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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.

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.

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The DUSP4 promoter is hypermethylated in nodal and extranodal DLBCL, which correlates with lack of DUSP4 protein expression. (A) Bisulfite sequencing of 16 CpG dinucleotides in island 28 was performed for 11 DLBCL cell lines and 2 peripheral blood B cell samples and a set of 18 clinical lymphoma and 3 tonsil samples. Closed and open circles represent methylated and unmethylated CpGs, respectively. Between two and five clones were sequenced per sample. The alignment of the DUSP4 promoter sequence of the indicated species was performed using ECR Browser and shows the high conservation of CpG island 28 in other mammalian species relative to the human sequence (top left). (B) DUSP4 expression by the indicated lymphoma entities was assessed by IHC of a lymphoma tissue microarray. The graph shows percentage of DUSP4-positive cases (>30% positive cells) and DUSP4-negative cases (<30% positive cells) for the indicated lymphoma entities (top left). Representative micrographs show staining of a tonsillar GC (with insets) as well as DUSP4-positive and -negative DLBCL and CLL cases (bars, 50 µm; insets bars, 25 µm). CLL, n = 20; MZL, n = 54; FL, n = 48; MCL, n = 9; nodal DLBCL, n = 170; extranodal DLBCL, n = 87; transformed DLBCL, n = 9; and tonsils, n = 3. (C) The correlation of DUSP4 promoter methylation (assessed by bisulfite sequencing) with DUSP4 protein expression (determined by IHC) was plotted for 16 lymphoma cases. **, P < 0.01, calculated using Mann–Whitney test; horizontal lines indicate medians. (D) CD138+ plasma cells were isolated from 9-mo-old C57BL/6 mice. T cells and B cell subsets were sorted from splenocyte preparations based on CD3, B220, CD21, and CD23 expression according to the depicted sorting strategy. DUSP4 expression in the indicated subsets was analyzed by qRT-PCR and depicted as mean + SEM. Plasma cells: two pools of three mice each; T cells: n = 2 individual mice (the results represent a limited number of repeats); spleen: n = 3 individual mice; IM, MZ, and FO: n = 5 individual mice. n.d., not detected; T, T cells; B, B cells; IM, immature splenic B cells (CD21low, CD23−); MZ, marginal zone B cells (CD21high, CD23−); FO, follicular B cells (CD21int, CD23+); PC, plasma cells. (E) DUSP4 copy number alterations were analyzed using the Progenetix database. The fractions of cases affected by deletions (of 100-Mb maximum length) targeting the DUSP4 locus are shown for the indicated lymphoma entities. A representative ABC-DLBCL array CGH profile showing a focal DUSP4 deletion is shown in the left panel.
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fig3: The DUSP4 promoter is hypermethylated in nodal and extranodal DLBCL, which correlates with lack of DUSP4 protein expression. (A) Bisulfite sequencing of 16 CpG dinucleotides in island 28 was performed for 11 DLBCL cell lines and 2 peripheral blood B cell samples and a set of 18 clinical lymphoma and 3 tonsil samples. Closed and open circles represent methylated and unmethylated CpGs, respectively. Between two and five clones were sequenced per sample. The alignment of the DUSP4 promoter sequence of the indicated species was performed using ECR Browser and shows the high conservation of CpG island 28 in other mammalian species relative to the human sequence (top left). (B) DUSP4 expression by the indicated lymphoma entities was assessed by IHC of a lymphoma tissue microarray. The graph shows percentage of DUSP4-positive cases (>30% positive cells) and DUSP4-negative cases (<30% positive cells) for the indicated lymphoma entities (top left). Representative micrographs show staining of a tonsillar GC (with insets) as well as DUSP4-positive and -negative DLBCL and CLL cases (bars, 50 µm; insets bars, 25 µm). CLL, n = 20; MZL, n = 54; FL, n = 48; MCL, n = 9; nodal DLBCL, n = 170; extranodal DLBCL, n = 87; transformed DLBCL, n = 9; and tonsils, n = 3. (C) The correlation of DUSP4 promoter methylation (assessed by bisulfite sequencing) with DUSP4 protein expression (determined by IHC) was plotted for 16 lymphoma cases. **, P < 0.01, calculated using Mann–Whitney test; horizontal lines indicate medians. (D) CD138+ plasma cells were isolated from 9-mo-old C57BL/6 mice. T cells and B cell subsets were sorted from splenocyte preparations based on CD3, B220, CD21, and CD23 expression according to the depicted sorting strategy. DUSP4 expression in the indicated subsets was analyzed by qRT-PCR and depicted as mean + SEM. Plasma cells: two pools of three mice each; T cells: n = 2 individual mice (the results represent a limited number of repeats); spleen: n = 3 individual mice; IM, MZ, and FO: n = 5 individual mice. n.d., not detected; T, T cells; B, B cells; IM, immature splenic B cells (CD21low, CD23−); MZ, marginal zone B cells (CD21high, CD23−); FO, follicular B cells (CD21int, CD23+); PC, plasma cells. (E) DUSP4 copy number alterations were analyzed using the Progenetix database. The fractions of cases affected by deletions (of 100-Mb maximum length) targeting the DUSP4 locus are shown for the indicated lymphoma entities. A representative ABC-DLBCL array CGH profile showing a focal DUSP4 deletion is shown in the left panel.

