characterization of the mechanism of action of the pan ... · for treatment of pi3k-dependent...

Preclinical Development

Characterization of the Mechanism of Action of the PanClass I PI3K Inhibitor NVP-BKM120 across a Broad Rangeof Concentrations

Saskia M. Brachmann1, Julia Kleylein-Sohn1, Swann Gaulis1, Audrey Kauffmann1, Marcel J.J. Blommers2,Malika Kazic-Legueux1, Laurent Laborde1, Marc Hattenberger1, Fabian Stauffer1, Juliane Vaxelaire1,Vincent Romanet1, Chryst�ele Henry3, Masato Murakami1, Daniel Alexander Guthy1, Dario Sterker1,Sebastian Bergling4, Christopher Wilson4, Thomas Br€ummendorf1, Christine Fritsch1,Carlos Garcia-Echeverria5, William R. Sellers6, Francesco Hofmann1, and Sauveur-Michel Maira1

AbstractThe pan-phosphoinositide 3-kinase (PI3K) inhibitor BKM120was found, at high concentrations, to cause cell

death in various cellular systems, irrespective of their level of PI3K addiction. Transcriptional and biochemical

profiling studieswereused to identify the origin of theseunexpected andapparentlyPI3K-independent effects.

At 5- to 10-fold, the concentration needed to half-maximally inhibit PI3K signaling. BKM120 treatment caused

changes in expression of mitotic genes and the induction of a robust G2–M arrest. Tubulin polymerization

assays and nuclear magnetic resonance-binding studies revealed that BKM120 inhibited microtubule dynam-

ics upon direct binding to tubulin. To assess the contribution of this off-target activity vis-�a-vis the antitumor

activity of BKM120 in PI3K-dependent tumors,weused amechanistic PI3K-a–dependentmodel.We observed

that, in vivo, daily treatment of mice with doses of BKM120 up to 40 mg/kg led to tumor regressions with no

increase in the mitotic index. Thus, strong antitumor activity can be achieved in PI3K-dependent models at

exposures that are below those necessary to engage the off-target activity. In comparison, the clinical data

indicate that it is unlikely that BKM120 will achieve exposures sufficient to significantly engage the off-target

activity at tolerated doses and schedules. However, in preclinical settings, the consequences of the off-target

activity start to manifest themselves at concentrations above 1 mmol/L in vitro and doses above 50 mg/kg in

efficacy studies using subcutaneous tumor–bearing mice. Hence, careful concentration and dose range

selection is required to ensure that any observation can be correctly attributed to BKM120 inhibition of PI3K.

Mol Cancer Ther; 11(8); 1747–57. �2012 AACR.

IntroductionThe phosphoinositide 3-kinase (PI3K) pathway plays a

pivotal role in cell growth, proliferation, survival, andmetabolism (1, 2). Lesions in key pathway componentscan lead to gain-of-function, pathway hyperactivation,aberrant cell proliferation, and subsequently to the pro-

motion and maintenance of cancer. For example, thePIK3CA gene encoding the p110a catalytic subunit hasbeen found to be amplified and frequently mutated in avariety of human cancers (3). Furthermore, the antago-nistic dual lipid/protein phosphatase PTEN is often inac-tivated by copy number loss, mutation, or epigeneticsilencing (4). In addition, the downstream target Akt hasbeen found to be amplified or mutated in human cancer(5). Over the last years, evidence of oncogenic mutationsin the gene coding for the regulatory subunit of PI3K, p85,has also been accumulating (6, 7). Last but not least,constitutively activated receptor tyrosine kinases, suchas for example, amplifiedHER2 (breast) can cause hyper-activation of the PI3K pathway (8).

The pharmaceutical industry heavily invested in the lastdecade to develop PI3K inhibitors with various profiles,such as dual mTOR/PI3K, pan-PI3K, and even isoform-specific PI3K inhibitors for clinical application. From thisplethora of molecules (9), efficacy and safety data fromphase I clinical trials have recently become available (10).

NVP-BKM120 (referred herein as BKM120) is a pan-PI3K inhibitor,whichhas recently entered clinical phase II

Authors' Affiliations: 1Disease Area Oncology, 2Center for ProteomicChemistry, 3Developmental and Molecular Pathways, Novartis Institutesfor Biomedical Research, Basel, Switzerland; 4Development andMolecularPathways, 5Oncology Drug Discovery and Preclinical Research, Sanofi-Aventis, Vitry-sur-Seine, France; and 6Disease Area Oncology, NovartisInstitutes for Biomedical Research, Cambridge, Massachusetts

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

Current address for C. Garcia-Echeverria: Oncology Drug Discovery andPreclinical Research, Sanofi-Aventis, Vitry-sur-Seine, France.

Corresponding Author: Saskia M. Brachmann, NIBR OncologyDisease Area, Novartis Pharma AG, Basel CH4002, Switzerland.Phone: 41-061-696-4063; Fax: 41-061-696-5511; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-11-1021

�2012 American Association for Cancer Research.

