Journal: The Journal of Clinical Investigation

Article Title: Glucocorticoids paradoxically facilitate steroid resistance in T cell acute lymphoblastic leukemias and thymocytes

doi: 10.1172/JCI130189

Figure Lengend Snippet: (A) Evaluation of STAT5A and STAT5B expression by Western blotting in independent scrambled control (S1–S4) and STAT5 single-KO (A1–A4 or B1–B4) and double-KO (AB1–AB4) CCRF-CEM cell clones (n = 4 per genotype). (B) Viability of independent scrambled control and STAT5-KO CCRF-CEM cell clones (n = 4 per genotype) treated with 100 nM DEX with or without 25 ng/mL IL-7 for 72 hours. (C) Venn diagram depicting the overlap between the top differentially expressed genes among scrambled control CCRF-CEM cell clones (n = 4) treated with DEX versus DEX plus IL-7 and STAT5B target genes. (D and E) Fold change in the fragments per kilobase per million mapped reads (FPKM) values for (D) BCL2 transcript and (E) ARHGEF3 transcript levels as determined by RNA-Seq analysis of scrambled control cells (n = 4) and STAT5A/B double-KO (n = 4) CCRF-CEM cell clones treated in the absence or presence of 100 ng/mL IL-7 and/or 1 μM DEX for 16 hours. (F) ΔMFI of BCL-2 protein expression in scrambled control (n = 4) and STAT5-KO (n = 4) CCRF-CEM cell clones treated with 100 ng/mL IL-7 and 1 μM DEX relative to 100 ng/mL IL-7 alone for 48 hours. (G) BCL2L11 and BCL2 transcript expression in CCRF-CEM cells cultured in the absence or presence of 1 μM DEX, 100 ng/mL IL-7, and/or 10 μg/mL CHX for 16 hours as determined by qPCR performed in technical triplicate. *P < 0.05, **P < 0.01, and ****P < 0.0001, by paired t test (B), 2-sample t test (D and E), or 1-way ANOVA with Tukey’s method for multiple comparisons adjustment (F and G). With the exception of the RNA-Seq experiment, all data are representative of 3 independent experiments.

Article Snippet: Consistent with their presence on both of these gene lists, targeted analysis of the RNA-Seq data revealed that these genes were induced by the combination of DEX and IL-7 relative to treatment with DEX or IL-7 alone only in the scrambled control clones but not in the STAT5A/B-KO clones ( ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 3 caption a7 caption a8 STAT5B, but not STAT5A, mediates the upregulation of BCL-2 expression in cells exposed to the combination of DEX and IL-7. ( A ) Evaluation of STAT5A and STAT5B expression by Western blotting in independent scrambled control (S1–S4) and STAT5 single-KO (A1–A4 or B1–B4) and double-KO (AB1–AB4) CCRF-CEM cell clones ( n = 4 per genotype). ( B ) Viability of independent scrambled control and STAT5-KO CCRF-CEM cell clones ( n = 4 per genotype) treated with 100 nM DEX with or without 25 ng/mL IL-7 for 72 hours. ( C ) Venn diagram depicting the overlap between the top differentially expressed genes among scrambled control CCRF-CEM cell clones ( n = 4) treated with DEX versus DEX plus IL-7 and STAT5B target genes. ( D and E ) Fold change in the fragments per kilobase per million mapped reads (FPKM) values for ( D ) BCL2 transcript and ( E ) ARHGEF3 transcript levels as determined by RNA-Seq analysis of scrambled control cells ( n = 4) and STAT5A/B double-KO ( n = 4) CCRF-CEM cell clones treated in the absence or presence of 100 ng/mL IL-7 and/or 1 μM DEX for 16 hours. ( F ) ΔMFI of BCL-2 protein expression in scrambled control ( n = 4) and STAT5-KO ( n = 4) CCRF-CEM cell clones treated with 100 ng/mL IL-7 and 1 μM DEX relative to 100 ng/mL IL-7 alone for 48 hours. ( G ) BCL2L11 and BCL2 transcript expression in CCRF-CEM cells cultured in the absence or presence of 1 μM DEX, 100 ng/mL IL-7, and/or 10 μg/mL CHX for 16 hours as determined by qPCR performed in technical triplicate.

