Context-Selective Death of Acute Myeloid Leukemia Cells Triggered by the Novel Hybrid Retinoid-HDAC Inhibitor MC2392

Published OnlineFirst February 24, 2014; DOI: 10.1158/0008-5472.CAN-13-2568

Cancer Therapeutics, Targets, and Chemical Biology Research

Floriana De Bellis1,4, Vincenzo Carafa1, Mariarosaria Conte1, Dante Rotili3, Francesca Petraglia1, Filomena Matarese4, Kees-Jan Francoijs¸ 4, Julien Ablain6, Sergio Valente3,Remy Castellano5, Armelle Goubard5, Yves Collette5, Amit Mandoli4, Joost H.A. Martens4, Hugues de The6, Angela Nebbioso1, Antonello Mai3, Hendrik G. Stunnenberg4, and Lucia Altucci1,2

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-RARa oncofusion protein is mediated by an HDAC-containing complex that can be dissociated by pharmacologic 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-RARa- 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-RARa, MC2392 induces changes in H3 acetylation at a small subset of PML-RARa–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-RARa conveys efficient MC2392-induced cell death. Our data suggest a model in which MC2392 binds to the RARa 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. Cancer Res; 74(8); 1–12. 2014 AACR.

Introduction promyelocytic leukemia gene (PML) in more than 95% of the Acute promyelocytic leukemia is characterized by the pres- cases (1, 2). Despite that the majority of APLs carry this t(15;17) ence of blasts blocked at the promyelocytic stage of myeloid translocation, rare alternative RARa-containing fusion pro- – differentiation and by the fusion protein of RARa with the teins 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 Authors' Affiliations: 1Dipartimento di Biochimica, Biofisica e Patologia 2 PML-RARa (6) and to recruit the multi-subunit HAT complex Generale, Seconda Universita degli Studi di Napoli; Istituto di Genetica e Biofisica, IGB, Adriano Buzzati Traverso, Naples; 3Dipartimento di Chimica on RARE. Besides having this desired therapeutic effect on APL e Tecnologie del Farmaco, Sapienza University of Rome, Rome, Italy; 4 5 blasts, ATRA binds to and activates the nuclear receptors NCMLS, Radboud University, Nijmegen, the Netherlands; Inserm, fi CRCM, U1068, TrGET & ISCB, University of Marseille; and 6Laboratoire (RARa, b,org) that bind to speci c DNA responsive elements U944 and UMR 7212, University Paris-Diderot, Paris, France (RARE; refs. 7, 8), thereby affecting a variety of biologic pro- Note: Supplementary data for this article are available at Cancer Research cesses in essential all cell types, such as cell proliferation, Online (http://cancerres.aacrjournals.org/). differentiation, and apoptosis (9, 10). – Corresponding Authors: Lucia Altucci, Dipartimento di Biochimica, Bio- The PML-RARa mediated epigenetic block of gene tran- fisica e Patologia Generale, Seconda Universita degli Studi di Napoli, Vico L. scription (11, 12) can also be overcome by HDAC inhibitors De Crecchio 7, 80138, Napoli, Italy. Phone: 390-8156-67569; Fax: 390- (HDACi) allowing cells to restart differentiation or proapoptotic 8145-0169; E-mail: [email protected]; Angela Nebbioso, Dipartimento di Biochimica, Biofisica e Patologia Generale, Seconda Università degli events (13). Activation of differentiation programs, inhibition of Studi di Napoli, Vico L. De Crecchio 7, 80138, Napoli, Italy. Phone: 390- the cell cycle, and eventually induction of apoptosis are among 8156-65681; Fax: 390-8145-0169; E-mail: [email protected]; Antonello Mai, Dipartimento di Chimica e Tecnologia del Farmaco the key antitumor activities of HDACi in cancer therapy (7, 11, "Sapienza" Università di Roma, P. le A. Moro 5, 00185 Roma; Phone: 14–16). HDACi cause enzymatic inhibition and/or the release of 390-649913392; Fax: 390-6649693268; E-mail: [email protected]; HDACs from repressive complexes permitting HAT recruit- and Hendrik G. Stunnenberg, Department of Molecular Biology, Faculty of Science, Nijmegen Centre for Molecular Life Sciences, Radboud University, ment, histone acetylation, chromatin decondensation and, ulti- Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. Phone: 31- mately, transcription activation of tumor-suppressor genes 243510524; Fax: 31-243610520; E-mail: [email protected] (TSG) or other genes crucial for the normal functioning of the doi: 10.1158/0008-5472.CAN-13-2568 cells (7). The major drawback of HDACi in treatment is the 2014 American Association for Cancer Research. broad inhibition of the plethora of HDAC-containing complexes.

