PI3K Pathway Inhibition Achieves Potent Antitumor Activity ...Cancer Therapy: Preclinical PI3K Pathway Inhibition Achieves Potent Antitumor Activity in Melanoma Brain Metastases In

Cancer Therapy: Preclinical

PI3K Pathway Inhibition Achieves PotentAntitumor Activity in Melanoma Brain MetastasesIn Vitro and In VivoHeike Niessner1, Jennifer Schmitz2, Ghazaleh Tabatabai3,4,5,6, Andreas M. Schmid2,Carsten Calaminus2, Tobias Sinnberg1, Benjamin Weide1, Thomas K. Eigentler1, ClausGarbe1, Birgit Schittek1, Leticia Quintanilla-Fend7, Benjamin Bender8, Marion Mai9,10,Christian Praetorius9,10, Stefan Beissert9,15, Gabriele Schackert11,15, Michael H. Muders12,15,Matthias Meinhardt12,15, Gustavo B. Baretton12,15, Reinhard Dummer13, Keith Flaherty14,Bernd J. Pichler2, Dagmar Kulms9,10, Dana Westphal9,10,15, and Friedegund Meier1,9,15

Abstract

Purpose: Great advances have recently been made in treat-ing patients with metastatic melanoma. However, existingtherapies are less effective on cerebral than extracerebralmetastases. This highlights the potential role of the brainenvironment on tumor progression and drug resistanceand underlines the need for "brain-specific" therapies. Wepreviously showed that the PI3K-AKT survival pathway ishyperactivated in brain but not extracerebral melanomametastases and that astrocyte-conditioned medium activatesAKT in melanoma cells in vitro. We therefore tested the PI3Kinhibitor buparlisib as an antitumor agent for melanomabrain metastases.

Experimental Design and Results: Buparlisib inhibited AKTactivity, decreased proliferation, and induced apoptosis in met-astatic melanoma cell lines and short-term brainmelanoma cells,irrespective of their BRAF and NRASmutation status. In addition,buparlisib inhibited hyperactivated AKT and induced apoptosisin melanoma cells that were stimulated with astrocyte-condi-tioned medium. The growth of tumors induced by injectinghuman BRAF- and NRAS-mutant metastatic melanoma cells intothe brain of mice was significantly inhibited by buparlisib.

Conclusions: These results emphasize the value of targeting thePI3K pathway as a strategy to develop drugs for melanoma brainmetastases. Clin Cancer Res; 22(23); 5818–28. �2016 AACR.

IntroductionMelanoma is one of the most common tumors that metasta-

sizes to the brain. Up to 75% of patients with stage IV melanomadevelop brainmetastases (1). Brainmetastases are associatedwithsignificantmorbidity and very poor prognosis, showing amedianoverall survival of 3 to 6 months (2). Patients with a goodperformance status, controlled extracerebral metastases, andbrain metastases that can be managed by neurosurgery or radio-surgery have a better prognosis, including long-term survival. Theapproval of effective systemic drugs has revolutionized the treat-ment of metastatic melanoma. BRAF inhibitors have demonstrat-ed efficacy in fighting active BRAFV600-mutatedmelanoma brainmetastases. In a phase II trial, the BRAF inhibitor dabrafenib led toan intracranial objective response rate (ORR) of 31% to 39%, amedian progression-free survival (PFS) of 16 to 17 weeks and amedian overall survival (OS) of 31 to 33 weeks in BRAFV600E-mutatedmelanoma patients with asymptomatic brainmetastases(3). Another phase II study with the BRAF inhibitor vemurafenibin patients with BRAFV600-mutated melanoma and asymptom-atic and symptomatic brain metastases displayed an intracranialORRof 18% to20%, amedianPFSof 16 to18weeks andamedianOS of 28 weeks (4). However, the benefit for these patients islimited by the short response duration and lack of completedurable responses. The cytotoxic T lymphocyte antigen-4(CTLA-4) antibody, ipilimumab, has also shown activity inasymptomatic melanoma brain metastases, with an intracranialORR of 16%, a median PFS of 6 weeks, and a median OS of

1Department of Dermatology, University Hospital T€ubingen, EberhardKarls University, T€ubingen, Germany. 2Werner Siemens Imaging Center,Department of Preclinical Imaging and Radiopharmacy, University Hos-pital T€ubingen, Eberhard Karls University, T€ubingen, Germany. 3Interdis-ciplinary Division of Neuro-Oncology, Departments of Vascular Neurol-ogy&Neurosurgery,Hertie Institute forClinicalBrainResearch,UniversityHospital T€ubingen, Eberhard Karls University, T€ubingen Germany.4Neuro-Oncology Center T€ubingen, Comprehensive Cancer CenterT€ubingen-Stuttgart, Germany. 5Center for Personalized Medicine, Eber-hard Karls University, T€ubingen, Germany. 6German Cancer Consortium(DKTK), DKFZ partner site, T€ubingen, Germany. 7Department of Pathol-ogy, University Hospital T€ubingen, Eberhard Karls University, T€ubingen,Germany. 8DepartmentofDiagnostic and InterventionalNeuroradiology,University Hospital T€ubingen, Eberhard Karls University, T€ubingen, Ger-many. 9Department of Dermatology, Medical Faculty and UniversityHospital Carl Gustav Carus,TU Dresden, Germany. 10Center for Regener-ativeTherapiesDresden,DFGResearchCenterandClusterofExcellence,TUDresden,Germany.11DepartmentofNeurosurgery,MedicalFacultyandUniversity Hospital Carl Gustav Carus, TU Dresden, Germany. 12Depart-ment of Pathology, Medical Faculty and University Hospital Carl GustavCarus, TU Dresden, Germany. 13Department of Dermatology, UniversityHospital Z€urich, Z€urich, Switzerland. 14Massachusetts General HospitalCancerCenter,HarvardMedical School, Boston,Massachusetts. 15Nation-al Center for Tumor Diseases (NCT), Partner Site Dresden, Germany.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

H. Niessner and J. Schmitz contributed equally as first authors; D. Westphal andF. Meier contributed equally as senior authors.

