Long-Read MDM4 Isoform Sequencing Reveals Aberrant Isoform Landscape in Metastatic Melanomas

The changes in splicing of MDM4 in human melanomagenesis are critical to p53 activity and represent potential therapeutic targets. Genomic characteristics of melanoma and precursor lesions, such as copy number variation and gene expression, have been investigated in detail. However, there has never been a detailed survey of splicing changes that occur during melanomagenesis. Splicing changes can be a very early event in melanoma tumor progression, with characteristic changes in the p53 pathway already in place in early nevi. MDM4 is upregulated in a strong majority of melanoma cases and has been described as a “key therapeutic target in cutaneous melanoma”. Identifying splicing changes in human tissue specimens will provide therapeutic targets during initiation, dysplasia, and progression toward metastatic melanoma.Splicing of MDM4 has been characterized in melanomas with increased MDM4 isoform expression over canonical MDM4-FL. However, these studies have utilized new or existing data obtained by next-generation sequencing (NGS) methods, typically Illumina sequencing, relying on short reads. A difficulty associated with NGS analysis is the loss of “connectivity” data in which full transcripts are inferred from the presence of splice junction reads. To address this problem, long-read, third generation sequencing, was utilized to read the entire length of transcripts, intact. Oxford Nanopore flow cell sequencing allowed for quantification and identification of the MDM4 transcripts, both alternative and canonical, present in melanoma specimens. RT-PCR and Nanopore sequencing provided a direct view into the isoform landscape of melanoma specimens while RT-qPCR quantified the expression of major MDM4 isoforms represented in the samples. This study identifies and quantifies MDM4 isoforms present in malignant melanoma tumor samples and provides evidence of a novel hybrid MDM4-A & MDM4-S isoform (MDM4-A/S).