Context-selective death of acute myeloid leukemia cells ... · 2/22/2014 · 1 Context-selective...

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Context-selective death of acute myeloid leukemia cells triggered by the novel hybrid retinoid-

HDAC inhibitor MC2392

Floriana De Bellis1,2, Vincenzo Carafa1, Mariarosaria Conte1, Dante Rotili3, Francesca Petraglia1,

Filomena Matarese2, Kees-Jan Francoijs2, Julien Ablain5, Sergio Valente3, Remy Castellano4,

Armelle Goubard4, Yves Collette4, Amit Mandoli2, Joost H. A. Martens2, Hugues de Thé5, Angela

Nebbioso1*, Antonello Mai3*, Hendrik G. Stunnenberg2*, Lucia Altucci 1,6*.

1 Dipartimento di Biochimica, Biofisica e Patologia Generale, Seconda Università degli Studi di

Napoli, Vico L. De Crecchio 7, 80138, Napoli, IT; 2 NCMLS, Radboud University, Nijmegen, NL;

3 Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza University of Rome, P.le Aldo

Moro 5, 00185, Rome, IT; 4 Inserm, CRCM, U1068, TrGET & ISCB, University of Marseille, F-

13009 France; 5 Laboratoire U944 and UMR 7212, University Paris-Diderot, Paris, FR; 6 Istituto di

Genetica e Biofisica, IGB, Adriano Buzzati Traverso, Via P. Castellino 111, 80131, Naples, IT.

Running title: MC2392 induces PML-RARα context-selective cell-death.

Keywords: HDAC inhibitors, retinoic acid, PML-RARα-HDAC complexes, APL targeted therapy,

cell death1

Financial support: Blueprint (n° 282510) to LA, HS, MA; Italian Flag Project: EPIGEN, AIRC n° 11812, PRIN_2009PX2T2E_004, PON0101227 to LA Corresponding authors: Prof. Lucia Altucci, Dipartimento di Biochimica, Biofisica e Patologia Generale, Seconda Università degli Studi di Napoli, Vico L. De Crecchio 7, 80138, Napoli, IT, Tel: +390815667569 Fax: +39081450169 [email protected]; Prof. Hendrik G. Stunnenberg, Department of Molecular Biology, Faculty of Science, Nijmegen Center for Molecular Life Sciences, Radboud University, 6500 HB Nijmegen Tel: +31243510524�Fax: +31243610520 [email protected]; Prof A. Mai: Dipartimento di Chimica e Tecnologie del Farmaco Sapienza Università di Roma, 00185 Roma, IT; Tel: +390649913392 Fax: +390649693268�[email protected]; Dr Angela Nebbioso Dipartimento di Biochimica, Biofisica e Patologia Generale, Seconda Università degli Studi di Napoli, Vico L. De Crecchio 7, 80138, Napoli, IT Tel: +390815665673 Fax: +39081450169. Disclosure of Potential Conflicts of Interest: No potential conflicts of interest were disclosed. Word count: 4629 Number of figures: 6

Research. on September 15, 2020. © 2014 American Association for Cancercancerres.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 February 24, 2014; DOI: 10.1158/0008-5472.CAN-13-2568

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ABSTRACT

HDAC inhibitors (HDACi) are widely used in the clinic to sensitize tumorigenic cells for

treatment with other anticancer compounds. The major drawback of HDACi is the broad

inhibition of the plethora of HDAC-containing complexes. In acute promyelocytic leukemia

(APL), repression by the PML-RARαoncofusion protein is mediated by an HDAC-containing

complex that can be dissociated by pharmacological doses of all trans retinoic acid (ATRA)

inducing differentiation and cell death at the expense of side effects and recurrence. We

hypothesized that the context-specific close physical proximity of a retinoid and HDACi

binding protein in the repressive PML-RARα-HDAC complex may permit selective targeting

by a hybrid molecule of ATRA with a 2-aminoanilide tail of the HDAC inhibitor MS-275,

yielding MC2392. We show that MC2392 elicits weak ATRA and essentially no HDACi

activity in vitro or in vivo. Genome-wide epigenetic analyses revealed that in NB4 cells

expressing PML-RARα, MC2392 induces changes in H3 acetylation at a small subset of

PML-RARα binding sites. RNA-seq reveals that MC2392 alters expression of a number of

stress-responsive and apoptotic genes. Concordantly, MC2392 induced rapid and massive,

caspase 8-dependent cell death accompanied by RIP1 induction and ROS production. Solid

and leukemic tumors are not affected by MC2392, but expression of PML-RARα conveys

efficient MC2392-induced cell death. Our data suggest a model in which MC2392 binds to

the RARα moiety and selectively inhibits the HDACs resident in the repressive complex

responsible for the transcriptional impairment in APLs. Our findings provide proof-of-

principle of the concept of a context-dependent targeted therapy.




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Introduction

Acute promyelocytic leukemia (APL) is characterized by the presence of blasts blocked at the

promyelocytic stage of myeloid differentiation and by the fusion protein of RARα with the

promyelocytic leukemia gene (PML) in more than 95% of the cases (1, 2). Despite that the majority

of APLs carry this t(15;17) translocation, rare alternative RARα-containing fusion proteins have

been described (3-5). In the treatment of APL, the efficacy of pharmacologic doses of ATRA is due

to its ability to release the HDAC-containing repressive complexes bound to PML-RARα (6) and to

recruit the multi-subunit HAT complex on RARE. Besides having this desired therapeutic effect on

APL blasts, ATRA binds to and activates the nuclear receptors (RARα, β or γ) that bind to specific

DNA responsive elements (RARE) (7, 8) thereby affect a variety of biological processes in essential

all cell types such as cell proliferation, differentiation and apoptosis (9, 10).

