Inactivating mutations of acetyltransferase genes in B-cell lymphoma

Abstract

B-cell non-Hodgkin's lymphoma comprises biologically and clinically distinct diseases the pathogenesis of which is associated with genetic lesions affecting oncogenes and tumour-suppressor genes. We report here that the two most common types--follicular lymphoma and diffuse large B-cell lymphoma--harbour frequent structural alterations inactivating CREBBP and, more rarely, EP300, two highly related histone and non-histone acetyltransferases (HATs) that act as transcriptional co-activators in multiple signalling pathways. Overall, about 39% of diffuse large B-cell lymphoma and 41% of follicular lymphoma cases display genomic deletions and/or somatic mutations that remove or inactivate the HAT coding domain of these two genes. These lesions usually affect one allele, suggesting that reduction in HAT dosage is important for lymphomagenesis. We demonstrate specific defects in acetylation-mediated inactivation of the BCL6 oncoprotein and activation of the p53 tumour suppressor. These results identify CREBBP/EP300 mutations as a major pathogenetic mechanism shared by common forms of B-cell non-Hodgkin's lymphoma, with direct implications for the use of drugs targeting acetylation/deacetylation mechanisms.

Figure 1. The CREBBP gene is mutated in DLBCL

a, Schematic diagram of the CREBBP gene (top) and protein (bottom). Exons are color coded according to the corresponding protein functional domains (CH, cysteine-histidine rich; KIX, CREB-binding; Bromo, bromodomain; HAT, histone acetyltransferase; Q, poly glutamine stretch). Color-coded symbols depict distinct types of mutations. b, Sequencing traces of representative mutated DLBCL tumor samples and paired normal DNA; arrows point to the position of the nucleotide change (amino acid change shown at the bottom). c, Distribution of CREBBP mutations in major DLBCL subtypes; on top, actual number of mutated samples over total analyzed.

Figure 2. Mutations and deletions of CREBBP are predominantly monoallelic

a, Map of the genomic region encompassing CREBBP. Blue lines below the map indicate the extent of the deletions identified in 9 DLBCL samples, with the dark blue segment corresponding to a homozygous loss. b, dChipSNP heatmap showing median-smoothed log2 CN ratio for 8 DLBCL biopsies harboring CREBBP deletions, and two normal DNAs (N). A vertical blue bar indicates the location of the CREBBP locus; in the red-blue scale, white corresponds to a normal (diploid) CN log-ratio, blue is deletion and red is gain. c, Allelic distribution of CREBBP genetic lesions in individual DLBCL samples. d, Overall frequency of CREBBP structural alterations in DLBCL subtypes (mutations and deletions, combined).

Figure 3. CREBBP and EP300 expression in normal and transformed B-cells

a, Immunofluorescence analysis of reactive tonsils. BCL6 identifies GC B-cells, and Dapi is used to detect nuclei. b, c, Immunohistochemistry analysis of CREBBP (b) and EP300 (c) protein expression in representative DLBCL biopsies (genomic status as indicated; scale bar, 100µm). Sample 2147, which harbors a homozygous CREBBP deletion, serves as negative control. d, Western blot and northern blot analysis of DLBCL cell lines carrying wild-type or aberrant CREBBP and EP300 alleles (color coded as indicated). The aberrant band in SUDHL10 corresponds in size to the predicted ~220kD CREBBP truncated protein. * non-specific bands. Tubulin and GAPDH control for total protein and RNA loading, respectively. e, Overall proportion of DLBCL biopsies showing defective CREBBP/EP300 function due to genetic lesions (red scale) and/or lack of protein expression (blue scale).

Figure 4. CREBBP missense mutations impair its ability to acetylate BCL6 and p53

a, Schematic diagram of the CREBBP HAT and CH domains, with the CREBBP point mutations tested in b–d (in green, residues located immediately outside the HAT domain). b, Acetylation levels of exogenous BCL6 in Flag immunoprecipitates obtained from HEK293T cells co-transfected with wild-type or mutant CREBBP expression vectors. Actin, input loading control. c, Luciferase reporter assays using a synthetic 5X-BCL6 reporter. Results are shown as relative activity compared to the basal activity of the reporter, set as 1 (mean +/− SD, n=2). Bottom, BCL6 and CREBBP-HA protein levels in the same lysates. Note that the amount of transfected BCL6 and CREBBP-encoding plasmids was adjusted to achieve equal protein amounts. d, p53 acetylation in HEK293T cells co-transfected with the indicated CREBBP expression vectors. The anti-p53 antibody documents comparable amounts of p53 (exogenous+endogenous). GFP monitors for transfection efficiency, and actin is used as loading control.

Figure 5. DLBCL-associated mutations in the CREBBP HAT domain decrease its affinity for Acetyl-CoA

a, Western blot analysis of in vitro acetyltransferase reactions performed using the wild-type or mutant Y1450C CREBBP-HA recombinant protein and a GST-p53 substrate in the presence of decreasing amounts of Acetyl-CoA. Anti-HA and anti-p53 antibodies document the presence of equivalent amounts of effector and substrate proteins in the reaction. b, In vitro acetyltransferase activity of the indicated CREBBP-HA mutant proteins in the same assay using 25nM Acetyl-CoA.