Combined HDAC and Bromodomain Protein ... · Orthotopic glioblastoma PDX and stem cell–like xenograft model FortheGBM12orthotopicGBMmodel(34),300,000cellsand forthestem-likeGBMmodel,20,000NCH644cellswereinjected

Translational Cancer Mechanisms and Therapy

Combined HDAC and Bromodomain ProteinInhibitionReprogramsTumorCellMetabolismandElicits Synthetic Lethality in GlioblastomaYiru Zhang1, Chiaki Tsuge Ishida1,Wataru Ishida2, Sheng-Fu L. Lo2, Junfei Zhao3,Chang Shu1, Elena Bianchetti1, Giulio Kleiner4, Maria J. Sanchez-Quintero4,Catarina M. Quinzii4, Mike-Andrew Westhoff5, Georg Karpel-Massler6, Peter Canoll1, andMarkus D. Siegelin1

Abstract

Purpose:Glioblastoma remains a challenge in oncology, inpart due to tumor heterogeneity.

Experimental Design: Patient-derived xenograft and stem-like glioblastoma cells were used as the primary modelsystems.

Results: Based on a transcriptome and subsequent geneset enrichment analysis (GSEA), we show by using clinicallyvalidated compounds that the combination of histone dea-cetylase (HDAC) inhibition and bromodomain protein(BRD) inhibition results in pronounced synergistic reduc-tion in cellular viability in patient-derived xenograft andstem-like glioblastoma cells. Transcriptome-based GSEAanalysis suggests that metabolic reprogramming is involvedwith synergistic reduction of oxidative and glycolytic path-ways in the combination treatment. Extracellular flux anal-ysis confirms that combined HDAC inhibition and BRDinhibition blunts oxidative and glycolytic metabolism of

cancer cells, leading to a depletion of intracellular ATPproduction and total ATP levels. In turn, energy deprivationdrives an integrated stress response, originating from theendoplasmic reticulum. This results in an increase in proa-poptotic Noxa. Aside from Noxa, we encounter a compen-satory increase of antiapoptotic Mcl-1 protein. Pharmaco-logic, utilizing the FDA-approved drug sorafenib, and genet-ic inhibition of Mcl-1 enhanced the effects of the combi-nation therapy. Finally, we show in orthotopic patient-derived xenografts of GBM, that the combination treatmentreduces tumor growth, and that triple therapy involving theclinically validated compounds panobinostat, OTX015, andsorafenib further enhances these effects, culminating in asignificant regression of tumors in vivo.

Conclusions: Overall, these results warrant clinical testingof this novel, efficacious combination therapy. Clin Cancer Res;24(16); 3941–54. �2018 AACR.

IntroductionOver the last decade, there were tremendous new insights in

the characteristics of solid cancers, such as melanoma andglioblastoma. Notable findings are for instance the discoveryof BRAF mutations in melanoma and IDH1 mutations in glio-mas (1–6). Regarding gliomas, comparable efforts have beentaken, albeit these approaches have not matured to the extent asin melanoma. Nevertheless, given the lack of durability new or

amended strategies are required to address the problem of solidtumors.

In conjunctionwith the discovery of drivermutations, there area number of small molecule inhibitors available that targetderegulated pathways in cancers. Similarly, transcription is alteredin cancer by various mechanisms, for example DNA methylationor histone acetylation. Very recently, mutations in the histone 3.3protein (H3) at codon 27 were identified and it was shown inpreclinical model systems that malignancies with this aberrationbenefit from treatment with a histone-deacetylase inhibitor. Inthis regard, broad histone deacetylase (HDAC) inhibitors, such aspanobinostat or vorinostat, have reachedFDA-approval andare inclinical testing for solid malignancies as well. However, as withother monotherapies, tumors are either primarily or becomesecondarily resistant to these agents. Therefore, combinationtherapies that targetmultiple pathwaysmay address this problem.One of these signaling cascades is c-myc, which is highly upre-gulated inmany cancers andwhichbecamedruggable through thediscovery of bromodomain (BRD) protein inhibitors (7, 8), suchas the prototype JQ1 and clinically more amenable chemicalderivatives, such as OTX015.

Here, we provide a mechanism-based drug combinationtherapy that synergistically reprograms tumor cell transcription,leading to metabolic reprogramming, energy starvation, andactivation of the integrated stress response followed by significant

1Department of Pathology andCell Biology, Columbia UniversityMedical Center,New York, New York. 2Department of Neurosurgery, The Johns Hopkins Uni-versity School of Medicine, Baltimore, Maryland. 3Department of BiomedicalInformatics, Columbia University, New York, New York. 4Department of Neu-rology, H. HoustonMerritt Neuromuscular Research Center, Columbia UniversityMedical Center, New York, NewYork. 5Department of Pediatrics and AdolescentMedicine, Ulm University Medical Center, Ulm, Germany. 6Department of Neu-rosurgery, Ulm University Medical Center, Ulm, Germany.

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

CorrespondingAuthor:Markus D. Siegelin, Columbia University Medical Center,630 West 168th Street, P&S 15-401, New York, NY 10032, Phone: 212-305-1993;Fax: 7748239187; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-18-0260

�2018 American Association for Cancer Research.

ClinicalCancerResearch

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intrinsic apoptosis. Moreover, the combination therapy causes apartial cyto-protective effect by elevating Mcl-1 and interferencewith Mcl-1 through an FDA-approved drug leads to significanttumor regression and survival extension in two glioblastomaxenograft models in vivo.

Materials and MethodsReagents

OTX015 (OTX), vorinostat (Vr), panobinostat (Pb), and sor-afenib (Sf) were purchased from Selleckchem.

Cell cultures and growth conditionsU87MG, LN229, U87-EGFRvIII, and T98G human glioblasto-

ma cell lines and A375 malignant melanoma cells were obtainedfrom the ATCC or the Coriell Institute for Medical Research,respectively. NCH644 and NCH421K stem cell-like glioma cellswere obtained from Cell Line Services (CLS). The GBM14 PDXcells have been described elsewhere (9–12). The respective cellline depository authenticated the cells. All cell lines were culturedas previously described (13–16).

Cell viability assaysViability assays were performed as previously described

(13–18).

Measurement of apoptosis and mitochondrial membranepotential

For Annexin V/propidium iodide staining the Annexin V Apo-ptosis Detection Kit (BD Pharmingen) was used as previouslydescribed (16, 19). Tetramethylrhodamine ethyl ester (TMRE)staining was performed according to the manufacturer's instruc-tions (Mitochondrial Membrane Potential Kit; Cell SignalingTechnology). The data were analyzed with the FlowJo software(version 8.7.1; Tree Star).

Extracellular flux analysisExtracellular flux analysis was performed on the Seahorse

XFe24 analyzer. The mitochondrial stress assay was utilized in

accordance with the instructions by the manufacturer and asdescribed earlier (14). The glycolytic stress test was used inaccordance with the instructions by the manufacturer. Briefly,40,000 cells were seeded and starved. During the assay,cells were exposed to glucose, followed by oligomycinand completed by 2-DG. The extracellular acidification rate(ECAR) was measured.

Transfections of siRNAs or transduction of shRNAsBriefly, cells were incubated for 6 hours with the formed

complexes of Oligofectamine 2000 (Invitrogen) and the respec-tive siRNA (12-well condition) in DMEM without FBS and anti-biotics. After 6 hours, FBS was added to a total concentrationof 1.5%. Transduction of lentiviral shRNAs is performed asdescribed earlier (14).

Western blot analysis, protein capillary electrophoresis, andimmunoprecipitation analysis

Specific protein expression in cell lines was determined byWestern blot analysis as described before (18). Co-immunopre-cipitations were performed as described earlier in ref. 13.

Subcutaneous xenograft modelA total of 1� 106 A375 cells, 1� 106 U87-EGFRvIII, or GBM12

PDX xenograft tumors were implanted subcutaneously into theflanks of 6- to 8-week-old SCID SHO mice as described before(16). Measurements were performed with a caliper and tumorsizes were calculated as (length � width2)/2. Treatments wereperformed intraperitoneally as described in the respective figurelegends of each individual experiment. Columbia IACUC hasapproved these studies.

