Chemical Biology Platforms to Define the Metabolism-Epigenetics Interface

Division Of Basic Sciences - Nci

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Abstract

The overarching goal of this project is to define how environmental cues that impact protein and nucleic acid-based signaling, including but not limited to the epigenetic regulation of gene expression, contribute to cancer development and progression. To facilitate these studies we are developing new technologies to define the physical interactions by which small molecule metabolites influence epigenetic and epitranscriptomic signaling, with the long-term goal of identifying novel strategies for cancer diagnosis, therapy, and prevention. This project aims to illuminate the fundamental biological principles linking metabolism and modification-based signaling, while simultaneously advancing cutting-edge technologies that can be used to accelerate drug discovery and facilitate next-generation cancer diagnostics. The goal of this project is to characterize and control novel modification-based regulatory mechanisms that drive altered gene activity in cancer. The majority of our initial efforts in this area have focused on the study of protein and nucleic acid acetylation. Using a suite of novel chemical biology platforms that allow us to study cellular acetylation mechanisms, we have discovered new enzymatic and non-enzymatic acetylation pathways that are highly elevated in cancer. In addition, we have made substantial progress characterizing the activity, druggability, and metabolic regulation of these mechanisms. These advances are grouped according to three specific aims. 1. Discovery and characterization of novel acetyltransferase enzymes. Chemoproteomic profiling studies in our lab led to the discovery that a relatively uncharacterized acetyltransferase, NAT10, is highly upregulated in a variety of cancer cell lines, and also sensitive to the metabolic state of the cell. Subsequent work has revealed the primary function of NAT10 is the catalysis of RNA cytidine acetylation, which evidence suggests extends to diverse elements of the transcriptome including ribosomal RNA, transfer RNA, and messenger RNA. Over the past year we have developed multiscale chemical approaches to biologically, biophysically, and biochemically characterize cytidine acetyltransferase activity, including the development of a method for directly sequencing substrates of these enzymes (Gamage et al., Nature Protocols 2021), installing modifications into synthetic RNAs (Bartee et al., JACS 2022), and sequencing related modifications (Link et al., Biochemistry 2022) . Our sequencing methods facilitated the recent unambiguous definition of the major substrates of NAT10 in human cells, as well as the identification of a promiscuous archaeal RNA acetyltransferase enzyme (Sas-Chen et al., Nature, 2020). We are currently using this methodological toolbox to dissect the mechanisms by which this modification is installed (Bortolin-Cavaille et al., Nucleic Acids Research 2021) and their role in cancer development and progression. Our expertise in this area is also lending itself to studies of how base modifications influence RNA-protein interactions in synthetic mRNAs such as those used in COVID-19 vaccines, a topic of substantial public interest (Nance et al., ACS Central Science 2021, Nance et al. Cell Chemical Biology 2022). 2. Characterization of acetyltransferase inhibitors. Targeting the cellular acetylation machinery is an emerging paradigm in oncology. However, relatively few small molecule inhibitors of acetyltransferases are known. To address this unmet need, our group has developed biochemical, chemoproteomic, and cell-based assays that can be used to unambiguously interrogate the activity of small molecule acetyltransferase inhibitors. These methods enabled the first evidence for cellular occupancy and on-target activity of a small molecule lysine acetyltransferase inhibitor as well as N-terminal acetyltransferase inhibitors. Currently we are applying these approaches in collaboration with industry to define the selectivity of novel classes of acetyltransferase inhibitors (Kung et al. ACS Med Chem Lett, 2020; Jing et al. ACS Chem Biol 2021) and aid the pre-clinical and clinical development of these compounds for cancer treatment. Finally, we continue to apply our methods to characterize the pan-assay interference features of reported acetyltransferase inhibitors (Shrimp et al. ACS Med Chem Lett 2021). These studies critical are crucial to aiding the interpretation of the activity of these molecules in cellular assays and providing impetus for the discovery of drug-like acetyltransferase inhibitors. 3. Metabolic regulation of epigenetics. Emerging evidence indicates that metabolism itself may function as a modification-based regulatory or "epigenetic" mechanism, through the ability of metabolites to modulate the activity of enzymes involved in epigenetic and epitranscriptomic regulation of gene expression, as well as directly react with amino acid residues leading to the deposition of non-enzymatic protein posttranslational modifications. Our previous work has focused on the development of technologies for studying the metabolic regulation of acetylation. Over the past two years, we have extended these methods to study the mechanism of action of oncometabolites, a class of cancer metabolites that can directly trigger tumorigenic signaling. Using the oncometabolite fumarate which accumulates in the hereditary cancer syndrome predisposition HLRCC as an initial model, we have shown that chemoproteomic methods can be used to identify hotspots of oncometabolite reactivity and quantify their level of protein modification on a proteome-wide scale (Perez et al., JBC, 2020). In our current and future work we are building on these insights by screening mechanistically well-annotated compounds as well as covalent ligands to identify pathways whose dysregulation is driven by aberrant oncometabolite accumulation in order to facilitate novel therapeutic strategies.