Using Chemical Epigenetics to Target Cancer.

Transcription is epigenetically regulated by the orchestrated function of chromatin-binding proteins that tightly control the expression of master transcription factors, effectors, and supportive housekeeping genes required for establishing and propagating the normal and malignant cell state. Rapid advances in chemical biology and functional genomics have facilitated exploration of targeting epigenetic proteins, yielding effective strategies to target transcription while reducing toxicities to untransformed cells. Here, we review recent developments in conventional active site and allosteric inhibitors, peptidomimetics, and novel proteolysis-targeted chimera (PROTAC) technology that have deepened our understanding of transcriptional processes and led to promising preclinical compounds for therapeutic translation, particularly in cancer.

Template-assisted covalent modification underlies activity of covalent molecular glues.

Molecular glues are proximity-inducing small molecules that have emerged as an attractive therapeutic approach. However, developing molecular glues remains challenging, requiring innovative mechanistic strategies to stabilize neoprotein interfaces and expedite discovery. Here we unveil a trans-labeling covalent molecular glue mechanism, termed 'template-assisted covalent modification'. We identified a new series of BRD4 molecular glue degraders that recruit CUL4DCAF16 ligase to the second bromodomain of BRD4 (BRD4BD2). Through comprehensive biochemical, structural and mutagenesis analyses, we elucidated how pre-existing structural complementarity between DCAF16 and BRD4BD2 serves as a template to optimally orient the degrader for covalent modification of DCAF16Cys58. This process stabilizes the formation of BRD4-degrader-DCAF16 ternary complex and facilitates BRD4 degradation. Supporting generalizability, we found that a subset of degraders also induces GAK-BRD4BD2 interaction through trans-labeling of GAK. Together, our work establishes 'template-assisted covalent modification' as a mechanism for covalent molecular glues, which opens a new path to proximity-driven pharmacology.

1,4-Dihydropyridinebutyrolactone-derived ring-opened ester and amide analogs targeting BET bromodomains.

Based on a previously reported 1,4-dihydropyridinebutyrolactone virtual screening hit, nine lactone ring-opened ester and seven amide analogs were prepared. The analogs were designed to provide interactions with residues at the entrance of the ZA loop of the testis-specific bromodomain (ZA) channel to enhance the affinity and selectivity for the bromodomain and extra-terminal (BET) subfamily of bromodomains. Compound testing by AlphaScreen showed that neither the affinity nor the selectivity of the ester and lactam analogs was improved for BRD4-1 and the first bromodomain of the testis-specific bromodomain (BRDT-1). The esters retained affinity comparable to the parent compound, whereas the affinity for the amide analogs was reduced 10-fold. A representative benzyl ester analog was found to retain high selectivity for BET bromodomains as shown by a BROMOscan. X-ray analysis of the allyl ester analog in complex with BRD4-1 and BRDT-1 revealed that the ester side chain is located next to the ZA loop and solvent exposed.

A Kinetic Scout Approach Accelerates Targeted Protein Degrader Development.

Bifunctional molecules such as targeted protein degraders induce proximity to promote gain-of-function pharmacology. These powerful approaches have gained broad traction across academia and the pharmaceutical industry, leading to an intensive focus on strategies that can accelerate their identification and optimization. We and others have previously used chemical proteomics to map degradable target space, and these datasets have been used to develop and train multiparameter models to extend degradability predictions across the proteome. In this study, we now turn our attention to develop generalizable chemistry strategies to accelerate the development of new bifunctional degraders. We implement lysine-targeted reversible-covalent chemistry to rationally tune the binding kinetics at the protein-of-interest across a set of 25 targets. We define an unbiased workflow consisting of global proteomics analysis, IP/MS of ternary complexes and the E-STUB assay, to mechanistically characterize the effects of ligand residence time on targeted protein degradation and formulate hypotheses about the rate-limiting step of degradation for each target. Our key finding is that target residence time is a major determinant of degrader activity, and this can be rapidly and rationally tuned through the synthesis of a minimal number of analogues to accelerate early degrader discovery and optimization efforts.

Group 3 medulloblastoma transcriptional networks collapse under domain specific EP300/CBP inhibition.

