Characterization of the mechanism of action of the pan ......May 30, 2012 · MCT-11-1021 BKM120 alters microtubule dynamics at high concentrations 1 Characterization of the mechanism

MCT-11-1021 BKM120 alters microtubule dynamics at high concentrations

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Characterization of the mechanism of action of the pan class I PI3K

inhibitor NVP-BKM120 across a broad range of 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, Chrystèle Henry6, Masato Murakami1, Daniel Alexander Guthy1, Dario Sterker1, Sebastian Bergling3, Christopher Wilson3, Thomas Brümmendorf1, Christine Fritsch1, Carlos Garcia-Echeverria4, William R. Sellers5, Francesco Hofmann1 and Sauveur-Michel Maira1 1Novartis Institutes for Biomedical Research, Disease Area Oncology, CH4002 Basel,

Switzerland

2Novartis Institutes for Biomedical Research, Center for Proteomic Chemistry, Novartis

Pharma AG, Forum 1, Novartis Campus, CH4056 Basel, Switzerland

3Novartis Institutes for Biomedical Research, Development and Molecular Pathways, 250

Massachusetts Avenue, Cambridge, MA02139, USA 4current address: Oncology Drug Discovery and Preclinical Research, Sanofi-Aventis, Vitry-

sur-Seine, France 5Novartis Institutes for Biomedical Research, Disease Area Oncology, 250 Massachusetts

Avenue, Cambridge, MA 02139, USA 6Novartis Institute for Biomedical Research, Developmental and Molecular Pathways,

Fabrikstrasse 22, Novartis Campus, CH4056 Basel, Switzerland

Running title: BKM120 alters microtubule dynamics at high concentrations.

on June 5, 2021. © 2012 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on May 31, 2012; DOI: 10.1158/1535-7163.MCT-11-1021

http://mct.aacrjournals.org/


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Corresponding author and requests for reprints: Saskia Brachmann, NIBR Oncology Disease

Area, Novartis Pharma AG, CH4002 Basel, Switzerland, tel +41 61 696 4063, e-mail:

[email protected]

Conflict of interest: all authors except of JKS and CGE are Novartis employees. JKS is now

employed at the FMI and CGE is now employed at Sanofi-Aventis.





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Abstract

The pan-PI3K inhibitor BKM120 was found, at high concentrations, to cause cell death in

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

biochemical profiling studies were used to identify the origin of these unexpected and

apparently PI3K 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

NMR binding studies revealed that BKM120 inhibited microtubule dynamics upon direct

binding to tubulin. To assess the contribution of this off-target activity vis-à-vis the anti-tumor

activity of BKM120 in PI3K-dependent tumors, we used a mechanistic PI3K-alpha dependent

model. We observed that, in vivo, daily treatment of mice with doses of BKM120 up to 40

mg/kg lead to tumour regressions with no increase in the mitotic index. Thus, strong anti-

tumor activity can be achieved in PI3K-dependent models at exposures that are below those

necessary to engage the off-target activity. By 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 μM 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.





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Introduction

The PI3K pathway plays a pivotal role in cell growth, proliferation, survival and metabolism

(1, 2). Lesions in key pathway components can lead to gain-of-function, pathway hyper-

activation, aberrant cell proliferation and subsequently to the promotion and maintenance of

cancer. For example, the PIK3CA gene encoding the p110α catalytic subunit has been found

to be amplified and frequently mutated in a variety of human cancers (3). Furthermore, the

antagonistic dual lipid/protein phosphatase PTEN is often inactivated by copy number loss,

mutation or epigenetic silencing (4). In addition, the downstream target Akt has been found

amplified or mutated in human cancer (5). Over the last years, evidence of oncogenic

mutations in 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, such

as for example amplified HER2 (breast) can cause hyper-activation of the PI3K pathway (8).

The pharmaceutical industry heavily invested in the last decade to develop PI3K inhibitors

with various profiles, such as dual mTOR/PI3K, pan-PI3K and even isoform specific PI3K

inhibitors for clinical application. From this plethora of molecules (9), efficacy and safety data

from phase I clinical trials has recently become available (10).

NVP-BKM120 (referred herein as BKM120) is a pan PI3K inhibitor which has recently

entered clinical phase II for treatment of PI3K dependent cancers (11). In mechanistic cellular

systems, BKM120 inhibits all Class IA PI3K paralogs (p110α, β and δ) that are generally

activated by receptor tyrosine signaling. In contrast, BKM120 does not significantly inhibit

class II and IV PI3K homologs or protein kinases.





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When tested in proliferation assays across a large panel of cell lines (the Cell Line

Encyclopedia, or CLE) encompassing different lineages and oncogenic addictions, BKM120

behaved differently compared to other PI3K inhibitors, at concentrations above 2 μM.

Specifically, the compound was efficacious against tumor lines that did not display PI3K

addiction. Hence, despite the fact that its biochemical profile is very specific, we suspected

that at concentrations 5- to 10-fold of those necessary to half-maximally modulate PI3K

signaling, other properties were acquired. Here we demonstrate that BKM120, at high

concentrations, can act as a microtubule destabilizer via direct tubulin binding. The

consequences of these findings for the interpretation of in vitro and in vivo data are presented

and discussed.





