PFI-1, a Highly Selective Protein Interaction Inhibitor, Targeting BET

Therapeutics, Targets, and Chemical Biology

PFI-1, a Highly Selective Protein Interaction Inhibitor,Targeting BET Bromodomains

Sarah Picaud1, David DaCosta3, Angeliki Thanasopoulou6, Panagis Filippakopoulos1, Paul V. Fish5, Martin Philpott1,Oleg Fedorov1, Paul Brennan1, Mark E. Bunnage5, Dafydd R. Owen5, James E. Bradner8,9, Philippe Taniere4,Brendan O'Sullivan4, Susanne M€uller1, Juerg Schwaller6, Tatjana Stankovic3, and Stefan Knapp1,2,7

AbstractBromo and extra terminal (BET) proteins (BRD2, BRD3, BRD4, and BRDT) are transcriptional regulators

required for efficient expression of several growth promoting and antiapoptotic genes as well as for cell-cycleprogression. BET proteins are recruited on transcriptionally active chromatin via their two N-terminal bromo-domains (BRD), a protein interaction module that specifically recognizes acetylated lysine residues in histonesH3 and H4. Inhibition of the BET–histone interaction results in transcriptional downregulation of a number ofoncogenes, providing a novel pharmacologic strategy for the treatment of cancer. Here, we present a potent andhighly selective dihydroquinazoline-2-one inhibitor, PFI-1, which efficiently blocks the interaction of BET BRDswith acetylated histone tails. Cocrystal structures showed thatPFI-1 acts as an acetyl-lysine (Kac)mimetic inhibitorefficiently occupying the Kac binding site in BRD4 and BRD2. PFI-1 has antiproliferative effects on leukemic celllines and efficiently abrogates their clonogenic growth. Exposure of sensitive cell lines with PFI-1 results in G1 cell-cycle arrest, downregulation of MYC expression, as well as induction of apoptosis and induces differentiation ofprimary leukemic blasts. Intriguingly, cells exposed to PFI-1 showed significant downregulation of Aurora Bkinase, thus attenuating phosphorylation of the Aurora substrate H3S10, providing an alternative strategy forthe specific inhibition of this well-established oncology target. Cancer Res; 73(11); 1–11. �2013 AACR.

IntroductionBromodomains (BRD) are protein interaction modules that

specifically recognize e-N-acetylated lysine residues (1, 2).BRDs are common interaction modules in nuclear proteins

that regulate gene transcription and chromatin organizationand play a key function recruiting these protein complexes toacetylated chromatin. Dysfunction of BRD-containing proteinshas been linked to the development of diverse diseases inparticular to the development of cancer (3).

Bromodomains are highly sequence diverse but they share aconserved fold that comprises a left-handed bundle of 4 alphahelices (aZ, aA, aB, aC; ref. 4). The acetyl-lysine side chain istypically anchored by a hydrogen bond to a conserved aspar-agine residue and water-mediated interactions with a con-served tyrosine (2, 5). Crystal structures of bromo and extraterminal (BET) complexes with di-acetylated histone 4 tailpeptides showed that the first BRDs of BRD4 and BRDT mayaccommodate 2 acetyl-lysines in a single binding site (2, 6).

The BET family of BRD proteins comprises 4 members inmammals (BRD2, BRD3, BRD4, and BRDT) each containing 2conserved N-terminal BRDs. BET proteins play critical roles incellular proliferation and cell-cycle progression (7). Geneticrearrangement of the BRD4 and BRD3 locus in which in-framechimeric proteins of the N-terminal BRDs of BRD4 or BRD3with the protein NUT (nuclear protein in testis) give rise to thedevelopment of NUT midline carcinoma (NMC), an incurableuniformly fatal subtype of squamous carcinoma (8). BRD4 hasbeen shown to be critical for survival of a number of diversetumors due to its function promoting transcription of growthpromoting and antiapoptotic genes (9) which prompted thedevelopment of potent and selective protein interaction inhi-bitors targeting BET BRDs. The potent pan-BET inhibitors(þ)-JQ1 and GSK1210151A (I-BET151) have shown significantantitumor activity in murine models of NMC (10), multiple

Authors' Affiliations: 1Structural Genomics Consortium and 2Target Dis-covery Institute, Nuffield Department of Clinical Medicine, University ofOxford, Oxford; 3School of Cancer Sciences, University of Birmingham;4Department of Cellular Pathology, Queen Elizabeth Medical Centre, Bir-mingham; 5Pfizer Worldwide Medicinal Chemistry, Pfizer Worldwide R&D,Ramsgate Road, Sandwich, United Kingdom; 6Laboratory for ChildhoodLeukemia, Department of Biomedicine, University of Basel and BaselUniversity Children's Hospital, Basel, Switzerland; 7Department of Bio-chemistry andMolecular Biology, School ofMedicine andHealth Sciences,George Washington University, Washington, DC; 8Department of MedicalOncology, Dana-Farber Cancer Institute; and 9Department of Medicine,Harvard Medical School, Boston, Massachusetts

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Data deposition: The crystal structures reported in this article have beendeposited in the Protein Data Bank, www.pdb.org.

