A Synthetic Cell-Penetrating Dominant-Negative ... · glioblastoma, melanoma, prostate cancer, and triple receptor– negative breast cancer xenograft models. Finally, the combina-tion

Cancer Therapy: Preclinical

A Synthetic Cell-Penetrating Dominant-NegativeATF5 Peptide Exerts Anticancer Activity against aBroad Spectrum of Treatment-Resistant CancersGeorg Karpel-Massler1, Basil A. Horst2, Chang Shu1, Lily Chau1, Takashi Tsujiuchi3,Jeffrey N. Bruce3, Peter Canoll1, Lloyd A. Greene1, James M. Angelastro4, andMarkus D. Siegelin1

Abstract

Purpose: Despite significant progress in cancer research, manytumor entities still have an unfavorable prognosis. Activatingtranscription factor 5 (ATF5) is upregulated in various malignan-cies and promotes apoptotic resistance. We evaluated the efficacyand mechanisms of the first described synthetic cell-penetratinginhibitor of ATF5 function, CP-d/n-ATF5-S1.

Experimental Design: Preclinical drug testing was performedin various treatment-resistant cancer cells and in vivo xenograftmodels.

Results: CP-d/n-ATF5-S1 reduced the transcript levels ofseveral known direct ATF5 targets. It depleted endogenousATF5 and induced apoptosis across a broad panel of treat-ment-refractory cancer cell lines, sparing non-neoplastic cells.CP-d/n-ATF5-S1 promoted tumor cell apoptotic susceptibilityin part by reducing expression of the deubiquitinase Usp9X and

led to diminished levels of antiapoptotic Bcl-2 family membersMcl-1 and Bcl-2. In line with this, CP-d/n-ATF5-S1 synergisti-cally enhanced tumor cell apoptosis induced by the BH3-mimetic ABT263 and the death ligand TRAIL. In vivo, CP-d/n-ATF5-S1 attenuated tumor growth as a single compound inglioblastoma, melanoma, prostate cancer, and triple receptor–negative breast cancer xenograft models. Finally, the combina-tion treatment of CP-d/n-ATF5-S1 and ABT263 significantlyreduced tumor growth in vivomore efficiently than each reagenton its own.

Conclusions: Our data support the idea that CP-d/n-ATF5-S1,administered as a single reagent or in combination with otherdrugs, holds promise as an innovative, safe, and efficient anti-neoplastic agent against treatment-resistant cancers.ClinCancer Res;22(18); 4698–711. �2016 AACR.

IntroductionAlthough recent significant responses for cancer therapy have

been achieved in some malignancies, these are often initiallyimpressive, but unfortunately not durable (1, 2). For other malig-nancies, such as high-grade primary brain cancers, the prognosis isunfavorable even with treatment (3). Therefore, efforts continueto identify new potential targets for tumor-specific treatments aswell as novel therapeutic strategies to exploit these targets.

ATF5 is an example that has been identified as a potentialtarget for cancer treatment but for which no specific therapy hasbeen developed (4, 5). Activating transcription factor 5 (ATF5;also termed ATFx) is a member of the activating transcriptionfactor/cyclic AMP–responsive element-binding (ATF/CREB)family. A common feature of this family is the presence of abasic leucine zipper (bZIP) domain that promotes DNA bind-ing via the basic region and interactions with other proteinsthrough the leucine zipper (5–7). ATF5 binds to several dif-ferent promoter elements including the nutrient-sensing reportelement (NRSE) and a novel motif to regulate gene transcrip-tion (6, 8). Full-length ATF5 appears to be rapidly degraded viathe proteasome and it has the unusual property that it is amonga small group of proteins that are selectively translated wheneiF2a is phosphorylated (9, 10).

ATF5 protein levels are increased in a variety of humanmalignancies, including glioblastoma, breast, pancreatic, lung,and colon cancers (11). In contrast, with few exceptions (liver,prostate, and testis), ATF5 expression is low in normal tissue ofthe respective organs. In several tumor types, including glio-blastoma and non–small cell lung cancer, ATF5 expressionnegatively correlates with survival (12, 13). In cell culturestudies, ATF5 promotes survival by counteracting apoptosis inpro-B lymphocytes deprived of IL3 or in HeLa cells after growthfactor withdrawal (14). In addition, ATF5 regulates transcrip-tion of antiapoptotic B-cell leukemia 2 (Bcl-2) and of Bcl-2family member, myeloid cell leukemia-1 (Mcl-1), presumably

1Department of Pathology&Cell Biology,ColumbiaUniversityMedicalCenter, New York, New York. 2Department of Dermatology, ColumbiaUniversity Medical Center, New York, New York. 3Department of Neu-rosurgery, Columbia University Medical Center, New York, New York.4Department of Molecular Biosciences, University of California, DavisSchool of Veterinary Medicine, Davis, California.

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

Current address for L. Chau: Department of Neurology, SUNY DownstateMedical Center, Brooklyn, New York, New York.

Corresponding Authors: Markus D. Siegelin, Department of Pathology & CellBiology, Columbia University Medical Center, 630 West 168th Street, P&S 15-401,New York, NY 10032. Phone: 212-305-1993; E-mail: [email protected];and [email protected] and James M. Angelastro, Department of MolecularBiosciences, University of California One Shields Avenue, Davis, CA 95616. Phone:530-752-1591; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-15-2827

�2016 American Association for Cancer Research.

ClinicalCancerResearch

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thereby promoting tumor cell survival (13, 15). Conversely,interference with ATF5 expression or activity yields a markedinduction of apoptosis in glioblastoma cells in vitro and in vivowithout affecting astrocytes (16, 17). Moreover, in a transgenicmurine model in which endogenous glioblastomas wereinduced by a PDGF/sh-p53–expressing virus, activation of adominant/negative (d/n)-ATF5 blocked tumor formation andresulted in regression of formed tumors (17). Antineoplasticactivity of d/n-ATF5 was also reported for breast cancer cellsand pancreatic cancer cells in vitro (11, 15, 18). These findingsthus suggest ATF5 as a promising target for a tailored anticancertherapy.

To provide a potentialmeans to target ATF5 in vivo, we designeda d/n-ATF5 linked to a cell-penetrating domain (Penetratin;ref. 19). This recombinant peptide passes the blood–brain barrier,enters tumor cells, and exerts antineoplastic activity in a rodenttransgenic glioma model. In this study, we assessed the activityand mechanism of action of a similar peptide (CP-d/n-ATF5-S1)that was furthermodified to reduce its size and that was generatedsynthetically. In in vitro studies and in in vivo murine xenograftmodels, CP-d/n-ATF5-S1 shows apoptosis induction over a broadrange of recalcitrant human malignancies without apparenteffects on nontransformed cells. A novel mechanism of actionwas found in which the peptide reduces expression of the deu-biquitinating enzyme Usp9X, which in turn leads to depletion ofMcl-1 and Bcl-2 and to consequent apoptotic death. The latterfindings led us to rationally design and carry out in vitro and in vivotests of several potential combination therapies with CP-d/n-ATF5-S1 that had enhanced efficacy compared with either agentalone.

Materials and MethodsEthics statement

All procedures were in accordance with Animal Welfare Reg-ulations and approved by the Institutional Animal Care and UseCommittee at Columbia University Medical Center (New York,NY).

ReagentsCP-d/n-ATF5-S1, mutated CP-d/n-ATF5-S1, and Penetratin

were purchased from CS Bio. Recombinant TRAIL was fromPeprotech. ABT263 was from Selleckchem.

Cell cultureCells were grown as described previously (20, 21). Cells were

obtained from the ATCC or Cell Line Services and authenticatedby the manufacturer. No cell line authentication was performedby the authors and details are found in the SupplementarySection.

Cell viability assaysTo examine cellular proliferation, MTT assays were performed

as described previously (21).

Measurement of apoptosis and mitochondrial membranepotential

Annexin V/propidium iodide (PI), PI, and JC-1 stainings wereperformed as described previously (20, 22).

Western blot analysisProtein expression was determined by Western blot analysis as

described previously (23).

Transfections of siRNAssiRNAs were transfected as described previously (22, 24).

cDNA synthesis and RT-PCRcDNA synthesis and RT-PCR were performed as described

previously (23).

Subcutaneous xenograft modelsSubcutaneous xenografts were implanted as described previ-

ously (20).

Statistical analysisStatistical significancewas assessed by Student t test using Prism

version 5.04 (GraphPad). P � 0.05 was considered statisticallysignificant.

ResultsCP-d/n-ATF5-S1

CP-d/n-ATF5-S1 is a synthetic 67-amino acid peptide that wasengineered to cross cellular membranes and to specifically inter-fere with the survival-promoting actions of ATF5 (Fig. 1A). TheN-terminal end has a 16-amino acid Penetratin domain thatfacilitates cellular penetration (25, 26). A dominant/negativesequence follows in which the DNA-binding domain of ATF5 issubstituted by an amphipathic sequence with a leucine repeat atevery seventh residue and then by the human ATF5 bZIP domaintruncated after the first valine (26–29). Parallel work has dem-onstrated that a similar recombinant tagged peptide passes theblood–brain barrier, enters intact cells both in vivo and in vitro, andpromotes selective death of glioma cells (19). Four independentbatches of the peptide (including one under GMP conditions)havehad comparable activity. For control purposes, peptideswerealso synthesizedwith a penetratin domain alone and inwhich keyleucine residues were mutated to glycine in the d/n portion toreduce binding to potential partners (Fig. 1A).

