identiﬁcation and characterization of nvp-bkm120, an ... · therapeutic discovery identiﬁcation...

Therapeutic Discovery

Identification and Characterization of NVP-BKM120,an Orally Available Pan-Class I PI3-Kinase Inhibitor

Sauveur-Michel Maira1, Sabina Pecchi3, Alan Huang4, Matthew Burger3, Mark Knapp3, Dario Sterker1,Christian Schnell1, Daniel Guthy1, Tobi Nagel3, Marion Wiesmann1, Saskia Brachmann1, Christine Fritsch1,MarionDorsch1, PatrickCh�ene1, KevinShoemaker3, AlainDePover1, DanielMenezes3,GeorgMartiny-Baron2,Doriano Fabbro2, Christopher J. Wilson5, Robert Schlegel4, Francesco Hofmann1, Carlos Garc��a-Echeverr��a1,William R. Sellers6, and Charles F. Voliva3

AbstractFollowing the discovery of NVP-BEZ235, our first dual pan-PI3K/mTOR clinical compound, we sought to

identify additional phosphoinositide 3-kinase (PI3K) inhibitors fromdifferent chemical classeswith a different

selectivity profile. The key to achieve these objectives was to couple a structure-based design approach with

intensive pharmacologic evaluation of selected compounds during the medicinal chemistry optimization

process. Here, we report on the biologic characterization of the 2-morpholino pyrimidine derivative pan-PI3K

inhibitor NVP-BKM120. This compound inhibits all four class I PI3K isoforms in biochemical assays with at

least 50-fold selectivity against other protein kinases. The compound is also active against the most common

somatic PI3Ka mutations but does not significantly inhibit the related class III (Vps34) and class IV (mTOR,

DNA-PK) PI3K kinases. Consistentwith itsmechanism of action, NVP-BKM120 decreases the cellular levels of

p-Akt inmechanistic models and relevant tumor cell lines, as well as downstream effectors in a concentration-

dependent and pathway-specific manner. Tested in a panel of 353 cell lines, NVP-BKM120 exhibited

preferential inhibition of tumor cells bearing PIK3CA mutations, in contrast to either KRAS or PTEN mutant

models. NVP-BKM120 shows dose-dependent in vivo pharmacodynamic activity as measured by significant

inhibition of p-Akt and tumor growth inhibition in mechanistic xenograft models. NVP-BKM120 behaves

synergistically when combined with either targeted agents such as MEK or HER2 inhibitors or with cytotoxic

agents such as docetaxel or temozolomide. The pharmacological, biologic, and preclinical safety profile of

NVP-BKM120 supports its clinical development and the compound is undergoing phase II clinical trials in

patients with cancer. Mol Cancer Ther; 11(2); 317–28. �2011 AACR.

Introduction

Because of their crucial role in signal transduction,the dysregulated metabolism of phosphoinositides

represents a key step in many disease settings. Multipleenzymes participate in the phosphorylation anddephosphorylation of the inositol head group of phos-phoinositide lipids (1). Among them, the phosphoinosi-tide 3-kinases (PI3K) have been the focus of extensiveresearch and drug discovery activities for the last 20years. Genetic alterations at multiple nodes in the PI3Kpathway have been implicated in oncogenesis and can-cer (2, 3). PI3K activation can occur in response to (i)constitutively active mutants and/or amplification ofgrowth factor receptor tyrosine kinases (RTK) as inbreast cancer with HER2 amplification (4), (ii) amplifi-cation of PI3K, (iii) gain of activating somatic mutationsin the PIK3CA gene encoding the p110a catalytic sub-unit (5), (iv) overexpression or activating mutations ofthe downstream effector kinase Akt (6), (v) loss orinactivating mutations of PTEN (the phosphatase thatbreaks down phosphatidylinositol-(3,4,5)-trisphosphate(PIP3), or (vi) constitutive recruitment and activation bymutant forms of the Ras oncogene. Given this pervasiveinvolvement in many cancers, the development of mole-cules that inhibit this pathway have recently been ini-tiated in first in man studies (7).

Authors' Affiliations: 1Oncology Disease Area, 2Center for ProteomicChemistry,Novartis Institutes forBiomedicalResearch,Basel, Switzerland;3Oncology Disease Area, Novartis Institutes for Biomedical Research,Emeryville, California; 4Oncology Translational Medicine, BU Oncology,Novartis Pharma Inc.; 5Developmental and Molecular Pathways, and6Oncology Disease Area, Novartis Institutes for Biomedical Research,Cambridge, Massachusetts

Note: Supplementary material for this article is available at MolecularCancer Therapeutics Online (http://mct.aacrjournals.org/).

S.-M. Maira and S. Pecchi contributed equally to this work.

Current address for C. Garc��a-Echeverr��a: Oncology Drug Discovery andPreclinical Research, Sanofi-Aventis, Vitry-sur-Seine, France; and currentaddress forM.Dorsch,OncologyDrugDiscovery andPreclinical Research,Sanofi-Aventis, Cambridge, Massachusetts.

Corresponding Author: Sauveur-Michel Maira, Novartis Institute for Bio-medical Research, Oncology Disease Area, Novartis Pharma AG, Klybeck-strasse 141, CH-4002 Basel, Switzerland. Phone: 41-61-69-67910; Fax:41-61-69-65571; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-11-0474

�2011 American Association for Cancer Research.

