhibitors but could not proliferate whensingle activity kinase inhibitors were combined with JQ1. How-ever, compound 3 alone completely suppressed UKE-R cellgrowth (Fig. 4D). To determine whether UKE-1 cells were moresensitive to the combination of JAK2 and BRD4 inhibition, cellgrowth was monitored over 6 days with JQ1 or ruxolitinib aloneand in different combination ratios (Fig. 4E; Supplementary Fig.S10). While cells treated with single inhibitors continued toproliferate, albeit at a slower rate, the drug combinationscompletely suppressed cell growth. Dose–effect analysis of thegrowth rate constants according toChou (22) indicates synergisticactivity of ruxolitinib and JQ1 in these cells. Finally, we assessedthe efficacy of dual BRD4-JAK2 inhibitors against erythroid col-ony formation in primary cells from JAK2-V617F–positive MPNpatients. A characteristic feature of primary myeloid progenitorcells of MPN patients is their ability to form erythropoietin-independent erythroid colonies in methylcellulose, which iswidely used to test MPN therapeutics. Compound 3 was highlyeffective against colony formation in 2 patient samples withIC50 values < 50 nmol/L, superior to ruxolitinib and TG101209(Fig. 4F). This was particularly evident for one patient samplethat showed reduced sensitivity to ruxolitinib. Taken together,the data on JAK2-driven cell lines and ex-vivo patient samplesindicate that dual BRD4-kinase inhibitors exhibit promisingactivity against MPNs.

Dual BET-kinase inhibitors display differential activity acrosscancer cell lineages

Compounds 2, 3, and 6 were screened for growth-inhibitoryactivity across a large cell line panel comprising 931 liquid andsolid tumor cell lines (Fig. 5). Compound 3 (mean IC50 ¼ 0.51mmol/L) was significantly more potent than compound 2 (meanIC50 ¼ 1.2 mmol/L), indicating that its higher kinase activitycontributes to increased cell kill potential (Fig. 5A). The 2 datasetsshowed high correlation with Pearson coefficient of 0.86, reflect-ing the same mechanism of action of these 2 compounds (Fig.5E). As expected, compound 6was substantially less active due toits reduced binding potential for BRD4 and kinases (mean IC50¼56 mmol/L). Comparisonwith cell line screening data for JQ1 andTG101348, separately collected under similar experimental con-ditions, revealed significantly weaker growth-inhibitory activitywith mean IC50 values of 10 mmol/L for both compounds against733 and 855 overlapping cell lines, respectively. The Pearsoncoefficients of the JQ1 and TG101348 datasets with that ofcompound 3 were 0.46 and 0.47, respectively (Fig. 5F and G).By tissue type, the cell lines most sensitive to compounds 2 and 3were bone and blood (mean IC50 ¼ 0.18 and 0.25 mmol/L,

respectively), and the least sensitivewere skin andpancreas (meanIC50 ¼ 0.88 and 1.1 mmol/L, respectively; Fig. 5B). The distribu-tion of cell line sensitivity grouped by tissue type and relative tothe respective overall IC50 values was similar for compound 3 andTG101348 but differed slightly from JQ1 (Fig. 5C and D; Sup-plementary Fig. S11). JQ1 showed increased activity for breast andnervous system cell lines but was significantly less active againstaerodigestive tract, bone, and lung cancer.

To identify potential gene–drug sensitivity correlations, theresources and databases of www.cancerrxgene.org, cancer.san-ger.ac.uk/cosmic, and www.cbioportal.org were used to assignthe mutational and overexpression/amplification status of rele-vant target proteins in the cell lines tested. Enhanced sensitivity fordual BRD4-kinase inhibitors was statistically significant in celllines overexpressingwild-type ormutant FLT3, RET, PDGFRb, andFGFR1 (Fig. 6A). Altered c-MYC status did not affect drug sensi-tivity, but cell lines overexpressing BRD4 were significantly moresensitive. Among blood cancer cell lines, those overexpressingFLT3 showed significantly increased sensitivity for the mostpotent FLT3 inhibitors, compound 3 and TG101348, exclusively(Fig. 6B). MYCN is an established target of BET inhibition inneuroblastoma (4), and consequently cell lines with alteredMYCN status showed significantly increased sensitivity only forthe most potent BRD4 inhibitors, compounds 2, 3, and JQ1 (Fig.6C). Unexpectedly, bone cancer cell lines harboring the EWS-FLI1fusion protein characteristic of Ewing sarcoma were significantlymore sensitive for dual BRD4-kinase inhibitors than for JQ1 (Fig.6D), suggesting the involvement of a kinase in cell killing.