Translational Relevance

Targeting treatment-resistant malignancies remains amajorchallenge in oncology. In this study, we introduce a noveltherapeutic compound targeting activating transcription factor5 (ATF5) through utilization of a novel cell-penetrating pep-tide, termed CP-d/n-ATF5-S1. CP-d/n-ATF5-S1 displays broadanticancer activity against glioblastoma, triple receptor–neg-ative breast cancer, prostatic carcinoma, pancreatic cancer,melanoma, non–small cell lung carcinoma, and hematologicmalignancies. Notably, we provide evidence that these anti-neoplastic effects are not only observed in vitro, but are alsoseen in six different animal models of glioblastoma, melano-ma, prostatic adenocarcinoma, triple receptor–negative breastcancer, and pancreatic carcinoma without any detectabletoxicity. Finally, CP-d/n-ATF5-S1 sensitizes tumor cells forBH3-mimetics and extrinsic apoptotic stimuli in vitro and invivo. Taken together, CP-d/n-ATF5-S1 is a novel highly effica-cious anticancer compound with minimal toxicity and poten-tially warrants clinical testing in patients.

Peptide-Based Inhibition of ATF5 for Cancer Therapy

www.aacrjournals.org Clin Cancer Res; 22(18) September 15, 2016 4699



LEGECQGLEARNRELKERAESV-COOHLEQRAEELARENEELLEKEAEELEQENAEH2N-RQIKIWFQNRRMKWKK

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Dominant/negative-sequencePenetratin zipper ATF5 Leucinetruncated after first valine

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GEGECQGGEARNREGKERAESV-COOHGEQRAEEGARENEEGGEKEAEEGEQENAEH2N-RQIKIWFQNRRMKWKK

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GFsamples n = 99All glioblastoma

ATF5-amplified (≥ 2.2 copies) n = 20ATF5-deleted (≤ 1.8 copies) n = 19

Log-rank P values:Amplified vs. deleted: 0.043 Amplified vs. all: 0.266Deleted vs. all: 0.012

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Figure 1.A, graphical representation showing the sequences of CP-d/n-ATF5-S1, mutated CP-d/n-ATF5-S1, and penetratin. B, T98G glioblastoma and MDA-MB-436breast cancer cellswere treated for 72 hourswith increasing concentrations of CP-d/n-ATF5-S1 under reduced serumconditions tomimic the nutrient-deprived stateof tumor cells in the tumor tissue (1.5% FBS) followed byWestern blot analysis for ATF5. ActinWestern blot analysiswas performed to confirm equal protein loading.Arrow indicates a specific band of ATF5. C, T98G glioblastoma cells were treated with CP-d/n-ATF5-S1 or solvent for 48 hours before adding 10 mg/mLcycloheximide and Western blot analysis for ATF5 and actin. D, graphical representation following densitometric analysis of the experiment described underC using ImageJ (NIH, Bethesda, MD; http://imagej.nih.gov/ij). (Continued on the following page.)

Karpel-Massler et al.

Clin Cancer Res; 22(18) September 15, 2016 Clinical Cancer Research4700



CP-d/n-ATF5-S1 depletes endogenous ATF5Western blot analyses revealed that treatment of cultured

tumor cell lines (T98G, MDA-MB-436, and GBM12) with CP-d/n-ATF5-S1 leads to a dose-dependent reduction of endoge-nous ATF5 protein levels by 3 days (Fig. 1B and SupplementaryFig. S1B). In T98G cells, this effect was present after 48 hours, butnot 24 hours (Supplementary Fig. S1A). RNAseq analysis ofT98G glioblastoma cells treated with CP-d/n-ATF5-S1 for 1 to3 days showed no significant alteration of ATF5 transcript levels(data not shown). Comparison of endogenous ATF5 levels inT98G cells treated with or without CP-d/n-ATF5-S1 in presenceof cycloheximide indicates that the peptide significantlydecreases ATF5 protein stability (Fig. 1C and D and Supplemen-tary Fig. S1C). In contrast, treatment with Penetratin peptide didnot affect ATF5 stability (Supplementary Fig. S1D and S1E).Thus, one action of CP-d/n-ATF5-S1 is loss of endogenous ATF5caused at least in part by enhanced turnover.

CP-d/n-ATF5-S1 interferes with transcription of known ATF5downstream targets

CP-d/n-ATF5-S1 treatment for 24 hours resulted in downregu-lation of three known ATF5 target genes [Mcl-1 (13), Bcl-2 (15,30), and asparagine synthetase (8)] at the mRNA level (Fig. 1E andSupplementary Fig. S2). However, for Mcl-1 and Bcl-2, thisdecrease was transitory with mRNA levels returning to baselineby 72 hours, presumably by compensatory mechanisms. In con-trast, CP-d/n-ATF5-S1 did not decrease mRNA levels of Usp9X, agene not described as transcriptionally regulated by ATF5(Fig. 1E).

CP-d/n-ATF5-S1 promotes apoptotic cell death across a widepanel of treatment-resistant human cancer cell lines

ATF5 is expressed in a variety of human cancers includingglioblastoma (11, 15, 18). In silico analysis of the Rembrandtdataset for glioblastoma shows a significantly worse overallsurvival in patients harboring an amplification of the ATF5 genecompared with those with �1.8 copies (Fig. 1F). Several studieshave also reported an inverse relationship between ATF5 proteinexpression and glioblastoma patient survival (5).

Inhibition of ATF5 function or expression has marked anti-neoplastic effects in vitro and in vivo (11, 15, 16, 18). Initially, toassess the activity of CP-d/n-ATF5-S1, T98G, U87MG glioblasto-ma, and HL-60 myeloid leukemia cells were treated for 72 hourswith increasing concentrations of the peptide. CP-d/n-ATF5-S1yielded a dose-dependent antiproliferative effect as determinedby MTT assay (Fig. 1G and Supplementary Fig. S3) as well asmarked changes in cellularmorphology observed by lightmicros-copy (Fig. 1H). To further assess themechanismof such effects, weperformed Annexin V/PI staining across a variety of therapy-refractory human cancer cell lines after treatment with increasingconcentrations of CP-d/n-ATF5-S1 for 48 hours. As shown in Fig.2A–C, Supplementary Figs. S4A, S4C, and S5A, the peptideyielded a strong and dose-dependent increase in the fraction of

Annexin V–positive cells, thus indicating an apoptotic responseacross a wide and diverse panel of solid and nonsolid cancer cells.Moreover, this effect was attenuated by treatment with the pan-caspase inhibitor z-VAD-Fmk in T98G cells (SupplementaryFig. S6), which is consistent with studies indicating that interfer-ence with ATF5 function or expression in tumor cells promotesapoptosis (9, 12).

To verify whether the effect of the peptide on survival isspecifically related to the dominant-negative domain and not tothe cell-penetrating domain, we treated T98G cells either withPenetratin alone or CP-d/n-ATF5-S1. In contrast to CP-d/n-ATF5-S1, Penetratin did not markedly increase the fraction of AnnexinV–positive cells (Fig. 2D and E). Moreover, neither Penetratin norCP-d/n-ATF5-S1 resulted in a significant induction of apoptosis inhuman fetal astrocyte cultures (Fig. 2F andG), suggesting that CP-d/n-ATF5-S1 possesses specificity toward cancer cells.

CP-d/n-ATF5-S1 leads to dissipation of mitochondrialmembrane potential and activates caspase-9

Next, we addressed whether activation of apoptosis by CP-d/n-ATF5-S1 is mediated at least in part through a mitochondrialpathway. JC1 staining revealed that peptide treatment leads toa marked reduction of mitochondrial membrane potential(Fig. 2H) and cleavage (activation) of caspase-9 (Fig. 2I) suggest-ing that CP-d/n-ATF5-S1 induces apoptosis in part through themitochondrially driven apoptotic pathway.

CP-d/n-ATF5-S1 downregulates antiapoptotic Bcl-2 and Mcl-1proteins

Because our findings pointed toward involvement of mito-chondria in apoptosis driven byCP-d/n-ATF5-S1, we next focusedon expression of Bcl-2 family proteins. ATF5 is reported toregulate transcription of antiapoptotic Bcl-2 and Mcl-1 (13,30). However, at least in T98G cells, there was a rebound ofMcl-1 and Bcl-2 mRNA expression by 3 days of peptide treatment(Fig. 1E), so it was important to assess protein levels at this time aswell. As shown in Fig. 3A and Supplementary Fig. S7A, Mcl-1 wasconsistently downregulated in all lines tested (U87MG, T98Gglioblastoma, and H1975 non–small cell lung cancer, PANC-1pancreatic carcinoma, A375melanoma, and PC3 prostate cancer)at 72 hours of peptide treatment and in some cases, by 48 hours.Bcl-2 protein was similarly downregulated in all but the PC3 cellline. Expressionof Bcl-xL, a thirdmember of the antiapoptotic Bcl-2 family, was altered in some lines (U87MGandT98G), but not inothers. Despite a rebound of Mcl-1 and Bcl-2 mRNA levels inT98G cells by 3 days of CP-d/n-ATF5-S1 treatment, expression ofthe corresponding proteins was still decreased in these cells after6 days of peptide treatment (Supplementary Fig. S1F).