Mentions: We focused on the dual-specificity phosphatase DUSP4 in all subsequent experimental efforts because it showed the highest expression in normal activated B cells and was strongly reactivated upon 5-aza-2′-deoxycytidine and PBA treatment in U2932 cells (Fig. 1 E; note that the other investigated cell line, Oci-Ly10, carries a deletion in one of two DUSP4 alleles, which attenuates the ability of this cell line to up-regulate DUSP4 upon 5-aza-2′-deoxycytidine and PBA treatment) and because the signaling pathways targeted by DUSP4 appeared likely to contribute to DLBCL pathogenesis. To validate our methylation array–based findings on a separate set of DLBCL samples using a different technical approach, we performed bisulfite sequencing of the CpG island 28 upstream of the DUSP4 transcription start site (Waha et al., 2010). All 11 examined cell lines and 10 of 13 primary nodal and extranodal DLBCL patient biopsies exhibited widespread DUSP4 promoter methylation, whereas normal B cells and tonsil samples were unmethylated (Fig. 3 A). Aberrant methylation of the DUSP4 locus is thus a unifying feature of DLBCL irrespective of the subtype and anatomical site of origin, but was not observed in CLL (Fig. 3 A). Preliminary data obtained by chromatin immunoprecipitation of various epigenetically modified histone variants followed by PCR-based amplification of the DUSP4 CpG island 28 suggest that repressive histone marks (H3K9me2, H3K27me3) are enriched and active histone marks (H3K9ac, H4ac) are underrepresented at the DUSP4 promoter in the DLBCL cell line SU-DHL16 relative to normal blood-derived B cells (not depicted). To examine whether promoter hypermethylation and repressive histone modifications at the DUSP4 promoter indeed result in loss of DUSP4 expression, we performed DUSP4-specific immunohistochemistry (IHC) on a B cell lymphoma tissue microarray featuring 397 cases that range in aggressiveness from indolent MZL of MALT type to primary nodal, extranodal, and secondary transformed DLBCL; several tonsil samples were examined for comparison. DUSP4 expression was exclusively nuclear in all DUSP4-positive cells (Fig. 3 B). Individual positive cells were detectable in the GCs of tonsils (Fig. 3 B). Interestingly, whereas 42% of MZL, 75% of CLL, 40% of FLs, and 56% of mantle cell lymphomas (MCLs) were positive for DUSP4 expression, that rate dropped to 20–25% for high-grade transformed DLBCL originating from gastric MZLs, FLs, or CLL (Fig. 3 B). Primary nodal and extranodal DLBCL had similar low rates of DUSP4 positivity. Whereas DUSP4 expression rates were thus significantly different between aggressive and indolent lymphomas (P = 0.01), no significant differences could be detected between the classified cases of ABC (26 of 83; 31%)- and GCB-type (11 of 33; 33%) nodal DLBCL. We further performed both DUSP4 IHC and bisulfite sequencing of 16 cases of DLBCL and CLL and indeed found a clear inverse association between DUSP4 expression and the extent of DUSP4 promoter methylation (Fig. 3 C). To examine in more detail which lymphocyte subsets express DUSP4 under physiological conditions, we sorted CD138+ plasma cells from mouse bone marrow and various lymphocyte subsets from mouse spleen. Interestingly, expression of DUSP4 was limited to splenic follicular and immature B cells and was not found in plasma cells, splenic marginal zone B cells, or splenic T cells (Fig. 3 D).