MolecularCancer

Therapeutics

www.aacrjournals.org 1747

on February 16, 2021. © 2012 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst May 31, 2012; DOI: 10.1158/1535-7163.MCT-11-1021

http://mct.aacrjournals.org/

for treatment of PI3K-dependent cancers (11). In mecha-nistic cellular systems, BKM120 inhibits all class IA PI3Kparalogs (p110a, b, and d) that are generally activated byreceptor tyrosine signaling. In contrast, BKM120 does notsignificantly inhibit class II and IV PI3K homologs orprotein kinases.

When tested in proliferation assays across a large panelof cell lines (the Cell Line Encyclopedia or CLE) encom-passing different lineages and oncogenic addictions,BKM120 behaved differently compared with other PI3Kinhibitors, at concentrations above 2 mmol/L. Specifically,the compoundwas efficacious against tumor lines thatdidnot display PI3K addiction. Hence, despite the fact that itsbiochemical profile is very specific, we suspected that atconcentrations 5- to 10-fold of those necessary to half-maximally modulate PI3K signaling, other propertieswere acquired. Here, we show that BKM120, at highconcentrations, can act as a microtubule destabilizer viadirect tubulinbinding. The consequences of thesefindingsfor the interpretation of in vitro and in vivo data arepresented and discussed.

Materials and MethodsCompounds, reagents, and antibodies

The structures of the compounds used in the manu-script are shown in Fig. 5B. BKM120 (Novartis), BEZ235(Novartis), and GDC-0941 (BioDuro) were prepared as 10mmol/L stock solutions in 100% dimethyl sulfoxide(DMSO).Working solutionswere freshly prepared beforeaddition to the cell media such that final DMSO concen-trations were kept constant at 0.1% in both control andcompound-treated cells. Nocodazole (#M1404) and poly-D-lysine (#P6407) were purchased from Sigma. MG-132and 40,6-diamidino-2-phenylindole (DAPI) were fromCalbiochem (#474790) and Invitrogen (#D3571), respec-tively. The origin of the primary antibodies used were asfollows: anti-S473P-Akt (#9271), anti-Histone 3-P (#9701),anti-caspase 7 (#9491), anti-alpha-tubulin (#T6199) fromCell Signaling Technologies. The anti-gamma tubulin(#T6557) and fluorescein isothiocyanate (FITC)-labeledanti-alpha tubulin (#F2128) antibodies were from Sigma.The secondary Alexa Fluor 568–conjugated anti-mouseantibody (#11031) was purchased from Invitrogen.

In vitro assaysTubulin polymerization assay. All assays were con-

ducted with the porcine tubulin polymerization kit fromCytoskeleton (#BK006-P), according to the manufac-turers’ protocol.

Nuclear magnetic resonance–binding studies. Beforestudies, lyophilized purified bovine brain tubulin (Cyto-skeleton, # TL238) was dissolved in 50 mmol/L PBS (pH7.0), without GTP and Mg2þ to prevent polymerization.BKM210 was freshly prepared as a 20 mmol/L stock solu-tion in d6-DMSO (Armar Chemicals/# 015200.2040). Thefinal concentration in nuclear magnetic resonance (NMR)samples was 0.2 mmol/L. The spectroscopy studies wereconducted on a Bruker AV-III-600 spectrometer equipped

with a QCI cryo-probe for sensitive detection of 1H and 19F.T1r experiments were recordedwith a 6 kHz spinlock pulseof 10 to 200 ms and acquisition using excitation sculptingfor water suppression. T2 experiments were measured witha CPMG pulse train of 200 ms. WaterLOGSY experimentswere measured in sensitive mode as described before (12).

Cellular biologyCell lines and cell culture. All human cell lines are

part of the Cancer Cell Line Encyclopedia from the BroadInstitute (Cambridge, MA) and have been authenticatedby 46SNP fingerprinting and expression arrays. Accord-ingly, these cell lines were obtained from the BroadInstitute (13). Cells from the original purchased vialswereexpanded and a reserve stock of 12 vials created. Masterand working stocks were prepared by the individualNovartis laboratories from 1 reserve vial and were usedfor the described studies. A2058, MDA-MB231, U87MG,MCF-7, and Rat1-myr-p110a cells (11) were cultured at37�C in 5% CO2 and 80% relative humidity in eitherDulbecco’s Modified Eagle’s Media (DMEM; MDA-MB231, A2058, MCF7, and Rat1-myr-p110a cells), EMEM(U87MG) high glucosemedia (Gibco) supplementedwith10% FBS, 2 mmol/L glutamine, 1% penicillin/streptomy-cin, and 1% sodium pyruvate. MCF7 pools expressing(MCF7-myr-Akt) or not (MCF7-BP) a hemagglutinin(HA)-tagged version of a dominant-active form, myris-toylated form of Akt, were generated upon infection ofparental MCF7 cells with viral particles generated from apBabe-puro–based retroviral expression vector (materialand sequences are available on request).

