Targeting APOBEC3A's genomic mutagenic activity with functionalized DNA dumbbells

项目摘要 Kataegic突变特征是在人类基因组中发现的定位的、超突变的簇 多种癌症类型。这些突变标记与肿瘤的发展和适应有关， 正在进行的研究旨在破译它们在患者预后和抗药性方面的更广泛作用 化疗药物。在努力确定kataegis来源的过程中，测序研究揭示了 这些突变大多是富含在5‘-TCN序列上下文中的C到T/G替换。鉴定 这一特征导致了对APOBEC3胞苷脱氨酶成员的怀疑和随后的验证 家族是卡塔吉斯基因组突变的来源。APOBEC3(A3)家族在防御中起着至关重要的作用 单链DNA脱氨基C到U抗逆转录病毒和反转录转座子元件 中间体。然而，A3A和A3B的错误调节可导致宿主基因组的病理性脱氨基。 在基因组DNA变成单链的事件中，例如在DNA复制或修复过程中，胞嘧啶 碱基容易脱氨基，导致靶向突变或促进双链DNA断裂。 因此，靶向这些基因组突变子为规避APOBEC提供了一种有吸引力的治疗策略。 被癌症驱使的卡特吉斯。然而，我们目前缺乏能够调节APOBEC活性的分子探针。 临床治疗学发展的实验室或策略。APOBEC3A最近被证明更喜欢 以茎环构象的单链DNA底物，靶标胞嘧啶位于环的3‘端，这是一个发现 在生化研究和遗传研究中都得到了证实，在这些研究中，APOBEC驱动的 肿瘤中的突变是在这种中尺度的结构背景下发生的。在这项提议中，我们试图利用这种底物 倾向于开发更有效的A3A抑制剂并将其转化为A3A的细胞靶向 基因组诱变活性。我们已经证明，将甲基泽布林，一种抑制碱基 胞苷脱氨酶，在环化的DNA哑铃中，一种模仿A3A首选底物结构的支架， 结果A3A在体外具有亚纳摩尔水平的抑制作用。在目标1中，我们将确定它们的抑制模式 DNA哑铃，并对一组结构不同的哑铃进行构效关系研究 以确定哪些特征可以转化为更有效的抑制剂。在目标2中，我们将推动这些成果朝着 可诱导A3A过表达的U2OS细胞的细胞研究及评价这些抑制剂是否能 阻断A3A介导的基因组DNA损伤和增加突变负荷。最后，在目标3中，我们将利用 哑铃的茎部分以一种方式调节对活性蛋白降解的抑制模式 类似于蛋白水解靶向嵌合体(PROTAC)。为此，我们将MZ哑铃与VH032共轭， E3连接酶招募配体，并评估其降解A3A和阻断其基因组诱变活性的能力。 这项提案的完成将促进合理设计的核酸类A3A抑制剂，提供两种 通过经典的抑制或蛋白质降解来扰乱癌症中APOBEC驱动的kataegis的新途径。

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.