The PML-RARα mediated epigenetic block of gene transcription (11, 12) can also be overcome by

HDAC inhibitors (HDACi) allowing cells to re-start differentiation or pro-apoptotic events (13).

Activation of differentiation programs, inhibition of the cell cycle and eventually induction of

apoptosis are among the key antitumor activities of HDACi in cancer therapy (7, 11, 14-16).

HDACi cause enzymatic inhibition and/or the release of HDACs from repressive complexes

permitting HAT recruitment, histone acetylation, chromatin decondensation and, ultimately,

transcription activation of tumor-suppressor genes (TSG) or other genes crucial for the normal

functioning of the cells (7). The major drawback of HDACi in treatment is the broad inhibition of

the plethora of HDAC-containing complexes.

MS-275/Entinostat is one of the most potent and widely used HDACi with micromolar affinity for

class I HDACs and with selectivity for HDAC1, HDAC3 and HDAC8 (17-19). MS-275 promotes

differentiation, apoptosis and inhibits the proliferation of multiple cancer cell lines (20). The effects

of MS-275 have been examined in human leukemia and lymphoma cells (U937, HL-60, K562, and

Jurkat). When administered at a low concentration (e.g., 1 μM), MS-275 exhibited potent




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antiproliferative activity inducing p21CIP1/WAF1-mediated growth arrest and expression of

differentiation markers (CD11b) in U937 cells (14, 21). However, at higher concentrations (e.g., 5

μM), MS-275 potently induced cell death and a very early increase in death receptor signaling (22)

as well as in reactive oxygen species (ROS), followed by the loss of mitochondrial membrane

potential and cytosolic release of cytochrome c (23).

The use of HDACi in combination with other anticancer agents (epi-drugs such as 5-aza-2′-

deoxycytidine and retinoic acid) is one way forward to a promising application against cancer. In

patients with leukemia, and particularly in the elderly, clinical studies combining ATRA treatment

with HDACi are in progress (24). For example, Valproic acid (VPA) has for many years been and

still is used as an anti-epileptic drug and inhibits preferably class I HDACs although in the high

micromolar to millimolar range. Moreover, even though there were a lot of concerns regarding toxic

side effects of HDACi in the clinical setting due to the roles of HDACs in multiples pathways, until

now clinical trials mostly showed manageable side effects (25). The combination of ATRA with an

HDACi could result in improved anti-tumorigenic activity (24).

Here, we exploited the synergy between HDACi and ATRA by generating and testing a single

hybrid molecule, named MC2392 on APL cells. Genome-wide epigenetic analysis revealed that

MC2392 is a weak retinoid. In contrast to the genome wide effects of MS-275 on histone H3

acetylation, MC2392 induces acetylation at only a small subset of PML-RARα binding sites in NB4

cells line. RNA-seq analysis showed that MC2392 alters the expression of a number of stress-

responsive and apoptotic genes differently from ATRA. Importantly, the hybrid compound acts in a

context and PML-RARα fusion protein-dependent fashion to induce rapid and massive cell death,

RIP1 induction and ROS production. We propose a model in which this hybrid compound binds to

the RARα via its retinoid-moiety and selectively inhibits the HDACs contained in the repressive

complex via the MS-275 part. Taken together, MC2392 is a promising candidate for apoptosis-

based therapy of APL, representing a new and effective, single hybrid drug able to modulate




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multiple cell death pathways. Our study convincingly shows that the targeting of multiple signaling

pathways by a single hybrid drug is a feasible and attractive paradigm for new cancer therapies.

Material and Methods

Cells. NB4, LnCap and U937 were purchased by DSMZ and ATCC respectively. Cell lines have

been tested and authenticated following manufacture’s instruction.

NB4, NB4-R4 (26, 27), U937, U937 PML/RAR, U937-PLZF/RAR Zn inducible (28, 29) and

LnCap cell lines were grown at 37°C in air and 5% CO2 in RPMI 1640 medium (EUROCLONE),

supplemented with 10% heat-inactivated fetal bovine serum (FBS, Sigma), 1% L-glutamine, 1%

ampicillin/streptomycin and 0.1% gentamicin (SIGMA). Cells were kept at the constant

concentration of 200000 cells per milliliter of culture medium. HeLa cell line, was grown at 37°C in

air and 5% CO2 in Dulbecco’s Modified Eagle Medium (GIBCO) with 10% FBS, 1% L-glutamine,

1% ampicillin/streptomycin and 0.1% gentamicin.

Chemicals. ATRA and MS-275 were dissolved in ethanol; MC2392, MC2677 and MC2678 were

dissolved in DMSO and used at 1 µM and 5 µM respectively. SAHA (Merck), resveratrol, and EX-

527 (Alexis) were dissolved in DMSO and used at 5, 100, and 5 µM, respectively.

Synthesis of MC2392, MC2677, MC2678. See Supplementary Materials and Methods.

Total Protein, Histone extraction and western blot. See Supplementary Materials and Methods.

Colony assay. Colony assay was carried out as in (22).

Cell cycle and differentiation analyses. See Supplementary Materials and Methods.