Proliferation assays, cell lysate preparation forWesternblotting, and S473P-Akt reverse-phase protein array phos-phorylation assays. Antiproliferative activities (GI50) aswell as cell death markers (LD0 and LD50) were quantifiedby methylene blue staining, as described (14). Biochemicalcharacterization upon compound exposure was conduct-ed on the mentioned cells seeded in 10-cm dishes at theindicated inoculum. Cells were exposed either for either1 hour (for PI3K pathway markers) or 6 hours (for G2–Mmarkers), before lysis for Western blotting or reverse-phase protein array (RPA) analysis as described (14).

Colony formation and fluorescence-activated cell-sort-ing assays. The fluorescence-activated cell-sorting(FACS) assays were conducted as described (15). Colonyformation assays were conducted by seeding 5 � 103

MCF7-BP of MCF7-myr-Akt cells in 6-well clusters. Six-teen hours later, the mediumwas discarded and replacedwith 2 mL of fresh medium containing the test items. Themedia were replaced every 3 days throughout the exper-iment. The experiment was stopped by adding 500 mL of20% glutaraldehyde to the media. Ten minutes later, thewells were washed with water and exposed to a 0.05%methylene blue solution for 15 minutes. Wells were thenwashed with water and colonies photographed with aCanosan 4400F scanner.

Immunofluorescence of tubulin networks. Cells wereseeded on 6-well dishes containing poly-D-lysine–treated

Brachmann et al.

Mol Cancer Ther; 11(8) August 2012 Molecular Cancer Therapeutics1748




coverslips. For investigating effects on the mitotic tubu-lin network, cells were treated with the indicatedinhibitors either for 24, 6, or 6 hours followed by an18-hour washout period. Cells were fixed for at least 15minutes with ice-cold methanol (�20�C), washed 3�with PBS, and blocked for 10 minutes with 3% bovineserum albumin (BSA)/PBS at room temperature. Theprimary antibody of choice was incubated in a moistchamber for 3 hours at room temperature (diluted 1:500in blocking solution), washed 3 times with PBS, andincubated for 1 hour at room temperature with thesecondary antibody (diluted 1:400 in blocking solution)and DAPI (diluted 1:1,000) in a moist, light-protectedchamber. The cells were washed 3 times with PBS andmounted with a drop of prolong gold-antifade (Invitro-gen, Ref# P36930) on glass. The next day, coverslips weresealed with nail polish, and the cells were analyzed andphotographed with a Zeiss Axioplan microscope. Fortubulin network in interphase cells, the microtubulenetwork of the cells was challenged by transferring theplates for 1 hour from 37�C to 4�C and switched back to37�C for 1 hour in presence or absence of the indicatedconcentration of the test item. Cells were fixed, stained,and analyzed for the effects of the treatment conditionson the microtubule network as above.

Gene expression analysismRNA extraction and microarray profiling. mRNA

was extractedwith the QiaShredder and RNeasyMini Kit(Qiagen/#79656 and #74104, respectively), according tothe manufacturer’s protocol. Synthesis of labeled cDNA,hybridization to HG-U133-plus2 arrays (Affymetrix Inc.),and quality control and processing using the MAS5 algo-rithm were done essentially as described previously (16).Microarray data are available at the Gene ExpressionOmnibus (GEO) database under the accession numberGSE33643.Expression data analysis. Analysis was restricted to

Affymetrix probe sets mapping unambiguously to singleEntrez gene IDs (NetAffx annotation version na29). Fur-thermore, when multiple probe sets were assigned to thesame Entrez gene IDs, only those with highest values(percentile 90) in an internal reference data set of 5216HG-U133-plus2 arrays were kept. Data were log2-trans-formed, and a subsequent filter (median > 2.25) wasapplied to exclude low expression genes, decreasing thetotal number of analyzed genes to 14,104. Principal com-ponent analysis was run using the Partek Genomics Suite6.4 (Partek Inc.) using the default parameters (dispersionmatrix, correlation; normalized eigenvectors). To gener-ate the BKM120 off-target effect gene list, the loadings ofthe second and third components, which maximized theseparation between the BKM120 IC90/max sample groupand the rest of the samples, were used. Gene scores werederived from the loadings by taking the absolute values ofthe sumof the loadings for each gene. The gene list rankedaccording to this score was submitted to a gene set enrich-ment analysis (GSEA).

GSEAwas conductedwith an in-house implementationof Mootha’s method using the 2-sample Wilcoxon rank-sum test (17, 18) using theMetaCore database byGeneGO,Inc. The enrichment score was divided by the square rootof the set size to adjust for the set size bias as suggested inthe work of Newton and colleagues (19). Calculationswere conducted with R (20), final results were plottedwith Spotfire (TIBCO Spotfire Inc.).

In vivo studiesCompound preparation. BKM120 was formulated in

NMP/PEG300 (10/90, v/v). Solutions were freshly pre-pared for each day of dosing by dissolving the powder,first in NMP with sonication and then by adding theremaining volume of PEG300.

