elicits Synthetic Lethality in Glioblastoma , Chiaki Tsuge ......1 Combined HDAC and Bromodomain Protein Inhibition Reprograms Tumor Cell Metabolism and elicits Synthetic Lethality

1

Combined HDAC and Bromodomain Protein Inhibition Reprograms Tumor Cell Metabolism and

elicits Synthetic Lethality in Glioblastoma

Yiru 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 and Markus D. Siegelin1

1 Department of Pathology & Cell Biology, Columbia University Medical Center, New York, NY,

U.S.A., 2 Department of Neurosurgery, The Johns Hopkins University School of Medicine,

Baltimore, Maryland, U.S.A., 3 Department of Biomedical Informatics, Columbia University, New

York, NY, U.S.A., 4 Department of Neurology, H. Houston Merritt Neuromuscular Research

Center, Columbia University Medical Center, New York, NY, U.S.A., 5 Department of Pediatrics

and Adolescent Medicine, Ulm University Medical Center, Ulm, Germany, 6 Department of

Neurosurgery, Ulm University Medical Center, Ulm, Germany

Running title: Targeting the Epigenome reprograms glioblastoma metabolism.

Correspondence to:

Markus D. Siegelin, MD, Dr. med., Department of Pathology & Cell Biology, Columbia University

Medical Center, 630 West 168th Street, P&S Rm. 15-415, New York, NY 10032, Phone: 212

305 1993, [email protected], [email protected]

Financial Support:

M.D. Siegelin: NIH NINDS R01NS095848, K08NS083732, Louis V. Gerstner, Jr. Scholars

Program (2017-2020) and American Brain Tumor Association Discovery Grant 2017

(DG1700013)

Conflict of interest statement: The authors have declared that no conflict of interest exists.

Research. on September 8, 2020. © 2018 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on May 15, 2018; DOI: 10.1158/1078-0432.CCR-18-0260

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Abstract

Purpose: Glioblastoma remain a challenge in oncology in part due to tumor heterogeneity.

Experimental Design: Patient-derived xenograft and stem-like glioblastoma cells were used as

the primary model systems. Results: Based on a transcriptome and subsequent gene set

enrichment analysis (GSEA), we show by using clinically validated compounds that combined

histone deacetylase (HDAC) inhibition and Bromodomain protein (BRD) inhibition results in

pronounced synergistic reduction in cellular viability in patient-derived xenograft and stem-like

glioblastoma cells. Transcriptome based GSEA analysis suggests that metabolic

reprogramming is involved with synergistic reduction of oxidative and glycolytic pathways in the

combination treatment. Extracellular flux analysis confirms that combined HDAC inhibition and

BRD inhibition blunts oxidative and glycolytic metabolism of cancer cells, leading to a depletion

of intracellular ATP production and total ATP levels. In turn, energy deprivation drives an

integrated stress response, originating from the endoplasmic reticulum. This results in an

increase in pro-apoptotic Noxa. Aside from Noxa, we encounter a compensatory increase of

anti-apoptotic Mcl-1 protein. Pharmacological, utilizing the FDA-approved drug sorafenib, and

genetic inhibition of Mcl-1 enhanced the effects of the combination therapy. Finally, we show in

orthotopic patient-derived xenografts of GBM, that the combination treatment reduces tumor

growth, and that the triple therapy, involving clinically validated compounds, panobinostat,

OTX015 and sorafenib further enhances these effects, culminating in a significant regression of

tumors in vivo. Conclusion: Overall, these results warrant clinical testing of this novel,

efficacious combination therapy.




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Introduction

Over the last decade, there were tremendous new insights in the characteristics of solid

cancers, such as melanoma and glioblastoma. Notable findings are for instance the discovery of

BRAF mutations in melanoma and IDH1 mutations in gliomas (1-6). Regarding gliomas,

comparable efforts have been taken, albeit these approaches have not matured to the extent as

in melanoma. Nevertheless, given the lack of durability new or amended strategies are required

to address the problem of solid tumors.

In conjunction with the discovery of driver mutations, there are a number of small molecule

inhibitors available that target deregulated pathways in cancers. Similarly, transcription is

altered in cancer by various mechanisms, e.g. DNA methylation or histone acetylation. Very

recently, mutations in the histone 3.3 protein (H3) at codon 27 were identified and it was shown

in preclinical model systems that malignancies with this aberration benefit from treatment with a

histone-deacetylase inhibitor. In this regard, broad HDAC-inhibitors, such as panobinostat or

vorinostat, have reached FDA-approval and are in clinical testing for solid malignancies as well.

However, as with other monotherapies, tumors are either primarily or become secondarily

resistant to these agents. Therefore, combination therapies that target multiple pathways may

address this problem. One of these signaling cascades is c-myc, which is highly upregulated in

many cancers and which became druggable through the discovery of bromodomain protein

inhibitors (BRDs) (7,8), such as the prototype JQ1 and clinically more amenable chemical

derivatives, such as OTX015.

Here, we provide a mechanism-based drug combination therapy that synergistically reprograms

tumor cell transcription, leading to metabolic reprogramming, energy starvation and activation of

the integrated stress response followed by significant intrinsic apoptosis. Moreover, the

combination therapy causes a partial cyto-protective effect by elevating Mcl-1 and interference




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with Mcl-1 through an FDA-approved drug leads to significant tumor regression and survival

extension in two glioblastoma xenograft models in vivo.




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Materials and Methods

Reagents

OTX015 (OTX), vorinostat (Vr), panobinostat (Pb) and sorafenib (Sf) were purchased from

Selleckchem.

Cell cultures and growth conditions

U87MG, LN229, U87-EGFRvIII and T98G human glioblastoma cell lines and A375 malignant

melanoma cells were obtained from the American Type Culture Collection (Manassas, VA) or

the Coriell Institute for Medical Research, respectively. NCH644 and NCH421K stem cell-like

glioma cells were obtained from Cell Line Services (CLS, Heidelberg, Germany). The GBM14

PDX cells have been described elsewhere (9-12). The respective cell line depository

authenticated the cells. All cell lines were cultured as previously described (13-16).

Cell viability assays

Viability assays were performed as previously described (13-18).

Measurement of apoptosis and mitochondrial membrane potential

For Annexin V/propidium iodide staining the Annexin V Apoptosis Detection Kit (BD

Pharmingen) was used as previously described (16,19). Tetramethylrhodamine ethyl ester

(TMRE) staining was performed according to the manufacturer’s instructions (Mitochondrial

Membrane Potential kit, Cell Signaling Technology, Danvers, MA). The data were analyzed with

the FlowJo software (version 8.7.1; Tree Star, Ashland, OR).

Extracellular flux analysis

Extracellular flux analysis was performed on the Seahorse XFe24 analyzer. The mitochondrial

stress assay was utilized in accordance with the instructions by the manufacturer and as




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described earlier (14). The glycolytic stress test was used in accordance with the instructions by

the manufacturer. Briefly, 40,000 cells were seeded and starved. During the assay, cells were

exposed to glucose, followed by oligomycin and completed by 2-DG. The extracellular

acidification rate (ECAR) was measured.

Transfections of siRNAs or transduction of shRNAs

Briefly, cells were incubated for 6h with the formed complexes of Oligofectamine® 2000

(Invitrogen, Carlsbad, CA) and the respective siRNA (12-well condition) in DMEM without FBS

and antibiotics. After 6h, FBS was added to a total concentration of 1.5%. Transduction of

lentiviral shRNAs is performed as described earlier (14).