MolecularCancer

Therapeutics

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on March 29, 2020. © 2012 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst December 21, 2011; DOI: 10.1158/1535-7163.MCT-11-0474

http://mct.aacrjournals.org/

Following the discovery of NVP-BEZ235, our firstdual pan-PI3K/mTOR clinical compound (8), wesought to identify additional PI3K inhibitors from dif-ferent chemical classes with different profiles againsttargets within the PI3K pathway. Because the PI3Kpathway is a complex signaling pathway composed ofmultiple and interconnected components, elucidation ofeffectiveness and tolerability of novel therapeuticmodalities is likely to depend upon clinical experiencewith a number of clinical candidates with varyingpathway profiles. An example of this pathway complex-ity is the regulation of the oncoprotein Akt. To beactivated, Akt is first recruited to the plasma membranethrough a direct interaction between its N-terminalpleckstrin homology domain and PIP3 molecules. Forfull activity, Akt is then phosphorylated on 2 distinctsites: on Thr308 by PDK1 and on Ser473 by the mTORC2complex (9). Once activated, Akt regulates key down-stream effectors, including the downregulation ofmTORC1, the master regulator of protein translation,growth, and autophagy. However, genetic and bio-chemical studies have shown that mTORC1 providesa negative feedback loop at the level of RTKs causingreactivation of the PI3K and the mitogen-activated pro-tein (MAP) pathway upon mTORC1 inhibition. Thesame negative feedback loop was showed in tumorbiopsies from patients treated with the mTORC1 inhib-itor RAD001 (10). Thus, in some but not all clinicalcontexts, the combined inhibition of both PI3K andmTOR might be necessary for effective therapy. Giventhat different wiring applies depending on the geneticalterations responsible for PI3K pathway constitutiveactivation (11, 12), compounds with varied and wellunderstood profiles will be necessary to design anddevelop tailored chemotherapy.

Here, we report on the biologic characterization of theclinical candidate NVP-BKM120. This molecule is a 2,6-dimorpholino pyrimidine derivative that is a potentpan-PI3K inhibitor. Unlike NVP-BEZ235, it does notsignificantly inhibit mTOR and is highly selectiveagainst other protein or lipid kinases. NVP-BKM120exhibits potent antiproliferative and proapoptotic activ-ities in tumor cell lines by specifically blocking thebiologic function of PI3K signaling components. NVP-BKM120 shows good oral bioavailability in mice and itson-target activity observed in cellular assays translateswell in in vivo models of human cancer in which NVP-BKM120 shows significant tumor growth inhibition orregression at tolerated doses.

Materials and Methods

Compounds and toolsNVP-BKM120was synthesized in the Global Discovery

Chemistry group (NIBR Oncology; Novartis). NVP-BKM120, RAD001 (Novartis), STI571 (Novartis), Wort-mannin (Alexis; #ALX-350–020-M001), LY294002 (Alexis;#ALX-270–038-M025), ZSTK474 (LC Laboratories; #Z-

1006),AZD6244(SelleckChemicals;#S1008),andAZD8055(Active Biochem; #A-1008) were prepared as 10 mmol/Lstock (except LY294002; 20 mmol/L) solutions in 100%dimethyl sulfoxide (DMSO). Working solutions werefreshly prepared before addition to the cell media suchthatfinalDMSOconcentrationswerekept constant at 0.1%in both control and compound-treated cells. Platelet-derivedgrowthfactor (PDGF)stocksolutionwaspreparedas recommended by the manufacturer (Sigma; #P4306).

In vitro kinase assaysPI3K assay. All the biochemical in vitro PI3K and pro-

tein kinase assays shown in Table 1 were previouslydescribed (13).

mTOR TR-FRET assay. Fifty nanoliters of compounddilutions were dispensed onto black 384-well low-volumenonbinding polystyrene plates (Corning; NBS#3676). Then5 mL of ATP and GFP-4EBP1 with 5 mL mTOR proteins(final assay volume: 10 mL) were added, and the reactionwas incubated at room temperature in 50 mmol/LHEPESpH 7.5, 10 mmol/L MnCl2, 50 mmol/L NaCl, 1 mmol/LEGTA, and 1 mmol/L DTT. Reactions were stopped with10 mL of a proprietary mixture (IVG), containing the Tb3þ-a-p4EBP1-(pT46) detection antibody, EDTA, in TR-FRETdilution buffer. Plates were then read 15 minutes later in aSynergy 2 reader using an integration time of 0.2 secondandadelayof 0.1 second. The control for 100% inhibitionofthe kinase reaction was created by replacing the mTORkinasewith an equal volume of reaction buffer. The controlfor 0% inhibition was created by substituting solvent vehi-cle (90% DMSO in H2O) without added test compounds.The TR-FRET mTOR kinase assay components were pur-chased from Invitrogen Corporation. Details are availableupon request.

DNA-dependent protein kinase assay. To determinethe potency of NVP-BKM120 on the phosphatidylinositol3-kinase–relatedkinases (PIKK) familymemberDNA-PK,an in vitro assay kit from Promega (SignaTECT; # V7870)was used in combination with the purified DNA-PKenzyme from Promega [DNA-dependent protein kinase(DNA-PK), V5811]. The in vitro kinase assay reactionswere conducted according to the manufacturer’s protocolbut modified as follows: 27 U of purified DNA-PK pro-tein/reaction, 1 mmol/L ATP/reaction, 1% DMSO, orindicated compound/reaction at 37�C for 30 minutes.

PI3Ka kinetic studies. PI3Ka was incubated for 60minutes in coated Maxisorp plates (14) in 50 mL mediumcontaining [g33P]-ATP (�6 kBq/well), 0.5 to 400 mmol/Lunlabelled ATP, 5 mmol/L MgCl2, 150 mmol/L NaCl, 25mmol/L Tris-HCl pH 7.4, and 1% DMSO. The reactionwas started by adding PI3Ka (0.4 mg/mL) and stoppedby adding 50 mL of 50 mmol/L EDTA. Plates werewashed twice with Tris-HCl buffer and dried, after which100 mL/well MicroScint PS was added and bound radio-activity was determined using a TopCount (Packard)counter. Curves were fitted to a mixed inhibition model(15) by nonlinear 3 dimensional regression. In this model,

Maira et al.

