TNFAIP3 (A20) is a tumor suppressor gene in Hodgkin lymphoma and primary mediastinal B cell lymphoma

Abstract

Proliferation and survival of Hodgkin and Reed/Sternberg (HRS) cells, the malignant cells of classical Hodgkin lymphoma (cHL), are dependent on constitutive activation of nuclear factor kappaB (NF-kappaB). NF-kappaB activation through various stimuli is negatively regulated by the zinc finger protein A20. To determine whether A20 contributes to the pathogenesis of cHL, we sequenced TNFAIP3, encoding A20, in HL cell lines and laser-microdissected HRS cells from cHL biopsies. We detected somatic mutations in 16 out of 36 cHLs (44%), including missense mutations in 2 out of 16 Epstein-Barr virus-positive (EBV(+)) cHLs and a missense mutation, nonsense mutations, and frameshift-causing insertions or deletions in 14 out of 20 EBV(-) cHLs. In most mutated cases, both TNFAIP3 alleles were inactivated, including frequent chromosomal deletions of TNFAIP3. Reconstitution of wild-type TNFAIP3 in A20-deficient cHL cell lines revealed a significant decrease in transcripts of selected NF-kappaB target genes and caused cytotoxicity. Extending the mutation analysis to primary mediastinal B cell lymphoma (PMBL), another lymphoma with constitutive NF-kappaB activity, revealed destructive mutations in 5 out of 14 PMBLs (36%). This report identifies TNFAIP3 (A20), a key regulator of NF-kappaB activity, as a novel tumor suppressor gene in cHL and PMBL. The significantly higher frequency of TNFAIP3 mutations in EBV(-) than EBV(+) cHL suggests complementing functions of TNFAIP3 inactivation and EBV infection in cHL pathogenesis.

Figure 1.

Inactivation of A20 in cHL and PMBL. (A) Mutations in TNFAIP3 correlate with the absence of detectable A20 protein (∼70 kD) in lymphoma cell lines. Immunoblotting using anti-A20 antibody was performed with each 100 µg of whole-cell extracts of PMBL and HL cell lines. β-Actin was used as loading control. No truncated proteins were detected. (B) Frequency and pattern of TNFAIP3 mutations in PMBL and cHL arranged by EBV status (A20 wild-type: cases carrying exclusively wild-type TNFAIP3 sequence; A20 premature stop: cases with destructive mutations [nonsense mutations and deletions or insertions causing premature stop codons]; A20 missense: cases with missense mutations). Numbers in brackets indicate the numbers of cases analyzed. p-values were calculated using Fisher’s exact test. Case 19 harbors both missense and premature stop, and is depicted among the group with premature stop. (C) Graphic representation of allelic distribution of TNFAIP3 mutations of cHL grouped according to EBV status. The columns represent the cases, and the rows represent the two alleles. ^a, the loss of one allele cannot be excluded as cases were not evaluable by FISH and lacked heterozygous sequence polymorphisms; ^b, the case harbors two mutations, but their allelic distribution could not be determined. (D) Schematic representation of location of TNFAIP3 mutations translated to A20 protein in cHL and PMBL, as described in Tables I and II. OTU, ovarian tumor domain; ZF, A20 zinc finger domains.

Figure 2.

Chromosomal deletion of TNFAIP3 in cHL detected by interphase cytogenetics. FICTION analyses of representative cHL cases combining CD30 expression (red) and FISH probes for TNFAIP3 and chromosome 6 centromere (blue [b]). In the double-color assays in A–C and E and F, the TNFAIP3 probe is labeled in green (g); in the triple-color assay applied in D, the TNFAIP3 probe gives a g/orange colocalized (co) signal. Two different strategies are used to display double- and triple-color FISH assays in combination with CD30 immunofluorescence; i.e., double-color assays (A–C and E and F) are shown using a triple-color display, whereas a false multicolor display as obtained by Isis software is applied for the triple-color assay (D) to simultaneously show four colors (i.e., CD30 [r], TNFAIP3 [g] and orange, and chromosome 6 centromere [b]). In contrast to the triple-color assay, the quadruple-color assay is based on the overlay of two displays generated by Isis software. To this end, the different channels needed to be individually enhanced so that the final integrated image shows multicolor signals. Fluorescent signals are shown in a false-color display. Arrows point to CD30⁺ HRS cells. For each case, between 6 and 26 evaluable HRS cells (mean = 12) were considered. (A) HRS cell (case 41) with normal diploid signal pattern (2g + 2b). (B) HRS cell (case 36) exhibiting one copy of TNFAIP3 (1g) and three copies of chromosome 6 centromere (3b), indicating the presence of a chromosomal deletion of TNFAIP3. (C) HRS cell (case 16) exhibiting one copy of TNFAIP3 (1g) and two copies of chromosome 6 centromere (2b), indicating the presence of a chromosomal deletion of TNFAIP3. (D) HRS cell (case 19) exhibiting one copy of TNFAIP3 (1co) and three copies of chromosome 6 centromere (3b), indicating the presence of a chromosomal deletion of TNFAIP3. (E) HRS cell (case 35) exhibiting two copies of TNFAIP3 (2g) and three copies of chromosome 6 centromere (3b), indicating the presence of a chromosomal deletion of TNFAIP3. (F) HRS cell (case 33) lacking signals for TNFAIP3 (0g) but showing two copies of chromosome 6 centromere (2b), indicating the presence of a chromosomal (homozygous?) deletion of TNFAIP3. Neighboring bystander CD30⁻ cells mostly show normal signal patterns (2g or 2co + 2b), although truncation artifacts do occur (some signals are out of the focus plane). Bars, 10 µm.

Figure 3.

A20 reconstitution in cHL cell lines harboring inactivating TNFAIP3 mutations results in reduction of transcriptional NF-κB activity and reduction of cellular metabolism. cHL cell lines harboring inactive TNFAIP3 (L-1236 and KM-H2) or wild-type TNFAIP3 (L-428) were lentivirally transduced with expression constructs encoding the reporter GFP and either A20, IκBα^S32,36A superrepressor or luciferase as control (Ctrl). (A) GFP⁺ cells were analyzed by qPCR for expression of representative NF-κB target genes (BIRC3, ICAM1, and LTA). Reduction of NF-κB target gene expression upon TNFAIP3 or IκBα^S32,36A gene transfer was determined by subtraction of ΔCts of luciferase-expressing cells, respectively (ΔΔCt method). Bars represent mean values of down-regulation resulting from at least three independent infections that were each analyzed in several independent replicates. p-values were calculated per gene using the Wilcoxon rank-sum test to determine the level of significance between negative regulation of NF-κB target genes in L-1236 and KM-H2 cells in comparison to L-428 cells in response to A20 expression. (B) GFP⁺ cells were FACS sorted, cultured for 48 h, and analyzed by MTS assay for metabolic activity. Mean OD values, reflecting conversion of MTS into a formazan product, are normalized to the empty medium control. The quantity of formazan product as measured by the amount of 490-nm absorbance is directly proportional to the number of living cells in culture. The experiments were performed in duplicates (Fig. S5). Each value is based on four measurements.