Corresponding Authors: Stefan Knapp, Structural Genomics Consortiumand Target Discovery Institute, Nuffield Department of Clinical Medicine,University of Oxford, Oxford, Oxon OX3 7DQ, United Kingdom. Phone: 44-1865-617584; Fax: 44-1865-617575; E-mail: [email protected];Juerg Schwaller, Laboratory of Childhood Leukemia, Department of Bio-medicine, University of Basel and Basel University Children's Hospital,Hebelstrasse 20 CH - 4031 Basel, Switzerland. Phone: 41-6126-53504;Fax: 41-6126-52350; E-mail: [email protected] and Tatjana Stanko-vic, School of Cancer Sciences, University of Birmingham; Edgbaston;Birmingham; B15 2TT; United Kingdom. Phone: 44-121-4144496; Fax: 44-121-414 4486; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-12-3292

�2013 American Association for Cancer Research.

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myeloma (11), acute myeloid leukemia (AML; ref. 12), andmixed lineage leukemia (MLL; ref. 13). Genetic knockdown byRNAi or exposure of cells with BET inhibitors resulted in asignificant transcriptional downregulation of MYC (11).

In contrast tomost transcriptional regulators that dissociatefrom chromatin during mitosis, BRD4 preferentially associateswithmitotic chromosomes (14) "bookmarking" G1 and growth-associated genes for efficient postmitotic transcription, pro-viding a mechanism for transcriptional memory during celldivision (15). BRD4 knockdown in primary human keratino-cytes by RNAi results in severe cytokinesis defects and down-regulation of Aurora B expression. Aurora kinases (A, B, and C)are essential formitotic entry andprogression (16). TheA andBisoforms play distinct roles duringmitosis: Aurora A is requiredformitotic spindle assembly during pro andmetaphase, where-as Aurora B is part of themitotic passenger complexmediatingchromosome segregation by ensuring proper biorientation ofsister chromatids during meta and anaphase (17, 18). BothAurora isoforms are highly expressed in cancer, augment Ras-induced transformation, and have therefore emerged as attrac-tive therapeutic targets (19, 20). Interestingly, transcription ofAurora A and B is strongly upregulated by the BRD4 targetgene Myc, whereas, in turn, Aurora kinases also have a criticalfunction in regulating c-Myc turnover by regulating proteinstability, suggesting that activity of these growth promotingproteins is tightly regulated by a feedback loop (21).

Here, we describe a novel highly potent inhibitor PFI-1 thatselectively targets BET BRDs. PFI-1 binds to BET BRDs withlow nanomole potency and is chemically distinct from previ-ously reported BET inhibitors. Exposure of leukemia cells toPFI-1 results in induction of caspase-dependent apoptosis,differentiation, and in downregulation of the Aurora B kinase.This highly selective chemical probe provides a versatile toolfor further validation of BRD4 in cancer and other diseases andsuggests synergism between 2 major oncogenes, c-Myc andAurora B that can be simultaneously targeted by selective BETinhibition.

Materials and MethodsBRD2, BRD3, BRD4, BRDT, and CREBBP BRDs were cloned,

expressed, and purified as previously described (10). For biotinlabeling, CREBBP (R1081-G1198) and BRD2 (K71-N195) weresubcloned into pNIC-BIO1 (Gene Bank: EF198106) andexpressed in BL21 (DE3)-R3-BirA. D-Biotine was dissolvedinto 10 mmol/L bicine pH 8.3 and added to the culture at500 mmol/L final. Biotinylated protein was immobilized onSuper Streptavidin Biosensors using 50mmol/L HEPES pH 7.4,100 mmol/L NaCl, and 0.01% Tween.

Isothermal titration calorimetry (ITC), temperature shiftassays, and fluorescence recovery after photobleaching werecarried out as previously described (22). All cell lines wereobtained from American Type Culture Collection andwere cultured in RPMI-1640 medium (Sigma) containing10% FBS. In vitro cytotoxicity assays were conducted in trip-licate using either Cell-Titer-Glo reagent (Promega) or WST-1(Roche) according to instructions provided by the vendor.

CD34þ human hematopoietic stem cells, obtained fromperipheral blood of healthy patients, were plated in methyl-

cellulose (StemAlpha) supplemented with human cytokines(H4535, Stem Cell Technologies) at a cell dose of 1 � 104 perplate.

B-ALL primary graphNOD/Shi-scid/IL-2Rgnull (NOG) mice were engrafted with

B-acute lymphoblastic leukemia (B-ALL) primary blastst(1, 19)þ using a sample obtained from a 9-year-old childwith high-risk B-ALL. The engraftment process was monitoredby staining peripheral blood samples for human CD45 andmouse CD45. At 5 weeks, hCD45 was at least 1% and a treat-ment cycle was initiated by injection of 50 mg/kg JQ1 4 timesper week into the peritoneum (IP) for 3 weeks. Bone marrowwas extracted from the femur of JQ1 and vehicle [dimethylsulfoxide (DMSO)]–treated mice, fixed in 10% formalin, andmounted in paraffin blocks.

Fixation, immunostaining, and confocal microscopyApproximately 5 � 104 cells were used for each cytospot.