Caspase-3, -8 and -9 assays. Caspase activity was detected within living cells using B-BRIDGE

Kits supplied with cell-permeable fluorescent substrates. The fluorescent substrates for caspase-3, -

8 and -9 were FAM-DEVD-FMK, FAM-LETD-FMK, FAM-LEHD-FMK, respectively. Cells were

washed twice in cold PBS and incubated for 1h in ice with the corresponding substrates, as

recommended by suppliers. Cells were analyzed using Cell Quest software applied to a

FACScalibur (BD). Experiments were performed in duplicate and values expressed in mean ± SD.




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Caspase 2 assay. Caspase-2 activity was detected within whole living cells, using Caspase-2

Fluorometric Kit following supplier’s instructions (R&D). The plate was incubated at 37°C for 1h

and Fluorescence quantified with a TECAN M200 station at a 400-505 nm wavelength. Results are

expressed as fold increase in caspase activity.

Analysis of mitochondrial membrane potential. Detection of the changes in mitochondrial

membrane potential (ΔΨm) was performed using the MITOPTTM JC-1 kit (Immunochemistry

Technologies) following the supplier’s suggestions and analyzed by flow cytometry with Cell Quest

Pro software.

ROS detection. NB4 cells were resuspended in pre-warmed PBS 1X, containing 5 μM of DCF-DA

(diclorodihydrofluorescein diacetate) probe and incubated at 37°C for 20 minutes. Cells were

analyzed after washing, using the Cell Quest software applied to a FACScalibur (BD). H2O2 was

used as positive control.

RNA extraction, reverse transcription (RT) and quantitative PCR in real time (RT-PCR). See

Supplementary Materials and Methods.

Immunoprecipitation. See Supplementary Materials and Methods.

HDAC assay. HDAC assays have been performed as in (30). Specifically, samples

immunoprecipitated with anti-PML-RAR or with IgG were pooled respectively to homogenize all

samples. The Fluor de Lys Substrate was incubated (1h) with the immunoprecipitated PML-RAR

(in presence or absence of SAHA or MC2392 5 μM) and fluorescence was quantified with a

TECAN M200 station.

SIRT assay. See Supplementary Materials and Methods.

Transfections and luciferase assay. See Supplementary Materials and Methods.

IkB alpha ELISA assay. Assay was performed after 48h from the treatment with ATRA and

MC2392 in NB4 cells following supplier’s instructions (Active Motif).

ChIP and ChIP-seq. NB4 cells were treated for 4, 6, 24 and 48h with 1 μM ATRA, 5 μM MS-275

and 5 μM MC2392. Chromatin was harvested as described previously (31). ChIP experiments were




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performed using the H3K9K14ac (Diagenode), Nrf2, c-Fos and c-Jun (Santa Cruz) antibodies.

ChIPed DNA was analyzed by real time qPCR with specific primers (Biolegio, see Supplementary

Methods) using the 2x SYBR Green mix (BIORAD) in a MyiQ thermocycler (BIORAD). Primers

amplifying myoglobin were used as negative control. In addition, ChIPed DNA was prepared for

sequencing and sequenced according to the manufacturer’s instructions (Illumina) in Supplementary

Materials and Methods and essentially as already described (32, 33).

Peak Detection, Clustering analysis and Motif Search are detailed in the Supplementary

Materials and Methods.

RNA-sequencing. See Supplementary Materials and Methods.

Results

MC2392 inhibits HDAC activity of the PML-RARα complex and maintains retinoid activity.

We set out to exploit the possibility to combine the active parts of the HDACi MS-275 and the

retinoid, ATRA, yielding MC2392 (Fig. 1A). To explore the activities of this hybrid compound,

U937 cells were treated for 24h with 5 μM MC2392, its parent compound MS-275 (Entinostat) a

class I inhibitor and the class I/II HDAC inhibitor SAHA (Vorinostat). In contrast to the reference

compounds, MC2392 did not result in a net increase of histone acetylation (Fig. 1B). Moreover

MC2392 did not induce p21 protein expression (Fig. 1B) corroborating that at this concentration the

molecule has reduced or no HDACi activity as compared to MS-275. MC2392 exerted only

minimal (if any) inhibitory action on HDAC1 and HDAC4, nor did it display SIRT1 modulating

activities in vitro (Fig. 1C). Our premise for the design of MC2392 was that the close physical

proximity of a retinoid (PML-RARα) and an HDACi binding moiety (HDAC1 and/or -4) could

convey context-specific inhibitory activity by a hybrid molecule spanning the physical distance. To

investigate this assertion, PML-RARα was immunoprecipitated from HeLa cells after transient




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transfection with PML-RARα and assayed for HDACi activity. Indeed, MC2392 inhibits the

HDAC(s) resident in the PML-RARα repressive complex similarly to SAHA (Fig. 1D).

To investigate whether MC2392 acts as a retinoid, its potential to activate the ATRA-responsive

luciferase reporter (RARE3tkluc) was first tested in transient transfected HeLa that expresses

endogenous RAR and RXR. MC2392 activates RARE3tkluc weakly compared to ATRA (Fig. 1E).

Consistent with these observations, quantitative real-time PCR in NB4 cells showed a similar low

response to MC2392 for the RNA expression of specific genes known to be retinoid modulated (34,

35) such as HOXA1, IRF1 and, to a lesser extent, TNFα, (Supplementary Fig. 1A) as compared to

ATRA suggesting that MC2392 still maintains at least part of its retinoid activity. Finally, western

blot analyses of PML-RARα following MC2392 treatment showed rapid degradation of PML-

RARα in NB4 cells (Fig. 1F) as has been reported for ATRA (36, 37). Taken together the results

show that MC2392 acts as a weak retinoid when compared to ATRA implying that it binds to

RARα, activates RARα signaling as well as induces the degradation of PML-RARα (Fig. 1F). We

tentatively conclude that MC2392 is an HDACi in the context of the PML-RARα-HDAC repressive

complex.