Cell growth–inhibitory activity of dual BRD4-kinase inhibitorsis similar to that of drug combinations

The synergistic lethality of JQ1 and ruxolitinib in UKE-1 cells(Fig. 4E) and the increased growth-inhibitory activity of com-pounds 2 and 3 over that of JQ1 (Fig. 5A) suggested positivecombination effects of BRD4 and kinase inhibition in certain celllines. We therefore performed drug combination studies of JQ1with the JAK2/FLT3 inhibitors ruxolitinib, quizartinib, andTG101209 in cell lines with different sensitivity for JQ1. MM1.S and UKE-1 cells as well as the lung cancer cell line HCC-78 andthe bone cancer cell line SAOS-2 were exposed to drugs alone and1:1 combinations with JQ1 for 72 hours (Fig. 7). HCC-78 waschosenbecause of its dependency onROS1 (37),which is stronglyinhibitedby compound3in vitro (IC50¼11nmol/L). SAOS-2 cellswere included because they overexpress several kinases potential-ly targeted by compound 3 including FLT3 (T382S), ROS1, andNTRK3. Ruxolitinib alone was effective only in UKE-1 cells, andcombinations of JQ1 with ruxolitinib, TG101209, or quizartinibwere synergistic (Fig. 7A), corroborating the results of Fig. 4E. ForMM1.S cells, the effect of JQ1 kinase inhibitor combinations wasadditive (Fig. 7B). BothHCC-78 and SAOS-2 cells showed limitedsensitivity for JQ1, as the maximum fraction affected was onlyapproximately 0.5 (Fig. 7C andD).However, both TG101209 andquizartinib showed strong synergism with JQ1 in these cells. Forthe cell lines tested here, the dose–response properties of com-pound 3 were similar to that of JQ1 in combination withTG101209, resulting in a complete suppression of cell viability.

DiscussionSeveral chemical warheads have been reported as acetyl-lysine

mimetics for the development of BRD inhibitors (5). Following

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Dual BET-kinase inhibitors show potent and differential growth inhibitory activity across cancer cell lineages. A, Compounds 2 and 3 were screened against931 cell lines, and the results were compared with separately obtained data for JQ1 and TG101348 using 733 and 885 overlapping cell lines, respectively. Eachcircle represents a single cell line, and the red bars represent the geometric mean along with the 95% confidence interval level. IC50 values are listed inSupplementary Table S5. B–D, Activity distribution of compound 3, JQ1, and TG101348 by tissue type. E–G, Pearson correlation analysis of the inhibitoryactivities of compound 2, JQ1, and TG101348 as a function of compound 3.

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thediscovery that certain kinase inhibitors also inhibit theKAc siteof BRD4 (7, 9), we focused our attention on the optimization ofthe dianilinopyrimidine-based JAK2 inhibitors fedratinib andTG101209, which are moderately active against BRD4. Dianili-nopyrimidine is a privileged scaffold for kinase inhibitor design,and several such drugs are in clinical trials including the ALKinhibitors ceritinib (38) and brigatinib (AP26113; ref. 39), theBTK inhibitor spebrutinib (AVL-292; ref. 40), the EGFR inhibitorrociletinib (41), and the Syk inhibitor fostamatinib (42).Our datademonstrate that the dianilinopyrimidine scaffold is also suitedto potently and selectively inhibit BET proteins (Figs. 1, 2).Current lead compounds have been designed as near equipotentinhibitors of BRD4 and a set of cancer-relevant tyrosine kinasesincluding JAK2, FLT3, RET, andROS1. These compounds potentlyinhibited the growth of diverse cancer cell lines particularly those

