Targeting APOBEC3A's genomic mutagenic activity with functionalized DNA dumbbells

点击翻译按钮获取中文摘要

Project Summary Kataegic mutational signatures are localized, hypermutated clusters found across the genomes of multiple cancer types. These mutational marks have been associated with tumor development and adaptation, and ongoing research is aimed at deciphering their wider role in patient prognosis and resistance to chemotherapeutic agents. In efforts to ascertain the source of kataegis, sequencing studies have revealed the majority of these mutations are C to T/G substitutions enriched within 5’-TCN sequence contexts. Identification of this feature led to the suspicion and subsequent validation that members of the APOBEC3 cytidine deaminase family are the source of genomic mutation in kataegis. The APOBEC3 (A3) family plays a crucial role in defense against retroviruses and retrotransposable elements by deaminating C to U in single-stranded DNA (ssDNA) intermediates. However, misregulation of A3A and A3B can lead to pathologic deamination of the host genome. During events where genomic DNA becomes single-stranded, such as in DNA replication or repair, cytosine bases become prone to deamination, leading to targeted mutations or promotion of double-stranded DNA breaks. Targeting of these genomic mutators thus presents an attractive therapeutic strategy for evading APOBEC- driven kataegis in cancer. However, we currently lack molecular probes that can modulate APOBEC activity in the lab or strategies for development of clinical therapeutics. APOBEC3A has recently been shown to prefer ssDNA substrates in a stem-loop conformation, with the target cytosine placed in the 3’ end of the loop, a finding verified in both biochemical studies and genetic studies where a predominant number of the APOBEC-driven mutations in tumors are in this mesoscale structural context. In this proposal, we seek to exploit this substrate preference to develop more potent inhibitors of A3A and translate them towards cellular targeting of A3A’s genomic mutagenetic activity. We have already shown that placing methylzebularine, an inhibitory base towards cytidine deaminases, within cyclized DNA dumbbells, a scaffold mimicking A3A’s preferred substrate structure, results in subnanomolar-level inhibition of A3A in vitro. In aim 1, we will identify the mode of inhibition of these DNA dumbbells and perform structure-activity relationship studies on a panel of structurally diverse dumbbells to determine which features translate to more potent inhibitors. In aim 2, we will advance these results towards cellular studies on U2OS cells with inducible A3A overexpression and evaluate whether these inhibitors can block A3A-mediated genomic DNA damage and increased mutational load. Finally, in aim 3, we will exploit the stem portion of the dumbbell to modulate the mode of inhibition towards active protein degradation in a manner analogous to proteolysis-targeting chimeras (PROTACs). To do this, we will conjugate an mZ dumbbell to VH032, an E3 ligase recruiting ligand, and assess its ability to degrade A3A and block its genomic mutagenic activity. Completion of this proposal will advance rationally designed nucleic-acid based inhibitors of A3A, providing two novel routes to perturb APOBEC-driven kataegis in cancer through classic inhibition or protein degradation.