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)

The DUSP4 promoter is hypermethylated in nodal and extranodal DLBCL, which correlates with lack of DUSP4 protein expression. (A) Bisulfite sequencing of 16 CpG dinucleotides in island 28 was performed for 11 DLBCL cell lines and 2 peripheral blood B cell samples and a set of 18 clinical lymphoma and 3 tonsil samples. Closed and open circles represent methylated and unmethylated CpGs, respectively. Between two and five clones were sequenced per sample. The alignment of the DUSP4 promoter sequence of the indicated species was performed using ECR Browser and shows the high conservation of CpG island 28 in other mammalian species relative to the human sequence (top left). (B) DUSP4 expression by the indicated lymphoma entities was assessed by IHC of a lymphoma tissue microarray. The graph shows percentage of DUSP4-positive cases (>30% positive cells) and DUSP4-negative cases (<30% positive cells) for the indicated lymphoma entities (top left). Representative micrographs show staining of a tonsillar GC (with insets) as well as DUSP4-positive and -negative DLBCL and CLL cases (bars, 50 µm; insets bars, 25 µm). CLL, n = 20; MZL, n = 54; FL, n = 48; MCL, n = 9; nodal DLBCL, n = 170; extranodal DLBCL, n = 87; transformed DLBCL, n = 9; and tonsils, n = 3. (C) The correlation of DUSP4 promoter methylation (assessed by bisulfite sequencing) with DUSP4 protein expression (determined by IHC) was plotted for 16 lymphoma cases. **, P < 0.01, calculated using Mann–Whitney test; horizontal lines indicate medians. (D) CD138+ plasma cells were isolated from 9-mo-old C57BL/6 mice. T cells and B cell subsets were sorted from splenocyte preparations based on CD3, B220, CD21, and CD23 expression according to the depicted sorting strategy. DUSP4 expression in the indicated subsets was analyzed by qRT-PCR and depicted as mean + SEM. Plasma cells: two pools of three mice each; T cells: n = 2 individual mice (the results represent a limited number of repeats); spleen: n = 3 individual mice; IM, MZ, and FO: n = 5 individual mice. n.d., not detected; T, T cells; B, B cells; IM, immature splenic B cells (CD21low, CD23−); MZ, marginal zone B cells (CD21high, CD23−); FO, follicular B cells (CD21int, CD23+); PC, plasma cells. (E) DUSP4 copy number alterations were analyzed using the Progenetix database. The fractions of cases affected by deletions (of 100-Mb maximum length) targeting the DUSP4 locus are shown for the indicated lymphoma entities. A representative ABC-DLBCL array CGH profile showing a focal DUSP4 deletion is shown in the left panel.
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fig3: The DUSP4 promoter is hypermethylated in nodal and extranodal DLBCL, which correlates with lack of DUSP4 protein expression. (A) Bisulfite sequencing of 16 CpG dinucleotides in island 28 was performed for 11 DLBCL cell lines and 2 peripheral blood B cell samples and a set of 18 clinical lymphoma and 3 tonsil samples. Closed and open circles represent methylated and unmethylated CpGs, respectively. Between two and five clones were sequenced per sample. The alignment of the DUSP4 promoter sequence of the indicated species was performed using ECR Browser and shows the high conservation of CpG island 28 in other mammalian species relative to the human sequence (top left). (B) DUSP4 expression by the indicated lymphoma entities was assessed by IHC of a lymphoma tissue microarray. The graph shows percentage of DUSP4-positive cases (>30% positive cells) and DUSP4-negative cases (<30% positive cells) for the indicated lymphoma entities (top left). Representative micrographs show staining of a tonsillar GC (with insets) as well as DUSP4-positive and -negative DLBCL and CLL cases (bars, 50 µm; insets bars, 25 µm). CLL, n = 20; MZL, n = 54; FL, n = 48; MCL, n = 9; nodal DLBCL, n = 170; extranodal DLBCL, n = 87; transformed DLBCL, n = 9; and tonsils, n = 3. (C) The correlation of DUSP4 promoter methylation (assessed by bisulfite sequencing) with DUSP4 protein expression (determined by IHC) was plotted for 16 lymphoma cases. **, P < 0.01, calculated using Mann–Whitney test; horizontal lines indicate medians. (D) CD138+ plasma cells were isolated from 9-mo-old C57BL/6 mice. T cells and B cell subsets were sorted from splenocyte preparations based on CD3, B220, CD21, and CD23 expression according to the depicted sorting strategy. DUSP4 expression in the indicated subsets was analyzed by qRT-PCR and depicted as mean + SEM. Plasma cells: two pools of three mice each; T cells: n = 2 individual mice (the results represent a limited number of repeats); spleen: n = 3 individual mice; IM, MZ, and FO: n = 5 individual mice. n.d., not detected; T, T cells; B, B cells; IM, immature splenic B cells (CD21low, CD23−); MZ, marginal zone B cells (CD21high, CD23−); FO, follicular B cells (CD21int, CD23+); PC, plasma cells. (E) DUSP4 copy number alterations were analyzed using the Progenetix database. The fractions of cases affected by deletions (of 100-Mb maximum length) targeting the DUSP4 locus are shown for the indicated lymphoma entities. A representative ABC-DLBCL array CGH profile showing a focal DUSP4 deletion is shown in the left panel.