Orthotopic glioblastoma PDX and stem cell–like xenograftmodel

For theGBM12 orthotopic GBMmodel (34), 300,000 cells andfor the stem-like GBMmodel, 20,000NCH644 cells were injectedin a manner as described earlier. The drugs for the indicatedtreatments were dissolved as described earlier (10). ColumbiaIACUC has approved these studies.

Microarray and gene set enrichment analysisTranscriptome and gene set enrichment analysis (GSEA) was

performed as previously described (14). For the present experi-ments, two biological replicates per condition were submitted.The experiment was deposited online with GEO with the follow-ing ID: GSE108958.

Statistical analysisStatistical significance was assessed by Student t test using

Prism version 7.00 (GraphPad). Data were consideredstatistically significant at �P < 0.05, ��P < 0.01, ��P <0.001, or ��P < 0.0001 level. n.s. indicates not significant.The CompuSyn software (ComboSyn, Inc.) was used todetect synergistic, additive or antagonistic effects as previ-ously described.

Study approvalAll procedures were in accordance with Animal Welfare

Regulations and approved by the Institutional Animal Careand Use Committee at the Columbia University MedicalCenter.

Translational Relevance

Due to their inherent heterogeneity, glioblastomas (GBM)are among the most difficult malignancies to treat. Targetingseveral relevant tumor pathways is a potential approach tocombat the recalcitrant phenomenon of heterogeneity. Here,we describe novel drug combinations, involving clinicallyvalidated histone deacetylase (HDAC) (panobinostat), bro-modomain protein (BRD) (OTX015), and multikinase inhi-bitors (sorafenib). The combination treatment of HDAC andBRD inhibitors elicits a synergistic reduction of cellular via-bility across a broad range of GBM model systems and exertsantiglioma activity in GBM xenograft models. Dual adminis-tration of HDAC and BRD inhibitors resulted in an increase inantiapoptotic Mcl-1 protein levels, reducing the efficacy of thecombination treatment. The FDA-approved drug sorafenibcounteracted the Mcl-1 increase, enhanced the potency of thedrug combination, and led to significant life extension inpatient-derived orthotopic xenograft models. Thus, the triplecombination of panobinostat, OTX015, and sorafenibmay beefficacious for the treatment of GBM

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Clin Cancer Res; 24(16) August 15, 2018 Clinical Cancer Research3942




ResultsHDAC and BRD inhibition leads to synergistic reduction intumor cell proliferation

To ensure that the utilized compounds act on-target, we val-idated as towhether ornotHDAC inhibition enhances acetylationof known targets. To this end, patient-derived xenograft cells,GBM14, were treated with panobinostat in the presence orabsence of OTX015. Protein expression analysis for total histoneH3 and acetylated histone H3 validated on-target activity (Sup-plementary Fig. S1A). To demonstrate that combined HDAC andBRD inhibition is a potential efficacious strategy to inhibit growthof glioblastoma cells in a synergistic manner, we tested twoclinically validated drug compounds that interfere with thesepathways, panobinostat (HDAC inhibitor) and OTX015 (BRDinhibitor), in established (U87, U87-EGFRvIII, T98G, LN229),patient-derived xenograft (GBM14), and stem-like glioma cells(NCH644). In all model systems tested, the drug combinationover a range of concentrations elicited a synergistic interaction asevaluated by combination index value analysis (CI values; Fig. 1Aand B). Notably, the most significant synergistic interaction wasidentified in patient-derived xenograft cells, GBM14. Similarresults were obtained when FDA-approved, vorinostat, wasadministered in lieu of panobinostat (Supplementary Fig. S1B).These results suggest that combined targeting of HDACs and BRDis efficacious.

HDAC and BRD inhibition causes enhanced apoptotic celldeath

The combination of panobinostat and OTX015 resulted inmorphological signs of apoptosis. In support of this finding,GSEA showed that the combination treatment activated a tran-scriptional proapoptotic state (Fig. 2C). Therefore, we determinedas to whether or not features of apoptotic cell death can beconfirmed biochemically. To this purpose, LN229, T98G, andU87 GBM cells or stem-cell like GBM cells, NCH421k, andNCH644 were treated with panobinostat (or vorinostat),OTX015, or the combination of both and stained with AnnexinV/propidium iodide and analyzed by multiparametric flow cyto-metric analysis. Consistently, we found that the combinationtreatment of OTX015 and Panobinostat led to more apoptoticcells than control or single treatments. Similar findingsweremadewhen vorinostat was used in lieu of panobinostat (Fig. 2A and B;Supplementary Fig. S2A–S2C). Intrinsic apoptosis is accompa-nied by loss of mitochondrial membrane potential. Consistently,the combination treatments (panobinostatþOTX015; vorinos-tatþOTX015) reduced the amount of TMREpositive cells strongerthan single treatments or control in LN229, T98G, and U87 cells(Fig. 2D andE; Supplementary Fig. S2D). To assess whether or notcaspases are involved in the death, we treated GBM cells in thepresence or absence of pan-caspase inhibitor, zVAD-fmk. Wefound that zVAD-fmk partially protected the cells from DNAfragmentation induced by the combination treatment, suggestingthat caspases are involved in the death (Supplementary Fig. S2E).This finding was also supported by enhanced cleavage of PARP bythe combination treatment (Fig. 2F; Supplementary Fig. S2H).

Combined HDAC and BRD inhibition modulates theexpression of pro- and antiapoptotic Bcl-2 family members

Given the activationof apoptosis, wewerewondering about theregulation of this form of cell death induced by the combination

treatment. To this purpose, we analyzed protein expression ofboth antiapoptotic Bcl-2 family members, Mcl-1, Bcl-xL, and Bcl-2, and proapoptotic Bcl-2 family members, Noxa and BIM, inestablished, stem-like and patient-derived xenograft cells of GBM.Our findings demonstrate that combined treatment withOTX015and HDAC inhibitors results in an increase in BIM and Noxaprotein levels (Fig. 2G and H; Supplementary Fig. S2F and 2G).Aside from these proapoptotic molecular changes, we found acompensatory, transitory upregulation of Mcl-1 and its deubi-quitinase Usp9X induced by the combination treatment (Fig. 2Gand H; Supplementary Fig. S2F and 2G). Next, we assessed thetranscriptional changes by real-time PCR analysis and found thatmRNA for Usp9x, Mcl-1, Noxa, and BIM were all increased withthe exception of BIM in NCH644 cells (Fig. 2I and J).

Next, we evaluated the functional impact of the observedprotein changes on the apoptotic efficacy of the combinationtreatment. Given the increase in Noxa and Mcl-1, we mainlyfocused on the MCl-1/Noxa/BAK cascade in which high levelsofNoxabind toMcl-1 andmediate the BAK/Mcl-1 dissociation. Inturn, BAK engages in mitochondrial outer membrane permeabi-lization. To this purpose, we silenced the expression of BAK withtwo siRNAs. Silencing of BAKwas confirmed by standardWesternblotting (Fig. 3A). Knockdown of BAK provided a partial protec-tion from panobinostatþOTX015-mediated apoptosis (Fig. 3B).Next, we tested the impact of Noxa on the combination treatmentand silencing of Noxa was confirmed by capillary electrophoresis(Fig. 3A). We found that silencing of Noxa provided a partialprotection from panobinostatþOTX015-mediated apoptosis(Fig. 3C; Supplementary Fig. S3E), in keeping with the results onBAK. To gain a further understanding about the molecular inter-actions implicated in the combination treatment, we conductedco-immunoprecipitation analysis by pulling down Mcl-1 in thepresence or absence of the combination treatment (Fig. 3D).Although the IgG control did not pull down any of the analyzedproteins, anti-Mcl-1 prominently precipitated Mcl-1 and its asso-ciated described binding partners, Usp9X, BIM, Noxa, BAK, butnot GAPDH (negative control), confirming high specificity of ourexperiment (Fig. 3D). Regarding the effects of the combinationtreatment on the binding partners of Mcl-1, we observed that thepanobinostatþOTX015 treatment resulted in an increased bind-ing of Noxa to Mcl-1 (Fig. 3D). Enhanced binding of Noxa toMcl-1 is accompanied by a dissociation of two pro-apoptotic,Bcl-2 family members, BIM and BAK, from Mcl-1 (20, 21).Moreover, a reduced interaction between Usp9x and Mcl-1 wasobserved (Fig. 3D).