Corresponding Author: Friedegund Meier, University of Dresden, Fetscherstr.74, Dresden 01307, Germany. Phone: 351-458-3677; Fax: 351-458-4338; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-16-0064

�2016 American Association for Cancer Research.

ClinicalCancerResearch

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30 weeks (5). However, the same survival benefit was not evidentfor patients with symptomatic melanoma brain metastases thatare dependent on a high dose of steroids (5). Thus, there is anurgent need for alternative treatments formelanomapatientswithbrain metastases.

Emerging data on the molecular characteristics of melanomabrain metastases suggest that brain metastases show significantdifferences compared to extracerebral metastases and primarytumors that may contribute to intracerebral therapy resistance.Although treatmentwith the BRAF inhibitor vemurafenib resultedin partial or complete remission of extracerebral metastases, brainmetastases appeared or progressed (6, 7). Immunohistochemicalanalysesof brainmetastases andmatchedextracerebralmetastasesrevealed the PI3K–AKT signaling pathway but not the RAF–MEK–ERK (MAPK) signaling pathway to be hyperactivated in the brain(6, 7). Although levels of ERK, pERK, and AKT appeared to beidentical, levels of pAKT were significantly increased in brainmetastases. This clinical difference is very significant, becauseERK, pERK,AKT, andpAKT expressionswere shown tobe identicalin monolayer cultures derived from melanoma brain and extra-cerebral metastases. In addition, melanoma cells cultured inastrocyte-conditioned medium again showed higher AKT activa-tion and invasiveness than when cultured in fibroblast-condi-tioned medium.

This suggests that brain-derived factors may hyperactivate theAKT survival pathway, promoting invasiveness and drug resis-tance of melanoma cells in the brain. Thus, inhibition of PI3K–AKT signaling with PI3K inhibitors such as buparlisib may helpdefeat melanoma brain metastases.

Buparlisib is a potent pan-class I PI3K inhibitor that selectivelytargets the catalytic isoforms of class IA (p110a, p110b, p110d)and class IB (p110g) PI3Ks. Buparlisib inhibits proliferation andinduces apoptosis in various tumor cell lines (8, 9). Furthermore,it inhibits the growth of human tumors in xenograft mousemodels in a well-tolerated manner and very efficiently penetratesthe blood–brain barrier (8–10). Buparlisib reduced glioblastomatumor spread in orthotopic xenograft models (11). A phase Iclinical trial using buparlisib on a variety of solid tumors showedgood disease control and tolerable toxicity (12). In addition, aclear clinical benefit was demonstrated in breast cancer patientstreatedwith buparlisib alone (13). Recently, phase II and phase IIIclinical trials have been extended to lung cancer, head and neck

cancer, and glioblastoma. Several studies suggest that the antitu-mor effects are enhanced when buparlisib is combined withother anticancer agents. For instance, combination of buparlisibwith fulvestrant in endocrine-resistant HRþ/HER2� breastcancer significantly improved PFS, ORR, and CBR (clinicalbenefit rate), particularly in patients with mutated phosphatidy-linositol-4,5-bisphosphate 3-kinase catalytic subunit alpha(PIK3CA; ref. 14).

Taken together, the favorable properties of the PI3K inhibitorbuparlisib in terms of potency, selectivity, brain penetrance, andthe reported clinical benefit in other cancers make it a goodcandidate for treating patients with melanoma brain metastases.We therefore wanted to investigate the antitumor activity ofbuparlisib in melanoma brain metastases in vitro and in vivo.

Materials and MethodsIsolation and culture of human cells

We used human metastatic melanoma cell lines, which wereBRAF-mutant (WM3734, 451Lu, A375, SKMel19), NRAS-mutant(SKMel147, WM1346, WM1366, MelJuso), or BRAF/NRAS wt(ZueMel1H). The cell lines were kindly provided byM. Herlyn, K.Smalley, and R. Dummer or purchased from the ATCC. In addi-tion, we used the patient derived brain tumor cell lines M10(BRAF-mutant), TueMel32H (NRAS-mutant), ZueMel2H, Tue-Mel20H, and TueMel41H, respectively. The cells were culturedin RPMI1640 medium supplemented with 10% FBS and 1%penicillin/streptomycin. Melanoma cell lines, cells isolated fromexcised brain or extracerebral melanoma metastases, and fibro-blasts were isolated and cultured as described previously (15). Allpatients provided written consent. The use of human tissues inthis study was approved by the medical ethical committee of theUniversities of Dresden, Z€urich, and T€ubingen, and performed inaccordance with the Declaration of Helsinki principles.

Signaling pathway inhibitors and treatmentsFor blockade of the RAF–MEK–ERK pathway, the BRAF inhib-

itor encorafenib (Novartis) and the MEK inhibitor binimetinib(Novartis) were employed. For inhibition of the PI3K–AKT path-way, the PI3K inhibitor buparlisib (Novartis) was used. Theinhibitors were added at concentrations ranging from 0.3 to 10mmol/L to the culture medium of cells with 50% to 70% con-fluency. Controls were incubated with DMSO alone.