CP-d/n-ATF5-S1 downregulates Bag3 and Usp9X proteinsThe observed decreases in Mcl-1 and Bcl-2 proteins at 72 hours

promoted by CP-d/n-ATF5-S1 under conditions in which mRNAlevels appear to be unaffected led us to next assess whether the

(Continued.) E, T98G glioblastoma cells were treated for the indicated durations with 100 mmol/L Penetratin (pen) or increasing concentrations of CP-d/n-ATF5-S1prior to performing qRT-PCR for Mcl-1, Bcl-2, and Usp9X. Columns, mean; error bars, SD. � , P < 0.05 versus treatment with Penetratin for 24 hours;#, P < 0.05 vs treatment with pen for 72 hours. F, in silico analysis on the survival of glioblastoma patients based on the amplification status of the ATF5 gene(National Cancer Institute, 2005; REMBRANDT home page, http://rembrandt.nci.nih.gov). G, T98G glioblastoma cells were treated with increasing concentrationsof CP-d/n-ATF5-S1 under low serum conditions (1.5% FBS). After 72 hours, a MTT assay was performed. Data presented are representative for at least twoindependent experiments. Columns, means; error bars, SEM. H, representative microphotographs of U87MG cells at 40� magnification after 72 hours oftreatment with CP-d/n-ATF5-S1. Morphologic changes such as decreased lengths and numbers of cell processes are especially seen in those cells treatedwith 200 mmol/L CP-d/n-ATF5-S1. Black arrow tip points at blebs.





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4.06 0.3950.31.7627.36.859.41 21.1

84.0 15.627.021.016.649.22.56 62.9

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6.36 0.7521.62.573.322.737.58 21.9

81.6 42.228.647.32.7391.24.42 35.1

U87MG

PC3

1.80 0.6351.82.3314.61.5311.6 17.0

79.3 14.223.122.829.054.87.37 68.1

MDA-MB-436

6.32 9.0935.116.214.010.39.61 51.3

82.9 34.42.6146.13.3872.31.20 5.19

DU145

2.05 1.1267.81.1161.51.396.52 45.8

87.4 25.517.114.017.519.64.06 27.7

MGPP-3

4.53 1.462.133.780.608.190.57 10.4

94.1 54.810.184.02.2788.90.84 33.3

H19752.10 3.9621.61.7811.22.497.41 44.4

85.3 28.125.351.36.1180.25.15 23.5

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1.06 6.6221.51.969.680.643.56 37.9

90.8 39.312.763.817.172.64.56 16.1

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88.4 10.66.4826.28.1665.13.05 1.63

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78.8 3.0041.829.94.0376.63.06 92.16.36 4.7616.010.39.815.713.05 18.5

86.7 51.417.955.820.164.43.92 25.3

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Figure 2.A, U87MG, T98G, U251, LN229, MGPP-3 (transgenic, proneural) glioblastoma, PC3 and DU1145 prostate cancer, PANC-1 pancreatic carcinoma, MDA-MB-436triple-negative breast cancer, H1975 non–small cell lung cancer, A375 malignant melanoma, HL-60 myeloid leukemia, SU-DHL4 diffuse large B-cell lymphoma,K562 chronic myeloid leukemia (in blast crisis), and Raji Burkitt lymphoma cells were treated for 48 hours with increasing concentrations of CP-d/n-ATF5-S1prior to staining with Annexin V/PI and flow cytometric analysis. (Continued on the following page.)





peptide affects expression of Bcl-2 family members by a posttran-scriptional mechanism. For instance, Mcl-1 is stabilized by itschaperone Bcl-2–associated athanogene 3 (Bag3) on one handand by deubiquitination through the ubiquitin-specific peptidase9, X-linked (Usp9X) on the other (31, 32). Therefore, we deter-mined protein levels of Bag3 and Usp9X in a panel of tumor linesfollowing treatment with increasing concentrations of CP-d/n-ATF5-S1 for 48 to 72 hours. Usp9X expressionwas greatly reducedby peptide treatment in a time- and dose-dependent manner,whereas Bag3 levels fell particularly in U87MG, T98G, and PC3cells (Fig. 3A and Supplementary Fig. S7Aand S7B).CP-d/n-ATF5-S1–mediated reduction in Usp9X protein levels was not rescuedby z-VAD-fmk, indicating that Usp9X depletion is most likelyindependent of apoptosis and activated caspases as well as of itsmRNA levels (Supplementary Fig. S8 and Fig. 1E).

Usp9X knockdown induces apoptosis, caspase activation, andrecapitulates effects of CP-d/n-ATF5-S1

The consistent effect of CP-d/n-ATF5-S1 on Usp9X expressionled us to examine whether silencing Usp9X by another meanswould be sufficient to phenocopy the proapoptotic effect of thepeptide. Similarly to cells treated with CP-d/n-ATF5-S1, T98G,U251, and LN229 glioblastoma cells inwhichUsp9Xwas silencedwith siRNA showed marked reduction in viability as indicated byAnnexin V/PI staining (Fig. 3B and Supplementary Fig. S9A). Thiswas accompanied by a substantial increase in cleavage of caspase-9 and -3 (Fig. 3C and Supplementary Fig. S9B). In addition, therewas significant reduction in Bag3, Mcl-1, and Bcl-2 proteinexpression in both U251 and T98G cells. Taken together, thesefindings indicate that loss of Usp9X expression promoted by CP-d/n-ATF5-S1 treatment is sufficient to diminish levels of keyantiapoptotic Bcl-2 family members and to induce cell death.

CP-d/n-ATF5-S1 sensitizes for apoptosis induced by BH3-mimetics

Mcl-1 is amajor resistance factor toward BH3-mimetics such asABT737/263/199, and a considerable number of solid malignanttumors, including gliomas, bear high levels of Mcl-1 (33, 34).Given that CP-d/n-ATF5-S1modulates antiapoptotic members ofthe Bcl-2 family, especially Mcl-1 and its interacting proteins, weexamined whether the peptide may act in a complementary orsynergistic fashion with BH3-mimetic agents. We accordinglytreated T98G cells with CP-d/n-ATF5-S1 and Bcl-2/Bcl-xL inhib-itor ABT263 or the Bcl-2/Bcl-xL/Mcl-1 inhibitor GX15-070. Inboth instances, combined treatment caused synergistic inhibitionof cell viability as assessed by MTT assay (Fig. 4A and Supple-mentary Fig. S10A). In concordance, cellular morphology wasmarkedly changed in cells subjected to the combination treat-ments and pointed, as anticipated, toward apoptosis as theunderlyingmechanism (Fig. 4B). BecauseGX15-070 has activities

in addition to Bcl-2 family inhibition (35), we focused furthercombinatorial studies on ABT263. Enhancement of ABT263-mediated apoptosis by CP-d/n-ATF5-S1 was confirmed byAnnexin V/PI staining. The combination treatment significantlyupregulated the fraction of Annexin V–positive cells in T98G,LN229, SF188 (pediatric), NCH644 (glioma stem-like), andGBM12 glioblastoma cultures as well as in PANC-1 pancreaticcarcinoma, A375melanoma, K562 chronic myeloid leukemia (inblast crisis), and HCT116 colorectal cancer cell cultures (Fig. 4Cand E and Supplementary Figs. S5B and S11). Consistent withthese findings, combined treatment with CP-d/n-ATF5-S1 andABT263 also enhanced caspase-9 cleavage in T98G cells (Fig. 4F).

In the contextof theseexperiments,wealsoassessedaPenetratin-only peptide and a form of CP-d/n-ATF5-S1 mutated in theextended leucine zipper (Fig. 1A) to diminish its interaction withother proteins. In comparison with CP-d/n-ATF5-S1, the mutatedpeptide showed markedly less effect on T98G cell morphologyeither aloneor in combinationwithABT263 (Fig. 4B). Themutatedpeptide also showed much less effect on apoptosis when appliedalone andminimally enhanced apoptosiswhencombinedwith theBH3-mimetic (Fig. 4D). Moreover, combined treatment with thePenetratin peptide and ABT263 resulted in an antagonistic anti-proliferative effect (Supplementary Fig. S10B).

Combined CP-d/n-ATF5-S1 and ABT263 treatment promotesenhanced downregulation of Mcl-1 and Bcl-2 which in turnresults in enhanced apoptotic death

Wenext examined the effect of combined treatment with CP-d/n-ATF5-S1 and ABT263 on expression of antiapoptotic Bcl-2familymembers. As illustrated in Fig. 4F, ABT263 alone increasedexpression of Mcl-1, a finding that represents a generally acceptedmechanismof resistance to BH3-mimetic compounds (34).How-ever, when ABT263 was combined with CP-d/n-ATF5-S1, Mcl-1expression was highly suppressed, as was expression of Bcl-2 andBcl-xL (Fig. 4F). In contrast, combined treatment with ABT263and mutated CP-d/n-ATF5-S1 yielded only a slight decrease inMcl-1 and Bcl-2 expression (Fig. 4F).

Part of the rationale for combining CP-d/n-ATF5-S1 withABT263 is that unlike the latter, the former promotes Mcl-1downregulation. To examine whether downregulation of Mcl-1, as occurs with CP-d/n-ATF5-S1, is sufficient to sensitize forABT263-mediated apoptosis, we silenced Mcl-1 in PANC-1 cellswith siRNA prior to treatment with ABT263 (Supplementary Fig.S12A). Mcl-1 knockdown combined with ABT263 yieldedmarkedly enhanced cleavage of caspase-9 and -3. In addition,Bag3, Usp9X, and Bcl-2 expression was significantly reducedunder these conditions compared with cells either silenced forMcl-1 and/or treatedwith ABT263 alone. These observations werealso reflected by an enhanced reduction in the fraction of viableLN229 cells remaining after silencing Mcl-1 and treating with

(Continued.) Quantitative representation of the fraction of viable cells (Annexin V and PI-negative cells). Columns, means of three serial measurements.Bars, SD.B andC, representative flowplots of cells treated as described forA. Bottom left quadrant, fraction of viable cells; top left quadrant, fraction of necrotic cells;bottom right quadrant, fraction of early apoptotic cells; and top right quadrant, fraction of late apoptotic cells. D, T98G glioblastoma cells were treated for48 hours eitherwith Penetratin or CP-d/n-ATF5-S1. Then, staining for Annexin V/PIwas performed to detect apoptosis. E,quantitative representation of cells treatedas described forD. Columns, means of three serial measurements. Bars, SD. F, human astrocytes were treated for 48 hours either with Penetratin or CP-d/n-ATF5-S1and then staining for Annexin V/PI was performed to detect apoptosis. G, quantitative representation of cells treated as described for F. Columns, meansof three serial measurements. Bars, SD. H, representative flow plots of T98G glioblastoma cells that were treated for 48 hours with indicated concentrationsof CP-d/n-ATF5-S1 prior to staining for JC-1 andflowcytometric analysis. I, T98Gglioblastoma cellswere treated for 48 hourswith CP-d/n-ATF5-S1 or control.Wholecell extracts were collected andWestern blot analysis for caspase-9 (CP9) and actin was performed. The cleaved form of CP9 is marked by cleaved fragment (CF).





