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Supplementary Data from EP300 Selectively Controls the Enhancer Landscape of MYCN-Amplified Neuroblastoma

Gene expression is regulated by promoters and enhancers marked by histone H3 lysine 27 acetylation (H3K27ac), which is established by the paralogous histone acetyltransferases (HAT) EP300 and CBP. These enzymes display overlapping regulatory roles in untransformed cells, but less characterized roles in cancer cells. We demonstrate that the majority of high-risk pediatric neuroblastoma (NB) depends on EP300, whereas CBP has a limited role. EP300 controls enhancer acetylation by interacting with TFAP2β, a transcription factor member of the lineage-defining transcriptional core regulatory circuitry (CRC) in NB. To disrupt EP300, we developed a proteolysis-targeting chimera (PROTAC) compound termed “JQAD1” that selectively targets EP300 for degradation. JQAD1 treatment causes loss of H3K27ac at CRC enhancers and rapid NB apoptosis, with limited toxicity to untransformed cells where CBP may compensate. Furthermore, JQAD1 activity is critically determined by cereblon (CRBN) expression across NB cells.Significance:EP300, but not CBP, controls oncogenic CRC-driven transcription in high-risk NB by binding TFAP2β. We developed JQAD1, a CRBN-dependent PROTAC degrader with preferential activity against EP300 and demonstrated its activity in NB. JQAD1 has limited toxicity to untransformed cells and is effective in vivo in a CRBN-dependent manner.This article is highlighted in the In This Issue feature, p. 587

The SARS-CoV-2 main protease (Mpro) is a major therapeutic target. The Mpro inhibitor, nirmatrelvir, is the antiviral component of Paxlovid, an orally available treatment for COVID-19. As Mpro inhibitor use increases, drug resistant mutations will likely emerge. We have established a non-pathogenic system, in which yeast growth serves as a proxy for Mpro activity, enabling rapid identification of mutants with altered enzymatic activity and drug sensitivity. The E166 residue is known to be a potential hot spot for drug resistance and yeast assays showed that an E166R substitution conferred strong nirmatrelvir resistance while an E166N mutation compromised activity. On the other hand, N142A and P132H mutations caused little to no change in drug response and activity. Standard enzymatic assays confirmed the yeast results. In turn, we solved the structures of Mpro E166R, and Mpro E166N, providing insights into how arginine may drive drug resistance while asparagine leads to reduced activity. The work presented here will help characterize novel resistant variants of Mpro that may arise as Mpro antivirals become more widely used.

Gene expression is regulated by promoters and enhancers marked by histone H3 lysine 27 acetylation (H3K27ac), which is established by the paralogous, multidomain-containing histone acetyltransferases (HAT) EP300 and CBP. These enzymes display overlapping regulatory roles in untransformed cells, but less characterized roles in cancer cells. We demonstrate that the majority of high-risk pediatric neuroblastoma (NB) depends on EP300, whereas CBP has a limited role. To disrupt EP300, we developed a proteolysis-targeting chimera (PROTAC) compound termed “JQAD1” that selectively targets EP300 for degradation. JQAD1 treatment causes loss of H3K27ac at the enhancers that regulate NB master transcription factors, and drives rapid NB apoptosis, with limited toxicity to untransformed cells where CBP may compensate. In parallel, we demonstrate that EP300 and CBP have subdomain-specific functions, with enriched activity for individual domains in distinct tumor lineages. These data provide a foundation for interrogation of these histone acetyltransferases in distinct tumor types and new strategies for therapeutic disruption of subdomain and scaffolding activities of these multidomain proteins in cancer. Citation Format: Adam D. Durbin, Noha Shendy, Audrey Mercier, Yang Zhang, Melissa J. Bikowitz, Logan H. Sigua, Sarah Robinson, Tingjian Wang, Barbara Jonchere, A. Thomas Look, Mark W. Zimmerman, Martine Roussel, Brian J. Abraham, Ernst Schonbrunn, Kimberly Stegmaier, Jun Qi. Targeting EP300 and CBP for therapeutic benefit in pediatric solid tumors. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr PR005.

The use of epigenetic bromodomain inhibitors as anti-cancer therapeutics has transitioned from targeting bromodomain extra terminal (BET) proteins into targeting non-BET bromodomains. The two most relevant non-BET bromodomain oncology targets are cyclic AMP response element-binding protein (CBP) and E1A binding protein P300 (EP300). To explore the growing CBP/EP300 interest, we developed a highly efficient two-step synthetic route for dimethylisoxazole-attached imidazo[1,2-a]pyridine scaffold-containing inhibitors. Our efficient two-step reactions enabled high-throughput synthesis of compounds designed by molecular modeling which together with structure activity relationship (SAR) studies facilitated overarching understanding of selective targeting of CBP/EP300 over non-BET bromodomains. This led to identification of a new potent and selective CBP/EP300 bromodomain inhibitor, UMB298 (compound 23, CBP IC(50) 72 nM and BRD4 IC(50) 5193 nM). The SAR we established is in good agreement with literature reported CBP inhibitors, such as CBP30, and demonstrates the advantage of utilizing our two-step approach for inhibitor development of other bromodomains.