Western blot analysesAfter 1 to 24hours incubationwith the inhibitors, the cellswere

lysed directly in the dish for 30 minutes on ice with buffercontaining 10 mmol/L Tris pH 7.5, 0.5% Triton X-100,5 mmol/L EDTA, 0.1 mmol/L phenylmethanesulphonylfluoride,10 mmol/L Pepstatin A, 10 mmol/L Leupeptin, 25 mmol/L apro-tinin, 20 mmol/L NaF, 1 mmol/L pyrophosphate, 1 mmol/Lorthovanadate. Lysates were cleared by centrifugation at13,000 g for 30 minutes and 15 to 60 mg protein was subjectedto SDS-PAGE and transferred to polyvinylidene difluoride(PVDF) membranes. Proteins were detected with Cell SignalingTechnology primary antibodies (AKT #9272, pAKTSer473 #4060,ERK #9102, pERKThr202/Tyr204 #4376, and b-actin #4970) andhorseradish peroxidase–conjugated secondary antibodies (Amer-sham), and membranes were exposed to X-ray film (EastmanKodak). All images were scanned and cropped in the AdobePhotoshop editing software.

Translational Relevance

Despite impressive advances with systemic therapies onpatients with metastatic melanoma, patients with melanomabrain metastases (MBM) still have a poor overall survival.Identifying and overcoming MBM-specific resistance mechan-isms is therefore a major aim in the search for successfultreatments. Recent studies have implicated a fundamental roleof the PI3K–AKT signaling pathway in survival and growth ofmelanoma cells in the brain microenvironment. Our studiesshow that inhibitionof the PI3K–AKTpathway leads to growtharrest and induction of apoptosis in brain metastatic melano-ma cells in vitro and in vivo. This provides a strong rationale fortargeting this pathway in melanoma patients with brainmetastases and will be investigated in a recently initiatedclinical trial.

PI3K Inhibition in Melanoma Brain Metastases

www.aacrjournals.org Clin Cancer Res; 22(23) December 1, 2016 5819



Growth assay (MUH assay)Cells seeded in quadruplicates in 96-well plates were treated

with the various inhibitors for 72 hours, washed twice with PBSand then incubated with 100 mL of a solution containing100 mg/mL 4-methylumbelliferyl-heptanoate (MUH) in PBS for1 hour at 37�C. The absolute fluorescence intensity at lex of355 nm and lem of 460 nm was measured using a FluoroskanII device (Labsystems). The intensity of fluorescence correspondsto the number of viable cells.

Cell-cycle analysisCells seeded in triplicates in 6-well plates were treated with

buparlisib for 72 hours. Floating and adherent cells were har-vested, washed with PBS, and fixed in ice-cold 70% ethanolovernight. After washing twice with cold PBS, cells were stainedwith 500 mL propidium iodide solution (propidium iodide40 mg/mL and RNase 100 mg/mL in PBS) for 20 min at 4�C. Thecell cycle was analyzed using flow cytometry and FACSDivasoftware (BD Biosciences).

IHCFor immunohistochemical analysis, human tumor tissue was

fixed in 4% formalin, embedded in paraffin, and stained withH&E, HMB45 (Dako #M0634), or pAKTSer473 (Cell SignalingTechnology, #4060). Bound antibodieswere detected usingUltra-View Universal Alkaline Phosphatase Red Detection Kits fromVentana (Tucson).

Immunofluorescent labelingHuman tumor tissue embedded in paraffin was prepared as

described above. Sectionswere blocked for 5minutes withUltra VBlock solution (Thermo Scientific) and then incubated overnightwith antibodies against cleaved PARP (Cell Signaling Technology,#5625) and MIB1 (Ki67, DAKO #M7240). Sections were washedwith PBS and incubated for 1 hour with a Cy3-coupled donkey-anti-mouse secondary antibody or a Cy5-coupled donkey-anti-rabbit antibody (Dianova). Nuclei were detected with YOPRO(Molecular Probes). Samples were analyzed using a Leica TCS SPconfocal microscope (Leica).

In vivo mouse modelStereotactic inoculation of melanoma cells into the brain of nudemice. In a stereotactic surgical procedure, 150,000 human mela-noma cells (WM3734 or SKMel147) were injected into the rightstriatum of 8-week-old female CD1nu/nu mice (Charles RiverLaboratories). All animals were housed under standardized envi-ronmental conditions (20 � 1�C room temperature, 50 � 10%relative humidity, and 12-hour light–dark phases) with free accessto food and water ad libitum. To achieve sufficient analgesia, themice were injected intraperitoneally with meditomidin/midazo-lam/fentanyl (0.5/5/0.05 mg/kg body weight). The anesthetizedanimals were positioned in the stereotactic apparatus by fixing thehead in the ear bars. After opening the skin and the skull, the cellswere injectedwith aHamilton syringe (150,000 cells in 3 mL) usinga stereotactic holder to insert the syringe into the mouse brains.Afterward, the wound was sutured and the mice were given car-profen (5 mg/kg body weight, twice daily, for 2–3 days s.c.)postoperatively.

All experiments were performed according to the animaluse and care protocol (x8 21.06.2012) of the German Animal

Protection Law and approved by the Regierungspr€asidiumT€ubingen.

Evaluation of the tumor volume. The tumor volume was evaluatedby MRI [BRAF-mutant tumors: Icon (1T-MRI; multislice multiecho (MSME) sequence, repetition time (TR) ¼ 2,931 millise-conds, echo time (TE) ¼ 60 milliseconds, 188 � 88 matrix, 22 �45 mm field of view, slice thickness (ST) ¼ 0.75 mm] and NRAS-mutant tumors: ClinScan [7T-MRI; turbo spin echo (TSE)sequence, TR ¼ 3,000 milliseconds, TE ¼ 205 milliseconds,256 �161 matrix, 35 � 57 mm field of view, ST ¼ 0.22 mmfrom Bruker BioSpin GmbH] and analyzed using the softwareInveon Research Workplace (Siemens Preclinical Solutions). Thefirst measurement was performed 15 to 16 days postinoculation.After the start of the therapy at day 20 postinoculation, theanimals were monitored twice weekly. All mice were sacrificedat the onset of neurologic symptoms, at 20% loss in weight or atthe latest 34 days after inoculation. The brains were excised andfixed in formalin for immunohistochemical analysis.