Mol Cancer Ther; 11(2) February 2012 Molecular Cancer Therapeutics318




the enzyme activity is related to S (ATP) and I (NVP-BKM120) by the following equation:

v ¼ Vmax � S½ �K1 � 1þ I½ �=K2ð Þ þ S½ � � 1þ I½ �=K3ð Þ ;

in which the apparent dissociation constant of ATP isrepresented byK1 and the apparent dissociation constantsof the inhibitor are represented by K2 (competitive com-ponent) and K3 (noncompetitive component).

Cellular biologyCell lines and cell culture: Mechanistic models. To

evaluate the isoform-specific potency of NVP-BKM120 ina cell-based system, an N-terminally myristoylated formof each PI3K class IA isoform was expressed in Rat1fibroblasts. The retroviral expression plasmid pBabePuro(16) containing human p110a, p110b, and p110d with anN-terminal myristoylation (myr) signal followed by anHA-tag were generated as described (17). Successfullyinfected Rat1 cells were selected in medium containing 4

mg/mL of puromycin, expanded and characterized forexpression of the p110 isoforms. Transgenic expression ofthe myristoylated protein was confirmed by increasedlevels of phosphorylated Akt. The TSC1�/�-null MEFsmechanistic model for mTORC1 constitutive activationare from Dr. D. Kwiatkowski (Brigham & Women’s Hos-pital, Boston, MA; ref. 18).

Cell lines and cell culture: Human tumor cell lines.The culture conditions used to maintain U87MG, SF268,HT-29, PC3M, HCT116, and BT474 are described else-where (8, 19). The U87MG (University of Basel, Basel,Switzerland), HCT116 (American Type Culture Collec-tion; #CCL-247), and BT-474 (Friedrich Miescher, Insti-tute, Basel, Switzerland) cell lines were authenticated bysingle-nucleotide polymorphism analysis. The SF268 cellline (National Cancer Institute, Bethesda, MD) was notauthenticated.

Biochemical characterization upon compound expo-sure and antibodies. A total of 2� 106 cells were seededper 10-cm dish. Eighteen hours later, the medium wasdiscarded and replaced with 10 mL of fresh medium

Table 1. Biochemical profile of NVP-BKM120 against selected protein kinases, class I and IV PI3Ks, andPI4Kb

Enzyme IC50, nmol/L Enzyme IC50, nmol/L

Class I PI3Ksa

p110a k52 � 37 (n ¼ 7) p110b 166 � 29 (n ¼ 3)p110a-H1047R 58 � 2 (n ¼ 2) p110d 116 (n ¼ 1)p110a-E545K 99 � 6 (n ¼ 2) p110g 262 � 94 (n ¼ 7)

Class IIIVps34b 2,410 � 150 (n ¼ 19)

Class IV PI3KsmTORc 2,866 � 1,671 DNA-PKa >10,000ATRd 8,091 � 2,038

PI4KPI4Kbb >25,000 (n ¼ 22)

Protein kinasesAxl >10,000 Fak >10,000VEGFR2/Kdr >10,000 Jak2 >10,000HER1/ErbB1 >10,000 c-Abl >10,000IGF1-R >10,000 c-Src >10,000EphB4 >10,000 PKA >10,000Ret >10,000 Akt1/PKBa >10,000Tie-2/Tek >10,000 PDK1 >10,000c-Met >10,000 B-RafV599E 9,200K650E-FGFR- >10,000 CDK2/cyclin A >10,000CSF1R 582

NOTE: In vitro kinase assayswere conductedwith the indicated recombinant PI3K lipid or protein kinases, in the presenceof increasingconcentrations of NVP-BKM120, as described in Materials and Methods. The concentration responsible for 50% inhibition of theactivity is reported (IC50) in nmol/L as determined in multiple experiments (n) is shown as an average � one SD.aFilter binding assay format.bKinase-Glo format.cTR-FRET assay format.dAlpha Screen assay format.

BKM120: A Potent and Specific Pan-PI3K Inhibitor

www.aacrjournals.org Mol Cancer Ther; 11(2) February 2012 319




containing the test items. Unless specified in the figurelegend, the incubation periodwas 30minutes. For agonistactivation, cells were first starved for 18 hours in 9 mL ofmedium containing only 0.05% FBS. Then cells werepretreated for 30 minutes by addition of 1 mL of mediumcontaining the test items and subsequently stimulatedfor 10 minutes with the agonist (final concentrations:50 ng/mL for PDGF, 100 ng/mL for epidermal growthfactor (EGF), 60 ng/mL interleukin (IL)-4, and 1 mmol/Lanisomycin). Cells were washed twice, lysed, and pro-cessed by Western blot analysis as described (8).

S473P- and T308P-Akt ELISA assays; S473P-Akt andS235/236P-RPS6 RPPA quantification. All the proce-dures have been described elsewhere (8, 20).

In vivo studiesCompound preparation. NVP-BKM120 andAZD6244

were formulated inNMP/PEG300 (10/90, v/v). Solutionswere freshly prepared for each day of dosing by dissol-ving the powder, first in N-Methyl-2-pyrrolidone (NMP)with sonication and thenby adding the remaining volumeof PEG300. The application volume was 10 mL/kg. Her-ceptin/trastuzumab (150 mg; Hammer Apotheke) wasprepared as a 0.22 mmol/L filtered sterile stock solutionat 15 mg/mL in PBS that was subsequently diluted to1 mg/mL aliquots and stored at �80�C. For each treat-ment (3 times per week), a new vial was thawed forimmediate intraperitoneal treatment in animals with anapplication volume of 10mL/kg (final dose of 10mg/kg).

