Bromodomain-Selective BET Inhibitors Are Potent Antitumor Agents Against MYC

Author Manuscript Published OnlineFirst on July 10, 2020; DOI: 10.1158/0008-5472.CAN-19-3934 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Bromodomain-selective BET inhibitors are potent antitumor agents against MYC-

driven pediatric cancer

P. Jake Slavish1,*, Liying Chi1,*, Mi-Kyung Yun2,*, Lyudmila Tsurkan1,*, Nancy E.

Martinez1, Barbara Jonchere3, Sergio C. Chai1, Michele Connelly1, M. Brett Waddell4,

Sourav Das1, Geoffrey Neale5, Zhenmei Li2, William R. Shadrick1,†, Rachelle R.

Olsen6,‡, Kevin W. Freeman6,♦, Jonathan A. Low1, Jeanine E. Price1, Brandon M.

Young1, Nagakumar Bharatham1, Vincent A. Boyd1,ǂ, Jun Yang7, Richard E. Lee1, Marie

Morfouace3,▲, Martine F. Roussel3, Taosheng Chen1, Daniel Savic8, R. Kiplin Guy1,▼,

Stephen W. White2, Anang A. Shelat1 and Philip M. Potter1

* - These authors contributed equally to this work.

† - Present address: Vaccine Production Program, VRC, NIAID, NIH, Gaithersburg, MD,

USA.

‡ - Present address: Department of Oncological Sciences, Huntsman Cancer Institute,

Salt Lake City, UT, USA.

♦ - Present address: Genetics, Genomics & Informatics, The University of Tennessee

Health Science Center, Memphis, TN, USA.

1 Department of Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA 2 Department of Structural Biology, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA 3 Department of Tumor Cell Biology, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA 4 Molecular Interaction Analysis Shared Resource, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA 5 Hartwell Center, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA 6 Department of Oncology, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA 7 Department of Surgery, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA 8 Department of Pharmaceutical Sciences, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA 1

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 10, 2020; DOI: 10.1158/0008-5472.CAN-19-3934 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

ǂ - Deceased.

▲ - Present address: EORTC, Avenue E. Mounier 83, 1200 Brussels, Belgium.

▼ - Present address: College of Pharmacy, University of Kentucky, Lexington, KY, USA.

Running title: Bromodomain-selective BET inhibitors

Keywords: Bromodomain, inhibitor, neuroblastoma.

Financial support: This work was supported in part by a NIH grant P01 CA096832 to

MFR, a NIH grant R01 CA225945 to AAS and PMP, a Cancer Center Core grant (NCI

P30 CA021765), and by the American Lebanese Syrian Associated Charities (ALSAC).

Corresponding authors:

Dr. Philip M. Potter, Department of Chemical Biology and Therapeutics,

St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105.

Tel: +1 901-595-5749; Email: [email protected].

Dr. Anang A. Shelat, Department of Chemical Biology and Therapeutics, St. Jude

Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105. Tel: +1

901-595-5749; Email: [email protected].

The authors declare no conflicts of interest.

Abbreviations

The abbreviation used in this article are: ALL – acute lymphoblastic leukemia; AML – acute myelogenous leukemia; BD – bromodomain; BET – Bromodomain and extra terminal protein; BETi – BET inhibitor; FRAP – fluorescence recovery after photobleaching; IPA – Ingenuity Pathway Analysis ; H3K27ac – histone H3, lysine 27 acetylation; i.p. – intra-peritoneal; ITC – isothermal calorimetry; JQ1 – tert-butyl 2-[(9S)-

7-(4-chlorophenyl)-4,5,13-trimethyl-3-thia-1,8,11,12-tetrazatricyclo[8.3.0.02,6]trideca-

2(6),4,7,10,12-pentaen-9-yl]acetate; MB – medulloblastoma; NB – neuroblastoma; RMS

– rhabdomyosarcoma; SJ018 – methyl 4-(((2S,4R)-6-(3-acetamidophenyl)-1-acetyl-2- methyl-1,2,3,4-tetrahydroquinolin-4-yl) amino) benzoate; SJ432 – 4-(((2S,4R)-1-acetyl-

2-methyl-6-(1H-pyrazol-3-yl)-1,2,3,4-tetrahydroquinolin-4-yl)amino)benzonitrile; SPR – surface plasmon resonance; THQ – tetrahydroquinoline; TF – transcription factor; TR-

FRET – time-resolved fluorescence energy transfer.

ABSTRACT Inhibition of members of the bromodomain and extra terminal (BET) family of proteins has proven a valid strategy for cancer chemotherapy. All BET identified to date contain two bromodomains (BD; BD1 and BD2) that are necessary for recognition of acetylated lysine residues in the N-terminal regions of histones. Chemical matter that targets BET

(BETi) also interact via these domains. Molecular and cellular data indicate that BD1 and BD2 have different biological roles depending upon their cellular context, with BD2 particularly associated with cancer. We have therefore pursued the development of

BD2-selective molecules both as chemical probes and as potential leads for drug development. Here we report the structure-based generation of a novel series of tetrahydroquinoline analogs that exhibit >50-fold selectivity for BD2 versus BD1. This selective targeting resulted in engagement with BD-containing proteins in cells, resulting in modulation of MYC proteins and downstream targets. These compounds were potent cytotoxins towards numerous pediatric cancer cell lines and were minimally toxic to non- tumorigenic cells. Additionally, unlike the pan BETi (+)-JQ1, these BD2-selective inhibitors demonstrated no rebound expression effects. Finally, we report a PK- optimized, metabolically stable derivative that induced growth delay in a neuroblastoma xenograft model with minimal toxicity. We conclude that BD2-selective agents are valid candidates for antitumor drug design for pediatric malignancies driven by the MYC oncogene.