Cells were centrifuged at 500 rpm for 5 minutes using aCytospin 3 SHANDON cytocentrifuge. Fixation of the cells wascarried out with 4% paraformaldehyde solution in PBS. Incu-bation was conducted for 16 hours at 4�C in 0.3% Triton X-100,0.5% BSA, and the indicated primary antibody. After washing,the cells were incubated with the secondary antibody for 2hours. The cells were washed and a coverslip was placed on topby adding a droplet of mounting solution (Clear-Mount, Invi-trogen). Confocal fluorescence images were obtained by aLSM710 microscope (Zeiss).

Chromatin immunoprecipitationChromatin immunoprecipitation (ChIP) assays were con-

ducted using the EZ-ChIP Kit by Millpore according to themanufacturer's protocol. Results were quantified both bysemiquantitative and quantitative PCR, conducted using TerraqPCR Direct SYBR Premix (Clonetech) on an ABI 7900HT.

ResultsIn a screen for putative acetyl-lysine mimetic compounds,

we identified the simple fragment 6-bromo-3-methyl-3,4-dihy-droquinazoline-2-one as a BRD4 and CREBBP BRD inhibitor.This inhibitor displaced tetra-acetylated histone 4 peptides inALPHA (amplified luminescent proximity homogeneousassay) screen assays with an IC50 value of approximately 50mmol/L for the first domain of BRD4 [BRD4(1)]. Expansion ofthis scaffold resulted in a series of highly potent and specificbenzenesulfonamide-quinazolin-2-one BET inhibitors. PFI-1was selected as one of the most potent and selective com-pounds of about 300 inhibitors profiled in ALPHA screenassays (Fig. 1A). The inhibitor was synthesized as outlined inSupplementary Fig. S1. Structure–activity relationship of thisclass of compounds will be described elsewhere (23).

PFI-1 is a potent and selective BET bromodomaininhibitor

ALPHA screen assays have shown that PFI-1 displaceshistone 4 peptide acetylated at lysines K5, K8, K12, and K16(H4K5acK8acK12acK16ac) with a potency (IC50) of 220 nmol/L

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for BRD4(1) and 98 nmol/L for BRD2(2). The tight interactionof PFI-1 with BET acetyl-lysine–binding sites was confirmed byITC, which revealed dissociation constants (KD) of PFI-1 of 47.4� 2.5 nmol/L [BRD4(1)] and 194.9� 6 nmol/L [BRD4(2)] for the2BRDs present in BRD4. ITC titrations against BRDs of the BETfamily showed that PFI-1 bound with similar affinities (Fig. 1Band Supplementary Table S1). Binding of PFI-1 was stronglydriven by large negative binding enthalpy change, suggestingthat polar interactions of PFI-1 with BET BRDs are highlyfavorable. Interestingly, binding enthalpies (DH) were between�4 and�7 kcal/mol larger for N-terminal BETBRDs. However,this large difference in favorable binding enthalpy was almostcompletely compensated by unfavorable changes in bindingentropy (DS) resulting in similar binding constants. The largestdifference in binding affinity (about 4 fold)was observed for the2 BRDs of BRD4.Comprehensive screening of PFI-1 against 42 human BRDs

using temperature shift assays (10, 24) have shown highspecificity of PFI-1 for the BET family of BRDs (Fig. 1C andSupplementary Table S2). The largest DTm shift observedoutside the BET family was 2.6�C for BRDs present in the

histone acetyl transferases CBP/EP300. The interaction withCBP was too weak for determination of an accurate IC50 valueusing ALPHA screen assays. However, we estimated an affinityof PFI-1 for CBP of approximately 11 mmol/L using Bio-LayerInterferometry (BLI; Supplementary Fig. S2; ref. 25), suggestingmore than 300-fold selectivity over the 2 BRDs that showed thelargest DTm shift outside the BET BRD family. In comparison,BLI determined an affinity of 111 nmol/L for BRD2(1) inagreement with ITC and ALPHA screen data. We also assessedactivity of PFI-1 outside the BRD family. Screening against 38protein kinases revealed no significant inhibitory activity ofPFI-1. Similarly, no significant activity was observed screeningPFI-1 against 40 human kinases and 14 human membranereceptors (Supplementary Tables S3 and S4).

PFI-1 binds to the acetyl-lysine–binding site of BETbromodomains

Cocrystallization of PFI-1 with BRD4(1) revealed the bindingmode of this highly specific BET BRD inhibitor. Crystals of theBRD4(1)/PFI-1 complex diffracted to high (1.52 Å) resolutionproviding a detailed view of the interactions formed by this

Figure 1. Potency and selectivity of PFI-1. A, chemical structure of PFI-1. B, selectivity screening data of PFI-1 using temperature shift assays. Screenedtargets are highlighted in bold. Temperature shifts are indicated by red filled circles with increasing radii for higher Tm values as indicated in thefigure. C, isothermal titration data measured on BRD4(1; black) and BRD4(2; red). Shown are heat effects for each injection and the normalized bindingisotherms (insert) including the fitted function (solid line). D, ALPHA-screen datameasured using isolated BRDs of BRD4(1; red triangle) BRD4(2; blue triangle)as well as the BRD of CREBBP (green circle). Fitted functions are show as solid lines.

