Bromodomain-Selective BET Inhibitors Are Potent Antitumor Agents Against MYC-Driven Pediatric Cancer P

Published OnlineFirst July 10, 2020; DOI: 10.1158/0008-5472.CAN-19-3934

CANCER RESEARCH | METABOLISM AND CHEMICAL BIOLOGY

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,†, 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

ABSTRACT ◥ Inhibition of members of the bromodomain and extraterminal MYC proteins and downstream targets. These compounds were (BET) family of proteins has proven a valid strategy for cancer potent cytotoxins toward numerous pediatric cancer cell lines and chemotherapy. All BET identiﬁed to date contain two bromodo- were minimally toxic to nontumorigenic cells. In addition, unlike mains (BD; BD1 and BD2) that are necessary for recognition of the pan BETi (þ)-JQ1, these BD2-selective inhibitors demonstrated acetylated lysine residues in the N-terminal regions of histones. no rebound expression effects. Finally, we report a pharmacoki- Chemical matter that targets BET (BETi) also interact via these netic-optimized, metabolically stable derivative that induced domains. Molecular and cellular data indicate that BD1 and BD2 growth delay in a neuroblastoma xenograft model with minimal have different biological roles depending upon their cellular context, toxicity. We conclude that BD2-selective agents are valid candidates with BD2 particularly associated with cancer. We have therefore for antitumor drug design for pediatric malignancies driven by the pursued the development of BD2-selective molecules both as MYC oncogene. chemical probes and as potential leads for drug development. Here we report the structure-based generation of a novel series of Signiﬁcance: This study presents bromodomain-selective BET tetrahydroquinoline analogs that exhibit >50-fold selectivity for inhibitors that act as antitumor agents and demonstrates that these BD2 versus BD1. This selective targeting resulted in engagement molecules have in vivo activity towards neuroblastoma, with essen- with BD-containing proteins in cells, resulting in modulation of tially no toxicity.

1Department of Chemical Biology and Therapeutics, St. Jude Children’s Research Introduction 2 Hospital, Memphis, Tennessee. Department of Structural Biology, St. Jude The targeting of chromatin-modifying proteins has become an Children’s Research Hospital, Memphis, Tennessee. 3Department of Tumor Cell fi ’ 4 attractive avenue for drug design, speci cally for antitumor Biology, St. Jude Children s Research Hospital, Memphis, Tennessee. Molecular – Interaction Analysis Shared Resource, St. Jude Children’s Research Hospital, agents (1 3). Consequently, there are considerable ongoing efforts Memphis, Tennessee. 5Hartwell Center, St. Jude Children’s Research Hospital, to develop chemical matter that demonstrates specificity for such Memphis, Tennessee. 6Department of Oncology, St. Jude Children’s Research proteins. The bromodomain and extraterminal (BET) family of Hospital, Memphis, Tennessee. 7Department of Surgery, St. Jude Children’s proteins, which includes BRD2, BRD3, BRD4, and BRDT, contains 8 Research Hospital, Memphis, Tennessee. Department of Pharmaceutical two domains (BD1 and BD2) that are required for the recognition of ’ Sciences, St. Jude Children s Research Hospital, Memphis, Tennessee. acetylated lysine residues in histones (4, 5). These bromodomains Note: Supplementary data for this article are available at Cancer Research (BD) demonstrate considerable amino acid and structural homol- Online (http://cancerres.aacrjournals.org/). ogy, and hence, it has been difficult to develop small molecules with Current address for W.R. Shadrick: Vaccine Production Program, VRC, NIAID, significant selectivity toward either BD1 or BD2 (6). Therefore, NIH, Gaithersburg, Maryland; current address for R.R. Olsen, Department of most clinical candidates that target BET inhibit both BD with Oncological Sciences, Huntsman Cancer Institute, Salt Lake City, Utah; current similar efficiencies. This includes the exemplar compound address for K.W. Freeman, Genetics, Genomics & Informatics, The University of þ Tennessee Health Science Center, Memphis, Tennessee; current address for M. ( )-JQ1 (1), based upon a benzodiazepine scaffold, which is Morfouace, EORTC, Brussels, Belgium; and current address for R.K. Guy, College currently the basis for at least four molecules in clinical trials (see of Pharmacy, University of Kentucky, Lexington, Kentucky. http://clinicaltrials.gov). P.J. Slavish, L. Chi, M.-K. Yun, and L. Tsurkan contributed equally to this article. However, biochemical and cellular studies have revealed that independently modulating these domains results in different down- †Deceased. stream sequelae, arguing that the recognition of acetylated lysine Corresponding Authors: Philip M. Potter and Anang A, Shelat, St. Jude Chil- residues by BD1 and BD2 is specific and leads to the regulation of ’ dren s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105-2729. disparate gene sets (7–11). For example, BRDT-BD1 (but not BRDT- Phone: 901-595-2825; Fax: 901-595-4293; E-mail: [email protected]; and Anang A. Shelat, [email protected] BD2) is required for spermatogenesis (8). Similarly, isothermal titra- tion calorimetry (ITC) studies indicate that BD1 preferentially binds to – Cancer Res 2020;80:3507 18 histone H3 sequences, whereas BD2 has higher affinity for histone H4 doi: 10.1158/0008-5472.CAN-19-3934 and acetylated lysine peptides derived from cyclin T1 (12). BRD3-BD1 2020 American Association for Cancer Research. has been shown to be important for the expression of erythroid and