In-life experimentation, analytic, and immunohis-tochemistry. All aspects of in-life experimentation, ana-lytic, preparation of tumors for immunohistochemistry aswell as section stainingweredescribedpreviously (14, 15).For pHistone H3 immunohistochemistry, tissue sectionsamples were stained with the anti-phospho Histone H3Ser10 antibody, coverslipped, and air-dried. Stained sec-tions were scanned (�20 magnification) using an Aperioscanner and the ImageScope software (Aperio) for imageacquisition and automatic exclusions of regions withdominant necrosis. Quantification of the staining usedthe Novartis in-house software ASTORIA and this wasused to establish the mitotic index [(number of pHistoneH3–positive nuclei/total number of nuclei) � 1,000].

ResultsComparison of BKM120 with another pan-PI3Kinhibitor,GDC-0941, across a largepanel of cell lines

We compared the sensitivity profile of BKM120 withanother class I PI3K inhibitor, GDC-0941 (21, 22) in a panelof 381 cell lines from the Novartis/Broad Institute CLE.The results are represented using the density distributionof theAmax (efficacy) and the crossing point (potency) forboth compounds (Fig. 1). We observed a shift to the rightof the density distribution of the crossing point forBKM120, indicating that the compound is generally lesspotent thanGDC-0941 butwe also noted a significant shiftof theAmaxdensitydistribution forGDC-0941 indicating,that BKM120 is overall more efficacious than GDC-0941.By setting thresholds of sensitivity using the medianefficacy and potency of BKM120, 21 cell lines are definedas sensitive to GDC-0941, whereas 131 cell lines are sen-sitive to BKM120 (Supplementary Fig. S1). However, asGDC-0941 is more potent than BKM120 in inhibiting Aktphosphorylation and proliferation of PI3K-addicted celllines (Figs. 2 and 3A), we speculated that BKM120 carriesactivities beyond targeting PI3K.

BKM120 exhibits an off-target activity at highconcentrations that is not related to PI3Kinhibition

To further characterize the potential off-target activitiesof BKM120, we determined the effects on pathway

BKM120 Alters Microtubule Dynamics at High Concentrations

www.aacrjournals.org Mol Cancer Ther; 11(8) August 2012 1749




inhibition and cell proliferation and viability in both PI3K(PIK3CA-mutant MCF7 cell line) and non–PI3K-addicted(PTEN-mutant/BRAF-mutant A2058 cell line)models. Asexpected, in MCF7 cells, both BKM120 (Fig. 2A, top left)and GDC-0941 (Fig. 2A, top right) displayed potent anti-proliferative activity (GI50 ¼ 160� 91 and 52� 8 nmol/L,respectively), as well as efficient cell killing, as judged bythe reduction of the cell number below the initial seedingnumber (LD0 ¼ 415 � 193 and 207 � 78 nmol/L, respec-tively; LD50 ¼ 980 � 273 and 678 � 220 nmol/L, respec-tively). In contrast, BKM120 (Fig. 2A, bottom left) but notGDC-0941 (Fig. 2A, bottom right)was capable of inducingrobust cell death in A2058 cells at high concentrations(LD50 ¼ 2,996 � 187 and >20,000 nmol/L, respectively),despite the fact that GDC-0941 was more efficient thanBKM120 in reducing Akt phosphorylation levels (IC50 ¼114 � 3 and 636 � 36 nmol/L, respectively).

To further elaborate on the hypothesis that additionalproperties besides PI3K inhibition were involved in thecell killing effects observed at high concentrations in non–PI3K-addicted models, similar studies were conducted ingenetically engineeredMCF7 cells overexpressing a dom-inant-active form (MCF7-myr-Akt) of the downstreamPI3K effector Akt (Supplementary Fig. S2A). In contrastto the MCF7 control cell pool (MCF7-BP), both BKM120and GDC-0941 were less efficient in inhibiting the path-way, showing that the exogenously expressed myr-Aktprotein was bypassing PI3K dependence for its activation(Supplementary Fig. S2B). Inproliferation assays (Fig. 2B),MCF7-myr-Akt cells were found to be less sensitive thanMCF7-BP cells to GDC-0941 (GI50 ¼ 270 � 18 and 29 � 10nmol/L, respectively) and BKM120 (GI50 ¼ 299 � 68 and76 � 17 nmol/L, respectively) resulting in a 9- and 4-foldshift in GI50, respectively. Moreover, while MCF7-myr-Akt cells were completely resistant to cell death whenexposed to GDC-0941 (LD0 and LD50 > 10,000 nmol/L),

BKM120 treatment still led to efficient cell killing (LD0 ¼1,535� 157 nmol/L). Similarly, the expression ofmyr-Aktcaused a shift in sensitivity to both GDC-0941 (Fig. 2C,top) and BKM120 (Fig. 2C, bottom) in colony formationinhibition. However, while treatment with 2 mmol/LBKM120 completely inhibited colony formation, the sameconcentration of GDC-0941 was less efficacious in thisPI3K-resistant model.

Overall, these data suggest that in cells, BKM120 dis-plays activities independent of PI3K inhibition at concen-trations equal or higher than 2 mmol/L.