MC2392 induces local H3 acetylation.

We showed that the HDACi activity of MC2392 is indeed apparent in the context of the PML-

RARα-contained HDAC complexes implying that acetylation maybe altered at PML-RARα

genomic binding sites. To examine whether MC2392 induces local epigenetic alterations, ChIP-seq

profiling was performed using the H3K9K14ac antibody in NB4 cells treated with ATRA, MS-275,

MC2392 or solvent, DMSO. In DMSO treated cells, H3K9K14 acetylation appeared in the typical

sharp narrow peaks. Treatment with the HDACi MS-275 had a dramatic effect on the genomic

acetylation landscape: H3K9K14ac peaks were dampened and spreading of acetylation occurred

(Fig. 2A). In line with the absence of global HDACi activity (Fig. 1B), MC2392 did not affect the

overall distribution of H3K9K14ac. Genomic annotation of the peaks called by MACS (38) with a




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P-value of 10-6 (32) revealed a small percentage of H3K9K14ac binding sites at promoter regions

(8.74%) while the major portion of the peaks were located in the gene body (52.11%) or intergenic

(34.19%) (Fig. 2B). Importantly, MC2392 did induce low but reproducible local changes in

acetylation at some PML-RARα binding sites such as at RARβ promoter or TGM2, ICAM1 gene

(Fig. 2C). The RARβ is avidly transcriptionally induced by ATRA and expectedly H3 acetylation is

induced at promoter region. These data suggest specificity and selectivity of MC2392 towards at

least some PML-RARα binding sites and reinforced the similar, although weaker, retinoid activity

of MC2392 as compared to ATRA (Fig. 1E, Supplementary Fig. 1A).

To substantiate our findings we examined the 1000 most significantly changed acetylation peaks

after ATRA induction in PML-RARα/RXR binding regions. The intensity plots covering the region

of 10 kb up- and downstream for cells treated with ATRA and MC2392 show acetylation increases

following MC2392 treatment although to a lower level and after longer treatment times as

compared to ATRA (Fig. 2D): a vivid increase is already apparent after 4h of ATRA treatment

whereas a similar response amplitude is reach only after 48h of MC2392 treatment.

The normalized tag numbers (in treated and untreated samples) were counted for all binding regions

and clustered using k-means. This analysis identified 20 clusters (Supplementary Fig. 1B), amongst

which in particular cluster 2 and cluster 9 showed H3K9K14ac peaks that responded similarly to

ATRA for 4h and 24h, and to MC2392 for 24h and 48h though with a strong timely delay (Fig. 3A).

Moreover, Gene Ontology (GO) analysis revealed that the regulated genes in cluster 2 are

functionally linked to leukocyte differentiation (Supplementary Fig. 1C). The overlap between

H3K9K14ac peaks in cluster 2 and PML-RARα binding sites (33) revealed a common set of 191

loci suggesting that indeed a subset of H3K9K14ac peaks are associated with PML-RARα/RXR.

The overlap between cluster 9 H3K9K14ac peaks and PML-RARα binding sites revealed a

common set of 183 regions, suggesting that also here a subset of induced H3K9K14ac peaks are

associated with PML-RARα/RXR (Fig. 3B, top).




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Recent studies analyzing the genome-wide PML-RARα binding sites not only identified DR2 and

DR5 elements as the primary PML-RARα response elements but also regions containing DR1, DR3

and DR4 motifs (33, 39). We found that both the DR1 and DR2 motifs are enriched within the

cluster 2 and 9 binding sites (Fig. 3B, middle) whereas DR3, DR4 and DR5 motifs are not enriched.

In addition, a de novo motif search identified the DR1 element in the 191 H3K9K14ac peaks of the

cluster 2 that are regulated by MC2392 (Fig. 3B, bottom). This suggest that a proper configuration,

i.e. spatial position of the PML-RARα with respect to the HDAC(s) in the repressive complex is an

important prerequisite for MC2392 to physically bridge the RAR and HDAC(s) moieties and

consequently cause inhibition of HDAC activity.

MC2392, but not ATRA, induces H3K9K14 acetylation at pro-apoptotic genes.

Our analysis also revealed cluster 8 consisting of 774 regulated genes that showed an opposite

epigenetic response to MC2392 as compared to ATRA treatment (Fig. 3C) that is induction by

MC2392 and reduction by ATRA. The assignment of genes in this cluster to biological functions

(Fig. 3C, bottom) revealed enrichment for regulation of cell death. De novo motif search showed

enrichment of the NF-E2-related factor-2 (NFE2L2, Nrf2) and Fos (AP1) response element each

present at about 10% of H3K9K14ac peaks (Fig. 3D). Note that the stress-responsive transcription

factors Nrf2 is reported to play a role in protection against oxidative stress-induced cellular damage.