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megakaryocyte-specific genes through interactions with the acetylated collected to establish a baseline. A single laser pulse lasting 0.09 transcription factor (TF) GATA1 (7). In contrast, BRD4-BD2 recruits seconds was then targeted onto the nucleus and single section images TWIST, and BRD4-BD1 serves to anchor the complex to chroma- were collected at 200 ms intervals. Fluorescence intensity plots were tin (9). Because there are greater sequence homologies among the BET generated using Slidebook 6 64 software (3i Technologies), and BD1 BDs and BD2 BDs, as compared with BD1 versus BD2, this FRAP determined as described previously (17). suggests that the separate bromodomains have different biological functions. We hypothesized therefore, that targeting either BD1 or Microarray BD2 may be exploited in anticancer drug design. Total RNA was hybridized to an Affymetrix Human Gene 2.0 ST We have developed BD-selective BET inhibitors (BETi) chemical Array (Thermo Fisher Scientific) and signals normalized using probes that target BD1 and BD2s, and assessed their activity in the multiarray average algorithm (18). Datasets were subsequently pediatric tumor models. Using the tetrahydroquinoline (THQ) analyzed by a one-factor ANOVA model (Partek Genomics Suite) scaffold (6, 13) and a combination of structure-based iterative drug where drug plus concentration were used as the treatment factor. A design, coupled with biochemical analyses, we have generated FDR threshold of <0.05 was used to identify differentially expressed highly potent, BD2-selective BETi. These molecules: bind BET in transcripts and were analyzed for functional enrichment using the cultured cells; downregulate MYC and associated targets; are cyto- Enrichr (19) and DAVID (20) bioinformatics databases. toxic to tumor, but less so to nontumorigenic cells; and are minimally toxic in vivo and induce antitumor activity. While Chromatin immunoprecipitation sequencing BD-selective probes have been synthesized previously (14–16), our Chromatin immunoprecipitation sequencing (ChIP-seq) was per- studies seek to validate the activity of such compounds toward formed as described previously (21). Differential histone 3, lysine pediatric tumors with dire prognoses. 27 acetylation (H3K27ac) read enrichment was identified using DESeq2 (22) on normalized read depth at the union of all reproducible H3K27ac sites. Principal component analysis was performed using the Materials and Methods prcomp function in R. CentriMo (23) and was used to identify enriched Reagents TF motifs within differentially enriched H3K27ac sites. The list of cell lines, their sources, culture conditions, and antibodies used in this study are reported in the Supplementary Information BRD2-BD1 and BRD2-BD2 expression (Supplementary Tables S1–S3). Three neuroblastoma lines were cDNAs encoding the human BRD2-BD1 (residues 67–200) and used for subsequent studies based upon their MYC expression status: BRD2-BD2 (amino acids 348–455) domains were expressed from SJ-N-AS—Amplified c-MYC, no expression of MYCN; SK-N-SH— pET28a(þ) containing a N-terminal His-tag. Detailed methods are Expresses c-MYC, no expression of MYCN; IMR32—No expression of provided in the Supporting Information. c-MYC, MYNC amplified (Supplementary Fig. S1). Cell lines were used directly without further testing. All synthetic reagents were of Crystallographic analyses ACS grade and purchased from Sigma-Aldrich, Combi-Blocks or Structure of BRD2-BD1/SJ432 complex was obtained by soaking Strem Chemicals. Female CB17SCID mice [CB17/Icr-Prkdc(scid)/ apo crystals in 1.5 mmol/L SJ432 for 2 days. BRD2-BD2/SJ432 IcrIcoCrl; Charles River Laboratories] were used for therapeutic complexes were preformed in solution and then crystallized. Crys- studies and were housed in an Association for Assessment and tals were grown using the sitting drop vapor diffusion method at Accreditation of Laboratory Animal Care International–accredited 18C and all diffraction data were collected at the SERCAT beam facility with food and water provided ad libitum. lines 22-BM and 22-ID at the Advanced Photon Source. The BD1/ SJ432 and BD2/SJ432 structures were solved by molecular replace- Biophysical analyses ment using, respectively, BD1 (PDB 4UYH) and BD2 (PDB 5IG6) Time-resolved fluorescence energy transfer (TR-FRET) was of BRD2 as search models, and refined and optimized using undertaken using commercially available kits (Cayman Chemical). PHENIX and COOT (24, 25). Data collection statistics are sum- Surface plasmon resonance (SPR) was undertaken on a Pioneer marized in the Supporting Information (BRD2-BD1/SJ432—PDB optical biosensor (ForteBio) using polyHis-tagged BD domains 2DVQ; BRD2-BD2/SJ432—PDB 2E3K). (BRD2-BD1 amino acids 74–194; BRD2-BD2 amino acids 348– 455). More detail is presented in the Supporting Information. ITC Immunoblotting analyses experiments were performed on an iTC200 (MicroCal Panalytical) Immunoreactivity of protein extracts to desired antibodies was and peak areas were integrated, normalized, and fitted using undertaken using standard approaches. See Supporting Information the independent sites model in Origin software (MicroCal Pana- for antibodies used and their respective working dilutions. lytical). BETi selectivity was validated using BROMOscan analysis (DiscoveRX). Pharmacokinetic studies Pharmacokinetic (PK) studies were conducted by SAI Life Sciences Cytotoxicity assays Ltd using female athymic nude mice (ACTREC). Molecules were Cytotoxicity of BETi was determined using either Alamar Blue or administered intraperitoneally, and at time intervals ranging from Cell Titer Glo (see Supporting Information for further details). 15 minutes to 24 hours, animals were humanely sacrificed and the levels of free drug present in the plasma and brain tissue were Fluorescence recovery after photobleaching determined. All data points were conducted in triplicate. Fluorescence recovery after photobleaching (FRAP) was determined in U2OS cells expressing BRD4 protein fused with GFP. Preclinical studies Microscopy images were acquired using a Yokogawa CSU-X spinning Six- to eight-week-old CB17SCID female mice were injected into the disc confocal microscope with multiple prebleach images of nuclei flank with 1 106 SK-N-AS cells resuspended in Matrigel matrix