Mentions: We focused on the dual-specificity phosphatase DUSP4 in all subsequent experimental efforts because it showed the highest expression in normal activated B cells and was strongly reactivated upon 5-aza-2′-deoxycytidine and PBA treatment in U2932 cells (Fig. 1 E; note that the other investigated cell line, Oci-Ly10, carries a deletion in one of two DUSP4 alleles, which attenuates the ability of this cell line to up-regulate DUSP4 upon 5-aza-2′-deoxycytidine and PBA treatment) and because the signaling pathways targeted by DUSP4 appeared likely to contribute to DLBCL pathogenesis. To validate our methylation array–based findings on a separate set of DLBCL samples using a different technical approach, we performed bisulfite sequencing of the CpG island 28 upstream of the DUSP4 transcription start site (Waha et al., 2010). All 11 examined cell lines and 10 of 13 primary nodal and extranodal DLBCL patient biopsies exhibited widespread DUSP4 promoter methylation, whereas normal B cells and tonsil samples were unmethylated (Fig. 3 A). Aberrant methylation of the DUSP4 locus is thus a unifying feature of DLBCL irrespective of the subtype and anatomical site of origin, but was not observed in CLL (Fig. 3 A). Preliminary data obtained by chromatin immunoprecipitation of various epigenetically modified histone variants followed by PCR-based amplification of the DUSP4 CpG island 28 suggest that repressive histone marks (H3K9me2, H3K27me3) are enriched and active histone marks (H3K9ac, H4ac) are underrepresented at the DUSP4 promoter in the DLBCL cell line SU-DHL16 relative to normal blood-derived B cells (not depicted). To examine whether promoter hypermethylation and repressive histone modifications at the DUSP4 promoter indeed result in loss of DUSP4 expression, we performed DUSP4-specific immunohistochemistry (IHC) on a B cell lymphoma tissue microarray featuring 397 cases that range in aggressiveness from indolent MZL of MALT type to primary nodal, extranodal, and secondary transformed DLBCL; several tonsil samples were examined for comparison. DUSP4 expression was exclusively nuclear in all DUSP4-positive cells (Fig. 3 B). Individual positive cells were detectable in the GCs of tonsils (Fig. 3 B). Interestingly, whereas 42% of MZL, 75% of CLL, 40% of FLs, and 56% of mantle cell lymphomas (MCLs) were positive for DUSP4 expression, that rate dropped to 20–25% for high-grade transformed DLBCL originating from gastric MZLs, FLs, or CLL (Fig. 3 B). Primary nodal and extranodal DLBCL had similar low rates of DUSP4 positivity. Whereas DUSP4 expression rates were thus significantly different between aggressive and indolent lymphomas (P = 0.01), no significant differences could be detected between the classified cases of ABC (26 of 83; 31%)- and GCB-type (11 of 33; 33%) nodal DLBCL. We further performed both DUSP4 IHC and bisulfite sequencing of 16 cases of DLBCL and CLL and indeed found a clear inverse association between DUSP4 expression and the extent of DUSP4 promoter methylation (Fig. 3 C). To examine in more detail which lymphocyte subsets express DUSP4 under physiological conditions, we sorted CD138+ plasma cells from mouse bone marrow and various lymphocyte subsets from mouse spleen. Interestingly, expression of DUSP4 was limited to splenic follicular and immature B cells and was not found in plasma cells, splenic marginal zone B cells, or splenic T cells (Fig. 3 D).

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