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MS-275/Entinostat is one of the most potent and widely been tested and authenticated following manufacturer's used HDACi with micromolar affinity for class I HDACs instruction. and with selectivity for HDAC1, HDAC3, and HDAC8 (17– NB4, NB4-R4 (26, 27), U937, U937 PML/RAR, U937-PLZF/ 19). MS-275 promotes differentiation, apoptosis, and inhibits RAR Zn inducible (28, 29), and LnCap cell lines were grown the proliferation of multiple cancer cell lines (20). The effects of at 37 Cinairand5%CO2 in RPMI-1640 medium (EURO- MS-275 have been examined in human leukemia and lympho- CLONE), supplemented with 10% heat-inactivated FBS (Sig- ma cells (U937, HL-60, K562, and Jurkat). When administered at ma), 1% L-glutamine, 1% ampicillin/streptomycin, and 0.1% a low concentration (e.g., 1 mmol/L), MS-275 exhibited potent gentamicin (SIGMA). Cells were kept at the constant con- antiproliferative activity inducing p21CIP1/WAF1-mediated centration of 200,000 cells per milliliter of culture medium. growth arrest and expression of differentiation markers HeLa cell line was grown at 37 C in air and 5% CO2 (CD11b) in U937 cells (14, 21). However, at higher concentra- in Dulbecco's Modified Eagle Medium (GIBCO) with 10% tions (e.g., 5 mmol/L), MS-275 potently induced cell death and a FBS, 1% L-glutamine, 1% ampicillin/streptomycin, and 0.1% very early increase in death receptor signaling (22) as well as in gentamicin. reactive oxygen species (ROS), followed by the loss of mitochondrial membrane potential and cytosolic release of cyto- Chemicals chrome c (23). ATRA and MS-275 were dissolved in ethanol; MC2392, The use of HDACi in combination with other anticancer MC2677, and MC2678 were dissolved in dimethyl sulfoxide agents (epi-drugs such as 5-aza-20-deoxycytidine and reti- (DMSO) and used at 1 and 5 mmol/L, respectively. SAHA noic acid) is one way forward to a promising application (Merck), resveratrol, and EX-527 (Alexis) were dissolved in against cancer. In patients with leukemia, and particularly in DMSO and used at 5, 100, and 5 mmol/L, respectively. the elderly, clinical studies combining ATRA treatment with HDACi are in progress (24). For example, valproic acid has Synthesis of MC2392, MC2677, MC2678 for many years been and still is used as an antiepileptic drug For details, see Supplementary Materials and Methods. and inhibits preferably class I HDACs although in the high micromolar to millimolar range. Moreover, even though Total protein, histone extraction, and Western blot there were a lot of concerns regarding toxic side effects of analysis HDACi in the clinical setting due to the roles of HDACs in For details, see Supplementary Materials and Methods. multiples pathways, until now clinical trials mostly showed manageable side effects (25). The combination of ATRA with Colony assay an HDACi could result in improved antitumorigenic activity Colony assay was carried out as described in ref. 22. (24). Here, we exploited the synergy between HDACi and ATRA by Cell cycle and differentiation analyses generating and testing a single hybrid molecule, named For details, see Supplementary Materials and Methods. MC2392 on APL cells. Genome-wide epigenetic analysis revealed that MC2392 is a weak retinoid. In contrast with the Caspase-3, -8, and -9 assays genome-wide effects of MS-275 on histone H3 acetylation, Caspase activity was detected within living cells using MC2392 induces acetylation at only a small subset of PML- B-BRIDGE Kits supplied with cell-permeable fluorescent sub- RARa–binding sites in NB4 cells line. RNA-seq analysis showed strates. The fluorescent substrates for caspase-3, -8, and -9 that MC2392 alters the expression of a number of stress- were FAM-DEVD-FMK, FAM-LETD-FMK, and FAM-LEHD- responsive and apoptotic genes differently from ATRA. Impor- FMK, respectively. Cells were washed twice in cold PBS and tantly, the hybrid compound acts in a context and PML-RARa incubated for 1 hour in ice with the corresponding substrates, fusion protein-dependent fashion to induce rapid and massive as recommended by suppliers. Cells were analyzed using Cell cell death, RIP1 induction, and ROS production. We propose a Quest software applied to a FACScalibur (BD Biosciences). model in which this hybrid compound binds to the RARa via its Experiments were performed in duplicate and values retinoid moiety and selectively inhibits the HDACs contained expressed in mean SD. in the repressive complex via the MS-275 part. Taken together, MC2392 is a promising candidate for apoptosis-based therapy Caspase-2 assay of APL, representing a new and effective, single hybrid drug Caspase-2 activity was detected within whole living cells, able to modulate multiple cell death pathways. Our study using Caspase-2 Fluorometric Kit following supplier's instruc- convincingly shows that the targeting of multiple signaling tions (R&D Systems). The plate was incubated at 37C for 1 pathways by a single hybrid drug is a feasible and attractive hour and fluorescence quantified with a TECAN M200 station paradigm for new cancer therapies. at a 400- to 505-nm wavelength. Results are expressed as fold increase in caspase activity.

Materials and Methods Analysis of mitochondrial membrane potential Cells Detection of the changes in mitochondrial membrane NB4, LnCap, and U937 were purchased by DSMZ and potential (Dym) was performed using the MITOPT JC-1 Kit American Type Culture Collection, respectively. Cell lines have (Immunochemistry Technologies) following the supplier's