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(Corning) and tumors were allowed to grow until they reached treatment protocol. All animal studies were approved by the St. Jude approximately 225 mm3. SJ432, formulated in 5% 1-methyl-2-pyrro- Children Research Hospital Institutional Animal Care and Use lidinone, 5% Solutol HS15 (Sigma Biochemicals), and 90% saline, was Committee. administered intraperitoneally dailyfor14days.JQ1,givenbythe same route and schedule, was formulated in 10% (2-hydroxypro- pil)-b-cyclodextrinsolution(SigmaBiochemicals),10%DMSO, Results and 80% saline. Ten mice per group were used. Tumors were Rational design of BD2-selective BETi measured using digital calipers and volumes were calculated [V Previously, we reported that amino acid residue variations ¼ (L W2)/2].Toxicitywasassessedprimarilybyweightloss,but between BD1 and BD2 induce differences in the water networks also by daily examination by individuals with no knowledge of the that could be exploited by heteroaryl-substituted THQ to achieve

Figure 1. A and 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 C.

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BD-selectivity (6, 13). However, the identified lead compound, position (9, 13–20). These compounds were more potent toward SJ599 (2), showed only modest BD2-selectivity and the 2-furan BRD2-BD2, with selectivity, as compared with BRD2-BD1, ranging group would be a liability for in vivo use. On the basis of our analysis from 6.9- to 66.5-fold. K ¼ of the cocrystal structure of 2 bound to BRD2-BD2 (PDB: 5EK9), On the basis of potency toward BD2 ( d 14 nmol/L), and we hypothesized that meta-substituted phenyl substituents (3–6) differential activity against BD1 (67-fold), we investigated 22 (SJ018) or indole (7) could stabilize the water network present in BD2 in more detail using three orthogonal biophysical assays: ITC, SPR, and (Fig. 1A–C). Unfortunately, no improvement in BD2-selectivity was a BD-binding assay (BROMOscan) (Fig. 2A–D; Supplementary obtained (Fig. 1C), although the m-acetamide (3) and m-aniline (4) Table S4). Excellent agreement between the outputs was observed analogs demonstrated increased potency toward BRD2-BD2. Increas- and the latter confirmed on-target BET inhibition, indicating that ing the steric bulk on the acetamide (8–12), improved BD2-selectivity, modifications made to the THQ scaffold did not alter BET specificity. resulting in higher lipophilicity and decreased ligand efficiency. Pre- To assess on-target engagement of SJ018 to BET in cells, we used viously, we found that replacing the isopropyl-carbamate at R1 with FRAP (17) and compared our results with those obtained when using an aryl group enhanced BD2-selectivity. Therefore, we generated the pan-BD BETi, JQ1 (1). In FRAP, a molecule that inhibits binding of fi fl analogs, holding the m-acetamide at R2 xed, and varying the R1 BET to chromatin increases the uorescence recovery process as the

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 mmol/L SJ018. D, ITC analysis with BRD2-BD2. E, Comparison of BETi-modulated FRAP in U2OS cells using GFP-labeled BRD4. Top, photobleaching recovery in nucleus after a pulse of laser light (area circled in yellow). Bottom, quantitation of FRAP datasets following incubation of cells with SJ018 (gray), JQ1 (gold), or DMSO (blue).

