Tumor cell-specific inhibition of MYC function using small molecule inhibitors of the HUWE1 ubiquitin ligase. Homo sapiens

Deregulated expression of MYC is a driver of colorectal carcinogenesis, necessitating novel strategies to inhibit MYC function. The ubiquitin ligase HUWE1 (HECTH9, ARFBP1, MULE) associates with both MYC and the MYC-associated protein MIZ1. We show here that HUWE1 is required for growth of colorectal cancer cells in culture and in orthotopic xenograft models. Using high throughput screening, we identify small molecule inhibitors of HUWE1, which inhibit MYC-dependent transactivation in colorectal cancer cells, but not in stem and normal colon epithelial cells. Inhibition of HUWE1 stabilizes MIZ1. MIZ1 globally accumulates on MYC target genes and contributes to repression of MYC-activated target genes upon HUWE1 inhibition. Our data show that transcriptional activation by MYC in colon cancer cells requires the continuous degradation of MIZ1 and identify a novel principle that allows for inhibition of MYC function in tumor cells. Overall design: MIZ1 and MYC ChIPseq experiments in HUWE1 inhibitor-treated Ls174T cells as well as RNAseq experiments in HUWE1- or MIZ1-depleted Ls174T cells after HUWE1 inhibitor treatment. Sequencing was performed on an Illumina Genome Analyzer IIx.

MYC的异常表达是结直肠癌发生的驱动因素，因此亟需开发抑制MYC功能的全新策略。泛素连接酶HUWE1（HECTH9、ARFBP1、MULE）可同时结合MYC以及MYC相关蛋白MIZ1。本研究证实，HUWE1是结直肠癌细胞在体外培养及原位异种移植模型中增殖所必需的。通过高通量筛选，我们鉴定出HUWE1的小分子抑制剂，该类抑制剂可在结直肠癌细胞中抑制MYC依赖的转录激活，但在干细胞及正常结肠上皮细胞中无此效应。抑制HUWE1可稳定MIZ1蛋白。MIZ1在MYC靶基因位点发生全局性富集，并在HUWE1抑制后参与抑制MYC激活的靶基因转录。本研究数据表明，结肠癌细胞中MYC介导的转录激活需要MIZ1的持续降解，并揭示了一种可在肿瘤细胞中抑制MYC功能的全新机制。
实验设计概况：对经HUWE1抑制剂处理的Ls174T细胞开展MIZ1与MYC的染色质免疫沉淀测序（ChIPseq）实验，同时对经HUWE1抑制剂处理后敲低HUWE1或MIZ1的Ls174T细胞开展RNA测序（RNAseq）实验。测序工作使用Illumina Genome Analyzer IIx平台完成。