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suggestions and analyzed by flow cytometry with Cell Quest Results Pro software. MC2392 inhibits HDAC activity of the PML-RARa complex and maintains retinoid activity ROS detection We set out to exploit the possibility to combine the active NB4 cells were resuspended in prewarmed PBS 1, contain- parts of the HDACi MS-275 and the retinoid, ATRA, yielding ing 5 mmol/L of DCF-DA (diclorodihydrofluorescein diacetate) MC2392 (Fig. 1A). To explore the activities of this hybrid probe and incubated at 37 C for 20 minutes. Cells were compound, U937 cells were treated for 24 hours with 5 analyzed after washing, using the Cell Quest software applied mmol/L MC2392, its parent compound MS-275 (Entinostat) to a FACScalibur (BD Biosciences). H O was used as positive 2 2 a class I inhibitor and the class I/II HDAC inhibitor SAHA control. (Vorinostat). In contrast to the reference compounds, MC2392 did not result in a net increase of histone acetylation (Fig. 1B). RNA extraction, reverse transcription, and quantitative Moreover MC2392 did not induce p21 protein expression (Fig. PCR in real time 1B) corroborating that at this concentration the molecule has For details, see Supplementary Materials and Methods. reduced or no HDACi activity as compared with MS-275. MC2392 exerted only minimal (if any) inhibitory action on Immunoprecipitation HDAC1 and HDAC4, nor did it display SIRT1-modulating For details, see supplementary Materials and Methods. activities in vitro (Fig. 1C). Our premise for the design of MC2392 was that the close physical proximity of a retinoid HDAC assay (PML-RARa) and an HDACi-binding moiety (HDAC1 and/or HDAC assays have been performed as described in ref. 30. 4) could convey context-specific inhibitory activity by a Specifically, samples immunoprecipitated with anti-PML-RAR hybrid molecule spanning the physical distance. To investigate or with IgG were pooled respectively to homogenize all sam- this assertion, PML-RARa was immunoprecipitated from HeLa ples. The Fluor de Lys Substrate was incubated (1 hour) with cells after transient transfection with PML-RARa and assayed the immunoprecipitated PML-RAR (in the presence or absence for HDACi activity. Indeed, MC2392 inhibits the HDAC(s) of SAHA or MC2392 5 mmol/L) and fluorescence was quantified resident in the PML-RARa repressive complex similarly to with a TECAN M200 station. SAHA (Fig. 1D). To investigate whether MC2392 acts as a retinoid, its poten- SIRT assay tial to activate the ATRA-responsive luciferase reporter (RAR- For details, see Supplementary Materials and Methods. E3tkluc) was first tested in transient transfected HeLa that expresses endogenous RAR and RXR. MC2392 activates RARE Transfections and luciferase assay 3 tkluc weakly compared with ATRA (Fig. 1E). Consistent with For details, see Supplementary Materials and Methods. these observations, quantitative real-time PCR in NB4 cells showed a similar low response to MC2392 for the RNA expres- IkB alpha ELISA assay sion of specific genes known to be retinoid modulated (34, 35) Assay was performed after 48 hours from the treatment with such as HOXA1, IRF1 and, to a lesser extent, TNFa (Supple- ATRA and MC2392 in NB4 cells following the supplier's mentary Fig. S1A), as compared with ATRA, suggesting that instructions (Active Motif). MC2392 still maintains at least part of its retinoid activity. Finally, Western blot analyses of PML-RARa following Chromatin immunoprecipitation and ChIP-seq MC2392 treatment showed rapid degradation of PML-RARa NB4 cells were treated for 4, 6, 24, and 48 hours with 1 mmol/L in NB4 cells (Fig. 1F) as has been reported for ATRA (36, 37). ATRA, 5 mmol/L MS-275, and 5 mmol/L MC2392. Chromatin Taken together the results show that MC2392 acts as a weak was harvested as described previously (31). Chromatin immuno- retinoid when compared with ATRA implying that it binds to precipitation (ChIP) experiments were performed using the RARa, activates RARa signaling, as well as induces the deg- H3K9K14ac (Diagenode), Nrf2, c-Fos and c-Jun (Santa Cruz radation of PML-RARa (Fig. 1F). We tentatively conclude that Biotechnology) antibodies. ChIPed DNA was analyzed by real- MC2392 is an HDACi in the context of the PML-RARa-HDAC time quantitative PCR with specific primers (Biolegio, see repressive complex. Supplementary Methods) using the 2 SYBR Green Mix (Bio-Rad) in a MyiQ thermocycler (Bio-Rad). Primers ampli- fying myoglobin were used as negative control. In addition, MC2392 induces local H3 acetylation ChIPed DNA was prepared for sequencing and processed We showed that the HDACi activity of MC2392 is indeed according to the manufacturer's instructions (Illumina) in apparent in the context of the PML-RARa-contained HDAC Supplementary Materials and Methods and essentially as complexes implying that acetylation may be altered at PML- already described (32, 33). RARa genomic binding sites. To examine whether MC2392 Peak detection, clustering analysis, and motif search are induces local epigenetic alterations, ChIP-seq profiling was detailed in the Supplementary Materials and Methods. performed using the H3K9K14ac antibody in NB4 cells treated with ATRA, MS-275, MC2392, or solvent, DMSO. In DMSO- RNA-sequencing treated cells, H3K9K14 acetylation appeared in the typical For details, see Supplementary Materials and Methods. sharp narrow peaks. Treatment with the HDACi MS-275 had

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MS-275 ATRA MC2392

B C HDAC1 SIRT1 HDAC4 AcH3 150 100 H1 80 100 Ctr 60 SAHA MS-275 MC2392 40 % Activity % Activity 50 p21 20 ERKs 24 h 48 h 0 0 Ctr Ctr Res Ctr SAHA Ex-527 MS-275 MS-275 MC2392 MC2392 MC2392 MC2392

D 100 E 80 F PML-RARα 70 80 HDAC2 60 50 60 Ctr 40 RLU 40 30

ATRA 24 h MC2392 24 h MC2392 48 h with PML-RAR α 20 20 10 % HDAC activity associated 0 0 Ctr Ctr ATRA SAHA MC2392 MC2392

Figure 1. MC2392 selectively inhibits the HDAC-repressive complexes associated to PML-RARa in NB4 cells. A, structure of MS-275, ATRA and MC2392. B, acetylation evaluation of histone H3 by SAHA and MC2392 at 5 mmol/L; p21 induction by MS-275 and MC2392 at 5 mmol/L in U937 cells. Histone H1 or ERKs were used for loading control. C, HDAC1-4 and SIRT1 assay in the presence of the indicated compounds. SAHA and EX-527 were used at 5 mmol/L, Resveratrol at 100 mmol/L. Data are expressed as the percentage of activity compared with control. Error bars, SDs based on three independent experiments. D, HDAC assay after immunoprecipitation of PML-RARa in HeLa cells overexpressing PML-RARa. Data are expressed as the percentage of activity compared with control; error bars, SD based on three independent experiments. E, activation of RARE3tkluc after transfection in HeLa cells treated as indicated. pMAX-EGFP cotransfection was used as an internal control; error bars, SD based on three independent experiments. F, Western blot analyses for PML-RARa in nuclei extracts in NB4 cells treated with ATRA 1 mmol/L and MC2392 5 mmol/L. HDAC2 was used as loading control.

