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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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In SS cell lines, inhibition of p38 with small molecules induces apoptosis and BIRB796 works through a p38 mechanism(a) H9 and Hut78 cells were treated with increasing concentrations of either SB202, SB203, or BIRB. Apoptosis was measured by flow cytometry, N=3. (b,c) H9 and Hut78 cells were cultured with either Enz+ARA plus BIRB or increasing concentrations of BIRB alone for 5 days. (b–d) Apoptosis was measured by Annexin V staining, N=3. Total cell death is represented as a fold change normalized to DMSO, in the absence of BIRB treatment. Comparisons were made between those cells with treated with BIRB alone and cells treated with BIRB in the presence of Enz+ARA. (b) H9 (c) Hut78 (d) Raw cell death (Annexin V positive cells), no BIRB treatment.
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Figure 4: In SS cell lines, inhibition of p38 with small molecules induces apoptosis and BIRB796 works through a p38 mechanism(a) H9 and Hut78 cells were treated with increasing concentrations of either SB202, SB203, or BIRB. Apoptosis was measured by flow cytometry, N=3. (b,c) H9 and Hut78 cells were cultured with either Enz+ARA plus BIRB or increasing concentrations of BIRB alone for 5 days. (b–d) Apoptosis was measured by Annexin V staining, N=3. Total cell death is represented as a fold change normalized to DMSO, in the absence of BIRB treatment. Comparisons were made between those cells with treated with BIRB alone and cells treated with BIRB in the presence of Enz+ARA. (b) H9 (c) Hut78 (d) Raw cell death (Annexin V positive cells), no BIRB treatment.

Mentions: There are numerous MAPK inhibitors that target p38 isoforms with varying efficacies; many have multiple known off-target effects. We chose a panel of three p38 inhibitors with established in vitro kinase properties and in vivo physiological functions. We treated H9 and Hut78 cells with increasing concentrations of either SB202190 (SB202), SB203580 (SB203), or BIRB796 (BIRB) for five days. Inhibition of p38-induced apoptosis in both cell lines, with a more robust inhibition observed in H9 cells (Figure 4a and Supplemental Figure S6, online). Both cell lines were more sensitive to SB202, an inhibitor documented to be p38β specific in vivo (Nemoto et al. 1998). Greater amplitude of response was observed with both SB compounds compared to BIRB treatment, perhaps due to the off-target inhibition of other kinases, such as GSK3. We established GSK3 as a driver of SS death in our microarray data (Figure 1). To a lesser extent, H9 and Hut78 cells were sensitive to BIRB (Figure 4a–c).


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)

In SS cell lines, inhibition of p38 with small molecules induces apoptosis and BIRB796 works through a p38 mechanism(a) H9 and Hut78 cells were treated with increasing concentrations of either SB202, SB203, or BIRB. Apoptosis was measured by flow cytometry, N=3. (b,c) H9 and Hut78 cells were cultured with either Enz+ARA plus BIRB or increasing concentrations of BIRB alone for 5 days. (b–d) Apoptosis was measured by Annexin V staining, N=3. Total cell death is represented as a fold change normalized to DMSO, in the absence of BIRB treatment. Comparisons were made between those cells with treated with BIRB alone and cells treated with BIRB in the presence of Enz+ARA. (b) H9 (c) Hut78 (d) Raw cell death (Annexin V positive cells), no BIRB treatment.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4289446&req=5

Figure 4: In SS cell lines, inhibition of p38 with small molecules induces apoptosis and BIRB796 works through a p38 mechanism(a) H9 and Hut78 cells were treated with increasing concentrations of either SB202, SB203, or BIRB. Apoptosis was measured by flow cytometry, N=3. (b,c) H9 and Hut78 cells were cultured with either Enz+ARA plus BIRB or increasing concentrations of BIRB alone for 5 days. (b–d) Apoptosis was measured by Annexin V staining, N=3. Total cell death is represented as a fold change normalized to DMSO, in the absence of BIRB treatment. Comparisons were made between those cells with treated with BIRB alone and cells treated with BIRB in the presence of Enz+ARA. (b) H9 (c) Hut78 (d) Raw cell death (Annexin V positive cells), no BIRB treatment.
Mentions: There are numerous MAPK inhibitors that target p38 isoforms with varying efficacies; many have multiple known off-target effects. We chose a panel of three p38 inhibitors with established in vitro kinase properties and in vivo physiological functions. We treated H9 and Hut78 cells with increasing concentrations of either SB202190 (SB202), SB203580 (SB203), or BIRB796 (BIRB) for five days. Inhibition of p38-induced apoptosis in both cell lines, with a more robust inhibition observed in H9 cells (Figure 4a and Supplemental Figure S6, online). Both cell lines were more sensitive to SB202, an inhibitor documented to be p38β specific in vivo (Nemoto et al. 1998). Greater amplitude of response was observed with both SB compounds compared to BIRB treatment, perhaps due to the off-target inhibition of other kinases, such as GSK3. We established GSK3 as a driver of SS death in our microarray data (Figure 1). To a lesser extent, H9 and Hut78 cells were sensitive to BIRB (Figure 4a–c).

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