Functional E3 ligase hotspots and resistance mechanisms to small-molecule degraders via chemical genetics [Hybrid capture]

Targeted protein degradation is a novel pharmacology established by drugs that recruit target proteins to E3 ubiquitin ligases. Based on the structure of the degrader and the target, different E3 interfaces are critically involved, thus forming defined "functional hotspots". Understanding disruptive mutations in functional hotspots informs on the architecture of the assembly, and highlights residues susceptible to acquire resistance phenotypes. Here, we employ haploid genetics to show that hotspot mutations cluster in substrate receptors of hijacked ligases, where mutation type and frequency correlate with gene essentiality. A Hybrid capture assay reveals resistance-conferring mutations after degrader treatment. 29 putative target genes were selected to be sequenced. Target gene enrichment from gDNA of treated cells was performed, followed by amplification and sequencing to identify mutations. Overall design: Total of 7 samples of KBM7 cells treated with BET PROTAC degraders ARV-771 or dBET6 (concentrations: 25x, 50x, 25x-50x pool). Amplicon next-generation sequencing of 29 genes after enrichment and amplification to detect genomic mutations.

靶向蛋白质降解（Targeted Protein Degradation）是一类由将靶蛋白招募至E3泛素连接酶（E3 Ubiquitin Ligase）的药物所确立的新型药理学范式。根据降解剂与靶标的结构差异，不同的E3结合界面发挥关键调控作用，进而形成明确的"功能热点区域"。解析功能热点区域中的破坏性突变，可揭示该复合物的组装架构，并明确易产生耐药表型的氨基酸残基位点。本研究采用单倍体遗传学（haploid genetics）方法，证实热点突变富集于被劫持的连接酶的底物受体中，且突变类型、发生频率与基因必需性呈显著相关。杂交捕获检测技术可揭示降解剂处理后产生的耐药相关突变。本研究选取29个推定靶基因进行测序：先从经处理细胞的基因组DNA（gDNA）中富集靶基因片段，随后进行扩增与测序以鉴定突变位点。实验整体设计：共7份经BET类蛋白水解靶向嵌合体（PROTAC）降解剂ARV-771或dBET6处理的KBM7细胞样本（处理浓度分别为25倍、50倍，以及25倍与50倍混合组）。通过对富集扩增后的29个基因进行扩增子下一代测序，以检测基因组突变。