In life experimentation and efficacy studies. A max-imumof 10 femaleHarlannude (HsdNpa:AthymicNude-nu) mice (8–10 weeks of age) were kept under sterileconditions (type III cage, in an Optimal Hygienic Condi-tions zone) with free access to food and water. Subcuta-neoustumorswereestablishedbyinjectionof100mLofPBScontaining 2� 106 tumor cells (Rat1-myr-p110a, U87MG,HCT116,NCI-N87,andPC3M), in leftflankofeachanimal.Orthotopic BT-474 tumorswere establishedby injection of5�106cellspreparedin30%Hank’sBalancedSaltSolution(HBSS) containing 10% Matrigel, in the third mammarygland. Treatments were initiated when the mean tumorvolume ineach randomizedgroup (n¼6–8) reached100 to150mm3. For BKM120, treatmentswere carried out orally,once per day, using an application volume of 10 mL/kg.Treatmentswere stoppedandanimals sacrificedwhen thetumor size in the vehicle control group reached 1,000 to1,200 mm3. Tumor volumes were determined by usingcalipers formeasurementof longest (considered as length)and shortest (considered as diameter) dimensions of eachtumor and according to the formula length� diameter2�p/6. Antitumor activity is expressed as T/C% (meanincrease of tumor volumes of treated animals divided bythe mean increase of tumor volumes of control animalsmultiplied by 100). Data are presented as means � oneSEM. Comparisons between groups and vehicle controlgroup were done using either one-way ANOVA orANOVA on ranks followed by the Dunnett tests whendatawere, respectively, eithernormallydistributedor not.

For all tests, the level of significance was set at P < 0.05.CalculationswerecarriedoutwithSigmaStatv2.03 (JandelScientific). All experimental procedures (approved by theKantonales Veterin€aramt Basel-Stadt under license #1769)strictly adhered to the Eidgen€ossisches Tierschutzgesetzand the Eidgen€ossische Tierschutzverordnung. For com-bination in vivo studies, the combination was determinedusing the Clarke method (21).

NVP-BKM120 tissue analytics, interstitial fluid pres-sure, and DCE-MRI. The methodologies used were asdescribed previously (22).

Results

NVP-BKM120: chemical structure andmechanismofaction

NVP-BKM120 (Fig. 1A) was identified upon optimiza-tion of drug-like and in vivoprotein kinaseproperties froma 2-morpholino-6-aminopyridyl-pyrimidine scaffold. Themolecule binds in the ATP-binding site of the lipid kinasedomain, as determined from its cocrystal structure in thePI3K p110g isoform (PDB accession code: 3SD5) and byhomology modeling in the PI3K p110a isoform (Fig. 1B).The alpha model suggests that there are 3 key hydrogenbond interactions (indicated by the dotted lines), formedby the oxygen of the 2-morpholino group and by theexocyclic nitrogen in the 6-position pyridyl substituent.The morpholine oxygen functions as a hydrogen bondacceptor with the backbone amino group of Val851 in thehinge domain of the PI3K p110a isoform. A similar inter-action is observed in all thepublished structureswithATPand with known inhibitors (23, 24). The 6-pyridyl exocy-clic nitrogen (as a donor) binds toAsp810 andAsp933 in thecatalytic region. A fourth interaction could be modeled,as high resolution structures of p110g are frequentlyobserved to contain awatermolecule forming a hydrogenbond bridge between Tyr836 and Asp810 (25). Its presencecould provide an additional hydrogen bond formed by itsinteraction with the 6-pyridyl endocyclic nitrogen actingas an acceptor.

Kinetic fittings of enzymatic results using a Michaelis–Mentenmodel revealed thatNVP-BKM120 inhibits PI3Kavia a mixed-type mode (15), affecting both the Vmax andtheKm forATP (Fig. 1C). Because theKm forATP increaseswith compound concentration (and correspondingly, thecompound IC50 value increases with ATP competition),NVP-BKM120 has greater affinity for the free, relative tothe ATP bound, PI3K enzyme. While further study of theATP-noncompetitive aspect has not been yet pursued, theATP-competitive facet of this inhibition is consistent withthe binding mode established by the crystallographyresults described earlier.

Biochemical selectivity of NVP-BKM120NVP-BKM120 is approximately equipotent against

the class IA PI3Ks a, b, and d and modestly less potentagainst the class IB g isoform (Table 1). The compoundalso shows comparable potency against activating

Maira et al.





p110a somatic mutations that have been described in awide array of human cancers (26). NVP-BKM120 issignificantly less potent in biochemical assays againstthe PI3K class III family member Vps34, the related classIV PIKK protein kinases mTOR, ATR, and DNA-PK,and the distinct lipid kinase PI4Kb. NVP-BKM120 wasshown to be mostly inactive against all the kinasestested in an in-house selectivity panel with the excep-tion of colony-stimulating factor 1 receptor (CSF1R).This inhibitory activity was confirmed at a concentra-tion of 5 mmol/L in the Invitrogen functional kinasepanel Supplementary Table S1 but no effect wasobserved in a cell-based CSF1R autophosphorylationassay (Supplementary Fig. S1A). NVP-BKM120 wasfurther profiled in the Ambit kinase competition panel(Supplementary Table S2), in which EphA2 and fibro-blast growth factor receptor 2 (FGFR2) kinases werefound to be inhibited by more than 90% at a concen-tration of 1 mmol/L. Such as for CSF1R, these hits werenot reconfirmed in FGFR2 (Supplementary Fig. S1B)and EphA2 (Supplementary Fig. S1C) cellular autopho-sphorylation assays. Therefore, NVP-BKM120 can beconsidered as a selective pan-class I PI3K inhibitor witha profile distinct from the previously described dualPI3K/mTOR inhibitor NVP-BEZ235 (8).