For mean value analyses, only the mice with a tumor volume�3 mm3 when treatment was commenced were included. For alltumors, the absolute tumor volume (Fig. 5) and the relative tumorvolume (Supplementary Fig. S6), normalized by the volume attreatment induction, were analyzed. Exponential fitting wasapplied on the tumor growth curves of the absolute tumorvolume. Kaplan–Meier plots were generated on the basis ofanimals reaching the endpoint criteria of 20% loss in weight.

Treatment of animals. Buparlisib was administered daily (30mg/kgbodyweight)on14 consecutivedays (8, 9), starting20daysafter the tumor inoculation, when the tumors were clearly visiblein the MRI scans.

Statistical analysisStatistical analysis was performed with GraphPad Prism ver-

sion 5.01 (GraphPad Prism Software Inc.). The differencesbetween the two groups were compared using the nonparametricMann–Whitney test (95% confidence interval). P values <0.05were considered statistically significant. N values represent thenumber of independent experiments unless stated otherwise.

Statistical analysis of the in vivoMRI growth datawas performedusing JMP Statistical Discovery version 11.0.0. The differencebetween the treated and the sham groups formultiple time pointswas analyzed using a Tukey-honest significant difference (HSD)post hoc test.

ResultsThe PI3K inhibitor buparlisib inhibits AKT phosphorylationand cell growth inmetastaticmelanoma cell lineswith differentmutation signatures

We and others previously showed that the PI3K–AKT pathwayis hyperactivated in melanoma brain metastases. Moreover, mel-anoma cells stimulated with astrocyte-conditioned mediumshowed increased AKT activation and invasiveness. On the basisof these results, we investigated whether the PI3K inhibitorbuparlisib could be implemented as an antitumor agent formelanoma brain metastases.

To verify that buparlisib inhibits the PI3K–AKT pathway, thephosphorylation status of AKT was investigated in three meta-static melanoma cell lines carrying different mutation signatures

Niessner et al.

Clin Cancer Res; 22(23) December 1, 2016 Clinical Cancer Research5820



(BRAFV600E: WM3734; NRASQ61R: SKMel147; BRAF/NRASwt:ZueMel1H). Buparlisib effectively inhibited the phosphorylationof AKT in a dose-dependent manner in all of the cell linesindependent of the mutation status (Fig. 1A). Consistently,buparlisib inhibited growth of all three cell lines by 60% to70%, as measured by the fluorimetric MUH assay (Fig. 1B).

Similar results were obtained with six other BRAF- or NRAS-mutated metastatic melanoma cell lines (Supplementary Fig. S1and S2), confirming that buparlisib effectively inhibits AKT phos-phorylation and proliferation of a variety of melanoma cell linesin vitro.

Growth inhibition is enhanced when the PI3K inhibitorbuparlisib is combined with a MEK inhibitor

To test whether the PI3K inhibitor buparlisib can augment theeffect of specific MEK and BRAF inhibitors, the three differentmetastatic melanoma cell lines were tested for growth inhibitionafter treatment with each of these inhibitors alone or incombination.

The MEK inhibitor binimetinib alone inhibited proliferationby 50% to 60% in the BRAF- and NRAS-mutated, and by 20% inthe BRAF/NRAS wild-type metastatic melanoma cells, whereascotreatment with buparlisib yielded significantly higher growthinhibition (80%; Supplementary Fig. S3A).

Because treatment of BRAFV600E metastatic melanoma cellswith increasing concentrations of the BRAF inhibitor encorafenibalone already inhibited cell growth by 80%, cotreatment withbuparlisib didnot yield additional improvement (SupplementaryFig. S3B).

Furthermore, AKT phosphorylation was inhibited by buparli-sib, with or without encorafenib or binimetinib, starting within1 hour after stimulation (Supplementary Fig. S3C). Inhibition ofAKT phosphorylation was still effective after 24 hours. Asexpected, pERK levels were inhibited by binimetinib and encor-afenib, but remained unaffected by buparlisib at 1 and 24 hoursafter treatment.

In summary, combining MEK and PI3K inhibitors inhibitsgrowth more effectively than specific MEK inhibitors alone,

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The PI3K inhibitor buparlisib inhibits AKT phosphorylation and cell growth inmetastatic melanoma cell lines.A,WM3734 (BRAF-mutant), SKMel147 (NRAS-mutant),and ZueMel1H (BRAF/NRASwt) metastatic melanoma cell lines were treated with increasing concentrations of the PI3K inhibitor buparlisib for 6 hoursand were subjected to Western Blot analysis of AKT and pAKT. b-Actin was detected as a loading control. Western Blot analysis is representative of threeindependent experiments. Western Blot analysis of eight other BRAF- or NRAS-mutant cell lines are shown in Fig. 3 and Supplementary Figs. S1 and S2.B, Metastaticmelanoma cells were treated with buparlisib for 72 hours and subjected to a growth inhibition assay (MUH assay). The percentage of growth inhibition wascompared to DMSO-treated controls. Error bars represent SD of three independent experiments. Growth inhibition assays of 11 other BRAF- or NRAS-mutantcell lines are shown in Fig. 3 and Supplementary Figs. S1, S2, and S4.





regardless of the mutation status of the target cell. In all cases,growth inhibition coincided with siginificant reduction ofpAKT and pERK levels. In contrast, the PI3K inhibitor did notaugment the efficacy of the BRAF inhibitor in the BRAF-mutatedcell line.