Western blot analysis, protein capillary electrophoresis and immunoprecipitation

analysis

Specific protein expression in cell lines was determined by Western blot analysis as described

before (18). Co-immunoprecipitations were performed as described earlier in (13).

Subcutaneous xenograft model

1 x 106 A375 cells, 1 x 106 U87-EGFRvIII or GBM12 PDX xenograft tumors were implanted

subcutaneously into the flanks of 6-8 week-old SCID SHO mice as described before (16).

Measurements were performed with a caliper and tumor sizes were calculated as (length x

width2)/2. Treatments were performed intraperitoneally as described in the respective figure

legends of each individual experiment. Columbia IACUC has approved these studies.

Orthotopic glioblastoma PDX and stem-cell like xenograft model

For the GBM12 orthotopic GBM model (34), 300,000 cells and for the stem-like GBM model,

20,000 NCH644 cells were injected in a manner as described earlier. The drugs for the




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indicated treatments were dissolved as described earlier (10). Columbia IACUC has approved

these studies.

Microarray and gene set enrichment analysis

Transcriptome and gene set enrichment analysis (GSEA) was performed as previously

described (14). For the present experiments, two biological replicates per condition were

submitted. The experiment was deposited online with GEO with the following ID: GSE108958.

Statistical analysis

Statistical significance was assessed by Student’s t-test using Prism version 7.00 (GraphPad,

La Jolla, CA). Data was considered statistically 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.,

Paramus, NJ) was used to detect synergistic, additive or antagonistic effects as previously

described.

Study approval

All procedures were in accordance with Animal Welfare Regulations and approved by the

Institutional Animal Care and Use Committee at the Columbia University Medical Center.




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Results

HDAC and BRD inhibition leads to synergistic reduction in tumor cell proliferation

To ensure that the utilized compounds act on-target, we validated as to whether or not HDAC

inhibition enhances acetylation of known targets. To this end, patient-derived xenograft cells,

GBM14, were treated with panobinostat in the presence or absence of OTX015. Protein

expression analysis for total histone H3 and acetylated histone H3 validated on-target activity

(Supplementary Figure 1A). To demonstrate that combined HDAC and BRD inhibition is a

potential efficacious strategy to inhibit growth of glioblastoma cells in a synergistic manner, we

tested two clinically validated drug compounds that interfere with these pathways, panobinostat

(HDAC inhibitor) and OTX015 (BRD inhibitor), in established (U87, U87-EGFRvIII, T98G,

LN229), patient-derived xenograft (GBM14) and stem-like glioma cells (NCH644). In all model

systems tested, the drug combination over a range of concentrations elicited a synergistic

interaction as evaluated by combination index value analysis (CI values) (Figure 1A and 1B).

Notably, the most significant synergistic interaction was identified in patient-derived xenograft

cells, GBM14. Similar results were obtained when FDA-approved, vorinostat, was administered

in lieu of panobinostat (Supplementary Figure 1B). These results suggest that combined

targeting of HDACs and BRD is efficacious.

HDAC and BRD inhibition causes enhanced apoptotic cell death

The combination of panobinostat and OTX015 resulted in morphological signs of apoptosis. In

support of this finding, GSEA showed that the combination treatment activated a transcriptional

pro-apoptotic state (Figure 2C). Therefore, we determined as to whether or not features of

apoptotic cell death can be confirmed biochemically. To this purpose, LN229, T98G and U87

GBM cells or stem-cell like GBM cells, NCH421k and NCH644 were treated with panobinostat

(or vorinostat), OTX015 or the combination of both and stained with Annexin V/propidium iodide

and analyzed by multi-parametric flow cytometric analysis. Consistently, we found that the




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combination treatment of OTX015 and Panobinostat led to more apoptotic cells than control or

single treatments. Similar findings were made when vorinostat was used in lieu of panobinostat

(Figure 2A and 2B, Supplementary Figure 2A-C). Intrinsic apoptosis is accompanied by loss of

mitochondrial membrane potential. Consistently, the combination treatments

(panobinostat+OTX015; vorinostat+OTX015) reduced the amount of TMRE positive cells

stronger than single treatments or control in LN229, T98G and U87 cells (Figure 2D and 2E and

Supplementary Figure 2D). To assess whether or not caspases are involved in the death, we

treated GBM cells in the presence or absence of pan-caspase inhibitor, zVAD-fmk. We found

that zVAD-fmk partially protected the cells from DNA fragmentation induced by the combination

treatment, suggesting that caspases are involved in the death (Supplementary Figure 2E). This

finding was also supported by enhanced cleavage of PARP by the combination treatment

(Figure 2F and Supplementary Figure 2H).

Combined HDAC and BRD inhibition modulates the expression of pro- and anti-apoptotic

Bcl-2 family members

Given the activation of apoptosis, we were wondering about the regulation of this form of cell

death induced by the combination treatment. To this purpose, we analyzed protein expression

of both anti-apoptotic Bcl-2 family members, Mcl-1, Bcl-xL and Bcl-2, and pro-apoptotic Bcl-2

family members, Noxa and BIM, in established, stem-like and patient derived xenograft cells of

GBM. Our findings demonstrate that combined treatment with OTX015 and HDAC-inhibitors

results in an increase in BIM and Noxa protein levels (Figure 2G and 2H and Supplementary

Figure 2F and 2G). Aside from these pro-apoptotic molecular changes, we found a

compensatory, transitory up-regulation of Mcl-1 and its deubiquitinase Usp9X induced by the

combination treatment (Figure 2G and 2H and Supplementary Figure 2F and 2G). Next, we

assessed the transcriptional changes by real-time PCR analysis and found that mRNA for




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Usp9x, Mcl-1, Noxa and BIM were all increased with the exception of BIM in NCH644 cells

(Figure 2I and 2J).

Next, we evaluated the functional impact of the observed protein changes on the apoptotic

efficacy of the combination treatment. Given the increase in Noxa and Mcl-1, we mainly focused

on the MCl-1/Noxa/BAK cascade in which high levels of Noxa bind to Mcl-1 and mediate the

BAK/Mcl-1 dissociation. In turn, BAK engages in mitochondrial outer membrane

permeabilization. To this purpose, we silenced the expression of BAK with two siRNAs.

Silencing of BAK was confirmed by standard western blotting (Figure 3A). Knockdown of BAK

provided a partial protection from panobinostat+OTX015 mediated apoptosis (Figure 3B). Next,

we tested the impact of Noxa on the combination treatment and silencing of Noxa was

confirmed by capillary electrophoresis (Figure 3A). We found that silencing of Noxa provided a

partial protection from panobinostat+OTX015 mediated apoptosis (Figure 3C and

Supplementary Figure 3E), in keeping with the results on BAK. To gain a further understanding

about the molecular interactions implicated in the combination treatment, we conducted co-

immunoprecipitation analysis by pulling down Mcl-1 in the presence or absence of the

combination treatment (Figure 3D). While the IgG control did not pull down any of the analyzed

proteins, anti-Mcl-1 prominently precipitated Mcl-1 and its associated described binding

partners, Usp9X, BIM, Noxa, BAK, but not GAPDH (negative control), confirming high specificity

of our experiment (Figure 3D). Regarding the effects of the combination treatment on the

binding partners of Mcl-1, we observed that the panobinostat+OTX015 treatment resulted in an

increased binding of Noxa to Mcl-1 (Figure 3D). Enhanced binding of Noxa to Mcl-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 was observed (Figure

3D).