Activity and specificity of NVP-BKM120 in cellsystems

Rat1 cell lines that overexpress activated versions ofeach class IA PI3K isoform were engineered and path-way activation was confirmed by the concomitantincrease in S473P-Akt levels (Supplementary Fig. S2).NVP-BKM120 inhibited S473P-Akt in a concentration-dependent manner (Fig. 2A) with IC50 values (RPPAdetermination) of 104 � 18, 234 � 47, and 463 � 87nmol/L (n ¼ 7) in lines that express myr-p110a, b, andd, respectively. These reductions in S473P-Akt levelsare probably not attributable to mTOR inhibition, as theIC50 value for S6RP S235/236 phospho levels was 922 �142 nmol/L (n ¼ 5) in the mTORC1 constitutivelyactivated TSC1�/� cells. In contrast, the dual PI3K/mTOR NVP-BEZ235 as well as the mTOR catalyticinhibitor AZD8055 (27) and NVP-BEZ235 were, asexpected, extremely active in this assay with IC50

values of 3.5 � 0.6 (n ¼ 4) and 2.0 � 0.8 (n ¼ 4),respectively (Fig. 2B).

In PTEN-null U87MG cells, NVP-BKM120 treatmentreduced both S473P-Akt (IC50 value: 130 � 44 nmol/L,ELISA) and T308P-Akt (IC50 value: 229 � 40 nmol/L,ELISA) levels with similar potency comparable withwhat is observed in the mechanistic Rat1-myr-p110

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Figure 1. NVP-BKM120: structure, binding mode, and mechanism of action. A, chemical structure of NVP-BKM120. B, the proposed binding mode ofNVP-BKM120 in the class 1A PI3K p110a isoform is shown. The PI3K p110a homology model was built with the Molecular Operating Environmentsoftware from the Chemical Computing Group using default settings in the alignment and homology modeling modules, a Novartis structure ofNVP-BKM120 bound to PI3K p110g (PDB deposition code: 3SD5) as a template and the input sequence of PI3K p110a (UNIPROT:P42336). All keyprotein–compound interactions were retained between the p110g structure and the p110amodel, and no new interactions were expected with the otherPI3K class I isoforms. C, PI3K p110a kinetic reactions in presence of NVP-BKM120 were done as described in Materials and Methods. NVP-BKM120concentrations (nmol/L) were as indicated in the graph. Curves were fitted to a mixed inhibition model by nonlinear 3 dimensional regression. Thepredicted Vmax and Km are plotted on the right top and bottom, respectively. The mixed model was highly significant as compared with the competition

model in a global fit (P < 0.001). The predicted Vmax and Km calculated with the equations Vappmax ¼ Vmax

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models (Fig. 2C). This effect is correlated in a dose-dependent manner with inhibition of downstreamdirect (FOXO3A/FKHRL1) and indirect (p70S6K) Akteffectors, showing profound Akt pathway modulationupon NVP-BKM120 exposure. Compound washoutstudies in U87MG cells showed that these inhibitoryeffects are fully and rapidly reversible upon compoundwithdrawal (Supplementary Fig. S3), suggesting rapidbinding kinetics and a short residence time of thecompound on the target.

To further evaluate the specificity of NVP-BKM120,the compound was tested in cellular models in the pres-ence of various agonists that activate a spectrum of well-characterized signaling pathways. In the presence ofmitogenic stimuli such as PDGF (Fig. 2D) or EGF (Sup-plementary Fig. S4A), NVP-BKM120 blocked S473P-Aktinduction by the respective growth factors but failed toaffect receptor activation (Y751P-PDGFR and Y845P-EGFR, respectively) and activation of the mitogen-activated protein kinase (MAPK) pathway. This is incontrast to the effects observed with PDGF receptor(STI571) or EGF receptor (NVP-AEE788) inhibitors. Sim-ilarly, NVP-BKM120 did not inhibit IL-4–induced activa-tion of STAT6 (Supplementary Fig. S4B), anisomycin-induced activation of c-jun-NH2-kinase (JNK) and p38(Supplementary Fig. S4C) nor class IV PIKK (ATM andDNA-PK) activation in response to DNA double-strandbreaks (Supplementary Fig. S4D). Cumulatively, theseresults further show the specificity of NVP-BKM120 ina cellular context.

NVP-BKM120 shows preferential inhibition oftumor cells bearing oncogenic mutations in thePIK3CA gene

Activation of the PI3K pathway is a common feature inhuman cancers, arising through various genetic abnor-malities (28). To test whether NVP-BKM120 would be aneffective treatment for tumors presenting these abnormal-ities, the antiproliferative activity of the molecule wasdetermined against a panel of 353 cell lines that vary withrespect to key genetic determinants such as the status ofthe PIK3CA, PTEN, and KRAS genes. Cell lines charac-terized by the presence of an oncogenic mutation in thePIK3CA gene showed greater and statistically significant(P¼ 0.01) sensitivity to NVP-BKM120 than cells bearing anonmutated wild-type form of the gene (Fig. 3A). Nodifferences in sensitivity could be observed when com-paring cells carrying a deletion or a mutation in the PTENgene (Fig. 3B) or an activating mutation in the KRAS gene(Fig. 3C) when compared with cells bearing wild-typePTEN and KRAS genes, respectively. However, in thelatter case, a trend toward more sensitivity in KRASwild-type cells is seen. PIK3CA and KRASmutation oftenco-occur in human colorectal adenocarcinoma. A multi-variate analysis looking at status of both genes revealedthat PIK3CA mutant/KRAS wild-type tumors are mostsensitive toNVP-BKM120 versus other possible combina-tions (Supplementary Fig. S5A) and most notably incomparison to PIK3CA mutant/KRAS mutant models(P ¼ 0.00005). These data show that the presence of aKRAS mutation could overcome the sensitivity to a PI3K

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Figure 2. NVP-BKM120 efficientlyand specifically inhibits PI3K inmechanistic and disease cell-based models. A and B,Rat1-myr-p110a, b, and d (A) andTSC1�/�-null MEFs (B) cell lineswere incubated for 30 minutes withthe indicated compound andconcentrations (in nmol/L). Cellswere then lysed and analyzed byRPPA for their S473P-Akt andS235/236P-RPS6 levels. Levelswere then plotted as percentage ofuntreated control cells and IC50

values determined. C,U87MGcellswere exposed for 30minutes to theindicated amount of NVP-BKM120and cell extracts subsequentlyanalyzed by Western blotting forthe indicated PI3K pathwaymarkers. D, SF268 cells werestarved for 18 hours, pretreated for30 minutes with 50, 250, or 1,250nmol/L of the indicatedcompounds and then stimulatedwith PDGF for 10 minutes. Cellswere then lysed and cell extractsanalyzed by Western blotting forthe indicated markers.