BET Bromodomain Inhibitor PFI-1

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inhibitor with the BRD acetyl-lysine–binding site. Details ondata collection and refinement are summarized in Supple-mentary Table S5. The overall structure of the PFI-1 in complexwith BRD4(1) revealed the typical helical fold and structuralelements of BRDs (Fig. 2A) and comparison with the apos-tructure (PDB-ID:2OSS) showed only minor structuralrearrangements in the ZA-loop regions. The inhibitor andcoordinating residues and water molecules were well definedby electron density (Fig. 2B). PFI-1 showed an extraordinaryshape complementarity with the Kac binding site. The quina-zolinone carbonyl and nitrogen acted as a hydrogen bonddonor/acceptor pair that interacted with the conserved aspar-agine N140. The quinazolinone carbonyl also formed a secondwater-mediated hydrogen bond with the conserved residueY97. The network of 5 tightly bound water molecules that aretypically found at the base of BRD acetyl-lysine–binding pocketwas also conserved in the BRD4(1) PFI-1 complex. Comparisonto BRD4(1) structures of diacetylated peptide complexes (2)confirmed the acetyl-lysine mimetic binding mode of PFI-1(Fig. 2C). The carbonyl moieties of the quinazolinone and the

acetyl-lysine are in similar position and mediate the sameinteractions in superimposition of both BRD4(1) ligand com-plexes. Superimposition with the BRD4(1)/(þ)-JQ1 complexrevealed a largely diverse binding mode. However, both com-plexes form an acetyl-lysinemimetic hydrogen bond toN140 aswell as aromatic stacking and hydrophobic interactions withW81 and the shelf region of the acetyl-lysine–binding site.

ITC data showed a significantly smaller binding enthalpychange to second BRDs of BET family members and we wereinterested if the cocrystal structure of PFI-1 with a second BRDwould explain these striking differences in the observed bind-ing thermodynamics. Comparison of the structure of the PFI-1complex of BRD4(1) and BRD2(2) showed that polar interac-tion of the inhibitor with BRD2(2) are less optimal (Fig. 2D andE). The hydrogen bond between the quinazolinoneNHwith theconserved asparagine residue [N140 in BRD4(1) and N429 inBRD2(2)] is less favorable (distance of 3.3 Å) in the case of BRD2(2)when comparedwith BRD4(1; distance of 2.9 Å). In addition,the water-mediated hydrogen bond between the sulfonamideNH function and the ZA loop backbone carbonyl observed in

Figure 2. PFI-1 cocrystal structurewith BRD4(1) and BRD2(2). A,structural overview of the PFI-1/BRD4(1) complex. PFI-1 is shownin ball and stick representation.Hydrogen bonds to the conservedasparagine (N140) are shown asdotted lines and water moleculesas semitransparent spheres. B,2FoFc omit electron density mapcontoured at 2s around the PFI-1.C, superimposition of the PFI-1complex with the di-acetylatedK5acK8ac peptide complex(2; top)and the BRD4(1)/(þ)-JQ1 complex(bottom; ref. 10). D, surfacerepresentation of the BRD4(1)acetyl-lysine–binding site.Residues that are differentbetween first and secondBRDs arelabeled in red and conservedresidues in black, respectively. Theconserved asparagine ishighlighted in blue. E, surfacerepresentation of the BRD2(2)acetyl-lysine–binding site.Residues were labeled using thesame color code as in D.

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the BRD4(1) complex (3.2 Å) is further away from the bridgingwatermolecule which was, however, still observed in the BRD2(2) cocrystal structure (3.8 Å). Interestingly, in contrast to(þ)-JQ1 BRD complexes, H433 reoriented and flipped into theacetyl-lysine–binding site to interact with the methoxybenzylmoiety of PFI-1. This large structural rearrangement mayprovide an explanation for the large difference in DS uponbinding of PFI-1 to the first and second BRDs. H433 is con-served in all second BRDs, whereas in N-terminal BET BRDs,this residue is always an aspartate [D144 in BRD4(1)]. Thisdifference in Kac site composition may be explored for thedevelopment of inhibitors that preferentially recognize eitherthe first or second BRD.

PFI-1 displaces BRD4 from chromatinTo establish whether PFI-1 dissociates full-length BRD4

from acetylated chromatin in cells, we developed fluorescencerecovery after photo-bleaching (FRAP) experiments in humanosteosarcoma cells (U2OS) transfected with GFP–BRD4. Theuse of FRAP in assessing the diffusion of BRD-containingproteins tagged with fluorescent fusion partners and thereforeproviding evidence of the level of chromatin association hasbeen previously established (22), andwe have successfully usedthis method to show the on-target effect of the BET inhibitorJQ1 inBRD4-dependentNMCcell lines (10). Cells treatedwith 1and 5 mmol/L PFI-1 showed significantly faster fluorescent

recovery times when compared with cells that have not beenexposed to this inhibitor, suggesting that full-length BRD4 wasdisplaced from chromatin in PFI-1–treated cells (Fig. 3). Dif-ferences in fluorescent recovery time of PFI-1–treated cellswere comparable with effects observed for the biochemicallyactive (þ)-JQ1 stereoisomer.

