Mutually Exclusive Structural Alterations of BLIMP1 and BCL6 Contribute to the Pathogenesis of Activated B Cell Type Diffuse Large B Cell Lymphoma.

Abstract 445

Diffuse large B-cell lymphoma (DLBCL), the most common type of B-cell non-Hodgkin lymphoma, is a heterogeneous disease comprising multiple biologically and clinically distinct subgroups, including germinal center B cell-like (GCB) and activated B cell-like (ABC) DLBCL. We have previously reported that the BLIMP1 gene, a master regulator of plasma cell differentiation normally expressed in a subset of germinal center (GC) B cells and in all plasma cells, is inactivated by truncating mutations in a fraction of ABC-DLBCL, but not in GCB-DLBCL (Pasqualucci et al, J Exp Med 2006). In addition, most ABC-DLBCL lack expression of the BLIMP1 protein despite the presence of IRF4, another key regulator of plasma cell differentiation known to be invariably co-expressed with BLIMP1 in normal B cells, thereby suggesting that additional genetic or epigenetic mechanisms may inactivate BLIMP1 in these tumors. Here we report the characterization of the full spectrum of genetic lesions affecting the BLIMP1 locus in DLBCL, as determined by genome-wide copy number analysis (Affymetrix SNP 6.0 array), fluorescence in situ hybridization, and direct sequencing of the entire BLIMP1 coding region in 158 primary biopsies, classified by gene expression profile analysis (51 ABC-DLBCL; 62 GCB-DLBCL; 11 unclassified) and/or by immunohistochemistry (24 non-GC and 10 GC-DLBCL). This analysis uncovered a total of 22 mutations, distributed in 21 cases and segregating with an activated DLBCL phenotype (13/51 ABC-, 3/11 unclassified and 5/24 non-GC-DLBCL, vs 0/71 GCB/GC-DLBCL). The vast majority of the mutations were represented by frameshift insertions/deletions (n=10), splice site mutations (n=7) and nonsense mutations (n=1) leading to severely truncated polypeptides that lack critical functional domains and have therefore lost their activity. Interestingly, in the remaining three cases, 4 missense mutations introduced amino acid changes that were documented to severely impair BLIMP1 function by either causing protein instability (n=3) or abrogating its ability to bind chromatin and repress its known target genes CIITA and ID3 (n=1). When transduced into the GCB-DLBCL cell line BJAB, the wild type, but not three of the BLIMP1 missense mutant constructs induced cell cycle arrest. Copy number analysis confirmed deletion of the second allele in 9/12 mutated ABC-DLBCL, and identified three additional cases harbouring biallelic loss of the gene, including a focal homozygous deletion of 274Kb, which encompasses the BLIMP1 gene but not the two proximal genes ATG5 and PREP. Thus, 31% (n=16/51) of ABC-DLBCL have inactivation of BLIMP1 due to mutations or biallelic deletions. Moreover, immunohistochemical analysis revealed the lack of Blimp1 protein expression in 90% (n=27/30) of IRF4+ ABC-DLBCL carrying normal BLIMP1 loci. Notably, ten of these samples (30%) were found to harbour chromosomal translocations affecting BCL6, a master regulator of the GC and a direct transcriptional repressor of BLIMP1, suggesting that deregulated BCL6 expression was responsible for the lack of BLIMP1 expression in these cases. With the exception of two cases, BCL6 translocations and BLIMP1 structural alterations were mutually exclusive. Collectively, these data identify a novel mechanism by which missense mutations of BLIMP1 can impair its function in human DLBCL, and demonstrate that the IRF4-BCL6-BLIMP1 pathway is inactivated by structural alterations in over half of ABC-DLBCLs, strongly suggesting that BLIMP1 inactivation and BCL6 translocations may represent alternative mechanisms contributing to the pathogenesis of this disease by blocking terminal B cell differentiation.