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inhibitor, probably by activation of alternate, KRAS-dependent signaling pathways such as the extracellularsignal-regulated kinase (ERK) pathway. Interestingly,cotreatment of NVP-BKM120 with a MAP/ERK kinase(MEK) inhibitor could induce cell death, as revealedby PARP cleavage, in KRAS mutant/PIK3CA mutantHCT116 cellswhen single-agent treatmentwas essentiallyineffective (Supplementary Fig. S5B). A similar observa-tion was made for ZSTK474, another pan-PI3K inhibitor(29), suggesting that this phenomenon is not unique toNVP-BKM120.

NVP-BKM120 efficiently inhibits the PI3K pathwayin tumor-bearing animals and displays strongantitumor activity in vivoTo determinewhetherNVP-BKM120 could reach expo-

sure leading topathwaymodulation in tumor tissue, Rat1-myr-p110a tumor-bearing animals were treated orally,once, at dose levels of either 30 or 60 mg/kg. Phosphor-ylated Akt levels (Ser 473) as well as compound concen-trations were then determined, at different time pointspostdrug administration (Fig. 4A). Maximum inhibitionof p-Akt levels in tumor tissue was achieved at the 1 hourtime point when the concentration in both tumor andplasma compartments was highest, for both dose levels.Phosphorylated Akt levels remained strongly inhibited 8hours postdose (the inhibition was equal or superior to80%, at 30 and 60 mg/kg, respectively) and at 16 hourspostdose for the 60 mg/kg dose level (70% inhibition). Asimilar relationship is observed in the PTEN-null U87MGmodel (Supplementary Fig. S6A). Overall, these datashow that NVP-BKM120 oral administration can achievepharmacodynamic effect in the tumor in a concentration-dependent manner, showing pharmacokinetic and phar-macodynamic relationship.

To show that the observed pharmacodynamic effectscould translate into effective antitumor activity, Rat1-myr-p110a tumor-bearing mice were treated orally withNVP-BKM120 once per day at doses of 40, 50, or 60 mg/kg. At these dose levels, the compoundwaswell tolerated(right) and a robust reduction in tumor mass (left)was observed (T/C values of �25%, �48%, and �46%,respectively; Fig. 4B), showing the ability ofNVP-BKM120to treat PI3K-addicted tumors. A similar response wasobserved in the MCF7 PIK3CA mutant model MCF7(Supplementary Fig. S6C). Consistent with the in vitrodata (Fig. 3), the compound was less efficacious againstU87MG PTEN-null tumors in mice compared withPIK3A-mutated cell lines, even at the dose of 60 mg/kg(Supplementary Fig. S6B).

Because clinical benefit in the treatment of solidtumors is often obtained as a combination therapy,NVP-BKM120 was combined with a number of stan-dards of care agents or with targeted inhibitors to modelrational combinatorial treatments. Genetically engi-neered lung KRAS mutant tumors have been shown tobe almost insensitive to PI3K inhibitor treatment, butcomplete efficacy was obtained when combined with aMEK inhibitor (30). Similarly, when grown in vivo,KRAS mutant/PIK3CA mutant HCT116 tumors werefound to be relatively insensitive to suboptimal doselevels of either NVP-BKM120 or the MEK inhibitorAZD6244. However, significant tumor regression wasobserved in a very well-tolerated manner when bothdrugs were combined (Fig. 4C and Supplementary Fig.S6D). These data show that the combination of bothdrugs lead to activity superior to that seen with eitheragent alone. In agreement with this, more apoptosis wasobserved in vitro, when HCT116 were exposed to bothdrugs (Supplementary Fig. S5B).

Figure 3. NVP-BKM120preferentially inhibits the proliferationof tumor cells with PIK3CA gene-bearing oncogenic mutations. Celllines with different genetic lesionswere binned and the percentages ofgrowth inhibition with treatment at0.8 mmol/L of NVP-BKM120 wereplotted as box plots and comparedbetween 23 PIK3CA mutant cancercell lines and 177 PIK3CA WT celllines (A), 36 PTEN mutant cell linesand 164 PTENwild-type cell lines (B),and 52 KRAS mutant cell lines and147 KRAS wild-type cell lines (C).P values were determined by theStudent t test; medians of the groupare labeled on each plot as the whitebars.

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As true for other pathways, the PI3K/Akt/mTORC1pathway is subjected to regulation by negative feedbackloops. Efficacy of PI3K inhibitors might, therefore, belimited by reactivation of critical upstream nodes.Such a phenomenon has recently been described inRTK-addicted tumor lines such as breast HER2-amplifiedmodels, in which PI3K inhibition led to ERK and PI3Kpathway reactivation through HER2/HER3 overexpres-sion and overactivation (31–34). Cotreatment with bothPI3K and RTK inhibitors in RTK-addicted tumors is,therefore, warranted to avoid this adaptive mechanismof resistance. To test this hypothesis, orthotopic breastHER2-amplified BT474 tumor-bearing mice were treatedwith NVP-BKM120 in presence or absence of the HER2antagonist trastuzumab (Fig. 4D and Supplementary Fig.S6E). Only partial responses could be obtained as singleagents (T/C of 49.7% and 18.7% for Herceptin andBKM120, respectively) but significant tumor regressionwas obtained when both drugs were combined (T/C of�15%) with no impact on body weight. Similar data wereobtained for the HER2-amplified gastric cancer tumormodel NCI-N87 (Supplementary Fig. S7A).