a dramatic effect on the genomic acetylation landscape: gene body (52.11%) or intergenic (34.19%; Fig. 2B). Importantly, H3K9K14ac peaks were dampened and spreading of acetyla- MC2392 did induce low but reproducible local changes in tion occurred (Fig. 2A). In line with the absence of global acetylation at some PML-RARa binding sites such as at RARb HDACi activity (Fig. 1B), MC2392 did not affect the overall promoter or TGM2, ICAM1 gene (Fig. 2C). The RARb is avidly distribution of H3K9K14ac. Genomic annotation of the peaks transcriptionally induced by ATRA and expectedly H3 acety- called by MACS (38) with a P value of 10 6 (32) revealed a small lation is induced at promoter region. These data suggest percentage of H3K9K14ac-binding sites at promoter regions speciﬁcity and selectivity of MC2392 toward at least some (8.74%) while the major portion of the peaks were located in the PML-RARa binding sites and reinforced the similar, although

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34.19%

A Scale B chr3: 45300000 45400000 45500000 45600000 45700000 45800000 45900000 46000000 46100000 46200000 Gene body 35 _ DMSO 52.11% Intergenic 1 36 _ ATRA Downstream near 1 11 _ MS_275 Upstream near 1 46 _ H3K9K14ac MC2392

1 CpGIslands CpG Islands (Islands < 300 Bases are Light Green) UCSC Genes Based on RefSeq, UniProt, GenBank, CCDS and Comparative Genomics CDCP1 TMEM158 LARS2 AK026206 SLC6A20 CCR9 AX747832 CCR3 CDCP1 LARS2 KIAA0851 LZTFL1 CCR9 XCR1 CCR1 CCR3 4.94% LIMD1 SACM1L LZTFL1 XCR1 CCR1 SACM1L CCR9 XCR1 CCR3 SLC6A20 CCR9 CXCR6 CCR3

8.74%

CDH3K9K14ac occupancy in PML-RARα/RXR–binding sites chr20: 34 0 22

DMSO 1_ 34 _

ATRA TGM2

1_ H3K9K14ac 34 _

MC2392 1 TGM2 Tag density Tag

chr19: chr3: 50 _ 42 _

20 kb DMSO 1 _ 1 _

50 _ 42 _ ctr

ATRA ATRA_4 h ATRA_4

H3K9K14ac 1 _ 1 _

50 _ 42 _ h ATRA_24 MC2392_4 h MC2392_24 h MC2392_48 h MC2392 1 _ 1 _ ICAM1 RAR

Figure 2. MC2392 selectively regulates acetylation at some PML-RARa binding sites in NB4 cells. A, genomic tracks of random regions displaying ChIP-seq data for H3K9K14ac in NB4 cells after 24 hours 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 þ5toþ25 kb; and distant, everything else; downstream near, end of untranslated region to þ5 kb. C, overview of the TGM2, ICAM1, RARb, PML-RARa/RXR–binding site in NB4 cells. D, tag density maps depicting the pattern of H3K9K14ac occupancy around (þ10 and 10 kb) the PML-RARa/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. weaker, retinoid activity of MC2392 as compared with ATRA though with a strong timely delay (Fig. 3A). Moreover, Gene (Fig. 1E and Supplementary Fig. S1A). Ontology (GO) analysis revealed that the regulated genes in To substantiate our findings we examined the 1,000 most cluster 2 are functionally linked to leukocyte differentiation significantly changed acetylation peaks after ATRA induction (Supplementary Fig. S1C). The overlap between H3K9K14ac in PML-RARa/RXR binding regions. The intensity plots cov- peaks in cluster 2, and PML-RARa–binding sites (33) revealed a ering the region of 10 kb up- and downstream for cells treated common set of 191 loci, suggesting that indeed a subset of with ATRA and MC2392 show acetylation increases following H3K9K14ac peaks are associated with PML-RARa/RXR. The MC2392 treatment although to a lower level and after longer overlap between cluster 9 H3K9K14ac peaks, and PML-RARa– treatment times as compared with ATRA (Fig. 2D): a vivid binding sites revealed a common set of 183 regions, suggesting increase is already apparent after 4 hours of ATRA treatment that also here a subset of induced H3K9K14ac peaks are whereas a similar response amplitude is reach only after 48 associated with PML-RARa/RXR (Fig. 3B, top). hours of MC2392 treatment. Recent studies analyzing the genome-wide PML-RARa– The normalized tag numbers (in treated and untreated binding sites not only identified DR2 and DR5 elements as samples) were counted for all binding regions and clustered the primary PML-RARa response elements but also regions using k-means. This analysis identified 20 clusters (Supple- containing DR1, DR3, and DR4 motifs (33, 39). We found that mentary Fig. S1B), among which in particular cluster 2 and both the DR1 and DR2 motifs are enriched within the cluster 2 cluster 9 showed H3K9K14ac peaks that responded similarly to and 9 binding sites (Fig. 3B, middle), whereas DR3, DR4, and ATRA for 4 and 24 hours, and to MC2392 for 24 and 48 hours DR5 motifs are not enriched. In addition, a de novo motif search

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Figure 3. A proper PML-RARa spatial position is a prerequisite for MC2392-mediated HDAC inhibition at PML-RARa–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 change with typical and similar epigenetic responses to ATRA and MC2392. The number of genes in each cluster is indicated. B, Venn diagram representing the overlap of cluster 2- or 9–regulated genes and PML-RARa/RXR–binding sites; analysis of the presence of DR motifs underlying the overlapped genes; response element for PPAR-RXRa 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, and 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.

