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 Ó2020 American Association for Cancer Research. has been shown to be important for the expression of erythroid and AACRJournals.org | 3507 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst July 10, 2020; DOI: 10.1158/0008-5472.CAN-19-3934 Slavish et al. 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 Â 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