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DUSP4 deficiency caused by promoter hypermethylation drives JNK signaling and tumor cell survival in diffuse large B cell lymphoma.

Schmid CA, Robinson MD, Scheifinger NA, Müller S, Cogliatti S, Tzankov A, Müller A - J. Exp. Med. (2015)

Bottom Line: The epigenetic dysregulation of tumor suppressor genes is an important driver of human carcinogenesis.We find that a CpG island in the promoter of the dual-specificity phosphatase DUSP4 is aberrantly methylated in nodal and extranodal DLBCL, irrespective of ABC or GCB subtype, resulting in loss of DUSP4 expression in 75% of >200 examined cases.This finding provides a mechanistic basis for the clinical development of JNK inhibitors in DLBCL, ideally in synthetic lethal combinations with inhibitors of chronic active B cell receptor signaling.

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Affiliation: Institute of Molecular Cancer Research, Institute of Molecular Life Sciences, and Swiss Institute of Bioinformatics (SIB), University of Zürich, 8057 Zürich, Switzerland.

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Ectopic DUSP4 expression decreases the viability of DLBCL cell lines by inducing apoptosis. (A) The viability of five different DLBCL cell lines was assessed 72 h after transfection with DUSP4 wt or DUSP4 p.C280S mutant constructs, relative to empty vector–transfected cells (control). Data represent means + SEM of at least three independent experiments. (B and C) Apoptosis of the same DLBCL cell lines as shown in A was assessed 72 h after transfection with DUSP4 wt or DUSP4 p.C280S mutant constructs relative to empty vector–transfected cells (control). A representative annexin V flow cytometry histogram of three independent experiments is shown in B. Data in C represent mean + SEM of at least three independent experiments per cell line. (A and C) *, P < 0.05; **, P < 0.01; ***, P < 0.001, calculated using two-tailed Student’s t test. (D) Edu-PI staining of SU-DHL16 cells was performed 72 h after transfection. Representative flow cytometry dot plots of three independent experiments are shown. The numbers indicate the percentage of live cells in the G1, S, and G2-M phases of the cell cycle, respectively.
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fig5: Ectopic DUSP4 expression decreases the viability of DLBCL cell lines by inducing apoptosis. (A) The viability of five different DLBCL cell lines was assessed 72 h after transfection with DUSP4 wt or DUSP4 p.C280S mutant constructs, relative to empty vector–transfected cells (control). Data represent means + SEM of at least three independent experiments. (B and C) Apoptosis of the same DLBCL cell lines as shown in A was assessed 72 h after transfection with DUSP4 wt or DUSP4 p.C280S mutant constructs relative to empty vector–transfected cells (control). A representative annexin V flow cytometry histogram of three independent experiments is shown in B. Data in C represent mean + SEM of at least three independent experiments per cell line. (A and C) *, P < 0.05; **, P < 0.01; ***, P < 0.001, calculated using two-tailed Student’s t test. (D) Edu-PI staining of SU-DHL16 cells was performed 72 h after transfection. Representative flow cytometry dot plots of three independent experiments are shown. The numbers indicate the percentage of live cells in the G1, S, and G2-M phases of the cell cycle, respectively.

Mentions: To assess the effects of DUSP4 re-expression on DLBCL viability in detail, five DLBCL cell lines were electroporated with a cDNA expression construct encoding DUSP4. Ectopic expression of wild-type DUSP4 reduced cell viability and induced apoptotic cell death as assessed by CellTiter-Blue assay and annexin V staining (Fig. 5, A–C). This effect was dependent on DUSP4 enzymatic activity, as ectopic expression of a point mutant (C280S) lacking phosphatase activity (Robinson et al., 2001) did not affect the viability of DLBCL cells (Fig. 5, A–C). In line with their shared, almost universal DUSP4 promoter methylation, GCB- and ABC-type DLBCL cell lines were equally susceptible to ectopic DUSP4 expression (Fig. 5, A–C; note that the RC-K8 subtype is controversial: although lumped with GCB-type DLBCL here based on Lenz et al. [2007], cells may actually be of the ABC subtype [Schmitz et al., 2012]). In contrast, progression through the cell cycle was not impaired upon ectopic DUSP4 expression, as the fraction of cells in the S, G1, and G2 phases of the cell cycle did not differ in any of the cell lines, as determined by EdU incorporation assay in conjunction with propidium iodide (PI) staining (Fig. 5 D). In summary, DUSP4 has proapoptotic activity in DLBCL cell lines of both subtypes, and its epigenetic silencing likely provides a significant growth advantage to DLBCL cells.