ChIP experiments coupled with qPCR showed enrichment of Nrf2 over specific responsive element

(ARE) of target genes after MC2392 treatment, confirming an early Nrf2-mediated transcriptional

response during oxidant-induced cell death, differently to ATRA which reduces the binding of Nrf2

to the ARE enhancer region (Supplementary Fig. 1D). Finally, we found an increase in transcription in the target genes that encode antioxidant proteins or

enzymes to buffer the intracellular redox activities, such as FTH1, GCLM, GCLC, NQO and

HMOX, STK17A. Conversely, ATRA inhibits/reduces the expression of these Nrf2 target genes

(Supplementary Fig. 2A).




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MC2392 and ATRA induce expression of apoptotic genes at transcriptional level.

To analyze the expression changes induced by MC2392, we performed transcriptome profiling on

MC2392 and ATRA-treated NB4 cells. The gene expression values from RNA-seq data were

calculated by quantifying the number of sequence tags for each genes using Genomatix (40, 41).

Scatter plots show the general differences of gene expression of MC2392 versus ATRA after 24 and

48h (Supplementary Fig. 2B). Moreover, functional annotation clustering of differentially expressed

genes by Gene Ontology (GO; PANTHER) analysis revealed that genes up-regulated in response to

MC2392 are enriched for terms associated with protein transport, response to stress and apoptosis

induction. Moreover ATRA up-regulated genes are enriched for terms associated mostly to protein

transport and metabolic processes (Supplementary Fig. 2C). The hierarchical clustering analysis

supports these data (Supplementary Fig. 2D).

Moreover, MC2392 down-regulated genes are enriched for terms associated with metabolic

processes and pathway analysis showed the decreased expression of components that drive cell

growth, proliferation and cell communication such as JAK/STAT and integrin pathway (data not

shown).

Analysis of the transcription level of the 148 genes associated with apoptosis

(http://www.reactome.org) revealed main differences between ATRA and MC2392 (Supplementary

Fig. 2E and Supplementary Materials) such as the fast induction of TRAF2, E2F1, DYNLL2 and

DBNL following MC2392 but not ATRA treatment. Next, we selected several MC2392-modulated genes related to apoptosis and oxidative stress, which are also modulated in the H3K914ac levels (Fig. 3 and Supplementary Fig. 1) upon MC2392 but not ATRA treatment and examined transcription factor binding. This revealed an increased occupancy of c-Jun, c-Fos and NRF2 in a time- and MC2392-dependent manner (Supplementary Fig. 3), corroborating and extending our observations that the apoptotic action of the MC2392 is causally bound to the modulation of cell death and redox pathways in response to modulation of AP1 and NRF2 bindings and Research.

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their downstream pathways. Taken together the transcriptome and histone H3 acetylome analysis

strongly point to a differential effect of MC2392 as opposed to ATRA on apoptotic genes.

MC2392 induces cell death in APL cells.

Genome-wide epigenetic and transcriptome analysis have provided strong indications that MC2392

effects are distinct from those elicited by ATRA in particular on apoptotic genes. Therefore, we

assessed the activity of MC2392 on NB4 proliferation, cell cycle progression, granulocytic

differentiation and apoptosis induction in comparison to MS-275, SAHA, and ATRA (Fig. 4).

Interestingly, analysis of NB4 clonogenicity in semisolid media upon MC2392 treatment (Fig. 4A,

left) revealed a clear loss of clonogenic activity at 10-5 M. In addition, two MC2392 analogues that

differ for the position of the amino group at the anilide ring (position 2, 3 and 4 in MC2392,

MC2678, and MC2677, respectively) (42) were used. The MC2678 and -2677 compounds do not

display HDAC inhibition (data not shown) and MC2677 has been previously described to inhibit

the growth of a number of tumor cell lines including prostate, head and neck, squamous carcinoma

and neuroblastoma (42, 43). In contrast to MC2392 and MS-275, MC2678 and MC2677 are unable

to induce a proliferation arrest of NB4 cells (Fig. 4A, right). MC2392 increased the pre-G1 fraction

(Fig. 4B), a measure of cell death induction but did not induce major changes in the cell cycle

phases (Fig. 4C). MC2392 did also induce an increase of the CD11c levels to 24%, thus inducing

maturation of APL cells similar to ATRA, MC2677 and MC2678 (Fig. 4D) but the MC2678 and

MC2677 compounds did not induce apoptosis (Fig. 4B).

Induction of cell death by MC2392 is not dependent on differentiation as MC2392 readily induces

cell death but not differentiation of the ATRA-resistant NB4 subclone, NB4R4 (26, 27) (Fig. 4E-F).

Note that as a result of the acquired mutation in the ligand-binding domain, PML-RARα can bind

ATRA but it is thought that the binding does not induce the conformational change needed to

dissociate the repressive complex and recruit activating proteins (26). Thus, the cell death activity

of MC2392 acts independently from maturation and is likely linked to the HDAC inhibition of the




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resident repressive complexes. Taken together, our analysis shows that MC2392 indeed acts as a

weak retinoid in line with the transcriptome and histone H3 acetylome data but clearly has acquired

additional properties or such that were ‘masked’ in ATRA and MS-275. Moreover, our results

corroborate and extend the rapid response to the apoptosis/cell death versus slow differentiation in

response to MC2392 treatment.

MC2392-induced cell death involves ROS and caspase-8.

