Novel Long Non-Coding RNAs Associated with BAP1 Mutations in Uveal Melanoma

Abstract

Purpose : Uveal melanoma (UM) is a highly aggressive eye cancer that leads to metastatic death in up to half of patients. Recent genetic discoveries have made tremendous advancements in our understanding of UM and are paving the way to more effective precision management. UMs can be divided into two prognostic groups based on their gene expression profile (GEP). Class 1 tumors are associated with EIF1AX or SF3B1 mutations and low metastatic risk, while Class 2 tumors are associated with inactivating mutations in the tumor suppressor BAP1 and are highly metastatic. Class 1 and Class 2 UMs have been shown to differ in their expression of numerous known long non-coding RNAs (lncRNA). Here, we sought to identify novel differentially expressed lncRNAs in UM tumors.

Methods : RNA-Seq from 103 UM samples were analyzed. Quality controlled using FastQC (v0.11.3), and trimmed using trim-galor (v0.4.1). Sequences were aligned to the human genome (GRCh38) using STAR (v2.5). Transcript discovery was performed. Transcripts with a high probability of protein coding were discarded. Transcripts predicted to be non-coding with transcript length >200bps were retained. Counts for all known and novel transcripts were generated. Random forests based surpervised machine learning algorithm was used to assign a class status for 12 unknown UM samples. Differential expression was performed using DESeq2. Tumor specific lncRNA expression was then assessed through single cell sequencing of 11 UM samples.

Results : A cutoff of FPKM > 1 in at least 30% of tumors was used as a threshold for transcripts of interest, resulting in 104 novel transcripts, 32 of which were differentially expressed at FDR < 0.05 between Class 1 and Class 2 tumors. UM specific expression was performed via single cell RNAseq analysis on 8 primary and 3 metastatic samples. When compared to the microenvironment 10 lead lncRNAs showed expression solely in tumor cells. BAP1 was knocked out via crispr cas9 in uveal melanocyte (UMC) cell lines. Cell lines were validated and novel lncRNA expression was assessed.

Conclusions : These methods confirm the presence of uncharacterized lncRNAs that are specific to UM. Some of these lncRNAs have a strong association with the functionality of BAP1. Further work is underway to elucidate the function of these novel transcripts in UM pathogenesis and their interplay with BAP1.

This is a 2020 ARVO Annual Meeting abstract.