The N6-methyladenosine (m6A) RNA binding protein YTHDF1 is frequently overexpressed in colorectal cancer (CRC) and drives chemotherapeutic resistance. To systematically identify druggable targets in CRC with high expression of YTHDF1, we employed a CRISPR/Cas9 screening strategy that revealed RUVBL1 and RUVBL2 as putative targets.RUVBL1/2 were overexpressed in primary CRC samples and represented independent predictors of poor patient prognosis. Functionally, loss of RUVBL1/2 preferentially impaired the growth ofYTHDF1-high CRC cells, patient-derived primary CRC organoids, and subcutaneous xenografts. Mechanistically, YTHFD1 and RUVBL1/2 formed a positive feed-forward circuit to accelerate oncogenic translation. YTHDF1 bound to m6A-modified RUVBL1/2 mRNA to promote translation initiation and protein expression. Co-IP and mass spectrometry identified that RUVBL1/2 reciprocally interacted with YTHDF1 at 40S translation initiation complexes. Consequently, RUVBL1/2 depletion stalled YTHDF1-driven oncogenic translation and nascent protein biosynthesis, leading to proliferative arrest and apoptosis. Ribo-seq revealed that RUVBL1/2 loss impaired the activation of MAPK, RAS and PI3K-AKT signaling induced by YTHDF1. Finally, blockade of RUVBL1/2 by the pharmacological inhibitor CB6644 or vesicle-like nanoparticle-encapsulated siRNAs preferentially arrested the growth of YTHDF1-expressing CRC in vitro and in vivo. Together, this study uncovered that RUVBL1/2 are potential prognostic markers and druggable targets that regulate protein translation in YTHDF1-high CRC.

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