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Materials and Methods

Compounds, reagents and antibodies

The structures of the compounds used in this manuscript are shown in Figure 5B. BKM120

(Novartis), BEZ235 (Novartis) and GDC-0941 (BioDuro, Beijing, China) were prepared as 10

mM stock solutions in 100% DMSO. Working solutions were freshly prepared prior to

addition to the cell media such that final DMSO concentrations were kept constant at 0.1% in

both control and compound-treated cells. Nocodazole (#M1404) and poly-D lysine (#P6407)

were purchased from Sigma. MG-132 and DAPI were from Calbiochem (#474790) and

Invitrogen (#D3571), respectively. The origin of the primary antibodies used were as follows:

anti-Akt-S473P (#9271), anti-Histone 3-P (#9701), anti-caspase 7 (#9491), anti-alpha-tubulin

(#T6199), were from Cell Signaling Technologies. The Anti-gamma tubulin (#T6557) and

FITC-labeled anti-alpha tubulin (#F2128) antibodies were from Sigma. The secondary Alexa

fluor 568 conjugated anti-mouse antibody (#11031) was purchased from Invitrogen.

In vitro assays

Tubulin polymerisation assay: All assays were performed with the porcine tubulin

polymerization kit from Cytoskeleton (#BK006-P), according to the manufacturers’ protocol.

NMR binding studies: Prior to studies, lyophilized purified bovine brain tubulin

(Cytoskeleton, # TL238) was dissolved in 50 mM PBS (pH 7.0), without GTP and Mg2+ to

prevent polymerization. BKM210 was freshly prepared as a 20 mM stock solution in d6-

DMSO (Armar Chemicals / # 015200.2040). The final concentration in NMR samples was 0.2

mM. The spectroscopy studies were performed on a Bruker AV-III-600 spectrometer





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equipped with a QCI cryo-probe for sensitive detection of 1H and 19F. T1ρ experiments were

recorded with a 6 kHz spinlock pulse of 10-200 ms and acquisition using excitation sculpting

for water suppression. T2 experiments were measured with a CPMG pulse train of 200 ms.

WaterLOGSY experiments were measured in sensitive mode as described before (12).

Cellular Biology

Cell lines and cell culture: All human cell lines are part of the Cancer Cell Line Encyclopedia

from the Broad Institute and have been authenticated by 46SNP fingerprinting and expression

arrays. Accordingly, these cell lines were obtained from the Broad Institute (13). Cells from

the original purchased vials were expanded and a reserve stock of 12 vials created. Out of this,

cells and only then expanded in master and working stocks. A2058, MDA-MB231, U87MG,

MCF-7, and Rat1-myr-p110α cells (11) were cultured at 37º C in 5% CO2 and 80% relative

humidity in either DMEM (MDA-MB231, A2058, MCF-7 and Rat1-myr-p110α cells),

EMEM (U87MG) high glucose media (Gibco) supplemented with 10% fetal bovine serum, 2

mM glutamine, 1% penicillin-streptomycin and 1% sodium-pyruvate. MCF7 pools expressing

(MCF7-myr-Akt) or not (MCF7-BP) a HA-tagged version of a dominant active form,

myristoylated form of Akt, were generated upon infection of parental MCF7 cells with viral

particles generated from a pBabe-puro based retroviral expression vector (material and

sequences are available on request).

Proliferation assays, cell lysate preparation for western-blotting and S473P-Akt RPA

phosphorylation assays: Antiproliferative activities (GI50) as well as cell death markers (LD0

and LD50) were quantified by methylene blue staining, as described (14). Biochemical

characterization upon compound exposure was performed on the mentioned cells seeded in 10





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cm dishes at the indicated inoculum. Cells were exposed either for either 1 h (for PI3K

pathway markers) or 6 h (for G2/M markers), prior to lysis for Western-blotting or RPA

analysis as described (14).

Colony formation and FACS assays: The FACS assays were performed as described (15).

Colony formation assays were conducted by seeding 5 x 103 MCF7-BP of MCF7-myr-Akt

cells in 6-well clusters. 16 h later, the medium was discarded and replaced with 2 mL of fresh

medium containing the test items. The media was replaced every 3 days throughout the

experiment. The experiment was stopped by adding 500 µl of 20% glutaraldehyde to the

media. Ten minutes later the wells were washed with water and exposed to a 0.05%

methylene blue solution 15 min. Wells were then washed with water and colonies

photographed with a Canosan 4400F scanner.

Immuno-fluorescence of tubulin networks: Cells were seeded on 6 well dishes containing

poly-D-Lysine treated coverslips. For investigating effects on the mitotic tubulin network,

cells were treated with the indicated inhibitors either for 24 h, 6 h or 6 h followed by an 18 h

washout period. Cells were fixed at least 15 min with ice cold methanol (-20°C), washed 3X

with PBS and blocked for 10 min with 3 % BSA / PBS at RT. The primary antibody of choice

was incubated in a moist chamber for 3 h at RT (diluted 1/500 in blocking solution) washed 3

times with PBS and incubated for 1 h at RT with the secondary antibody (diluted 1/400 in

blocking solution) and DAPI (diluted 1/1000) in a moist, light-protected chamber. The cells

were washed 3 times with PBS and mounted with a drop of prolong gold-antifade (Invitrogen,

Ref# P36930) on glass. The next day, coverslips were sealed with nail polished, and the cells

were analyzed and photographed with a Zeiss Axioplan microscope. For tubulin network in





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interphase cells, the microtubule network of the cells was challenged by transferring the plates

for 1 h from 37º C to 4º C and switched back to 37°C for 1 h in presence or absence of the

indicated concentration of the test item. Cells were fixed, stained and analyzed for the effects

of the treatment conditions on the microtubule network as above.