INTRODUCTION

The targeting of chromatin modifying proteins has become an attractive avenue for drug design, specifically for antitumor agents (1-3). Consequently, there are considerable ongoing efforts to develop chemical matter that demonstrates specificity for such proteins. The bromodomain and extra terminal (BET) family of proteins, which includes

BRD2, BRD3, BRD4 and BRDT, contains two domains (BD1 and BD2) that are required for the recognition of acetylated lysine residues in histones (4,5). These bromodomains

(BD) demonstrate considerable amino acid and structural homology, and hence, it has been difficult to develop small molecules with significant selectivity towards either BD1 or BD2 (6). Therefore, most clinical candidates that target BET inhibit both BD with similar efficiencies. This includes the exemplar compound (+)-JQ1 (1) (1), based upon a benzodiazepine scaffold, which is currently the basis for at least 4 molecules in clinical trials (see http://clinicaltrials.gov).

However, biochemical and cellular studies have revealed that independently modulating these domains results in different downstream sequelae, arguing that the recognition of acetylated lysine residues by BD1 and BD2 is specific and leads to the regulation of disparate gene sets (7-11). For example, BRDT-BD1 (but not BRDT-BD2) is required for spermatogenesis (12). Similarly, isothermal titration calorimetry (ITC) studies indicate that BD1 preferentially binds to histone H3 sequences, whereas BD2 has higher affinity for histone H4 and acetylated lysine peptides derived from cyclin T1 (13).

BRD3-BD1 has been shown to be important for the expression of erythroid and megakaryocyte-specific genes through interactions with the acetylated transcription

factor (TF) GATA1 (7). In contrast, BRD4-BD2 recruits TWIST, and BRD4-BD1 serves

to anchor the complex to chromatin (14). Since there are greater sequence homologies among the BET BD1 BDs and BD2 BDs, as compared to BD1 versus BD2, this

suggests that the separate bromodomains have different biological functions. We

hypothesized therefore, that targeting either BD1 or BD2 may be exploited in anticancer drug design.

We have developed BD-selective BET inhibitors (BETi) chemical probes that target BD1

and BD2s, and assessed their activity in pediatric tumor models. Using the

tetrahydroquinoline (THQ) scaffold (6,15) and a combination of structure-based iterative

drug design, coupled with biochemical analyses, we have generated highly potent, BD2- selective BETi. These molecules: bind BET in cultured cells; downregulate MYC and associated targets; are cytotoxic to tumor, but less so to non-tumorigenic, cells; and, are minimally toxic in vivo and induce antitumor activity. While BD-selective probes have been synthesized previously (16-18), our studies seek to validate the activity of such

compounds towards pediatric tumors with dire prognoses.

MATERIALS AND METHODS

Reagents

The list of cell lines, their sources, culture conditions, and antibodies used in this study

are reported in the Supplementary Information (Tables S1, S2 and S3). Three

neuroblastoma (NB) lines were used for subsequent studies based upon their MYC expression status: SJ-N-AS – Amplified C-MYC, no expression of MYCN; SK-N-SH –

Expresses C-MYC, no expression of MYCN; IMR32 – No expression of C-MYC, MYNC

amplified (Fig. S1). Cell lines were used directly without further testing. All synthetic

reagents were of ACS grade and purchased from Sigma-Aldrich (St. Louis, MO),

Combi-Blocks (San Diego, CA) or Strem Chemicals (Newburyport, MA). Female

CB17SCID mice (CB17/Icr-Prkdc(scid)/IcrIcoCrl; Charles River Laboratories,

Wilmington, MA) were used for therapeutic studies and were housed in an AAALAC-

accredited facility with food and water provided ad libitum.

Biophysical analyses

Time-resolved fluorescence energy transfer (TR-FRET) was undertaken using

commercially available kits (Cayman Chemical, Ann Arbor, MI). Surface plasmon

resonance (SPR) was undertaken on a Pioneer optical biosensor (ForteBio) using

polyHis-tagged BD domains (BRD2-BD1 amino acids 74-194; BRD2-BD2 amino acids

348-455). More detail is presented in the Supporting Information. Isothermal calorimetry

(ITC) experiments were performed on an iTC200 (MicroCal Panalytical, Malvern, UK) and peak areas were integrated, normalized, and fitted using the independent sites

model in Origin software (MicroCal Panalytical). BETi selectivity was validated using

BROMOscan analysis (DiscoveRX, Fremont, CA).

Cytotoxicity assays

Cytotoxicity of BETi was determined using either Alamar Blue or Cell Titer Glo (see

Supporting Information for further details).

Fluorescence recovery after photobleaching

Fluorescence recovery after photobleaching (FRAP) was determined in U2OS cells expressing BRD4 protein fused with GFP. Microscopy images were acquired using a

Yokogawa CSU-X spinning disc confocal microscope with multiple pre-bleach images of nuclei collected to establish a baseline. A single laser pulse lasting 0.09 s was then targeted onto the nucleus and single section images were collected at 200ms intervals.

Fluorescence intensity plots were generated using Slidebook 6 x64 software (3i

Technologies, Denver, CO), and FRAP determined as previously described (19).

Microarray

Total RNA was hybridized to an Affymetrix Human Gene 2.0 ST Array (ThermoFisher

Scientific) and signals normalized using the multi-array average algorithm (20).

Datasets were subsequently analyzed by a one-factor ANOVA model (Partek Genomics

Suite) where drug plus concentration were used as the treatment factor. A FDR threshold of <0.05 was used to identify differentially expressed transcripts and were

analyzed for functional enrichment using the Enrichr (21) and DAVID (22) bioinformatics

databases.