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Given that we observed an increased expression of Mcl-1 and Usp9x at early time points upon

treatment with the combination treatment, we hypothesized that these effects mediate a

transitory “pro-survival” effect. Indeed, later time points (>24h after treatment) show a decline in

Mcl-1, coinciding with enhanced death (Figure 3G and 3H). To test this hypothesis, we utilized

two Mcl-1 specific siRNAs and one Mcl-1 pool siRNA, which potently suppressed Mcl-1 protein

levels (Figure 3E and Supplementary Figure 3A-D). Indeed, silencing of Mcl-1 significantly

enhanced apoptosis in LN229 GBM cells (a cell line that responds slower to the combination

treatment) and T98G GBM cells (Figure 3E and 3F and Supplementary Figure 3A-D). In like

manner, we evaluated the effects of USP9X silencing on the combination treatment and found

that Usp9X silencing enhanced apoptosis induced by the combination treatment further (Figure

3F and Supplementary Figure 3C).

These results intimate that inhibition of Mcl-1 might further enhance the efficacy of the drug

combination therapy. To further evaluate this point, we tested whether the kinase inhibitor,

sorafenib, a known modulator of Mcl-1 levels and FDA-approved drug (22,23), is capable of

counteracting the combination treatment mediated increase of Mcl-1 protein levels. In keeping

with this expectation, sorafenib blunted Mcl-1 up-regulation mediated by panobinostat and

OTX015 (Figure 3G and 3H). Remarkably, sorafenib also curtailed Usp9X expression,

suggesting that sorafenib abrogated two main pro-survival effects elicited by the combination

treatment (Figure 3G and 3H). To evaluate whether sorafenib also enhances apoptosis

induction by the combination treatment, LN229 or GBM14 cells were treated with the

combination treatment in the presence or absence of sorafenib. While in the absence of

sorafenib the combination treatment displayed some apoptosis induction, this effect was

significantly enhanced in the presence of the multi-kinase inhibitor (Figure 3J and

Supplementary Figure 3F). Similar results were obtained in T98G GBM cells (DNA –




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fragmentation) (Supplementary Figure 3G). These findings establish a novel, rational triple drug

therapy, called OTX+Pb+Sf (OTX015 + panobinostat + sorafenib).

Combined HDAC and BRD inhibition suppresses oxidative phosphorylation and

glycolysis in a synergistic manner

Given the pronounced synergistic reduction of cellular proliferation over a broad range of

different cell lines, we wondered about the underlying molecular mechanisms, orchestrating and

driving these changes. To this end, GSEA with subsequent interaction analysis assisted us to

identify pathways that are synergistically down regulated by panobinostat and OTX015. This

analysis was proved powerful since it permits to elucidate which molecular processes require

both compounds for most potent pathway inhibition. It turned out that the key energy producing

pathways in tumor cells, glycolysis and oxidative phosphorylation, were drastically suppressed

(Figure 4A), presumably leading to an intracellular energy crisis. Consistently, the combination

treatment suppressed protein levels of transporters and enzymes, involved in glycolysis

(GLUT1, LDHA, GAPDH) (Figure 4B).

Next, we determined the underlying molecular mechanism, governing these significant

metabolic changes mediated by HDAC and BRD inhibition. We hypothesized that these

metabolic aberrations might likely be a result of interference with the expression of the master-

regulator, c-Myc. First, c-Myc is pivotal for the regulation of tumor cell metabolism (24). Second,

c-Myc is a 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 inhibition of BRD and HDAC results in silencing of MYC transcript as shown by real-

time PCR and GSEA analysis in glioblastoma stem-like cells, NCH644 (Figure 4C, 4D and




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Supplementary Figure 4A). Along with the reduction in c-Myc, the combination treatment also

affected markers of stemness, such as SOX2 and Nanog. Although in these model systems

CD133 and Nestin cannot be considered as bona-fide stem-cell markers, their expression was

suppressed as well (Supplementary Figure 4B). Most relevantly, c-Myc was down-regulated on

protein level, showing the strongest c-Myc suppression in the combination treatment of OTX015

and panobinostat (Figure 4B).

The above findings support the hypothesis that glioblastoma cells treated with the combination

of OTX015 and panobinostat have low energy levels. Indeed, the combination treatment

depletes GBM14 patient-derived xenograft cells of ATP more potently than single treatments or

vehicle (Figure 4E). This is also in keeping with the related gene set enrichment analysis,

showing a transcriptional signature of “starvation” (GO_RESPONSE_TO_STARVATION)

(Figure 5A).

Given the impact on the transcripts and proteins related to energy metabolism, we conducted

extracellular flux analysis to validate whether or not the combination treatment affects oxidative

phosphorylation and glycolysis in glioblastoma cell cultures (Figure 4F-M)). To this end, patient-

derived xenograft cells, GBM14, were treated with OTX015, panobinostat, the combination

treatment in the presence or absence of sorafenib and were subjected to analysis for oxygen

consumption rate (OCR) in the context of a mitochondrial stress test. We found a significant

reduction in basal OCR (mitochondrial oxygen consumption rate) in the combination treatment,

which was mirrored also by the amount of OXPHOS related ATP production (Figure 4F, 4J and

4K). Similarly, we found that the maximal respiration and spare respiratory capacity was

prominently reduced by the combination treatment, which was further enhanced by the

presence of sorafenib, suggesting that sorafenib further inhibits oxidative phosphorylation, in

keeping with its suppression on Mcl-1 protein levels (Figure 4L-M). Next, we assessed the




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impact of the combination treatment on glycolysis by determining ECAR (extracellular

acidification rate) in the context of a glycolysis stress test. Akin to OXPHOS, we found that the

combination treatment reduced key indicators of glycolysis more potently than each compound

alone (Figure 4G-I). In the presence of sorafenib, the combination treatment completely

abrogated the glycolytic reserve, suggesting that sorafenib further enhances the inhibitory

effects of the combination treatment on energy metabolism (Figure 4I). To address the

applicability of these findings to other model systems, we performed the same experiments in

another glioblastoma cell culture, LN229. The findings in LN229 are overall similar to GBM14

except that the triple therapy (OTX+Pb+Sf) had a more drastic impact on OXPHOS related ATP

production and that the combination treatment was in some instances less efficacious

(Supplementary Figure 5A-D). Finally, we evaluated the impact of c-Myc levels on glycolysis in

patient-derived xenograft GBM cells, GBM14, and found that silencing of c-Myc levels

suppressed glycolysis (Supplementary Figure 5E). These results are in keeping with our

findings related to the combination treatment of OTX015 and panobinostat, showing enhanced

suppression of glycolytic key indicators and confirm that c-Myc suppression results in an

inhibition of glycolysis. All in all, our findings suggest that the combination treatment interferes

with energy metabolism, and these effects are further enhanced in the presence of sorafenib.