To gain insight into the mechanism(s) by which MC2392 initiates cell death, caspases-8, -9 and -3/7

activities in NB4 cells were analyzed. Similarly to SAHA, MC2392 notably induced caspase

activation with caspase-8, -9 (initiator caspases), caspase-3/7 (effector caspases) (Fig. 5A-B) and

mitochondrial potential activated in NB4 cells (Fig. 5C), thus indicating that the cell death induced

by MC2392 is caspase dependent. MS-275 induced cell death is caspase-8 dependent and related to

TRAIL induction (22). In order to identify the molecular pathway we evaluated the levels of known

players of apoptosis, such as FAS, TRAIL, FADD, caspase-8 and -3/7, Bcl2 and Bid in NB4 cells

after 24h and 48h treatment. As shown in Fig. 5D and Supplementary Fig. 3A, MC2392 induced the

extrinsic apoptotic pathway, increased the expression of FasL, Fas and TRAIL similarly to what has

been reported for MS-275 (22). MC2392 also induced degradation of Bid into the apoptotic factor t-

Bid, mitochondrial depolarization (Fig. 5D) and Bcl2 down-regulation (Supplementary Fig. 4B).

Next, NB4 cells were treated with MC2392 and H2O2 with and without the antioxidant N-

acetylcysteine (NAC). The data suggest that MC2392 mediates a ROS-dependent cell death

associated with the generation of reactive oxygen species (Fig. 5E and 5F). We further observed

increased H2AX phosphorylation which is a hallmark of ongoing DNA damage as a result of

increased reactive oxygen species (ROS) (44, 45). The response of APL cells to MC2392 includes

the generation of ROS as a consequence of Bid cleavage, which triggers mitochondrial membrane

permeabilization. As both caspase-2 and caspase-8 are capable of cleaving the pro-apoptotic Bid we

set out to investigate whether caspase-2 or caspase-8 are responsible. MC2392 induced caspase-2




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activation, which is fully blocked by the caspase-2 inhibitor Z-VDVAD-FMK (Fig. 6A). However,

cell death mediated by MC2392 stayed unaltered (Fig. 6A, right). MC2392-mediated cell death is

also not NOX-dependent, given that Apocynin - a NOX specific inhibitor - is not able to block

apoptosis (Supplementary Fig. 3C). Furthermore, NF-kB is neither induced nor transcriptionally

activated by MC2392 (Supplementary Fig. 4D-4E). In contrast, when the pan-caspase inhibitor

ZVAD (data not shown) or the caspase-8 inhibitor IETD are used together with MC2392, NB4 cell

death is fully abolished (Fig. 6B), strongly suggesting that the MC2392 mechanism of cell death is

caspase 8-dependent. Surprisingly, necrostatin-1 (NEC-1), the known RIP1 kinase and necroptosis

inhibitor, completely abrogated MC2392 cell death (Fig. 6C), thus suggesting a RIP1 mediated cell

death. Consistent with these observations, analysis for RIPK1 expression showed both a strong up-

regulation upon MC2392 and a full abrogation with NEC-1 (Fig. 6D). Finally, in line with our

hypothesis, MC2392 induces cell death specifically in a PML-RARα but not in RARα or PML

driven way (Fig. 4F and data not shown).

Taken together our data revealed that at the level of acetylation, MC2392 induces much fewer

changes as compared to ATRA. Moreover, MS-275 resulted in genome wide dampening and

broadening of all acetylation sites (Fig. 2A) whereas MC2392 affected acetylation only at a subset

of PML-RARα sites (DR1-2 type). Finally, transcriptome analysis corroborated and extended that

MC2392 affects apoptotic genes.

Discussion

Epigenetic modifications can be reversed by epi-drugs that are promising for antitumor therapy. In

patients with leukemia, HDACi are widely used in combination with ATRA resulting in improved

anti-tumorigenic activity. Here we identified and characterized the mechanism of action of

MC2392, a compound merging the HDACi part of the well-known MS-275 to ATRA. When

compared to both ATRA and MS-275, MC2392 displays similar as well as divergent

characteristics. Despite being inactive as an HDACi standard in vitro biochemical assays, MC2392




15

specifically inhibits the HDACs contained in the PML-RARα repressive complex (Fig. 1D).

MC2392 induces degradation of PML-RARα (Fig. 1E) and differentiation of NB4 cells (Fig. 4D).

Genome-wide epigenetic analysis shows that MC2392 induces increased acetylation at a small

subset of PML-RARα sites with preponderance for DR1 and -2 type (Fig. 3B) distinct from

acetylation at PML-RARα sites induced by ATRA. This MC2392 characteristic is particularly

interesting and classifies it as a novel class of drugs that need precise protein-protein ‘positioning’

to function. Moreover a specific configuration, i.e. spatial position of the PML-RARα with respect

to the HDAC(s) in the repressive complex is likely an important prerequisite for MC2392 to bridge

the RARα and HDAC(s) moieties and consequently inhibition of HDAC activity.

The fact that MC2392 may represent the first RARα-driven HDACi is corroborated by i) its

inactivity on human recombinant HDACs in vitro, ii) its inability to modify general histone and/or

tubulin acetylation; iii) its selective HDACi action on PML-RARα complexes; iv) its enhanced

ability to induce apoptosis in APL cells but not in other leukemia or solid cancer models. The fact

that MC2392 induces apoptosis in NB4 cells and increases caspases-8, -2 and -9 activity suggests

that MC2392 activate both mitochondrial and death receptor apoptotic pathways. Although

MC2392 is able to activate the extrinsic pathway by inducing FAS and TRAIL, the death response

to MC2392 includes also the generation of ROS as a possible consequence of the cleavage of Bid

(Fig. 5D, F). Indeed, the cleavage of Bid in APL cells occurs concurrently with early generation of