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identified the DR1 element in the 191 H3K9K14ac peaks of Analysis of the transcription level of the 148 genes associated the cluster 2 that are regulated by MC2392 (Fig. 3B, bottom). with apoptosis (http://www.reactome.org) revealed main dif- This suggest that a proper configuration, that is, spatial ferences between ATRA and MC2392 (Supplementary Fig. S2E position of the PML-RARa with respect to the HDAC(s) and Supplementary Materials) such as the fast induction of in the repressive complex is an important prerequisite for TRAF2, E2F1, DYNLL2, and DBNL following MC2392 but not MC2392 to physically bridge the RAR and HDAC(s) moieties ATRA treatment. Next, we selected several MC2392-modulated and consequently cause inhibition of HDAC activity. genes related to apoptosis and oxidative stress, which are also modulated in the H3K914ac levels (Fig. 3 and Supplementary MC2392, but not ATRA, induces H3K9K14 acetylation at Fig. S1) upon MC2392 but not ATRA treatment and examined proapoptotic genes transcription factor binding. This revealed an increased occu- Our analysis also revealed cluster 8 consisting of 774 regu- pancy of c-Jun, c-Fos, and NRF2 in a time- and MC2392- lated genes that showed an opposite epigenetic response to dependent manner (Supplementary Fig. S3), corroborating MC2392 as compared with ATRA treatment (Fig. 3C) that is and extending our observations that the apoptotic action of induction by MC2392 and reduction by ATRA. The assignment the MC2392 is causally bound to the modulation of cell death of genes in this cluster to biologic functions (Fig. 3C, bottom) and redox pathways in response to modulation of AP1 and revealed enrichment for regulation of cell death. De novo motif NRF2 bindings and their downstream pathways. Taken togeth- search showed enrichment of the NF-E2–related factor-2 er, the transcriptome and histone H3 acetylome analysis (NFE2L2, Nrf2) and Fos (AP1) response element each present strongly point to a differential effect of MC2392 as opposed at about 10% of H3K9K14ac peaks (Fig. 3D). Note that the to ATRA on apoptotic genes. stress-responsive transcription factors Nrf2 is reported to play a role in protection against oxidative stress-induced cellular MC2392 induces cell death in APL cells damage. ChIP experiments coupled with quantitative PCR Genome-wide epigenetic and transcriptome analysis have showed enrichment of Nrf2 over specific responsive element provided strong indications that MC2392 effects are distinct (ARE) of target genes after MC2392 treatment, confirming an from those elicited by ATRA in particular on apoptotic genes. early Nrf2-mediated transcriptional response during oxidant- Therefore, we assessed the activity of MC2392 on NB4 prolif- induced cell death, differently from ATRA, which reduces the eration, cell-cycle progression, granulocytic differentiation, binding of Nrf2 to the ARE enhancer region (Supplementary and apoptosis induction in comparison with MS-275, SAHA, Fig. S1D). and ATRA (Fig. 4). Interestingly, analysis of NB4 clonogenicity Finally, we found an increase in transcription in the target in semisolid media upon MC2392 treatment (Fig. 4A, left) genes that encode antioxidant proteins or enzymes to buffer revealed a clear loss of clonogenic activity at 10 5 mol/L. In the intracellular redox activities, such as FTH1, GCLM, GCLC, addition, two MC2392 analogs that differ for the position of the NQO and HMOX, STK17A. Conversely, ATRA inhibits/reduces amino group at the anilide ring (position 2, 3, and 4 in MC2392, the expression of these Nrf2 target genes (Supplementary MC2678, and MC2677, respectively; ref. 42) were used. The Fig. S2A). MC2678 and -2677 compounds do not display HDAC inhibition (data not shown) and MC2677 has been previously described to MC2392 and ATRA induce expression of apoptotic genes inhibit the growth of a number of tumor cell lines, including at transcriptional level prostate, head and neck, squamous carcinoma, and neuroblas- To analyze the expression changes induced by MC2392, we toma (42, 43). In contrast to MC2392 and MS-275, MC2678 and performed transcriptome profiling on MC2392 and ATRA- MC2677 are unable to induce a proliferation arrest of NB4 cells treated NB4 cells. The gene expression values from RNA-seq (Fig. 4A, right). MC2392 increased the pre-G1 fraction (Fig. 4B), data were calculated by quantifying the number of sequence a measure of cell death induction but did not induce major tags for each genes using Genomatix (40, 41). Scatter plots changes in the cell-cycle phases (Fig. 4C). MC2392 did also show the general differences of gene expression of MC2392 induce an increase of the CD11c levels to 24%, thus inducing versus ATRA after 24 and 48 hours (Supplementary Fig. S2B). maturation of APL cells similar to ATRA, MC2677, and MC2678 Moreover, functional annotation clustering of differentially (Fig. 4D) but the MC2678 and MC2677 compounds did not expressed genes by Gene Ontology (GO; PANTHER) analysis induce apoptosis (Fig. 4B). revealed that genes upregulated in response to MC2392 are Induction of cell death by MC2392 is not dependent on enriched for terms associated with protein transport, response differentiation as MC2392 readily induces cell death but not to stress and apoptosis induction. Moreover, ATRA upregu- differentiation of the ATRA-resistant NB4 subclone, NB4R4 lated genes are enriched for terms associated mostly to protein (Fig. 4E and F; refs. 26, 27). Note that as a result of the acquired transport and metabolic processes (Supplementary Fig. S2C). mutation in the ligand-binding domain, PML-RARa can bind The hierarchical clustering analysis supports these data (Sup- ATRA but it is thought that the binding does not induce the plementary Fig. S2D). conformational change needed to dissociate the repressive Moreover, MC2392 downregulated genes are enriched for complex and recruit activating proteins (26). Thus, the cell terms associated with metabolic processes and pathway anal- death activity of MC2392 acts independently from maturation ysis showed the decreased expression of components that and is likely linked to the HDAC inhibition of the resident drive cell growth, proliferation, and cell communication such repressive complexes. Taken together, our analysis shows that as JAK/STAT and integrin pathway (data not shown). MC2392 indeed acts as a weak retinoid in line with the

