Epigenetic alterations are increasingly recognized as playing a key role in the pathogenesis of B-cell lymphomas, and genome-wide studies continue to yield important insights into the process, highlighting new opportunities for treatment based on the molecular phenotype. This study from the group led by Dr. Anne Müller at the University of Zurich has identified a dual-specificity phosphatase (DUSP4) that is suppressed by aberrant methylation in the majority of cases of diffuse large B-cell lymphoma (DLBCL). Functional studies in cell lines and xenografts confirmed that it can play a role in B-cell apoptosis via c-JUN N-terminal kinase (JNK). The discovery of the DUSP4 tumor suppressor gene opens up the possibility of novel therapy by inhibition of JNK, which may also synergize with existing inhibitors of B-cell receptor signaling.
An initial screen for tumor suppressors that are silenced by methylation was performed on DNA from a range of lymphomas of mucosa-associated lymphoid tissue, gastric DLBCL, and DLBCL cell lines. All showed similar patterns of promoter methylation compared to normal B-cells. To identify the genes that might be re-expressed following demethylation, gene expression profiles were carried out on DLBCL lines and activated peripheral blood B-cells before and after treatment with 5-aza-2’-deoxycytidine and the histone deacetylase inhibitor 4-phenylbutyric acid. By comparing the results of these screens, 35 genes of interest were identified, and expression constructs were made for 30 of them. In eight cases, ectopic expression of the gene resulted in reduced viability in DLBCL lines, of which five were transcription factors and three had enzymatic activity (DUSP4, the kinase PRKCH, and the phospholipase C β1, PLCB1).
The group chose to study DUSP4 in more detail because it was strongly expressed in activated B-cells, was reactivated on demethylation in DLBCL cells, and has potentially important downstream targets, including JNK. Targeted bisulphite sequencing of the CpG island upstream of the DUSP4 start site confirmed that it was extensively methylated in all 11 B-cell lymphoma lines studied, and in 10 of 13 primary DLBCL samples, irrespective of subtype. By contrast, normal B-cells from blood and tonsils, and chronic lymphocytic leukemia (CLL) cells showed no methylation. Using an antibody specific to DUSP4, the group was able to examine a tissue array of several hundred different lymphomas; it appeared that the nuclear expression of the protein is much less common in DLBCL than in indolent types such as marginal-zone follicular lymphoma or CLL, with an inverse correlation between promoter methylation and protein expression in a subset of 16 cases of DLBCL and CLL. A search of comparative genomic hybridization databases showed that DUSP4 deletion is relatively uncommon, with 6.4 percent of DLBL samples showing loss of the locus. A study of DUSP4 transcript levels in three different cohorts previously analyzed for gene expression profiling showed a significant correlation of DUSP4 expression and prognosis, which applied across DLBCL subtypes.
![B-cell activation (eg, via B-cell receptor [BCR] and CD40 engagement) induces the expression of dual-specificity phosphatase (DUSP4), which negatively regulates c-JUN N-terminal kinase (JNK) 1/2 by dephosphorylation, resulting in apoptosis. Inhibition of BTK and JNK1/2 acts synergistically to promote apoptosis of lymphoma cells.© Rickert R., 2015. Originally published in J Exp Med. doi:10.1084/ jem.2125insight2.](https://ash.silverchair-cdn.com/ash/content_public/journal/thehematologist/12/5/10.1182_hem.v12.5.4320/2/m_3dc2dc35-420f-469e-8b60-b33aa4d9177e.jpeg?Expires=1724148053&Signature=tcJKERNuMuldZSDsPI4~NW68x6aE3gtE8NGguQ8JhdP86EvZM7PotqAM8S4QIgBqZN4skk409CViqf~55pPepRKjFEBIoW0d8I19euBdOHXmpj0TJ-5Tdn5ZfHL3aFXAy8fxGixHpHDQTij7rNuWnSLCTXDTY-cMsB-Fu4nWJl2VoEEzPvs2r6xxEHEO13ZzeqHZpVdFsQbbj~tIRKF6fSbsyDIF5gY95zMb-WjgkwvSlJF0QA459RlfAT5ViJnntZcRD675X6X~d5PPX6ul9y2J32c2mTvTq3Yax2g7Vph9pxuocArksUu9Q3RcdMyvMLjlcHMgnxEnYJhpJKwnEg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
B-cell activation (eg, via B-cell receptor [BCR] and CD40 engagement) induces the expression of dual-specificity phosphatase (DUSP4), which negatively regulates c-JUN N-terminal kinase (JNK) 1/2 by dephosphorylation, resulting in apoptosis. Inhibition of BTK and JNK1/2 acts synergistically to promote apoptosis of lymphoma cells.© Rickert R., 2015. Originally published in J Exp Med. doi:10.1084/ jem.2125insight2.
B-cell activation (eg, via B-cell receptor [BCR] and CD40 engagement) induces the expression of dual-specificity phosphatase (DUSP4), which negatively regulates c-JUN N-terminal kinase (JNK) 1/2 by dephosphorylation, resulting in apoptosis. Inhibition of BTK and JNK1/2 acts synergistically to promote apoptosis of lymphoma cells.© Rickert R., 2015. Originally published in J Exp Med. doi:10.1084/ jem.2125insight2.
To assess the effects of DUSP4 loss and re-expression in DLBCL, expression constructs were transfected into five DLBCL lines; this produced reduced viability and increased apoptosis but no disturbance of progression through the cell cycle. A point mutant construct lacking enzymatic activity was used as a control to show that a functional DUSP4 protein was required to exert this effect. DUSP4 has a number of mitogen-activated protein kinase targets for its dephosphorylation activity, including the extracellular signal-regulated kinase, p38, and JNK family members. JNK1 and JNK2 were selected for further study because they are phosphorylated in both subtypes of DLBCL, and phospho-JNK levels were shown to be reduced by ectopic DUSP4 expression. A small-molecule JNK inhibitor reduced the viability of DLBCL cell lines, as did a genetic approach using dominant negative JNK1 and JNK2 constructs. Combination therapy using ibrutinib to inhibit Bruton tyrosine kinase in addition to a JNK inhibitor resulted in synergistic activity on ABC-type DLBCL lines in vitro.
In Brief
This study gives further evidence that epigenetic dysregulation is important in the development of B-cell lymphomas. DUSP4, which is silenced by methylation in many cases of DLBCL and by allelic loss in a few, seems to function normally as a tumor suppressor gene, providing negative feedback following activation via the B-cell receptor, CD40, or other pathways. Inhibition of JNK family members is a strategy that can effectively restore one of the physiologic functions of absent DUSP4. It is interesting that this approach is effective across the molecular DLBCL subtypes, and the finding of synergy between JNK inhibition and ibrutinib in ABC cell lines has important implications for the clinical development of combination therapy approaches.
Figure. B-cell activation (eg, via B-cell receptor [BCR] and CD40 engagement) induces the expression of dual-specificity phosphatase (DUSP4), which negatively regulates c-JUN N-terminal kinase (JNK) 1/2 by dephosphorylation, resulting in apoptosis. Inhibition of BTK and JNK1/2 acts synergistically to promote apoptosis of lymphoma cells. ©Rickert R., 2015. Originally published in J Exp Med. doi:10.1084/jem.2125insight2.