The alkylating agent temozolomide or the microtubulestabilizer taxotere are 2 standard of care agents used totreat glioblastoma multiforme (GBM) or prostate cancer,

respectively. In both indications, the PTEN gene is fre-quently inactivated, suggesting some dependencies onthe PI3K signaling. Suboptimal dosage regimen of NVP-BKM120 (30 mg/kg) was used to check whether it couldgive advantages in combination studies to these agents.Whereas only partial activity is observed as single agent(as was the single agent use of temozolamide or taxotere),the combination of temozolamide or taxotere with NVP-BKM120 against GBM U87MG (Supplementary Fig. S7B)and prostate PC3M (Supplementary Fig. S7C) tumors,respectively, caused robust tumor shrinkage in a well-tolerated manner. These results show the potential for atleast additive activities with these cytotoxic agents andNVP-BKM120 in selected settings.

Overall, these in vivo efficacy data show that NVP-BKM120 is an orally available PI3K inhibitor that displaysa good correlation between its pharmacokinetic and spe-cific pharmacodynamic readouts, resulting in a strongantitumor activity either alone or in combination withtargeted or cytotoxic therapies in relevant diseasemodels.

NVP-BKM120 possesses antiangiogenic activitiesin vivo

PI3K can be actively recruited and activated by angio-genic factors (35, 36). PI3K inhibitors are, therefore,

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expected to display some degree of antiangiogenicactivity. As described for NVP-BEZ235 (22), NVP-BKM120 treatment readily blocks VEGF induced neo-vascularization in vivo (Supplementary Fig. S8A).Angiogenic tumors are characterized by tortuous, cha-otic, and highly permeable vessels. This phenomenon isin part due to VEGF-induced eNOS activation, througha PI3K-dependent mechanism involving Akt (22). Treat-ment of highly angiogenic rat mammary BN472 tumor-bearing rats with NVP-BKM120 led to significant reduc-tion of tumor vasculature leakiness from the tumortissue, as reflected by the strong reduction of Evansblue–mediated fluorescence (Supplementary Fig. S8B).As a consequence, NVP-BKM120 treatment also pro-duced a strong reduction in tumor interstitial fluidpressure (IFP), which depends on the vasculature per-meability (Fig. 5A). Reduction in permeability and IFPcould be measured in a noninvasive manner by dynam-ic contrast enhanced (DCE)-MRI, in the presence of thecontrasting agent Vistarem. Treatment of BN472 tumor-bearing rats with NVP-BKM120 strongly reduced tumorpermeability as reflected by the rapid drop in Ktrans

factor (Fig. 5B). These data confirm that NVP-BKM120possesses strong antiangiogenic properties and thesecan be visualized and quantified by DCE-MRI imagingtechnology.

Discussion

A plethora of preclinical evidence predicts that inhibi-tors targeting various components of the PI3K pathwaywill have therapeutic benefit in cancer. However, a keyquestion that remains to be answered is: what is the mosteffective and tolerated inhibitory profile of a small mol-ecule cancer therapeutic targeting this pathway? Becausecancer is not one disease, but many, it is unlikely that alltumor types, encompassing different lineages and geneticstatus, would be similarly "wired" in this signal transduc-tionmachinery. Therefore, tomaximize chances of clinicalsuccess, following the discovery of the dual PI3K/mTORinhibitor and imidazoquinoline derivative NVP-BEZ235,we pursued the development of other clinical candidatesfrom a different chemical space and with a differentbiologic profile.

This effort led to the discovery of NVP-BKM120,identified from the pan-PI3K inhibitor lead series 2-mor-pholino-4-amino-6-pyrimidinyl pyrimidines (MAPP)through a combination of structure-based drug designand optimization of in vivo properties (37). NVP-BKM120inhibits PI3K in anATPcompetitivemanner, but this is theresult of a mixed effect on Km and Vmax. NVP-BKM120spares tyrosine or serine/threonine protein kinases, andunlike NVP-BEZ235, is not an effective mTOR inhibitor.

Figure 5. NVP-BKM120 reducesvasculature permeability and IFP intumors. A, orthotopic BN472 tumor-bearing rats containing a pressuresensing catheter inserted in thetumor were treated for 3 consecutivedays with NVP-BKM210 at 5 mg/kg(n ¼ 5; initial IFP: 20.4 � 2.6 mm Hg)orwith the vehicle control (n¼ 9; 18.2� 1.5 mm Hg). The IFP wascontinuously recorded for 72 hoursand variations (D toward initialvalues) plotted (gray lines, untreatedanimals; orange line, appliedtreatment as indicated in the graph).Data presented are means � SEM.The arrows represent theadministration schedule. Gray barsrepresent night time. B, quantitativeevaluation on normalizedKtrans (right)and Ktrans maps (left) in BN472tumors at days 0, 2, and 7 followingonce daily treatment with vehicle andBKM120 at 2.5 mg/kg.

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This distinction can be explained by the bindingmodes ofthe 2 molecules: the proposed binding mode of NVP-BEZ235 in PI3Ka showed a key interaction with the Ser774 residue of PI3K that was proposed to also be impor-tant for mTOR inhibition, due to the H-bond interactionwith themTOR corresponding residue, Ser 2665 (8). NVP-BKM120, on the other hand, lacks this contact with the Ser774 residue providing a rationale why it is a much lesseffective mTOR inhibitor.