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A B C G2–M 200 35 80 S 8 G MC2678 30 70 1

) 7 5 150 MC2677 1 60 6 25 MC2392 50 5 20 100 4 ATRA 40

15 % Cells 30 3 MS-275 10 50 2 20 % Cells in pre-G

Number of colonies Ctr 1 5 10 Number of cells (×10 0 0 0 0 Time (h) 240 48 (Time 48 h) (Time 48 h) Ctr Ctr mol/L mol/L ATRA ATRA SAHA SAHA 6 5 Untreat MS-275 MS-275 MC2677 MC2678 MC2392 MC2392 MC2677 MC2678 10 10 D E F PML-RARα PML-RARα PLZF-RARα LnCap 50 + Zn + Zn 50 NB4 60 30 50 80 50 NB4R4 70 40 50 40 25 40 1 40 1 1 60 40 20 30 30 30 50 30 /PI-cells /PI-cells + + 30 15 40 20 20 20 20 20 10 30

20 % Cells in pre-G % Cells in pre-G % CD11c % Cells in pre-G

% CD11c 10 10 10 5 10 10 10 0 0 0 0 0 0 0 Ctr Ctr Ctr (Time 48 h) Ctr (Time 72 h) MS Ctr Ctr Ctr MS ATRA ATRA ATRA

MC2392 MC2677 MC2678 MC2392 MC2392

MC2392 (Time 48 h) MC2392 48 h MC2392 24 h MC2392 48 h MC2392 24 h MC2392 48 h MC2392 24 h

Figure 4. MC2392 induces PML-RARa context-speciﬁc apoptosis in APL cells. A, clonogenicity 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 48 hours with the indicated compounds. C, cell-cycle analysis of NB4 cells treated for 48 hours with the indicated compounds. MS-275, SAHA, MC2677, and MC2678 were used at 5 mmol/L; ATRA was used at 1 mmol/L. 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-RARa and PLZF-RARa cells after 24 and 48 hours of treatment with the indicated compounds in the presence or absence of zinc. All experiments have been performed at least three times and error bars represent SD based on three independent experiments.

transcriptome and histone H3 acetylome data but clearly has ptotic pathway, increased the expression of FasL, Fas, and acquired additional properties or such that were "masked" in TRAIL similarly to what has been reported for MS-275 (22). ATRA and MS-275. Moreover, our results corroborate and MC2392 also induced degradation of Bid into the apoptotic extend the rapid response to the apoptosis/cell death versus factor t-Bid, mitochondrial depolarization (Fig. 5D) and Bcl2 slow differentiation in response to MC2392 treatment. downregulation (Supplementary Fig. 4B). Next, NB4 cells were treated with MC2392 and H2O2 with and without the antiox- MC2392-induced cell death involves ROS and caspase-8 idant N-acetylcysteine (NAC). The data suggest that MC2392 To gain insight into the mechanism(s) by which MC2392 mediates a ROS-dependent cell death associated with the initiates cell death, caspases-8, -9, and -3/7 activities in NB4 generation of ROS (Fig. 5E and F). We further observed cells were analyzed. Similarly to SAHA, MC2392 notably induc- increased H2AX phosphorylation, which is a hallmark of ed caspase activation with caspase-8, -9 (initiator caspases), ongoing DNA damage as a result of increased ROS (44, 45). caspase-3/7 (effector caspases; Fig. 5A and B) and mitochon- The response of APL cells to MC2392 includes the generation of drial potential activated in NB4 cells (Fig. 5C), thus indicating ROS as a consequence of Bid cleavage, which triggers mito- that the cell death induced by MC2392 is caspase dependent. chondrial membrane permeabilization. As both caspase-2 and MS-275–induced cell death is caspase-8 dependent and related caspase-8 are capable of cleaving the proapoptotic Bid we set to TRAIL induction (22). To identify the molecular pathway out to investigate whether caspase-2 or caspase-8 are respon- we evaluated the levels of known players of apoptosis, such sible. MC2392 induced caspase-2 activation, which is fully as FAS, TRAIL, FADD, caspase-8, and -3/7, Bcl2 and Bid in NB4 blocked by the caspase-2 inhibitor Z-VDVAD-FMK (Fig. 6A). cells after 24 and 48 hours treatment. As shown in Fig. 5D and However, cell death mediated by MC2392 stayed unaltered Supplementary Fig. S3A, MC2392 induced the extrinsic apo- (Fig. 6A, right). MC2392-mediated cell death is also not NOX-

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MC2392 Induces PML-RARa Context-Selective Cell Death

ABC 80 Caspase-8 Caspase-9 60 70 50 60 30 50 40 25 40 30 20

% Activity 30 20 15 20 10 10 10 % Caspase-3/7 activity 5 % MTP dissipation % MTP 0 0 0 Ctr Ctr Ctr Figure 5. MC2392 leads to caspase ATRA SAHA ATRA SAHA SAHA MS-275 MC2392 MC2392 MC2677

activation and ROS production in MC2678 MC2677 MC2678 MC2392 NB4 cells. A and B, caspase-8, -9, and -3/7 assay in NB4 cells treated DE for 48 hours with the indicated FasL Casp-3 25 compounds. C, mitochondrial Fas membrane potential dissipation of ERKs NB4 treated with MS-275, SAHA, ERKs 20 and MC2392 used at 5 mmol/L. D, TRAIL 1 Western blot analysis of apoptosis ERKs Bid players in NB4 cells treated with tBid 15 Casp-8 ATRA and MC2392 for 24 and 48 tub hours. ERKs and tubulin were Time (h) 48 24 48 24 used as control for equal protein ERKs MC2392 ATRA Ctr 10 loading. E, pre-G1 fraction in NB4 % Cells in pre-G cells treated with the indicated FADD 5 compounds. F, analysis of ROS ERKs production in conditions as in A. 48 24 48 24 Twenty mmol/L of NAC was used Time (h) MC2392 ATRA Ctr 0 and 500 mmol/L H2O2; Western (Time 48 h) 2 O blot analysis for phosphoH2AX Ctr 2 NAC (S139). Error bars in A, B, C, E, and H +NAC

