Unravelling the transcriptome in mantle cell lymphoma

Many studies in the incurable B-cell hematological malignancy, mantle cell lymphoma (MCL), have focused on identifying mutations in oncogenes and tumor suppressor genes. Next-generation sequencing technologies have opened the potential to discover other global changes that contribute to the malignancy. In particular, sequencing the RNA can help us uncover transcriptome diversity resulting from alternative splicing, alternative polyadenylation (resulting in different 3'UTRs), generation of fusion transcripts as well as non-coding RNAs. The goal of this study was to characterize the transcriptome of MCL. We performed long-read Iso Sequencing and short-read RNA Sequencing on RNA samples from three MCL cell lines. The data was analyzed to identify novel transcripts as well as alternative splicing and alternative polyadenylation patterns. Select transcripts were validated using different types of PCR, Sanger Sequencing and other molecular biology techniques. Our heatmap showed that while there was always overlap between highly upregulated and downregulated transcripts in two cell lines, each cell line had its own unique gene signature. We were able to identify different variants arising from alternative splicing and alternative promoter usage in several genes including glutaminase (GLS1) by overlaying our long-read sequencing and short-read sequencing data. Our qRT-PCR and Western blot results confirmed the presence of the two most prevalent GLS1 alternatively spliced variants GAC and KGA. At the protein level, all the MCL cell lines expressed higher levels of the GAC isoform than B-cells. The GAC isoform is the form most commonly associated with cancer since the mRNA lacks a target site for miR-23 and an AU rich element (ARE) which are both potent modulators of the KGA isoform. When we looked at alternative polyadenylation, we found that the ATM gene, which is a major contributor to MCL pathogenesis, undergoes shortening of the 3'UTR in MCL cell lines. Using SQANTI3 on our long-read sequencing data, we found that transcripts were in 9 different structural categories and only ~30% of our transcripts mapped to annotated genes. We also identified fusion transcripts from our long-read sequencing data. As a proof of concept, we validated the ubiquitous fusion transcript, CTBS::GNG5, in our MCL cell lines using PCR and Sanger Sequencing. We also detected it in MCL patient samples using PCR. So far, we have identified heterogeneity as well as overlaps in levels of the differentially expressed genes in MCL. Use of both short-read and long-read sequencing technology has uncovered transcripts in MCL cell lines arising from alternative splicing, alternative polyadenylation as well as fusion transcripts. This comprehensive analysis of the MCL transcriptome can provide potential biomarkers and therapeutic targets. Our analysis is currently ongoing.