The off-target activity of BKM120 is linked tomitosis

To identify additional targets of BKM120, global geneexpression profiles for BKM120, GDC-0941, and forthe dual mTOR/PI3K inhibitor NVP-BEZ235 (BEZ235)were established upon exposure to concentrations cor-responding to different degrees of pathway inhibition(50% or 90% inhibition, as judged by reduction of pAktlevels) in the A2058 cell line (Fig. 3A, left). Principalcomponent analysis of the microarray data revealed thatconcentrations of BKM120 leading to 50% pathwaymodulation induced similar expression profiles as con-centrations of GDC-0941 leading to either 50% or 90%pathway modulation. Treatment with BEZ235 causedsimilar (at IC50) or even stronger changes (at IC90) tothose caused by GDC-0941 (at IC90), but within the samedirectionality (Fig. 3A, middle). However, the maximalconcentration of BKM120 tested (IC90, dark red sym-bols) displayed a strong outlier behavior characterizedby changes in gene expression not related to thoseobserved with the 2 other inhibitors at any concentra-tion (Fig. 3A, right). Thus, high concentrations ofBKM120 elicit changes in additional sets of transcriptscompared with other PI3K inhibitors.

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Figure 1. Density distributions of the Amax (A) and the crossing point on log scale (B) for BKM120 (solid line) and GDC-0941 (dashed line). A, the shiftto the right of the peak of GDC-0941 indicates that BKM120 is more efficacious than GDC-0941. B, the small shift to the right of the peak of BKM120indicates that BKM120 is less potent than GDC-0941. The higher peak for BKM120 indicates again that BKM120 is killing more cell lines overall.Cell lines that were not responding to GDC-0941 and did not reach a crossing point in the dose–response curve were assigned the maximumconcentration tested value (i.e., 8 mmol/L), explaining the second peak at crossing point of 8 mmol/L for GDC-0941.

Brachmann et al.





To identify gene sets linked to the transcriptional effectsinduced upon exposure to high concentrations ofBKM120, the most significantly changed transcripts(versus all other conditions)were identified and subjectedto a GSEA (Fig. 3B). Interestingly, the gene sets withhighest scorewere found to be related to cell cycle, spindleassembly, and the metaphase checkpoint. Overall, theseresults suggest that at high concentrations, BKM120 dis-plays activities that might have an impact on G2–Mprogression.

BKM120 blocks the prometaphase to metaphasetransition?To testwhetherBKM120 could cause amitotic block, the

effects on the cell cyclewere analyzed inA2058 cells usingBKM120orGDC-0941 at concentrations sufficient to cause

complete pathway inhibition (10-fold the IC50 for phos-pho-Akt inhibition). Treatment with GDC-0941 had noeffect on the cell cycle, whereas treatment with BKM120led to a significant increase in the G2–M population, incomparison with control untreated cells (Fig. 4A). Theincrease in G2–M occurred in a dose-dependent manner,but the concentration required to achieve half of this effect(EC50) was 8-fold higher than the concentration needed toreach the EC50 on PI3K pathway inhibition (measured bypAkt levels; Supplementary Fig. S3A). Furthermore, treat-ment with either BEZ235 or GDC-0941 at concentration ashigh as 5 mmol/L had no effect on the cell-cycle distribu-tion (Supplementary Fig. S3B).

Phenotypic analysis of the A2058 cells using immuno-fluorescence analysis revealed that treatment with 5mmol/L of BKM120 (but not with GDC-0941) induced the

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Figure 2. BKM120 elicits activities in non–PI3K-addicted models independent of its PI3K-inhibitory functions. A, the indicated cell lines were seeded either in96-well clusters (20,000 cells per well, A2058 andMCF7 for viability assays, and A2058 for pAkt level determination) or in 10-cm dishes (MCF7 cells, 5� 106

cells per plates, for pAkt level determination) and incubatedwith the indicated compound for either 72or 1hour. At this stage, cellswerefixed (viability assay) orlysed (S473P-Akt RPAquantification assay) and effects on either viability (left, y-axis) or onS473P-Akt levels (right, y-axis), respectively,were plotted. B andC,engineered MCF7-BP and MCF-myr-Akt were seeded (10 � 104 cells) either in 96-well clusters (B) or in a 6-well cluster (C) and incubated with theindicated compoundsat the indicatedconcentrations either for 72hours (B) or 2weeks (C). At this stage, cellswere fixedandeffects onviability (B) aswell asoncolony formation (C)were assessed. Thedashedgray line represents theLD0 value, that is, to say the concentration of the compound responsible for completegrowth inhibition (i.e., values below 100% are reflective of active cell killing) as this represents the amount of cells initially present at the addition of thecompound as well as complete pAkt level reduction. The dark plain line represents the LD50 value, that is, to say the concentration of the compound neededto kill 50% of the cells present at the addition of the compound.






accumulation of mitotic cells. Most cells displayed dupli-cated centrosomes (determined by g-tubulin staining),early bi- and multipolar spindles (determined by a-tubu-lin staining), and condensed but not fully aligned DNA,indicating early mitotic phases (Fig. 4B). Similar effectswere also observed with BKM120 in the K-RAS mutantMDA-MB231 and PTEN null U87MG cell lines (Supple-mentary Fig. S4). Interestingly, treatment of these cellswith the microtubule destabilizer nocodazole caused aremarkably similar phenotype. These results suggest thatat high concentrations, BKM120 causes a prometaphase tometaphase arrest in a PI3K-independent manner.