ABT263, as compared with treating with either alone (Supple-mentary Fig. S12B). Similarly, when Usp9X was silenced, LN229cells became more susceptible to the cytotoxic effects of ABT263(Supplementary Fig. S12C).

Thus, when combined with ABT263, specific knockdown ofMcl-1 and Usp9X (as seen after treatment with CP-d/n-ATF5-S1)suffices to reproduce themolecular profile of combined treatmentwith CP-d/n-ATF5-S1 and ABT263.

CP-d/n-ATF5-S1 enhances apoptosis induced by the deathreceptor ligand TRAIL

Next, we examined whether combined treatment with CP-d/n-ATF5-S1 also enhances apoptosis triggered by the extrinsicpathway. Our reasoning was that if the peptide increases sensi-tivity to the mitochondrial apoptotic pathway, it might comple-ment or enhance mitochondrial-dependent and/or mitochondri-al-independent apoptotic actions of a death-promoting ligand.

A

B C

Bag3

Mcl-1

Bcl-2

Bcl-xL

Actin

CP-d/n-ATF5-S1

T98G

-

72 h48 hPANC-1

--

72 h48 h

-

U87MG

-

72 h48 h

-

H197572 h48 h

Usp9X

2.933.93

5.3987.8

35.448.4

6.759.43

n.t.-siRNA Usp9X-siRNAU251

7.235.35

10.277.2

38.42.39

50.09.19

n.t.-siRNA Usp9X-siRNA

T98G

LN229

17.0

4.62

10.5 5.65

82.0 1.93

19.8

58.6

n.t.-siRNA Usp9X-siRNA

.1.6.61

.41.1.81

.61.2.81

.4.4.91

.7.9.81

.2.52.51

.3.2.91

1.61.41

.1.2.61

.3.311

.2.2.21

.8.8.81

.511.71

.61.11.31

.2.6.51

.2.7.81

.1.2.51

.31.91

.01.1.21

.1.3.71

.6.711

.1.81.11

.911.21

.4.5.71

.8.91.41

.2.4.71

.2.711

.6.71.11

.1.51.41

.4.611

.8.71.21

.5.81.21

1.21.11.31

5.63.53.31

.4.5.91

.3.61.21

.1.61.11

2.62.32.41

.4.6.81

.6.811

+

–

–

+

β-Actin

CP9

cCP3

Actin

Usp9X

Bag3

Mcl-1

Bcl-2

Bcl-xL

U251

+

–

–

+

n.t.-siRNA

Usp9X-siRNA

1 1.1

1 0.2

1 0.2

1 0.04

1 0.05 1 171

0.06/261/1

n.t.-siRNA

Usp9X-siRNA

+

–

T98G

–

+

Actin

Usp9X

Bag3

Mcl-1

Bcl-2

Bcl-xL

1 0.9

1 0.03

1 0.03

1 0.7

1 0.06

Figure 3.A, U87MG and T98 glioblastoma as well as H1975 non–small cell lung cancer and PANC-1 pancreatic cancer cells were treated with increasing concentrationsof CP-d/n-ATF5-S1 for 48 and 72 hours under reduced serum conditions. Whole-cell extracts were examined by Western blot analysis for Mcl-1, Bcl-2, Bcl-xL,Usp9X, and Bag3. Actin Western blot analysis was performed to confirm equal protein loading. pen indicates the usage of 100 mmol/L Penetratin as control.Densitometric analysis was performed using ImageJ (NIH, Bethesda, MD; http://imagej.nih.gov/ij). B, T98G, U251, and LN229 glioblastoma cells weretreated with nontargeting (n.t.)-siRNA or Usp9X-siRNA prior to staining with Annexin V/PI and flow cytometric analysis. C, U251 and T98G glioblastomacells were treated with n.t.-siRNA or Usp9X-siRNA followed by Western blot analysis for Usp9X, Bag3, Mcl-1, Bcl-2, Bcl-xL, and in U251 glioblastoma cells inaddition for cleaved caspase-3 (cCP3) and caspase-9 (CP9). Actin Western blot analysis was performed to confirm equal protein loading. Densitometric analysiswas performed using ImageJ (NIH, Bethesda, MD; http://imagej.nih.gov/ij).





BA

Control

ABT263 0.5 μmol/LCP-d/n-ATF5-S1+ ABT263

CP-d/n-ATF5-S1100 μmol/L

100 μm

T98GmutatedCP-d/n-ATF5-S1 100 μmol/L

Mutated CP-d/n-ATF5-S1 + ABT263

50

100

ABT263CP-d/n-ATF5-S1 +

+- + - +- -

T98G

Cel

lula

r via

bilit

y (%

) 150

0

P < 0.02P < 0.002

P < 0.02P < 0.002

-+ - -

Mutated CP-d/n-ATF5-S1 100 μmol/L

Mutated CP-d/n-ATF5-S1 100 μmol/L + ABT263 0.5 μmol/L

Annexin V

Prop

idiu

mio

dide 7.29 10.8 9.99 25.4

74.6 7.22 44.1 20.5

C

Annexin V

CP-d/n-ATF5-S1 (μmol/L)ABT263 (μmol/L)

- 100 - 100- 0.5- 0.5

T98G

8.13 11.2 4.18 18.9

50.2 30.5 18.8 58.2

3.67 7.06 10.3 29.2

84.4 4.91 40.3 20.3

LN229

2.35 11.2 5.68 53.2

80.3 6.16 26.9 14.3

2.86 4.18 8.73 21.9

88.8 4.14 58.2 11.2

PANC-1

6.85 28.7 2.26 35.4

53.1 11.4 34.8 27.6

3.03 14.5 1.43 16.3

73.5 8.97 48.4 33.9

1.72 10.3 0.59 24.4

76.6 11.4 32.1 42.9

2.87 4.62 0.36 15.3

84.8 7.73 54.4 29.9

A375


- 100 - 1002- 2-


- 100 - 1001- 1-

1.55 33.8 2.45 24.8

33.6 31.0 23.4 49.4

0.34 8.91 0.53 32.6

84.6 6.17 39.5 27.3

K562

SF1882.81 39.3 4.38 60.2

51.4 6.52 13.7 21.8

2.83 22.6 5.01 40.0

71.7 2.87 40.9 14.0

NCH6445.80 7.22 12.2 24.4

59.8 27.2 31.4 32.0

11.9 1.66 14.8 12.6

80.4 6.00 48.0 24.6Prop

idiu

mio

dide

D

F

E

GX15-070 ++- - - -

Frac

tion

of A

nnex

inV

-an

d/or

PI-p

ositi

ve

cells

in %

T98G LN229 PANC-1

0

2040

60

80100

SF188 A375 K562NCH644

ABT263CP-d/n-ATF5-S1

+ +- + - +- - + +

- + - +- - + +

- + - +- - + +

- + - +- - + +

- + - +- - + +

- + - +- - + +

- + - +- -

+ +- + - +- -- - - -

--+

+-+

Mcl-1

Bcl-2

Bcl-xL

Actin

Actin

CP9

T98G

MutatedABT263

+ +- + - +- -

CP-d/n-ATF5-S1- - - -

--+

+-+

CF

1 .69 1.2 .31 1.3 1.3

1 1.0 .81 .37 1.1 .67

1 .81 1.0 .43 1.0 1.2

** *

*

** *

***

**

**

Figure 4.A, T98G glioblastoma cells were treated for 72 hours under reduced serum conditions (1.5% FBS) with CP-d/n-ATF5-S1 (100 mmol/L), ABT263 (0.5 mmol/L), orGX15-070 (50 nmol/L) at the indicated combinations prior to performing MTT assays. Columns, means; error bars, SD. B, representative microphotographsat 40� magnification of T98G glioblastoma cells treated with CP-d/n-ATF5-S1, ABT263, the combination of both or solvent for 48 hours. In addition,microphotographs of cells treatedwithmutated CP-d/n-ATF5-S1 (100 mmol/L) alone or combinedwith ABT263 (0.5 mmol/L) are shown.C, representative flowplotsof T98G, LN229, SF188 (pediatric), NCH644 (glioma stem-like) glioblastoma and PANC-1 pancreatic carcinoma, A375 melanoma, K562 chronic myeloid leukemia(in blast crisis) cells that were treated for 72 hours with CP-d/n-ATF5-S1, ABT263, the combination of both or solvent as indicated prior to staining withAnnexin V/PI, and flow cytometric analysis.D, representative flowplots of T98G glioblastoma cells subjected to treatment withmutated CP-d/n-ATF5-S1 alone or incombination with ABT263. E, quantitative representation of the fraction of Annexin V and/or PI-positive cells treated as described for C. � , P < 0.05. Columns,means of three serial measurements; bars, SD. F, T98G glioblastoma cells were treated with CP-d/n-ATF5-S1 (100 mmol/L), mutated CP-d/n-ATF5-S1(100 mmol/L), and ABT 263 (0.5 mmol/L) at indicated combinations for 48 hours under serum starvation. Whole-cell extracts were examined by Western blotanalysis for caspase-9 [CP9, ¼ cleaved fragment (CF)], Mcl-1, Bcl-2, and Bcl-xL. Actin Western blot analysis was performed to confirm equal protein loading.Densitometric analysis was performed using ImageJ (NIH, Bethesda, MD; http://imagej.nih.gov/ij). Data were normalized first to the respective actin controland second to the respective treatment control.