of blood, bone, nervous system, and lung cancer. The increasedgrowth-inhibitory activity of dual BRD4-kinase inhibitors overJQ1 suggested synergistic lethality caused by the concomitantinhibition of BRD4 and kinase(s) in certain cell lines. Drugcombination studies revealed synergism of BRD4 and JAK2 inhi-bition inUKE-1MPN cells (Figs. 4E and 7A), a finding in linewiththe recently reported synergistic lethality of BRD4 and JAK2inhibitors against cultured and patient-derived secondary AMLblast progenitor cells (43). Synergismof JQ1 andkinase inhibitorsin cell killing was also observed in solid tumor cell lines that weresensitive to our inhibitors (Fig. 7C andD). These findings indicatethe potential of dual BRD4-kinase inhibitors against cancer cellsthat depend on aberrant TK activity and BRD4 functionality.

Despite strong initial responses, cancer cells frequently developresistance to kinase inhibitors by acquiring active site mutations

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Figure 6.

Dual BET-kinase inhibitors exhibit characteristic gene–drug sensitivity correlations. A, Databases (www.cancerrxgene.org, cancer.sanger.ac.uk/cosmic, andwww.cbioportal.org) were analyzed for cell lines that overexpress wild-type or mutated genes of interest. For potential target kinases, cell lines with altered FLT3,RET, PDGFRb, or FGFR1 showed significantly increased sensitivity for compound 3 whereas those with altered JAK2, NTRK3, and ROS1 did not. Cell lines withaltered BRD4 status were significantly more sensitive whereas those with altered c-MYC were not. B, Blood cancer cell lines with altered FLT3 status weresignificantly more sensitive for drugs with highest kinase activity, that is, compound 3 and TG101348. C, Cell lines with altered MYCN status were significantlymore sensitive for drugs with highest BRD4 activity, that is, compounds 2, 3, and JQ1.D, Bone cancer cell lines that harbor the EWS-FLI1 oncogene were significantlymore sensitive for dual BRD4-kinase inhibitors than JQ1. In all instances, compound 3was significantly more potent than the other drugs. Statistical significance wasevaluated by the Student t test (2-tailed distribution) as indicated (� , P � 0.05; �� , P � 0.01; ��� , P � 0.001; ���� , P � 0.0001; ns, P > 0.05).

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or exploiting the intrinsic redundancy of kinase signaling path-ways (44). This shift to alternative kinase signaling nodes occursthrough a process termed "adaptive kinome reprogramming"resulting in transcriptional upregulation and activation of com-pensatory kinases and their adaptor proteins. The failure of single-agent targeted therapy to prevent adaptive kinome reprogram-ming is seen in almost all cancer types, including in those driven

by chromosomal translocations or somatic mutations. For exam-ple, gain-of-function somatic and internal tandem duplicationmutations of FLT3 are the most common denominators in AML(28). Although FLT3 inhibitors can induce short-term remissionin clinical trials, the onset of resistant clones remains a significantchallenge (45). Similarly, although JAK2 inhibitor therapy ofMPNs can reduce disease burden, it does not reverse the disease

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Figure 7.

Growth inhibition by dual BRD4-kinase inhibitors is similar to that of drug combinations. Shown are drug combination studies using the cell lines (A) UKE-1,(B) MM1.S, (C) HCC78, and (D) SAOS2 upon 72-hour drug treatment. The effect of single drug was determined in parallel with constant 1:1 combinations of JQ1and TG101209, ruxolitinib, or quizartinib. Left, Growth inhibition relative to DMSO as a function of dose. To account for differences in the maximum inhibitorypotential of a drug, data were fit to a 3-parameter Hill equation (Min ¼ 0). Using the resultant curve parameters, dose–effect analysis was performed according toChou (22) and is described in Materials and Methods. Middle, Corresponding combination index (CI) plot, where synergism is indicated for CI values < 1.0.Right, Corresponding normalized IC50 isobologram where synergism is indicated for combination data points below the diagonal line.