The combined inhibition of BRD and HDAC leads to endoplasmic reticulum stress

followed by enhanced expression of pro-apoptotic Noxa

Based on the findings related to energy metabolism, we hypothesized that the state of energy

deprivation elicited by the combination treatment should affect the homeostasis of the

endoplasmic reticulum (ER) since ATP is necessary for protein folding and loss of ATP will

result in the accumulation of unfolded proteins. GSEA confirms this notion, which was also

validated by RT-PCR analysis for common ER-stress related markers, e.g. GRP78, ATF3, ATF4




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and CHOP in both NCH644 and LN229 GBM cells (Figure 5A-C). To confirm that the

transcriptional changes are also found at protein level, we conducted western blot analysis as

well as capillary electrophoresis for common ER-stress related markers. The combination

treatment of OTX015 and panobinostat displayed the most significant ER-stress related

signature with up-regulation of GRP78, phosphorylated eIF2, total eIF2, ATF3, ATF4 and

CHOP (Figure 5D and Supplementary Figure 2H). Moreover, the induction of ER-stress by the

indicated treatments correlated with the cleavage of PARP, suggesting that ER-stress is

important for the induction of apoptosis by the combination treatment (Supplementary Figure

2H). Overall, these results confirm the notion that the combination treatment elicits a potent ER-

stress response likely related to energy starvation.

To determine the down-stream consequences of ER-stress, we hypothesized that the ER-stress

signaling is responsible for the enhanced 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-Noxa cascade and hypothesized that the combination treatment utilizes this

stress pathway to upregulate Noxa expression. To evaluate this claim, we silenced the

expression of PERK by siRNA in LN229 GBM cells. Silencing of PERK decreased ATF4 levels

and suppressed OTX015, panobinostat and the combination treatment mediated increase of

Noxa protein levels (Figure 5E). Another transcription factor related to ER-stress signaling and

to Noxa regulation is ATF3 (26,27). Given that ATF3 was potently increased by the combination

treatment, we tested the hypothesis that silencing of ATF3 will attenuate Noxa up-regulation by

the combination treatment. To this purpose, we silenced ATF3 in U87 and LN229 GBM cells

and subsequently treated the cells with OTX015 and panobinostat. In agreement with our

hypothesis, we found that silencing of ATF3 suppressed Noxa increase in response to treatment

with the drug combination (Figure 5F).




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Combined inhibition of BRD and HDAC facilitates the stability of Mcl-1 in a GSK3

dependent manner

Aside from the pro-apoptotic changes induced by the combination treatment, we detected an

increase of Mcl-1 and its associated deubiquitinase Usp9X at early time points, which based on

our mechanistic analysis exerts pro-survival features since interference with both targets

enhances the apoptotic effects of the combination treatment. Therefore, we sought to establish

a deeper understanding by which mechanisms Mcl-1 is up-regulated. Given the increase of

Usp9X, it appears obvious that the combination treatment modulates the stability of Mcl-1 aside

from its effects on mRNA levels. To this purpose, we conducted a cycloheximide (CHX) block

experiment in the presence or absence of OTX015 and panobinostat combination in LN229 and

T98G cells. While control treated cells displayed a rapid decline in Mcl-1 protein levels, the

combination treatment enhanced the stability of Mcl-1 in both cell lines (Figure 5G and 5H),

indicating that OTX015 and panobinostat combination affects Mcl-1 half-life.

Concerning protein stability, it is well known that Mcl-1 possesses a short-half life and is prone

to rapid degradation by proteasomes. Proteasomal degradation is preceded by phosphorylation

of Mcl-1 through several kinases. Amongst those regulators, GSK3is known to phosphorylate

Mcl-1 at serine 159 (28), preceding ubiquitin conjugation and subsequent energy dependent

degradation by proteasomes. Given these implications, we hypothesized that the combination

treatment inhibits the activity of GSK3 (phosphorylated form at serine 9) and that in turn Mcl-1

becomes dephosphorylated at serine 159, rendering Mcl-1 more stable (29). To this purpose,

GBM14 and LN229 cells were treated with OTX015, panobinostat or the combination. Protein

expression analysis revealed that both cell lines demonstrate an increase in Mcl-1, which was

most pronounced in the combination treatment, which was accompanied by a reduction in Mcl-1




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phosphorylation at serine 159 (Figure 5I). In agreement with the reduction of Mcl-1

phosphorylation, the combination treatment led to an enhanced phosphorylation of GSK3 at

serine 9 (Figure 5I), rendering the kinase less active. These findings reinforce the notion that

Mcl-1 is regulated at the posttranslational level by the combination treatment.

To address the related up-stream mechanisms that govern the Mcl-1 increase mediated by the

combination treatment, we reasoned that it may be related to the effects of the combination

treatment on the transcription factor, c-Myc, since suppression of c-Myc has been linked to

inhibit PP2A (30), which binds to and de-phosphorylates GSK3. To assess whether this

previously described relationship of c-Myc and GSk3 exists in the setting of glioblastoma

models as well, we created two stable cell lines derived from the patient-derived xenograft cell

culture, GBM14, either infected with a lentiviral shRNA targeting c-Myc, or a lentiviral driving

ectopic over-expression of c-Myc. The activity of the corresponding constructs was verified by

protein capillary electrophoresis, confirming the silencing or over-expression of c-Myc in the

respective cell cultures (Figure 5J). In agreement with our earlier hypothesis, we found that

while silencing of c-Myc results in an enhancement of phosphorylation of GSK3, coupled with

an increase in total Mcl-1 and reduced phosphorylation of Mcl-1 at serine 159, over-expression

of c-Myc results in a reciprocal expression phenotype with reduced phosphorylation of GSK3

and lower protein levels of Mcl-1 (Figure 5J). Similar results were observed in LN229 GBM cells

in which stable knockdown of c-Myc results in increased levels of Mcl-1 and over expression

leads to down-regulation of Mcl-1. To exclude that c-Myc modulation affects Mcl-1 mRNA levels,

we conducted real-time PCR analysis in c-Myc over-expressing and silenced cells. In both

situation, we found that c-Myc modulation only marginally affected Mcl-1 mRNA, suggesting that

most likely c-Myc regulates Mcl-1 in a posttranslational manner (Figure 5K). These results




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confirm that c-Myc levels affect Mcl-1 protein levels likely through enhanced stability mediated

by reduced GSK3 activity.

The triple therapy of OTX015, panobinostat and sorafenib leads to tumor regression and

significant host survival extension

To determine the efficacy of the combination treatment of OTX015 and panobinostat, we utilized

several xenograft model systems, involving glioblastoma and malignant melanoma. To this

purpose, we implanted U87-EGFRvIII cells in the subcutis of immunocompromised mice. After

establishment of tumors, treatment groups were formed, consisting of vehicle, OTX015,

panobinostat, OTX015+panobinostat, sorafenib and the triple combination of

OTX015+panobinostat+sorafenib. Our findings show that the combination treatment of OTX015

and panobinostat reduced tumor growth much more potently than each compound alone (Figure

6A, 6B and Supplementary Figure 7A-B). We found similar results in a BRAF V600E mutated

melanoma xenograft model (A375 cells), albeit less efficient compared to the U87-EGFRvIII

model (Supplementary Figure 7E-F). Given the limited efficacy of the combination treatment in

vivo, we addressed whether or not sorafenib would further enhance the combination treatment.