ChIP-seq

ChIP-seq was performed as previously described (23). Differential histone 3, lysine 27

acetylation (H3K27ac) read enrichment was identified using DESeq2 (24) on normalized

read depth at the union of all reproducible H3K27ac sites. Principal component analysis

was performed using the prcomp function in R. CentriMo (25) and was used to identify

enriched TF motifs within differentially enriched H3K27ac sites.

BRD2-BD1 and BRD2-BD2 expression

cDNAs encoding the human BRD2-BD1 (residues 67-200) and BRD2-BD2 (amino acids

348-455) domains were expressed from pET28a(+) containing a N-terminal His-tag.

Detailed methods are provided in the Supporting Information.

Crystallographic analyses

Structure of BRD2-BD1/SJ432 complex was obtained by soaking apo crystals in 1.5mM

SJ432 for 2 days. BRD2-BD2/SJ432 complexes were pre-formed in solution and then

crystallized. Crystals were grown using the sitting drop vapor diffusion method at 18C and all diffraction data were collected at the SERCAT beam lines 22-BM and 22-ID at the Advanced Photon Source. The BD1/SJ432 and BD2/SJ432 structures were solved by molecular replacement using, respectively, BD1 (PDB 4UYH) and BD2 (PDB 5IG6) of BRD2 as search models, and refined and optimized using PHENIX and COOT

(26,27). Data collection statistics are summarized in the Supporting Information (BRD2-

BD1/SJ432 - PDB 2DVQ; BRD2-BD2/SJ432 - PDB 2E3K).

Immunoblotting analyses

Immunoreactivity of protein extracts to desired antibodies was undertaken using

standard approaches. See Supporting Information for antibodies used and their

respective working dilutions.

Pharmacokinetic studies

Pharmacokinetics studies were conducted by SAI Life Sciences Ltd (Pune, India) using

female athymic nude mice (ACTREC, Mumbai, India). Molecules were administered i.p., and at time intervals ranging from 15 min to 24 h, animals were humanely sacrificed and the levels of free drug present in the plasma and brain tissue were determined. All data points were conducted in triplicate.

Pre-clinical studies

Six- to eight-week old CB17SCID female mice were injected into the flank with 1 x106

SK-N-AS cells resuspended in Matrigel matrix (Corning, Manassas, VA) and tumors

were allowed to grow until they reached ~225 mm3. SJ432, formulated in 5% 1-methyl-

2-pyrrolidinone, 5% Solutol HS15 (Sigma Biochemicals) and 90% saline, was

administered intra-peritoneally (i.p.) daily for 14 days. JQ1, given by the same route and

schedule, was formulated in 10% (2-hydroxypropil)-β-cyclodextrin solution (Sigma

Biochemicals), 10% DMSO and 80% saline. Ten mice per group were used. Tumors

were measured using digital calipers and volumes were calculated (V = (L x W2)/2).

Toxicity was assessed primarily by weight loss, but also by daily examination by individuals with no knowledge of the treatment protocol. All animal studies were approved by the St. Jude Children’s Research Hospital Institutional Animal Care and

Use Committee.

RESULTS

Rational design of BD2-selective BETi

Previously, we reported that amino acid residue variations between BD1 and BD2

induce differences in the water networks that could be exploited by heteroaryl-

substituted THQ to achieve BD-selectivity (6,15). However, the identified lead

compound, SJ599 (2), showed only modest BD2-selectivity and the 2-furan group would

be a liability for in vivo use. Based on our analysis of the co-crystal structure of 2 bound

to BRD2-BD2 (PDB: 5EK9), we hypothesized that meta-substituted phenyl substituents

(3-6) or indole (7) could stabilize the water network present in BD2 (Figs. 1A-C).

Unfortunately, no improvement in BD2-selectivity was obtained (Fig. 1C), although the m-acetamide (3) and m-aniline (4) analogs demonstrated increased potency towards

BRD2-BD2. Increasing the steric bulk on the acetamide (8-13), improved BD2- selectivity, resulting in higher lipophilicity and decreased ligand efficiency (LiPE).

Previously, we found that replacing the isopropyl-carbamate at R1 with an aryl group enhanced BD2-selectivity. Therefore, we generated analogs, holding the m-acetamide at R2 fixed, and varying the R1 position (14-22). These compounds were more potent towards BRD2-BD2, with selectivity, as compared to BRD2-BD1, ranging from 6.9- to

66.5-fold.

Based on potency towards BD2 (Kd=14nM), and differential activity against BD1 (~67-

fold), we investigated 22 (‘SJ018’) in more detail using three orthogonal biophysical

assays: ITC, SPR, and a BD binding assay (BROMOscan) (Figs. 2A-D, Table S4).

Excellent agreement between the outputs was observed and the latter confirmed on-

target BET inhibition, indicating that modifications made to the THQ scaffold did not

alter BET specificity. To assess on-target engagement of SJ018 to BET in cells, we used FRAP (19) and compared our results to those obtained when using the pan-BD

BETi, JQ1 (1). In FRAP, a molecule that inhibits binding of BET to chromatin increases

the fluorescence recovery process as the protein is no longer bound to histones and

can diffuse much faster. Hence, this approach provides a direct measure of in vivo

displacement of BET from chromatin. SJ018 was more effective than JQ1 in increasing

FRAP (Fig. 2E), suggesting that BD2-binding alone is enough to evict BRD4 from

chromatin.

Cytotoxicity studies comparing SJ018 and JQ1

Having established intracellular binding of SJ018 to BRD4, we evaluated the cytotoxicity

of this molecule compared to JQ1 in a diverse panel of pediatric tumor cell lines (Fig.