The PI3K inhibitor buparlisib induces apoptosis in metastaticmelanoma cell lines

To investigate whether growth inhibition induced by buparli-sib also coincides with induction of cell death, cell-cycle analysiswas performed for all three mutation-specific melanoma celllines. Buparlisib treatment caused a cell-cycle arrest in theG2–M phase in the BRAF-mutated cell line, and additionally a20% to 50% increase of cells in the sub-G1 phase (apoptotic cells)in all cell lines tested (Fig. 2A andB). Similar results were obtainedwith six other BRAF- and NRAS-mutant metastatic melanoma celllines, confirming the ability of buparlisib to drive a variety ofmelanoma cell lines into apoptotic cell death (SupplementaryFigs. S1C and S2C).

The PI3K inhibitor buparlisib inhibits proliferation andinduces apoptosis in short-term brain melanoma cells

To add more in vivo relevance to the growth inhibitory andcell death inducing effects of buparlisib, two melanoma celllines (BRAF and NRAS mutated) directly isolated from excisedbrain metastases of patients were investigated. In accordancewith our previous observations, activated AKT was significant-ly decreased after buparlisib treatment as shown by WesternBlot analysis (Fig. 3A). Buparlisib also inhibited growth by upto 70% in these as well as three other short-term brainmelanoma cell lines (Fig. 3B and Supplementary Fig. S4).Furthermore, buparlisib caused G2–M arrest and a 15%increase in apoptosis in the BRAF-mutated cell line, and a40% increase in apoptosis in the NRAS-mutated cell line(Fig. 3C and D).

In contrast, nontumor-derived cells of human skin such asfibroblasts stayed largely resistant to buparlisib, with growthbeing inhibited by only 40% at the highest buparlisib concen-tration and no increased apoptosis (Supplementary Fig. S5A).

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The PI3K inhibitor buparlisib induces apoptosis in metastatic melanoma cell lines. WM3734 (BRAF-mutant), SKMel147 (NRAS-mutant), and ZueMel1H (BRAF/NRASwt) metastatic melanoma cell lines were treated with increasing concentrations of the PI3K inhibitor buparlisib for 72 hours and subjected to cell-cycleanalysis by flow cytometry. Histograms in A Are representative of triplicate experiments. The percentage of cells in <G1, G1, S, and G2–M phase was quantified anddisplayed in a stacked bar graph (B). Error bars represent SD of triplicate experiments. Cell-cycle analysis of eight other BRAF- or NRAS-mutant cell lines isshown in Fig. 3 and Supplementary Figs. S1 and S2.

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The PI3K inhibitor buparlisib inhibits AKT phosphorylation and cell growth and induces cell-cycle arrest and apoptosis in short-term brain melanoma celllines. A, Brain-derived melanoma cells were treated with increasing concentrations of the PI3K inhibitor buparlisib for 6 to 12 hours and were subjected toWestern Blot analysis of AKT andpAKT.b-Actinwas detected as a loading control.Western Blot analysis is representative of three independent experiments.B, Brainmelanoma cells were treated with buparlisib for 72 hours and then subjected to a growth inhibition assay (MUH assay). The percentage of growthinhibition was compared with DMSO-treated controls. Error bars represent SD of three independent experiments. C and D, Brain melanoma cells weretreated with buparlisib for 72 hours and then subjected to cell-cycle analysis by flow cytometry. Histograms in C are representative of triplicate (TueMel32H) orthree independent (M10) experiments. The percentage of cells in <G1, G1, S, and G2–M phase was quantified and displayed in a stacked bar graph (D). Errorbars represent SD.





Together, these findings strongly imply that buparlisib selectivelyacts on tumor cells.

The PI3K inhibitor buparlisib inhibits AKT activation andinduces apoptosis inmetastaticmelanoma cell lines stimulatedwith astrocyte-conditioned medium

To evaluate buparlisib for the treatment of brainmetastases, wemimicked the brain environment in vitro by culturing WM3734brain-metastatic melanoma cells in astrocyte-conditioned medi-um. As seen before, astrocyte- but not fibroblast-conditionedmedium increased the pAKT levels in WM3734 cells, which werecompletely inhibited by addition of 1 mmol/L buparlisib(Fig. 4A). Moreover, the PI3K inhibitor buparlisib induced apo-ptosis in approximately 20% of melanoma cells (Fig. 4B and C),indicating that buparlisib can successfully combat the changesbeing induced by the brain microenvironment.

The PI3K inhibitor buparlisib inhibits growth of BRAF- andNRAS-mutant melanoma in the brain of nude mice

The encouraging results of buparlisib antitumor activity onmelanoma cells placed in an artificial brain microenvironmentprompted us to assess its effects on intracranial tumor growthusing an in vivo mouse model of orthotopically transplantedhuman BRAF-mutant and NRAS-mutant melanoma brainmetastases.

Excitingly, mice treated with the PI3K inhibitor buparlisibshowed no further tumor growth, whereas mice treated only withthe vehicle developed rapidly growing tumors in the brain, in

both, the BRAF- and the NRAS-mutated tumors (Fig. 5A). Signif-icant differences in relative tumor volumes (Supplementary Fig.S6A) as well as in absolute tumor volumes (Fig. 5B) between thebuparlisib and sham-treated mice were observed 7 days aftertreatment onset (day 27 postinoculation) for the BRAF-mutantcells and 6 days after treatment induction (day 26 postinocula-tion) for the NRAS-mutant cells. For both cell lines, exponentialfittingwas applied to the growth curves of all tumors, verifying theinhibition of the exponential growth pattern of all tumors in thebuparlisib-treated animals against the sham-treated controls(Supplementary Fig. S6B). After 34 days, a Kaplan–Meier plotshowed moderate survival for both the sham and the treatmentgroup in the BRAF-mutant tumormice but dramatically improvedsurvival in the treatment group for the NRAS-mutant tumors(Fig. 5C).