Given that we observed an increased expression of Mcl-1 andUsp9x at early time points upon treatment with the combina-tion treatment, we hypothesized that these effects mediate atransitory "prosurvival" effect. Indeed, later time points (>24hours after treatment) show a decline in Mcl-1, coinciding withenhanced death (Fig. 3G and H). To test this hypothesis, weutilized two Mcl-1–specific siRNAs and one Mcl-1 pool siRNA,which potently suppressed Mcl-1 protein levels (Fig. 3E; Sup-plementary Fig. S3A–S3D). Indeed, silencing of Mcl-1 signifi-cantly enhanced apoptosis in LN229 GBM cells (a cell line thatresponds slower to the combination treatment) and T98G GBMcells (Fig. 3E and F; Supplementary Fig. S3A–S3D). In likemanner, we evaluated the effects of USP9X silencing on thecombination treatment and found that Usp9X silencingenhanced apoptosis induced by the combination treatmentfurther (Fig. 3F; Supplementary Fig. S3C).

Targeting the Epigenome Reprograms Glioblastoma Metabolism

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Figure 1.

Dual inhibition of HDAC (Pb) and BRD (OTX) causes synergistic reduction in cellular proliferation of a broad range of glioblastoma model systems in vitro.A, Established glioblastoma cells, U87, U87-EGFRvIII, T98G, and LN229; patient-derived xenograft cells, GBM14; and stem-like GBM cells, NCH644, weretreated with OTX, Pb, or the combination over a broad range of concentrations for 72 hours. Thereafter, cells were analyzed for cellular viability: OTX (blue),Pb (red), and the combination OTXþPb (green). Shown are mean and SD, n ¼ 3 biological replicates. All concentrations are in mmol/L. #, Combination vs. OTX,P < 0.05; þ, Combination vs. Pb, P < 0.05; �, Combination vs. OTX or Pb, P > 0.05. B, Combination index (CI) is plotted for the cells treated as in A. A CI valueof less than 1.0 indicates synergy, whereas a CI value larger than 1.0 shows antagonism. A CI value of 1.0 defines additivity. The average CI value of all datapoints is provided in the upper portion of each diagram.

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Figure 2.

Dual inhibition of HDAC (Pb) and BRD (OTX) causes synergistic activation of apoptosis in glioblastoma cells. A and B, LN229 GBM cells were treated withindicated drugs for 24 hours, stained with Annexin V/PI, and analyzed by flow cytometry; n ¼ 3 biological replicates. Shown are mean and SD. C, Microarraywas performed with NCH644 stem-like GBM cells treated with the combination of OTXþPb and vehicle DMSO control. Shown is GSEA enrichment plotwith FDR q-values, normalized enrichment score (NES), and P-values; n ¼ 2 biological replicates for each condition. D and E, LN229 GBM cells were treated withindicated drugs and stained with TMRE, and analyzed by flow cytometry for the change in mitochondrial membrane potential; n ¼ 3 biological replicates. Shownare mean and SD. F,Western blotting analysis of LN229 GBM cells treated with indicated drugs. TF, total form; CF, cleaved form. All concentrations are in mmol/L.G andH, LN229, U87, NCH644, or GBM14 cellswere treatedwith indicated drugs and analyzed for levels of the indicated proteins by conventionalWestern blotting orcapillary electrophoresis. All concentrations are in mmol/L. Blots or capillary electrophoresis were quantified for the levels of Mcl-1 and Noxa normalized with itsrelated loading control. I and J, LN229 or NCH644 GBM cells were treated with indicated drugs and analyzed by real-time PCR for the indicated markers. Shown aremean and SD (n ¼ 3), and statistical analysis was performed. � , P < 0.05; �� , P < 0.01; �� , P < 0.001; �� , P < 0.0001; n.s., not significant.






Figure 3.

Molecular requirements of apoptosis induction by combined inhibition of HDAC and BRD. A, LN229 GBM cells were transfected with nontargeting (siNT),BAK, Noxa siRNA. Indicated protein levels are shown. B and C, The same transfected LN229 GBM cells from A were subjected to the combination treatment,OTXþPb, stained with propidium iodide for flow cytometric analysis. D, T98G GBM cells were treated with vehicle or the OTXþPb combination for 16 hours.Thereafter, protein lysates were immunoprecipitated with a species control IgG or Mcl-1–specific antibody and detected by conventional Western blotting. The leftportion shows the immunoprecipitated lysates, whereas on the right side 1% input lysates were loaded. All concentrations are in mmol/L. E, LN229 GBM cellswere transfected with indicated siRNA for 3 days, then treated with indicated drugs for 24 hours and analyzed for the expression of the indicated proteins by eitherstandard Western blotting or capillary electrophoresis. Arrows indicate total and cleavage forms of PARP. All concentrations are in mmol/L. F, LN229 cellstreated as inEwere stainedwith propidium iodide andanalyzedbyflowcytometry forDNA-fragmentation;n¼ 3 biological replicates. Shownaremean andSD.G andH, LN229 or NCH644 GBM cells were treated with indicated drugs for 24 and 48 hours and analyzed for the indicated proteins by capillary electrophoresis.All concentrations are in mmol/L. Quantifications are provided for Mcl-1 normalized with vinculin. I, LN229 GBM cells were treated with indicated drugs and stainedwith Annexin V/propidium iodide and analyzed by flow cytometry. Displayed are representative flow plots and quantification of the results; n ¼ 3 biologicalreplicates. Shown are mean and SD. � , P < 0.05; ��, P < 0.001; �� , P < 0.0001.

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These results intimate that inhibition of Mcl-1 might furtherenhance the efficacy of the drug combination therapy. To furtherevaluate this point, we tested whether the kinase inhibitor, sor-afenib, a known modulator of Mcl-1 levels and FDA-approveddrug (22, 23), is capable of counteracting the combinationtreatment mediated increase of Mcl-1 protein levels. In keepingwith this expectation, sorafenib blunted Mcl-1 upregulationmediated by panobinostat andOTX015 (Fig. 3G andH). Remark-ably, sorafenib also curtailed Usp9X expression, suggesting thatsorafenib abrogated two main pro-survival effects elicited by thecombination treatment (Fig. 3G and H). To evaluate whethersorafenib also enhances apoptosis induction by the combinationtreatment, LN229 or GBM14 cells were treated with the combi-nation treatment in the presence or absence of sorafenib.Although in the absence of sorafenib the combination treatmentdisplayed some apoptosis induction, this effect was significantlyenhanced in the presence of the multi-kinase inhibitor (Fig. 3I;Supplementary Fig. S3F). Similar results were obtained in T98GGBM cells (DNA-fragmentation; Supplementary Fig. S3G). Thesefindings establish a novel, rational triple drug therapy, calledOTXþPbþSf (OTX015 þ panobinostat þ sorafenib).

Combined HDAC and BRD inhibition suppresses oxidativephosphorylation and glycolysis in a synergistic manner

Given the pronounced synergistic reduction of cellular prolif-eration over a broad range of different cell lines, we wonderedabout the underlying molecular mechanisms, orchestrating, anddriving these changes. To this end, GSEA with subsequent inter-action analysis assisted us to identify pathways that are synergis-tically down regulated by panobinostat and OTX015. This anal-ysis was proved powerful because it permits to elucidate whichmolecular processes require both compounds for most potentpathway inhibition. It turned out that the key energy producingpathways in tumor cells, glycolysis and oxidative phosphoryla-tion, were drastically suppressed (Fig. 4A), presumably leading toan intracellular energy crisis. Consistently, the combination treat-ment suppressed protein levels of transporters and enzymes,involved in glycolysis (GLUT1, LDHA, GAPDH; Fig. 4B).