PFI-1 inhibits proliferation of a subset of leukemic cellsWe used a luminescent ATP-based cytotoxicity assay to

examine the sensitivity of a panel of established leukemia celllines to PFI-1 in a dose-dependent manner (Table 1). Inagreementwith earlier studies, we found that cell lines carryingoncogenic rearrangements in the MLL locus (26) such asMV4;11 (MLL-AF4), NOMO-1 (MLL-AF9), SEMKH2 (MLL-AF4)RS4;11 (MLL-AF4), or THP-1 (MLL-AF9) were highly sensitiveto BET inhibition (12, 13, 27). Comparable activity was alsoobserved for the AML1–ETO fusion oncogenes bearing AMLcell line Kasumi. Significantly less sensitive (5–10 mmol/L) wasthe ALL-derived cell line KOCL-45 despite its MLL–AF4rearrangement and the human histiocytic lymphoma cellline U937. No significant activity was detected for K-562[BCR-ABL–positive blast crisis chronic myelogenous leukemia(CML)] and PL-21 (AML) showing that the growth inhibitionobserved in certain leukemia cell lines is not due to ageneral cytotoxicity of PFI-1. However, the activity of PFI-1on cell proliferation of leukemia cell lines was between 5- and

Figure 3. FRAP data showingdissociation of GFP-BRD4 fromchromatin. A, nuclei of PFI-1–treated(top) and untreated (bottom) cells.The bleached area is indicated by aread polygon. B, time dependence offluorescent recovery in the bleachedarea for DMSO-treated, (þ)-JQ1–treated, and PFI-1 (1, 5 mmol/L)-treated cells. C, half times offluorescence recovery of DMSO,(þ)-JQ1, and PFI-1 (1, 5 mmol/L)-treated cells. The data shownrepresent the average values of 20experiments.






10-fold weaker than that observed for the pan-BET inhibitor(þ)-JQ1.

We also investigated the efficiency of PFI-1 to suppress theclonogenic growth of leukemic cells in methylcellulose. Inagreement with the cytotoxicity assays in liquid culture, weobserved strong ablation of clonogenic growth in the PFI-1–sensitive cell lines MV4;11 and THP-1 (Fig. 4). However,clonogenic growth of human CD34-positive human hemato-poietic stem cells from 2 different healthy donors was alsosignificantly impaired. Interestingly, colony formation of thePFI-1 insensitive cell line K562 was not affected, but exposureto PFI-1 did significantly reduce cell numbers, suggesting thatgrowth is still compromised in this cell line. This effect was alsoobserved using (þ)-JQ1 (Fig. 4C–E). The sensitivity of cell linesto BET-dependent growth inhibitionwas independent of BRD4mRNA expression levels. We conducted quantitative real-timePCR (qRT-PCR) experiments quantifying the levels of the 2main BRD4 isoforms [long (GI:19718731) and short(GI:7657218)]. We found comparable levels of the short BRD4isoforms in all cell lines. mRNA expression levels of the longisoform were particularly high in K-562 and THP-1 cells(Supplementary Fig. S3).

PFI-1 induces cell-cycle arrest and apoptosis in sensitivecell lines

Annexin V staining combined with FACS analysis estab-lished strong induction of apoptosis by BRD4 knockdown orchemical inhibition of BET proteins (12, 13, 27). Here, we wereinterested to characterize the mechanism of PFI-1–inducedapoptosis using Western blot analysis of proteins known toplay key roles in apoptosis. In the BET inhibitor–sensitive cellline MV4;11, we observed strong induction of PARP1 andprocaspase 7 cleavage after 24-hour exposure with PFI-1,whereas protein levels as well as the phosphorylation state ofthe proapoptotic protein BAD were unaffected. In contrast,neither PARP1 nor procaspase 7 cleavage was observed in the

PFI-1 insensitive cell line K-562 (Fig. 5). In agreement withearlier studies using (þ)-JQ1, we also detected significantlylower c-Myc protein levels in PFI-1–treated MV4;11 cells, butonly a minor reduction in c-Myc levels was observed in K-562cells (12, 27). This analysis shows that the canonical caspase-induced apoptotic pathway is activated in cell lines that aresensitive to BET BRD inhibition by low molecular weightinhibitors. As predicted from RNAi knock down studies andstudies on (þ)-JQ1 (10) that have shown a key role of BRD4 incell-cycle progression, we found that exposure of cells sensitiveto PFI-1 cause cell-cycle arrest. In contrast, the cell cycle in PFI-1–insensitive cell lines (K-562) was not affected as shown byflow cytometry (Supplementary Fig. S4). Because c-Myc drivesexpression of a number of genes that are essential for cell-cycleprogression (e.g., E2Fs, CDC25A, CDK2, CDK4, and Rb), theobserved G1–S arrest might be a consequence of c-Myc deple-tion in sensitive MV4;11 but not K-562 cells (28–31).

PFI-1 downregulates Aurora B and attenuates H3S10phosphorylation

BRD4 is one of the few transcriptional regulators that arerecruited to chromatin during mitosis providing a mechanismfor transcriptional memory during cell division (14, 15, 22).Mitotic entry and progression are principally regulated byserine/threonine kinases of the Aurora family (16). Aurorakinases are highly expressed in diverse cancer types and arealso frequently upregulated in leukemia (32, 33). In addition,Aurora B expression levels are modulated by BRD4 andare stimulated by c-Myc, whereas c-Myc stability is regulatedby Aurora kinase–dependent degradation (21). We weretherefore interested to study whether inhibition of BET BRDsby the inhibitors PFI-1 and (þ)-JQ1 would affect Aurora Bprotein levels resulting in indirect inhibition of the Aurora Boncogene.