Gene expression analysis

mRNA extraction and microarray profiling: mRNA was extracted with the QiaShredder and

RNA easy mini kit (Qiagen/ #79656 and #74104, respectively) according to the

manufacturer’s protocol. Synthesis of labeled cDNA, hybridization to HG-U133-plus2 arrays

(Affymetrix Inc, Santa Clara, CA, USA), quality control and processing using the MAS5

algorithm was done essentially as described previously (16). Microarray data are available at

the Gene Expression Omnibus (GEO) database under the accession number GSE33643.

Expression data analysis: Analysis was restricted to Affymetrix probe-sets mapping

unambiguously to single Entrez gene IDs (NetAffx annotation version na29). Furthermore,

when multiple probe-sets were assigned to the same Entrez gene IDs, only those with highest

values (percentile 90) in an internal reference data set of 5216 HG-U133-plus2 arrays were

kept. Data was log2-transformed and a subsequent filter (median >2.25) was applied to

exclude low expression genes, decreasing the total number of analyzed genes to 14104.

Principal Component Analysis was run using the Partek Genomics Suite 6.4 (Partek Inc, St.

Louis, MO, USA) using the default parameters (dispersion matrix, correlation; normalized

eigenvectors). To generate the BKM120 off-target effect gene list, the loadings of the second

and third components, which maximized the separation between the BKM120 IC90/Max

sample group and the rest of the samples, were used. Gene scores were derived from the





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loadings by taking the absolute values of the sum of the loadings for each gene. The gene list

ranked according to this score was submitted to a gene set enrichment analysis (GSEA).

GSEA was performed with an in-house implementation of Mootha's method using the two-

sample Wilcoxon rank-sum test (17, 18) using the MetaCore database by GeneGO, Inc, St.

Joseph, MI, USA. The enrichment score was divided by the square root of the set size to

adjust for the set size bias as suggested in (19). Calculations were performed with R (20), final

results were plotted with Spotfire (TIBCO Spotfire Inc, Somerville, MA, USA).

In vivo studies

Compound preparation: BKM120 was formulated in NMP/PEG300 (10/90, V/V). Solutions

were freshly prepared for each day of dosing by dissolving the powder, first in NMP with

sonication and then by adding the remaining volume of PEG300.

In life experimentation, analytic and immunohistochemistry: All aspects of in life

experimentation, analytic, preparation of tumors for immunohistochemistry (IHC) as well as

section staining were described previously (14, 15). For pHistoneH3 IHC, tissue section

samples were stained with the anti-phospho Histone H3 Ser10 antibody, cover-slipped and

air-dried. Stained sections were scanned (20 X magnification) using an Aperio scanner and

the ImageScope software (Aperio, SanDiego, CA) for image acquisition and automatic

exclusions of regions with dominant necrosis. Quantification of the staining used the Novartis

in house software ASTORIA, and this was used to established the Mitotix Index ((number of

pHistone H3 positive nuclei / total number of nuclei) x 1000).





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Results

Comparison of BKM120 with another pan-PI3K inhibitor, GDC-0941,

across a large panel of cell lines

We compared the sensitivity profile of BKM120 to another class I PI3K inhibitor, GDC-0941

(21, 22) in a panel of 381 cell lines from the Novartis / Broad Institute Cell Line Encyclopedia

(CLE). The results are represented using the density distribution of the Amax (efficacy) and

the crossing point (potency) for both compounds (Figure 1). We observed a shift to the right

of the density distribution of the crossing point for BKM120 indicating that the compound is

generally less potent than GDC-0941, but we also noted an significant shift of the Amax

density distribution for GDC-0941 indicating that BKM120 is overall more efficacious than

GDC-0941. By setting thresholds of sensitivity using the median efficacy and potency of

BKM120, 21 cell lines are defined as sensitive to GDC-0941 whereas 131 cell lines are

sensitive to BKM120 (supplementary figure 1). However, as GDC-0941 is more potent than

BKM120 in inhibiting Akt phosphorylation and proliferation of PI3K addicted cell lines

(Figures 2 and 3A), we speculated that BKM120 carries activities beyond targeting PI3K.

BKM120 exhibits an off target activity at high concentrations which is not

related to PI3K inhibition

In order to further characterize the potential off target activities of BKM120, we determined

the effects on pathway inhibition and cell proliferation and viability in both PI3K (PIK3CA

mutant MCF7 cell line) or non-PI3K addicted (PTEN mutant/BRAF mutant A2058 cell line)

models. As expected, in MCF7 cells, both BKM120 (Figure 2A, top left panel) and GDC-





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0941 (Figure 2A, top right panel) displayed potent anti-proliferative activity (GI50 = 160 ± 91

and 52 ± 8 nM, respectively), as well as efficient cell killing, as judged by the reduction of the

cell number below the initial seeding number (LD0 = 415 ± 193 and 207 ± 78 nM,

respectively; LD50 = 980 ± 273 and 678 ± 220 nM, respectively). In contrast, BKM120 (Figure

2A, bottom left panel) but not GDC-0941 (Figure 2A, bottom right panel) was capable of

inducing robust cell death in A2058 cells at high concentrations (LD50 = 2996 ± 187 and >

20000 nM, respectively), despite the fact that GDC-0941 was more efficient than BKM120 in

reducing Akt-phosphorylation levels (IC50 = 114 ± 3 and 636 ± 36 nM, respectively).