Next, we determined the underlying molecular mechanism,governing these significant metabolic changes mediated byHDAC and BRD inhibition. We hypothesized that these meta-bolic aberrations might likely be a result of interference with theexpression of the master-regulator, c-Myc. First, c-Myc is pivotalfor the regulation of tumor cellmetabolism (24). Second, c-Myc isa master-regulator in glioma stem cells (7). Third, both HDAC-and BRD-inhibitors have been reported to suppress c-Myc levels(25). Based on this reasoning, we found that combined inhibitionof BRD andHDAC results in silencing ofMYC transcript as shownby real-time PCR and GSEA analysis in glioblastoma stem-likecells, NCH644 (Fig. 4C and D; Supplementary Fig. S4A). Alongwith the reduction in c-Myc, the combination treatment alsoaffectedmarkers of stemness, such as SOX2 andNanog. Althoughin these model systems CD133 and Nestin cannot be consideredas bona-fide stem-cellmarkers, their expressionwas suppressed aswell (Supplementary Fig. S4B). Most relevantly, c-Myc was down-regulated on protein level, showing the strongest c-Myc suppres-sion in the combination treatment of OTX015 and panobinostat(Fig. 4B).

The above findings support the hypothesis that glioblastomacells treated with the combination of OTX015 and panobinostathave lowenergy levels. Indeed, thecombination treatmentdepletes

GBM14 patient-derived xenograft cells of ATPmore potently thansingle treatmentsorvehicle(Fig.4E).This is also inkeepingwith therelated GSEA, showing a transcriptional signature of "starvation"(GO_RESPONSE_TO_STARVATION; Fig. 5A).

Given the impact on the transcripts and proteins related toenergy metabolism, we conducted extracellular flux analysis tovalidate whether or not the combination treatment affects oxi-dative phosphorylation and glycolysis in glioblastoma cell cul-tures (Fig. 4F–M)). To this end, patient-derived xenograft cells,GBM14, were treated with OTX015, panobinostat, the combina-tion treatment in the presence or absence of sorafenib and weresubjected to analysis for oxygen consumption rate (OCR) in thecontext of a mitochondrial stress test. We found a significantreduction in basal mitochondrial oxygen consumption rate(OCR) in the combination treatment, which was mirrored alsoby the amount ofOXPHOS-relatedATPproduction (Fig. 4F, J, andK). Similarly, we found that the maximal respiration and sparerespiratory capacitywas prominently reduced by the combinationtreatment, which was further enhanced by the presence of sor-afenib, suggesting that sorafenib further inhibits oxidative phos-phorylation, in keeping with its suppression on Mcl-1 proteinlevels (Fig. 4L and M). Next, we assessed the impact of thecombination treatment on glycolysis by determining ECAR inthe context of a glycolysis stress test. Akin to OXPHOS, we foundthat the combination treatment reduced key indicators of glycol-ysis more potently than each compound alone (Fig. 4G–I). In thepresence of sorafenib, the combination treatment completelyabrogated the glycolytic reserve, suggesting that sorafenib furtherenhances the inhibitory effects of the combination treatment onenergy metabolism (Fig. 4I). To address the applicability of thesefindings to other model systems, we performed the same experi-ments in another glioblastoma cell culture, LN229. The findingsin LN229 are overall similar to GBM14 except that the tripletherapy (OTXþPbþSf) had a more drastic impact on OXPHOSrelated ATP production and that the combination treatment wasin some instances less efficacious (Supplementary Fig. S5A–S5D).Finally, we evaluated the impact of c-Myc levels on glycolysis inpatient-derived xenograft GBM cells, GBM14, and found thatsilencing of c-Myc levels suppressed glycolysis (SupplementaryFig. S5E). These results are in keeping with our findings related tothe combination treatment of OTX015 and panobinostat, show-ing enhanced suppression of glycolytic key indicators and confirmthat c-Myc suppression results in an inhibition of glycolysis. All inall, our findings suggest that the combination treatment interfereswith energymetabolism, and these effects are further enhanced inthe presence of sorafenib.

The combined inhibition of BRD and HDAC leads toendoplasmic reticulum stress followed by enhanced expressionof proapoptotic Noxa

Based on the findings related to energy metabolism, wehypothesized that the state of energy deprivation elicited by thecombination treatment should affect the homeostasis of theendoplasmic reticulum (ER) because ATP is necessary for proteinfolding and loss of ATP will result in the accumulation of unfold-ed proteins. GSEA confirms this notion, which was also validatedby RT-PCR analysis for common ER-stress–related markers, forexample GRP78, ATF3, ATF4, and CHOP in both NCH644 andLN229 GBM cells (Fig. 5A–C). To confirm that the transcriptionalchanges are also found at protein level, we conductedwestern blotanalysis as well as capillary electrophoresis for common ER-






Figure 4.

Metabolic reprogramming of energy metabolism elicited by combined inhibition of HDAC and BRD. A, Microarray was performed with NCH644 stem-likeGBMcells treatedwith thecombinationofOTXþPbandDMSOcontrol.ShownareGSEAenrichmentplotswithFDRq-values,normalizedenrichmentscore(NES),andPvalues; n ¼ 2 biological replicates for each condition. B, GBM14, LN229, and NCH644 GBM cells were treated as indicated and analyzed for the expression of theindicatedproteins by capillary electrophoresis. � indicates 14-3-3 protein.C,NCH644 stem-likeGBMcellswere treatedwith indicateddrugs and analyzedby real-timePCR for the levels of c-Myc; n ¼ 3 biological replicates. Shown are mean and SD. D, GSEA enrichment plot for MYC targets from the same cells in A. FDRq-values,normalizedenrichment score (NES), andPvalues.E,GBM14GBMcellswere treatedas indicated for24hours, andATP levelsweredetermined;n¼3biologicalreplicates. Shown are mean and SD. F, GBM14 GBM cells were treated with indicated drugs and subjected to extracellular flux analysis for oxygen consumptionrate(OCR) inthecontextofamitochondrialstressassay.O,oligomycin;F,FCCP;A/R,antimycin/rotenone.ShownaremeanandSD;n¼3biological replicates.G,GBM14cells were treated as in F and subjected to extracellular flux analysis in the setting of glycolysis stress test. G, glucose; O, oligomycin; 2-DG, 2-deoxyglucose.ShownaremeanandSD;n¼3biological replicates.Hand I,Theglycolytic capacity andglycolytic reservewere calculatedbasedon theexperiment shown inG. Shownare mean and SD; n ¼ 3 biological replicates. J–M, Functional OXPHOS related parameters were calculated based on the experiment shown in F, ATP production,basalOCR,maximal respiration,andsparerespiratorycapacity.ShownaremeanandSD;n¼3biological replicates. � ,P<0.05; �� ,P<0.01; �� ,P<0.001; �� ,P<0.0001;n.s., not significant.

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stress–related markers. The combination treatment of OTX015andpanobinostat displayed themost significant ER-stress–relatedsignature with upregulation of GRP78, phosphorylated eIF2a,total eIF2a, ATF3, ATF4, and CHOP (Fig. 5D; Supplementary Fig.S2H). Moreover, the induction of ER-stress by the indicatedtreatments correlated with the cleavage of PARP, suggesting thatER-stress is important for the induction of apoptosis by thecombination treatment (Supplementary Fig. S2H). Overall, theseresults confirm the notion that the combination treatment elicits apotent ER-stress response likely related to energy starvation.