2A). SJ018 was more active against all evaluated cells types, as compared to JQ1,

except for the ALL lines, Nalm16 and Loucy. Toxicity was generally time-dependent,

with activity increasing with longer drug exposure times. We used extended periods of

treatment in these assays since it was not clear how long it would take for compounds

that modified chromatin to induce their cytotoxic action. Consistent with our previous

studies, little toxicity was observed with the THQ BETi scaffold against two non-

tumorigenic cell lines (BJ – fibroblast; LHCN-M2 – myoblast), even after long drug

exposure times, arguing that BETi demonstrate inherent antitumor selectivity.

Modulation of gene expression by SJ018 and JQ1

To assess the molecular consequences of selective BD2 inhibition, we exposed three different and diverse pediatric tumor cell lines (MV4-11 – AML; HDMB03 – Group 3 medulloblastoma; Kelly – NB) to SJ018 or JQ1 at 6 different concentrations (25-4000 nM), and performed microarray analyses after 3 hours. Data were pooled across cell lines and normalized to eliminate cell line specific effects. Genes differentially expressed relative to DMSO, following drug treatment at each concentration, were identified using ANOVA (FDR<0.05). Consequently, data obtained from these three experiments were independent of cell type and better represented the most consistent changes of expression induced by each drug and the respective dose.

Changes in transcription following treatment with SJ018 occurred at significantly lower concentrations than that observed with JQ1. At the lowest dose tested (25nM), 628 of the 651 differences (97%) were exclusively due to SJ018, 20 were common (3%), and only 3 (<1%) were unique to JQ1 (Fig. 3A). Importantly, it required 100nM JQ1 to induce statistically significant changes in most (545/628=87%) of the genes perturbed by SJ018 at 25nM. At 100nM, JQ1 induced changes in a total of 2042 genes, indicating that potency alone, as opposed to BD-selectivity, was unlikely to account for the differential gene expression pattern associated with this compound. As the dose of each agent increased, the pattern of gene expression changes converged. At 4000nM, both drugs differentially expressed 68% of all perturbed genes (6917/10216).

Yet, it is important to note that there was still strong concordance between gene expression changes at each drug concentration when looking at all genes, not just

those meeting the criteria for statistical significance (Fig. 3B). The concordance improved steadily (as reflected by Pearson R-squared values) with increasing molecule concentration, consistent with the hypothesis that SJ018 behaved more like a pan-BD inhibitor at higher concentrations. However, while the slope of the regression line at 25 nM was not unity (slope = 1.64), the R-squared was 0.86, indicating that most of the genes that were differentially expressed by SJ018 were also perturbed by JQ1.

Therefore, the extent to which SJ018 selectively perturbed BD2-responsive genes at low drug concentration was modest.

To further investigate changes in RNA expression induced by SJ018 and JQ1, the list of genes that significantly changed (FDR<0.05) were divided into two groups: (a) “25 nM”, which contained all genes altered at the lowest concentration tested (25nM), and was

primarily driven by SJ018; and (b) “≥200 nM”, which contained all genes perturbed by

SJ018, JQ1, or both at concentrations greater than or equal to 200nM (Tables S5 and

S6). Analysis using Enrichr (21,28) indicated that the 25 nM gene set was significantly

enriched for biological processes associated with modification of chromatin, namely

histone lysine acetyltransferase and histone deacetylase activities, as well as genes

involved in RNA transcription. In contrast, the ≥200 nM gene set was significantly

enriched for functions associated with the cell cycle, DNA repair and replication, RNA

metabolism (ncRNA, rRNA, tRNA), stress response, and ubiquitin-associated

proteolysis. This also included chromatin modification genes with methyltransferase

activity. Furthermore, analysis of both gene sets predicted alteration of two different

transcription factor protein:protein interaction networks (Tables S7 and S8). Both

contained TP53 and FOXP3, but the 25 nM set contained histone acetyltransferases

(CLOCK, HDAC8), while the ≥200 nM set contained MYC, and other stimulus-response

genes (ESR1, ESR2, STAT1, SMAD2).

To better explore the differences between the two drugs, a 2-factor (compound and

concentration) ANOVA was performed on the batch-adjusted data sets after removing

the untreated and DMSO samples. This analysis identified 382 transcripts (291 unique

genes) that were differentially expressed between SJ018 and JQ1 (FDR<0.05). The

magnitude of the ratio of expression (fold changes) in this set was small and ranged

from 0.86 to 1.25 (Table S9). The top DAVID (29) function enrichment cluster contained

the terms “Transcription”, “Transcription regulation”, and “DNA-binding” (Table S10).

“DNA Methylation and Transcriptional Repression Signaling” was the top pathway

returned by Ingenuity Pathway Analysis (IPA) Canonical Pathway (30) (Fig. S2).

Moreover, the top gene identified by IPA as an upstream regulator was CLOCK, which was also identified earlier in the comparison between genes expressed at low and high drug concentrations.

ChIP-seq studies with SJ018 and JQ1

To further explore differences between the epigenetic changes induced by SJ018 and

JQ1, we undertook ChIP-seq studies (31) on drug-treated MV4-11 cells to identify loci across the genome that are perturbed by BETi exposure. H3K27ac, a marker for

transcriptionally active chromatin (32), was monitored as microarray showed BETi induced both activation and repression of gene expression. ChIP-seq experiments

showed high concordance between triplicate samples, and principal component

analysis showed clear delineation of BETi treatment groups (Figs. S3 and S4).

Interestingly, both compounds at 25nM caused a similar change in the number of

H3K27ac sites that demonstrated either a decrease, or increase, in ChIP read

enrichment, relative to DMSO (Fig. 4A, 1,259 versus 1,359 H3K27ac sites at FDR<0.01

for SJ018 and JQ1, respectively). Moreover, JQ1 induced 71.2% more changes at 1µM

(7,553 versus 12,952 H3K27ac sites at FDR<0.01 for SJ018 and JQ1, respectively), even though both compounds showed a similar number of differentially-expressed

genes and higher concordance at this concentration (Fig. 3). These observations

indicate that JQ1 disrupts chromatin to a greater extent than SJ018.