As anticipated with our in vitro findings, the triple combination therapy had the strongest impact

on tumor growth, leading to a regression of tumors (Figure 6A, 6B and Supplementary Figure

7A-B). To determine as to whether or not single, combination and triple therapies display similar

efficacy in a patient-derived xenograft GBM model, we tested the GBM12 PDX model and found

that in agreement with the findings obtained from the U87-EGFRvIII model system, the triple

therapy was most effective and leads to a significant suppression of tumor growth in this model

system as well (Figure 6C-D and Supplementary Figure 7D). Next, we evaluated the impact of

the various treatment on the histopathological level. We found that the triple therapy results in a

significantly reduced cellularity and mitotic rate, while at the same time an increase in

apoptosis/necrosis was encountered in the U87-EGFRvIII and GBM12 PDX model system




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(Figure 6E-H, and Supplementary Figure 6A-C and 7A). Consistently, we found a reduced

amount of Ki67 staining in tumors that received the triple therapy, consistent with a reduced

mitotic rate (Figure 6F and 6H and Supplementary Figure 6B and 7A). To confirm cell death, we

conducted TUNEL-staining and found the highest amount of TUNEL positive cells in the triple

therapy treated tumors (Figure 6G-H and Supplementary Figure 6C and 7A). In addition, we

confirmed that the triple combination therapy suppresses c-Myc protein levels in vivo

(Supplementary Figure 7C).

Finally, we determine as to whether or not this treatment approach is active in orthotopic models

of glioblastoma, which bear a closer resemblance to the clinical situation and are more

challenging to treat. First, since the combination treatment regulated c-Myc and several other

transcription factors that orchestrate the stem-cell like phenotype in NCH644 GBM cells

(Supplementary Figure 4B and Figure 4B-C), we used NCH644 cells and implanted them in into

the right striatum of immunocompromised mice (Figure 6I). Treatment groups were formed and

animals were treated three times a week over the course of three weeks. While single

treatments (OTX or Pb) and the combination treatment (OTX+Pb) did not result in a significant

life extension for the host animals (compared to vehicle treated animals), the triple therapy

(OTX+Pb+Sf) resulted in a statistical significant survival benefit compared to the control and

combination treatment (Figure 6I). Next, we evaluated our drug treatments in the current gold-

standard for preclinical drug therapy, an orthotopic patient-derived xenograft of glioblastoma. To

this end, we utilized the GBM12 PDX model (9-12) and implanted cells into the right striatum of

nude mice (Figure 6J). Thereafter, animals were assigned to groups and treated according to

the same schedule of the NCH644 orthotopic model system. The triple therapy resulted in a

significant extension of host survival akin to the results obtained in the heterotopic xenograft

model (Figure 6J). These findings suggest that our treatment strategy is active in orthotopic




20

xenograft models, 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 involved mechanisms of the combination and

triple therapy (Figure 6K).

Discussion

Although multi-combination 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 clinical

burden of proof that even a triple therapy is more efficacious and safe than single treatments or

combination therapies, consisting of only two drugs. However, a lesson again might be learnt

from HIV therapy. While in the early 1980’s HIV constituted a death sentence, this changed

dramatically of the course of several decades when HIV was started to be treated with “drug

combination cocktails”. In our study, we have taken a similar approach by combining two drug

compound classes, involving HDAC inhibitors and BRD inhibitors, and in the course of

mechanism studies we identified a rational triple therapy, involving HDAC inhibitors

(panobinostat, vorinostat), BRD inhibitor (OTX015) and multikinase inhibitors (sorafenib).

Our findings reveal that combined inhibition of HDAC by panobinosat or vorinostat, and BRD

proteins by OTX015 results in a significant synergistic reduction of cell growth that is

predominantly mediated through enhanced cell death in the form of apoptosis, which was

supported by gene set enrichment analysis showing up-regulation of multiple apoptosis

promoting genes. Cell death was protected by the pan-caspase inhibitor, zVAD-fmk, which




21

demonstrating that caspases are involved in the death pathway elicited by the combination

treatment. These findings are in agreement with earlier studies, involving single treatments with

HDAC inhibitors or BRD inhibitors (25,33-35), that demonstrated activation of intrinsic

apoptosis. We hypothesized that the enhanced apoptotic cell death by the combination

treatment is driven by an alteration of pro- and anti-apoptotic factors and identified Noxa as one

of the key players to mediate apoptosis. Noxa is a pro-apoptotic Bcl-2 family member that is

known for its inhibitory activity on anti-apoptotic Mcl-1. Induced by several stress responses,

Noxa is increased in the context of various stimuli and known to be down-stream of the

integrated stress response when ATF4 is up regulated. In keeping with this, the drug

combination treatment resulted in an activation of a profound ER-stress response and increased

Noxa via at least two mechanisms, involving either the PERK-ATF4 pathway or ATF3

(13,27,36,37).

Based on our transcriptome analysis, we found evidence for energy deprivation induced by the

combination treatment, which was further supported by the fact that gene sets related to energy

metabolisms were synergistically down-regulated. We validated this observation by determining

total ATP levels and confirmed these findings by extracellular flux analysis, which supported the

notion that the combination treatment suppressed glycolysis and oxidative energy metabolism.

To the best of our knowledge, our findings are the first to show that simultaneous inhibition of

BRD and HDAC proteins results in an energy crisis in glioblastoma cells, which then in turn

elicits a profound stress response with activation of ER-stress.

To provide an explanation for the energy suppression mediated by the combination treatment,

we hypothesized that c-Myc is involved in this context since c-Myc is known to modulate energy

metabolism in cancer cells (24,38-40). Indeed, our findings unanimously showed that the

combination treatment reduced c-Myc mRNA and protein levels in a synergistic manner and




22

along with c-Myc suppression we found a decline in enzymes and transporters related to

glycolysis, which are known transcriptional targets of c-Myc. Indeed, selective stable knockdown

of c-Myc suppressed glycolysis in patient-derived xenograft cells. Although c-Myc seems to be a

major player in the regulation of our observed metabolic changes, we cannot exclude that

others factors regulate energy metabolism in the combination treatment.

Another major aspect of our findings relates to the observation that the combination treatment

not only increased the levels of pro-apoptotic molecules, but also anti-apoptotic factors, such as

Mcl-1 and its deubiquitinase Usp9X, which were detected in essentially every cell culture tested,

suggesting that this response appears to be robust and broadly relevant. Given this implication,

it was tempting to speculate as to whether or not the combination treatment can be further

enhanced through selective genetic or pharmacological inhibition. Our findings confirmed the

anti-apoptotic role of Mcl-1, and sorafenib (23) potently enhanced the effects of the combination

treatment, giving rise to a novel, rational triple therapy. Interestingly, the combination treatment

affected Mcl-1 levels both at the transcriptional as well as the posttranslational level. At the post-

translational level, the combination treatment increased Mcl-1 through a feed-forward

mechanism, involving the suppression of c-Myc with a subsequent inactivation of GSK3 and

stabilization of Mcl-1. Being an FDA-approved drug, sorafenib (23,41,42) appeared to be the

ideal compound to counteract the Mcl-1 increase mediated by the combination treatment

because sorafenib has been shown to suppress Mcl-1 levels through multiple mechanisms,

involving inhibition of GSK3, but also suppression of MCL1 transcripts (22,43). In keeping with

this, sorafenib suppressed Mcl-1 up-regulation by the combination treatment in our model

systems and in turn facilitated apoptosis induced by the combination treatment. Most relevantly,

our in vivo studies showed that adding sorafenib to the combination treatment resulted in a

potent enhancement of efficacy in the highly aggressive U87-EGFRvIII model as well as in a




23

patient-derived xenograft system. While initially we examined only heterotopic xenografts,

similar impressive results were obtained with the triple combination therapy in orthotopic

models, significantly extending overall survival. These findings intimate that the triple therapy is

feasible, well-tolerated and efficacious. In the broader picture, this research suggests that akin

to infectious disease the consideration of multi-combination therapies might be a viable

approach to enhance treatment efficacy of recalcitrant malignancies.