To determine the downstream consequences of ER-stress, wehypothesized that the ER-stress signaling is responsible for theenhanced induction of Noxa by the combination treatment,which is supported by earlier findings by others and our group(26, 27). In this context, we first focused on the PERK-ATF4-Noxacascade andhypothesized that the combination treatment utilizesthis stress pathway toupregulateNoxa expression. To evaluate thisclaim, we silenced the expression of PERK by siRNA in LN229GBM cells. Silencing of PERK decreased ATF4 levels and sup-pressed OTX015, panobinostat, and the combination treatmentmediated increase of Noxa protein levels (Fig. 5E). Anothertranscription factor related to ER-stress signaling and to Noxaregulation is ATF3 (26, 27). Given that ATF3 was potentlyincreased by the combination treatment, we tested the hypothesisthat silencing of ATF3 will attenuate Noxa upregulation by thecombination treatment. To this purpose, we silenced ATF3 inU87and LN229 GBM cells and subsequently treated the cells withOTX015 and panobinostat. In agreement with our hypothesis, wefound that silencing of ATF3 suppressed Noxa increase inresponse to treatment with the drug combination (Fig. 5F).

Combined inhibition of BRD andHDAC facilitates the stabilityof Mcl-1 in a GSK3b-dependent manner

Aside from the proapoptotic changes induced by the combi-nation treatment, we detected an increase of Mcl-1 and its asso-ciated deubiquitinase Usp9X at early time points, which based onourmechanistic analysis exerts prosurvival features because inter-ference with both targets enhances the apoptotic effects of thecombination treatment. Therefore, we sought to establish a dee-per understanding by which mechanisms Mcl-1 is upregulated.Given the increase of Usp9X, it appears obvious that the combi-nation treatment modulates the stability of Mcl-1 aside from itseffects on mRNA levels. To this purpose, we conducted a cyclo-heximide (CHX) block experiment in the presence or absence ofOTX015 and panobinostat combination in LN229 and T98Gcells. Although control-treated cells displayed a rapid decline inMcl-1 protein levels, the combination treatment enhanced thestability ofMcl-1 in both cell lines (Fig. 5G andH), indicating thatOTX015 and panobinostat combination affects Mcl-1 half-life.

Concerning protein stability, it is well known that Mcl-1possesses a short-half life and is prone to rapid degradation byproteasomes. Proteasomal degradation is preceded by phosphor-ylation ofMcl-1 through several kinases. Among those regulators,GSK3b is known to phosphorylate Mcl-1 at serine 159 (28),preceding ubiquitin conjugation and subsequent energy depen-dent degradation by proteasomes. Given these implications, wehypothesized that the combination treatment inhibits the activityof GSK3b (phosphorylated form at serine 9) and that in turnMcl-1 becomes dephosphorylated at serine 159, renderingMcl-1morestable (29). To this purpose, GBM14 and LN229 cells were treatedwith OTX015, panobinostat or the combination. Protein expres-

sion analysis revealed that both cell lines demonstrate an increasein Mcl-1, which was most pronounced in the combination treat-ment, which was accompanied by a reduction in Mcl-1 phos-phorylation at serine 159 (Fig. 5I). In agreement with the reduc-tion of Mcl-1 phosphorylation, the combination treatment led toan enhanced phosphorylation of GSK3b at serine 9 (Fig. 5I),rendering the kinase less active. These findings reinforce thenotion that Mcl-1 is regulated at the posttranslational level bythe combination treatment.

To address the related up-stream mechanisms that govern theMcl-1 increase mediated by the combination treatment, we rea-soned that it may be related to the effects of the combinationtreatment on the transcription factor, c-Myc, since suppression ofc-Myc has been linked to inhibit PP2A (30), which binds to andde-phosphorylates GSK3b. To assess whether this previouslydescribed relationship of c-Myc and GSk3b exists in the settingof glioblastoma models as well, we created two stable cell linesderived from the patient-derived xenograft cell culture, GBM14,either infected with a lentiviral shRNA targeting c-Myc, or alentiviral driving ectopic over-expression of c-Myc. The activityof the corresponding constructs was verified by protein capillaryelectrophoresis, confirming the silencing or over-expression of c-Myc in the respective cell cultures (Fig. 5J). In agreement with ourearlier hypothesis, we found that while silencing of c-Myc resultsin an enhancement of phosphorylation of GSK3b, coupled withan increase in total Mcl-1 and reduced phosphorylation of Mcl-1at serine 159, over-expression of c-Myc results in a reciprocalexpression phenotype with reduced phosphorylation of GSK3band lower protein levels of Mcl-1 (Fig. 5J). Similar results wereobserved in LN229 GBM cells in which stable knockdown of c-Myc results in increased levels of Mcl-1 and over expression leadsto down-regulation of Mcl-1. To exclude that c-Myc modulationaffects Mcl-1 mRNA levels, we conducted real-time PCR analysisin c-Myc over-expressing and silenced cells. In both situation, wefound that c-Myc modulation only marginally affected Mcl-1mRNA, suggesting that most likely c-Myc regulates Mcl-1 in aposttranslational manner (Fig. 5K). These results confirm thatc-Myc levels affect Mcl-1 protein levels likely through enhancedstability mediated by reduced GSK3b activity.

The triple therapy of OTX015, panobinostat, and sorafenibleads to tumor regression and significant host survivalextension

To determine the efficacy of the combination treatment ofOTX015 and panobinostat, we utilized several xenograft modelsystems, involving glioblastoma and malignant melanoma. Tothis purpose, we implanted U87-EGFRvIII cells in the subcutis ofimmunocompromisedmice. After establishment of tumors, treat-ment groups were formed, consisting of vehicle, OTX015, pano-binostat, OTX015þpanobinostat, sorafenib, and the triple com-bination of OTX015þpanobinostatþsorafenib. Our findingsshow that the combination treatment of OTX015 and panobino-stat reduced tumor growth much more potently than each com-poundalone (Fig. 6A andB; Supplementary Fig. S7AandS7B).Wefound similar results in a BRAF V600E mutated melanomaxenograft model (A375 cells), albeit less efficient compared withthe U87-EGFRvIII model (Supplementary Fig. S7E and S7F).Given the limited efficacy of the combination treatment in vivo,we addressed whether or not sorafenib would further enhance thecombination treatment. As anticipated with our in vitro findings,the triple combination therapy had the strongest impact on tumor






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growth, leading to a regression of tumors (Fig. 6A and B; Sup-plementary Fig. S7A and S7B). To determine as to whether or notsingle, combination and triple therapies display similar efficacy ina patient-derived xenograft GBM model, we tested the GBM12PDX model and found that in agreement with the findingsobtained from theU87-EGFRvIII model system, the triple therapywasmost effective and leads to a significant suppression of tumorgrowth in this model system as well (Fig. 6C and D; Supplemen-tary Fig. S7D). Next, we evaluated the impact of the varioustreatment on the histopathological level. We found that the tripletherapy results in a significantly reduced cellularity and mitoticrate, while at the same time an increase in apoptosis/necrosis wasencountered in theU87-EGFRvIII andGBM12PDXmodel system(Fig. 6E–H; Supplementary Fig. S6A–S6C and S7A). Consistently,we found a reduced amount of Ki67 staining in tumors thatreceived the triple therapy, consistent with a reduced mitotic rate(Fig. 6F and H; Supplementary Fig. S6B and S7A). To confirm celldeath, we conducted TUNEL-staining and found the highestamount of TUNEL positive cells in the triple therapy treatedtumors (Fig. 6G and H; Supplementary Fig. S6C and S7A). Inaddition, we confirmed that the triple combination therapysuppresses c-Myc protein levels in vivo (Supplementary Fig. S7C).

