Nucleosomes are the basic units of chromatin, comprised of a histone octamer made up of two copies of each of the histone subunits (H2A, H2B, H3, and H4). Changes in chromatin structure and function are mediated through histone post-translational modifications (PTMs), such as methylation and ubiquitination. Alterations of specific PTMs are highly associated with human diseases, including numerous forms of cancer. Some histone PTMs work in intranucleosomal groups or systems to regulate the function of histone methyltransferases (HMTs), a concept referred to as the ?histone code?. For instance, mono-ubiquitination at lysine 120 of histone H2B (H2Bub1) dramatically enhances enzymatic activity of HMTs that target histone H3, including DOT1L and the SET1-family (SETD1A, SETD1B, MLL1, -2, -3, -4). These striking observations provide a unique opportunity for targeted therapeutic development. PTM enzyme inhibitor assays currently use modified histone proteins or fragments, and therefore fail to screen enzymes in the context of PTM combinations, which are inherent to chromatin regulation in vivo. We hypothesize that modified semi-synthetic nucleosomes carrying specific PTMs (i.e. `designer nucleosomes' or `dNucs' for brevity) will provide a biologically-relevant substrate for identification of inhibitors in the context of both chromatin architecture and unique epigenetic signatures, making them optimal substrates for PTM enzyme inhibitor assays. In this proposal, EpiCypher will develop an innovative dNuc-based HMT inhibitor screening platform, which capitalizes on cooperative interactions between HMTs and histone ubiquitination to identify context-specific inhibitors for cancer therapy. Further, we will enable the use of dNucs as substrates in high-throughput drug screening assays by developing the commercial potential of Amber suppression technology to generate large quantities of high quality ubiquitinated histones. We will focus our study on the HMT activity of DOT1L and SETD1A, enzymes that are highly dependent on the presence of H2Bub1 and are upregulated in numerous cancers, including leukemia and colorectal cancer. While several potent and specific DOT1L inhibitors have been identified, there are no known specific inhibitors of SETD1A. Thus, DOT1L provides a unique molecular avenue to examine how known inhibitors with varying specificities function in our dNuc-based assay, whereas SETD1A provides an opportunity to identify new inhibitor compounds with immediate and unmet clinical relevance. The H2Bub1-dependent HTM inhibitor assay proposed here represents a first step toward not only a new research platform but also a potentially powerful, novel drug screening tool. Pending success of our Phase I efforts, the Phase II program will ramp up commercial manufacturing of H2Bub1-modified nucleosomes. In addition, we will focus on optimizing high-throughput assay development for DOT1L and SETD1A as well as establishing additional H2Bub1-dependent inhibitor assays using other SET1 family members including SETD1B, MLL1, -2, -3, and -4.
The progression and onset of numerous of cancers have been linked to modifications of histones, the structural proteins of chromosomes that serve as modification-dependent regulators of gene expression. Understanding how one histone modification relates to the presence, absence, or function of another is challenging given the dozens of potential modifications that can appear on any of the 8 histone subunits found in a mature nucleosome complex. Here, we propose a yet undeveloped and novel designer nucleosome- based platform, comprising specifically modified histones to screen for context-dependent inhibitors of histone- modifying enzymes.