To further elaborate on the hypothesis that additional properties besides PI3K inhibition were

involved in the cell killing effects observed at high concentrations in non-PI3K addicted

models, similar studies were performed in genetically engineered MCF7 cells over-expressing

a dominant active form (MCF7-myr-Akt) of the downstream PI3K effector Akt (Sup Figure

2A). In contrast to the MCF7 control cell pool (MCF7-BP), both BKM120 and GDC-0941

were less efficient in inhibiting the pathway, demonstrating that the exogenously expressed

myr-Akt protein was by-passing PI3K dependence for its activation (Sup Figure 2B). In

proliferation assays (Figure 2B), MCF7-myr-Akt cells were found to be less sensitive than

MCF7-BP cells to GDC-0941 (GI50 = 270 ± 18 and 29 ± 10 nM, respectively) and BKM120

(GI50 = 299 ± 68 and 76 ± 17 nM, respectively) resulting in a 9- and 4-fold shift in GI50,

respectively. Moreover, while MCF7-myr-Akt cells were completely resistant to cell death

when exposed to GDC-0941 (LD0 and LD50 >10000 nM), BKM120 treatment still led to

efficient cell killing (LD0 = 1535 ± 157 nM).Similarly, the expression of myr-Akt caused a

shift in sensitivity to both GDC-0941 (Figure 2C, upper panel) and BKM120 (Figure 2C,

lower panel) in colony formation inhibition. However, while treatment with 2µM BKM120





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completely inhibited colony formation, the same concentration of GDC-0941 was less

efficacious in this PI3K resistant model.

Overall, these data suggest that in cells, BKM120 displays activities independent of PI3K

inhibition at concentrations equal or higher than 2 μM.

The off-target activity of BKM120 is linked to mitosis

In order to identify additional targets of BKM120, global gene expression profiles for

BKM120, GDC-0941 and for the dual mTOR/PI3K inhibitor NVP-BEZ235 (BEZ235) were

established upon exposure to concentrations corresponding to different degrees of pathway

inhibition (50 or 90% inhibition, as judged by reduction of pAkt levels) in the A2058 cell line

(Figure 3A, left panel). Principal component analysis of the microarray data revealed that

concentrations of BKM120 leading to 50% pathway modulation induced similar expression

profiles as concentrations of GDC-0941 leading to either 50 or 90% pathway modulation.

Treatment with BEZ235 caused similar (at IC50) or even stronger changes (at IC90) to those

caused by GDC-0941 (at IC90), but within the same directionality (Figure 3A, middle panel).

However, the maximal concentration of BKM120 tested (IC90, dark red symbols) displayed a

strong outlier behavior characterized by changes in gene expression not related to those

observed with the two other inhibitors at any concentration (Figure 3A, right panel). Thus,

high concentrations of BKM120 elicit changes in additional sets of transcripts compared to

other PI3K inhibitors.

To identify gene sets linked to the transcriptional effects induced upon exposure to high

concentrations of BKM120, the most significantly changed transcripts (versus all other





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conditions) were identified and subjected to a gene set enrichment analysis (Figure 3B).

Interestingly, the gene sets with highest score were found to be related to cell cycle, spindle

assembly and the metaphase checkpoint. Overall, these results suggest that at high

concentrations, BKM120 displays activities that might have an impact on G2/M progression.

BKM120 blocks the prometaphase to metaphase transition?

In order to test whether BKM120 could cause a mitotic block, the effects on the cell cycle

were analyzed in A2058 cells using BKM120 or GDC-0941 at concentrations sufficient to

cause complete pathway inhibition (10 fold the IC50 for phospho-Akt inhibition). Treatment

with GDC-0941 had no effect on the cell cycle, whereas treatment with BKM120 led to a

significant increase in the G2/M population, in comparison to control untreated cells (Figure

4A). The increase 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

to reach the EC50 on PI3K pathway inhibition (measured by pAkt levels, Sup Figure 3A).

Furthermore, treatment with either BEZ235 or GDC-0941 at concentration as high as 5 μM,

had no effect on the cell cycle distribution (Sup Figure 3B).

Phenotypic analysis of the A2058 cells using immune-fluorescence analysis revealed that

treatment with 5 μM of BKM120 (but not with GDC-0941) induced the accumulation of

mitotic cells. Most cells displayed duplicated centrosomes (determined by gamma-tubulin

staining), early bi- and multi-polar spindles (determined by alpha-tubulin staining), and

condensed but not fully aligned DNA, indicating early mitotic phases (Figure 4B). Similar

effects were also observed with BKM120 in the K-RAS mutant MDA-MB231 and PTEN null





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U87MG cell lines (Sup Figure 4). Interestingly, treatment of these cells with the microtubule

destabilizer Nocodazole caused a remarkably similar phenotype. These results suggest that at

high concentrations, BKM120 causes a prometaphase to metaphase arrest in a PI3K-

independent manner.

BKM120 inhibits tubulin polymerization

To test whether BKM120 might influence microtubule dynamics, potential effects on tubulin

polymerization were analyzed. Cells were pre-incubated at 4°C to cause peripheral

microtubule depolymerization followed by a switch back to 37°C, either in presence or

absence of inhibitors, to allow re-polymerization of the microtubule network to the rim of the

cells (Figure 5A). In contrast to GDC-0941, incubation with BKM120 or Nocodazole

enhanced the loss of the microtubule network in the cell periphery, demonstrating that

BKM120 exhibits microtubule destabilizing activity.

