Cell state is governed by master regulatory transcription factors which establish coordinated gene expression programs. The robustness of these programs is maintained by covalent modification to nuclear chromatin. In cancer, aberrant transcriptional growth pathways are enforced and normal differentiation pathways are suppressed. Previous research from our laboratory established the feasibility of targeting epigenetic reader proteins for open, active chromatin (bromodomains). This proposal defines a research strategy expected to discover first inhibitors of chromatin-binding modules (chromodomains) found in regions of silenced chromatin. Suppression of differentiation is accomplished in cancer cells, in great part, by covalent modification of histone 3 lysine 27 by side-chain trimethylation (H3K27me3), which is a mark placed by the polycomb repressive complex (PRC). Already, human cancer genome sequencing has identified activating, oncogenic mutations in the PRC catalytic lysine methyltransferase, EZH2, as well as inactivating mutations in the H3K27me3 lysine demethylase, UTX. Biological validation of deregulated PRC biochemistry in numerous cancer types (e.g. non- Hodgkin lymphoma, prostate cancer, multiple myeloma, acute myeloid leukemia and breast cancer), has established a pressing need for drugs targeting H3K27me3 signaling. The centrality of PRC function in developmental and cellular biology renders chemical probes of H3K27me3 signaling broadly desirable.
The Specific Aims defining this research proposal will employ high-throughput screening to discover chemotypes capable of disrupting the H3K27me3 interaction with CBX-family chromodomain epigenetic reader proteins. Our laboratory has developed a robust, efficient and highly sensitive assay allowing detection of this molecular recognition event for CBX7, using a nanoparticle-based proximity technology (AlphaScreen; PerkinElmer). Already, we have miniaturized the CBX7 AlphaScreen Assay, observing adequate robustness (Z' = 0.88) and signal (signal-to-noise ratio of 71.9) to prompt immediate use in high-throughput screening. Indeed, we have completed a pilot HTS study of CBX7 in the Bradner laboratory with a 3,000 feature chemical library, observing excellent assay performance but as yet no confirmed inhibitors. We therefore propose to implement high- throughput screening of a large and chemically diverse compound collection at an experienced and specialized screening facility at Harvard Medical School, called the Institute for Chemical and Cell Biology (ICCB). Assay positives will be retested in dose-ranging format for specific activity against CBX7, and further characterized in a panel of secondary biochemical and biological assays of relevance to cancer biology. Based on a compelling biological hypothesis, these studies will focus on a genetically-defined subtype of non-Hodgkin lymphoma, featuring activating mutations in EZH2 which result in hypermethylation of chromatin at H3K27. In keeping with an open-innovation model of small-molecule discovery, we will place all screening data into the public domain and provide access to chemical probes (structures and samples) discovered and developed within this Project.
Cellular memory is maintained at the molecular level by proteins which mark active ('on') and inactive ('off') regions of the genome. In cancer, these marks are misplaced throughout genome causing endless growth and an inability to differentiate into normal tissue. Based on our prior successful research which led to the first inhibitors of active bookmarks, we here propose a strategy to discover first inhibitors of bookmarks located near inactivated genes.