BLIMP1 is a tumor suppressor gene frequently disrupted in activated B cell-like diffuse large B cell lymphoma

Abstract

Diffuse large B cell lymphoma (DLBCL) is a heterogeneous disease composed of at least two distinct subtypes: germinal center B cell-like (GCB) and activated B cell-like (ABC) DLBCL. These phenotypic subtypes segregate with largely unique genetic lesions, suggesting the involvement of different pathogenetic mechanisms. In this report we show that the BLIMP1/PRDM1 gene is inactivated by multiple mechanisms, including homozygous deletions, truncating or missense mutations, and transcriptional repression by constitutively active BCL6, in ∼53% of ABC-DLBCL. In vivo, conditional deletion of Blimp1 in mouse B cells promotes the development of lymphoproliferative disorders recapitulating critical features of the human ABC-DLBCL. These results demonstrate that BLIMP1 is a bona fide tumor-suppressor gene whose loss contributes to lymphomagenesis by blocking plasma cell differentiation.

Figure 1. Inactivation of BLIMP1 by truncating mutations and biallelic deletions in ABC-DLBCL

(A) Distribution of truncating mutations along the BLIMP1 protein, with known functional domains annotated. Aci=acidic domain; PR=PR domain; Pro-rich=proline-rich domain (see also Table S1). (B) Percentage of cases with truncating BLIMP1 mutations in various DLBCL subtypes classified by gene expression profiling (GEP) or by immunohistochemistry (IHC)(see text). (C) dChip SNP inferred copy number heatmap of the 6q15-q22.1 region in ABC-DLBCL cases and three normal DNA controls. Cases harboring homozygous deletions or truncating mutations are denoted by symbols (see also Figure S1). The region indicated by an asterisk is shown at higher magnification in panel (D) for the three homozygously deleted cases (and two normal DNAs), with the approximate position of the BLIMP1, ATG5 and PREP genes on the left.

Figure 2. Missense mutations affect BLIMP1 protein stability and trans-repression activity

(A) Distribution of missense mutations along the BLIMP1 protein; in red, mutants that showed an effect in any of the assays performed in (B–D)(see also Table S2). (B) Western blot (top) and semi-quantitative RT-PCR (bottom) analysis of exogenous BLIMP1 expression in 293T cells transfected with equimolar amounts of vectors expressing HA-tagged wild-type or mutant BLIMP1 alleles. (C) Analysis of exogenous BLIMP1 protein expression in 293T cells transfected with the indicated mutant alleles and treated with cycloheximide for 2, 4 or 8 hours. Data were quantitated by densitometric analysis, normalized to β-actin levels, and graphed relative to time zero (top). The western blot analysis is shown on the bottom panel. (D) Trans-repression activity of wild-type and mutant BLIMP1 proteins in 293T cells co-transfected with a luciferase reporter construct driven by the human CIITA promoter (region −545 to +123, encompassing a consensus BLIMP1 binding site at position −180)(top). Luciferase activities are represented as percent change relative to the basal activity of the reporter (set to 100), after normalization to Renilla luciferase activity (mean ± SD, as obtained from three independent experiments). In the bottom panel, western blot analysis using anti-HA antibodies monitors for the corresponding exogenous BLIMP1 expression levels; note that, for the three unstable mutants, higher amounts of plasmid DNA were transfected to achieve comparable levels. Nevertheless, expression of the P48R mutant protein remained significantly lower than wild-type, presumably due to its marked instability (see also Figure S2).

Figure 3. BLIMP1 missense mutations impair its ability to induce cell cycle arrest and promote plasma cell differentiation

(A) Proliferative capacity of BJAB B cells transduced with lentiviral vectors expressing the indicated BLIMP1 proteins along with GFP, as assessed by an MTT assay on sorted GFP+ cells. Shown are representative data from one of two independent experiments performed in duplicate (mean ± SD) (see also Figure S3). (B) Left, representative flow cytometric analysis of BJAB B cells transduced with the indicated vectors and stained for incorporated BrdU and 7-amino-actinomycin D (7-AAD). Region gates define cells residing in G0-G1, S and G2-M phases of the cell cycle. The G0-G1 population was quantitated relative to empty vector-transduced cells (set at 1), and the mean ± SD from two independent experiments is shown below. (C) Expression of the BLIMP1 targets ID3 and CIITA in sorted GFP+ BJAB B cells, as determined by quantitative real-time RT-PCR (n=3; mean ± SD). Levels were normalized to both BLIMP1 and GAPDH, and are shown as fold changes relative to vector-transduced cells (set as 1). (D) Representative flow cytometric analysis of CD138 and B220 staining in Blimp1^CD19KO splenic B cells, reconstituted with the indicated vectors and stimulated to undergo plasma cell differentiation by LPS treatment for 3 days, as compared to wild-type (Blimp1^CD19WT) B cells. The percentage of cells in the gated (plasma cell) population is shown. Data from two independent experiments are quantitated in the bottom panel (mean ± SD). Mutants that showed an effect in any of the assays (A-D) are indicated in red.