Chemical discovery efforts commonly target individual protein domains. Many proteins, including the EP300/CBP histone acetyltransferases (HATs), contain several targetable domains. EP300/CBP are critical gene-regulatory targets in cancer, with existing high potency inhibitors of either the catalytic HAT domain or protein-binding bromodomain (BRD). A domain-specific inhibitory approach to multidomain-containing proteins may identify exceptional-responding tumor types, thereby expanding a therapeutic index. Here, we discover that targeting EP300/CBP using the domain-specific inhibitors, A485 (HAT) or CCS1477 (BRD) have different effects in select tumor types. Group 3 medulloblastoma (G3MB) cells are especially sensitive to BRD, compared with HAT inhibition. Structurally, these effects are mediated by the difluorophenyl group in the catalytic core of CCS1477. Mechanistically, bromodomain inhibition causes rapid disruption of genetic dependency networks that are required for G3MB growth. These studies provide a domain-specific structural foundation for drug discovery efforts targeting EP300/CBP and identify a selective role for the EP300/CBP bromodomain in maintaining genetic dependency networks in G3MB.

SALL4B, not targeted by IMiD, is important for SALL4-mediated tumorigenesis.

Oncofetal transcription factor SALL4 is essential for cancer cell survival. 1-5 Recently, several groups reported that immunomodulatory imide drugs (IMiDs) could degrade SALL4 in a proteasome-dependent manner. 6,7 Intriguingly, we observed that IMiDs had no effect on SALL4-positive cancer cells. Further studies demonstrated that IMiDs could only degrade SALL4A, one of the SALL4 isoforms. This finding raises the possibility that SALL4B, the isoform not affected by IMiDs, may be essential for SALL4-mediated cancer cell survival. SALL4B knockdown led to an increase in apoptosis and inhibition of cancer cell growth. SALL4B gain-of-function alone led to liver tumor formation in mice. Our observation that protein degraders can possess isoform-specific effects exemplifies the importance of delineating drug action and oncogenesis at the isoform level to develop more effective cancer therapeutics.

Template-assisted covalent modification of DCAF16 underlies activity of BRD4 molecular glue degraders.

Small molecules that induce protein-protein interactions to exert proximity-driven pharmacology such as targeted protein degradation are a powerful class of therapeutics1-3. Molecular glues are of particular interest given their favorable size and chemical properties and represent the only clinically approved degrader drugs4-6. The discovery and development of molecular glues for novel targets, however, remains challenging. Covalent strategies could in principle facilitate molecular glue discovery by stabilizing the neo-protein interfaces. Here, we present structural and mechanistic studies that define a trans-labeling covalent molecular glue mechanism, which we term "template-assisted covalent modification". We found that a novel series of BRD4 molecular glue degraders act by recruiting the CUL4DCAF16 ligase to the second bromodomain of BRD4 (BRD4BD2). BRD4BD2, in complex with DCAF16, serves as a structural template to facilitate covalent modification of DCAF16, which stabilizes the BRD4-degrader-DCAF16 ternary complex formation and facilitates BRD4 degradation. A 2.2 Å cryo-electron microscopy structure of the ternary complex demonstrates that DCAF16 and BRD4BD2 have pre-existing structural complementarity which optimally orients the reactive moiety of the degrader for DCAF16Cys58 covalent modification. Systematic mutagenesis of both DCAF16 and BRD4BD2 revealed that the loop conformation around BRD4His437, rather than specific side chains, is critical for stable interaction with DCAF16 and BD2 selectivity. Together our work establishes "template-assisted covalent modification" as a mechanism for covalent molecular glues, which opens a new path to proximity driven pharmacology.

Dihydropyridine Lactam Analogs Targeting BET Bromodomains.