Finally, we determine as to whether or not this treatmentapproach is active in orthotopic models of glioblastoma, whichbear a closer resemblance to the clinical situation and are morechallenging to treat. First, because the combination treatmentregulated c-Myc and several other transcription factors that orches-trate the stem-cell like phenotype in NCH644 GBM cells (Sup-plementary Fig. S4B; Fig. 4B and C), we used NCH644 cells andimplanted them in into the right striatum of immunocompro-mised mice (Fig. 6I). Treatment groups were formed and animalswere treated three times a week over the course of three weeks.Although single treatments (OTX or Pb) and the combinationtreatment (OTXþPb) did not result in a significant life extensionfor the host animals (compared with vehicle treated animals), thetriple therapy (OTXþPbþSf) resulted in a statistical significantsurvival benefit compared with the control and combinationtreatment (Fig. 6I). Next, we evaluated our drug treatments in thecurrent gold-standard for preclinical drug therapy, an orthotopicpatient-derived xenograft of glioblastoma. To this end,we utilizedthe GBM12 PDXmodel (9–12) and implanted cells into the rightstriatum of nudemice (Fig. 6J). Thereafter, animals were assignedto groups and treated according to the same schedule of theNCH644 orthotopic model system. The triple therapy resulted ina significant extension of host survival akin to the results obtainedin theheterotopic xenograftmodel (Fig. 6J). Thesefindings suggest

that our treatment strategy is active in orthotopic xenograft mod-els, suggesting that these agents are capable of crossing the blood-brain barrier and exert efficacy in the brain microenvironment.

Finally, we provide a summarizing scheme of the involvedmechanisms of the combination and triple therapy (Fig. 6K).

DiscussionAlthough multicombination therapies are a logical step ahead

due to the inherent heterogeneity of most solid tumors (31, 32),one of the concerns remains toxicity and the challenging clinicalburden of proof that even a triple therapy is more efficacious andsafe than single treatments or combination therapies, consistingof only two drugs. However, a lesson again might be learnt fromHIV therapy. Although in the early 1980s HIV constituted a deathsentence, this changed dramatically of the course of severaldecades when HIV was started to be treated with "drug combi-nation cocktails." In our study, we have taken a similar approachby combining two drug compound classes, involving HDACinhibitors and BRD inhibitors, and in the course of mechanismstudies we identified a rational triple therapy, involving HDACinhibitors (panobinostat, vorinostat), BRD inhibitor (OTX015),and multi-kinase inhibitors (sorafenib).

Our findings reveal that combined inhibition of HDAC bypanobinosat or vorinostat, and BRD proteins by OTX015 resultsin a significant synergistic reduction of cell growth that is pre-dominantly mediated through enhanced cell death in the form ofapoptosis, which was supported by GSEA showing upregulationof multiple apoptosis promoting genes. Cell death was protectedby the pan-caspase inhibitor, zVAD-fmk, which demonstratingthat caspases are involved in the death pathway elicited by thecombination treatment. These findings are in agreement withearlier studies, involving single treatments with HDAC inhibitorsor BRD inhibitors (25, 33–35), that demonstrated activation ofintrinsic apoptosis. We hypothesized that the enhanced apoptoticcell death by the combination treatment is driven by an alterationof pro- and antiapoptotic factors and identifiedNoxa as one of thekey players to mediate apoptosis. Noxa is a proapoptotic Bcl-2family member that is known for its inhibitory activity on anti-apoptotic Mcl-1. Induced by several stress responses, Noxa isincreased in the context of various stimuli and known to bedownstream of the integrated stress response when ATF4 isupregulated. In keeping with this, the drug combination treat-ment resulted in an activation of a profound ER-stress responseand increased Noxa via at least two mechanisms, involving eitherthe PERK-ATF4 pathway or ATF3 (13, 27, 36, 37).

Figure 5.Dual inhibition of HDAC (Pb) and BRD (OTX) causes endoplasmic reticulum stress and mediates enhanced stability of Mcl-1 in a GSK3b-dependent manner.A and B, NCH644 stem-like GBM cells were treated with the combination of OTX and Pb and subjected to microarray analysis. Shown are GSEA enrichmentplots with FDR q-values, normalized enrichment score (NES), and P values; n ¼ 2 biological replicates for each condition. C, NCH644 and LN229 GBMcells were treated with indicated drugs, and real-time PCR analysis was performed for the expression of the ER stress–related transcripts. Shown are meanand SD; n¼ 3 biological replicates.D,U87 and LN229 cells were treated with indicated drugs for 7 hours and analyzed for the protein expression of ER stress–relatedmarkers (ATF3 and Vinculin were run on capillary electrophoresis). All concentrations are in mmol/L. E, LN229 GBM cells were transfected with nontargetingor PERK specific siRNA for 3 days following treatment with the indicated drugs for 7 hours, and analyzed for expression of the indicated markers by conventionalWestern blotting. All concentrations are in mmol/L. F, U87 and LN229 GBM cells were transfected with nontargeting or ATF3-specific siRNA for 3 days followingtreatment with the indicated drugs for 7 hours, and analyzed by capillary electrophoresis for the indicatedmarkers. All concentrations are in mmol/L.G andH, LN229or T98GGBMcellswere pretreatedwith combinedOTX and Pbor Ctrl, and then cycloheximide (CHX)was added for indicated time (minutes). Lysateswere analyzedfor the expression of the indicated markers by capillary electrophoresis. I, GBM14 and LN229 GBM cells were treated with indicated drugs and analyzed forthe expression of the indicatedmarkers. J,GBM14 and LN229 GBM cells were transducedwith indicated lentivirus coding nontargeting or c-myc shRNA (sh, shRNA),c-myc overexpression (O/E, overexpression). Whole-cell protein lysates were analyzed for the indicated markers. K, GBM14 or LN229 GBM cells were transducedas indicated in J and analyzed for the mRNA levels of c-Myc and Mcl-1.






Figure 6.

Dual inhibition of HDAC (Pb) and BRD (OTX) causes synergistic reduction in tumor growth and extends host survival of animals bearing orthotopic patient-derivedglioblastoma in the presence of the FDA-approved multikinase inhibitor sorafenib. A and B, U87-EGFRvIII GBM cells were implanted in the subcutis ofimmunocompromised mice (Nu/Nu). After establishment of tumors, groups were formed as indicated. Tumor volumes were plotted; n ¼ 9–15 tumors per group.C and D, GBM12 patient-derived xenograft cells were implanted in the subcutis of immunocompromised mice (Nu/Nu). After establishment of tumors, groups wereformed as indicated. Tumor volumeswere plotted; n¼ 6–12 tumors per group. E–G, Shown are H&E, Ki67, and Tunel-stained representative sections from a vehicle orOTX015þpanobinostatþsorafenib-treated GBM12 tumor (from D). H, Quantifications of mitosis and apoptotic/necrotic cells from the GBM12 tumor sectionscollected at day 20 after tumor transplantation. Five fields of each sectionwere counted. Shown aremean and SD. I, Stem-like GBM cells, NCH644, were implanted inthe right striatum of nude mice. Thereafter, mice were randomly assigned to indicated groups. Treatments were performed three times a week for 3 weeks[OTX015 (OTX): 50 mg/kg; panobinostat (Pb): 7.5 mg/kg; sorafenib (Sf): 100 mg/kg]. Kaplan–Meier survival curves were plotted. Median survival days ofeach group were: DMSO: 24.5, OTX: 26, Pb: 23, OTXþPb: 25, and OTXþPbþSf: 34. J, Kaplan–Meier survival curves of mice xenograft with GBM12patient-derived GBM cells. Treatments were performed three times a week for 5 weeks until animals became moribund. Median survival days of eachgroup were: DMSO: 23, OTX: 31, Pb: 31, OTXþPb: 35, and OTXþPbþSf: 50. K, Graphical summary of the proposed mechanisms of action by the OTX, Pb,and Sf drug combinations. � , P < 0.05; �� , P < 0.01; �� , P < 0.001; �� , P < 0.0001; n.s., not significant.

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Based on our transcriptome analysis, we found evidence forenergy deprivation induced by the combination treatment, whichwas further supported by the fact that gene sets related to energymetabolisms were synergistically down-regulated. We validatedthis observation by determining total ATP levels and confirmedthese findings by extracellular flux analysis, which supported thenotion that the combination treatment suppressed glycolysis andoxidative energy metabolism. To the best of our knowledge, ourfindings are the first to show that simultaneous inhibition of BRDand HDAC proteins results in an energy crisis in glioblastomacells, which then in turn elicits a profound stress response withactivation of ER-stress.

