BCL11B-Mediated Epigenetic Repression Is a Crucial Target for Histone Deacetylase Inhibitors in Cutaneous T-Cell Lymphoma  Wenjing Fu, Shengguo Yi, Lei Qiu, Jingru Sun, Ping Tu, Yang Wang  Journal of Investigative Dermatology  Volume 137, Issue 7, Pages 1523-1532 (July 2017) DOI: 10.1016/j.jid.2017.02.980 Copyright © 2017 The Authors Terms and Conditions

Figure 1 BCL11B expression levels and the response to SAHA in seven CTCL lines. (a) BCL11B mRNA and (b) protein expression levels in each CTCL line. GAPDH was used as endogenous reference. (c) SAHA inhibits cell viability of CTCL lines. Seven CTCL lines were treated with increasing concentrations of SAHA for 48 hours and 72 hours. Relative cell viabilities were calculated with results from MTS assays. (d) Correlation analysis between the IC50 and BCL11B protein expression levels in CTCL lines, using Spearman’s (r2) rank test. P < 0.05 was considered significant. (e) SAHA-induced apoptosis in CTCL lines as measured by flow cytometry (1 μmol/L SAHA for 48 hours). (f) Specific apoptosis induced by SAHA in CTCL lines. (g) Protein expression levels of HDAC1 and HDAC2 in each CTCL line. Either mean ± standard deviation of triplicate measurements, or a representative result of three independent experiments is shown. t test, ∗∗∗∗P < 0.0001. CTCL, cutaneous T-cell lymphoma; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; h, hour; HDAC, histone deacetylase; IC50, half maximal inhibitory concentration; SAHA, suberoylanilide hydroxamic acid. Journal of Investigative Dermatology 2017 137, 1523-1532DOI: (10.1016/j.jid.2017.02.980) Copyright © 2017 The Authors Terms and Conditions

Figure 2 Suppression of BCL11B in H9 and HH cells and the synergistic effect in inducing cell apoptosis with SAHA treatment. (a) Suppression of BCL11B protein expression in H9 cell by lentiviral transduction with two independent shRNA sequences (Sh1, Sh2). Mock-transduction (MOCK) and nonsilencing scrambled shRNA (Sh0) were used as controls. (b) Inhibition of cell growth in H9 after BCL11B suppression as measured by MTS assays. (c) Increased sensitivity to SAHA treatment in BCL11B-suppressed H9 cells. Cells were incubated with increasing concentrations of SAHA, and relative cell viabilities were calculated at 48 hours. (d) SAHA induced more cell apoptosis in BCL11B-suppressed H9 and HH cells compared with control cells. Cells were incubated with 0.3 μmol/L SAHA for 48 hours before proceeding to apoptosis assays. (e) Specific apoptosis induced by combined BCL11B suppression (Sh1) and SAHA treatment was higher than the sum of the apoptosis induced by BCL11B suppression (Sh1) or SAHA alone, indicating a synergistic effect. Either mean ± standard deviation of triplicate measurements or a representative result of three experiments is shown. t test, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗∗P < 0.0001. M, mol/L; SAHA, suberoylanilide hydroxamic acid; shRNA, short hairpin RNA. Journal of Investigative Dermatology 2017 137, 1523-1532DOI: (10.1016/j.jid.2017.02.980) Copyright © 2017 The Authors Terms and Conditions

Figure 3 Physical interaction between BCL11B and HDAC1/2 and their functional interaction on regulating cell apoptosis. (a) Co-immunoprecipitation of nuclear extracts from H9 and HH cells using an anti-BCL11B mAb (lane 3) or normal mouse IgG (lane 2) with protein A/G agarose. HDAC1, HDAC2, and HDAC3 were identified by Western blot. Lane 1 shows input control. (b) Reverse immunoprecipitation with specific antibodies against HDACs or IgG and the following immunoblot analysis with the anti-BCL11B antibody. (c) Schematic diagram of structure of wild-type BCL11B (WT) and N-terminal–deletion mutant BCL11B (ΔN) vectors. (d) BCL11B protein levels on H9 cells transduced with different vectors, as determined by a mAb specific to the N-terminal epitope of BCL11B. (e) BCL11B-WT, but not BCL11B-ΔN, rescued cell growth in BCL11B-suppressed H9 cells. (f) The response to SAHA was rescued by BCL11B-WT, but not BCL11B-ΔN, in BCL11B-suppressed H9 cells. (g) Cell apoptosis and (h) specific apoptosis induced by 0.3 μmol/L SAHA for 48 hours in different transduced H9 cells. Sh0 indicates scrambled shRNA; V0 indicates control overexpression vector. Sh0 + V0 indicates cells transduced with Sh0 and V0, etc. Either mean ± standard deviation of triplicate measurements or a representative result of three experiments is shown. t test, ∗P < 0.05, ∗∗∗P < 0.001. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HDAC, histone deacetylase; IP, immunoprecipitation; M, mol/L; SAHA, suberoylanilide hydroxamic acid; shRNA, short hairpin RNA. Journal of Investigative Dermatology 2017 137, 1523-1532DOI: (10.1016/j.jid.2017.02.980) Copyright © 2017 The Authors Terms and Conditions

Figure 4 BCL11B expression levels in MF patients. (a) The relative BCL11B mRNA expressions in different T classifications of MF in 46 lesional skin biopsy samples. The relative transcript levels are expressed as copies of BCL11B per 10,000 copies of CD4 transcripts. By t test, ∗P < 0.05 was designated as indicating a significant difference between appointed groups. (b) The relative mRNA expressions of BCL11B in four MF patients (Pt1, Pt22, Pt23, Pt40) who have received vorinostat treatment for more than 6 months. The patient who responded well to vorinostat (Pt23) displays higher BCL11B expression than the other three nonresponders. Error bars represent mean ± standard deviation of triplicate measurements. MF, mycosis fungoides; Pt, patient; SAHA, suberoylanilide hydroxamic acid. Journal of Investigative Dermatology 2017 137, 1523-1532DOI: (10.1016/j.jid.2017.02.980) Copyright © 2017 The Authors Terms and Conditions