Figure 4. BCL6 translocations and BLIMP1 inactivation are mutually exclusive in ABC-DLBCL

(A) Immunofluoresence analysis of BLIMP1 (green), IRF4 (red) and CD20 (blue) expression in normal GC cells of a human tonsil (scale bar, 325μm; inset, 150μm). (B) IRF4 (brown) and BLIMP1 (blue) immunostaining in representative ABC-DLBCL cases displaying a normal expression pattern (IRF4⁺BLIMP1⁺)(left panels) or specific lack of BLIMP1 expression (IRF4⁺BLIMP1⁻)(right panels)(scale bar: 125 μm). The percentage of cases in each group is provided below. Six additional cases (12%) were negative for expression of both proteins (not shown; see also Table S1 and S2 for a detailed characterization of individual cases). (C) Distribution of BLIMP1 and BCL6 structural alterations in IRF4+ ABC-DLBCL. Columns represent individual patients, with color-codes indicating the presence or absence of the corresponding feature. (D) BLIMP1 mRNA (left) and protein (right) levels in the BCL6-translocated RCK8 cell line, transduced with lentiviral vectors expressing a control shRNA (shCtrl) or a BCL6-specific shRNA (shBCL6). BLIMP1 mRNA levels were determined by quantitative real-time RT-PCR and are shown as fold change relative to shCtrl-transduced cells, after normalization for GAPDH (n=3; mean ±SD). Western blot analysis of BCL6 controls for efficient BCL6 knockdown (see also Figure S4). (E) Overall frequency of BLIMP1 and BCL6 structural alterations in ABC-DLBCL. The unknown category denotes cases that lack BLIMP1 protein expression, in the absence of BLIMP1 or BCL6 structural alterations. Two cases carrying both BCL6 translocations and biallelic BLIMP1 inactivation were included into the “BLIMP1 mutation + deletion” category.

Figure 5. Blimp1 B cell conditional knockout mice develop lymphoproliferative disorders

(A) Representative flow cytometric analysis of splenic B cell suspensions isolated from mice of the indicated genotypes and stained for CD21/CD23 (top) and B220/PNA (bottom). A significant increase in both GC (B220⁺PNA^hi) and extra follicular, MZ (CD21⁺CD23⁻) B cell subpopulations can be seen in the Blimp1^CD19KO animals, as compared to their control littermates. Data are quantitated on the right (mean ± SD; n=3). (B) Spleen/body weight ratio in mice of the indicated genotypes, analyzed between 10–16 months of age. Splenomegaly was defined as an increase in the spleen/body weight ratio above 0.7% (dashed line). Solid lines indicate the mean value in each genotype. (C) Frequency of lymphoproliferative disorders in the three cohorts shown in (B). MZBCH=marginal zone B cell hyperplasia; LPD=lymphoproliferative disease; DLBCL=diffuse large B cell lymphoma; p values (student’s t-test) are provided if significant (<0.05). See also Figure S5 and Table S3.

Figure 6. Conditional deletion of Blimp1 in the GC promotes lymphomagenesis

(A) Representative flow cytometric analysis of the CD21⁺CD23⁻ MZ B cell and B220⁺PNA^hi GC B cell compartments in mice of the indicated genotypes, analyzed ten days after SRBC immunization (left). Data from three mice per genotype are quantitated on the right (mean ± SD). (B) Percentage of mice developing lymphoproliferative disorders in the CD19-Cre and Cγ1-Cre models, sacrificed between 10–18 months of age. See also Table S4.

Figure 7. Blimp1 B cell conditional knockout mice develop DLBCL with an activated phenotype and constitutive NF-κB activation

Representative spleen and lymph node sections from Blimp1 knock-out mice presenting with a spectrum of lymphoproliferative disorders, including MZBCH, LPD, and overt DLBCL, as compared to a wild-type control (see results for a detailed histological description, and Figure S6 for additional data). Tissues were stained with hematoxylin and eosin (H&E) or immunostained with antibodies against the B220 pan-B cell marker, IRF4 and the NF-κB subunit p50, as indicated (Scale bar, 1250 μm; inset, 125 μm; scale is 50 μm for p50 stain).