To provide an explanation for the energy suppression medi-ated by the combination treatment, we hypothesized that c-Mycis involved in this context since c-Myc is known to modulateenergy metabolism in cancer cells (24, 38–40). Indeed, ourfindings unanimously showed that the combination treatmentreduced c-Myc mRNA and protein levels in a synergistic mannerand along with c-Myc suppression we found a decline inenzymes and transporters related to glycolysis, which areknown transcriptional targets of c-Myc. Indeed, selective stableknockdown of c-Myc suppressed glycolysis in patient-derivedxenograft cells. Although c-Myc seems to be a major player inthe regulation of our observed metabolic changes, we cannotexclude that others factors regulate energy metabolism in thecombination treatment.

Another major aspect of our findings relates to the observationthat the combination treatment not only increased the levels ofproapoptotic molecules, but also antiapoptotic factors, such asMcl-1 and its deubiquitinase Usp9X, which were detected inessentially every cell culture tested, suggesting that this responseappears to be robust and broadly relevant. Given this implication,it was tempting to speculate as to whether or not the combinationtreatment can be further enhanced through selective genetic orpharmacological inhibition. Our findings confirmed the antia-poptotic role of Mcl-1, and sorafenib (23) potently enhanced theeffects of the combination treatment, giving rise to a novel,rational triple therapy. Interestingly, the combination treatmentaffected Mcl-1 levels both at the transcriptional as well as theposttranslational level. At the posttranslational level, the combi-nation treatment increased Mcl-1 through a feed-forward mech-anism, involving the suppression of c-Myc with a subsequentinactivation of GSK3b and stabilization of Mcl-1. Being anFDA-approved drug, sorafenib (23, 41, 42) appeared to be theideal compound to counteract theMcl-1 increasemediated by thecombination treatment because sorafenib has been shown to

suppress Mcl-1 levels through multiple mechanisms, involvinginhibition of GSK3b, but also suppression of MCL1 transcripts(22, 43). In keeping with this, sorafenib suppressed Mcl-1 upre-gulation by the combination treatment in ourmodel systems andin turn facilitated apoptosis induced by the combination treat-ment. Most relevantly, our in vivo studies showed that addingsorafenib to the combination treatment resulted in a potentenhancement of efficacy in the highly aggressive U87-EGFRvIIImodel as well as in a patient-derived xenograft system. Althoughinitiallywe examined only heterotopic xenografts, similar impres-sive results were obtained with the triple combination therapy inorthotopic models, significantly extending overall survival. Thesefindings intimate that the triple therapy is feasible, well-tolerated,and efficacious. In the broader picture, this research suggests thatakin to infectious disease the consideration of multicombinationtherapies might be a viable approach to enhance treatmentefficacy of recalcitrant malignancies.

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

Authors' ContributionsConception and design: Y. Zhang, M.D. SiegelinDevelopment of methodology: Y. Zhang, C.T. Ishida, G. Karpel-Massler,M.D. SiegelinAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. Zhang, C.T. Ishida, W. Ishida, G. Kleiner,M.J. Sanchez-QuinteroAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y. Zhang, J. Zhao, M.D. SiegelinWriting, review, and/or revision of the manuscript: Y. Zhang, S.-F.L. Lo,E. Bianchetti, G. Kleiner, C.M. Quinzii, M.-A. Westhoff, G. Karpel-Massler,P. Canoll, M.D. SiegelinAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): C.T. Ishida, C. Shu, E. Bianchetti, M.D. SiegelinStudy supervision: M.D. Siegelin

AcknowledgmentsThis work was supported by NIH NINDS grants R01NS095848,

R01NS102366, and K08NS083732; the Louis V. Gerstner, Jr. Scholars Program(2017–2020); and American Brain Tumor Association Discovery Grant 2017(DG1700013) (M.D. Siegelin).

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 23, 2018; revised April 19, 2018; accepted May 10, 2018;published first May 15, 2018.

References1. Fu X, Chin RM, Vergnes L, Hwang H, Deng G, Xing Y, et al. 2-

Hydroxyglutarate inhibits ATP synthase and mTOR signaling. CellMetab 2015;22:508–15.

2. Sasaki M, Knobbe CB, Itsumi M, Elia AJ, Harris IS, Chio II, et al. D-2-hydroxyglutarate produced by mutant IDH1 perturbs collagen matu-ration and basement membrane function. Genes Dev 2012;26:2038–49.

3. Gross S, Cairns RA, MindenMD, Driggers EM, Bittinger MA, Jang HG, et al.Cancer-associated metabolite 2-hydroxyglutarate accumulates in acutemyelogenous leukemia with isocitrate dehydrogenase 1 and 2 mutations.J Exp Med 2010;207:339–44.

4. Yan H, Parsons DW, Jin G, McLendon R, Rasheed BA, Yuan W, et al.IDH1 and IDH2 mutations in gliomas. N Engl J Med 2009;360:765–73.

5. Parsons DW, Jones S, Zhang X, Lin JC, Leary RJ, Angenendt P, et al. Anintegrated genomic analysis of human glioblastoma multiforme. Science2008;321:1807–12.

6. Balss J, Meyer J, Mueller W, Korshunov A, Hartmann C, von Deimling A.Analysis of the IDH1 codon 132 mutation in brain tumors. Acta Neuro-pathol 2008;116:597–602.

7. Fang X, Zhou W, Wu Q, Huang Z, Shi Y, Yang K, et al. DeubiquitinaseUSP13 maintains glioblastoma stem cells by antagonizing FBXL14-medi-ated Myc ubiquitination. J Exp Med 2017;214:245–67.

8. Wang X, Cunningham M, Zhang X, Tokarz S, Laraway B, Troxell M, et al.Phosphorylation regulates c-Myc's oncogenic activity in the mammarygland. Cancer Res 2011;71:925–36.

9. Parrish KE, Cen L, Murray J, Calligaris D, Kizilbash S, Mittapalli RK, et al.Efficacy of PARP inhibitor rucaparib in orthotopic glioblastoma xenografts






is limited by ineffective drug penetration into the central nervous system.Mol Cancer Ther 2015;14:2735–43.

10. Gupta SK, Kizilbash SH, Carlson BL, Mladek AC, Boakye-Agyeman F,Bakken KK, et al. Delineation of MGMT hypermethylation as a biomarkerfor veliparib-mediated temozolomide-sensitizing therapy of glioblastoma.J Natl Cancer Inst 2015;108:pii: djv369.

11. AllenC,OpyrchalM,Aderca I, SchroederMA, Sarkaria JN,DomingoE, et al.Oncolytic measles virus strains have significant antitumor activity againstglioma stem cells. Gene Ther 2013;20:444–9.

12. Sarkaria JN, Carlson BL, Schroeder MA, Grogan P, Brown PD, Giannini C,et al. Use of an orthotopic xenograft model for assessing the effect ofepidermal growth factor receptor amplification on glioblastoma radiationresponse. Clin Cancer Res 2006;12:2264–71.

13. Karpel-Massler G, Ishida CT, Bianchetti E, Shu C, Perez-Lorenzo R,Horst B,et al. Inhibition ofmitochondrialmatrix chaperones and antiapoptotic Bcl-2 family proteins empower antitumor therapeutic responses. Cancer Res2017;77:3513–26.

14. Karpel-Massler G, Ishida CT, Bianchetti E, Zhang Y, ShuC, Tsujiuchi T, et al.Induction of synthetic lethality in IDH1-mutated gliomas through inhi-bition of Bcl-xL. Nat Commun 2017;8:1067.

15. Karpel-Massler G, Ramani D, Shu C, Halatsch ME, Westhoff MA, Bruce JN,et al. Metabolic reprogramming of glioblastoma cells by L-asparaginasesensitizes for apoptosis in vitro and in vivo. Oncotarget 2016;7:33512–28.

