LAUSR

Drug resistance critically limits the long-term efficacy of MAPK-targeted therapy in melanoma. While resistance mechanisms at genetic, epigenetic, and transcriptional scales are well-documented, post-transcriptional splicing regulation remains poorly understood. By analyzing patient-matched pre-treatment and resistant melanoma biopsies, we uncover widespread alternative splicing alterations during therapy resistance. Splicing perturbations are most pronounced in MAPK and PI3K-AKT pathway genes. We identify a splicing switch of AKT2 from isoform 210 to 206 in 29.55% (13/44) of disease-progressive biopsies. This splicing switch induces AKT2 kinase hyperactivity by restoring the activated fragment A-loop. Functional validations confirm that AKT2-206 confers BRAF inhibitor resistance in melanoma cells by activating S6 kinase. Further, the splicing factor hnRNPK likely drives the splicing switch of AKT2 during acquired resistance. Our results not only provide insights into splicing-mediated regulation of drug resistance but also highlight the importance of alternative splicing isoforms as targets for clinical diagnosis and therapy. The splicing factor hnRNPK drives a splicing switch of AKT2 (isoform 210 to 206), restoring kinase hyperactivity via retention of the A-loop fragment and conferring MAPK-targeted therapy resistance in melanoma through PI3K-AKT pathway activation. Splicing perturbation is a key mechanism of resistance to targeted therapy. AKT2 splicing switch (isoform 210 to 206) is a driver of MAPKi resistance. hnRNPK mediates retention of AKT2 exon 10 in acquired resistance. Splicing perturbation is a key mechanism of resistance to targeted therapy. AKT2 splicing switch (isoform 210 to 206) is a driver of MAPKi resistance. hnRNPK mediates retention of AKT2 exon 10 in acquired resistance. The splicing factor hnRNPK drives a splicing switch of AKT2 (isoform 210 to 206), restoring kinase hyperactivity via retention of the A-loop fragment and conferring MAPK-targeted therapy resistance in melanoma through PI3K-AKT pathway activation.