High-throughput small molecule screen identifies inhibitors of aberrant chromatin accessibility

Abstract

Mutations in chromatin-modifying proteins and transcription factors are commonly associated with a wide variety of cancers. Through gain-or loss-of-function, these mutations may result in characteristic alterations of accessible chromatin, indicative of shifts in the landscape of regulatory elements genome-wide. The identification of compounds that reverse a specific chromatin signature could lead to chemical probes or potential therapies. To explore whether chromatin accessibility could serve as a platform for small molecule screening, we adapted formaldehyde-assisted isolation of regulatory elements (FAIRE), a chemical method to enrich for nucleosomedepleted genomic regions, as a high-throughput, automated assay. After demonstrating the validity and robustness of this approach, we applied this method to screen an epigenetically targeted small molecule library by evaluating regions of aberrant nucleosome depletion mediated by EWSR1-FLI1, the chimeric transcription factor critical for the bone and soft tissue tumor Ewing sarcoma. As a class, histone deacetylase inhibitors were greatly overrepresented among active compounds. These compounds resulted in diminished accessibility at targeted sites by disrupting transcription of EWSR1-FLI1. Capitalizing on precise differences in chromatin accessibility for drug discovery efforts offers significant advantages because it does not depend on the a priori selection of a single molecular target and may detect novel biologically relevant pathways.chromatin | Ewing sarcoma | high throughput screening | FAIRE | histone deacetylase inhibitor A growing range of human cancers have been associated with mutations in genes encoding proteins that regulate chromatin, the assembly of proteins and DNA that control DNAtemplated processes, including transcription and replication (1, 2). Small molecule drugs and chemical probes offer an approach to explore the biological consequences of these mutations and are emerging as a therapeutic strategy to target disease pathways. Drugs targeting histone deacetylase (HDAC) enzymes, the bromodomain reader BRD4, and DNA methylation have already received regulatory approval or have entered clinical testing, and chemical probes have been developed against a broad range of chromatin regulators, such as the methyltransferases (3) DOT1L (4), EZH2 (5-8), and G9a (9, 10), and the reader proteins L3MBTL3 (11) and BRD4 (12-14). However, transcription factors that lack enzymatic activity or binding pockets with targetable molecular features have been considered "undruggable," and a reductionist approach based on identification of their molecular targets has largely failed.The majority of Ewing sarcomas, highly malignant pediatric bone and soft tissue tumors, harbor a chromosomal translocation that joins the amino-terminal domain of EWSR1 with the DNA binding domain of the ETS transcription factor family member FLI1 to generate the chimeric transcription factor EWSR1-FLI1 (15). Translocations with other ETS genes are detected in most of the remaining tumors, y...

Targeted inhibition of histone deacetylase leads to suppression of Ewing sarcoma tumor growth through an unappreciated EWS-FLI1/HDAC3/HSP90 signaling axis

“…As such, epigenetic therapy has been considered as an attractive treatment strategy in ES. Pattenden et al [35] previously identified that a cluster of HDAC inhibitors including entinostat significantly decreased EWS-FLI1-dependent chromatin accessibility by decreasing EWS-FLI1 levels, which explained our predictions in in silico screen that entinostat can reverse the ES disease signature and EWS-FLI1-mediated transcriptional signature. Besides entinostat, other HDAC inhibitors (e.g., romidepsin, vorinostat, sodium butyrate) have also shown potent anti-ES activity [22,36,37], which provides convincing evidence of the epigenome as a therapeutic target in ES.…”

Chromatin remodeling controls Kaposi's sarcoma-associated herpesvirus reactivation from latency

“…FAIRE relies on differential crosslinking efficiency between DNA bound by nucleosomes versus DNA in nucleosome-depleted regions, where an increase in FAIRE signal indicates a reduction in nucleosome density [ 33 – 35 ]. As a control, we probed the FAIRE signal at AURKAIP1, a region of host chromatin that is consistently nucleosome depleted [ 36 ]. Romidepsin and Panobinostat treatment resulted in a 1.9- to 5.9-fold increase in the FAIRE signal at four regions of the RTA promoter compared to DMSO treated cells ( Fig 6A ).…”

The Jumonji-domain histone demethylase inhibitor JIB-04 deregulates oncogenic programs and increases DNA damage in Ewing Sarcoma, resulting in impaired cell proliferation and survival, and reduced tumor growth

“…A number of different pharmacologic manipulations have been shown to alter EWS/Fli1 oncofusion levels in Ewing Sarcoma [ 37 – 39 ]. Since the oncofusion is known to promote cell proliferation and survival [ 40 ], downregulation of EWS/Fli1 levels could account for the observed opposing effects of JIB-04 on the EWS/Fli1-controlled transcriptome.…”