High throughput RNA sequencing has revealed enormous diversity in mRNA transcript expression whereby alternative splicing produces multiple mRNAs from a single gene. Most human genes are regulated by splicing, yet mRNA diversity largely remains an understudied and hidden layer of cell regulation in health and disease. In melanoma, the extent of splicing is prognostic in patients indicating a relationship between mRNA diversity and more aggressive disease, whilst aberrantly spliced BRAF confers resistance to MAPK targeted therapies. To explore this further, we have applied both short- and long-read RNA sequencing approaches to experimental models and patient samples during disease progression and after treatment with MAPK targeted therapies. Analysis of existing short-read data from patients on-therapy identified thousands of exon-skipping events and pathway analysis identified cellular processes required for drug tolerance and resistance. One of the most significant splicing events was exon inclusion in the RNA processing factor RBM39, and we demonstrate this produces multiple protein isoforms with distinct sub-cellular localisations and functions. Notably, a clinically relevant RBM39 inhibitor synergised with MAPK targeted therapies, reversed biomarkers of drug tolerance and plasticity, and overcame resistance to improve survival in multiple in vivo models. These observations expand our current understanding of therapy-induced transcriptional reprogramming by revealing global changes at the mRNA isoform level, and importantly, we show that inhibiting a key isoform switch can overcome adaptive resistance.
To overcome limitations of short-read sequencing which lacks resolution of long-range exon connectivity, we have now comprehensively profiled the mRNA isoform repertoire in melanoma using Nanopore long read sequencing. Excitingly, we have identified thousands of novel transcripts in both primary and metastatic melanoma patient samples, and hundreds of significant isoform-switching events in melanoma tumours during distinct phases of the adaptive response to targeted therapy. These analyses provide new insights into the post-transcriptional landscape of melanoma progression and reveal new biology underpinning therapy-induced plasticity and acquired resistance. Collectively, we expect these approaches will provide new targets for both small molecule and mRNA-based therapies to improve outcomes for melanoma patients resistant to current therapies.