16. Karpel-Massler G, Horst BA, Shu C, Chau L, Tsujiuchi T, Bruce JN, et al.A synthetic cell-penetrating dominant-negative ATF5 peptide exerts anti-cancer activity against a broad spectrumof treatment-resistant cancers. ClinCancer Res 2016;22:4698–711.

17. Karpel-Massler G, Ba M, Shu C, Halatsch ME, Westhoff MA, Bruce JN, et al.TIC10/ONC201 synergizeswith Bcl-2/Bcl-xL inhibition in glioblastomabysuppression of Mcl-1 and its binding partners in vitro and in vivo.Oncotarget 2015;6:36456–71.

18. Pareja F, Macleod D, Shu C, Crary JF, Canoll PD, Ross AH, et al. PI3K andBcl-2 inhibition primes glioblastoma cells to apoptosis through down-regulation of Mcl-1 and Phospho-BAD. Mol Cancer Res 2014;12:987–1001.

19. Karpel-Massler G, Ramani D, Shu C, Halatsch ME, Westhoff MA, BruceJN, et al. Metabolic reprogramming of glioblastoma cells by L-aspar-aginase sensitizes for apoptosis in vitro and in vivo. Oncotarget2016;7:33512–28.

20. Elgendy M, Abdel-Aziz AK, Renne SL, Bornaghi V, Procopio G, ColecchiaM, et al. Dualmodulation ofMCL-1 andmTORdetermines the response tosunitinib. J Clin Invest 2017;127:153–68.

21. Sheridan C, Brumatti G, Elgendy M, Brunet M, Martin SJ. An ERK-depen-dent pathway to Noxa expression regulates apoptosis by platinum-basedchemotherapeutic drugs. Oncogene 2010;29:6428–41.

22. Wang R, Xia L, Gabrilove J,Waxman S, Jing Y. Sorafenib inhibition ofMcl-1accelerates ATRA-induced apoptosis in differentiation-responsive AMLcells. Clin Cancer Res 2016;22:1211–21.

23. Kiprianova I, Remy J, Milosch N, Mohrenz IV, Seifert V, Aigner A, et al.Sorafenib sensitizes glioma cells to the BH3Mimetic ABT-737 by targetingMCL1 in a STAT3-dependent manner. Neoplasia 2015;17:564–73.

24. Xiao ZD, Han L, Lee H, Zhuang L, Zhang Y, Baddour J, et al. Energy stress-induced lncRNA FILNC1 represses c-Myc-mediated energy metabolismand inhibits renal tumor development. Nat Commun 2017;8:783.

25. Nagaraja S, Vitanza NA, Woo PJ, Taylor KR, Liu F, Zhang L, et al. Tran-scriptional dependencies in diffuse intrinsic pontine glioma. Cancer Cell2017;31:635–52 e6.

26. Dey S, Sayers CM, Verginadis II, Lehman SL, Cheng Y, Cerniglia GJ, et al.ATF4-dependent induction of heme oxygenase 1 prevents anoikis andpromotes metastasis. J Clin Invest 2015;125:2592–608.

27. Fels DR, Koumenis C. The PERK/eIF2alpha/ATF4 module of the UPR inhypoxia resistance and tumor growth. Cancer Biol Thera 2006;5:723–8..

28. Pan R, Ruvolo V, Mu H, Leverson JD, Nichols G, Reed JC, et al. Syntheticlethality of combined Bcl-2 inhibition and p53 activation inAML:mechan-isms and superior antileukemic efficacy. Cancer Cell 2017;32:748–60.e6.

29. Nifoussi SK, Ratcliffe NR, Ornstein DL, Kasof G, Strack S, Craig RW.Inhibition of protein phosphatase 2A (PP2A) prevents Mcl-1 proteindephosphorylation at the Thr-163/Ser-159 phosphodegron, dramaticallyreducing expression in Mcl-1-amplified lymphoma cells. J Biol Chem2014;289:21950–9.

30. Liu L, Eisenman RN. Regulation of c-Myc protein abundance by a proteinphosphatase 2A-glycogen synthase kinase 3beta-negative feedback path-way. Genes Cancer 2012;3:23–36.

31. Wei W, Shin YS, Xue M, Matsutani T, Masui K, Yang H, et al. Single-Cellphosphoproteomics resolves adaptive signaling dynamics and informstargeted combination therapy in glioblastoma. Cancer Cell 2016;29:563–73.

32. Prados MD, Byron SA, Tran NL, Phillips JJ, Molinaro AM, Ligon KL, et al.Toward precision medicine in glioblastoma: the promise and the chal-lenges. Neuro-oncol 2015;17:1051–63.

33. LeeDH, RyuHW,WonHR, Kwon SH. Advances in epigenetic glioblastomatherapy. Oncotarget 2017;8:18577–89.

34. Chen S, Zhang Y, Zhou L, Leng Y, Lin H, Kmieciak M, et al. A Bim-targetingstrategy overcomes adaptive bortezomib resistance in myeloma through anovel link between autophagy and apoptosis. Blood 2014;124:2687–97.

35. Gammoh N, Lam D, Puente C, Ganley I, Marks PA, Jiang X. Role ofautophagy in histone deacetylase inhibitor-induced apoptotic and non-apoptotic cell death. PNAS 2012;109:6561–5.

36. Ishida CT, ShuC,HalatschME,WesthoffMA, Altieri DC, Karpel-Massler G,et al. Mitochondrial matrix chaperone and c-myc inhibition causesenhanced lethality in glioblastoma. Oncotarget 2017;8:37140–53.

37. Ishida CT, Bianchetti E, Shu C, Halatsch ME, Westhoff MA, Karpel-MasslerG, et al. BH3-mimetics and BET-inhibitors elicit enhanced lethality inmalignant glioma. Oncotarget 2017;8:29558–73.

38. Satoh K, Yachida S, SugimotoM,OshimaM,Nakagawa T, Akamoto S, et al.Global metabolic reprogramming of colorectal cancer occurs at adenomastage and is induced by MYC. PNAS 2017;114:E7697–E706.

39. Mertz JA, Conery AR, Bryant BM, Sandy P, Balasubramanian S, Mele DA,et al. Targeting MYC dependence in cancer by inhibiting BET bromodo-mains. PNAS 2011;108:16669–74.

40. Wang J, Wang H, Li Z, Wu Q, Lathia JD, McLendon RE, et al. c-Myc isrequired for maintenance of glioma cancer stem cells. PLoS One 2008;3:e3769.

41. Allen JE, Prabhu VV, TalekarM, van denHeuvel AP, Lim B, Dicker DT, et al.Genetic and pharmacological screens converge in identifying FLIP, BCL2,and IAP proteins as key regulators of sensitivity to the TRAIL-Inducinganticancer agent ONC201/TIC10. Cancer Res 2015;75:1668–74.

42. Sheng Z, Li L, Zhu LJ, Smith TW, Demers A, Ross AH, et al. A genome-wideRNA interference screen reveals an essential CREB3L2-ATF5-MCL1 survivalpathway in malignant glioma with therapeutic implications. Nat Med2010;16:671–7.

43. Tong J, Tan S, Zou F, Yu J, Zhang L. FBW7 mutations mediate resistance ofcolorectal cancer to targeted therapies by blocking Mcl-1 degradation.Oncogene 2017;36:787–96.


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2018;24:3941-3954. Published OnlineFirst May 15, 2018.Clin Cancer Res Yiru Zhang, Chiaki Tsuge Ishida, Wataru Ishida, et al. GlioblastomaTumor Cell Metabolism and Elicits Synthetic Lethality in Combined HDAC and Bromodomain Protein Inhibition Reprograms

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Combined HDAC and Bromodomain Protein ... · Orthotopic glioblastoma PDX and stem cell–like xenograft model FortheGBM12orthotopicGBMmodel(34),300,000cellsand forthestem-likeGBMmodel,20,000NCH644cellswereinjected