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Identification of p38β as a therapeutic target for the treatment of Sézary syndrome.

Bliss-Moreau M, Coarfa C, Gunaratne PH, Guitart J, Krett NL, Rosen ST - J. Invest. Dermatol. (2014)

Bottom Line: Gene set enrichment analysis uncovered candidate genes enriched for an immune-cell signature, specifically the T-cell receptor and mitogen-activated protein kinase signaling pathways.Further analysis identified p38 as a potential therapeutic target that is overexpressed in SS patients and decreased by synergistic-inhibitor treatment.This target was verified through small-molecule inhibition of p38, leading to cell death in both SS cell lines and patient cells.

View Article: PubMed Central - PubMed

Affiliation: Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

ABSTRACT
Cutaneous T-cell lymphomas (CTCLs) represent a group of hematopoietic malignancies that home to the skin and have no known molecular basis for disease pathogenesis. Sézary syndrome (SS) is the leukemic variant of CTCL. Currently, CTCL is incurable, highlighting the need for new therapeutic modalities. We have previously observed that combined small-molecule inhibition of protein kinase C-β (PKCβ) and glycogen synthase kinase 3 (GSK3) causes synergistic apoptosis in CTCL cell lines and patient cells. Through microarray analysis of a SS cell line, we surveyed global gene expression following combined PKCβ-GSK3 treatment to elucidate therapeutic targets responsible for cell death. Clinically relevant targets were defined as genes differentially expressed in SS patients that were modulated by combination-drug treatment of SS cells. Gene set enrichment analysis uncovered candidate genes enriched for an immune-cell signature, specifically the T-cell receptor and mitogen-activated protein kinase signaling pathways. Further analysis identified p38 as a potential therapeutic target that is overexpressed in SS patients and decreased by synergistic-inhibitor treatment. This target was verified through small-molecule inhibition of p38, leading to cell death in both SS cell lines and patient cells. These data establish p38 as a SS biomarker and a potential therapeutic target for the treatment of CTCL.

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SS cell lines and patient samples express all p38 isoforms. p38β (MAPK11) is differentially regulated by small-molecule inhibitor treatment(a,b) H9 and Hut78 cells were treated with Enz and ARA as previously described, n=3. (a) MAPK14, MAPK11, MAPK12, and MAPK13 mRNA expression measured by qRT-PCR. (b) p38 isoforms protein expression, normalized to α-tubulin, were measured by immunoblot. Representative immunoblot images of three replicates (Quantification, Supplemental Figure 5, online). (c,d) Endogenous CTCL patient and healthy donor cell samples. (c) MAPK isoform mRNA expression measured by qRT-PCR, normalized to housekeeping genes, and normalized to donor cells. (d) Quantitative representation of p38 isoform immunoblots. Protein expression normalized to loading control (α-tubulin) expression and then normalized to healthy volunteers. H9/Hut78 cells as positive controls.
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Figure 3: SS cell lines and patient samples express all p38 isoforms. p38β (MAPK11) is differentially regulated by small-molecule inhibitor treatment(a,b) H9 and Hut78 cells were treated with Enz and ARA as previously described, n=3. (a) MAPK14, MAPK11, MAPK12, and MAPK13 mRNA expression measured by qRT-PCR. (b) p38 isoforms protein expression, normalized to α-tubulin, were measured by immunoblot. Representative immunoblot images of three replicates (Quantification, Supplemental Figure 5, online). (c,d) Endogenous CTCL patient and healthy donor cell samples. (c) MAPK isoform mRNA expression measured by qRT-PCR, normalized to housekeeping genes, and normalized to donor cells. (d) Quantitative representation of p38 isoform immunoblots. Protein expression normalized to loading control (α-tubulin) expression and then normalized to healthy volunteers. H9/Hut78 cells as positive controls.

Mentions: p38 MAPK signaling converts numerous extracellular signals into a spectrum of cellular responses (such as stimulation of proliferation or apoptosis) through changes in transcriptional regulation, posttranslational modifications, or tissue-specific expression of the four p38 isoforms (Cuenda and Rousseau 2007; Cuadrado and Nebreda 2010). In the healthy T-cell setting, reports document that p38α is highly expressed, and p38β and p38δ to lesser extents (Wang et al. 1997; Hale et al. 1999). p38γ and p38δ expression are largely restricted to non-hematopoietic tissues (Li et al. 1996; Wang et al. 1997). Surprisingly, all four p38 isoforms were endogenously expressed in SS cell lines with high mRNA and protein expression levels (Figure 3a and b, Supplemental Figure S5, online), as reported in few other malignancies (Pramanik et al. 2003; O'Callaghan et al. 2013). Using qRT-PCR, we confirmed our drug treatment microarray data and determined that MAPK11 (p38β) was significantly down-regulated following Enz+ARA treatment in both H9 and Hut78 cell lines after five-day treatment (Figure 3a). MAPK12 (p38γ) gene and protein expression was also significantly down-regulated after drug treatment in cell lines (Figure 3a and b). p38δ (MAPK13) protein expression in Hut78 cells post Enz+ARA treatment was modestly reduced by 28.86% (p=0.0287; Supplemental Figure S5, online). In contrast, p38β was decreased by 88.83% (p=0.0054) upon inhibitor treatment.