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by eliminating themalignant MPN clone (46). Overcoming theseproblems will require attacking cancer cells at multiple levels,either by drug combinations or single drugs targeting multipleproteins. For example, it has been demonstrated that combinedtargeting of the JAK2 and Bcl-2/Bcl-xL pathways via administra-tion of multiple JAK2 and Bcl-2/Bcl-xL inhibitors overcomesacquired resistance to single-agent JAK2 inhibitor treatment(47). Resistance to BET inhibitors has recently been reported inleukemia as a consequence of increased Wnt/b-catenin signaling(48, 49). As indicated by their high efficacy against MPN cells andpatient samples (Fig. 4), dual BRD4-kinase inhibitors may beparticularly useful in the treatment of cancers with evolvedresistance to single activity TK or BET inhibitors.

Disclosure of Potential Conflicts of InterestH.R. Lawrence has Ownership Interest (including patents) as an inventor

and has provided licensing fees. N.J. Lawrence has Ownership Interest(including patents) as an inventor on a patent and has provided licensingfees. E. Schonbrunn has Ownership Interest (including patents) as aninventor and has provided licensing fees. The IP described in patent applica-tions concerning the dual BET-kinase inhibitors was licensed from theMoffitt Cancer Center to Aptose Biosciences. No potential conflicts of interestwere disclosed by the other authors.

Authors' ContributionsConception and design: S.W. Ember, M. Ayaz, C.C. Lynch, N.J. Lawrence,E. Sch€onbrunnDevelopment of methodology: S.W. Ember, M. Ayaz, M. Tauro, J.-Y. Zhu,H.R. Lawrence, N.J. Lawrence, E. Sch€onbrunnAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S.W. Ember,N. Berndt,M.Ayaz,M. Tauro, P.J. Cranfill,P. Greninger, C.C. Lynch, C.H. Benes, G.W. Reuther, N.J. Lawrence,E. Sch€onbrunn

Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S.W. Ember, N. Berndt, M. Ayaz, M. Tauro, J.-Y. Zhu,P.J. Cranfill, C.C. Lynch, H.R. Lawrence, G.W. Reuther, N.J. Lawrence,E. Sch€onbrunnWriting, review, and/or revision of the manuscript: S.W. Ember, N. Berndt,M. Ayaz, J.-Y. Zhu, C.C. Lynch, H.R. Lawrence, G.W. Reuther, N.J. Lawrence,E. Sch€onbrunnAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S.W. Ember, Q.T. Lambert, J.-Y. Zhu, P.J. Cran-fill, P. Greninger, C.C. Lynch, H.R. Lawrence, E. Sch€onbrunnStudy supervision: S.W. Ember, C.C. Lynch, N.J. Lawrence, E. Sch€onbrunnOther (synthesis and characterization/analysis of key compounds used inthis study): S. GunawanOther (directed the synthesis of compounds/analogs, interpretation of data):H.R. Lawrence

AcknowledgmentsWe thank the Southeast Regional Collaborative Access Team (SER-CAT,

University of Georgia) for assistance with Synchrotron data collection atArgonne National Laboratory. We also thank Mathew J. Garnett and UltanMcDermott (Wellcome Trust Sanger Institute, Hinxton, UK) for assistance withcell line screening setup.

Grant SupportThis work was supported in part by the Chemical Biology Core at the Moffitt

Cancer Center (NIH/NCI: P30-CA076292; H.R. Lawrence, E. Sch€onbrunn), theNational Institute of Child Health and Human Development (NIH/NICHD:HHSN275201300017C; E. Sch€onbrunn), and a Moffitt Team Science Award(G.W. Reuther, N.J. Lawrence, and E. Sch€onbrunn). C.H. Benes and P.Greningerwere supported by a grant from the Wellcome Trust (102696).

Received September 6, 2016; revised November 1, 2016; accepted March 2,2017; published OnlineFirst March 23, 2017.

References1. Filippakopoulos P, Knapp S. The bromodomain interactionmodule. FEBS

Lett 2012;586:2692–704.2. Romero FA, Taylor AM, Crawford TD, Tsui V, Cote A, Magnuson S.