ROS, thereby contributing to cell death by promoting mitochondrial release of cytochrome c. For

this reason, normally the death receptor pathway (extrinsic pathway) converges with the

mitochondrial pathway (intrinsic pathway) through caspase-mediated cleavage and activation of

Bid. Furthermore, both the induction of RIP1 by MC2392 and the abrogation of cell death operated

by NEC-1, an inhibitor of RIP1, dictate a complex scenario in which, alternatively to apoptosis,

necroptotic pathways may lead to mitochondrial depolarization and cell death of NB4 APL cells. Of

note, both ATRA and MS-275 have been previously shown to function via TRAIL and FAS

induction (16, 22). Moreover, the fact that the MC2392 caspase 8-dependent apoptosis can be




16

abrogated by NEC-1 supports a model in which mixed types of cell death can occur, displaying

shared features of both necroptosis and apoptosis. Clearly, MC2392-induced apoptosis occurs in

response to oxidative stress and is orchestrated by stress-responsive transcription factors. Nuclear

translocation of NRf2 and subsequent induction of its target genes via antioxidant stress response

element (ARE) may function to buffer oxidative stress response. Moreover, MC2392 induces the

modulation of a large number of stress-responsive genes (Supplementary Fig. 2A) and apoptotic

genes (see Supplementary Materials and Methods). Finally, the evidence that apoptosis occurs in

APL independently from an ‘classical’ ATRA response is suggested both by the MC2392 action in

Zn-inducible PLZF-RARα expression in U937 cells (28, 29) as well as by its activity on NB4R4

cells (26, 27). Note that in both PLZF-RARα U937 cells and in NB4R4 cells ATRA signaling stays

impaired upon treatment (27, 46, 47). Excitingly, given that MC2392 is not an HDACi in vitro,

MC2392 is able to exert its HDAC inhibition only when driven by its ATRA moiety to PML-RARα

and thereby co-localized to HDAC-containing repressive complexes (Fig. 4). In this way, MC2392

relieves the HDAC repressive complex and allows induction of cell death in ATRA responsive and

in bona fide APL ATRA-resistant cells. Accordingly, MC2392 is not inducing apoptosis in solid

tumor such as prostate or breast cancer (data not shown). Moreover, ChIP-seq profiles show that

MC2392 induces changes in H3 acetylation at a small subset of PML-RARα/RXR binding regions

but also in regions not regulated by ATRA (Fig. 2, 3). Unlike natural retinoids, MC2392 exerts

distinct biological effects, inducing partial differentiation and promoting apoptosis, possibly

similarly to As2O3. MC2392 displays a minimal or no cytotoxicity in other kind of tumor cells.

Collectively these data underpin the specificity and selectivity of the MC2392. In conclusion,

MC2392 represents the creation of a single chemical entity with dual action thus reflecting the

concept of a laboratory-created ad hoc drug, which as a single drug can overcome resistance in

cancer.

Acknowledgements

We apologize to the authors whose work we could not cite due to reference restriction.




17

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Figure 1. MC2392 selectively inhibits the HDAC-repressive complexes associated to PML-RARα

in NB4 cells. A, structure of MS-275, ATRA and MC2392. B, acetylation evaluation of histone H3




20

by SAHA and MC2392 at 5 μM; p21 induction by MS-275 and MC2392 at 5 μM in U937 cells.

Histone H1 or ERKs were used for loading control. C, HDAC1-4 and SIRT1 assay in presence of

the indicated compounds. SAHA and EX-527 were used at 5 μM, Resveratrol at 100 μM. Data are

expressed as % of activity compared to control. Error bars represent standard deviations (SD) based

on three independent experiments. D, HDAC assay after immunoprecipitation of PML-RARα in

HeLa cells overexpressing PML-RARα. Data are expressed as % of activity compared to control,

error bars represent SD based on three independent experiments. E, activation of RARE3tkluc after

transfection in HeLa cells treated as indicated. pMAX-EGFP co-transfection was used as an internal

control, error bars represent ST based on three independent experiments. F, Western blot analyses

for PML-RARα in nuclei extracts in NB4 cells treated with ATRA 1 μM and MC2392 5 μM.

HDAC2 was used as loading control.

Figure 2. MC2392 selectively regulates acetylation at some PML-RARα binding sites in NB4 cells.

A, genomic tracks of random regions displaying ChIP-seq data for H3K9K14ac in NB4 cells after

24h of treatment with MS-275, ATRA and MC2392. B, distribution of the H3K9K14ac enrichment.

Locations are divided in gene body (gene), upstream near (-5 kb to the transcription start site),

intergenic (upstream far from -25 to -5 kb of the transcription start site, downstream far, end of

untranslated region +5 to +25 kb, and distant (everything else)), downstream near (end of

untranslated region to +5 kb). C, overview of the TGM2, ICAM1, RARβ, PML-RARα/RXR binding

site in NB4 cells. D, tag density maps depicting the pattern of H3K9K14ac occupancy around (+ 10

and –10 kb) the PML-RARα/RXR sites. The intensity plot indicates the level of H3K9K14ac

occupancy (log2 of tag density; see scale on the top) in a 500-bp window. The position of one

example (TGM2) is indicated.