50

100

T98GC

ellu

lar v

iabi

lity

(%)

mutatedCP-d/n-ATF5-S1

+- + - +- -

+ + - - -- - -+ + +- - -

TRAIL 1.25TRAIL 2.5

TRAIL 5

- - -- - -- - -- - -

+ - +-+ - +-

+ - +-

+ - -- -+ -- --

- - - +-

A B

CD

Annexin V

Prop

idiu

mio

dide

T98G

MDA-MB-436

LN229

CP-d/n-ATF5-S1 (μmol/L)TRAIL (ng/mL)

- 100 - 1005- 5-

4.94 7.85 11.6 44.5

76.1 11.1 20.9 23.0

2.05 3.40 7.90 29.9

90.1 4.44 38.5 23.7

1.24 5.54 12.8 55.0

88.8 4.40 26.5 5.78

2.52 4.76 14.4 22.2

89.3 3.46 55.0 8.42

7.84 36.1 12.4 72.1

53.9 2.19 13.4 2.05

12.9 10.4 12.7 36.5

74.7 2.01 41.9 8.94


- 100 - 100- 500- 500


- 100 - 10001- 01-

MutatedCP-d/n-ATF5-S1 100 μmol/L

Mutated CP-d/n-ATF5-S1 100 μmol/L + TRAIL 5 ng/mL

Annexin VProp

idiu

mio

dide 4.80 8.07 9.12 5.95

74.4 12.7 76.6 8.34

F

G

100 μm

ControlCP-d/n-ATF5-S1 100 μmol/L

TRAIL 5 ng/mLCP-d/n-ATF5-S1+ TRAIL

mutated CP-d/n-ATF5-S1 100 μmol/L

T98G

Mutated CP-d/n-ATF5-S1 + TRAIL

E

Bag3-siRNA +TRAIL 250 ng/mL

Mcl-1Bcl-2

Bcl-xLActin

Usp9XBag3

cCP3

CP9

Actin

Bag3-siRNATRAIL

+- +- -

n.t.-siRNA

-+

+

- -+ +LN229

6.20 7.45

84.0 2.34

8.58 13.3

74.6 3.52

13.1 34.9

47.8 4.20

8.17 20.2

67.2 4.43

LN229

Annexin V

Prop

idiu

mio

dide

n.t.-siRNA Bag3-siRNA

n.t.-siRNA +TRAIL 250 ng/mL

H JI

n.t.-siRNA Mcl-1-siRNA

n.t.-siRNA +TRAIL 200 ng/mL

Mcl-1-siRNA +TRAIL 200 ng/mL

Annexin V

Prop

idiu

mio

dide

6.91 9.86

79.3 3.96

5.12 17.9

73.1 3.87

44.5 25.4

27.3 2.80

10.4 34.0

51.1 4.49

LN229

Mcl-1Bcl-2

Bcl-xLActin

Usp9XBag3

cCP3

CP9

Actin

Mcl-1-siRNATRAIL

+- +- -

n.t.-siRNA

-+

+

- -+ +LN229

Frac

tion

of A

nnex

inV

-an

d/or

PI-p

ositi

ve

cells

in %

T98G LN229 MDA-MB-436

020406080

100

TRAILCP-d/n-ATF5-S1

+ +- + - +- - + +

- + - +- - + +

- + - +- -

CFCF

CP-d/n-ATF5-S1

Actin

CP3

T98G

MutatedTRAIL

+ +- + - +- -- - - -

--+

+-+

Mcl-1

Bcl-2

Bcl-xL

Actin

0.370.870.130.200.691

0.800.880.470.360.941

1.31.50.090.230.671

0.290.610.10.421.21

P < 0.001

P < 0.005

Figure 5.A, T98G glioblastoma cells were treated with CP-d/n-ATF5-S1 (50 mmol/L), mutated CP-d/n-ATF5-S1 (50 mmol/L) and increasing concentrations of TRAIL asindicated. After 72 hours MTT assays were performed. Columns, means; error bars, SD. B, representative microphotographs at 40� magnification of T98Gglioblastoma cells treated with CP-d/n-ATF5-S1, TRAIL, the combination of both or solvent for 48 hours. In addition, microphotographs of cells treated withmutated CP-d/n-ATF5-S1 (100 mmol/L) alone or combined with TRAIL (5 ng/mL) are shown. C, representative flow plots of T98G, LN229 glioblastoma, andMDA-MB-436 breast cancer cells treated for 72 hours with CP-d/n-ATF5-S1, TRAIL, the combination of both or solvent at the indicated concentrations prior tostaining with Annexin V/PI and flow cytometric analysis. (Continued on the following page.)





We therefore treated T98G cells with CP-d/n-ATF5-S1 andincreasing concentrations of TNF-related apoptosis-inducingligand (TRAIL). As shown in Fig. 5A, treatment with thiscombination results in an enhanced antiproliferative effect inthe MTT assay compared with control or single treatments. Incontrast, the combination of TRAIL with mutated CP-d/n-ATF5-S1 did not show this effect. Representative microphotographsin Fig. 5B illustrate these findings at the level of morphology.Annexin V/PI staining also showed that CP-d/n-ATF5-S1enhances TRAIL-mediated apoptosis in T98G cells as well asin LN229 glioblastoma cells and MDA-MB-436 breast cancercells (Fig. 5C and E). In this assay, mutated CP-d/n-ATF5-S1alone only slightly increased apoptotic cells when comparedwith controls and did not enhance TRAIL-induced apoptosis(Fig. 5D). In concordance with these findings, the combinationtherapy led to reduced expression of full-length caspase-3 inT98G cells, presumably due to elevated cleavage of this protein(Fig. 5F). Mutated CP-d/n-ATF5-S1 did not have this effect. Inaddition, combined treatment with CP-d/n-ATF5-S1 and TRAILenhanced downregulation of Mcl-1 and Bcl-2 expression (Fig.5F). Treatment with TRAIL alone in this cell line reducedexpression of Bcl-xL, although this effect was neither matchednor enhanced by CP-d/n-ATF5-S1 (Fig. 5F).

CP-d/n-ATF5-S1 sensitizes for TRAIL-mediated apoptosis atleast in part by downregulating Bag3 and Mcl-1

Decreased expression of Mcl-1 following treatment with CP-d/n-ATF5-S1 represents a mechanism likely to contribute to theCP-d/n-ATF5-S1–mediated sensitization toward TRAIL.Because Bag3 stabilizes Mcl-1 (23) and our data indicate thatCP-d/n-ATF5-S1 downregulates Bag3 in most cell lines tested(Fig. 5G), we examined whether Bag3 knockdown wouldphenocopy the sensitizing effect of CP-d/n-ATF5-S1 towardTRAIL. Silencing Bag3 in LN229 glioblastoma cells results indownregulation of Mcl-1 (Fig. 5G). When combined withTRAIL, Bag3 knockdown markedly increased cleavage of cas-pase-9 and -3. Consistent with these observations, treatmentwith Bag3-siRNA and TRAIL yields a significant increase inapoptosis of LN229 cells as determined by Annexin V/PIstaining (Fig. 5H). Moreover, TRAIL combined with silencingof Mcl-1 with siRNA also markedly increased cleavage ofcaspase-9 and -3 and apoptosis in LN229 cells (Fig. 5I andJ). In contrast, Bag3 and Usp9X levels were not affected by Mcl-1 knockdown alone or when combined with TRAIL (Fig. 5I).Taken together, these findings indicate that CP-d/n-ATF5-S1sensitizes tumor cells to TRAIL and that this occurs at least inpart by loss of Mcl-1 due to reduction of Bag3 expression.

CP-d/n-ATF5-S1 significantly attenuates tumor growth in vivoWe next assessed the therapeutic efficacy of CP-d/n-ATF5-S1 in

multiple murine xenograft models. U87MG glioblastoma, A375melanoma, PC3 prostate cancer, PANC-1 pancreatic cancer, andHCT116 colorectal cancer cells were implanted subcutaneously;MDA-MB-231 triple-negative breast cancer cells were implantedin the mammary fat pad; and GBM12 patient-derived xenograftcells were implanted intracranially. Once tumors formed, micewere randomized and treated with CP-d/n-ATF5-S1, vehicle, orpenetratin peptide as outlined in Fig. 6A–C and SupplementaryFigs. S5C, S13, and S14. Under these conditions, except for thecases of PANC-1 cells (P¼ 0.25) and HCT116 cells (P¼ 0.18), inall tumor types animals that received treatment with CP-d/n-ATF5-S1 had significantly smaller tumors than the animals treatedwith vehicle or Penetratin (Fig. 6B and C and Supplementary Figs.S5C, S13, and S14). Moreover, in a GBM12 intracranial patient-derived xenograft model, animals treated with CP-d/n-ATF5-S1showed a median survival of 38 days, which was significantlyprolonged compared with 22.5 days in animals receiving vehicle(Fig. 6A). Although the treatments used here affected tumorgrowth rate, for the most part, they did not result in statisticallysignificant regression of tumors. However, there was a statisticallysignificant tumor regression inmice bearingMDA-MB-231 breastcancer mammary fat pad xenografts (Supplementary Fig. S14E–S14G). This effect was not observed when mice were treated withPenetratin alone (Supplementary Fig. S14E–S14G).