BKM120 inhibits tubulin polymerizationTo test whether BKM120 might influence microtubule

dynamics, potential effects on tubulin polymerizationwere analyzed. Cells were preincubated at 4�C to causeperipheral microtubule depolymerization followed by aswitch back to 37�C, either in presence or absence of

inhibitors, to allow repolymerization of the microtubulenetwork to the rim of the cells (Fig. 5A). In contrast toGDC-0941, incubation with BKM120 or nocodazoleenhanced the loss of the microtubule network in the cellperiphery, showing that BKM120 exhibits microtubule-destabilizing activity.

TodeterminewhetherBKM120woulddirectly interferewith microtubule polymerization, in vitro polymerizationassays using purified tubulin were conducted. Asexpected, themicrotubule stabilizer paclitaxel significant-ly increased the tubulin polymerization kinetics, whereasnocodazole caused the opposite effects (SupplementaryFig. S5A). Interestingly, and in contrast to GDC-0941,BKM120 decreased the tubulin polymerization kineticsin a concentration-dependent manner (Fig. 5C).

To further show direct binding of BKM120 to tubulin,assessment of direct interactions upon changes in relax-ation of resonances was conducted by NMR spectro-scopy (Fig. 5D). T1r (left) and T2 (19F, right) relaxation

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Figure 3. Transcriptomic profile of A2058 cells upon treatment of PI3K inhibitors BEZ235, GDC-0941, and BKM120. A, A2058 cells were treated withequipotent concentrations of the PI3K inhibitors BKM120 (IC30, IC50, IC90), GDC-0941 (IC50, IC90, and a third concentration called "max" corresponding to theIC90 of BKM120), aswell as BEZ235 (IC50, IC90,max¼ IC90 of BKM120) based on S473P-Akt inhibition. Principal component analysis of transcript expressiondata showing good reproducibility of biologic replicates and clear clustering of different treatment conditions. Left, plotting PC1 versus PC2 shows aclear continuous effect of the compounds along PC1 dimension in line with increasing pathway inhibition. BKM120 at off-target concentrations (dark red)formsanoutlier cluster. Right, plottingPC2 versusPC3strongly supports outlier behavior of highBMK120 concentrations, comparedwith all other conditions.B, to identify gene sets linked to the BKM120 off-target effect genes most strongly associated with PC2 and PC3 of the principal component analysis havebeen submitted to aGSEA.Size of thegenesets (horizontal axis) is plotted against strengthof enrichment (vertical axis). Circles,GeneGOprocesses; triangles,GeneGO pathway maps.

Brachmann et al.





experiments upon addition of freshly prepared tubulin toBKM120 (20-fold excess) showed an enhancement of therelaxation. These effects were tubulin concentration–dependent (Supplementary Table S1) and were furtherconfirmed by waterLOGSY relaxation experiments (Fig.5C, left). The tubulin/BKM120 interactionwas found tobein fast exchange as observed for other tubulin ligands (23).Furthermore, relaxation competition studies could notshow binding to the colchicine site, when well-describedtubulin colchicine site binders were used as competitors(Supplementary Fig. S5B).

The microtubule-destabilizing activity of BKM120does not translate to antitumor activity in vivoWe previously showed that BKM120 was able to cause

significant regressions in themechanistic Rat1-myr-p110ain vivo model, when dosed once per day at doses of40 mg/kg and above (T/C of �25% and �48% at 40 and50 mg/kg, respectively; ref. 11). To test whether at thesedose levels, the exposure of BKM120 would have reachedconcentrations to engage its off-target (tubulin-binding)activity, tumors were fixed and stained for phospho-

Histone H3 levels as a mitotic marker, and the mitoticindex was calculated. In cellular assays, a strong increasein phospho-Histone H3 levels could be observed as earlyas 6 hours (Supplementary Fig. S6). In vivo, (Rat1-myr-p110a tumor model), no mitotic index increase was evi-dent at the 40 mg/kg dose [plasma area under the curve(AUC): 65 hmmol/L), up to 16 hours after last doseadministration. This result suggests that the tumor regres-sion (which is accompanied with a robust increase incaspase-7 cleavage) seen upon the exposure to BKM120at the dose of 40 mg/kg (Fig. 6A, right) is due to the solemerit of PI3K inhibition. However, a 2.5-fold transient(6 hours but not anymore at 16-hour time point) andstatistically significant increase in mitotic index to 5%could be observed for the 50 mg/kg dose (plasma AUC:75 hmmol/L; Fig. 6A, left). To assess whether such a mildand transient increase in mitotic index at 50 mg/kg trans-lates into efficacy, a similar study was repeated in thePTEN-null U87MG tumors. Daily treatment of BKM120resulted in antitumor activity with T/C of 20% and 7% at40 and 50mg/kg, respectively, but these differences werenot statistically different (P > 0.05; Supplementary

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Figure4. BKM120provokesaprometaphase tometaphaseblock.A, a total of 2�106A2058cellswere seeded in 10-cmdishes and incubated for 24hourswiththe indicated compounds. Cells were then fixed and prepared as described for quantification of the population in the different phases of the cell cycleby FACS. B, A2058 cells grown on coverslips were treated for 24 hours either with BKM120 (5 mmol/L) or nocodazole (100 nmol/L). Effects on microtubuledynamics and G2–M arrest was monitored by immunofluorescence staining of a-tubulin (microtubules), g-tubulin (centrosomes), and DAPI (DNA). Pictureswere taken with a �100 objective.