The 7T TSE MRI data of the NRAS-mutated tumors allowed usto identify T2 hyperintense regions indicative of hemorrhages andcysts as well as peritumoral edema (Supplementary Fig. S6C).Peritumoral edema was mild and not seen in all animals. Inter-estingly, there was a remarkable increase of T2 hyperintenseregions in the therapy group, with multifocal intratumoral areasof liquid indicative of hemorrhages or cysts. In the sham group,most tumors appeared relatively homogeneous and with distinc-tively fewer T2 hyperintense areas.

H&E staining of untreated and treatedBRAF- andNRAS-mutanttumors confirmed what was seen in the MRI (Supplementary Fig.S7A). Intratumoral bleeding was seen in both treatment groups,but was considerably higher in the buparlisib-treated group(Supplementary Fig. S7B). Necrosis was detected in all untreated

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Figure 4.

The PI3K inhibitor buparlisib inhibits AKT activation and induces apoptosis in metastatic melanoma cell lines stimulated with astrocyte-conditioned medium. TheWM3734 (BRAF-mutant) metastatic melanoma cell line was cultured in RPMI, astrocyte- (ACM), and fibroblast-conditioned medium (FCM) without FCS andtreated with or without 1 mmol/L PI3K inhibitor buparlisib for 30 hours.A, Samples were harvested forWestern blot analysis and analyzed for AKT and pAKT. b-Actinwas detected as a loading control. Western Blot analysis is representative of three independent experiments. B and C, Samples were subjected to cell-cycleanalysis by flow cytometry. Histograms in B are from three independent experiments. The percentage of cells in <G1, G1, S, and G2–M phase was quantified anddisplayed in a stacked bar graph (C). Error bars represent SD of three independent experiments.

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The PI3K inhibitor buparlisib inhibits growth of metastatic melanoma in the brain of nude mice. Representative MRI data of injected WM3734 (BRAF-mutant)humanmelanomabrainmetastasis under buparlisib or sham treatment (A).MRIwas performed 16, 20, 23, 27, and30days after inoculation.Onday20postinoculation(p.i.), the treatment was started. Buparlisib induced stable disease, compared to rapid tumor growth in the sham-treated animals for both BRAF-mutant(WM3734) and NRAS-mutant (SKMel147) brain metastasis, shown by mean values� SD of the absolute tumor volume (B). For mean value analyses, the mice withsecure tumor indication at day 19 to 20were included (tumor volume�3mm3; n¼ 8/8 in the BRAF-mutated group; n¼ 5/5 in the NRAS-mutated group); even if notvisible at day 15 in the NRAS-mutated group (n ¼ 3/3 at day 15). Significant differences were reached 7 and 6 days after treatment induction for the BRAF- andNRAS-mutant tumors respectively, as determined by Tukey-HSD post hoc tests. Kaplan–Meier plots showed moderate survival for both the treatmentand sham group in BRAF-mutant tumors but dramatically improved survival of the treatment group in NRAS-mutant tumors (C).





NRAS-mutant tumors, highlighting the aggressiveness of theNRAS-mutant tumors (Supplementary Fig. S7C). However, therewere also areas of tumor regression, in which the tumor shrankand was replaced by water-filled pseudo-cysts (SupplementaryFig. S7D). Buparlisib caused a substantial increase of these pseu-do-cysts in the treatedNRAS-mutant tumors, withM12 exhibitingup to 70% of tumor regression.

The effectiveness of buparlisib could also be visualized inimmunohistochemical stainings for pAKT and immunofluores-cence stainings for Ki67 (proliferation marker) and cleaved PARP(apoptosis marker; Supplementary Fig. S8). Although buparlisibcould not induce sufficient apoptosis to achieve clearance of thetumors (Supplementary Fig. S8A and S8C), it almost completelyinhibited proliferation and tumor growth (Supplementary Fig.S8B and S8D). As expected, AKT activation was completely pre-vented by buparlisib (Supplementary Fig. S8E).

In summary, the PI3K inhibitor buparlisib inhibited growth ofBRAF-mutant, and even more effectively of NRAS-mutant mela-noma cells in the brain of mice, providing evidence for its potentantitumor activity on melanoma brain metastases in vivo.

DiscussionRecently, impressive novel treatment strategies have been devel-

oped for patients with metastatic melanoma, particularly throughinhibiting the RAF–MEK–ERK signaling pathway and by immu-notherapy. Although these systemic therapies have been remark-ably successful infightingmetastaticmelanoma, patients frequent-ly develop brain metastases during these therapies. So far, little isknown about how melanoma brain metastases evade existingtherapies. Investigatingmatched samples frommelanomapatientswith both cerebral and extracerebral metastases, the PI3K–AKTsignaling pathway was found to be upregulated in cerebral com-pared with extracerebral metastases (6, 16). This prompted us toinvestigate the antitumor activity of the PI3K inhibitor buparlisibin melanoma brain metastases in vitro and in vivo.

We showhere that buparlisib prevented AKT activation and cellgrowth of a variety of metastatic melanoma cell lines in vitro. Inaddition, it caused an arrest in the G2–M phase and inducedapoptosis to varying degrees in these cell lines.