Inhibitors of Bromodomain and Extra Terminal (BET) proteins are investigated for various therapeutic indications, but selectivity for BRD2, BRD3, BRD4, BRDT and their respective tandem bromodomains BD1 and BD2 remains suboptimal. Here we report selectivity-focused structural modifications of previously reported dihydropyridine lactam 6 by changing linker length and linker type of the lactam side chain in efforts to engage the unique arginine 54 (R54) residue in BRDT-BD1 to achieve BRDT-selective affinity. We found that the analogs were highly selective for BET bromodomains, and generally more selective for the first (BD1) and second (BD2) bromodomains of BRD4 rather than for those of BRDT. Based on AlphaScreen and BromoScan results and on crystallographic data for analog 10 j, we concluded that the lack of selectivity for BRDT is most likely due to the high flexibility of the protein and the unfavorable trajectory of the lactam side chain that do not allow interaction with R54. A 15-fold preference for BD2 over BD1 in BRDT was observed for analogs 10 h and 10 m, which was supported by protein-based 19 F NMR experiments with a BRDT tandem bromodomain protein construct.

BRD9-containing non-canonical BAF complex maintains somatic cell transcriptome and acts as a barrier to human reprogramming.

Epigenetic reprogramming to pluripotency requires extensive remodeling of chromatin landscapes to silence existing cell-type-specific genes and activate pluripotency genes. ATP-dependent chromatin remodeling complexes are important regulators of chromatin structure and gene expression; however, the role of recently identified Bromodomain-containing protein 9 (BRD9) and the associated non-canonical BRG1-associated factors (ncBAF) complex in reprogramming remains unknown. Here, we show that genetic or chemical inhibition of BRD9, as well as ncBAF complex subunit GLTSCR1, but not the closely related BRD7, increase human somatic cell reprogramming efficiency and can replace KLF4 and c-MYC. We find that BRD9 is dispensable for human induced pluripotent stem cells under primed but not under naive conditions. Mechanistically, BRD9 inhibition downregulates fibroblast-related genes and decreases chromatin accessibility at somatic enhancers. BRD9 maintains the expression of transcriptional regulators MN1 and ZBTB38, both of which impede reprogramming. Collectively, these results establish BRD9 as an important safeguarding factor for somatic cell identity whose inhibition lowers chromatin-based barriers to reprogramming.

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 TFAP2ß, 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 TFAP2ß. 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.

BAF Complex Maintains Glioma Stem Cells in Pediatric H3K27M Glioma.

Diffuse midline gliomas are uniformly fatal pediatric central nervous system cancers that are refractory to standard-of-care therapeutic modalities. The primary genetic drivers are a set of recurrent amino acid substitutions in genes encoding histone H3 (H3K27M), which are currently undruggable. These H3K27M oncohistones perturb normal chromatin architecture, resulting in an aberrant epigenetic landscape. To interrogate for epigenetic dependencies, we performed a CRISPR screen and show that patient-derived H3K27M-glioma neurospheres are dependent on core components of the mammalian BAF (SWI/SNF) chromatin remodeling complex. The BAF complex maintains glioma stem cells in a cycling, oligodendrocyte precursor cell-like state, in which genetic perturbation of the BAF catalytic subunit SMARCA4 (BRG1), as well as pharmacologic suppression, opposes proliferation, promotes progression of differentiation along the astrocytic lineage, and improves overall survival of patient-derived xenograft models. In summary, we demonstrate that therapeutic inhibition of the BAF complex has translational potential for children with H3K27M gliomas. SIGNIFICANCE: Epigenetic dysregulation is at the core of H3K27M-glioma tumorigenesis. Here, we identify the BRG1-BAF complex as a critical regulator of enhancer and transcription factor landscapes, which maintain H3K27M glioma in their progenitor state, precluding glial differentiation, and establish pharmacologic targeting of the BAF complex as a novel treatment strategy for pediatric H3K27M glioma. See related commentary by Beytagh and Weiss, p. 2730. See related article by Mo et al., p. 2906.

Development of Dimethylisoxazole-Attached Imidazo[1,2-a]pyridines as Potent and Selective CBP/P300 Inhibitors.

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.

Peptide-Based PROTAC: The Predator of Pathological Proteins.

Qu et al. (2020) demonstrate a peptide-induced targeted degradation of the alpha-synuclein protein, a hallmark of Parkinson's disease. Using a modular three-component design, the target-protein-specific, cell-permeable peptide disposed of alpha-synuclein via the ubiquitin-proteasome pathway rather than the standard autophagy-lysosome pathway.