To detect possible toxic effects due to peptide treatment,histologic analysis was performed on various tumor-free tissuesof animals treated with either vehicle or CP-d/n-ATF5-S1 accord-ing to the dosing schedule described in Fig. 6C. No tissue altera-tions in brain, lung, kidney, heart, liver, spleen, and intestine werefound (Supplementary Fig. S15A). Moreover, the body weights ofthe animals did not vary between the treatment groups toward theend of the experiment (Supplementary Fig. S15B).

Combined treatment with CP-d/n-ATF5-S1 and ABT263significantly enhances attenuation of tumor growth in vivo

Our in vitro studies indicated that combined CP-d/n-ATF5-S1and ABT263 treatment enhanced tumor cell death due to additiveand complementary effects on antiapoptotic Bcl-2 family mem-bers. To assess whether the combination is more effective in vivothan either treatment alone, we utilized a U251 heterotopicglioblastoma xenograft model and a HCT116 heterotopic colo-rectal cancer xenograft model (Fig. 6D and Supplementary Fig.S5C). The mice with xenografted tumors were divided into fourgroups: vehicle, ABT263, CP-d/n-ATF5-S1, or the combination ofABT263 and CP-d/n-ATF5-S1. As shown in Fig. 6D, in the U251

(Continued.)D, representative flow plots of T98G glioblastoma cells subjected to treatment with mutated CP-d/n-ATF5-S1 alone or in combination with TRAIL priorto staining for Annexin V/PI and flow cytometric analysis. E, quantitative representation of the fraction of Annexin V and/or PI-positive cells for T98G,LN229, and MDA-MB-436 cells that were treated as in C. Columns, means of three serial measurements; bars, SD. F, T98G glioblastoma cells were treatedwith CP-d/n-ATF5-S1 (100 mmol/L), mutated CP-d/n-ATF5-S1 (100 mmol/L), and TRAIL (5 ng/mL) at indicated combinations for 24 hours under reducedserum conditions. Whole-cell extracts were examined by Western blot analysis for caspase-3 (CP3), Mcl-1, Bcl-2, and Bcl-xL. Actin Western blot analysis wasperformed to confirm equal protein loading. Densitometric analysis was performed using ImageJ (NIH, Bethesda, MD; http://imagej.nih.gov/ij). Data werenormalized first to the respective actin control and second to the respective treatment control. G, LN229 glioblastoma cells were treated with nontargeting(n.t.)-siRNA, Bag3-siRNA, and TRAIL as indicated. Whole-cell extractswere examined byWestern blot analysis for caspase-9 [CP9, cleaved fragment (CF)], cleavedcaspase-3 (cCP3), Usp9X, Bag3, Mcl-1, Bcl-2, and Bcl-xL. Actin served as loading control. H, representative flow plots of LN229 glioblastoma cells treatedwith n.t.-siRNAor Bag3-siRNA prior to additional treatment with TRAIL or solvent for 24 hours and stainingwith Annexin V/PI plus flow cytometric analysis. I, LN229glioblastoma cells were treated with n.t.-siRNA, Mcl-1-siRNA and TRAIL as indicated. Whole-cell extracts were examined by Western blot analysis for CP9 (CF),cCP3, Usp9X, Bag3, Mcl-1, Bcl-2, and Bcl-xL. Actin served as loading control. J, representative flow plots of LN229 glioblastoma cells treated with n.t.-siRNAor Mcl-1-siRNA prior to additional treatment with TRAIL or solvent for 24 hours and staining with Annexin V/PI plus flow cytometric analysis.





A375

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Figure 6.A, a total of 3 � 105 GBM12 glioblastoma cells were implanted intracranially. After tumor formation animals were treated intraperitoneally with vehicle (n ¼ 6)or CP-d/n-ATF5-S1 (n ¼ 7). Treatment was started on day 5 with a dose escalation from 50 to 150 mg/kg over the first 4 days followed by a de-escalationto a maintenance therapy of 75 mg/kg, three times per week. Microphotographs at 2� and 40� magnification of a representative tumor from thevehicle-treated group are shown as well as representative brain MRIs of animals treated with vehicle or CP-d/n-ATF5-S1. (Continued on the following page.)





model, at the endpoint of the study, animals that received thecombination treatment had significantly smaller tumors com-pared with those treated either with ABT263 or CP-d/n-ATF5-S1alone and showed a decrease in tumor size over time whencompared with the beginning of treatment. Similarly, in theHCT116 model, the combination treatment led to significantreduction in tumor growth rate when compared with vehicle orsingle-agent treatments (Supplementary Fig. S5C). The combina-tion treatment also showed no clinical signs of toxicity, indicatingthat although the combined treatment is more efficient, it doesnot increase the occurrence of evident side effects.

DiscussionCancer cells typically develop primary or secondary resistance

to apoptosis (36). Therefore, means to manipulate the apoptoticmachinery are pivotal to restore therapeutic sensitivity. Deregu-lation of the apoptotic machinery is mediated through numerousfactors, such as the Bcl-2 family of proteins, the Inhibitor ofApoptosis Proteins, and expression of death receptors, initiatorcaspases, and endogenous caspase inhibitors (37, 38). The aber-rant expression of such molecules is regulated by various means,including the actions of transcription factors. ATF5 is an exampleof a transcription factor with oncogenic potential that affects Bcl-2family member expression (13, 16). ATF5 is upregulated invarious malignancies, including highly prevalent tumors, suchas breast carcinoma (11), but it is also increased in less commonmalignancies, such as low- andhigh-grade gliomas (13, 16). In thecontext of low- andhigh-grade gliomas, ATF5 expression levels arenot only increased, but also correlate with survival (13). Thus,ATF5 represents a potential target in treatment-refractory cancers.

Here, we show that a novel synthetic cell-penetrating domi-nant-negative ATF5 peptide induces apoptosis in a broad range oftumor types, including glioblastoma, triple-negative breast cancer(MDA-MB-436), hormone-refractory prostate cancer (PC3 andDU145), EGFR kinase inhibitor resistant non–small cell lungcancer (H1975), BRAF (V600E)-mutated melanoma (A375), andpancreatic carcinoma (PANC-1). CP-d/n-ATF5-S1 also showed invivo efficacy in reducing growth of a range of tumor types inxenograft models. We have yet to optimize dosing or regimens ofadministration. Our in vitro and in vivo studies indicate that thepeptide does not kill nontransformed cells and causes no evidenthistologic or behavioral signs of toxicity in mice at levels up to atleast 150 mg/kg. With respect to specificity, a Penetratin peptideand a peptide in which key leucine residues in CP-d/n-ATF5-S1were mutated showed no or little apoptotic activity alone or incombination studies. The fact that ATF5, as a prosurvival factor isoverexpressed in cancer cells, may cause a state of cellular ATF5dependency in the sense of oncogene addiction. This wouldexplain the selective response toward CP-d/n-ATF5-S1 treatmentin cancer cells as a consequence of a sudden CP-d/n-ATF5-S1–mediated loss of ATF5 function.

CP-d/n-ATF5-S1–mediated cell death was accompanied bydepletion of endogenous ATF5 protein, suggesting that CP-d/n-

ATF5-S1 may induce cell death in part through depletion of totalATF5 levels. In that context, earlier results showed that depletionof ATF5 by siRNA/shRNA leads to cell death in a broad variety oftumor cells (11, 13, 15). It remains to be determined by whatunderlying mechanisms CP-d/n-ATF5-S1 controls ATF5 proteinlevels. Our observations suggest that this is a nontranscriptionalevent. Given that ATF5 possesses a short-half life (39) and isstabilized by chaperones (39), it is conceivable that CP-d/n-ATF5-S1 enhances ATF5's degradation through disruption of its inter-actions with other binding partners.

Consistent with its activation of the intrinsic apoptotic path-way, CP-d/n-ATF5-S1 modulated the expression of the antiapop-totic Bcl-2 protein family, including Mcl-1, Bcl-2, and, in someinstances, Bcl-xL. Particularly, at earlier time points, there is anoccasional CP-d/n-ATF5-S1–mediated increase in Bcl-xL, which,however, for most cell lines (except for PANC1) tested is notsustained. Additional research will shed light on why CP-d/n-ATF5-S1 causes this biphasic modulation of Bcl-xL. Nevertheless,one possible consequence might be that the increase in Bcl-xLmight render tumor cells more sensitive to Bcl-xL inhibitors, suchas ABT263. These are known to counteract Bax-dependent apo-ptosis by preventing mitochondrial outer membrane permeabi-lization and subsequent cytochrome c release and caspase-9activation.

Although Bcl-2 and Mcl-1 have been described as transcrip-tional targets of ATF5, our findings indicate that CP-d/n-ATF5-S1causes only a transient decrease in their transcript levels and thatMcl-1, in particular, is subject to sustained downregulation at theposttranscriptional level by CP-d/n-ATF5-S1. Several moleculeshave been described that control Mcl-1 levels posttranslationally.Examples includeMULE (40), Bag3 (31), andUsp9X (32). Bag3 isa co-chaperone of Hsp70 (41) and binds Mcl-1 to prevent itsdegradation, whereas Usp9X is a deubiquitinase that removesubiquitin chains fromMcl-1, rendering it resistant to proteasomaldegradation and thereby in turn increasing its half-life (32). BothBag3 and Usp9X have been shown to counteract intrinsic apo-ptosis. For instance, Bag3 is upregulated inmalignant gliomas andinterferes with Bax-mediated apoptosis (42), whereas Usp9Xknockdown enhances ABT263-mediated cell death in glioblasto-ma (20, 33). Usp9X interacts with a variety of molecules inaddition to Bcl-2 proteins that may also affect cell survival (43,44) and these too may thus play a role in tumor cell deathpromoted by CP-d/n-ATF5-S1.