The effects of NVP-BKM120 on the PI3K pathwaydownstream biomarkers p-Akt and pRPS6 inmechanisticPI3K (Rat1-transduced cells) and mTORC1 (TSC1�/�

minusMEFs) cell systems, respectively, show the predict-ability of thebiochemical profile in a cellular environment.These effectswere also observed in diseasemodels, show-ing the potency, specificity, as well as the dose depen-dency of the compound to block the PI3K pathway inrelevant cell-based models. NVP-BKM120 inhibitoryeffects in cells canbe rapidly revertedupondrugwashout,in agreement with the fast kinetics of the drug on PI3Kobserved in vitro. Pathway inhibitionbyNVP-BKM120 ledto proliferation inhibition in a variety of cell lines, repre-senting different lineages and genetic abnormalities thatpromote PI3K pathway activation. Interestingly, PIK3CAmutant lines were found to be statistically more sensitivethan PIK3CA wild-type lines. However, equivalent sen-sitivity was observed between cells bearing a functionalPTEN gene versus cells with phosphatase inactivatingmutations or deletions. Identical observations were madefor the pan-class I PI3K inhibitor GDC-0941, in an analysisrestricted to a panel of 54 breast cancer lines (38). How-ever, another study conducted with different PI3K inhi-bitors carried out in a panel of 39 lines from 9 differentlineages did not show enhanced activity in either PIK3CAor PTEN mutant lines (39). These conflicting data mightreflect the fact that, in the latter study, only a small numberof lines for each lineage and genetic alteration wereanalyzed, with a strong imbalance in the representationof each characteristic. Moreover, evidences for an impor-tant function for p110b in PTEN-null cell and tumormodels have been reported (40, 41). BKM120 is slightlyless potent on p110b and the concentration used to estab-lish the sensitivity profile (0.8 mmol/L) might have beeninsufficient to completely inhibit this isoform inour study.

Overall, pharmacologic inhibition of PI3K shows that:(i) a PIK3CAmutation is certainly not identical to a PTENmutation/deletion in terms of PI3K addiction and (ii)PTEN mutant or deleted tumors might have addiction toother pathways for proliferation and survival. Under-standing what these pathways would be could provideinsightful information on which combination partnerscould be used to complement the efficacy of PI3K inhibi-tors. Although PIK3CA mutant tumors seemed to bemore sensitive to PI3K inhibitors, one cannot concludethat all PIK3CAmutant tumors will respond to them. Forinstance, the cooccurrence of KRAS activating mutationsdramatically reduces the sensitivity toNVP-BKM120.Oneexplanation to this phenomenon is that pathways often

converge into similar and important downstream keyeffectors, and efficacy is dependent upon silencing of allcritical inputs to such downstream node. This was recent-ly elegantly illustrated in KRAS mutant/PIK3CA mutantcolorectal cancer models, in which efficacy could beachieved only when cyclin D1 levels could be reducedby cotreatment with MEK and Akt inhibitors (42). Simi-larly, apoptotic events in the HCT116 lines could bedetected only when NVP-BKM120 was combined withthe MEK inhibitor AZD6244, and these effects translatedin strong enhanced in vivo antitumor activity.

Target modulation and good pharmacokinetic andpharmacodynamic correlation was achieved with NVP-BKM120 in tumor-bearing mice. Significant and dose-dependent antitumor activity was observed at dose levelssufficient to shut down the PI3K pathway, showing thestrong relationship between compound concentration,pathway inhibition and efficacy. Suboptimal dose ofNVP-BKM120 was enough to strongly enhance the anti-tumor activity of standard of care (cytotoxic or targetedagents) in various cancer types such as prostate (withTaxotere),HER2-amplifiedbreast cancer, or gastric cancer(with Herceptin/trastuzumab) and GBM (with temozo-lomide). The fact that enhanced activitywas also observedin vivo against KRAS mutant HCT116 tumors whenBKM120 was combinedwith theMEK inhibitor AZD6244and opens newopportunities in indicationswith high andunmet medical need, in which KRAS is frequently mutat-ed (such as pancreatic cancers), but confirmatory studiesare warranted to test and eventually validate this para-digm in the relevant preclinical models.

As found with other PI3K inhibitors (22, 43), NVP-BKM120 possesses strong antiangiogenic activity. Hence,highly angiogenic tumors could be very sensitive to thecompound. NVP-BKM120 could, therefore, be used effi-ciently as second line treatment upon failure of approvedantiangiogenic drugs such as sutent, or sorafenib, in thecase of metastatic renal cell carcinoma. Of interest is thefact that NVP-BKM120 possesses excellent brain penetra-tion, hence, there is the possibility to treat advancednonresectable highly angiogenic GBM, mostly in combi-nation with temozolomide-based therapies.

>In conclusion, we have identified NVP-BKM120 as aselective pan-class I PI3K inhibitor. This compound pos-sesses excellent drug-like properties that have allowed itsselection for full clinical development. NVP-BKM120 iscurrently in phase II trials, in parallel with our dual PI3K/mTOR inhibitor NVP-BEZ325. The outcome of thesestudies is eagerly awaited, as they will be of primaryimportance in understanding the clinical relevance ofPI3K or dual PI3K/mTOR inhibition in different cancerindications.

Disclosure of Potential Conflicts of Interest

S.-M. Maira, S. Pecchi, A. Huang, M. Burger, M. Knapp, D. Sterker, C.Schnell, D. Guthy, T. Nagel, M. Wiesmann, S. Brachmann, C. Fritsch, P.Ch�ene, K. Shoemaker, A.D. Pover, D. Menezes, G. Martiny-Baron, D.Fabbro, C.L.Wilson, R. Schlegel, F. Hofmann,W.R. Sellers, andC.F. Voliva

Maira et al.





are Novartis employees. M. Dorsch and C. Garcia-Echeverria are Sanofi-Aventis employees.

Acknowledgments

The authors thank Marc Hattenberger, Fabian Stauffer, Sandra Moll�e,Laurent Laborde, and Juliane Vaxelaire for excellent technical assistance.

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 July 8, 2011; revised October 17, 2011; accepted November 17,2011; published OnlineFirst December 21, 2011.

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2012;11:317-328. Published OnlineFirst December 21, 2011.Mol Cancer Ther Sauveur-Michel Maira, Sabina Pecchi, Alan Huang, et al. Available Pan-Class I PI3-Kinase InhibitorIdentification and Characterization of NVP-BKM120, an Orally

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