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protein is no longer bound to histones and can diffuse much faster. most consistent changes of expression induced by each drug and the Hence, this approach provides a direct measure of in vivo displacement respective dose. of BET from chromatin. SJ018 was more effective than JQ1 in Changes in transcription following treatment with SJ018 occurred increasing FRAP (Fig. 2E), suggesting that BD2-binding alone is at significantly lower concentrations than that observed with JQ1. At enough to evict BRD4 from chromatin. the lowest dose tested (25 nmol/L), 628 of the 651 differences (97%) were exclusively due to SJ018, 20 were common (3%), and only 3 (<1%) Cytotoxicity studies comparing SJ018 and JQ1 were unique to JQ1 (Fig. 3A). Importantly, it required 100 nmol/L JQ1 Having established intracellular binding of SJ018 to BRD4, we to induce statistically significant changes in most (545/628 ¼ 87%) of evaluated the cytotoxicity of this molecule compared with JQ1 in a the genes perturbed by SJ018 at 25 nmol/L. At 100 nmol/L, JQ1 diverse panel of pediatric tumor cell lines (Fig. 2A). SJ018 was more induced changes in a total of 2,042 genes, indicating that potency alone, active against all evaluated cells types, as compared with JQ1, except for as opposed to BD-selectivity, was unlikely to account for the differ- the ALL lines, Nalm16, and Loucy. Toxicity was generally time- ential gene expression pattern associated with this compound. As the dependent, with activity increasing with longer drug exposure times. dose of each agent increased, the pattern of gene expression changes We used extended periods of treatment in these assays because it was converged. At 4,000 nmol/L, both drugs differentially expressed 68% of not clear how long it would take for compounds that modified all perturbed genes (6,917/10,216). chromatin to induce their cytotoxic action. Consistent with our Yet, it is important to note that there was still strong concordance previous studies, little toxicity was observed with the THQ BETi between gene expression changes at each drug concentration when scaffold against two nontumorigenic cell lines (BJ—fibroblast; looking at all genes, not just those meeting the criteria for statistical LHCN-M2—myoblast), even after long drug exposure times, arguing significance (Fig. 3B). The concordance improved steadily (as that BETi demonstrate inherent antitumor selectivity. reflected by Pearson R-squared values) with increasing molecule concentration, consistent with the hypothesis that SJ018 behaved Modulation of gene expression by SJ018 and JQ1 more like a pan-BD inhibitor at higher concentrations. However, To assess the molecular consequences of selective BD2 inhibition, while the slope of the regression line at 25 nmol/L was not unity we exposed three different and diverse pediatric tumor cell lines (slope ¼ 1.64), the R-squared was 0.86, indicating that most of the (MV4–11—AML; HDMB03—Group 3 medulloblastoma (MB); genes that were differentially expressed by SJ018 were also perturbed Kelly—neuroblastoma) to SJ018 or JQ1 at six different concentrations by JQ1. Therefore, the extent to which SJ018 selectively perturbed (25–4,000 nmol/L), and performed microarray analyses after 3 hours. BD2-responsive genes at low drug concentration was modest. Data were pooled across cell lines and normalized to eliminate cell To further investigate changes in RNA expression induced by SJ018 line–specific effects. Genes differentially expressed relative to DMSO, and JQ1, the list of genes that significantly changed (FDR < 0.05) were following drug treatment at each concentration, were identified using divided into two groups: (i) “25 nmol/L,” which contained all genes ANOVA (FDR < 0.05). Consequently, data obtained from these three altered at the lowest concentration tested (25 nmol/L), and was experiments were independent of cell type and better represented the primarily driven by SJ018 and (ii) “≥200 nmol/L,” which contained

Figure 3. Microarray studies comparing the changes in gene expression induced after 3 hours by increasing doses of SJ018 or JQ1 across three pediatric cancer cell lines (MV4–11, Kelly, and HDMB03). A, Venn diagrams depict- ing the number of signiﬁcant changes in gene expression and the number of transcripts modulated over the dose range (FDR < 0.05). B, Scatter plots 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.