Our in vivo experiments demonstrated robust inhibition of AKTactivation as well as proliferation of human melanoma cellsimplanted into the brain of mice, indicating that buparlisibcrosses the blood–brain barrier, as suggested by earlier xenograftexperiments (8, 9). The inhibitory effect of buparlisib prevailedirrespective of the BRAF or NRASmutation status. In particular, inmice with NRAS-mutated brain tumors treatment with buparlisibprovided a survival benefit compared with sham treatment.Although the tumors were smaller in the BRAF-mutated group,mice of both the sham and the treatment group showed rapidweight loss, reaching the endpoint of 20% loss in weight fasterthan the NRAS-mutated group. Evaluation of the tumor sectionssuggested a fundamental difference between the NRAS- andBRAF-mutated tumors. Although the NRAS-mutated tumorsseemed to be more aggressive, the inhibitory effect of buparlisibwas much more prominent in these tumors compared to BRAF-mutated tumors. As RASbut not RAF can signal through the PI3K–AKT pathway, NRAS-mutated tumors are probablymore sensitiveto PI3K pathway inhibition than BRAF-mutated tumors. Howev-er, more extensive experiments would be needed to furtherinvestigate this prediction.

In our in vitro experiments, the combination of the PI3Kinhibitor buparlisib with a MEK or BRAF inhibitor led to robustinhibition of AKT and ERK activation. Furthermore, combiningthe PI3K inhibitor buparlisib with theMEK inhibitor binimetinibinhibited the growth of the melanoma cell lines significantlybetter than the PI3K or MEK inhibitor alone, irrespective of themutation status. These data are in line with recent in vivo studies.In a BRAF inhibitor-resistant patient-derived xenograft model,tumor growth was significantly better controlled by buparlisib incombination with encorafenib plus binimetinib compared tobuparlisib or encorafenib plus binimetinib alone (17). Similarresults were obtained when the PI3K inhibitor buparlisib wascombined with the ERK inhibitor VX-11e (17).

In our BRAF-mutated brain metastatic melanoma cells, thePI3K inhibitor buparlisib did not enhance the potent growthinhibition by the BRAF inhibitor encorafenib. However, in a pilotstudy, combinations of buparlisib and encorafenib improved thesurvival of mice with intracranial BRAF-mutated tumors com-pared to encorafenib alone (16). Accordingly, similar findingswere obtained by using another BRAF inhibitor (PLX4720) andpan-PI3K inhibitor (PX-866) (18).

These data suggest that combining the PI3K inhibitor buparli-sib with a BRAF/MEK inhibitor may prolong response durationand survival in melanoma patients with BRAF-mutated brainmetastases. However, a preliminary study of eight cases in whichbuparlisib was combined with the BRAF inhibitor vemurafenibwas not as successful, as half the patients developed dose-limitingtoxicities such as myalgia, DRESS syndrome, and febrile neutro-penia, andonly oneof the eight patients showed amixed responseto the treatment (19). Given that in breast cancer clinical trialscombination treatment with buparlisib and the estrogen-receptorantagonist fulvestrant significantly improved the OS in theabsence of severe side effects (14), further clinical trials of bupar-lisib or other PI3K inhibitors in combination with RAF/MEKinhibitors will be required to determine the actual clinical benefit.

Since buparlisib inhibits class I PI3 kinases with similar poten-cy, the present data donot resolvewhich PI3K isoformswithin thePI3K–AKT signaling cascade are being activated by the brainmicroenvironment. Isoform selective inhibitors are currentlybeing clinically investigated, and could offer an alternative strat-egy if a pan-PI3K inhibitor such as buparlisib is not tolerated incombination with MAP kinase pathway inhibitors.

Our data on buparlisib inhibiting AKT activation and decreasinggrowth of human melanoma cells in the brain of mice highlightsthe importance of the PI3K–AKT pathway in the survival andgrowth of brain metastatic melanoma cells and is nicely in linewith previous reports. Multidimensional molecular profiling ofmatched cerebral and extracerebral melanoma metastases foundno differences in hotspot mutations, copy number variations,mRNA, and protein expression, but detected increased expressionof several activation-specific protein markers of the PI3K–AKTpathway in brain metastases (16). Furthermore, in a retrospectiveanalysis, complete loss of the phosphatase PTEN, which inhibitsPI3K, correlated with more rapid brain metastasis formation anddecreased OS in stage IIIB/C melanoma patients with BRAFV600mutations (20). A recent study suggested a functional crosstalkbetween PI3K–AKT signaling and PLEKHA5 to exist in melanomabrain metastases (21). PLEKHA5 was overexpressed in cerebrotro-pic A375 melanoma cells, and its knockdown stopped the cellsfrom proliferating and migrating across the blood–brain barrier invitro. PLEKHA5 possesses a pleckstrin homology domain that


Niessner et al.



facilitates recruitment to the plasma membrane and binding tophosphoinositides such as PIP3 (22). As PIP3 is generated by thePI3K, a PI3K inhibitor such as buparlisib might inhibit PLEKHA5membrane association and its function.When staining the untreat-ed versus treated NRAS- and BRAF-mutated tumors for PLEKHA5,we found that PLEKHA5 was significantly decreased in the bupar-lisib-treated tumors (Supplementary Fig. S9). One may speculatethat after loss of itsmembrane association, PLEKHA5 is targeted forproteasomal degradation. As PLEKHA5 expression correlates withthe earlyonset ofmelanomabrainmetastases (21), PI3K inhibitionmay be a new strategy to affect melanoma brain metastasis for-mation via targeting PLEKHA5.

