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Large-scale identification and functional analysis of the m6A reader YTHDF2 as a therapeutic target for triple negative breast cancer


RNA-binding proteins (RBPs) are critical regulators of post-transcriptional gene expression and aberrant RBP-RNA interactions can cause cancer. However, RBPs are often overlooked as therapeutically relevant targets because unlike transcription factors, altered RBP activity is more frequently caused by changes in the expression levels of RBPs and their underlying targets, as opposed to somatic mutations. It has therefore been challenging to systematically evaluate the function of RBPs in disease. We addressed the lack of characterization of RBPs in cancer by developing an approach to interrogate the function of RBPs using pooled CRISPR-Cas9 screening. We identified 57 putative RBPs with distinct roles in supporting MYC-driven oncogenic pathways and we found that disrupting YTHDF2-dependent degradation of its target transcripts triggers apoptosis of MYC-dependent cancer cells and tumors. Next, we profiled YTHDF2 function using enhanced CLIP (eCLIP) and m6A-seq analysis, which revealed extensive interactions with mRNAs encoding MAPK pathway genes. We found that mRNA stabilization of upstream MAPK pathway genes drives epithelial-to-mesenchymal transition in MYC-dependent cells and is accompanied by profound oxidative cellular stress due to increased protein synthesis. We next explored the cellular stress response elicited by YTHDF2 depletion by surveying the activation of the intrinsic and extrinsic apoptotic pathways. Loss of YTHDF2 caused activation of the unfolded protein response and subsequent mitochondrial dysfunction in MYC-dependent cells, which are especially sensitive to increases in oxidative stress. Finally, we determined that PRSS23 mRNA stabilization is necessary for driving apoptosis by promoting TCF12-mediated transcription of cancer lineage-specific transcripts and translation factors. Thus, this dissertation 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.

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