Our findings show that in PC3, PANC-1, T98G, H1975, A375,and U87MG cells, CP-d/n-ATF5-S1 significantly affects proteinlevels of Usp9X starting as early as 48 hours and continuing at 72hours after treatment, when apoptotic death ismanifest. To assessthe impact ofUsp9XdepletionbyCP-d/n-ATF5-S1,we transfectedglioblastoma cells with Usp9X siRNA and found that this wassufficient to induce significant apoptotic death. Mechanistically,Usp9X knockdown caused concomitant suppression of Bag3,Mcl-1, and Bcl-2 expression, which remarkably recapitulates theeffects of CP-d/n-ATF5-S1 on thesemolecules. These observations

(Continued.) B, a total of 1 � 106 A375 malignant melanoma cells were implanted subcutaneously. After tumor formation animals were treated intraperitoneallywith vehicle (n ¼ 12 tumors) or CP-d/n-ATF5-S1 (n ¼ 12 tumors) as indicated. C, a total of 1 � 106 PC3 prostate cancer cells were implanted subcutaneously.After tumor formation animals were treated intraperitoneally with vehicle (n ¼ 12 tumors) or CP-d/n-ATF5-S1 (n ¼ 12 tumors) as indicated. D, a total of1 � 106 U251 glioblastoma cells were implanted subcutaneously. After tumor formation animals were treated intraperitoneally with vehicle (n ¼ 9 tumors),CP-d/n-ATF5-S1 (n¼9 tumors), ABT263 (n¼9 tumors), or the combination of CP-d/n-ATF5-S1 andABT263 (n¼9 tumors) as indicated. Data are presented asmeanand SEM. The Student t test was used for statistical analysis and a P < 0.05 was considered statistically significant. Representative photographs of the tumorsare provided.





suggest that CP-d/n-ATF5-S1–mediated suppression of Usp9Xlevels may be an instrumental mechanism by which it mediatesdeath of neoplastic cells. Although Usp9X is known to modulateMcl-1 expression, our observed effects of Usp9Xmanipulation onBag3 and Bcl-2 have not been previously described and maysuggest that Usp9X also interacts with Bag3 and Bcl-2. Themechanism(s) bywhichCP-d/n-ATF5-S1decreasesUsp9Xexpres-sion remain to be explored. Our data indicate a posttranscrip-tional mechanism in that the peptide does not affect Usp9XmRNA levels. The strong antineoplastic activity related to Usp9Xdownregulation warrants further studies directed at identificationof specific small-molecule inhibitors of Usp9X function.

Given our observation that CP-d/n-ATF5-S1 strongly affects theintrinsic apoptotic machinery and the possibility that treatmentwith a single drug may fall short in the clinic, we investigatedwhether rational drug combination therapies could enhance theefficacy of CP-d/n-ATF5-S1. For that purpose, we utilized theorally available BH3-mimetic ABT263 (43). This class of com-pounds has received great attention because they target both Bcl-2and Bcl-xL, which are upregulated in many malignancies, espe-cially in hematologic malignancies, such as follicular lymphoma(45), and also in solid neoplasms, such as glioblastoma (46).Although certain tumors demonstrate remarkable sensitivity forBH3-mimetics, others reveal resistance, which in the vastmajorityof cases is attributed to high-levels of Mcl-1 expression (47).Therefore, means to counteract high Mcl-1 protein levels maysensitize resistant tumors to BH3-mimetic treatment (48, 49). Inthe current case,we found thatCP-d/n-ATF5-S1 stronglydepressesexpression of two Mcl-1–interacting proteins, Bag3 and Usp9X,and this in turn leads toMcl-1 depletion. These considerations ledus to assess the combination of CP-d/n-ATF5-S1 and ABT263 invitro and in in vivo xenograft tumor models. We found enhancedcell death by this combination in a variety of tumor cell lines,including LN229 and A375 that are relatively resistant to ABT263.In the in vivo models, the combination was highly effectivecompared with the single treatments and blocked/reduced tumorgrowth over the course of the studies.

Because the Bcl-2 family is also implicated in extrinsic apopto-sis, we tested whether CP-d/n-ATF5-S1 overcomes resistance toTRAIL. TRAIL has received attention for its ability to kill a broadvariety of cancer cells in vitro and in vivo. One main obstacle forTRAIL-related therapies is that although a subset of tumorsrespond, the majority display resistance (50). Therefore, effortshave aimed to identify treatments that sensitize cancer cells toTRAIL therapeutics. Our results suggest that CP-d/n-ATF5-S1 is apotent sensitizer for TRAIL-mediated apoptosis. Mechanistically,this is most likely linked to the ability of CP-d/n-ATF5-S1 tosuppress Bag3 andMcl-1, as specific knockdown of Bag3 or Mcl-1was sufficient to sensitize TRAIL-resistant LN229 glioblastomacells to apoptosis.

Overall, our results serve as a proof-of-principle and suggestthat the strategy of treatment with a cell-penetrating dominant-negative form of ATF5 is efficacious, selective, and nontoxic andtherefore holds promise for cancer therapy, either alone or in amultitargeting approach.

Disclosure of Potential Conflicts of InterestL.A. Greene is a consultant/advisory board member for and reports receiving

commercial research grants from Sapience Therapeutics. L.A. Greene and J.M.Angelastro are listed as coinventors on patents entitled "Methods for promotingapoptosis and treating tumor cells by inhibiting the expressionor functionof thetranscription factor ATF5" and "Methods for inhibiting the differentiation ofproliferative telencephalic cells in vitro by addition of ATF5," which are ownedby Columbia University, and on a provisional patent application for "Compo-sitions and Methods for Inhibiting Tumor Cells by Inhibiting the TranscriptionFactor ATF5," which is owned by Columbia University and University ofCalifornia, Davis. No potential conflicts of interest were disclosed by the otherauthors.

Authors' ContributionsConception and design: G. Karpel-Massler, B.A. Horst, L.A. Greene, J.M.Angelastro, M.D. SiegelinDevelopment ofmethodology:G.Karpel-Massler, L.A. Greene, J.M. Angelastro,M.D. SiegelinAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): G. Karpel-Massler, C. Shu, L. Chau, T. Tsujiuchi,M.D. SiegelinAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): G. Karpel-Massler, L. Chau, L.A. Greene, M.D.SiegelinWriting, review, and/or revision of the manuscript: G. Karpel-Massler, B.A.Horst, J.N. Bruce, P. Canoll, L.A. Greene, J.M. Angelastro, M.D. SiegelinAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): G. Karpel-Massler, B.A. Horst, C. Shu, L. Chau,J.N. Bruce, M.D. SiegelinStudy supervision: L.A. Greene, M.D. Siegelin

AcknowledgmentsThe authors thank Yanping Sun for excellent assistance with theMRI studies.

Grant SupportThis work was supported by a scholarship from the Dr. Mildred Scheel

Foundation of the German Cancer Aid (to G. Karpel-Massler) and AmericanBrain Tumor Association, Translational Grant 2013 (ABTACU13-0098), the2013 AACR-National Brain Tumor Society Career Development Award forTranslational Brain Tumor Research (13-20-23-SIEG), NIH NINDS awardK08NS083732 (to M.D. Siegelin), and NIH NINDS grant R01NS083795 (toJ.M. Angelastro and L.A. Greene).

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.

ReceivedNovember 23, 2015; revisedMarch 21, 2016; accepted April 9, 2016;published OnlineFirst April 28, 2016.

References1. Sosman JA, Kim KB, Schuchter L, Gonzalez R, Pavlick AC, Weber JS, et al.

Survival in BRAF V600-mutant advanced melanoma treated with vemur-afenib. N Engl J Med 2012;366:707–14.

2. Tsao MS, Sakurada A, Cutz JC, Zhu CQ, Kamel-Reid S, Squire J, et al.Erlotinib in lung cancer—molecular and clinical predictors of outcome. NEngl J Med 2005;353:133–44.

3. Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ,et al. Radiotherapy plus concomitant and adjuvant temozolomide forglioblastoma. N Engl J Med 2005;352:987–96.

4. Greene LA, Lee HY, Angelastro JM. The transcription factor ATF5: role inneurodevelopment and neural tumors. J Neurochem 2009;108:11–22.

5. Sheng Z, Evans SK, Green MR. An activating transcription factor 5-mediated survival pathway as a target for cancer therapy? Oncotarget2010;1:457–60.

6. Li G, Li W, Angelastro JM, Greene LA, Liu DX. Identification of a novelDNA binding site and a transcriptional target for activating transcriptionfactor 5 in c6 glioma and mcf-7 breast cancer cells. Mol Cancer Res2009;7:933–43.





7. VinsonC,AcharyaA, TaparowskyEJ.DecipheringB-ZIP transcription factorinteractions in vitro and in vivo. Biochim Biophys Acta 2006;1759:4–12.

8. Al Sarraj J, Vinson C, Thiel G. Regulation of asparagine synthetase genetranscription by the basic region leucine zipper transcription factors ATF5and CHOP. Biol Chem 2005;386:873–9.