Disrupting Acetyl-Lysine Recognition: Progress in the Development ofBromodomain Inhibitors. J Med Chem 2016;59:1271–98.

3. Krueger BJ, Varzavand K, Cooper JJ, Price DH. Themechanism of release ofP-TEFb and HEXIM1 from the 7SK snRNP by viral and cellular activatorsincludes a conformational change in 7SK. PLoS ONE 2010;5:e12335.

4. Puissant A, Frumm SM, Alexe G, Bassil CF, Qi J, Chanthery YH, et al.Targeting MYCN in neuroblastoma by BET bromodomain inhibition.Cancer Discov 2013;3:308–23.

5. Filippakopoulos P, Knapp S. Targeting bromodomains: epigenetic readersof lysine acetylation. Nat Rev Drug Discov 2014;13:337–56.

6. Zhang G, Smith SG, Zhou MM. Discovery of Chemical Inhibitors ofHuman Bromodomains. Chem Rev 2015;115:11625–68.

7. Ember SW, Zhu JY, Olesen SH,MartinMP, Becker A, Berndt N, et al. Acetyl-lysine binding site of bromodomain-containing protein 4 (BRD4) interactswith diverse kinase inhibitors. ACS Chem Biol 2014;9:1160–71.

8. Martin MP, Olesen SH, Georg GI, Schonbrunn E. Cyclin-dependent kinaseinhibitor dinaciclib interacts with the acetyl-lysine recognition site ofbromodomains. ACS Chem Biol 2013;8:2360–5.

9. Ciceri P, Muller S, O'Mahony A, Fedorov O, Filippakopoulos P, Hunt JP,et al. Dual kinase-bromodomain inhibitors for rationally designed poly-pharmacology. Nat Chem Biol 2014;10:305–12.

10. Lee DH, Qi J, Bradner JE, Said JW, Doan NB, Forscher C, et al. Synergisticeffect of JQ1 and rapamycin for treatment of human osteosarcoma. Int JCancer 2015;136:2055–64.

11. Fiskus W, Sharma S, Qi J, Shah B, Devaraj SG, Leveque C, et al. BET proteinantagonist JQ1 is synergistically lethal with FLT3 tyrosine kinase inhibitor(TKI) and overcomes resistance to FLT3-TKI in AML cells expressing FLT-ITD. Mol Cancer Ther 2014;13:2315–27.

12. Stratikopoulos EE, Dendy M, Szabolcs M, Khaykin AJ, Lefebvre C, ZhouMM, et al. Kinase and BET Inhibitors Together Clamp Inhibition of PI3KSignaling and Overcome Resistance to Therapy. Cancer Cell 2015;27:837–51.

13. Sun B, Shah B, Fiskus W, Qi J, Rajapakshe K, Coarfa C, et al. Synergisticactivity of BET protein antagonist-based combinations in mantle celllymphoma cells sensitive or resistant to ibrutinib. Blood 2015;126:1565–74.

14. Baker EK, Taylor S, Gupte A, Sharp PP, Walia M, Walsh NC, et al. BETinhibitors induce apoptosis through a MYC independent mechanism andsynergise with CDK inhibitors to kill osteosarcoma cells. Sci Rep2015;5:10120.

15. Stuhlmiller TJ,Miller SM, Zawistowski JS,NakamuraK, BeltranAS,DuncanJS, et al. Inhibition of lapatinib-induced kinome reprogramming in ERBB2-positive breast cancer by targeting BET family bromodomains. Cell Rep2015;11:390–404.

16. Liu S,Walker SR, Nelson EA, Cerulli R, XiangM, Toniolo PA, et al. TargetingSTAT5 in hematologic malignancies through inhibition of the bromodo-main and extra-terminal (BET) bromodomain protein BRD2. Mol CancerTher 2014;13:1194–205.

17. Schonbrunn E, Lawrence NJ, Lawrence HR, inventors. Potent dual brd4-kinase inhibitors as cancer therapeutics. World patent WO 2016022460.A1. H. LeeMoffitt Cancer Center and Research Institute, Inc.; Feb 11, 2016.