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all genes perturbed by SJ018, JQ1, or both at concentrations greater nmol/L, a larger percentage of sites showed decreases in H3K27ac than or equal to 200 nmol/L (Supplementary Tables S5 and S6). enrichment versus increases, as compared with JQ1 (59.5% vs. 38.7%). Analysis using Enrichr (19, 26) indicated that the 25 nmol/L gene At 1 mmol/L of each compound, this disparity was reduced (58.8% vs. set was significantly enriched for biological processes associated with 53.4%). Examples where chromatin was found to be selectively acti- modification of chromatin, namely, histone lysine acetyltransferase vated by JQ1 or repressed by SJ018 include the NT5C2 gene locus and and histone deacetylase activities, as well as genes involved in RNA LINC01565 and RPN1 gene loci, respectively (Fig. 4C and D). Finally, transcription. In contrast, the ≥200 nmol/L gene set was significantly an analysis of TF motif enrichment at H3K27ac sites indicated that the enriched for functions associated with the cell cycle, DNA repair and E26 transformation-specific family of TFs (ETV6, ELK3, ELF5, ERG, replication, RNA metabolism (ncRNA, rRNA, tRNA), stress response, GABPA, etc.), RUNX family TFs (RUNX2 and RUNX3), AP-2 family and ubiquitin-associated proteolysis. This also included chromatin TFs (TFAP2A, TFAP2B, and TFAP2C), and ZIC family TFs (ZIC1, modification genes with methyltransferase activity. Furthermore, ZIC3, and ZIC4) demonstrated the greatest increases. However, at both analysis of both gene sets predicted alteration of two different TF 25 nmol/L and 1 mmol/L, JQ1 sites that exhibited increased H3K27ac protein–protein interaction networks (Supplementary Tables S7 and enrichment were also highly enriched for CCAAT-enhancer-binding S8). Both contained TP53 and FOXP3, but the 25 nmol/L set contained family TF motifs (CEBPB, CEBPE, CEBPG, and CEBPD). This was not histone acetyltransferases (CLOCK, HDAC8), while the ≥200 nmol/L observed with SJ018. In summary, these ChIP-seq experiments suggest set contained MYC, and other stimulus-response genes (ESR1, ESR2, that SJ018 induces greater selectivity with respect to chromatin STAT1, SMAD2). modification than JQ1. To better explore the differences between the two drugs, a two- factor (compound and concentration) ANOVA was performed on Design, synthesis, and biological activity of a metabolically the batch-adjusted datasets after removing the untreated and stable, PK-optimized, BD2-selective BETi DMSO samples. This analysis identified 382 transcripts (291 unique While SJ018 demonstrated excellent selectivity for BD2 versus BD1, genes) that were differentially expressed between SJ018 and JQ1 it contained significant liabilities that would preclude its use in vivo. (FDR < 0.05). The magnitude of the ratio of expression (fold These were eliminated by replacement of the ester on the A ring with a changes) in this set was small and ranged from 0.86 to 1.25 nitrile group, and substitution of the m-phenylacetamide (D ring) with (Supplementary Table S9). The top DAVID (27) function enrich- 2,3-pyrazole. The resulting molecule, SJ432 (Fig. 5A), maintained ment cluster contained the terms “Transcription,”“Transcription potency and selectivity for BD2 versus BD1 in the TR-FRET assay for regulation,” and “DNA-binding” (Supplementary Table S10). both BRD2 and BRD4 (6 nmol/L and 2 nmol/L, respectively, >80-fold “DNA Methylation and Transcriptional Repression Signaling” was more potent that that seen with BD1), was considerably more water the top pathway returned by Ingenuity Pathway Analysis (IPA) soluble than SJ018 (40 mmol/L vs. <0.1 mmol/L), and was stable in Canonical Pathway (Supplementary Fig. S2; ref. 28). Moreover, the biological samples (Supplementary Table S11 and S12). BD2-selec- top gene identified by IPA as an upstream regulator was CLOCK, tivity toward BRD2, 3, 4, and BRDT was independently confirmed which was also identified earlier in the comparison between genes using BROMOScan and SPR (Supplementary Table S13 and S14; expressed at low and high drug concentrations. Supplementary Fig. S5–S7). The cytotoxicity of SJ432 toward MV4-11 was within <3-fold of SJ018 (8 nmol/L vs. 3 nmol/L), and almost 2-fold ChIP-seq studies with SJ018 and JQ1 higher than JQ1; in contrast, the enantiomer of SJ432, SJ432-neg, was To further explore differences between the epigenetic changes >42-fold less potent (Supplementary Fig. S8). induced by SJ018 and JQ1, we undertook ChIP-seq studies (29) on drug-treated MV4–11 cells to identify loci across the genome that are Structural analysis of SJ432 binding to BRD2-BD1 and perturbed by BETi exposure. H3K27ac, a marker for transcriptionally BRD2-BD2 active chromatin (30), was monitored as microarray showed BETi To better understand the basis of BD-selectivity for SJ432, induced both activation and repression of gene expression. ChIP-seq we undertook X-ray crystallography of the complexes with isolated experiments showed high concordance between triplicate samples, BRD2-BD1 and BRD2-BD2 domains. Following previous stud- and principal component analysis showed clear delineation of BETi ies (31, 32), we obtained well-diffracting crystals from constructs treatment groups (Supplementary Figs. S3 and S4). encompassing residues 67–200 of BD1 and 348–455 of BD2, Interestingly, both compounds at 25 nmol/L caused a similar and determined high-quality complex structures at 1.5 Å and change in the number of H3K27ac sites that demonstrated either a 1.05 Å, respectively (Supplementary Table S15; Supplementary decrease, or increase, in ChIP read enrichment, relative to DMSO Fig. S9; PDB 2DVQ and PDB 2E3K). SJ432 binds BD1 and BD2 (Fig. 4A; 1,259 vs. 1,359 H3K27ac sites at FDR < 0.01 for SJ018 and similarly (Fig. 5B and C) and the three component moieties each JQ1, respectively). Moreover, JQ1 induced 71.2% more changes at 1 engage three distinct pockets. The pyrazole moiety occupies an open mmol/L (7,553 vs. 12,952 H3K27ac sites at FDR < 0.01 for SJ018 and pocket bounded by Trp97/370 and Leu108/381. The ring is oriented JQ1, respectively), even though both compounds showed a similar such that the two nitrogen atoms are exposed to the solvent, but the ring number of differentially expressed genes and higher concordance at is flipped by 180 due to crystal packing in the one of the three this concentration (Fig. 3). These observations indicate that JQ1 BD1 complex structures in the asymmetric unit, reflecting the loose disrupts chromatin to a greater extent than SJ018. interactions of this moiety (Supplementary Fig. S10). As consistent activation or repression of a site is an indicator of an The central THQ moiety binds within a tight conserved hydro- on-target effect, both molecules demonstrated near perfect perturba- phobic pocket bounded by Pro98/371 (BD1/BD2), Phe99/372, and tion: 952 of 953 (99.9%) of SJ018-modulated sites and 1,171 of 1,173 Ile162/Val435 on one side, and Val103/376, Leu108/381, and Leu110/ (99.8%) of JQ1-modulated sites. Moreover, we observed consistently 383 on the opposite side. The methyl group at the C2 position of THQ stronger perturbations at 1 mmol/L compared with 25 nmol/L for both ring contributes to the latter interactions, but also extends out to make drugs: 902 of 952 (94.7%) of SJ018-modulated sites and 1,071 of 1,171 additional van der Waals interactions with Tyr113/386 and Tyr155/ (91.4%) of JQ1-modulated sites (Fig. 4B). However, with SJ018 at 25 428. The oxygen atom of the acetyl group at the N1 position forms a