As consistent activation or repression of a site is an indicator of an on-target effect, both

molecules demonstrated near perfect perturbation: 952 of 953 (99.9%) of SJ018

modulated sites and 1171 of 1173 (99.8%) of JQ1 modulated sites. Moreover, we

observed consistently stronger perturbations at 1µM compared to 25nM for both drugs:

902 of 952 (94.7%) of SJ018 modulated sites and 1071 of 1171 (91.4%) of JQ1

modulated sites (Fig. 4B). However, with SJ018 at 25nM, a larger percentage of sites

showed decreases in H3K27ac enrichment versus increases, as compared to JQ1

(59.5% vs 38.7%). At 1µM of each compound, this disparity was reduced (58.8% vs

53.4%). Examples where chromatin was found to be selectively activated by JQ1 or repressed by SJ018 include the NT5C2 gene locus and LINC01565 and RPN1 gene

loci, respectively (Figs. 4C and 4D). Finally, an analysis of TF motif enrichment at

H3K27ac sites indicated that the E26 transformation-specific (ETS) family of TFs

(ETV6, ELK3, ELF5, ERG, GABPA, etc.), RUNX family TFs (RUNX2 and RUNX3), AP-

2 family TFs (TFAP2A, TFAP2B and TFAP2C), and ZIC family TFs (ZIC1, ZIC3 and

ZIC4) demonstrated the greatest increases. However, at both 25nM and 1µM, JQ1 sites that exhibited increased H3K27ac enrichment were also highly enriched for CCAAT- enhancer-binding family TF motifs (CEBPB, CEBPE, CEBPG and CEBPD). This was

not observed with SJ018. In summary, these ChIP-seq experiments suggest that SJ018

induces greater selectivity with respect to chromatin modification than JQ1.

Design, synthesis and biological activity of a metabolically stable, PK-optimized,

BD2-selective BETi

While SJ018 demonstrated excellent selectivity for BD2 versus BD1, it contained

significant liabilities that would preclude its use in vivo. These were eliminated by

replacement of the ester on the A ring with a nitrile group, and substitution of the m-

phenylacetamide (D ring) with 2,3-pyrazole. The resulting molecule, SJ432 (Fig. 5A),

maintained potency and selectivity for BD2 versus BD1 in the TR-FRET assay for both

BRD2 and BRD4 (6nM and 2nM, respectively, >80-fold more potent that that seen with

BD1), was considerably more water soluble than SJ018 (~40µM vs <0.1µM), and was

stable in biological samples (Table S11-S12). BD2 selectivity towards BRD2, 3, 4 and

BRDT was independently confirmed using BROMOScan and SPR (Table S13-S14, Fig.

S5-S7). The cytotoxicity of SJ432 toward MV4-11 was within <3-fold of SJ018 (8nM vs

3nM), and almost 2-fold higher than JQ1; in contrast, the enantiomer of SJ432, SJ432-

neg, was >42-fold less potent (Fig. S8).

Structural analysis of SJ432 binding to BRD2-BD1 and BRD2-BD2

To better understand the basis of BD selectivity for SJ432, we undertook X-ray

crystallography of the complexes with isolated BRD2-BD1 and BRD2-BD2 domains.

Following previous studies (33,34), we obtained well diffracting crystals from constructs encompassing residues 67-200 of BD1 and 348-455 of BD2, and determined high quality complex structures at 1.5Å and 1.05Å, respectively (Table S15, Fig. S9; PDB

2DVQ and PDB 2E3K). SJ432 binds BD1 and BD2 similarly (Figs. 5B and 5C) and the

three component moieties each engage three distinct pockets. The pyrazole moiety

occupies an open pocket bounded by Trp97/370 and Leu108/381. The ring is oriented

such that the two nitrogen atoms are exposed to the solvent, but the ring is flipped by

180° due to crystal packing in the one of the three BD1 complex structures in the

asymmetric unit, reflecting the loose interactions of this moiety (Fig. S10).

The central THQ moiety binds within a tight conserved hydrophobic pocket bounded by

Pro98/371 (BD1/BD2), Phe99/372 and Ile162/Val435 on one side, and Val103/376,

Leu108/381 and Leu110/383 on the opposite side. The methyl group at the C2 position

of THQ ring contributes to the latter interactions, but also extends out to make additional

van der Waals interactions with Tyr113/386 and Tyr155/428. The oxygen atom of the

acetyl group at the N1 position forms a hydrogen bond with the ND2 nitrogen of

Asn156/429 and the methyl group makes van der Waals interactions with

Ile162/Val435, Val103/376, Pro98/371 and Phe99/372. Comparison with the published

structures of BD1 and BD2 in complex with their cognate peptides reveals that the THQ

moiety largely occupies the conserved binding pocket for the acetylated lysine side

chain in which the terminal acetyl oxygen forms a hydrogen bond with the ND2 nitrogen

of Asn156/429 (Figs. 5D and 5E).

The 4-(methylamino) benzonitrile moiety of SJ432 also binds to a conserved pocket

bounded by Trp97/370, Pro98/371, Asp161/434, Met165/438 and Ile162/Val435. In

addition, there is a hydrogen bond interaction between the linker nitrogen atom and the

main chain carbonyl oxygen of Leu108/381 that is mediated by two bridging water

molecules. However, the moiety adopts a small but significantly different conformation

that suggests a tighter binding in the BD2 complex (Fig. 5F). Specifically, His433 forms

a π-π stacking interaction with the phenyl ring in the BD2 complex whereas its

counterpart Asp160 does not in the BD1 complex. The smaller Val435 in BD2, as

compared to Ile162 in BD1, allows the moiety to penetrate deeper into the pocket. In

addition, the conformations of Trp370 and Pro371 in BD2 create a slightly smaller

pocket than Trp97 and Pro98 in BD1 that is also apparent when comparing the apo

structures. One caveat to the His433/Asp160 difference is that a crystallographic

ethylene glycol molecule occupies the His433 location in the BD1 structure that may

prevent the equivalent interaction with Asp160. His433 forms a hydrogen bond with

amide nitrogen atom of Val435 in the BD2 complex, and ethylene glycol and a water

molecule make hydrogen bind interactions with amide nitrogen atoms of Asp161 and

Ile162 in the BD1 complex (Fig. S10). However, there are three complexes in the asymmetric unit of the BD1 structure and Asp160 adopts the same orientation in all three including one that has water molecules and not ethylene glycol at this location.