References

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

ATP Synthase and mTOR Signaling. Cell Metab 2015;22(3):508-15 doi

10.1016/j.cmet.2015.06.009.

2. Sasaki M, Knobbe CB, Itsumi M, Elia AJ, Harris IS, Chio, II, et al. D-2-hydroxyglutarate

produced by mutant IDH1 perturbs collagen maturation and basement membrane function.

Genes Dev 2012;26(18):2038-49 doi 10.1101/gad.198200.112.

3. Gross S, Cairns RA, Minden MD, Driggers EM, Bittinger MA, Jang HG, et al. Cancer-

associated metabolite 2-hydroxyglutarate accumulates in acute myelogenous leukemia with

isocitrate dehydrogenase 1 and 2 mutations. J Exp Med 2010;207(2):339-44 doi

10.1084/jem.20092506.

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(8):765-73 doi 10.1056/NEJMoa0808710.




24

5. Parsons DW, Jones S, Zhang X, Lin JC, Leary RJ, Angenendt P, et al. An integrated

genomic analysis of human glioblastoma multiforme. Science 2008;321(5897):1807-12 doi

10.1126/science.1164382.

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 Neuropathol 2008;116(6):597-602 doi

10.1007/s00401-008-0455-2.

7. Fang X, Zhou W, Wu Q, Huang Z, Shi Y, Yang K, et al. Deubiquitinase USP13 maintains

glioblastoma stem cells by antagonizing FBXL14-mediated Myc ubiquitination. J Exp Med

2017;214(1):245-67 doi 10.1084/jem.20151673.

8. Wang X, Cunningham M, Zhang X, Tokarz S, Laraway B, Troxell M, et al.

Phosphorylation regulates c-Myc's oncogenic activity in the mammary gland. Cancer research

2011;71(3):925-36 doi 10.1158/0008-5472.CAN-10-1032.

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. Molecular cancer therapeutics 2015;14(12):2735-

43 doi 10.1158/1535-7163.MCT-15-0553.

10. Gupta SK, Kizilbash SH, Carlson BL, Mladek AC, Boakye-Agyeman F, Bakken KK, et al.

Delineation of MGMT Hypermethylation as a Biomarker for Veliparib-Mediated Temozolomide-

Sensitizing Therapy of Glioblastoma. J Natl Cancer Inst 2015;108(5) doi 10.1093/jnci/djv369.

11. Allen C, Opyrchal M, Aderca I, Schroeder MA, Sarkaria JN, Domingo E, et al. Oncolytic

measles virus strains have significant antitumor activity against glioma stem cells. Gene Ther

2013;20(4):444-9 doi 10.1038/gt.2012.62.

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 of epidermal growth factor receptor

amplification on glioblastoma radiation response. Clinical cancer research : an official journal of




25

the American Association for Cancer Research 2006;12(7 Pt 1):2264-71 doi 10.1158/1078-

0432.CCR-05-2510.

13. Karpel-Massler G, Ishida CT, Bianchetti E, Shu C, Perez-Lorenzo R, Horst B, et al.

Inhibition of Mitochondrial Matrix Chaperones and Antiapoptotic Bcl-2 Family Proteins Empower

Antitumor Therapeutic Responses. Cancer research 2017;77(13):3513-26 doi 10.1158/0008-

5472.CAN-16-3424.

14. Karpel-Massler G, Ishida CT, Bianchetti E, Zhang Y, Shu C, Tsujiuchi T, et al. Induction

of synthetic lethality in IDH1-mutated gliomas through inhibition of Bcl-xL. Nat Commun

2017;8(1):1067 doi 10.1038/s41467-017-00984-9.

15. Karpel-Massler G, Ramani D, Shu C, Halatsch ME, Westhoff MA, Bruce JN, et al.

Metabolic reprogramming of glioblastoma cells by L-asparaginase sensitizes for apoptosis in

vitro and in vivo. Oncotarget 2016 doi 10.18632/oncotarget.9257.

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 Anticancer Activity against a Broad

Spectrum of Treatment-Resistant Cancers. Clinical cancer research : an official journal of the

American Association for Cancer Research 2016;22(18):4698-711 doi 10.1158/1078-

0432.CCR-15-2827.

17. Karpel-Massler G, Ba M, Shu C, Halatsch ME, Westhoff MA, Bruce JN, et al.

TIC10/ONC201 synergizes with Bcl-2/Bcl-xL inhibition in glioblastoma by suppression of Mcl-1

and its binding partners in vitro and in vivo. Oncotarget 2015;6(34):36456-71 doi

10.18632/oncotarget.5505.

18. Pareja F, Macleod D, Shu C, Crary JF, Canoll PD, Ross AH, et al. PI3K and Bcl-2

inhibition primes glioblastoma cells to apoptosis through downregulation of Mcl-1 and Phospho-

BAD. Molecular cancer research : MCR 2014;12(7):987-1001 doi 10.1158/1541-7786.MCR-13-

0650.




26

19. Karpel-Massler G, Ramani D, Shu C, Halatsch ME, Westhoff MA, Bruce JN, et al.

Metabolic reprogramming of glioblastoma cells by L-asparaginase sensitizes for apoptosis in

vitro and in vivo. Oncotarget 2016;7(23):33512-28 doi 10.18632/oncotarget.9257.

20. Elgendy M, Abdel-Aziz AK, Renne SL, Bornaghi V, Procopio G, Colecchia M, et al. Dual

modulation of MCL-1 and mTOR determines the response to sunitinib. J Clin Invest 2016 doi

10.1172/JCI84386.

21. Sheridan C, Brumatti G, Elgendy M, Brunet M, Martin SJ. An ERK-dependent pathway

to Noxa expression regulates apoptosis by platinum-based chemotherapeutic drugs. Oncogene

2010;29(49):6428-41 doi 10.1038/onc.2010.380.

22. Wang R, Xia L, Gabrilove J, Waxman S, Jing Y. Sorafenib Inhibition of Mcl-1

Accelerates ATRA-Induced Apoptosis in Differentiation-Responsive AML Cells. Clinical cancer

research : an official journal of the American Association for Cancer Research 2016;22(5):1211-

21 doi 10.1158/1078-0432.CCR-15-0663.

23. Kiprianova I, Remy J, Milosch N, Mohrenz IV, Seifert V, Aigner A, et al. Sorafenib

Sensitizes Glioma Cells to the BH3 Mimetic ABT-737 by Targeting MCL1 in a STAT3-

Dependent Manner. Neoplasia 2015;17(7):564-73 doi 10.1016/j.neo.2015.07.003.

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 metabolism and inhibits renal tumor

development. Nat Commun 2017;8(1):783 doi 10.1038/s41467-017-00902-z.

25. Nagaraja S, Vitanza NA, Woo PJ, Taylor KR, Liu F, Zhang L, et al. Transcriptional

Dependencies in Diffuse Intrinsic Pontine Glioma. Cancer cell 2017;31(5):635-52 e6 doi

10.1016/j.ccell.2017.03.011.