We found high expression levels in Western blots usingAurora B-specific antibodies in both MV4;11 and K-562 cells

Table 1. Sensitivity of leukemic cell lines to PFI-1

Cell line Cell type GI50 [mmol/L]

MV4;11 Childhood B-cell myelomonocytic acute leukemia (MLL/AF4) 1.5 � 1THP-1 Acute monocytic leukemia (MLL/AF9) 4 � 2KASUMI-1 Acute myeloid leukemia with t(8;21) translocation (AML1-ETO) 0.8 � 1NALM-17 Pre B-blast ALL 8 � 4TOM-1 Ph1-positive ALL 3 � 5REH Adult ALL 9 � 4SD-1 Epstein-Barr virus immortalizedimmortalized Ph-1 positive ALL 8 � 3NALM-6 Pre-B ALL [t(5; 12) (q33; p13)] 8 � 3NOMO-1 Acute monocytic leukemia (MLL/AF9) 3 � 3SEMKH2 Acute monocytic leukemia (MLL/AF9) 2 � 2RS4;11 Acute monocytic leukemia (MLL/AF4) 2 � 2KOCL-45 Acute monocytic leukemia (MLL/AF4) 10 � 5U937 Leukemic monocytic lymphoma 5 � 3PL21 Acute promyelolytic leukemia 10 � 5SUPB-15 Phþ childhood B-cell ALL 11 � 4K-562 Erythroleukemia CML blast (Bcr-ABL) >20

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(Fig. 6A). Aurora B often appeared as a double band possiblydue to different phosphorylation states of the protein. Expo-sure of these cells to PFI-1 and (þ)-JQ1 resulted in significantreduction of Aurora B protein levels after 8-hour exposure inthe BET inhibitor–sensitive cell line MV4;11. In K-562 cells, areduction of Aurora B has been observed after 4 hours but theprotein levels were increased again after 8 hours. This is likelydue to differences of BET inhibitors on Aurora B mRNAexpression and Aurora B degradation in these 2 cell lines. Asa consequence of Aurora B downregulation, we observedsignificantly reduced phosphorylation of the Aurora substratehistone 3 S10 (H3S10) in cells stained with H3S10p-specificantibodies (Fig. 6B); H3S10 phosphorylation was completelyablated in Western blots (Fig. 6C) in MV4;11 cells. Immuno-cytochemistry and Western blots showed that reduction ofH3S10 phosphorylation levels were significantly less dramaticin K-562 cells than observed in MV4;11 cells. In conclusion,BRD4 inhibition results in significant Aurora B inactivation

offering an alternative strategy for inhibition of this attractivekinase target.

Synthetic lethality of BET and Aurora kinase inhibitionAs efficacious killing of tumor cells by Aurora inhibitors is

often constrained by dose-limiting toxicity, we were interestedif dual inhibition of BET proteins and Aurora kinases would besynergistic. We selected for these studies the potent andreversible small-molecule Aurora inhibitor VX-680 (MK-0457) that inhibits all 3 Aurora kinase isoforms with lownanomole potency (0.6, 18, and 4.6 nmol/L for AuroraA, AuroraB, and Aurora C, respectively). This inhibitor has shown in vivoefficacy in xenograft models in human AML and other cancersand has entered clinical testing (34–37). Aurora kinases inter-act with many key regulators of the cell cycle and cell survival,including p53, cyclin B, and Cdc2. As a consequence, inhibitionof Aurora kinases affects different stages of the cell cycle:Aurora A inhibition has been associated with G2–M phase

Figure 4. Effects of PFI-1 on cellsurvival and clonogenic growth. A,dose response of cell survival ofMV4;11 and K-562 cell lines in thepresence of PFI-1 and (þ)-JQ1.B and C, effects of PFI-1 onclonogenic growth of MV4;11 andhuman CD34þ stem cells (HSC; B)and K-562 cells (C). D and E, bardiagram showing the number ofcolonies (D) or cells (E) measuredin clonogenic growth assays ofleukemic cells and HSCs. Themeasurements were carried out induplicates. However, due tovariations of the colony numbersobtained from different donors, onlyone set of data is shownfor CD34þ stem cells. CFU,colony-forming units.






arrest, whereas Aurora B inhibition leads to failure in celldivision, abnormal exit from mitosis, polyploidy cells, andultimately induction of apoptosis.We observed at low inhibitorconcentration (lower than 20 nmol/L) cell-cycle arrest inG2–Mphase consisted with Aurora A inhibition. A higher concen-tration, VX680, caused predominantly G0–G1 arrest. Apoptosiswas observed at concentrations higher than 160 nmol/L (Sup-plementary Fig. S5). Interestingly, combination PFI-1 or(þ)-JQ1 and VX-680 at concentration that do not causeobservable toxicity as single agents led to strong cancer celltoxicity (Fig. 7A). Intriguingly, the optimal concentration ofVX-680 was 40 nmol/L for both BET inhibitors tested. At thisconcentration, the G2–M arrest observed at lower VX-680 isreleased leading to sensitization of the tested cell lines to dualAurora and BET inhibition. At higher VX-680 concentration,however, this synergistic effect is lost. It is therefore likely thatthe strong induction of apoptosis at 40 nmol/L VX-680 withboth BET inhibitors is highly dependent on differences in BET-dependent gene expression and cell-cycle effects induced byAurora kinase inhibitors. No synergy between VX-680 and BETinhibitors was observed in K-562 cells that are insensitive toBET inhibition (data not shown).