Our data indicate that the selectivity of SJ432 for BD2 over BD1 relates to the different peptide specificities. The almost identical interactions of the THQ core with BD1 and

BD2, together with the loose interactions of the pyrazole moiety, suggest that the selectivity resides in the different binding modes of the benzonitrile moiety. Comparison with the peptide complexes of BD1 and BD2 reveals that the benzonitrile binds adjacent to the peptide binding locale. The specificity can be rationalized by Asp160 in BD1 and

His433 in BD2 that both have central roles in binding the respective peptides, but have different contributions in binding the benzonitrile. Thus, whereas His433 forms the favorable π-π stacking interaction with the phenyl ring, a similar interaction would not expected for Asp160. We also note that His433 contributes to the water bridging hydrogen bonding interaction between the linker nitrogen atom and the main chain carbonyl oxygen of Leu381 via a third water molecule (Fig. 5E). Computational analysis revealed that in the BD2/SJ432 complex, where the benzonitrile moiety more tightly engages the pocket, SJ432 demonstrates a lower intrinsic strain energy than in the

BD1/SJ432 complex (28.9kcal/mol vs 31.3 kcal/mol, respectively). Furthermore, ΔG for

SJ432 with BD2 was calculated to be -67.11 kcal/mol, as compared to -65.21 kcal/mol for BD1. These structural and computational studies provide a detailed rationale for the selectivity of SJ432 with BD2.

Modulation of MYC expression and downstream target genes by SJ432

Since BETi are known to modulate MYC expression, we assessed the ability of SJ432 to alter C-MYC and MYCN in a series of NB cell lines. Three lines were chosen (SK-N-

AS – C-MYC amplified; SK-N-SH – C-MYC overexpressed, but not amplified; and

IMR32 – MYCN amplified) and were exposed to SJ432 or JQ1. SJ432 was more

effective than JQ1 at reducing levels of MYC protein in NB cell lines (see Fig. 6 and

Fig. S11). This occurs at both lower drug concentrations and over shorter time frames as compared to JQ1. It should also be noted that SJ432 did not induce a rebound effect in C-MYC expression, in contrast to JQ1 (e.g., see the 0.04µM data points in Figs. 6A and 6B, and 0.80µM value in Fig. S11). Additionally, C-MYC target genes that were

either up- (p21 and GADD45H) or down- (CCND2 and ODC1) regulated following

exposure to SJ432 were readily observed in SK-N-AS following treatment (Fig. 6C).

Finally, the upregulation of HEXIM1, a validated target for BET inhibition, was more

robust and durable in SJ432-treated cells (Fig. 6D).

We also analyzed the levels of BRD2 and BRD4 in SK-N-AS cells following drug treatment. Both compounds induced the rapid loss of both proteins, with continued and

extended suppression of BRD4 (Figs. 6E and 6F). Similar results were also observed in

a C-MYC amplified human pediatric Group 3 medulloblastoma line, HDMB03 (35) (Fig.

S12). Treatment of cells with MG132 did not reduce the loss of BRD4, indicating that this event is not mediated via the proteasome (Fig. S13).

Pharmacokinetic parameters for SJ432

Having developed SJ432, we assessed its pharmacokinetic parameters in female athymic/nude mice (Table S16). These results indicated that the molecule was readily bioavailable, and yielded greater Cmax and AUC0→∞ values (~4.5-fold and ~4-fold,

respectively), but reduced clearance (~3.5-fold), as compared to JQ1. The differences in

these values indicated that SJ432 levels would be maintained at therapeutic

concentrations for longer than JQ1 in animals and should therefore be more effective in preclinical studies (see Fig. S14). We also noted that the brain penetration for SJ432

was >100-fold lower than that for the pan BETi (Table S16), arguing that SJ432 would

not demonstrate the inherent neurotoxicity seen with JQ1. No serious adverse toxicity

was seen in these animals and hence MTD studies were undertaken. These

experiments indicated that 15 mg/kg was minimally toxic when given i.p. using a daily

dosing schedule for 14 consecutive days.

Antitumor activity of BD2-selective BETi

We assessed the activity of SJ432 towards the pediatric NB xenograft SK-N-AS.

Female CB17SCID mice bearing flank tumors, were dosed with i.p. with SJ432 daily for

14 days. We observed a pharmacological dose response that resulted in significant

tumor growth delays (Fig. 7A). Direct comparison of the activity of SJ432 (15 mg/kg)

with JQ1 (50 mg/kg) resulted in a significant reduction in tumor volumes by the former,

and increased animal survival, with median survivals of 31 (p=0.0002 vs control) and 26

(p=0.0064 vs control) days, respectively (Fig. 7B and 7C). More impressively however,

no serious toxicities were observed in SJ432-treated mice, with essentially no loss in body weight for the duration of the study (Fig. 7D). This contrasts with studies with JQ1

where mice demonstrated morbidity, weight loss and a failure to thrive.