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Figure 4. ChIP-seq experiments identifying areas of active chromatin in MV4–11 cells following 3 hours treatment with either SJ018 or (þ)-JQ1. A, Number of H3K27ac sites perturbed by SJ018 or JQ1 at 25 nmol/L and 1 mmol/L. B, Histograms indicating that both SJ018 and JQ1 exhibit consistent, but more pronounced changes in H3K27ac activated and repressed sites at 1 mmol/L versus 25 nmol/L. 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.

hydrogen bond with the ND2 nitrogen of Asn156/429 and the methyl gen bond interaction between the linker nitrogen atom and the group makes van der Waals interactions with Ile162/Val435, Val103/ main chain carbonyl oxygen of Leu108/381 that is mediated by two 376, Pro98/371, and Phe99/372. Comparison with the published bridging water molecules. However, the moiety adopts a small but structures of BD1 and BD2 in complex with their cognate peptides signiﬁcantly different conformation that suggests a tighter binding reveals that the THQ moiety largely occupies the conserved binding in the BD2 complex (Fig. 5F). Speciﬁcally, His433 forms a p–p pocket for the acetylated lysine side chain in which the terminal acetyl stacking interaction with the phenyl ring in the BD2 complex oxygen forms a hydrogen bond with the ND2 nitrogen of Asn156/429 whereas its counterpart Asp160 does not in the BD1 complex. The (Fig. 5D and E). smaller Val435 in BD2, as compared with Ile162 in BD1, allows the The 4-(methylamino) benzonitrilemoietyofSJ432alsobindsto moiety to penetrate deeper into the pocket. In addition, the a conserved pocket bounded by Trp97/370, Pro98/371, Asp161/ conformations of Trp370 and Pro371 in BD2 create a slightly 434, Met165/438, and Ile162/Val435. In addition, there is a hydro- smaller pocket than Trp97 and Pro98 in BD1 that is also apparent

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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).

when comparing the apo structures. One caveat to the His433/ the benzonitrile. Thus, whereas His433 forms the favorable p–p Asp160 difference is that a crystallographic ethylene glycol mol- stacking interaction with the phenyl ring, a similar interaction ecule occupies the His433 location in the BD1 structure that may would not expected for Asp160. We also note that His433 con- prevent the equivalent interaction with Asp160. His433 forms a tributes to the water bridging hydrogen-bonding interaction hydrogen bond with amide nitrogen atom of Val435 in the BD2 between the linker nitrogen atom and the main chain carbonyl complex, and ethylene glycol and a water molecule make hydrogen oxygen of Leu381 via a third water molecule (Fig. 5E). Computa- bind interactions with amide nitrogen atoms of Asp161 and tional analysis revealed that in the BD2/SJ432 complex, where Ile162 in the BD1 complex (Supplementary Fig. S10). However, the benzonitrile moiety more tightly engages the pocket, SJ432 there are three complexes in the asymmetric unit of the BD1 demonstrates a lower intrinsic strain energy than in the BD1/SJ432 structure and Asp160 adopts the same orientation in all three complex (28.9kcal/mol vs. 31.3 kcal/mol, respectively). Further- including one that has water molecules and not ethylene glycol at more, DG for SJ432 with BD2 was calculated to be 67.11 kcal/mol, this location. as compared with 65.21 kcal/mol for BD1. These structural and Our data indicate that the selectivity of SJ432 for BD2 over computational studies provide a detailed rationale for the selectivity BD1 relates to the different peptide specificities. The almost iden- of SJ432 with BD2. tical interactions of the THQ core with BD1 and BD2, together with the loose interactions of the pyrazole moiety, suggest that the Modulation of MYC expression and downstream target genes by selectivity resides in the different binding modes of the benzonitrile SJ432 moiety. Comparison with the peptide complexes of BD1 and Because BETi are known to modulate MYC expression, BD2 reveals that the benzonitrile binds adjacent to the peptide we assessed the ability of SJ432 to alter c-MYC and MYCN in a binding locale. The specificity can be rationalized by Asp160 in series of neuroblastoma cell lines. Three lines were chosen (SK-N- BD1 and His433 in BD2 that both have central roles in binding the AS—c-MYC–amplified; SK-N-SH—c-MYC–overexpressed, but not respective peptides, but have different contributions in binding amplified; and IMR32—MYCN-amplified) and were exposed to

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loss of BRD4, indicating that this event is not mediated via the proteasome (Supplementary Fig. S13).

PK parameters for SJ432 Having developed SJ432, we assessed its PK parameters in female athymic/nude mice (Supplementary 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 with 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 Sup- plementary Fig. S14). We also noted that the brain penetration for SJ432 was >100-fold lower than that for the pan BETi (Supple- mentary 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 intraperitoneally using a daily dosing schedule for 14 consecutive days.