BET inhibition causes downregulation of Aurora Bin vivo

To study downregulation of Aurora B in vivo, we establisheda xenograft model of high-risk primary childhood B-cell ALL.Pharmacokinetics studies inmice suggested that the necessaryeffective dose of PFI-1 cannot be obtained in vivo (23) whichprevented us from using PFI-1 for long-term in vivo studies inmice and (þ)-JQ1 was therefore used. Engraftment was mon-itored by staining peripheral blood samples for human andmouse CD45. After 5 weeks of engraftment in NOG mice, the

level of hCD45 was at least 1% and mice were treated for 3weeks with 50 mg/kg (þ)-JQ1 as described (10). Bone marrowwas extracted from the femur of tumor-bearing mice andstained with specific antibodies for Aurora B and c-Myc. Theimmunofluorescent images revealed strong downregulation ofboth Aurora B as well as c-Myc (Fig. 7B) showing that BET

Figure 5. Western blots showing induction of apoptosis anddownregulation of c-Myc in MV4;11 but not K-562 cells. Shown areWestern blot data on the BET inhibitor sensitive cell line MV4;11 and theinsensitive cell line K-562 (cleavage of procaspase 7, PARB activation,BAD phosphorylation, and c-Myc downregulation) using cell extracts ofPFI-1–treated cells after 0, 24, and 48 hours of incubation times.

Figure 6. Downregulation of Aurora B. A,Western blot analysis of Aurora Bexpression in MV4;11 and K-562 cells treated with PFI-1 and (þ)-JQ1. B,immunohistochemistry showing reduction of phosphorylation of theAurora B substrate H3S10. DNA is stained with 40, 6-diamidino-2-phenylindole (DAPI). C, Western blot analysis of phosphor H3S10 afterPFI-1 and (þ)-JQ1 treatment of MV4;11 and K-562 cells.

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inhibition significantly reduces Aurora B levels in this in vivomodel.

PFI-1 and JQ1 dissociate BRD4 from HOXA9 andpromotes differentiationEvolutionarily conserved HOX genes are transcription fac-

tors that play an important role during hematopoiesis inregulating apoptosis, receptor signaling, differentiation, motil-ity, and angiogenesis. Aberrant expression of HOX genes hasbeen implicated in the development of leukemia and othercancers (38–40). During hematopoietic cell development,clusters of HOX genes are highly expressed in primitivehematopoietic cells and in poorly differentiated leukemic cells,whereas they are largely downregulated in differentiated cells.In particular, HOXA9 has been identified as a marker of poorprognosis in patients with acute myeloid leukemia (41), and

overexpression of HOXA9 leads to immortalization of bonemarrow cells and development of leukemia in mice (42). Wewere therefore interested to know whether BRD4 inhibitiondirectly influences HOXA9 expression and induces differenti-ation of primary leukemic blasts. ChIP assays showed that both(þ)-JQ1 as well as PFI-1 displace BRD4 from the HOXA9promoter region (Fig. 7C, Supplementary Fig. S6). QuantitativePCR showed that HOXA9 mRNA is upregulated after 4 hoursbut strongly downregulated after incubation times of 8 and 24hours in PFI-1–treated MV4;11 cells but not in BET inhibitor–insensitive K-562 cells that follow, however, the same biphasicpattern of the detected mRNA levels (Fig. 7D). The delayedresponse of HOXA9 transcriptional downregulation suggestsan indirect mechanism that is possibly regulated by reducedc-Myc levels. As PFI-1 inhibits all BET family members, differ-ences in BET protein levels or levels of the target genes may

Figure 7. Synergy of BET andAurora inhibition in vitro and in vivoand effects onHOXA9 expression. A,cytotoxicity of MV4;11 cells usingcombinations of BET inhibitors (PFI-1, JQ1) and the pan-Aurora inhibitorVX-680. Values shown representdata from 4 independentexperiments. B, staining for Aurora Band c-Myc of bonemarrow extractedfrom JQ1- and vehicle-treated B-ALL–bearing mice. C, ChIP of BRD4binding to the HOXA9 promoter.THP-1 cells were treated with eithervehicle (DMSO) or PFI-1. D, qRT-PCR of HOXA9 levels in K-562 andMV4;11 cells. E, MLL-AF9 murineleukemic blast cells treated withvehicle (dimethyl sulfoxide; DMSO)and PFI-1.






result in the different overall response of HOXA9 expression inthese 2 cell lines. In agreement with the known key function ofHOXA9 in suppressing differentiation, murine MLL-AF9 leu-kemic blasts treated with PFI-1 showed rapid differentiationinto cells with polymorphonuclear neutrophil–like appear-ances (Fig. 7E).