To determine whether BKM120 would directly interfere with microtubule polymerization, in

vitro polymerization assays using purified tubulin were performed. As expected, the

microtubule stabilizer Paclitaxel significantly increased the tubulin polymerization kinetics,

whereas Nocodazole caused the opposite effects (Sup Figure 5A). Interestingly, and in

contrast to GDC-0941, BKM120 decreased the tubulin polymerization kinetics in a

concentration-dependent manner (Figure 5C).

To further demonstrate direct binding of BKM120 to tubulin, assessment of direct interactions

upon changes in relaxation of resonances was performed by NMR spectroscopy (Figure 5D).

T1ρ (left panels) and T2 (19F, right panel) relaxation experiments upon addition of freshly

prepared tubulin to BKM120 (20-fold excess) demonstrated an enhancement of the relaxation.





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These effects were tubulin concentration dependent (Sup Table 1) and were further confirmed

by waterLOGSY relaxation experiments (Figure 5C, left panels). The tubulin/BKM120

interaction was found to be in fast exchange as observed for other tubulin ligands (23).

Furthermore, relaxation competition studies could not demonstrate binding to the colchicine

site, when well described tubulin colchicine site binders were used as competitors (Sup Figure

5B).

The microtubule destabilizing activity of BKM120 does no translate to

antitumor activity in vivo.

We previously demonstrated that BKM120 was able to cause significant regressions in the

mechanistic Rat1-myr-p110α in vivo model, when dosed once per day at doses of 40 mg/kg

and above (T/C of -25 and -48% at 40 and 50 mg/kg, respectively) (11). To test whether at

these dose levels the exposure of BKM120 would have reached concentrations to engage its

off-target (tubulin binding) activity, tumors were fixed and stained for phospho-Histone H3

levels as a mitotic marker and the mitotic index (MI) was calculated. In cellular assays, a

strong increase in phospho-Histone H3 levels could be observed as early as 6 h (Sup Figure

6). In vivo, (Rat1-myr-p110α tumor model), no MI increase was evident at the 40 mg/kg dose

(plasma AUC: 65 h*μM), up to 16 h post last dose administration. This result suggests that

the tumor regression (which is accompanied with a robust increase in caspase 7 cleavage)

seen upon the exposure to BKM120 at the dose of 40 mg/kg (Figure 6A, right panel), is due to

the sole merit of PI3K inhibition. However, a 2.5-fold transient (6 h but not anymore at 16 h

time point) and statistically significant increase in MI to 5% could be observed for the 50

mg/kg dose (plasma AUC: 75 h*μM) (Figure 6A, left panel). To assess whether such a mild





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and transient increase in MI at 50 mg/kg translates into efficacy, a similar study was repeated

in the PTEN null U87MG tumors. Daily treatment of BKM120 resulted in antitumor activity

with T/C of 20% and 7% at 40 and 50 mg/kg, respectively, but these differences were not

statistically different (p>0.05, Sup Figure 6B). As in the Rat1-myr-p110α model, a similar

transient and statistically significant increase in MI to 3% was observed at the 50 mg/kg dose

level (Figure 6B, left panel). Importantly, no increase in caspase 7 cleavage was observed at

the 40 and the 50 mg/kg dose levels (Figure 6B, right panel). Altogether, these data suggest

that the exposure of BKM120 at a dose of 50 mg/kg might reach sufficient blood/tumor levels

to engage the off-target activity in the first 6 hours following administration which then cause

a mild and transient mitotic arrest MI (3 to 5%). In contrast to BKM120, other microtubule

binding agents such as Paclitaxel cause peaks of MI which range between 10 to 25% (Milas

1996) (24). Furthermore, BKM120 induced mitotic block seemed to be reversible as soon as

the compound gets cleared as no increase of MI was observed 16h after compound

administration. This reversibility was also observed in in vitro pulse-chase studies where

BKM120 was washed out (Sup Figure 7).





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Discussion

Our studies show that sustained exposure to BKM120 at concentrations above 1 micromolar

engages PI3K independent activities, resulting in enhanced antiproliferative and cell killing

effects. Biochemical and transcriptome profiling studies with BKM120 and other PI3K

inhibitors such as BEZ235 or GDC-0941 pointed to a unique role of BKM120 in regulating

microtubule dynamics causing a prometaphase to metaphase block in cell lines without strong

PI3K addiction where on-target PI3K inhibition is not able to induce apoptosis.

Microtubule stabilizers (such as paclitaxel and derivatives) and destabilizers (such as vinca-

alcaloïds or Nocodazole) are known to activate the spindle-assembly checkpoint leading to an

arrest of cells in mitosis and subsequent cell killing probably as a result of induction of mitotic

catastrophe. These agents have been used for many years as anti-neoplastic therapy for

various types of cancers (25). The phenotype detected upon BKM120 treatment at high

concentration was highly reminiscent of that observed upon Nocodazole treatment, suggesting

that BKM120 also interferes with microtubule dynamics directly (i.e. by tubulin binding

capacities) or indirectly (i.e. by blocking the activities of factors associated to the functions of

microtubules such as the kinesin Eg5).

Direct binding of BKM120 to pure tubulin was demonstrated using in vitro tubulin

polymerization assays and further confirmed by NMR studies.