Antitumor activity of BD2-selective BETi We assessed the activity of SJ432 toward the pediatric neuroblastoma xenograft SK-N-AS. Female CB17SCID mice bearing flank tumors, were dosed intraperitoneally with SJ432 daily for 14 days. We observed a pharmacologic 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 ¼ 0.0002 vs. control) and 26 (P ¼ 0.0064 vs. control) days, respectively (Fig. 7B and C). 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. Figure 6. An analysis of tumor material harvested from SJ432-treated mice, Biological activity of SJ432. A, Western blot analysis indicating loss of c-MYC either 4 or 24 hours after the last dose of a 14-day consecutive protein in SK-N-SH cells after 16 hours exposure to either SJ432 or JQ1. B, treatment, demonstrated a reduction in the level of c-MYC, loss of 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. BRD4, and upregulation of HEXIM1 (Fig. 7E). At 24 hours after the E, Loss of BRD4, and to a lesser extent BRD2, following exposure of cells to last dose of drug, BRD4 was essentially undetectable and HEXIM1 was SJ432 or JQ1. F, Quantitation of BRD4 and BRD2 protein levels from E. increased by approximately 5.4-fold (Fig. 7F). Levels of BRD2 were transiently elevated in the tumor harvested at 4 hours postdosing, but SJ432 or JQ1. SJ432 was more effective than JQ1 at reducing levels of essentially returned to control levels after 24 hours. These results are MYC protein in neuroblastoma cell lines (see Fig. 6; Supplementary consistent with our in vitro studies using this same drug and cell line Fig. S11). This occurs at both lower drug concentrations and over (Fig. 6D and E). shorter time frames as compared with 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 0.04 mmol/L data points in Fig. 6A and B, and 0.80 Discussion mmol/L value in Supplementary Fig. S11). In addition, c-MYC target All BET proteins contain two BDs, indicating that the duplication genes that were either upregulated (CDKN1A-p21 and GADD45H) or and architecture of these domains are required for their biological downregulated (CCND2-cyclin D2 and ODC1) regulated following function. However, the previous targeting of BET has focused on the exposure to SJ432 were readily observed in SK-N-AS following treat- development of small molecules that interact with these proteins ment (Fig. 6C). Finally, the upregulation of HEXIM1, a validated target without regard to BD-selectivity. Because evidence in the literature for BET inhibition, was more robust and durable in SJ432-treated cells indicates differential effects of the different BD domains (7–11), we (Fig. 6D). have generated BD2-selective BETi based upon a THQ scaffold. We also analyzed the levels of BRD2 and BRD4 in SK-N-AS cells Using SJ018 as a tool compound, we evaluated the molecular and following drug treatment. Both compounds induced the rapid loss of cellular consequence of selective inhibition of the BD2 domain. both proteins, with continued and extended suppression of BRD4 Microarray studies indicated that modulation of expression of (Fig. 6E and F). Similar results were also observed in a c-MYC– different gene sets was observed in comparison with JQ1 in three amplified human pediatric Group 3 MB line, HDMB03 (33; Supple- tumor cell lines of different histologic origin (AML, neuroblastoma, mentary Fig. S12). Treatment of cells with MG132 did not reduce the and MB). By selecting a diverse panel of lines for these analyses, the

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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 with 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 with 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 datasets using log rank test were as follows: SJ432 vs. control, P < 0.0007; JQ1 vs. control, P ¼ 0.067; SJ432 vs. JQ1, P ¼ 0.0791. D, Weights of mice from the study presented in B and C. E, Western blot analyses indicating the levels of c-MYC, HEXIM1, BRD4, and BRD2 protein in SK-N-AS xenografts harvested either 4 hours or 24 hours after SJ432 dosing. C, control, untreated tumors. F, Quantitation of the levels of protein in E. Data were corrected for gel loading differences using b-actin expression.

derived datasets would be agnostic of cell of origin and more tion and design by these investigators, our results argue that the former reflective of drug activity. Further validation using ChIP-seq con- is not the only consideration that should be evaluated for development firmed changes in H3K27ac in chromatin, indicating that these of this class of inhibitors. molecular approaches allowed discrimination and identification of In growth inhibition assays, we observed that the THQ analogs were genes and pathways that are specifically regulated by BD2 interac- universally active (Fig. 2A), and generally more potent than JQ1. This tion with histones. occurred in essentially all tumor histotypes and indicated that BD2- During the course of this project, another THQ-based BETi, selective compounds have broad spectrum activity. It is not known IBET726, was reported (34, 35). This molecule demonstrated <100 whether this is a consequence of the selective targeting of BD2, an nmol/L potency and 5-fold and 8-fold selectivity for BD2 versus inherent susceptibility of the pediatric lines to such molecules, or a BD1 in BRD2 and BRD4, respectively. In addition, recent work superior chemicophysical and biological profile associated with the by Law and colleagues (36) identified compounds with comparable THQ scaffold. However, our results argue that the development of BD2-selectivity with SJ018. However, no cytotoxicity or in vivo data such agents for pediatric tumors is warranted. While numerous clinical was presented for the series of compounds. While molecule potency, trials with BETi are ongoing in adults diagnosed with a variety of rather than BD-selectivity, may have been used for compound selec- malignancies, we are aware of only one being undertaken in children