18. Filippakopoulos P, Qi J, Picaud S, Shen Y, Smith WB, Fedorov O, et al.Selective inhibition of BET bromodomains. Nature 2010;468:1067–73.

19. Kabsch W. Xds. Acta Crystallogr D Biol Crystallogr 2010;66:125–32.20. Adams PD, Afonine PV, Bunkoczi G, Chen VB, Davis IW, Echols N,

et al. PHENIX: a comprehensive Python-based system for macromo-lecular structure solution. Acta Crystallogr D Biol Crystallogr 2010;66:213–21.

Ember et al.

Mol Cancer Ther; 16(6) June 2017 Molecular Cancer Therapeutics1066

on March 14, 2021. © 2017 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst March 23, 2017; DOI: 10.1158/1535-7163.MCT-16-0568-T

21. Emsley P, Lohkamp B, Scott WG, Cowtan K. Features and development ofCoot. Acta Crystallogr D Biol Crystallogr 2010;66:486–501.

22. Chou TC. Theoretical basis, experimental design, and computerized sim-ulation of synergism and antagonism in drug combination studies. Phar-macol Rev 2006;58:621–81.

23. Garnett MJ, Edelman EJ, Heidorn SJ, Greenman CD, Dastur A, Lau KW,et al. Systematic identification of genomic markers of drug sensitivity incancer cells. Nature 2012;483:570–5.

24. Futreal PA, Coin L, Marshall M, Down T, Hubbard T, Wooster R, et al. Acensus of human cancer genes. Nat Rev Cancer 2004;4:177–83.

25. Graczyk PP. Gini coefficient: a new way to express selectivity of kinaseinhibitors against a family of kinases. J Med Chem 2007;50:5773–9.

26. Davis MI, Hunt JP, Herrgard S, Ciceri P, Wodicka LM, Pallares G, et al.Comprehensive analysis of kinase inhibitor selectivity. Nat Biotechnol2011;29:1046–51.

27. Delmore JE, Issa GC, Lemieux ME, Rahl PB, Shi J, Jacobs HM, et al. BETbromodomain inhibition as a therapeutic strategy to target c-Myc. Cell2011;146:904–17.

28. Quentmeier H, Reinhardt J, Zaborski M, Drexler HG. FLT3 mutations inacute myeloid leukemia cell lines. Leukemia 2003;17:120–4.

29. QuentmeierH,MacLeodRA, ZaborskiM,DrexlerHG. JAK2V617F tyrosinekinase mutation in cell lines derived from myeloproliferative disorders.Leukemia 2006;20:471–6.

30. Mullighan CG, Collins-Underwood JR, Phillips LA, Loudin MG, Liu W,Zhang J, et al. Rearrangement of CRLF2 in B-progenitor- and Downsyndrome-associated acute lymphoblastic leukemia. Nat Genet 2009;41:1243–6.

31. Van Roosbroeck K, Cox L, Tousseyn T, Lahortiga I, Gielen O, CauwelierB, et al. JAK2 rearrangements, including the novel SEC31A-JAK2 fusion,are recurrent in classical Hodgkin lymphoma. Blood 2011;117:4056–64.

32. James C, Ugo V, Le Couedic JP, Staerk J, Delhommeau F, Lacout C, et al. Aunique clonal JAK2 mutation leading to constitutive signalling causespolycythaemia vera. Nature 2005;434:1144–8.

33. Ghoreschi K, Laurence A,O'Shea JJ. Janus kinases in immune cell signaling.Immunol Rev 2009;228:273–87.

34. Wyspianska BS, Bannister AJ, Barbieri I, Nangalia J, Godfrey A, Calero-Nieto FJ, et al. BET protein inhibition shows efficacy against JAK2V617F-driven neoplasms. Leukemia 2014;28:88–97.

35. Koppikar P, Bhagwat N, Kilpivaara O, Manshouri T, Adli M, Hricik T, et al.Heterodimeric JAK-STAT activation as a mechanism of persistence to JAK2inhibitor therapy. Nature 2012;489:155–9.