Fig. S6B). As in the Rat1-myr-p110a model, a similartransient and statistically significant increase in mitoticindex to 3% was observed at the 50 mg/kg dose level(Fig. 6B, left). Importantly, no increase in caspase-7 cleav-age was observed at the 40 and the 50 mg/kg dose levels(Fig. 6B, right). Altogether, these data suggest that theexposure of BKM120 at a dose of 50 mg/kg might reachsufficient blood/tumor levels to engage the off-targetactivity in the first 6 hours following administration,which then cause a mild and transient mitotic index(3% to 5%). In contrast to BKM120, other microtubule-binding agents such as paclitaxel cause peaks of mitoticindex that range between 10% and 25% (Milas and col-leagues; ref. 24). Furthermore, BKM120-induced mitoticblock seemed to be reversible as soon as the compoundgets cleared as no increase of mitotic index was observed

16 hours after compound administration. This reversibil-ity was also observed in in vitro pulse chase studies whereBKM120 was washed out (Supplementary Fig. S7).

DiscussionOur studies show that sustained exposure to BKM120 at

concentrations above 1 mmol/L engages PI3K-indepen-dent activities, resulting in enhanced antiproliferative andcell killing effects. Biochemical and transcriptomic profil-ing studies with BKM120 and other PI3K inhibitors suchas BEZ235 or GDC-0941 pointed to a unique role ofBKM120 in regulating microtubule dynamics causing aprometaphase to metaphase block in cell lines withoutstrong PI3K addiction where ontarget PI3K inhibition isnot able to induce apoptosis.

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Figure 5. BKM120 directly binds to tubulin and inhibits tubulin polymerization. A, rat-myr-p110a cells grown on coverslips were switched from37�C to 4�C for 1 hour and then switched back for 1 hour to 37�C, in the presence of either DMSO control, BKM120 (5 mmol/L), nocodazole(100 nmol/L), or GDC-0941 (5 mmol/L). Cells were then fixed, and effects on microtubule stability were visualized by immunofluorescence staining ofa-tubulin. B, chemical structures of BEZ235, GDC-0941, nocodazole, BKM120, and labeled BKM120 used in D. C, purified tubulin was mixed withthe indicated compounds at the indicated concentrations in the presence of GTP. The polymerization of monomeric tubulin into microtubulewas started by transferring the reaction tubes from 4�C to 37�C and monitored by the increase in absorbance (l ¼ 340 nm) over a period of60 minutes. Of note, this assay requires high concentration of compound to be in stoichiometry to the high amount of purified tubulin that is used inthis low-sensitivity polymerization assay. D, NMR spectroscopy shows binding of BKM120 to tubulin. Weak T1r and negative waterLOGSY signalswere observed for the compound in the absence of tubulin (bottom left), whereas significant T1r relaxation and positive waterLOGSY signalswere observed in the presence of tubulin (top). �, impurities in the buffer. The assignment of BKM120 is indicated. T2-relaxation enhancement of19F due to tubulin binding is evident after adding tubulin in a concentration-dependent manner (right).

Brachmann et al.





Microtubule stabilizers (such as paclitaxel and deriva-tives) and destabilizers (such as vinca-alkaloids ornocodazole) are known to activate the spindle assemblycheckpoint leading to an arrest of cells in mitosis andsubsequent cell killing probably as a result of induction ofmitotic catastrophe. These agents have been used formany years as antineoplastic therapy for various typesof cancers (25). The phenotype detected upon BKM120treatment at high concentrationwas highly reminiscent ofthat observeduponnocodazole treatment, suggesting thatBKM120 also interferes with microtubule dynamicsdirectly (i.e., by tubulin-binding capacities) or indirectly(i.e., by blocking the activities of factors associated to thefunctions of microtubules such as the kinesin Eg5).Direct binding of BKM120 to pure tubulin was showed

using in vitro tubulin polymerization assays and furtherconfirmed by NMR studies.Different microtubule-targeting agents have distinct

binding modes andmechanisms of actions. Taxanes pref-erentially bind to polymerized b-tubulin and more pre-cisely at the inner surface of the microtubules (26). Vinca-

alkaloids bind at the interface of a 2 a/b tubulin hetero-dimers, at theþ endmicrotubules (27). A third category oftubulin interactors bind to the so-called colchicinedomain, which mostly lies within the b-tubulin subunit(28, 29). Taxanes and vinca-alkaloids are both high-molec-ular-weight molecules and derivatives of natural pro-ducts. In contrast, colchicine site binders are generallysmall molecules (30), hence, we hypothesized thatBKM120 might share similar binding modalities. How-ever, NMR competition studies did not confirm thishypothesis. Further structural studies will be needed toelucidate BKM120-binding mode to tubulin.