An analysis of tumor material harvested from SJ432-treated mice, either 4 or 24 hours

after the last dose of a 14-day consecutive treatment, demonstrated a reduction in the

level of C-MYC, loss of BRD4, and upregulation of HEXIM1 (Fig. 7E). At 24 hours after

the last dose of drug, BRD4 was essentially undetectable and HEXIM1 was increased

by ~5.4 fold (Fig. 7F). Levels of BRD2 were transiently elevated in the tumor harvested

at 4h post dosing, but essentially returned to control levels after 24h. These results are consistent with our in vitro studies using this same drug and cell line (Figs. 6D and 6E).

DISCUSSION

All BET proteins contain two BDs, indicating that the duplication and architecture of

these domains are required for their biological function. However, the previous targeting

of BET has focused on the development of small molecules that interact with these

proteins without regard to BD-selectivity. Since evidence in the literature indicates

differential effects of the different BD domains (7-11), we have generated BD2-selective

BETi based upon a THQ scaffold. Using SJ018 as a tool compound, we evaluated the

molecular and cellular consequence of selective inhibition of the BD2 domain.

Microarray studies indicated that modulation of expression of different gene sets was

observed in comparison to JQ1 in 3 tumor cell lines of different histologic origin (AML,

NB, and MB). By selecting a diverse panel of lines for these analyses, the derived

datasets would be agnostic of cell of origin and more reflective of drug activity. Further

validation using ChIPseq confirmed changes in H3K27ac in chromatin indicating that

these molecular approaches allowed discrimination and identification of genes and

pathways that are specifically regulated by BD2 interaction with histones.

During the course of this project, another THQ-based BETi, IBET726, was reported

(36,37). This molecule demonstrated <100nM potency and 5-fold and 8-fold selectivity for BD2 vs BD1 in BRD2 and BRD4, respectively. In addition, recent work by Law and colleagues (38) identified compounds with comparable BD2 selectivity to SJ018.

However, no cytotoxicity or in vivo data was presented for the series of compounds.

While molecule potency, rather than BD selectivity, may have been used for compound

selection and design by these investigators, our results argue that the former is not the

only consideration that should be evaluated for development of this class of inhibitors.

In growth inhibition assays, we observed that the THQ analogs were universally active

(Fig. 2A), and generally more potent than JQ1. This occurred in essentially all tumor

histotypes and indicated that BD2-selective compounds have broad spectrum activity. It

is not known whether this is a consequence of the selective targeting of BD2, an

inherent susceptibility of the pediatric lines to such molecules, or a superior chemico-

physical and biological profile associated with the THQ scaffold. However, our results

argue that the development of such agents for pediatric tumors is warranted. While

numerous clinical trials with BETi are ongoing in adults diagnosed with a variety of

malignancies, we are aware of only one being undertaken in children (NCT03936465).

Coupled with our in vivo data demonstrating the efficacy of SJ432 with minimal toxicity, we urge the medical community to consider the use of BD2-selective BETi in pediatric patients.

The THQ analogs induced cytotoxicity in pediatric lines and potently suppressed C-

MYC and MYCN expression. Whether these effects are directly mediated via the

inhibition of these cellular pathways is unclear, but our data argue that BD2-selective compounds are more efficacious than JQ1 at modulating MYC protein levels.

Furthermore, we observed no re-expression of C-MYC protein, consistent with the lack

of a rebound effect. Since upregulation of BET proteins following exposure to BETi has

been observed (39), it is likely that this may act as a potential mechanism of drug

resistance, whereby cells transiently overcome the effects of BET inhibition by

enhanced transcription/translation of genes encoding these proteins (e.g. C-MYC). In

the NB lines we used, we observed an upregulation of C-MYC following JQ1 exposure, but not with SJ432. Presumably, the former arises from increased expression of BETs following JQ1 treatment, but we conclude that resistance to THQ-derived molecule is

unlikely to occur by this same mechanism. We also noted that BRD4 was rapidly lost

following SJ432 exposure, whereas BRD2 was not. It is not known whether the

modulation of BET proteins by this drug is important for biological activity, but THQs

may have inherent targeting of BRD4. This would explain the potent antitumor activity of

these drugs with minimal off-target toxicity.

In summary, our data indicate that dual targeting of BD domains in BET is not

necessary to induce cytotoxicity and antitumor activity. Hence, the use of BD-specific

assays, rather than those that assess binding to BET proteins as a whole, should be

sufficient to identify potent, novel BETi. We have exploited these approaches and

developed unique reagents that are effective in vitro and in vivo. These data suggest

that selective BD targeting should allow the design of more effective, and potentially,

less toxic BETi with antitumor activity.

DECLARATIONS OF INTEREST

The authors declare no conflict of interest.

AUTHORS’ CONTRIBUTIONS

Design of study – PJS, BMY, REL, MFR, TC, DS, RKG, SWW, AAS, PMP

Generation of data – PJS, LC, M-KY, LT, NEM, BJ, SCC, MC, MBW, SD, GN, ZL,

WRS, RRO, KWF, JAL, JEP, BMY, NB, VAB, JY, MM, DS

Analysis of data - PJS, LC, M-KY, LT, NEM, BJ, SCC, MC, MBW, SD, GN, ZL, WRS,

RRO, KWF, JAL, JEP, BMY, NB, MM, MFR, DS, RKG, SWW, AAS, PMP

Authorship of manuscript – PJS, GN, MFR, DS, SWW, AAS, PMP

Securing of funding – MFR, AAS, PMP

Study supervision – REL, KWF, MFR, TC, RKG, SWW, AAS, PMP

ACKNOWLEDGMENTS

We would like to acknowledge Dr. Aaron Pitre from the Cellular Imaging Shared

Resource at St. Jude Children’s Research Hospital for his assistance with the FRAP

experiments. High resolution microscopy images were acquired in the Cell & Tissue

Imaging Center at St. Jude. This work was supported in part by a NIH grant P01

CA096832 to MFR, a NIH grant R01 CA225945 to AAS and PMP, a Cancer Center

Core grant (NCI P30 CA021765), and by the American Lebanese Syrian Associated

Charities (ALSAC). The content is solely the responsibility of the authors and does not

necessarily represent the official views of the National Institutes of Health.

