AID and TET2 co-operation modulates FANCA expression by active demethylation in diffuse large B cell lymphoma

Abstract

Diffuse large B cell lymphoma (DLBCL) is traced to a mature B malignance carrying abnormal activation-induced cytidine deaminase (AID) expression. AID activity initially focuses on deamination of cytidine to uracil to generate somatic hypermutation and class-switch recombination of the immunoglobulin (Ig), but recently it has been implicated in DNA demethylation of genes required for B cell development and proliferation in the germinal centre (GC). However, whether AID activity on mutation or demethylation of genes involves oncogenesis of DLBCL has not been well characterized. Our data demonstrate that the proto-oncogene Fanconi anaemia complementation group A (FANCA) is highly expressed in DLBCL patients and cell lines, respectively. AID recruits demethylation enzyme ten eleven translocation family member (TET2) to bind the FANCA promoter. As a result, FANCA is demethylated and its expression increases in DLBCL. On the basis of our findings, we have developed a new therapeutic strategy to significantly inhibit DLBCL cell growth by combination of the proteasome inhibitor bortezomib with AID and TET2 depletion. These findings support a novel mechanism that AID has a crucial role in active demethylation for oncogene activation in DLBCL.

Keywords: 10-11 translocation-2; Fanconi anaemia complementation group A; activation-induced cytidine deaminase; active demethylation; diffuse large B cell lymphoma.

Figure 1

Activation‐induced cytidine deaminase (AID) promotes DLBCL cell survival in vitro. (a) mRNA level of AID in non‐neoplastic lymphatic tissue (control) (n = 3) and neoplastic lymph node diffuse large B cell lymphoma (DLBCL) patients (n = 8) were measured by real‐time polymerase chain reaction (PCR). (b,c) AID depletion by transducing a transgene including CRISPR‐Cas9 targeting AID into OCI‐LY7 cells was confirmed by real‐time PCR (b) and immunoblot (c). (d) The cell divisions of AIDWT and AIDKO OCI‐LY7 cells were detected by flow cytometry detecting carboxyfluorescein succinimidyl ester (CFSE)‐stained DLBCL cells. (e) AIDWT (left) and AIDKO (right) OCI‐LY7 cells were treated by starvation culture in vitro. Cell apoptosis was assessed by flow cytometry detecting the apoptosis maker annexin V and 7‐aminoactinomycin D (7‐AAD) signals (left); statistical charts for percentages of apoptosis cells (right). Small horizontal lines indicate the mean [± standard deviation (s.d.)]. P‐values were analysed by two‐tailed t‐test with 95% confidence intervals. ***P < 0·001; *P < 0·05 compared with the AIDWT group.

Figure 2

Activation‐induced cytidine deaminase (AID) contributes to tumorigenesis in the diffuse large B cell lymphoma (DLBCL) cell engraftment mouse model. (a) Tumour volumes were monitored every week (n = 5). (b) Tumour weight measurement following tumour excision from the euthanized mice (n = 5). Small horizontal lines indicate the mean [± standard deviation (s.d.)]. P‐values were analysed by two‐tailed t‐test with 95% confidence intervals. ***P < 0·001 compared with the AIDWT group.

Figure 3

Activation‐induced cytidine deaminase (AID) establishes FANCA demethylation in diffuse large B cell lymphoma (DLBCL) cells. (a) mRNA level of Fanconi anaemia complementation group A (FANCA) in non‐neoplastic lymphatic tissue (control) (n = 3) and neoplastic lymph node DLBCL patients (DLBCL) (n = 8) were measured by real‐time polymerase chain reaction (PCR). (b) FANCA expression following AID deficiency was detected by immunoblot. (c) 10–11 translocation‐2 (TET2), FANCA and AID expression in AIDWT and AIDKO OCI‐LY7 cells were detected by immunoblot. (d) Statistical percentage of point mutation in AIDWT and AIDKO OCI‐LY7 cells by single clone sequencing for T vector with FANCA promoter sequences. (e) The methylation status of FANCA promoter in AIDWT and AIDKO OCI‐LY7 cells by single clone sequencing for T vector with FANCA promoter sequence after bisulphite treatment. The graph of methylation or demethylation CpG sites (left) were indicated in the schematic diagram (right). Black circles signify methylated cytosine–phosphate–guanosine (CpG) and white circles indicate demethylated CpG. Small horizontal lines indicate the mean [± standard deviation (s.d.)]. P‐values were analysed by two‐tailed t‐test with 95% confidence intervals. ***P < 0·001; *P < 0·05 compared with the AIDWT group.

Figure 4

Activation‐induced cytidine deaminase (AID) recruits TET2 to bind to its promoter. (a) TET2 expression following AID deficiency was detected by immunoblot. (b) OCI‐LY7 cells were treated with DMOG (1500 µm) for 96 h in vitro; Fanconi anaemia complementation group A (FANCA) expression was detected by real‐time polymerase chain reaction (PCR). (c) OCI‐LY7 cells were treated as in (b), 10–11 translocation‐2 (TET2), AID and FANCA expression were detected by immunoblot. (d,e) Immunoblotting of TET2 and AID in purified extracts immunoprecipitation (IP) by anti‐AID (d) and anti‐TET2 (e). (f) DNA recovery in immunoprecipitates from chromatin immunoprecipitation (ChIP) experiments was measured by quantitative polymerase chain reaction (PCR) using primers that detect the FANCA promoter. (g) DNA recovery obtained as (f) was measured by PCR using primers that detect the FANCA promoter. Small horizontal lines indicate the mean [± standard deviation (s.d.)]. P‐values were analysed by two‐tailed t‐test with 95% confidence intervals. **P < 0·01 compared with the AIDWT group.

Figure 5

In‐vitro‐cultured diffuse large B cell lymphoma (DLBCL) cells tend to be apoptotic after combination treatment with activation‐induced cytidine deaminase (AID) deficiency, dimethyloxaloylglycine (DMOG) and bortezomib. (a) OCI‐LY7 cells were cultured with DMOG (1·5 mM) treatment for 96 h, bortezomib (5 nM), with combined treatment by bortezomib (5 nM) for another 5 h, after being pretreated with DMOG (1·5 mM) for 91 h. Apoptosis of AIDWT (upper) and AIDKO (lower) was assessed by detecting signals of the apoptosis maker annexin V and 7‐aminoactinomycin D (7‐AAD) (left); and statistical diagram of percentage for apoptosis cells (right). Bor = bortezomib. (b) Relative Fanconi anaemia complementation group A (FANCA) mRNA expression in OCI‐LY7 cells treated as in (a) was detected by real time polymerase chain reaction (PCR). (c) Heat‐map for relative mRNA expression of AID, 10–11 translocation‐2 (TET2), anti‐apoptosis and apoptosis‐associated genes in OCI‐LY7 cells treated as in (a). Small horizontal lines indicate the mean [± standard deviation (s.d.)]. P‐values were analysed by two‐tailed t‐test with 95% confidence intervals; ***P < 0·001; **P < 0·01; *P < 0·05 compared with the AIDWT group.

Figure 6

Model of activation‐induced cytidine deaminase (AID) recruiting 10–11 translocation‐2 (TET2) to bind to the Fanconi anaemia complementation group A (FANCA) promoter to mediate demethylation. (a) AID recruits TET2 to bind to the FANCA promoter and sequentially drive its demethylation and enhance FANCA expression. (b) AID deficiency and TET2 inhibition by dimethyloxaloylglycine (DMOG) compromise the demethylation of AID and TET2 to the FANCA promoter; the methylation status indicates the down‐regulation of FANCA expression.