Discovery of Small Molecules That Inhibit MYC mRNA Translation Through hnRNPK and Induction of Stress Granule-Mediated mRNA Relocalization

<i>MYC</i> is a key oncogenic driver frequently overexpressed in non-small cell lung carcinoma (NSCLC) and other cancers, where its protein levels often exceed what would be expected from <i>MYC</i> mRNA levels alone, suggesting post-transcriptional regulation. Strategies to inhibit <i>MYC</i> function by targeting mRNA translation hold potential for therapeutics utility in Myc-dependent cancers. We developed TranslationLight, a high-content imaging platform which detects <i>MYC</i> mRNA translation in human cells. Using this system, we conducted a high-throughput screen of ~100,000 compounds to identify small molecules that selectively modulate <i>MYC</i> translation. Candidate compounds were evaluated by immunofluorescence, ribosome profiling, RNA sequencing, cellular thermal shift assays (CETSA), and subcellular localization studies of mRNA and RNA-binding proteins. We identified a lead compound, CMP76, that potently reduces Myc protein without substantially decreasing its mRNA abundance. Mechanistic investigations showed that the compound induces relocalization of <i>MYC</i> mRNA into stress granules, accompanied by translational silencing. CETSA identified hnRNPK as a primary protein target, and compound treatment triggered its cytoplasmic relocalization together with formation of hnRNPK-containing granules colocalizing with <i>MYC</i> mRNA. Analysis across cancer cell lines revealed that sensitivity to CMP76 was significantly associated with RBM42 dependency. This work establishes a novel therapeutic strategy to inhibit <i>MYC</i> translation mediated by hnRNPK, offering a translationally targeted approach to cancer therapy.