Identification of p38β as a therapeutic target for the treatment of Sézary syndrome.

Bliss-Moreau M, Coarfa C, Gunaratne PH, Guitart J, Krett NL, Rosen ST - J. Invest. Dermatol. (2014)

SS cell lines and patient samples express all p38 isoforms. p38β (MAPK11) is differentially regulated by small-molecule inhibitor treatment(a,b) H9 and Hut78 cells were treated with Enz and ARA as previously described, n=3. (a) MAPK14, MAPK11, MAPK12, and MAPK13 mRNA expression measured by qRT-PCR. (b) p38 isoforms protein expression, normalized to α-tubulin, were measured by immunoblot. Representative immunoblot images of three replicates (Quantification, Supplemental Figure 5, online). (c,d) Endogenous CTCL patient and healthy donor cell samples. (c) MAPK isoform mRNA expression measured by qRT-PCR, normalized to housekeeping genes, and normalized to donor cells. (d) Quantitative representation of p38 isoform immunoblots. Protein expression normalized to loading control (α-tubulin) expression and then normalized to healthy volunteers. H9/Hut78 cells as positive controls.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4289446&req=5

Figure 3: SS cell lines and patient samples express all p38 isoforms. p38β (MAPK11) is differentially regulated by small-molecule inhibitor treatment(a,b) H9 and Hut78 cells were treated with Enz and ARA as previously described, n=3. (a) MAPK14, MAPK11, MAPK12, and MAPK13 mRNA expression measured by qRT-PCR. (b) p38 isoforms protein expression, normalized to α-tubulin, were measured by immunoblot. Representative immunoblot images of three replicates (Quantification, Supplemental Figure 5, online). (c,d) Endogenous CTCL patient and healthy donor cell samples. (c) MAPK isoform mRNA expression measured by qRT-PCR, normalized to housekeeping genes, and normalized to donor cells. (d) Quantitative representation of p38 isoform immunoblots. Protein expression normalized to loading control (α-tubulin) expression and then normalized to healthy volunteers. H9/Hut78 cells as positive controls.
Mentions: p38 MAPK signaling converts numerous extracellular signals into a spectrum of cellular responses (such as stimulation of proliferation or apoptosis) through changes in transcriptional regulation, posttranslational modifications, or tissue-specific expression of the four p38 isoforms (Cuenda and Rousseau 2007; Cuadrado and Nebreda 2010). In the healthy T-cell setting, reports document that p38α is highly expressed, and p38β and p38δ to lesser extents (Wang et al. 1997; Hale et al. 1999). p38γ and p38δ expression are largely restricted to non-hematopoietic tissues (Li et al. 1996; Wang et al. 1997). Surprisingly, all four p38 isoforms were endogenously expressed in SS cell lines with high mRNA and protein expression levels (Figure 3a and b, Supplemental Figure S5, online), as reported in few other malignancies (Pramanik et al. 2003; O'Callaghan et al. 2013). Using qRT-PCR, we confirmed our drug treatment microarray data and determined that MAPK11 (p38β) was significantly down-regulated following Enz+ARA treatment in both H9 and Hut78 cell lines after five-day treatment (Figure 3a). MAPK12 (p38γ) gene and protein expression was also significantly down-regulated after drug treatment in cell lines (Figure 3a and b). p38δ (MAPK13) protein expression in Hut78 cells post Enz+ARA treatment was modestly reduced by 28.86% (p=0.0287; Supplemental Figure S5, online). In contrast, p38β was decreased by 88.83% (p=0.0054) upon inhibitor treatment.

Bottom Line: Gene set enrichment analysis uncovered candidate genes enriched for an immune-cell signature, specifically the T-cell receptor and mitogen-activated protein kinase signaling pathways.Further analysis identified p38 as a potential therapeutic target that is overexpressed in SS patients and decreased by synergistic-inhibitor treatment.This target was verified through small-molecule inhibition of p38, leading to cell death in both SS cell lines and patient cells.

View Article: PubMed Central - PubMed

Affiliation: Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

ABSTRACT
Cutaneous T-cell lymphomas (CTCLs) represent a group of hematopoietic malignancies that home to the skin and have no known molecular basis for disease pathogenesis. Sézary syndrome (SS) is the leukemic variant of CTCL. Currently, CTCL is incurable, highlighting the need for new therapeutic modalities. We have previously observed that combined small-molecule inhibition of protein kinase C-β (PKCβ) and glycogen synthase kinase 3 (GSK3) causes synergistic apoptosis in CTCL cell lines and patient cells. Through microarray analysis of a SS cell line, we surveyed global gene expression following combined PKCβ-GSK3 treatment to elucidate therapeutic targets responsible for cell death. Clinically relevant targets were defined as genes differentially expressed in SS patients that were modulated by combination-drug treatment of SS cells. Gene set enrichment analysis uncovered candidate genes enriched for an immune-cell signature, specifically the T-cell receptor and mitogen-activated protein kinase signaling pathways. Further analysis identified p38 as a potential therapeutic target that is overexpressed in SS patients and decreased by synergistic-inhibitor treatment. This target was verified through small-molecule inhibition of p38, leading to cell death in both SS cell lines and patient cells. These data establish p38 as a SS biomarker and a potential therapeutic target for the treatment of CTCL.

Show MeSH
Related in: MedlinePlus