26. Dey S, Sayers CM, Verginadis, II, Lehman SL, Cheng Y, Cerniglia GJ, et al. ATF4-

dependent induction of heme oxygenase 1 prevents anoikis and promotes metastasis. J Clin

Invest 2015;125(7):2592-608 doi 10.1172/JCI78031.




27

27. Fels DR, Koumenis C. The PERK/eIF2alpha/ATF4 module of the UPR in hypoxia

resistance and tumor growth. Cancer biology & therapy 2006;5(7):723-8.

28. Pan R, Ruvolo V, Mu H, Leverson JD, Nichols G, Reed JC, et al. Synthetic Lethality of

Combined Bcl-2 Inhibition and p53 Activation in AML: Mechanisms and Superior Antileukemic

Efficacy. Cancer cell 2017;32(6):748-60 e6 doi 10.1016/j.ccell.2017.11.003.

29. Nifoussi SK, Ratcliffe NR, Ornstein DL, Kasof G, Strack S, Craig RW. Inhibition of

protein phosphatase 2A (PP2A) prevents Mcl-1 protein dephosphorylation at the Thr-163/Ser-

159 phosphodegron, dramatically reducing expression in Mcl-1-amplified lymphoma cells. The

Journal of biological chemistry 2014;289(32):21950-9 doi 10.1074/jbc.M114.587873.

30. Liu L, Eisenman RN. Regulation of c-Myc Protein Abundance by a Protein Phosphatase

2A-Glycogen Synthase Kinase 3beta-Negative Feedback Pathway. Genes Cancer

2012;3(1):23-36 doi 10.1177/1947601912448067.

31. Wei W, Shin YS, Xue M, Matsutani T, Masui K, Yang H, et al. Single-Cell

Phosphoproteomics Resolves Adaptive Signaling Dynamics and Informs Targeted Combination

Therapy in Glioblastoma. Cancer cell 2016;29(4):563-73 doi 10.1016/j.ccell.2016.03.012.

32. Prados MD, Byron SA, Tran NL, Phillips JJ, Molinaro AM, Ligon KL, et al. Toward

precision medicine in glioblastoma: the promise and the challenges. Neuro-oncology 2015 doi

10.1093/neuonc/nov031.

33. Lee DH, Ryu HW, Won HR, Kwon SH. Advances in epigenetic glioblastoma therapy.

Oncotarget 2017;8(11):18577-89 doi 10.18632/oncotarget.14612.

34. Chen S, Zhang Y, Zhou L, Leng Y, Lin H, Kmieciak M, et al. A Bim-targeting strategy

overcomes adaptive bortezomib resistance in myeloma through a novel link between autophagy

and apoptosis. Blood 2014;124(17):2687-97 doi 10.1182/blood-2014-03-564534.

35. Gammoh N, Lam D, Puente C, Ganley I, Marks PA, Jiang X. Role of autophagy in

histone deacetylase inhibitor-induced apoptotic and nonapoptotic cell death. Proceedings of the




28

National Academy of Sciences of the United States of America 2012;109(17):6561-5 doi

10.1073/pnas.1204429109.

36. Ishida CT, Shu C, Halatsch ME, Westhoff MA, Altieri DC, Karpel-Massler G, et al.

Mitochondrial matrix chaperone and c-myc inhibition causes enhanced lethality in glioblastoma.

Oncotarget 2017;8(23):37140-53 doi 10.18632/oncotarget.16202.

37. Ishida CT, Bianchetti E, Shu C, Halatsch ME, Westhoff MA, Karpel-Massler G, et al.

BH3-mimetics and BET-inhibitors elicit enhanced lethality in malignant glioma. Oncotarget

2017;8(18):29558-73 doi 10.18632/oncotarget.16365.

38. Satoh K, Yachida S, Sugimoto M, Oshima M, Nakagawa T, Akamoto S, et al. Global

metabolic reprogramming of colorectal cancer occurs at adenoma stage and is induced by

MYC. Proceedings of the National Academy of Sciences of the United States of America

2017;114(37):E7697-E706 doi 10.1073/pnas.1710366114.

39. Mertz JA, Conery AR, Bryant BM, Sandy P, Balasubramanian S, Mele DA, et al.

Targeting MYC dependence in cancer by inhibiting BET bromodomains. Proceedings of the

National Academy of Sciences of the United States of America 2011;108(40):16669-74 doi

10.1073/pnas.1108190108.

40. Wang J, Wang H, Li Z, Wu Q, Lathia JD, McLendon RE, et al. c-Myc is required for

maintenance of glioma cancer stem cells. PloS one 2008;3(11):e3769 doi

10.1371/journal.pone.0003769.

41. Allen JE, Prabhu VV, Talekar M, van den Heuvel 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-Inducing Anticancer Agent ONC201/TIC10. Cancer

research 2015;75(8):1668-74 doi 10.1158/0008-5472.CAN-14-2356.

42. Sheng Z, Li L, Zhu LJ, Smith TW, Demers A, Ross AH, et al. A genome-wide RNA

interference screen reveals an essential CREB3L2-ATF5-MCL1 survival pathway in malignant

glioma with therapeutic implications. Nature medicine 2010;16(6):671-7 doi 10.1038/nm.2158.




29

43. Tong J, Tan S, Zou F, Yu J, Zhang L. FBW7 mutations mediate resistance of colorectal

cancer to targeted therapies by blocking Mcl-1 degradation. Oncogene 2017;36(6):787-96 doi

10.1038/onc.2016.247.

Figures and figure legends:

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, were treated with OTX, Pb or the

combination over a broad range of concentrations for 72h. Thereafter, cells were analyzed for

cellular viability: OTX (blue), Pb (red) and the combination OTX+Pb (green). Shown are means

and SD. n=3 biological replicates. All concentrations are in M. #: Combination vs OTX, p<0.05;

+: 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 value of 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 data points is provided in the upper portion of each diagram.




30

Figure 2: Dual inhibition of HDAC (Pb) and BRD (OTX) causes synergistic activation of

apoptosis in glioblastoma cells

A, B, LN229 GBM cells were treated with indicated drugs for 24h, stained with Annexin V/PI and

analyzed by flow cytometry. n=3 biological replicates. Shown are means and SD. C, Microarray

was performed with NCH644 stem-like GBM cells treated with the combination of OTX+Pb and

vehicle DMSO control. Shown is GSEA enrichment plot with FDR q-values (false discovery

rate), NES (normalized enrichment score) and p-values. n=2 biological replicates for each

condition. D, E, LN229 GBM cells were treated with indicated drugs and stained with

tetramethylrhodamine, ethyl ester (TMRE) and analyzed by flow cytometry for the change in

mitochondrial membrane potential. n=3 biological replicates. Shown are means and SD. F,

Western blotting analysis of LN229 GBM cells treated with indicated drugs. TF: Total form, CF:

cleaved form. All concentrations are in M. G, H, LN229, U87, NCH644 or GBM14 cells were

treated with indicated drugs and analyzed for the levels of the indicated proteins by conventional

western blotting or capillary electrophoresis. All concentrations are in M. Blots or capillary

electrophoresis were quantified for the levels of Mcl-1 and Noxa normalized with its related

loading control. I, J, LN229 or NCH644 GBM cells were treated with indicated drugs and

analyzed by real-time PCR for the indicated makers. Shown are means and SD (n=3), and

statistical analysis was performed.