The pronounced spindle assembly defects in mitosisseen in vitro were not observed in vivo after multipleadministrations of BKM120 at a dose of 40 mg/kg.Interestingly, at a dose of 50 mg/kg, a small and tran-sient increase of the mitotic index was observed. Thesefindings suggest that BKM120 displays tubulin-bindingand microtubule-destabilizing activities only above acertain concentration/exposure threshold. Further-more, the fast binding kinetics to tubulin as well as the

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Figure 6. Treatment with BKM120 leads to a transient increase in mitotic markers. A and B, rat1-myr-p110a (A) or U87MG (B) tumor–bearing animalswere treated orally with the indicated dose of BKM120, once per day for a period of 6 days. Upon last treatment, animals were sacrificed at the indicatedtime points for collection of plasma and tumor tissues. Compound concentration in plasma (left, left y-axis) as well as quantification in tumors ofpHistone H3 levels and subsequent determination of the mitotic index (MI; left, right y-axis) were plotted; effects on apoptosis were also assessed byimmunohistochemistry by staining of tumor sections with an anti-cleaved caspase-7 antibody (right).






intrinsic compound clearance probably result in revers-ibility of the mitotic effects, as no accumulation of G2–Marrested cells could be detected following chronicBKM120 administration. It therefore appears thatBKM120 can cause regression in PI3K-dependenttumors when administered orally to animals bearingsubcutaneous tumors without engaging the tubulin off-target activity to a sufficient level to contribute to thetherapeutic effect.

It is interesting to observe that the plasma exposure inpatients treated with BKM120 at the efficacious maxi-mum tolerated dose (100 mg, AUC: 56 hmmol/L; ref. 31),lies below the exposure necessary to transiently engagethe off-target in a mouse model (AUC > 65 hmmol/L).These findings strongly argue that in patients, thethreshold for microtubule-destabilizing activity of thiscompound is not reached. Therefore, it is anticipatedthat efficacy in patients will solely stem from PI3Kinhibition. Further analysis of clinical data, such as theassessment of mitotic markers in tumor biopsies frompatients treated with BKM120, will be required to fullyconfirm that the compound off-target activity is notclinically relevant.

Disclosure of Potential Conflicts of InterestW.R. Sellers is employed as VP/Global Head of Oncology and has

ownership interest (including patents) in Novartis Institutes for BioMed-ical Research.All authors except J. Kleylein-Sohn andC.Garcia-Echeverriaare Novartis employees. J. Kleylein-Sohn is now employed at the FMI andC. Garcia-Echeverria is now employed at Sanofi-Aventis.

Authors' ContributionsConception and design: S.M. Brachmann, J. Kleylein-Sohn, S. Gaulis, C.Fritsch, S.-M. MairaDevelopment of methodology: M. Hattenberger, V. Romanet, D. Guthy,C. Wilson, S.-M. MairaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S.M. Brachmann, M.J.J. Blommers, M. Kazic-Legueux, L. Laborde, F. Stauffer, J. Vaxelaire, V. Romanet, C. Henry, M.Murakami, D. Sterker, C. Wilson, S.-M. MairaAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis):S.M. Brachmann, J.Kleylein-Sohn, S.Gaulis,A. Kauffmann,M.J.J. Blommers, V. Romanet,M.Murakami, S. Bergling, C.Wilson, T. Bruemmendorf, F. Hofmann, S.-M. MairaWriting, review, and/or revision of the manuscript: S.M. Brachmann, A.Kauffmann, M.J.J. Blommers, T. Bruemmendorf, C. Garcia-Echeverria, W.R. Sellers, F. Hofmann, S.-M. MairaAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): L. Laborde, M. Hattenberger, J.Vaxelaire, D. Sterker, S.-M. MairaStudy supervision: S.M. Brachmann, C. Garcia-Echeverria, S.-M. Maira

AcknowledgmentsThe authors thank Andreas Bauer, Hans Voshol, Christian Schnell,

Markus Wartmann, Patrick Chene, Thomas Radimerski, and Pascal Furetfor helpful discussions; Dr. Levi Garraway (Dana-Farber Cancer Institute,Boston, MA) for the inspiring comments and recommendations to usetools such as themyr-Akt transduced cells for better characterization of theBKM120 activities in cells; the Genomics Technology group of NIBR Baselfor the excellent collaboration, in particular, Nicole Hartmann, ClarisseWache-Mainier, and Frank Staedtler; and Sabina Cosulich for revising themanuscript.

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

Received December 15, 2011; revised March 30, 2012; accepted May 9,2012; published OnlineFirst May 31, 2012.

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2012;11:1747-1757. Published OnlineFirst May 31, 2012.Mol Cancer Ther Saskia M. Brachmann, Julia Kleylein-Sohn, Swann Gaulis, et al. ConcentrationsPI3K Inhibitor NVP-BKM120 across a Broad Range of Characterization of the Mechanism of Action of the Pan Class I

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