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(NCT03936465). Coupled with our in vivo data demonstrating the Authors’ Contributions efficacy of SJ432 with minimal toxicity, we urge the medical commu- P.J. Slavish: Conceptualization, data curation, formal analysis, investigation, nity to consider the use of BD2-selective BETi in pediatric patients. methodology, writing-original draft, writing-review and editing. L. Chi: Data The THQ analogs induced cytotoxicity in pediatric lines and curation, formal analysis, investigation. M.-K. Yun: Data curation, formal analysis, investigation, visualization, writing-original draft, writing-review and potently suppressed c-MYC and MYCN expression. Whether these editing. L. Tsurkan: Data curation, formal analysis, investigation. N.E. Martinez: effects are directly mediated via the inhibition of these cellular path- Data curation, formal analysis, investigation. B. Jonchere: Data curation, formal ways is unclear, but our data argue that BD2-selective compounds are analysis, investigation. S.C. Chai: Data curation, formal analysis, investigation. more efficacious than JQ1 at modulating MYC protein levels. Fur- M. Connelly: Data curation, formal analysis, investigation. M.B. Waddell: thermore, we observed no reexpression of c-MYC protein, consistent Data curation, formal analysis, investigation. S. Das: Data curation, formal with the lack of a rebound effect. Because upregulation of BET proteins analysis, investigation. G. Neale: Data curation, formal analysis, investigation, methodology. Z. Li: Data curation, investigation. W.R. Shadrick: following exposure to BETi has been observed (37), it is likely that this Conceptualization, data curation, formal analysis, investigation. R.R. Olsen: Data may act as a potential mechanism of drug resistance, whereby cells curation, formal analysis, investigation. K.W. Freeman: Conceptualization, formal transiently overcome the effects of BET inhibition by enhanced analysis, supervision, investigation. J.A. Low: Data curation, formal analysis, transcription/translation of genes encoding these proteins (e.g., investigation. J.E. Price: Data curation, investigation. B.M. Young: c-MYC). In the neuroblastoma lines we used, we observed an Conceptualization, data curation, formal analysis, supervision, investigation, upregulation of c-MYC following JQ1 exposure, but not with methodology. N. Bharatham: Data curation, formal analysis, investigation. V.A. Boyd: Data curation, investigation. J. Yang: Data curation, investigation, SJ432. Presumably, the former arises from increased expression of methodology. R.E. Lee: Conceptualization, supervision, methodology. BETs following JQ1 treatment, but we conclude that resistance to M. Morfouace: Data curation, formal analysis, investigation. M.F. Roussel: THQ-derived molecule is unlikely to occur by this same mecha- Conceptualization, formal analysis, supervision, project administration, writing- nism. We also noted that BRD4 was rapidly lost following SJ432 review and editing. T. Chen: Formal analysis, supervision, methodology. D. Savic: exposure, whereas BRD2 was not. It is not known whether the Data curation, formal analysis, investigation, methodology, writing-review modulation of BET proteins by this drug is important for biological and editing. R.K. Guy: Conceptualization, formal analysis, supervision, methodology, project administration, writing-review and editing. S.W. White: activity, but THQs may have inherent targeting of BRD4. This Conceptualization, formal analysis, supervision, visualization, methodology, would explain the potent antitumor activity of these drugs with writing-original draft, project administration, writing-review and editing. minimal off-target toxicity. A.A. Shelat: Conceptualization, data curation, formal analysis, supervision, In summary, our data indicate that dual targeting of BD domains in funding acquisition, investigation, visualization, methodology, writing-original BET is not necessary to induce cytotoxicity and antitumor activity. draft, project administration, writing-review and editing. P.M. Potter: Hence, the use of BD-specific assays, rather than those that assess Conceptualization, data curation, formal analysis, supervision, funding acquisition, writing-original draft, project administration, writing-review and editing. 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 Acknowledgments suggest that selective BD targeting should allow the design of more 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 effective, and potentially, less toxic BETi with antitumor activity. experiments. High-resolution microscopy images were acquired in the Cell & Tissue Imaging Center at St. Jude. This work was supported, in part, by NIH grants (P01 Disclosure of Potential Conflicts of Interest CA096832 to M.F. Roussel; R01 CA225945 to A.A. Shelat and P.M. Potter), a Cancer P.J. Slavish reports a patent for US20200079739A1 pending. W.R. Shadrick Center Core grant (NCI, P30 CA021765), and by the American Lebanese Syrian has a patent for US20200079739A1 pending, and support for this research came Associated Charities (ALSAC). from the donors to St. Jude Children’s Research Hospital (SJCRH), and the pending patent will belong to SJCRH. J.E. Price reports a patent for US20200079739A1 pending. B.M. Young reports a patent for US20200079739A1 The costs of publication of this article were defrayed in part by the pending. R.K. Guy reports a patent for US20200079739A1 pending. A.A. Shelat paymentofpagecharges.Thisarticlemustthereforebeherebymarked advertisement has a patent for tetrahydroquinoline-based bromodomain inhibitors pending. No in accordance with 18 U.S.C. Section 1734 solely to indicate potential conflicts of interest were disclosed by the other authors. this fact.

Disclaimer The content is solely the responsibility of the authors and does not necessarily Received December 17, 2019; revised April 27, 2020; accepted June 29, 2020; ﬁ represent the ofﬁcial views of the NIH. published rst July 10, 2020.

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3518 Cancer Res; 80(17) September 1, 2020 CANCER RESEARCH

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 2020;80:3507-3518. Published OnlineFirst July 10, 2020.

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