Different microtubule targeting agents have distinct binding modes and mechanisms of

actions. Taxanes preferentially bind to polymerized β-tubulin, and more precisely at the inner

surface of the microtubules (26). Vinca-alkaloids bind at the interface of a 2 α/β tubulin

heterodimers, at the + end microtubules (27). A third category of tubulin interactors bind to





19

the so-called colchicine domain, which mostly lies within the β-tubulin subunit (28, 29).

Taxanes and vinca-alkaloids are both high molecular weight molecules and derivatives of

natural products. In contrast, colchicine-site binders are generally small molecules (30),hence

we hypothesized that BKM120 might share similar binding modalities. However, NMR

competition studies did not confirm this hypothesis. Further structural studies will be needed

to elucidate BKM120 binding mode to tubulin.

The pronounced spindle assembly defects in mitosis seen in vitro were not observed in vivo

after multiple administrations of BKM120 at a dose of 40 mg/kg. Interestingly, at a dose of 50

mg/kg, a small and transient increase of the mitotic index was observed. These findings

suggest that BKM120 displays tubulin binding and microtubule destabilizing activities only

above a certain concentration / exposure threshold. Furthermore, the fast binding kinetics to

tubulin as well as the intrinsic compound clearance probably results in reversibility of the

mitotic effects, as no accumulation of G2/M arrested cells could be detected following chronic

BKM120 administration. It therefore appears that BKM120 can cause regression in PI3K-

dependent tumors when administered orally to animals bearing subcutaneous tumors without

engaging the tubulin off-target activity to a sufficient level to contribute to the therapeutic

effect.

It is interesting to observe that the plasma exposure in patients treated with BKM120 at the

efficacious MTD (100 mg, AUC: 56 h*μM) (31), lies below the exposure necessary to

transiently engage the off-target in a mouse model (AUC > 65 h*μM). These findings

strongly argue that in patients, the threshold for microtubule destabilizing activity of this

compound is not reached. Therefore, it is anticipated that efficacy in patients will solely stem

from PI3K inhibition. Further analysis of clinical data, such as the assessment of mitotic





20

markers in tumor biopsies from patients treated with BKM120 will be required to fully

confirm that the compound off-target activity is not clinically relevant.





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Acknowledgements

We would like to thank Andreas Bauer, Hans Voshol, Christian Schnell, Markus Wartmann,

Patrick Chene, Thomas Radimerski and Pascal Furet for helpful discussions. We would also

like to acknowledge the inspiring comments and recommendations from Dr Levi Garraway

(DFCI, Boston, USA) to use tools such as the myr-Akt transduced cells for better

characterization of the BKM120 activities in cells. We also gratefully acknowledge the

excellent collaboration with the Genomics Technology group of NIBR Basel, in particular

Nicole Hartmann, Clarisse Wache-Mainier and Frank Staedtler. We also would like to thank

Sabina Cosulich for revising the manuscript.





22

References 1. Engelman JA, Luo J, Cantley LC. The evolution of phosphatidylinositol 3-kinases as

regulators of growth and metabolism. Nat Rev Genet 2006;7:606-19.

2. Zhao L, Vogt PK. Class I PI3K in oncogenic cellular transformation. Oncogene 2008;27:5486-96.

3. Samuels Y, Wang Z, Bardelli A, Silliman N, Ptak J, Szabo S, et al. High Frequency of Mutations of the PIK3CA Gene in Human Cancers. Science 2004;304:554.

4. Sansal I, Sellers WR. The Biology and Clinical Relevance of the PTEN Tumor Suppressor Pathway. J Clin Oncol 2004;22:2954-63.

5. Hers I, Vincent EE, Tavaro JM. Akt signalling in health and disease. Cell Signal 2011;23:1515-27.

6. Urick ME, Rudd ML, Godwin AK, Sgroi D, Merino M, Bell DW. PIK3R1 (p85) Is Somatically Mutated at High Frequency in Primary Endometrial Cancer. Cancer Res 2011;71:4061-7.

7. Sjödahl G, Lauss M, Gudjonsson S, Liedberg F, Halldén C, Chebil G, et al. A Systematic Study of Gene Mutations in Urothelial Carcinoma; Inactivating Mutations in TSC2 and PIK3R1. PLoS ONE 2011;6:e18583.

8. Alimandi M, Romano A, Curia MC, Muraro R, Fedi P, Aaronson SA, et al. Cooperative signaling of ErbB3 and ErbB2 in neoplastic transformation and human mammary carcinomas. Oncogene 1995;10:1813-21.

9. Shuttleworth SJ, Silva FA, Cecil AR, Tomassi CD, Hill TJ, Raynaud FI, et al. Progress in the preclinical discovery and clinical development of class I and dual class I/IV phosphoinositide 3-kinase (PI3K) inhibitors. Curr Med Chem 2011;18:2686-714.

10. Markman B, Atzori F, Perez-Garcia J, Tabernero J, Baselga J. Status of PI3K inhibition and biomarker development in cancer therapeutics. Ann Oncol 2010;21:683-91.

11. Maira SM, Pecchi S, Huang A, Burger M, Knapp M, Sterker D, et al. Identification and characterization of NVP-BKM120, an orally available pan class I PI3-Kinase inhibitor. Mol Cancer Ther 2012;11:317-28.

12. Gossert A, Henry C, Blommers M, Jahnke W, Fernandez C. Time efficient detection of protein−ligand interactions with the polarization optimized PO-WaterLOGSY NMR experiment. J Biomol NMR 2009;43:211-7.