Figure 3. A proper PML-RARα spatial position is a prerequisite for MC2392 mediated HDAC

inhibition at PML-RARα binding sites in NB4 cells. A, k-means clustering using a Pearson

correlation metric and the log2 value of the tag density. Cluster 2 and cluster 9 of H3K9K14ac

changes with typical and similar epigenetic responses to ATRA and to MC2392. The number of




21

genes in each cluster is indicated. B, Venn diagram representing the overlap of cluster 2 or 9

regulated genes and PML-RARα/RXR binding sites; analysis of the presence of DR motifs

underlying the overlapped genes; response element for PPAR-RXRα identified as the predominant

motif in the 191 genes of the cluster 2. Motif was identified by a de novo motif search. C, Cluster 8

of H3K9K14ac changes with different epigenetic responses to ATRA and to MC2392. The number

of genes is indicated. Functional annotation clustering (GO) of the clustered genes. D, Response

element for NFE2L2, FOS, AP1 identified like predominant motifs in the H3K9K14ac binding sites

of the regulated genes of Cluster 8. Motifs were identified by a de novo motif search.

Figure 4. MC2392 induces PML-RARα-context specific apoptosis in APL cells. A, clonogenity

assay in NB4 cells upon MC2392; proliferation curves in NB4 cells at the indicated time. B, pre-G1

fraction analysis of NB4 cells treated for 48h with the indicated compounds. C, cell cycle analysis

of NB4 cells treated for 48h with the indicated compounds. MS-275, SAHA, MC2677 and MC2678

were used at 5 μM; ATRA was used at 1 μM. D, granulocytic differentiation in NB4 by the CD11c

positive cells. PI positive cells have been excluded from the analysis. E, granulocytic differentiation

by the CD11c positive cells in NB4R4 cells. PI positive cells, have been excluded from the analysis;

pre-G1 fraction in NB4R4 cells treated with the indicated compounds. F, analysis of the pre-G1

fraction in U937 PML-RARα and PLZF-RARα cells after 24h and 48h of treatment with the

indicated compounds in presence or absence of zinc. All experiments have been performed at least

three times and error bars represent SD based on three independent experiments.

Figure 5. MC2392 leads to caspase activation and ROS production in NB4 cells. A, B, Caspase-8, -

9 and -3/7 assay in NB4 cells treated 48h with the indicated compounds. C, mitochondrial

membrane potential dissipation of NB4 treated with MS-275, SAHA and MC2392 used at 5 μM. D,

Western blot analysis of apoptosis players in NB4 cells treated with ATRA and MC2392 for 24h

and 48h. ERKs and tubulin have been used as control for equal protein loading. E, pre-G1 fraction

in NB4 cells treated with the indicated compounds. F, analysis of ROS production in conditions as




22

in A. NAC was used 20 mM and H2O2 500 μM; Western blot analysis for phosphoH2AX(S139).

Error bars shown in A, B, C, E, F represent SD based on three independent experiments.

Figure 6. MC2392-mediated apoptosis is caspase 8 dependent. A, Caspase-2 activity on NB4 cells

treated for 48h with the indicated compounds; pre-G1 pick detection in identical settings; the

caspase-2 inhibitor VDVAD was used at 100 μM. B, Caspase-8 activity on NB4 cells treated for

48h with the indicated compounds; pre-G1 pick detection in identical settings. The caspase-8

inhibitor IETD was used at 100 μM. C, pre-G1 fraction in NB4 cells treated with the indicated

compounds; NEC-1 was used at 50 μM, D, Western blot analysis on RIP1 after 24h and 48h of

treatment with the indicated compounds. Blot against ERKs represents control for equal protein

loading. Error bars shown in A, B, C, represent SD based on three independent experiments.




% H

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CpGIslands

45300000 45400000 45500000 45600000 45700000 45800000 45900000 46000000 46100000 46200000

CpG Islands (Islands < 300 Bases are Light Green)

UCSC Genes Based on RefSeq, UniProt, GenBank, CCDS and Comparative GenomicsCDCP1CDCP1

TMEM158 LARS2LARS2

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Ctr(time 48h)

% o

xid

ate

d R

OS


casp3

ERKs

Bid

tubtBid

48 24 48 24time (h)MC2392 ATRA Ctr

Fas

ERKs

FasL

TRAILERKs

casp8

ERKs

FADD

ERKs

48 24 48 24

MC2392 ATRA Ctr

time (h)




A B


0

5

10

15

20

25

% c

ells

in

pre

G1

ne

c-1

+ M

C2

39

2

ne

c-1

MC

23

92

Ctr

C

0,0

0,5

1,0

1,5

2,0

2,5

3,0ca

sp

ase

2 a

ctiva

tio

n (

fold

)

VD

VA

D+

MC

23

92

MC

23

92

SA

HA

Ctr

Ctr(time 48h)

Z-V

DV

AD

MC

23

92

Z-V

DV

AD

VD

VA

D+

MC

23

92

% c

ells

in

pre

-G1

0

10

20

30

40

ca

sp

ase

8 a

ctivita

tio

n (

fold

)

MC

23

92

Ctr(time 48h) D

TEI

IET

D+

MC

23

92

0

1

2

3

0

10

20

30

% c

ells

in

pre

-G1

D

MC

23

92

Ctr D

TEI

IET

D+

MC

23

92

RIPK1ERK1

Ctr MSATRAMC2392

48 24

RIPK1ERK1

Ctr

(time h)

M

C2

39

2

MC

23

92

+n

ec-1

48 24 48 24

(time 48h)




Published OnlineFirst February 24, 2014.Cancer Res Floriana De Bellis, Vincenzo Carafa, Mariarosaria Conte, et al. triggered by the novel hybrid retinoid-HDAC inhibitor MC2392Context-selective death of acute myeloid leukemia cells

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