F 2

F, SD based on three independent MC2392

100 O 2 experiments. pH2AX H 80

H4 MC2392 +NAC

60 Ctr

20 ATRA 48 h ATRA 24 h % Oxidated ROS MC2392 48 h MC2392 24 h

(Time 48 h) 2 O Ctr 2 NAC H +NAC 2 MC2392 O 2 H MC2392+NAC

dependent, given that Apocynin, a NOX-speciﬁc inhibitor, is sion showed both a strong upregulation upon MC2392 and a not able to block apoptosis (Supplementary Fig. S3C). Further- full abrogation with NEC-1 (Fig. 6D). Finally, in line with our more, NF-kB is neither induced nor transcriptionally activated hypothesis, MC2392 induces cell death speciﬁcally in a PML- by MC2392 (Supplementary Fig. S4D and S4E). In contrast, RARa but not in RARa or PML-driven way (Fig. 4F and data not when the pan-caspase inhibitor ZVAD (data not shown) or the shown). caspase-8 inhibitor IETD are used together with MC2392, NB4 Taken together, our data revealed that at the level of cell death is fully abolished (Fig. 6B), strongly suggesting that acetylation, MC2392 induces much fewer changes as compared the MC2392 mechanism of cell death is caspase-8–dependent. with ATRA. Moreover, MS-275 resulted in genome-wide damp- Surprisingly, necrostatin-1 (NEC-1), the known RIP1 kinase ening and broadening of all acetylation sites (Fig. 2A), whereas and necroptosis inhibitor, completely abrogated MC2392 cell MC2392 affected acetylation only at a subset of PML-RARa death (Fig. 6C), thus suggesting a RIP1-mediated cell death. sites (DR1-2 type). Finally, transcriptome analysis corroborat- Consistent with these observations, analysis for RIPK1 expres- ed and extended that MC2392 affects apoptotic genes.

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De Bellis et al.

3.0 3 2.5 40 2.0 2 1 30 30

1.5 1 Figure 6. MC2392-mediated 20 20 apoptosis is caspase-8 1.0 1 dependent. A, caspase-2 activity 0.5 10 10 on NB4 cells treated for 48 hours Caspase-2 activation (fold) % Cells in pre-G

Caspase-8 activation (fold) with the indicated compounds; pre-G1 pick detection in identical

0.0 0 0 % Cells in pre-G 0 settings; the caspase-2 inhibitor

D (Time 48 h) (Time 48 h) D

Ctr T Ctr Ctr Ctr VDVAD was used at 100 mmol/L.

E E

I I

SAHA B, caspase-8 activity on NB4 cells MC2392 MC2392 MC2392 MC2392 was treated for 48 hours with the Z-VDVAD Z-VDVAD indicated compounds; pre-G1 pick detection in identical settings. The IETD+MC2392 IETD+MC2392

VDVAD+MC2392 caspase-8 inhibitor IETD was used VDVAD+MC2392 at 100 mmol/L. C, pre-G1 fraction in C D NB4 cells treated with the 25 indicated compounds; NEC-1 was RIPK1 used at 50 mmol/L. D, Western blot

1 20 ERK1 analysis on RIP1 after 24 and (Time h) 48 24 48 24 48 24 48 hours of treatment with the 15 MC2392 ATRA MS Ctr indicated compounds. Blot against ERKs represents control 10 RIPK1 for equal protein loading. Error ERK1 bars in A, B, and C, SD based on % Cells in pre-G 5 (Time 48 h) three independent experiments. Ctr 0 MC2392 Ctr nec-1 MC2392+nec-1 MC2392 nec-1 + MC2392

Discussion respect to the HDAC(s) in the repressive complex is likely an Epigenetic modifications can be reversed by epi-drugs that important prerequisite for MC2392 to bridge the RARa and are promising for antitumor therapy. In patients with leuke- HDAC(s) moieties and consequently inhibition of HDAC mia, HDACi are widely used in combination with ATRA, activity. resulting in improved antitumorigenic activity. Here, we iden- The fact that MC2392 may represent the first RARa-driven tified and characterized the mechanism of action of MC2392, a HDACi is corroborated by (i) its inactivity on human recom- compound merging the HDACi part of the well-known MS-275 binant HDACs in vitro, (ii) its inability to modify general to ATRA. When compared with both ATRA and MS-275, histone and/or tubulin acetylation; (iii) its selective HDACi MC2392 displays similar as well as divergent characteristics. action on PML-RARa complexes; (iv) its enhanced ability to Despite being inactive as an HDACi standard in vitro biochem- induce apoptosis in APL cells but not in other leukemia or solid ical assays, MC2392 specifically inhibits the HDACs contained cancer models. The fact that MC2392 induces apoptosis in NB4 in the PML-RARa repressive complex (Fig. 1D). MC2392 cells and increases caspases-8, -2, and -9 activity suggests that induces degradation of PML-RARa (Fig. 1E) and differentiation MC2392 activate both mitochondrial and death receptor apo- of NB4 cells (Fig. 4D). Genome-wide epigenetic analysis shows ptotic pathways. Although MC2392 is able to activate the that MC2392 induces increased acetylation at a small subset of extrinsic pathway by inducing FAS and TRAIL, the death PML-RARa sites with preponderance for DR1 and -2 type (Fig. response to MC2392 includes also the generation of ROS as 3B) distinct from acetylation at PML-RARa sites induced by a possible consequence of the cleavage of Bid (Fig. 5D and F). ATRA. This MC2392 characteristic is particularly interesting Indeed, the cleavage of Bid in APL cells occurs concurrently and classifies it as a novel class of drugs that need precise with early generation of ROS, thereby contributing to cell protein-protein "positioning" to function. Moreover a specific death by promoting mitochondrial release of cytochrome c. configuration, i.e., spatial position of the PML-RARa with For this reason, normally the death receptor pathway (extrinsic

