Diffuse large B-cell lymphoma with combined TP53 mutation and MIR34A methylation: Another "double hit" lymphoma with very poor outcome?

Abstract

MiR34A, B and C have been implicated in lymphomagenesis, but information on their role in normal CD19+ B-cells (PBL-B) and de novo diffuse large B-cell lymphoma (DLBCL) is limited. We show that in normal and activated B-cells miR34A-5p plays a dominant role compared to other miR34 family members. Only miR34A-5p is expressed in PBL-B, and significantly induced in activated B-cells and reactive lymph nodes. In PBL-B, the MIR34A and MIR34B/C promoters are unmethylated, but the latter shows enrichment for the H3K4me3/H3K27me3 silencing mark. Nine de novo DLBCL cases (n=150) carry both TP53 mutation and MIR34A methylation ("double hit") and these patients have an exceedingly poor prognosis with a median survival of 9.4 months (P<0.0001), while neither TP53 mutation, MIR34A or MIR34B/C promoter methylation alone ("single hit") influence on survival. The TP53/MIR34A "double-hit" is an independent negative prognostic factor for survival (P=0.0002). In 2 DLBCL-cell lines with both TP53 mutation and promoter methylation of MIR34A, miR34A-5p is upregulated by 5-aza-2'deoxycytidine. Thus, the TP53/MIR34A "double hit" characterizes a very aggressive subgroup of DLBCL, which may be treatable with epigenetic therapy prior to or in combination with conventional immunochemotherapy.

Figure 1. MiR34A, miR34B, and miR34C expression and regulation in normal B-cells, T-cells and reactive lymph nodes

(A) Normal peripheral blood CD19+ B-lymphocytes (PBL-B) have an unmethylated MIR34A promoter and show low expression of miR34A-5p, which, however, is significantly upregulated in reactive lymph nodes (P<0.001) and in vitro activated B-cells (P<0.017). MiR34A-5p is not expressed in normal CD3+ T-cells. The MIR34B/C promoter is unmethylated and miR34B and miR34C is not expressed in normal or in vitro activated B-cells, T-cells or reactive lymphnodes. (B) No enrichment of the H3K27me3 or H3K4me3 marks was observed at the TSS of MIR34A. (C) The downregulation of miR34B/C in PBL-B may be due to enrichment for the bivalent H3K27me3/H3K4me3 silencing mark at the MIR34B/C promoter.

Figure 2. MIR34A, MIR34B/C and TP53 regulation in lymphoma cell lines

(A) MS-MCA showing that all cell lines are methylated at the MIR34B/C promoter in the DLBCL cell lines. (B) In the DLBCL cell lines Toledo, DB1 and HT, the MIR34A promoter shows a biphasic methylation pattern (methylated and unmethylated), while Farage and DOHH2 are completely unmethylated. (C) Bisulfite sequencing of the MIR34A promotor region showing methylation of ~50% of the MIR34A alleleles, methylated cytosines, unmethylated cytosines. (D) MIR34A promoter methylation correlates with expression. (E) Direct sequencing showing the concomitant TP53 mutation (V216M) in the MIR34A/B/C methylated cell line HT (F) Upregulation of miR34A in the MIR34A methylated and TP53 mutant cell line HT after treatment with the hypomethylating agent 5-aza-2'deoxycytidine (5-aza-CdR 0.5uM). No induction of miR34A was observed after treatment with cytosine arabinoside (araC 20nM).

Figure 3. MIR34A, MIR34B/C and TP53 deregulation in primary DLBCLs

(A) MIR34A methylation in primary DLBCL by MS-MCA. (B) MIR34B/C methylation in primary DLBCL (MS-MCA). (C) Representative image of DGGE gel and Sanger sequence of TP53 mutation in primary DLBCL.

Figure 4. Overall survival of DLBCL patients with and without MIR34A methylation and TP53 mutation

Patients are divided into 3 groups: Patients with no alterations of TP53 or MIR34A, patients with either MIR34A methylation or TP53 mutation only, and patients with concomitant MIR34A methylation and TP53 mutation. (A) 5 year overall survival for the three groups in the entire cohorte (n=150). The group with concomitant MIR34A methylation and TP53 mutation (n=9) shows significantly poor overall survival (P<0.0001). (B) Among the Rituximab treated patients (n=62) concomitant MIR34A methylation and TP53 mutation (n=6) leads to significantly inferior survival as well (P<0.0001).

Figure 5. A schematic model of the p53-miR34 axis

Oncogenic stress or DNA damage may activate p53 leading to cell cycle arrest, senescense and/or apoptosis. This is mediated via its downstream effectors miR34A -B and -C, which in turn downregulate known DLBCL proto-oncogenes. Our data indicate that DLBCL cells with concomitant TP53 mutation and MIR34A methylation have a growth advantage compared to “single-hit” tumors, suggesting that in DLBCL miR-34A may also be upregulated independently of p53 by oncogenic stress, probably via the ERK/ELK pathway as suggested by Christoffersen et al [17].