We observed that mimicking the brain environment in vitro bystimulating metastatic melanoma cells with astrocyte-condi-tioned medium increased the pAKT levels. Buparlisib completelyprevented this increase and induced apoptosis in these cells. Theseresults illustrate how the brain microenvironment activates thePI3K–AKT pathway, potentially allowing brain metastases toresist therapy. Several studies have demonstrated interactionsbetween astrocytes and tumor cells including melanoma cells.The interaction with astrocytes upregulates multiple genes inmelanoma cells, including several survival genes that increasetheir resistance to cytotoxic drugs (23). It has been demonstratedrecently that astrocytes epigenetically repress expression of thenegative regulator of PI3K signaling, PTEN, in metastatic tumorcells (24). Another experimental study showed that brain-meta-static melanoma cells acquire neuron-like characteristics (25),whichmay intensify their interactionswith normal brain cells andpromote survival and growth (25). This brain-specific signatureoverlaps with previous data showing significant transcript differ-ences between melanoma cells harvested from the brain com-pared to other metastatic sites (26). Moreover, interactionsbetween astrocytes and melanoma cells seem to be reciprocal,as melanoma cells can reprogram astrocytes to express IL23 (27).This inflammatory cytokine in turn causes the melanoma cells tosecrete the matrix metalloproteinase MMP2, thereby enhancingtheir invasiveness (27).

In conclusion, our data provide evidence that hyperactivationof the PI3K–AKT pathway is a prominent feature of melanomabrain metastases. Even though themolecular mechanisms under-lying PI3K–AKT hyperactivation remain to be determined, ourdata provide a strong rationale for targeting this pathway inmelanoma patients with brainmetastases. Combinations of PI3Kinhibitors with BRAF orMEK inhibitorsmay significantly increaseantitumor activity and prolong survival. A phase II trial of bupar-lisib in patients with stage IVmelanoma and brainmetastases hasbeen initiated (NCT02452294). The results will further increaseour knowledge about the potential of buparlisib for treatingmelanoma brain metastases.

Disclosure of Potential Conflicts of InterestT.K. Eigentler reports receiving speakers bureau honoraria from and is a

consultant/advisory board member for Bristol-Myers Squibb. C. Garbe reportsreceiving other commercial research support fromBristol-Myers Squibb, Novar-tis, and Roche; and is a consultant/advisory board member for Amgen, Bristol-Myers Squibb, LEO, MSD, Novartis, and Roche. F. Meier reports receiving othercommercial research support from Novartis. No potential conflicts of interestwere disclosed by the other authors.

Authors' ContributionsConception and design: H. Niessner, J. Schmitz, G. Tabatabai, A.M. Schmid,C. Garbe, R. Dummer, B.J. Pichler, D. Westphal, F. MeierDevelopment of methodology: H. Niessner, J. Schmitz, A.M. Schmid,C. Calaminus, R. Dummer, B.J. Pichler, D. Westphal, F. MeierAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): H. Niessner, J. Schmitz, G. Tabatabai, B. Weide,C. Garbe, L. Quintanilla-Fend, M. Mai, G. Schackert, M.H. Muders, M. Mein-hardt, G.B. Baretton, R. Dummer, B.J. Pichler, D. Westphal, F. MeierAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): H. Niessner, J. Schmitz, A.M. Schmid, T. Sinnberg,C. Garbe, L. Quintanilla-Fend, B. Bender, M.Mai, C. Praetorius, S. Beissert, M.H.Muders, R. Dummer, K. Flaherty, B.J. Pichler, D. Kulms, D. Westphal, F. MeierWriting, review, and/or revision of the manuscript: H. Niessner, J. Schmitz,G. Tabatabai, A.M. Schmid, C. Calaminus, T. Sinnberg, B. Weide, T.K. Eigentler,C. Garbe, B. Schittek, B. Bender, M. Mai, C. Praetorius, S. Beissert, G. Schackert,M. Meinhardt, G.B. Baretton, R. Dummer, K. Flaherty, B.J. Pichler, D. Kulms,D. Westphal, F. MeierAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J. Schmitz, A.M. Schmid, C. Calaminus,C. Garbe, B. Schittek, M. Mai, G. Schackert, M. Meinhardt, D. Kulms,D. Westphal, F. MeierStudy supervision: A.M. Schmid, R. Dummer, F. MeierOther (performed and interpreted the histological analysis, supervised thecorrect presentation of the information): L. Quintanilla-Fend

AcknowledgmentsThe authors would like to thank Funda Cay, Laura K€ubler, Sandro Aidone,

and Julia Lagler, for excellent technical assistance. We thankMichelleMeredyth-Stewart for critical reading of the manuscript.

Grant SupportThis work was supported by the German Research Foundation (SFB 773;

F. Meier, H. Niessner, B. Schittek, and G. Tabatabai), Novartis (F. Meier,H. Niessner), the University of T€ubingen (fortuene, H. Niessner; Demonstra-torprojekt, G. Tabatabai), the Federal Ministry of Education and Research (FKZ031A423B; C. Praetorius, D. Kulms), the German Cancer Aid (110210;B. Schittek), and the National Center for Cancer/NCT Program and Infrastruc-ture Grant (F. Meier, M.H. Muders).

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

Received January 11, 2016; revised May 3, 2016; accepted May 25, 2016;published OnlineFirst June 15, 2016.

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Correction

Correction: PI3K Pathway Inhibition AchievesPotent Antitumor Activity in Melanoma BrainMetastases In Vitro and In Vivo

In this article (Clin Cancer Res 2016;22:5818–28), which was published in the Decem-ber 1, 2016, issue of Clinical Cancer Research (1), contributions for two authors weremistakenly omitted from the Authors' Contributions section of the published article.Tobias Sinnberg should be listed under "Conception and design" and "Development ofmethodology," andHeikeNiessner should be listed under "Administrative, technical, ormaterial support (i.e., reporting or organizing data, constructing databases)." Theauthors regret this error.

Reference1. Niessner H, Schmitz J, Tabatabai G, Schmid AM, Calaminus C, Sinnberg T, et al. PI3K pathway

inhibition achieves potent antitumor activity in melanoma brain metastases in vitro and in vivo.Clin Cancer Res 2016;22:5818–28.

Published online March 1, 2017.doi: 10.1158/1078-0432.CCR-16-3165�2017 American Association for Cancer Research.

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