13. http://www.BroadInstitute.org/ccle.





23

14. Brachmann SM, Hofmann I, Schnell C, Fritsch C, Wee S, Lane H, et al. Specific apoptosis induction by the dual PI3K/mTor inhibitor NVP-BEZ235 in HER2 amplified and PIK3CA mutant breast cancer cells. Proc Natl Acad Sci 2009;106:22299-304.

15. Maira SM, Stauffer F, Brueggen J, Furet P, Schnell C, Fritsch C, et al. Identification and characterization of NVP-BEZ235, a new orally available dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor with potent in vivo antitumor activity. Mol Cancer Ther 2008;7:1851-63.

16. Jagani Z, Wiederschain D, Loo A, He D, Mosher R, Fordjour P, et al. The Polycomb Group Protein Bmi-1 Is Essential for the Growth of Multiple Myeloma Cells. Cancer Res 2010;70:5528-38.

17. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, et al. Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci 2005;102:15545-50.

18. Szustakowski JD, Lee JH, Marrese CA, Kosinski PA, Nirmala NR, Kemp DM. Identification of novel pathway regulation during myogenic differentiation. Genomics 2006;87:129-38.

19. Newton M, Quintana F, den Boon J, Sengupta S, Ahlquist P. Random-set methods identify distinct aspects of the enrichment signal in gene-set analysis. Ann Appl Stat 2007;1:85-106.

20. http://www.R-project.org/

21. Folkes AJ, Ahmadi K, Alderton WK, Alix S, Baker SJ, Box G, et al. The identification of 2-(1H-indazol-4-yl)-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin -4-yl-thieno[3,2-d]pyrimidine (GDC-0941) as a potent, selective, orally bioavailable inhibitor of class I PI3 kinase for the treatment of cancer. J Med Chem 2008;51:5522-32.

22. Raynaud FI, Eccles SA, Patel S, Alix S, Box G, Chuckowree I, et al. Biological properties of potent inhibitors of class I phosphatidylinositide 3-kinases: from PI-103 through PI-540, PI-620 to the oral agent GDC-0941. Mol Cancer Ther 2009;8:1725-38.

23. Carlomagno T, Blommers MJJ, Meiler J, Jahnke W, Schupp T, Petersen F, et al. The High-Resolution Solution Structure of Epothilone A Bound to Tubulin: An Understanding of the Structure to Activity Relationships for a Powerful Class of Antitumor Agents. Angewandte Chem Int Ed 2003;42:2511-5.

24. Milas L, Hunter NR, Kurdoglu B, Mason KA, Meyn RE, Stephens LC, et al. Kinetics of mitotic arrest and apoptosis in murine mammary and ovarian tumors treated with taxol. Cancer Chem Pharm 1995;35:297-303.





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25. Weaver BA, Cleveland DW. Decoding the links between mitosis, cancer, and chemotherapy: The mitotic checkpoint, adaptation, and cell death. Cancer cell 2005;8:7-12.

26. Nogales E, Wolf S, Khan IA, Luduena R, Downing K. Structure of tubulin at 6.5 A and location of the taxol-binding site. Nature 1995;375:424-7.

27. Gigant B, Wang C, Ravelli RBG, Roussi F, Steinmetz MO, Curmi PA, et al. Structural basis for the regulation of tubulin by vinblastine. Nature 2005;435:519-22.

28. Ravelli RBG, Gigant B, Curmi PA, Jourdain I, Lachkar S, Sobel A, et al. Insight into tubulin regulation from a complex with colchicine and a stathmin-like domain. Nature 2004;428:198-202.

29. Nguyen TL, McGrath C, Hermone AR, Burnett JC, Zaharevitz DW, Day BW, et al. A Common Pharmacophore for a Diverse Set of Colchicine Site Inhibitors Using a Structure-Based Approach. J Med Chem 2005;48:6107-16.

30. Chen J, Liu T, Dong X, Hu Y. Recent Development and SAR Analysis of Colchicine Binding Site Inhibitors . Mini Rev Med Chem 2009;9:1174-90.

31. Baselga J, De Jonge MJ, Rodon J, Burris HA, Birle DC, De Buck SS, et al. A first-in-human phase I study of BKM120, an oral pan-class I PI3K inhibitor, in patients with advanced solid tumors. ASCO Meeting Abstracts 2010 Jun 14;28:3003.





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Figure legends

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 shift to 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 BKM120 indicate that BKM120 is less potent than GDC-0941. The

higher peak for BKM120 indicates again that BKM120 is killing more cell lines on overall.

Cell lines that are not responding to GDC-0941 and do not reach a crossing point in the dose

response curve, are assigned the maximum concentration tested value (i.e. 8µM), explaining

the second peak at crossing point 8µM for GDC-0941.