36. Mazzacurati L, Lambert QT, Pradhan A, Griner LN, Huszar D, Reuther GW.The PIM inhibitor AZD1208 synergizes with ruxolitinib to induce apo-ptosis of ruxolitinib sensitive and resistant JAK2-V617F-driven cells and

inhibit colony formation of primary MPN cells. Oncotarget 2015;6:40141–57.

37. Bergethon K, ShawAT,Ou SH, Katayama R, Lovly CM,McDonaldNT, et al.ROS1 rearrangements define a unique molecular class of lung cancers.J Clin Oncol 2012;30:863–70.

38. Li S, Qi X, Huang Y, Liu D, Zhou F, Zhou C. Ceritinib (LDK378): a potentalternative to crizotinib for ALK-rearranged non-small-cell lung cancer.Clin Lung Cancer 2015;16:86–91.

39. Ceccon M, Mologni L, Giudici G, Piazza R, Pirola A, Fontana D, et al.Treatment efficacy and resistancemechanisms using the second-generationALK Inhibitor AP26113 in human NPM-ALK-positive anaplastic large celllymphoma. Mol Cancer Res 2015;13:775–83.

40. Burger JA. Bruton's tyrosine kinase (BTK) inhibitors in clinical trials. CurrHematol Malig Rep 2014;9:44–9.

41. Sequist LV, Soria JC, Goldman JW,Wakelee HA, Gadgeel SM, Varga A, et al.Rociletinib in EGFR-mutated non-small-cell lung cancer. N Engl J Med2015;372:1700–9.

42. Kuiatse I, Baladandayuthapani V, Lin HY, Thomas SK, Bjorklund CC,Weber DM, et al. Targeting the spleen tyrosine kinase with fostamatinibas a strategy against Waldenstrom macroglobulinemia. Clin Cancer Res2015;21:2538–45.

43. Saenz DT, Fiskus W, Manshouri T, Rajapakshe K, Krieger S, Sun B, et al.BET protein bromodomain inhibitor-based combinations are highlyactive against post-myeloproliferative neoplasm secondary AML cells.Leukemia 2017;31:678–87. doi: 10.1038/leu.2016.260.

44. Murray BW, Miller N. Durability of kinase-directed therapies-A networkperspective on response and resistance. Mol Cancer Ther 2015;14:1975–84.

45. Sweet K, Lancet JE. Novel therapeutics in acute myeloid leukemia. CurrHematol Malig Rep 2014;9:109–17.

46. Verstovsek S, Kantarjian H, Mesa RA, Pardanani AD, Cortes-Franco J,Thomas DA, et al. Safety and efficacy of INCB018424, a JAK1 and JAK2inhibitor, in myelofibrosis. N Engl J Med 2010;363:1117–27.

47. Waibel M, Solomon VS, Knight DA, Ralli RA, Kim SK, Banks KM, et al.Combined targeting of JAK2 and Bcl-2/Bcl-xL to cure mutant JAK2-drivenmalignancies and overcome acquired resistance to JAK2 inhibitors. CellRep 2013;5:1047–59.

48. Fong CY, Gilan O, Lam EY, Rubin AF, Ftouni S, Tyler D, et al. BET inhibitorresistance emerges from leukaemia stem cells. Nature 2015;525:538–42.

49. Rathert P, Roth M, Neumann T, Muerdter F, Roe JS, Muhar M, et al.Transcriptional plasticity promotes primary and acquired resistance toBET inhibition. Nature 2015;525:543–7.

50. Quintas-Cardama A, Vaddi K, Liu P, Manshouri T, Li J, Scherle PA, et al.Preclinical characterization of the selective JAK1/2 inhibitor INCB018424:therapeutic implications for the treatment of myeloproliferative neo-plasms. Blood 2010;115:3109–17.

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2017;16:1054-1067. Published OnlineFirst March 23, 2017.Mol Cancer Ther   Stuart W. Ember, Que T. Lambert, Norbert Berndt, et al.   Multitargeted Chemical Probes and Cancer TherapeuticsPotent Dual BET Bromodomain-Kinase Inhibitors as Value-Added

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