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Figure legends Figure 1. (A-B) Docking studies of THQ analogs of 1. (C) Synthetic route to aryl

substituted THQ and BETi BD binding affinities for derived analogs. Reagents and conditions: (a) TFA, CH2Cl2, quantitative; (b) R1-Br, K2CO3, BrettPhos Palladacycle

Gen. 3, BrettPhos, THF 100 ºC.

Figure 2. Biophysical characterization of SJ018. (A) Chemical structure, biophysical parameters and growth inhibition data. (B) SPR analysis with BRD2-BD2. (C)

BROMOscan analysis using 1 µM SJ018. (D) ITC analysis with BRD2-BD2. (E)

Comparison of BETi-modulated FRAP in U2OS cells using GFP labelled BRD4. Upper panel depicts photobleaching recovery in nucleus after a pulse of laser light (area circled in yellow). Lower panel indicates quantitation of FRAP datasets following incubation of cells with SJ018 (gray), JQ1 (gold), or DMSO (blue).

Figure 3. Microarray studies comparing the changes in gene expression induced after

3h by increasing doses of SJ018 or JQ1 across three pediatric cancer cell lines (MV4-

11, Kelly and HDMB03). (A) Venn diagrams depicting the number of significant changes

in gene expression and the number of transcripts modulated over the dose range

(FDR<0.05). (B) Scatterplots comparing all gene changes between the two drugs. All

datasets were normalized to gene expression obtained from treatment of the relevant

cell line with DMSO.

Figure 4. ChIP-seq experiments identifying areas of active chromatin in MV4-11 cells

following 3h treatment with either SJ018 or (+)-JQ1. (A) Number of H3K27ac sites

perturbed by SJ018 or JQ1 at 25 nM and 1 µM. (B) Histograms indicating that both

SJ018 and JQ1 exhibit consistent, but more pronounced changes in H3K27ac activated

and repressed sites at 1 µM versus 25 nM. (C) ChIP-seq H3K27ac read enrichment plot

at the NT5C2 gene locus where chromatin was found to be selectively activated by JQ1.

(D) ChIP-seq H3K27ac read enrichment plots at the LINC01565 and RPN1 gene loci

where chromatin was found to be selectively repressed by SJ018.

Figure 5. Crystal structures of the BRD2 complexes with SJ432. (A) SJ432 chemical

structure and indicated selectivity for BD2 with BRD2 and BRD4. (B) BRD2-BD1: SJ432

(orange). (C) BRD2-BD2: SJ432 (blue). (D) BRD2-BD1: acetylated histone H4 peptide

(magenta, PDB 2DVQ). (E) BRD2-BD2: acetylated histone H4 peptide (green, PDB

2E3K). (F) Overlay of the BRD2-BD1:SJ432 complex (orange/yellow) and BRD2-BD2

(blue/cyan).

Figure 6. Biological activity of SJ432. (A) Western analysis indicating loss of C-MYC

protein in SK-N-SH cells after 16hr exposure to either SJ432 or JQ1. (B) Quantitation of

C-MYC levels from A. (C) Modulation of MYC targets by SJ432 in SK-N-AS cells. (D)

Upregulation of HEXIM1 by SJ432 or JQ1 in SK-N-AS cells. (E) Loss of BRD4, and to a

lesser extent BRD2, following exposure of cells to SJ432 or JQ1. (F) Quantitation of

BRD4 and BRD2 protein levels from panel E.

Figure 7. In vivo activity of SJ432. In all experiments, 10 mice were used per group. (A)

Flank SK-N-AS tumor volumes in cohorts of mice treated with increasing daily doses of

SJ432 (5, 10 or 15 mg/kg). Statistical significance versus control tumors is indicated by

asterisks (blue asterisk – 15 mg/kg vs control; red asterisk – 10 mg/kg vs control; green

asterisk – 5 mg/kg vs control). (B) Comparison of tumor volumes of animals bearing SK-

N-AS xenografts following treatment with either JQ1 (50 mg/kg; blue line) or SJ432 (15

mg/kg; red line). Asterisks above the data points indicate statistical significance as

compared to control animals (red asterisk – 15 mg/kg SJ432 vs control; blue asterisk –

JQ1 vs control). Asterisks (red) below the data points indicate statistical significance between JQ1- and SJ432-treated animals. (C) Kaplan Meier curves demonstrating increased survival of animals bearing SK-N-AS tumors treated with SJ432 (15 mg/kg; red line) as compared to JQ1 (50 mg/kg; blue line), or control mice (black line). Median survivals were 22.5, 26 and 31 days for control, JQ1-treated, and SJ432-treated mice, respectively. Significance for these data sets using log rank test were as follows: SJ432 vs control – p < 0.0007; JQ1 vs control – p = 0.067; SJ432 vs JQ1 – p = 0.0791. (D)

Weights of mice from the study presented in panels B and C. (E) Western analyses indicating the levels of C-MYC, HEXIM1, BRD4 and BRD2 protein in SK-N-AS xenografts harvested either 4h or 24 h after SJ432 dosing. C – Control, untreated tumors. (F) Quantitation of the levels of protein in panel E. Data was corrected for gel loading differences using β-actin expression.

Bromodomain-selective BET inhibitors are potent antitumor agents against MYC-driven pediatric cancer

P. Jake Slavish, Liying Chi, Mi-Kyung Yun, et al.

Cancer Res Published OnlineFirst July 10, 2020.

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