GSE137258
Inhibition of YTHDF2 triggers proteotoxic cell death in MYC-driven breast cancer
We found in this study through targeted CRISPR-Cas9 screening of RBPs that disrupting YTHDF2-dependent degradation of its target transcripts triggers apoptosis of MYC-dependent cancer cells and tumors. Enhanced CLIP (eCLIP), m6A-sequencing (m6A-seq) and YTHDF2 knockdown followed by RNA-seq analysis of YTHDF2-m6A-mRNA interactions revealed extensive associations with mRNAs encoding MAPK pathway genes that when stabilized, induce epithelial-to-mesenchymal transition and stress-induced apoptosis. scRibo-STAMP profiling of translating mRNAs revealed global alterations in the translatome of single cells in YTHDF2-depleted solid tumors. Thus, our work highlights the therapeutic relevance of RBPs by uncovering the critical role of YTHDF2 in counteracting the global increase of mRNA synthesis in MYC-driven cancers. Overall design: We constructed and applied an RBP targeting CRISPR-Cas9 library to MYC-inducible human mammary epithelial cells 8 and 16 days after transduction by sequencing of gDNA in biological duplicate. YTHDF2 binding and m6A profiles were examined using eCLIP-seq and m6A-seq by immunoprecipitating mRNA fragments bound to either endogenous YTHDF2 or m6A antibody in biological duplicates with input controls. In addition, we examined changes in mRNA expression following depletion of YTHDF2 using two independent shRNAs in biological duplicate. Lastly, we identified translational changes in the YTHDF2-depleted solid tumors by directing C-to-U editing of translating transcripts by transducing cells with RPS2-APOBEC1 fusions prior to subcutaneous xenograft.