Figure 3: Molecular requirements of apoptosis induction by combined inhibition of HDAC

and BRD.

A, LN229 GBM cells were transfected with non-targeting (siNT), BAK, Noxa siRNA. Indicated

protein levels were shown. B, C, The same transfected LN229 GBM cells from A were subjected

to the combination treatment, OTX+Pb, stained with propidium iodide for flow cytometric

analysis. D, T98G GBM cells were treated with vehicle or the OTX+Pb combination for 16h.




31

Thereafter, protein lysates were immunoprecipitated with a species control IgG or Mcl-1 specific

antibody and detected by conventional western blotting. The left portion shows the

immunoprecipitated lysates, whereas on the right side 1% input lysates were loaded. All

concentrations are in M. E, LN229 GBM cells were transfected with indicated siRNA for 3

days, then treated with indicated drugs for 24h and analyzed for the expression of the indicated

proteins by either standard western blotting or capillary electrophoresis. Arrows indicate total

and cleavage forms of PARP. All concentrations are in M. F, LN229 cells treated as in E were

stained with propidium iodide and analyzed by flow cytometry for DNA – fragmentation. n=3

biological replicates. Shown are means and SD. G, H, LN229 or NCH644 GBM cells were

treated with indicated drugs for 24h and 48h and analyzed for the indicated proteins by capillary

electrophoresis. All concentrations are in M. Quantifications are provided for Mcl-1 normalized

with vinculin. J, LN229 GBM cells were treated with indicated drugs and stained with Annexin

V/PI and analyzed by flow cytometry. Displayed are representative flow plots and quantification

of the results. n=3 biological replicates. Shown are means and SD.

Figure 4: Metabolic reprogramming of energy metabolism elicited by combined inhibition

of HDAC and BRD.

A, Microarray was performed with NCH644 stem-like GBM cells treated with the combination of

OTX+Pb and DMSO control. Shown are GSEA enrichment plots with FDR q-values (false

discovery rate), NES (normalized enrichment score) and p-values. n=2 biological replicates for

each condition. B, GBM14, LN229 and NCH644 GBM cells were treated as indicated and

analyzed for the expression of the indicated proteins by capillary electrophoresis. * indicates 14-

3-3 protein. C, NCH644 stem-like GBM cells were treated with indicated drugs and analyzed by

real-time PCR for the levels of c-Myc. n=3 biological replicates. Shown are means and SD. D,

GSEA enrichment plot for MYC targets from the same cells in A. FDR q-values (false discovery

rate), NES (normalized enrichment score) and p-values. E, GBM14 GBM cells were treated as




32

indicated for 24h and ATP levels were determined. n=3 biological replicates. Shown are means

and SD. F, GBM14 GBM cells were treated with indicated drugs and subjected to extracellular

flux analysis for oxygen consumption rate (OCR) in the context of a mitochondrial stress assay.

O: Oligomycin, F: FCCP, A/R: Antimycin/rotenone. Shown are means and SD, n=3 biological

replicates. G, GBM14 cells 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. Shown

are means and SD, n=3 biological replicates. H, I, The glycolytic capacity and glycolytic reserve

were calculated based on the experiment, shown in G. Shown are means and SD. n=3

biological replicates. J-M, Functional OXPHOS related parameters were calculated based on

the experiment shown in F, ATP production, basal OCR, maximal respiration and spare

respiratory capacity. Shown are means and SD. n=3 biological replicates.

Figure 5: Dual inhibition of HDAC (Pb) and BRD (OTX) causes endoplasmic reticulum

stress and mediates enhanced stability of Mcl-1 in a GSK3 dependent manner.

A, B NCH644 stem-like GBM cells were treated with the combination of OTX and Pb and

subjected to microarray analysis. Shown are GSEA enrichment plots with with FDR q-values

(false discovery rate), NES (normalized enrichment score) and p-values. n=2 biological

replicates for each condition. C, NCH644 and LN229 GBM cells were treated with indicated

drugs, and Real-time PCR analysis was performed for the expression of the ER-stress related

transcripts. Shown are means and SD. n=3 biological replicates. D, U87 and LN229 cells were

treated with indicated drugs for 7h and analyzed for the protein expression of ER-stress related

markers (ATF3 and Vinculin were run on capillary electrophoresis). All concentrations are in

M. E, LN229 GBM cells were transfected with non-targeting or PERK specific siRNA for 3 days

following with the treatment of indicated drugs for 7h, and analyzed for expression of the

indicated makers by conventional western blotting. All concentrations are in M. F, U87 and




33

LN229 GBM cells were transfected with non-targeting or ATF3 specific siRNA for 3 days

following with the treatment of indicated drugs for 7h, and analyzed by capillary electrophoresis

for the indicated markers. All concentrations are in M. G, H, LN229 or T98G GBM cells were

pretreated with combined OTX and Pb or Ctrl, and then cycloheximide (CHX) was added for

indicated time (minutes). Lysates were analyzed for the expression of the indicated markers by

capillary electrophoresis. I, GBM14 and LN229 GBM cells were treated with indicated drugs and

analyzed for the expression of the indicated markers. J, GBM14 and LN229 GBM cells were

transduced with indicated lentivirus coding non-targeting 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 transduced as 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 derived-

glioblastoma in the presence of the FDA-approved multi-kinase inhibitor, sorafenib.

A, B, U87-EGFRvIII GBM cells were implanted in the subcutis of immunocompromised mice

(Nu/Nu). After establishment of tumors, groups were formed as indicated. Tumor volumes were

plotted. n=9-15 biological replicates. C, D, GBM12 patient-derived xenograft cells were

implanted in the subcutis of immunocompromised mice (Nu/Nu). After establishment of tumors,

groups were formed as indicated. Tumor volumes were plotted. n=6-12 biological replicates. E,

F, G Shown are H&E, Ki67, and Tunel stained representative sections from a vehicle or

OTX015+panobinostat+sorafenib treated GBM12 tumor (from D). H, Quantifications of mitosis

and apoptotic/necrotic cells from the GBM12 tumor sections collected at day 20 post tumor

transplantation. 5 fields of each section were counted. Shown are means and SD. I, Stem-like

GBM cells, NCH644, were implanted in the right striatum of nude mice. Thereafter, mice were

randomly assigned to indicated groups. Treatments were performed three times a week for 3




34

weeks. Kaplan-meier survival curves were plotted. Median survival days of each group were:

DMSO: 24.5, OTX: 26, Pb: 23, OTX+Pb: 25 and OTX+Pb+Sf: 34. J, Kaplan-meier survival

curves of mice xenograft with GBM12 patient derived GBM cells. Treatments were performed

three times a week for 5 weeks until animals became moribund. Median survival days of each

group were: DMSO: 23, OTX: 31, Pb: 31, OTX+Pb: 35 and OTX+Pb+Sf: 50. K, Graphical

summary of the proposed mechanisms of action by the OTX, Pb and Sf drug combinations.






















Published OnlineFirst May 15, 2018.Clin Cancer Res Yiru Zhang, Chiaki Tsuge Ishida, Wataru Ishida, et al. Lethality in GlioblastomaReprograms Tumor Cell Metabolism and elicits Synthetic Combined HDAC and Bromodomain Protein Inhibition

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