DiscussionThis study presents a chemically diverse BET inhibitor PFI-1

with high potency and selectivity for this subfamily of BRD-containing proteins. Here, we used PFI-1 to study the role of BETproteins in acute leukemia and in regulating HOXA9 expressionas well as Aurora B kinase activity. Inhibition of BET BRDsresults in the change of transcription of diverse target genes,many of them linked to cellular proliferation and prevention ofapoptosis. The set of BET-regulated target genes is highlycorrelated in sensitive cell lines. For instance, in the study byDawsonandcolleagues (13), the top100genes thatwere foundtobe decreased in the sensitive MLL cell lines MOLM-13 andMV4;11 overlapped and contained many known MLL targetgenes such as key regulators of proliferation (MYC, CDK6) andantiapoptotic genes (BCL2), suggesting that BET proteins arerequired for efficient expression of genes driven by oncogenicMLL-fusionproteins. Expression of these geneswas less affectedin inhibitor insensitive K-562 cells expressing the BCR-ABLfusion gene product. A recent study by Mertz and colleagueshas shown thatMYC expression was affected by BET inhibitiononly in the context of natural, chromosomally translocated oramplified gene loci, but not if expression is driven by non-endogenous promoters or viral insertions providing a possibilityfor stratification for BET-sensitive cancer types (27).

In this study, we have also shown that BET inhibition resultsin significant downregulation of Aurora B kinase in vitro aswellas in vivo. However, whether Aurora B expression is directlymediated by BET or indirectly through downregulation ofMYCexpression remains to be shown. A recent report showed thatknockdown of the BET family member BRD4 results in down-regulation of Aurora B expression, whereas exogenous over-expression of BRD4 increases Aurora B protein levels (43). Insynchronized cells, Aurora B protein levels have been signif-icantly reduced during mitosis after RNAi knockdown of BRD4(43) and abnormal chromosomal segregation has beenobserved in BRD4-depleted primary human foreskin keratino-cytes leading to high frequency of binuclear tetraploid andoctoploid nuclei (43). Aurora B activity is also tightly linkedto c-Myc function. Yang and colleagues have recently shownthat the pan-Aurora inhibitor VX-680 preferentially kills cellsthat overexpress c-Myc (44) and that both oncogenes arefrequently amplified in colorectal carcinomas and medullo-blastomas (45, 46). Here, we showed that VX-680 inhibitionis strongly synergistic with BET inhibition showing effective

induction of apoptosis at concentration that did not showany cytotoxic effects of the single agents. Interestingly,synergism of VX-680 has also been observed with the HDACinhibitor vorinostat as a result of reactivated proapoptoticgenes and enhanced cancer cell death. It is likely thatsuppression of antiapoptotic and reactivation of preapop-totic genes that have been reported for BET inhibitors (13)leads to the strong induction of apoptosis that have beenobserved in our synergy study.

The 8 BRDs present in the 4 human BET family membersshare high sequence similarity in their acetyl-lysine–bindingsites making the design of target selective inhibitors a chal-lenging task. Although we believe that it will be difficult toachieve high selectivity for any of the isoforms, we observedthat all first and second BRDs contain a set of diverse residues.These differences could be explored for the development ofsubdomain-specific inhibitors, which is an ongoing researchactivity in our laboratory.

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

Authors' ContributionsConception and design: P. Filippakopoulos, P.V. Fish, P. Brennan, M.E.Bunnage, J. Schwaller, S. KnappDevelopment ofmethodology:M. Philpott, B. O'Sullivan, J. Schwaller, S. KnappAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S.S. Picaud, P. Filippakopoulos, M. Philpott, O.Fedorov, M.E. Bunnage, D.R. Owen, P. Taniere, J. Schwaller, T. StankovicAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S.S. Picaud, D.D. Costa, A. Thanasopoulou, P. Filip-pakopoulos, P.V. Fish, M. Philpott, O. Fedorov, M.E. Bunnage, J.E. Bradner, S.Muller, J. Schwaller, S. KnappWriting, review, and/or revision of the manuscript: S.S. Picaud, P. Filippa-kopoulos, O. Fedorov, P. Brennan, D.R. Owen, J.E. Bradner, S. Muller, J. Schwaller,T. Stankovic, S. KnappAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): B. O'Sullivan, S. MullerStudy supervision: P.V. Fish, M.E. Bunnage, S. Knapp

AcknowledgmentsS. Picaud, M. Philpott, O. Fedorov, P. Brennan, S. Müller, and S. Knapp thank

the Structural Genomics Consortium (SGC).

Grant SupportS. Picaud, M. Philpott, O. Fedorov, P. Brennan, S. Müller, and S. Knapp are

supported by the SGC, a registered charity (number 1097737) that receives fundsfrom the Canadian Institutes for Health Research, the Canada Foundation forInnovation, Genome Canada, GlaxoSmithKline, Pfizer, Eli Lilly, Takeda, AbbVie,the Novartis Research Foundation, the Ontario Ministry of Research andInnovation, and the Wellcome Trust. P. Filippakopoulos is supported by aWellcome Trust Career-Development Fellowship (095751/Z/11/Z). J. Schwallerwas supported by the Gertrude von Meissner Foundation, the Swiss NationalResearch Foundation (SNF, 31003A-130661), the Swiss Cancer League (OCS-02357-2009), and the Swiss Bridge Award.

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 24, 2012; revised February 6, 2013; accepted February 24, 2013;published OnlineFirst April 10, 2013.

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Published OnlineFirst April 10, 2013.Cancer Res Sarah Picaud, David Da Costa, Angeliki Thanasopoulou, et al. Targeting BET BromodomainsPFI-1, a Highly Selective Protein Interaction Inhibitor,

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