Nanoscale photoproximity labeling of c-Myc predicts cancer specific therapeutic targets

Transcription factors (TFs) have long been aspirational therapeutic targets for the treatment of diseases, as their dysregulation is a common mechanism for altered cell states. Despite this, many TFs implicated in disease have disordered structures and lack canonical binding pockets, rendering them non-trivial targets for small molecule-based therapies. Directly inhibiting TF function has proven difficult, but indirect inhibition by targeting the effector molecules that modulate TF function is a promising alternative strategy. Here we report a unified strategy for capturing cancer-specific protein-protein interactions using µMap photoproximity labeling. Using a recently reported intein-based strategy for catalyst conjugation in biochemically intact nuclei, we demonstrate that we can capture unique protein interactomes of c-Myc in androgen receptor dependent and independent prostate cancer cell lines, and that these unique interactors can be mined to identify druggable vulnerabilities. We find that a PC-3 specific Myc interactor, SLK, selectively promotes Myc stabilization and drives epithelial morphology only in androgen receptor negative prostate cancer. Overall design: RNA-seq profiling of total mRNA in WPMY-1, LNCaP, and PC-3 wild-type cells. The goal of this experiment is to evaluate alternative splicing of SLK in each cell line. Additionally, PC-3 cells transfected with 0.75µg c-Myc-CfaN or c-Myc-V5-6xHis plasmids are compared against PC-3 wild-type mRNA profiles to validate that c-Myc target gene transcription is increased after transient transfection of both constructs.

转录因子（Transcription factors, TFs）长期以来一直是疾病治疗领域极具吸引力的治疗靶点，因其表达失调是细胞状态改变的常见致病机制。尽管如此，诸多与疾病相关的转录因子存在结构紊乱的问题，且缺乏经典结合口袋，使其难以作为小分子疗法的靶向靶点。直接抑制转录因子功能已被证实颇具挑战，而通过靶向调控其功能的效应分子实现间接抑制，则是一项颇具前景的替代策略。本文报道了一种利用µMap光邻近标记技术捕获癌症特异性蛋白质-蛋白质相互作用的统一策略。借助近期报道的、可在生化完整的细胞核中实现催化剂偶联的内含肽（intein）介导策略，我们证实可在雄激素受体依赖性与非依赖性前列腺癌细胞系中，捕获c-Myc的独特蛋白质相互作用组，并可通过挖掘这些独特互作蛋白，识别可成药的靶点脆弱性。我们发现，PC-3细胞特异性的Myc互作蛋白SLK仅在雄激素受体阴性的前列腺癌细胞中，选择性促进Myc的稳定化，并驱动上皮细胞形态发生。实验总体设计：对WPMY-1、LNCaP及PC-3野生型细胞中的总mRNA进行RNA测序（RNA-seq）谱分析，本实验旨在评估SLK在各细胞系中的可变剪接情况。此外，将转染了0.75μg c-Myc-CfaN或c-Myc-V5-6xHis质粒的PC-3细胞的mRNA表达谱，与PC-3野生型细胞的mRNA谱进行对比，以验证两种质粒经瞬时转染后，c-Myc靶基因的转录水平均得到上调。