Proximity, or the physical closeness of molecules, is a pervasive regulatory mechanism in biology. For example, most posttranslational modifications such as phosphorylation, methylation, and acetylation promote proximity of molecules to play deterministic roles in cellular processes. To understand the role of proximity in biologic mechanisms, chemical inducers of proximity (CIPs) were developed to synthetically model biologically regulated recruitment. Chemically induced proximity allows for precise temporal control of transcription, signaling cascades, chromatin regulation, protein folding, localization, and degradation, as well as a host of other biologic processes. A systematic analysis of CIPs in basic research, coupled with recent technological advances utilizing CRISPR, distinguishes roles of causality from coincidence and allows for mathematical modeling in synthetic biology. Recently, induced proximity has provided new avenues of gene therapy and emerging advances in cancer treatment.

Small-molecule inhibition of extracellular proteins that activate membrane receptors has proved to be extremely challenging. Diversity-oriented synthesis and small-molecule microarrays enabled the discovery of robotnikinin, a small molecule that binds the extracellular Sonic Hedgehog (Shh) protein and blocks Shh-signaling in cell lines, human primary keratinocytes and a synthetic model of human skin. Shh pathway activity is rescued by small-molecule agonists of Smoothened, which functions immediately downstream of the Shh receptor Patched.

Mutation of SMARCA4 (BRG1), the ATPase of BAF (mSWI/SNF) and PBAF complexes, contributes to a range of malignancies and neurologic disorders. Unfortunately, the effects of SMARCA4 missense mutations have remained uncertain. Here we show that SMARCA4 cancer missense mutations target conserved ATPase surfaces and disrupt the mechanochemical cycle of remodeling. We find that heterozygous expression of mutants alters the open chromatin landscape at thousands of sites across the genome. Loss of DNA accessibility does not directly overlap with Polycomb accumulation, but is enriched in “A compartments” at active enhancers, which lose H3K27ac but not H3K4me1. Affected positions include hundreds of sites identified as superenhancers in many tissues. Dominant-negative mutation induced pro-oncogenic expression changes, including increased expression of Myc and its target genes. Together, our data suggest that disruption of enhancer accessibility represents a key source of altered function in SMARCA4-mutated disorders in a wide variety of tissues.

Trithorax-group genes and mammalian homologues, including BAF (mSWI/SNF) complexes, have been known for nearly 30 years to oppose Polycomb repressive activity1–5. This opposition underlies the tumor-suppression role of BAF3,5–7 and is expected to contribute to neurodevelopmental disorders, as evidenced by frequent driving mutations8,9. However, the mechanisms underlying opposition to Polycomb silencing are poorly understood. Here we report that recurrent disease mutations of BAF subunits induce genome-wide increases in Polycomb complex deposition and activity. We show that point mutations of the Smarca4 (Brg) ATPase domain cause loss of direct binding between BAF and PRC1 that occurs independently of chromatin. Release of this direct interaction occurs via an ATP-dependent mechanism, consistent with a role as a transient intermediate of eviction. Using a new in vivo assay, we find that BAF directly evicts Polycomb factors within minutes of its occupancy, together establishing a new mechanism for the widespread opposition underlying development and disease.

Core regulatory transcription factors (CR TFs) orchestrate the placement of super-enhancers (SEs) to activate transcription of cell-identity specifying gene networks, and are critical in promoting cancer. Here, we define the core regulatory circuitry of rhabdomyosarcoma (RMS) and identify critical CR TF dependencies. These CR TFs build SEs that have the largest levels of histone acetylation, yet paradoxically SEs also harbor the highest amounts of histone deacetylases Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Identity determining transcription factors (TFs), or core regulatory (CR) TFs, are governed by cell-type specific super enhancers (SEs). Drugs to selectively inhibit CR circuitry are of high interest for cancer treatment. In alveolar rhabdomyosarcoma, PAX3-FOXO1 activates SEs to induce the expression of other CR TFs, providing a model system for studying cancer cell addiction to CR transcription. Using chemical genetics, the systematic screening of chemical matter for a biological outcome, here we report on a screen for epigenetic chemical probes able to distinguish between SE-driven transcription and constitutive transcription. We find that chemical probes along the acetylation-axis, and not the methylation-axis, selectively disrupt CR transcription. Additionally, we find that histone deacetylases (HDACs) are essential for CR TF transcription. We further dissect the contribution of HDAC isoforms using selective inhibitors, including the newly developed selective HDAC3 inhibitor LW3. We show HDAC1/2/3 are the co-essential isoforms that when co-inhibited halt CR transcription, making CR TF sites hyper-accessible and disrupting chromatin looping.

Fragment hopping, a new fragment-based approach for de novo inhibitor design focusing on ligand diversity and isozyme selectivity, is described. The core of this approach is the derivation of the minimal pharmacophoric element for each pharmacophore. Sites for both ligand binding and isozyme selectivity are considered in deriving the minimal pharmacophoric elements. Five general-purpose libraries are established: the basic fragment library, the bioisostere library, the rules for metabolic stability, the toxicophore library, and the side chain library. These libraries are employed to generate focused fragment libraries to match the minimal pharmacophoric elements for each pharmacophore and then to link the fragment to the desired molecule. This method was successfully applied to neuronal nitric oxide synthase (nNOS), which is implicated in stroke and neurodegenerative diseases. Starting with the nitroarginine-containing dipeptide inhibitors we developed previously, a small organic molecule with a totally different chemical structure was designed, which showed nanomolar nNOS inhibitory potency and more than 1000-fold nNOS selectivity. The crystallographic analysis confirms that the small organic molecule with a constrained conformation can exactly mimic the mode of action of the dipeptide nNOS inhibitors. Therefore, a new peptidomimetic strategy, referred to as fragment hopping, which creates small organic molecules that mimic the biological function of peptides by a pharmacophore-driven strategy for fragment-based de novo design, has been established as a new type of fragment-based inhibitor design. As an open system, the newly established approach efficiently incorporates the concept of early "ADME/Tox" considerations and provides a basic platform for medicinal chemistry-driven efforts.Agman@chem.northwestern.edu.

Diffuse midline gliomas (DMGs) are universally lethal malignancies occurring chiefly during childhood and involving midline structures of the central nervous system, including thalamus, pons, and spinal cord. These molecularly related cancers are characterized by high prevalence of the histone H3K27M mutation. In search of effective therapeutic options, we examined multiple DMG cultures in sequential quantitative high-throughput screens (HTS) of 2706 approved and investigational drugs. This effort generated 19,936 single-agent dose responses that inspired a series of HTS-enabled drug combination assessments encompassing 9195 drug-drug examinations. Top combinations were validated across patient-derived cell cultures representing the major DMG genotypes. In vivo testing in patient-derived xenograft models validated the combination of the multi–histone deacetylase (HDAC) inhibitor panobinostat and the proteasome inhibitor marizomib as a promising therapeutic approach. Transcriptional and metabolomic surveys revealed substantial alterations to key metabolic processes and the cellular unfolded protein response after treatment with panobinostat and marizomib. Mitigation of drug-induced cytotoxicity and basal mitochondrial respiration with exogenous application of nicotinamide mononucleotide (NMN) or exacerbation of these phenotypes when blocking nicotinamide adenine dinucleotide (NAD+) production via nicotinamide phosphoribosyltransferase (NAMPT) inhibition demonstrated that metabolic catastrophe drives the combination-induced cytotoxicity. This study provides a comprehensive single-agent and combinatorial drug screen for DMG and identifies concomitant HDAC and proteasome inhibition as a promising therapeutic strategy that underscores underrecognized metabolic vulnerabilities in DMG.