Alternative splicing is a major mechanism of gene regulation in diffuse large B cell lymphoma

Abstract

Diffuse large B-cell lymphomas (DLBCL) is the most common form of lymphoma and these tumors demonstrate a striking molecular and clinical heterogeneity. Gene expression profiling has shown that these tumors can be divided into at least two groups, activated B cell-like (ABC) and germinal B cell-like (GCB), as well as a number of other cellular processes underlying survival and tumor biology. However, the molecular mechanisms which lead to the differential gene expression patterns are not well understood. Alternative splicing is a process through which individual exons that comprise genes that are assembled into different gene isoforms with potentially different function. Alternative splicing has been shown to be a ubiquitous mechanism of gene regulation in eukaryotes and a number of cancers. The role of alternative splicing in DLBCL is unknown. We hypothesized that alternative splicing might play an important role in DLBCL. We measured genome-wide expression of over 1 million exons in 106 primary DLBCL tumors using Affymetrix Exon 1.0 ST microarrays. The same cases were also profiled for gene expression using a conventional Affymetrix Gene microarray for comparison, and further sub-classified as the molecular subgroups of DLBCL and for independent assessment of gene expression associated with known biological processes. We identified those genes as alternatively spliced which had at least one exon that was significantly different (P<0.01) compared with the comparison group. Through examination of exon-level expression data, we found evidence for splicing events in over 10,000 genes that affect at least 10% of DLBCL cases. We identified over 200 genes that have differential exon usage between ABC and GCB DLBCL. The expression of selected alternatively spliced exons was confirmed by real-time PCR. We further examined the occurrence of alternative splicing in a number of cellular processes including each of the survival associated gene expression signatures. We found that alternative splicing regulates a significant number of genes underlying the survival-associated proliferation, stromal response and germinal center differentiation gene expression signatures (P<10–6 in all cases). In addition, a number of processes that are known to be important in oncogenesis appeared to be highly regulated by alternative splicing including transcription factor activity, DNA-repair and apoptosis (P<0.001). These data confirm that alternative splicing plays a significant role in regulating genes that are important mediators of DLBCL biology. We further investigated whether lineage-specific effects were responsible for some of the observed differences in splicing in the molecular subgroups of DLBCLs. We obtained normal resting B cells from healthy donors and stimulated them with IgM and CD40-ligand to generate activated B cells. We found that the gene-isoforms expressed highly in activated normal B cells were significantly enriched in ABC-DLBCLS (P<0.01), suggesting that lineage-derived differences in isoform splicing are preserved in their malignant counterparts. Our data indicate that alternative splicing provides an additional and significant component of regulation that encompasses nearly every biological process that is known to be important in DLBCL. Future studies that examine the role gene expression will need to recognize the specific isoforms that result in proteins with altered function.