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

Lysine demethylase 5A (KDM5A) is a negative regulator of histone H3K4 trimethylation, a histone mark associated with activate gene transcription. We identify that KDM5A interacts with the P-TEFb complex and cooperates with MYC to control MYC targeted genes in multiple myeloma (MM) cells. We develop a cell-permeable and selective KDM5 inhibitor, JQKD82, that increases histone H3K4me3 but paradoxically inhibits downstream MYC-driven transcriptional output in vitro and in vivo. Using genetic ablation together with our inhibitor, we establish that KDM5A supports MYC target gene transcription independent of MYC itself, by supporting TFIIH (CDK7)-and P-TEFb (CDK9)mediated phosphorylation of RNAPII.These data identify KDM5A as a unique vulnerability in MM functioning through regulation of MYC-target gene transcription, and establish JQKD82 as a tool compound to block KDM5A function as a potential therapeutic strategy for MM.

No abstract

Proteasome inhibition is an effective treatment for multiple myeloma (MM); however, targeting different components of the ubiquitin-proteasome system (UPS) remains elusive. Our RNAinterference studies identified proteasome-associated ubiquitin-receptor Rpn13 as a mediator of MM cell growth and survival. Here, we developed the first degrader of Rpn13, WL40, using a small-molecule-induced targeted protein degradation strategy to selectively degrade this component of the UPS. WL40 was synthesized by linking the Rpn13 covalent inhibitor RA190 with the cereblon (CRBN) binding ligand thalidomide. We show that WL40 binds to both Rpn13 and CRBN and triggers degradation of cellular Rpn13, and is therefore first-in-class in exploiting a covalent inhibitor for the development of degraders. Biochemical and cellular studies show that WL40-induced Rpn13 degradation is both CRBN E3 ligase-and Rpn13-dependent. Importantly, WL40 decreases viability in MM cell lines and patient MM cells, even those resistant to bortezomib. Mechanistically, WL40 interrupts Rpn13 function and activates caspase apoptotic cascade, ER stress response and p53/p21 signaling. In animal model studies, WL40 inhibits

Although the exact cause(s) of Parkinson's disease (PD) is not fully understood, it is believed that environmental factors play a major role. The discovery that the synthetic chemical, 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP)-derived N-methyl-4-phenylpyridinium (MPP + ), recapitulates major pathophysiological characteristics of PD in humans, has provided the strongest support for this possibility. While the mechanism of the selective dopaminergic toxicity of MPP + has been extensively studied and is in most respects well accepted, several key aspects of the mechanism are still debatable. In the present study, we use a series of structurally related, novel, and lipophilic MPP + derivatives [N-(2-phenyl-1-propene)-4-phenyl-pyridinium (PP-PP + )] to probe the mechanism of action of MPP + using dopaminergic MN9D and non-neuronal HepG2 cells in vitro. Here we show that effective mitochondrial complex I inhibition is necessary and that the specific uptake through DAT is not essential for dopaminergic toxicity of MPP + and related toxins. We also provide strong evidence to support our previous proposal that the selective vulnerability of dopaminergic cells to MPP + and similar toxins is likely due to the high inherent propensity of these cells to produce excessive ROS as a downstream effect of complex I inhibition. Based on the current and previous findings, we propose that MPP + is the simplest of a larger group of unidentified environmental dopaminergic toxins, a possibility that may have major public health implications.

The double membrane architecture of Gram-negative bacteria forms a barrier that is impermeable to most extracellular threats. Bacteriocin proteins evolved to exploit the accessible, surface-exposed proteins embedded in the outer membrane to deliver cytotoxic cargo. Colicin E1 is a bacteriocin produced by, and lethal to, Escherichia coli that hijacks the outer membrane proteins TolC and BtuB to enter the cell. Here we capture the colicin E1 translocation domain inside its membrane receptor, TolC, by high-resolution cryoEM to obtain the first reported structure of a bacteriocin bound to TolC. Colicin E1 binds stably to TolC as an open hinge through the TolC pore-an architectural rearrangement from colicin E1's unbound conformation. This binding is stable in live E. coli cells as indicated by single-molecule fluorescence microscopy. Finally, colicin E1 fragments binding to TolC plug the channel, inhibiting its native efflux function as an antibiotic efflux pump and heightening susceptibility to three antibiotic classes. In addition to demonstrating that these protein fragments are useful starting points for developing novel antibiotic potentiators, this method could be expanded to other colicins to inhibit other outer membrane protein functions.

The use of epigenetic bromodomain inhibitors as anticancer therapeutics has transitioned from targeting bromodomain extraterminal domain (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 an overarching understanding of selective targeting of CBP/EP300 over non-BET bromodomains. This led to the identification of a new potent and selective CBP/EP300 bromodomain inhibitor, UMB298 (compound 23, CBP IC50 72 nM and bromodomain 4, BRD4 IC50 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.

High-risk neuroblastoma (NB) is an aggressive tumor of the peripheral sympathetic nervous system. Patients with NB have poor overall survival despite increases in intensity of anti-NB therapy, and survivors are typically left with long-term treatment-related morbidities. Thus, there is a need to develop novel targeted therapies that kill tumor cells without toxicity to normal tissues. We recently demonstrated that NB relies on a set of genes for survival, termed “dependencies.” One NB dependency that regulates numerous other dependencies is the histone acetyltransferase (HAT) enzyme, EP300. EP300 catalyzes the acetylation of histone H3, lysine-27 (H3K27ac) that defines active enhancer and promoter elements. This mark can also be catalyzed by the paralogous protein CBP; however, CBP is not required for NB survival despite generally being expressed in NB. Thus, selective inhibition of EP300 may result in anti-NB effects with minimal toxicity to normal tissues where CBP compensates. Here, we demonstrate that EP300, but not CBP, controls NB cell survival through regulation of the oncogenic enhancer landscape of NB. Conventional small-molecule inhibition of EP300/CBP or CRISPR-cas9-mediated knockout of EP300, but not CBP, results in neuroblastic differentiation associated with loss of the NB lineage-defining and oncogenic core transcriptional regulatory circuitry. All agents targeting EP300 equivalently target CREBBP due to their extensive protein homology. Thus, to pharmacologically eliminate EP300 and spare CBP, we designed a novel proteolysis-targeting chimera (PROTAC) agent (“JQAD1”). JQAD1 is a cereblon-dependent, selective degrader of EP300 with minimal off-target effects on CBP in NB cell lines, low passage primary cells, and in xenografts in vivo. JQAD1 is exceedingly stable and well tolerated in vivo. JQAD1 treatment results in loss of the transcriptional circuitry driving NB, transcriptional collapse. and irreversible commitment of NB cells to apoptosis in vitro and in vivo. This study defines the mechanism by which EP300 centrally regulates NB cell fate through epigenetic regulation of the transcriptional state and provides the first EP300-selective pharmacologic agent for evaluation in a myriad of other EP300-dependent malignancies. This abstract is also being presented as Poster B11. Citation Format: Adam D. Durbin, Virangika Wimalasena, Mark W. Zimmerman, Li Deyao, Elizabeth S. Frank, Paul Park, Ken Morita, Neekesh V. Dharia, Ken N. Ross, Ernst Schonbrunn, Richard A. Young, Brian J. Abraham, Kimberly Stegmaier, A. Thomas Look, Jun Qi. EP300 controls the oncogenic enhancer landscape of high-risk neuroblastoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr PR11.