Figure 2: BKM120 elicits activities in non-PI3K addicted models independent of its PI3K

inhibitory functions. A. The indicated cell lines were seeded either in 96-well clusters (20000

cells/well, A2058 and MCF7 for viability assays and A2058 for pAkt level determination) or

in 10 cm dishes (MCF7 cells, 5x106 cells/plates, for pAkt level determination), and incubated

with the indicated compound for either 72 or 1 h. At this stage, cells were fixed (viability

assay) or lysed (S473P-Akt RPA quantification assay) and effects on either viability (left Y

axis) or on S473P-Akt levels (right Y axis), respectively, were plotted.. B and C. Engineered

MCF7-BP and MCF-myr-Akt were seeded (10x104 cells) either in 96-well clusters (B) or in a

6-well cluster (C), and incubated with the indicated compounds at the indicated

concentrations either for 72 h (B) or 2 weeks (C). At this stage cells were fixed and effects on

viability (B) as well as on colony formation (C) were assessed. The dashed grey line

represents the LD0 value that is to say the concentration of the compound responsible for





26

complete growth 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 the compound) as well as

complete pAkt level reduction. The dark plain line represents the LD50 value that is to say the

concentration of the compound needed to kill 50% of the cells present at the addition of the

compound.

Figure 3: Transcriptome profile of A2058 cells upon treatment of PI3K inhibitors BEZ235,

GDC-0941 and BKM120. A. A2058 cells were treated with equipotent concentrations of the

PI3K inhibitors BKM120 (IC30, IC50, IC90), GDC0941 (IC50, IC90 and a third

concenctration called “max” corresponding to the IC90 of BKM120) as well as BEZ235

(IC50, IC90, max = IC90 of BKM120) based on Akt-S473P inhibition. Principal component

analysis of transcript expression data demonstrating good reproducibility of biological

replicates and clear clustering of different treatment conditions. Left: plotting PC1 versus PC2

shows a clear continuous effect of the compounds along PC1 dimension in line with

increasing pathway inhibition. BKM120 at off-target concentrations (dark red) forms an

outlier cluster. Right: plotting PC2 versus PC3 strongly supports outlier behavior of high

BMK120 concentrations, compared with 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 have been submitted to a gene set enrichment analysis. Size of

the gene sets (horizontal axis) is plotted against strength of enrichment (vertical axis). Circles:

GeneGO processes, triangles: GeneGO pathway maps.





27

Figure 4: BKM120 provokes a prometaphase to metaphase block. A. 2x106 A2058 cells were

seeded in 10 cm dishes and incubated for 24 h with the indicated compounds. Cells were then

fixed, prepared as described for quantification of the population in the different phases of the

cell cycle by fluorescence-activated cell sorting. B. A2058 cells grown on coverslips were

treated for 24 h either with BKM120 (5 μM) or Nocodazole (100 nM). Effects on microtubule

dynamics and G2/M arrest was monitored by immuno-fluorescence staining of alpha-tubulin

(microtubules), gamma tubulin (centrosomes) and DAPI (DNA). Pictures were taken with a

100X objective.

Figure 5: BKM120 directly binds to tubulin and inhibits tubulin polymerisation. A. Rat-myr-

p110α cells grown on coverslips were switched from 37°C to 4°C for 1 h and then switched

back for 1 h to 37°C, in the presence of either DMSO control, BKM120 (5 µM), Nocodazole

(100 nM) or GDC-0941 (5 μM). Cells were then fixed and effects on microtubule stability

were visualized by immuno-fluorescence staining of alpha-tubulin. B. Chemical structures of

BEZ235, GDC0941, nocodazole, BKM120 and labeled BKM120 used in D. C. Purified

Tubulin was mixed with the indicated compounds at the indicated concentrations in the

presence of GTP. The polymerization of monomeric tubulin into microtubule was started by

transferring the reaction tubes from 4°C to 37°C, and monitored by the increase in absorbance

(λ=340 nM) over a period of 60 min. Of note, this assay requires high concentration of

compound in order to be in stoichiometry to the high amount of purified tubulin that is used in

this low-sensitivity polymerization assay. D. NMR spectroscopy shows binding of BKM120

to tubulin. Weak T1ρ and negative waterLOGSY signals were observed for the compound in

the absence of tubulin (bottom left panel) whereas significant T1ρ relaxation and positive





28

waterLOGSY signals were observed in the presence of tubulin (top panel). *, denote

impurities in the buffer. The assignment of BKM120 is indicated. T2-Relaxation enhancement

of 19F due to tubulin binding is evident after adding tubulin in a concentration dependent

manner (right panel).

Figure 6: Treatment with BKM120 lead to a transient increase in mitotic markers. A and B.

Rat1-myr-p110a (A) or U87MG (B) tumor bearing animals were treated p.o with the indicated

dose of BKM120, once per day for a period of 6 days. Upon last treatment, animals were

sacrificed at the indicated time points, for collection of plasma and tumor tissues. Compound

concentration in plasma (left panel, left Y axis) as well as quantification in tumors of pHistone

H3levels and subsequent determination of the Mitotic Index (MI) (left panel right Y axis)

were plotted; effects on apoptosis were also assessed by immuno-histochemistry by staining

of tumor sections with an anti-cleaved caspase 7 antibody (right panels).




Density

Density

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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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Article FileDensity distributions of the Amax (A) and the Crossing Point on log scale (b) fo...BKM120 elicits activities in non-PI3K addicted models independent of its PI3K in...Figure 3 - Transcriptome profile of A2058 cells upon treatment of PI3K inhibitor...Figure 4 - BKM120 provokes a prometaphase to metaphase block.Figure 5 - BKM120 directly binds to tubulin and inhibits tubulin polymerisation.Figure 6 - Treatment with BKM120 lead to a transient increase in mitotic markers...

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Characterization of the mechanism of action of the pan ......May 30, 2012 · MCT-11-1021 BKM120 alters microtubule dynamics at high concentrations 1 Characterization of the mechanism