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MC2392 Induces PML-RARa Context-Selective Cell Death

pathway) converges with the mitochondrial pathway (intrinsic natural retinoids, MC2392 exerts distinct biologic effects, pathway) through caspase-mediated cleavage and activation inducing partial differentiation and promoting apoptosis, pos- of Bid. Furthermore, both the induction of RIP1 by MC2392 sibly similarly to As2O3. MC2392 displays a minimal or no and the abrogation of cell death operated by NEC-1, an cytotoxicity in other kind of tumor cells. Collectively, these data inhibitor of RIP1, dictate a complex scenario in which, alter- underpin the specificity and selectivity of the MC2392. In natively to apoptosis, necroptotic pathways may lead to mito- conclusion, MC2392 represents the creation of a single chem- chondrial depolarization and cell death of NB4 APL cells. Of ical entity with dual action thus reflecting the concept of a note, both ATRA and MS-275 have been previously shown to laboratory-created ad hoc drug, which as a single drug can function via TRAIL and FAS induction (16, 22). Moreover, the overcome resistance in cancer. fact that the MC2392 caspase-8–dependent apoptosis can be abrogated by NEC-1 supports a model in which mixed types of Disclosure of Potential Conflicts of Interest cell death can occur, displaying shared features of both necrop- No potential conflicts of interest were disclosed. tosis and apoptosis. Clearly, MC2392-induced apoptosis occurs in response to oxidative stress and is orchestrated by stress- Authors' Contributions Conception and design: D. Rotili, R. Castellano, A. Mai, H.G. Stunnenberg, responsive transcription factors. Nuclear translocation of NRf2 L. Altucci and subsequent induction of its target genes via antioxidant Development of methodology: F.D. Bellis, V. Carafa, M. Conte, D. Rotili, stress response element (ARE) may function to buffer oxidative F. Petraglia, J. Ablain, S. Valente, R. Castellano, A. Goubard, A. Nebbioso, L. Altucci Acquisition of data (provided animals, acquired and managed patients, stress response. Moreover, MC2392 induces the modulation of provided facilities, etc.): F.D. Bellis, J. Ablain, A. Goubard, Y. Collette, A. Mandoli, a large number of stress-responsive genes (Supplementary Fig. H. de The, L. Altucci S2A) and apoptotic genes (see Supplementary Materials and Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): F.D. Bellis, V. Carafa, K.-J. Francoijs, S. Valente, Methods). Finally, the evidence that apoptosis occurs in APL R. Castellano, J.H.A. Martens, H. de The, L. Altucci independently from a "classical" ATRA response is suggested Writing, review, and/or revision of the manuscript: F.D. Bellis, J.H.A. Martens, H. de The, A. Mai, H.G. Stunnenberg, L. Altucci both by the MC2392 action in Zn-inducible PLZF-RARa Administrative, technical, or material support (i.e., reporting or orga- expression in U937 cells (28, 29) as well as by its activity on nizing data, constructing databases): F. Matarese, K.-J. Francoijs, S. Valente, NB4R4 cells (26, 27). Note that in both PLZF-RARa U937 cells L. Altucci Study supervision: Y. Collette, H.G. Stunnenberg, L. Altucci and in NB4R4 cells ATRA signaling stays impaired upon treatment (27, 46, 47). Excitingly, given that MC2392 is not an in vitro Acknowledgments HDACi , MC2392 is able to exert its HDAC inhibition The authors apologize to those whose work could not be cited due to only when driven by its ATRA moiety to PML-RARa and restriction in reference citation. thereby colocalized to HDAC-containing repressive complexes (Fig. 4). In this way, MC2392 relieves the HDAC repressive Grant Support complex and allows induction of cell death in ATRA responsive This work was supported by grants from Blueprint (282510; H.G. bona fide Stunnenberg, L. Altucci) and Italian Flag Project: EPIGEN (AIRC no. 11812, and in APL ATRA-resistant cells. Accordingly, PRIN_2009PX2T2E_004, PON0101227; L. Altucci). MC2392 is not inducing apoptosis in solid tumor such as The costs of publication of this article were defrayed in part by the payment prostate or breast cancer (data not shown). Moreover, ChIP- of page charges. This article must therefore be hereby marked advertisement seq profiles show that MC2392 induces changes in H3 acety- in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. lation at a small subset of PML-RARa/RXR binding regions but Received September 5, 2013; revised February 6, 2014; accepted February 9, also in regions not regulated by ATRA (Figs. 2 and 3). Unlike 2014; published OnlineFirst February 24, 2014.

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OF12 Cancer Res; 74(8) April 15, 2014 Cancer Research

Context-Selective Death of Acute Myeloid Leukemia Cells Triggered by the Novel Hybrid Retinoid-HDAC Inhibitor MC2392

Floriana De Bellis, Vincenzo Carafa, Mariarosaria Conte, et al.

Cancer Res Published OnlineFirst February 24, 2014.

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