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

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 mRNA levels alone, suggesting post-transcriptional regulation. Strategies to inhibit function by targeting mRNA translation hold potential for therapeutics utility in Myc-dependent cancers. We developed TranslationLight, a high-content imaging platform which detects 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 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 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 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 translation mediated by hnRNPK, offering a translationally targeted approach to cancer therapy.