9. Watatani Y, Ichikawa K, Nakanishi N, Fujimoto M, Takeda H, Kimura N,et al. Stress-induced translation of ATF5 mRNA is regulated by the 50-untranslated region. J Biol Chem 2008;283:2543–53.

10. Zhou D, Palam LR, Jiang L, Narasimhan J, Staschke KA, Wek RC. Phos-phorylation of eIF2 directs ATF5 translational control in response todiverse stress conditions. J Biol Chem 2008;283:7064–73.

11. Monaco SE, Angelastro JM, SzabolcsM,Greene LA. The transcription factorATF5 is widely expressed in carcinomas, and interference with its functionselectively kills neoplastic, but not nontransformed, breast cell lines. Int JCancer 2007;120:1883–90.

12. Ishihara S, Yasuda M, Ishizu A, Ishikawa M, Shirato H, Haga H. Activatingtranscription factor 5 enhances radioresistance and malignancy in cancercells. Oncotarget 2015;6:4602–14.

13. 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.

14. Persengiev SP, Devireddy LR, GreenMR. Inhibition of apoptosis by ATFx: anovel role for a member of the ATF/CREB family of mammalian bZIPtranscription factors. Genes Dev 2002;16:1806–14.

15. Chen A, Qian D, Wang B, Hu M, Lu J, Qi Y, et al. ATF5 is overexpressed inepithelial ovarian carcinomas and interference with its function increasesapoptosis through the downregulation of Bcl-2 in SKOV-3 cells. Int JGynecol Pathol 2012;31:532–7.

16. Angelastro JM, Canoll PD, Kuo J, Weicker M, Costa A, Bruce JN, et al.Selective destruction of glioblastoma cells by interference with the activityor expression of ATF5. Oncogene 2006;25:907–16.

17. Arias A, Lame MW, Santarelli L, Hen R, Greene LA, Angelastro JM.Regulated ATF5 loss-of-function in adultmice blocks formation and causesregression/eradication of gliomas. Oncogene 2012;31:739–51.

18. HuM,Wang B, Qian D, Li L, Zhang L, Song X, et al. Interference with ATF5function enhances the sensitivity of human pancreatic cancer cells topaclitaxel-induced apoptosis. Anticancer Res 2012;32:4385–94.

19. Cates CC, Arias AD, Nakayama Wong LS, Lame MW, Sidorov M, CayananG, et al. Regression/eradication of gliomas in mice by a systemically-deliverable ATF5 dominant-negative peptide. Oncotarget. 2016. [Epubahead of print].

20. 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. Onco-target 2015;6:36456–71.

21. Karpel-Massler G,Westhoff MA, Zhou S, Nonnenmacher L, Dwucet A, KastRE, et al. Combined inhibition of HER1/EGFR and RAC1 results in asynergistic antiproliferative effect on established and primary culturedhuman glioblastoma cells. Mol Cancer Ther 2013;12:1783–95.

22. Siegelin MD, Dohi T, Raskett CM, Orlowski GM, Powers CM, GilbertCA, et al. Exploiting the mitochondrial unfolded protein response forcancer therapy in mice and human cells. J Clin Invest 2011;121:1349–60.

23. 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.

24. Karpel-MasslerG, Pareja F, AimeP, ShuC,ChauL,WesthoffMA, et al. PARPinhibition restores extrinsic apoptotic sensitivity in glioblastoma. PLoSONE 2014;9:e114583.

25. Derossi D, Chassaing G, Prochiantz A. Trojan peptides: the penetratinsystem for intracellular delivery. Trends Cell Biol 1998;8:84–7.

26. Greene LA, Angelastro JM, inventors; The Trustees Of Columbia UniversityIn The City Of New York, assignee. Compositions and methods forinhibiting tumor cells by inhibiting the transcription factor ATF5. UnitedStates patent US 2016/0046686 A1. 2016 Feb 18.

27. Angelastro JM, Ignatova TN, Kukekov VG, Steindler DA, Stengren GB,Mendelsohn C, et al. Regulated expression of ATF5 is required for theprogression of neural progenitor cells to neurons. J Neurosci 2003;23:4590–600.

28. Moll JR, OliveM, Vinson C. Attractive interhelical electrostatic interactionsin the proline- and acidic-rich region (PAR) leucine zipper subfamilypreclude heterodimerization with other basic leucine zipper subfamilies.J Biol Chem 2000;275:34826–32.

29. Vinson CR, Hai T, Boyd SM. Dimerization specificity of the leucine zipper-containing bZIP motif on DNA binding: prediction and rational design.Genes Dev 1993;7:1047–58.

30. Dluzen D, Li G, Tacelosky D, Moreau M, Liu DX. BCL-2 is a downstreamtarget of ATF5 thatmediates the prosurvival function of ATF5 in a cell type-dependent manner. J Biol Chem 2011;286:7705–13.

31. BoianiM,Daniel C, Liu X,HogartyMD,Marnett LJ. The stress proteinBAG3stabilizesMcl-1 protein andpromotes survival of cancer cells and resistanceto antagonist ABT-737. J Biol Chem 2013;288:6980–90.

32. Schwickart M, Huang X, Lill JR, Liu J, Ferrando R, French DM, et al.Deubiquitinase USP9X stabilizes MCL1 and promotes tumour cell surviv-al. Nature 2010;463:103–7.

33. Karpel-Massler G, ShuC, Chau L, BanuM,HalatschME,WesthoffMA, et al.Combined inhibition of Bcl-2/Bcl-xL and Usp9X/Bag3 overcomes apopto-tic resistance in glioblastoma in vitro and in vivo. Oncotarget 2015;6:14507–21.

34. Konopleva M, Contractor R, Tsao T, Samudio I, Ruvolo PP, Kitada S, et al.Mechanisms of apoptosis sensitivity and resistance to the BH3 mimeticABT-737 in acute myeloid leukemia. Cancer Cell 2006;10:375–88.

35. McCoy F, Hurwitz J, McTavish N, Paul I, Barnes C, O'Hagan B, et al.Obatoclax induces Atg7-dependent autophagy independent of beclin-1and BAX/BAK. Cell Death Dis 2010;1:e108.

36. Holohan C, Van Schaeybroeck S, Longley DB, Johnston PG. Cancer drugresistance: an evolving paradigm. Nat Rev Cancer 2013;13:714–26.

37. Marini ES, Giampietri C, Petrungaro S, Conti S, Filippini A, Scorrano L,et al. The endogenous caspase-8 inhibitor c-FLIP regulates ERmorphologyand crosstalk with mitochondria. Cell Death Differ 2015;22:1131–43.

38. Yip KW, Reed JC. Bcl-2 family proteins and cancer. Oncogene 2008;27:6398–406.

39. Li G, Xu Y, Guan D, Liu Z, Liu DX. HSP70 protein promotes survival of C6and U87 glioma cells by inhibition of ATF5 degradation. J Biol Chem2011;286:20251–9.

40. Zhong Q, Gao W, Du F, Wang X. Mule/ARF-BP1, a BH3-only E3 ubiquitinligase, catalyzes the polyubiquitination of Mcl-1 and regulates apoptosis.Cell 2005;121:1085–95.

41. Colvin TA, Gabai VL, Gong J, Calderwood SK, Li H, Gummuluru S, et al.Hsp70-Bag3 interactions regulate cancer-related signaling networks. Can-cer Res 2014;74:4731–40.

42. Festa M, Del Valle L, Khalili K, Franco R, Scognamiglio G, Graziano V, et al.BAG3 protein is overexpressed in human glioblastoma and is a potentialtarget for therapy. Am J Pathol 2011;178:2504–12.

43. Tse C, Shoemaker AR, Adickes J, AndersonMG,Chen J, Jin S, et al. ABT-263:a potent and orally bioavailable Bcl-2 family inhibitor. Cancer Res2008;68:3421–8.

44. Xie Y, Avello M, Schirle M, McWhinnie E, Feng Y, Bric-Furlong E, et al.Deubiquitinase FAM/USP9X interacts with the E3 ubiquitin ligaseSMURF1 protein and protects it from ligase activity-dependent self-deg-radation. J Biol Chem 2013;288:2976–85.

45. Mahadevan D, Fisher RI. Novel therapeutics for aggressive non-Hodgkin'slymphoma. J Clin Oncol 2011;29:1876–84.

46. Cristofanon S, Fulda S. ABT-737 promotes tBid mitochondrial accumula-tion to enhance TRAIL-induced apoptosis in glioblastoma cells. Cell DeathDis 2012;3:e432.

47. LucasKM,Mohana-KumaranN,LauD,ZhangXD,HerseyP,HuangDC, etal.ModulationofNOXAandMCL-1 as a strategy for sensitizingmelanomacellsto the BH3-mimetic ABT-737. Clin Cancer Res 2012;18:783–95.

48. Preuss E,HugleM, Reimann R, SchlechtM, Fulda S. Pan-mammalian targetof rapamycin (mTOR) inhibitor AZD8055 primes rhabdomyosarcomacells for ABT-737-induced apoptosis by down-regulating Mcl-1 protein.J Biol Chem 2013;288:35287–96.

49. Vaillant F,MerinoD, Lee L, Breslin K, Pal B, RitchieME, et al. TargetingBCL-2 with the BH3 mimetic ABT-199 in estrogen receptor-positive breastcancer. Cancer Cell 2013;24:120–9.

50. Dimberg LY, Anderson CK, Camidge R, Behbakht K, Thorburn A, FordHL. On the TRAIL to successful cancer therapy? Predicting and counter-acting resistance against TRAIL-based therapeutics. Oncogene 2013;32:1341–50.





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