Epigenetic proteins are intently pursued targets in ligand discovery. To date, successful efforts have been limited to chromatin modifying enzymes, or so-called epigenetic “writers” and “erasers”. Potent inhibitors of histone binding modules have not yet been described. Here we report a cell-permeable small molecule (JQ1) which binds competitively to acetyl-lysine recognition motifs, or bromodomains. High potency and specificity toward a subset of human bromodomains is explained by co-crystal structures with BRD4, revealing excellent shape complementarity with the acetyl-lysine binding cavity. Recurrent translocation of BRD4 is observed in a genetically-defined, incurable subtype of human squamous carcinoma. Competitive binding by JQ1 displaces the BRD4 fusion oncoprotein from chromatin, prompting squamous differentiation and specific anti-proliferative effects in BRD4-dependent cell lines and patient-derived xenograft models. These data establish proof of concept for targeting protein-protein interactions of epigenetic “readers” and provide a versatile chemical scaffold for the development of chemical probes more broadly throughout the bromodomain family.

Epigenetic pathways can regulate gene expression by controlling and interpreting chromatin modifications. Cancer cells are characterized by altered epigenetic landscapes, and commonly exploit the chromatin regulatory machinery to enforce oncogenic gene expression programs1. Although chromatin alterations are, in principle, reversible and often amenable to drug intervention, the promise of targeting such pathways therapeutically has been limited by an incomplete understanding of cancer-specific dependencies on epigenetic regulators. Here we describe a non-biased approach to probe epigenetic vulnerabilities in acute myeloid leukaemia (AML), an aggressive haematopoietic malignancy that is often associated with aberrant chromatin states2. By screening a custom library of small hairpin RNAs (shRNAs) targeting known chromatin regulators in a genetically defined AML mouse model, we identify the protein bromodomain-containing 4 (Brd4) as being critically required for disease maintenance. Suppression of Brd4 using shRNAs or the small-molecule inhibitor JQ1 led to robust antileukaemic effects in vitro and in vivo, accompanied by terminal myeloid differentiation and elimination of leukaemia stem cells. Similar sensitivities were observed in a variety of human AML cell lines and primary patient samples, revealing that JQ1 has broad activity in diverse AML subtypes. The effects of Brd4 suppression are, at least in part, due to its role in sustaining Myc expression to promote aberrant self-renewal, which implicates JQ1 as a pharmacological means to suppress MYC in cancer. Our results establish small-molecule inhibition of Brd4 as a promising therapeutic strategy in AML and, potentially, other cancers, and highlight the utility of RNA interference (RNAi) screening for revealing epigenetic vulnerabilities that can be exploited for direct pharmacological intervention.

Small-molecule antagonists disable discrete biochemical properties of protein targets. For multi-domain protein targets, the pharmacologic consequence of drug action is limited by selective disruption of one domain-specific activity. More broadly, target inhibition is kinetically limited by the durability and degree of target engagement. These features of traditional drug molecules are challenging to the development of inhibitors targeting transcription factors and chromatin-associated epigenetic proteins, which function as multi-domain biomolecular scaffolds and generally feature rapid association and dissociation kinetics. We therefore devised a chemical strategy to prompt ligand-dependent target protein degradation, via chemical conjugation with derivatized phthalimides that hijack the function of the Cereblon E3 ubiquitin ligase complex. Using this approach, we converted an acetyl-lysine competitive antagonist that displaces BET bromodomains from chromatin (JQ1) to a phthalimide-conjugated ligand that prompts immediate Cereblon-dependent BET protein degradation (dBET1). Expression proteomics confirms high specificity for BET family members BRD2, BRD3 and BRD4 among 7429 proteins detected. Degradation of BET bromodomains is associated with a more rapid and robust apoptotic response compared to bromodomain inhibition in primary human leukemic blasts, and dBET1 exhibits in vivo efficacy in a human leukemia xenograft. The reach of this approach is illustrated by a second series of probes that degrade the cytosolic signaling protein, FKBP12. Together, these findings identify a facile and general new strategy to control target protein stability, with implications for approaching previously intractable protein targets.

Thalidomide-like drugs such as lenalidomide are clinically important treatments for multiple myeloma and show promise for other B cell malignancies. The biochemical mechanisms underlying their antitumor activity are unknown. Thalidomide was recently shown to bind to, and inhibit, the cereblon ubiquitin ligase. Cereblon loss in zebrafish causes fin defects reminiscent of the limb defects seen in children exposed to thalidomide in utero. Here we show that lenalidomide-bound cereblon acquires the ability to target for proteasomal degradation two specific B cell transcription factors, Ikaros family zinc finger proteins 1 and 3 (IKZF1 and IKZF3). Analysis of myeloma cell lines revealed that loss of IKZF1 and IKZF3 is both necessary and sufficient for lenalidomide's therapeutic effect, suggesting that the antitumor and teratogenic activities of thalidomide-like drugs are dissociable.

The broad study of histone deacetylases in chemistry, biology and medicine relies on tool compounds to derive mechanistic insights. A phylogenetic analysis of Class I and II HDACs as targets of a comprehensive, structurally diverse panel of inhibitors revealed unexpected isoform selectivity even among compounds widely perceived as non-selective. The synthesis and study of a focused library of cinnamic hydroxamates allowed the identification of a first non-selective HDAC inhibitor. These data will guide a more informed use of HDAC inhibitors as chemical probes and therapeutic agents.

Targeted therapies that inhibit receptor tyrosine kinases (RTKs) and the downstream phosphatidylinositol 3-kinase (PI3K) signaling pathway have shown promising anticancer activity, but their efficacy in the brain tumor glioblastoma multiforme (GBM) and other solid tumors has been modest. We hypothesized that multiple RTKs are coactivated in these tumors and that redundant inputs drive and maintain downstream signaling, thereby limiting the efficacy of therapies targeting single RTKs. Tumor cell lines, xenotransplants, and primary tumors indeed show multiple concomitantly activated RTKs. Combinations of RTK inhibitors and/or RNA interference, but not single agents, decreased signaling, cell survival, and anchorage-independent growth even in glioma cells deficient in PTEN, a frequently inactivated inhibitor of PI3K. Thus, effective GBM therapy may require combined regimens targeting multiple RTKs.

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Dissection of complex biological systems requires target-specific control of the function or abundance of proteins. Genetic perturbations are limited by off-target effects, multicomponent complexity, and irreversibility. Most limiting is the requisite delay between modulation to experimental measurement. To enable the immediate and selective control of single protein abundance, we created a chemical biology system that leverages the potency of cell-permeable heterobifunctional degraders. The dTAG system pairs a novel degrader of FKBP12 with expression of FKBP12 in-frame with a protein of interest. By transgene expression or CRISPR-mediated locus-specific knock-in, we exemplify a generalizable strategy to study the immediate consequence of protein loss. Using dTAG, we observe an unexpected superior antiproliferative effect of pan-BET bromodomain degradation over selective BRD4 degradation, characterize immediate effects of KRAS loss on proteomic signaling, and demonstrate rapid degradation in vivo. This technology platform will confer kinetic resolution to biological investigation and provide target validation in the context of drug discovery.