DUSP4 deficiency caused by promoter hypermethylation drives JNK signaling and tumor cell survival in diffuse large B cell lymphoma.

Schmid CA, Robinson MD, Scheifinger NA, Müller S, Cogliatti S, Tzankov A, Müller A - J. Exp. Med. (2015)

Ectopic DUSP4 expression decreases the viability of DLBCL cell lines by inducing apoptosis. (A) The viability of five different DLBCL cell lines was assessed 72 h after transfection with DUSP4 wt or DUSP4 p.C280S mutant constructs, relative to empty vector–transfected cells (control). Data represent means + SEM of at least three independent experiments. (B and C) Apoptosis of the same DLBCL cell lines as shown in A was assessed 72 h after transfection with DUSP4 wt or DUSP4 p.C280S mutant constructs relative to empty vector–transfected cells (control). A representative annexin V flow cytometry histogram of three independent experiments is shown in B. Data in C represent mean + SEM of at least three independent experiments per cell line. (A and C) *, P < 0.05; **, P < 0.01; ***, P < 0.001, calculated using two-tailed Student’s t test. (D) Edu-PI staining of SU-DHL16 cells was performed 72 h after transfection. Representative flow cytometry dot plots of three independent experiments are shown. The numbers indicate the percentage of live cells in the G1, S, and G2-M phases of the cell cycle, respectively.
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fig5: Ectopic DUSP4 expression decreases the viability of DLBCL cell lines by inducing apoptosis. (A) The viability of five different DLBCL cell lines was assessed 72 h after transfection with DUSP4 wt or DUSP4 p.C280S mutant constructs, relative to empty vector–transfected cells (control). Data represent means + SEM of at least three independent experiments. (B and C) Apoptosis of the same DLBCL cell lines as shown in A was assessed 72 h after transfection with DUSP4 wt or DUSP4 p.C280S mutant constructs relative to empty vector–transfected cells (control). A representative annexin V flow cytometry histogram of three independent experiments is shown in B. Data in C represent mean + SEM of at least three independent experiments per cell line. (A and C) *, P < 0.05; **, P < 0.01; ***, P < 0.001, calculated using two-tailed Student’s t test. (D) Edu-PI staining of SU-DHL16 cells was performed 72 h after transfection. Representative flow cytometry dot plots of three independent experiments are shown. The numbers indicate the percentage of live cells in the G1, S, and G2-M phases of the cell cycle, respectively.
Mentions: To assess the effects of DUSP4 re-expression on DLBCL viability in detail, five DLBCL cell lines were electroporated with a cDNA expression construct encoding DUSP4. Ectopic expression of wild-type DUSP4 reduced cell viability and induced apoptotic cell death as assessed by CellTiter-Blue assay and annexin V staining (Fig. 5, A–C). This effect was dependent on DUSP4 enzymatic activity, as ectopic expression of a point mutant (C280S) lacking phosphatase activity (Robinson et al., 2001) did not affect the viability of DLBCL cells (Fig. 5, A–C). In line with their shared, almost universal DUSP4 promoter methylation, GCB- and ABC-type DLBCL cell lines were equally susceptible to ectopic DUSP4 expression (Fig. 5, A–C; note that the RC-K8 subtype is controversial: although lumped with GCB-type DLBCL here based on Lenz et al. [2007], cells may actually be of the ABC subtype [Schmitz et al., 2012]). In contrast, progression through the cell cycle was not impaired upon ectopic DUSP4 expression, as the fraction of cells in the S, G1, and G2 phases of the cell cycle did not differ in any of the cell lines, as determined by EdU incorporation assay in conjunction with propidium iodide (PI) staining (Fig. 5 D). In summary, DUSP4 has proapoptotic activity in DLBCL cell lines of both subtypes, and its epigenetic silencing likely provides a significant growth advantage to DLBCL cells.

Bottom Line: The epigenetic dysregulation of tumor suppressor genes is an important driver of human carcinogenesis.We find that a CpG island in the promoter of the dual-specificity phosphatase DUSP4 is aberrantly methylated in nodal and extranodal DLBCL, irrespective of ABC or GCB subtype, resulting in loss of DUSP4 expression in 75% of >200 examined cases.This finding provides a mechanistic basis for the clinical development of JNK inhibitors in DLBCL, ideally in synthetic lethal combinations with inhibitors of chronic active B cell receptor signaling.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Molecular Cancer Research, Institute of Molecular Life Sciences, and Swiss Institute of Bioinformatics (SIB), University of Zürich, 8057 Zürich, Switzerland.

Show MeSH
Related in: MedlinePlus