Techniques: Expressing, Western Blot, Control, Clone Assay, RNA Sequencing, Cell Culture

Journal: The Journal of Clinical Investigation

Article Title: Glucocorticoids paradoxically facilitate steroid resistance in T cell acute lymphoblastic leukemias and thymocytes

doi: 10.1172/JCI130189

Figure Lengend Snippet: (A) Percentage of priming of CCRF-CEM cells treated in the absence or presence of 1 μM DEX and/or 100 ng/mL IL-7 in technical triplicate for 16 hours followed by BH3 profiling with 0.5 μM ABT-199 for 90 minutes. (B) Viability of CCRF-CEM cells treated with DEX in the absence or presence of 25 ng/mL IL-7 and increasing concentrations of ABT-199 for 72 hours in technical triplicate. The no IL-7 (black line) and the 25 ng/mL IL-7 (red line) conditions were replotted from Figure 1C. (C) Heatmap of Bliss independence scores calculated as the average of technical triplicates for the combination of DEX and ABT-199 in the presence of 25 ng/mL IL-7. (D) MFI of BCL-2 protein expression assessed in technical triplicate in untransduced CCRF-CEM cells and CCRF-CEM cells transduced with a nontargeting shRNA control (shControl) or a BCL2-targeting shRNA (shBCL2-1-5). Statistical significance is relative to the untransduced cells. (E) Viability of untransduced or shRNA-transduced CCRF-CEM cells treated with DEX in the absence or presence of 25 ng/mL IL-7 in technical triplicate for 72 hours. (F) FPKM values for BCL2 transcript levels according to published RNA-Seq data from diagnostic samples from patients enrolled in the COG AALL0434 trial, stratified on the basis of day-29 bone marrow MRD. (G) Schematic of the proposed model for the mechanism by which DEX paradoxically induces steroid resistance in T-ALL cells in the presence of IL-7. In the presence of DEX (right), the GR induces an increase in IL-7R expression (i), leading to an increase in IL-7R at the cell surface (ii). This in turn leads to an increase in STAT5 transcriptional activity (iii) that ultimately results in the upregulation of BCL-2 (iv). GRE, glucocorticoid response element. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 1-way ANOVA with Tukey’s method for multiple comparisons adjustment (A, D, and F). All CCRF-CEM cell data are representative of 3 independent experiments.

Article Snippet: Consistent with their presence on both of these gene lists, targeted analysis of the RNA-Seq data revealed that these genes were induced by the combination of DEX and IL-7 relative to treatment with DEX or IL-7 alone only in the scrambled control clones but not in the STAT5A/B-KO clones ( ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 3 caption a7 caption a8 STAT5B, but not STAT5A, mediates the upregulation of BCL-2 expression in cells exposed to the combination of DEX and IL-7. ( A ) Evaluation of STAT5A and STAT5B expression by Western blotting in independent scrambled control (S1–S4) and STAT5 single-KO (A1–A4 or B1–B4) and double-KO (AB1–AB4) CCRF-CEM cell clones ( n = 4 per genotype). ( B ) Viability of independent scrambled control and STAT5-KO CCRF-CEM cell clones ( n = 4 per genotype) treated with 100 nM DEX with or without 25 ng/mL IL-7 for 72 hours. ( C ) Venn diagram depicting the overlap between the top differentially expressed genes among scrambled control CCRF-CEM cell clones ( n = 4) treated with DEX versus DEX plus IL-7 and STAT5B target genes. ( D and E ) Fold change in the fragments per kilobase per million mapped reads (FPKM) values for ( D ) BCL2 transcript and ( E ) ARHGEF3 transcript levels as determined by RNA-Seq analysis of scrambled control cells ( n = 4) and STAT5A/B double-KO ( n = 4) CCRF-CEM cell clones treated in the absence or presence of 100 ng/mL IL-7 and/or 1 μM DEX for 16 hours. ( F ) ΔMFI of BCL-2 protein expression in scrambled control ( n = 4) and STAT5-KO ( n = 4) CCRF-CEM cell clones treated with 100 ng/mL IL-7 and 1 μM DEX relative to 100 ng/mL IL-7 alone for 48 hours. ( G ) BCL2L11 and BCL2 transcript expression in CCRF-CEM cells cultured in the absence or presence of 1 μM DEX, 100 ng/mL IL-7, and/or 10 μg/mL CHX for 16 hours as determined by qPCR performed in technical triplicate.

Techniques: Expressing, Transduction, shRNA, Control, RNA Sequencing, Diagnostic Assay, Activity Assay

Journal: The Journal of Clinical Investigation

Article Title: Glucocorticoids paradoxically facilitate steroid resistance in T cell acute lymphoblastic leukemias and thymocytes

doi: 10.1172/JCI130189

Figure Lengend Snippet: (A) Percentage of thymocyte subpopulations in thymi isolated from mice treated with vehicle control (n = 3) or DEX (n = 3) at 2 mg/kg/day for 3 days. (B) Percentage of priming of thymocytes in the basal state following BH3 profiling with 1 μM synthetic BIM peptide in technical triplicate for 90 minutes. (C) Histograms of the basal expression of IL-7Rα in the major murine thymocyte subpopulations. (D) Histograms of p-STAT5 in the major murine thymocyte subpopulations in the basal state (white histograms) and following a 15-minute stimulation with 100 ng/mL IL-7 (colored histograms). (E) Viability of murine thymocyte subpopulations following ex vivo treatment for 24 hours with DEX in the absence or presence of increasing concentrations of IL-7. (F) MFI of IL-7Rα in murine thymocytes treated ex vivo in the presence of 100 pg/mL IL-7 with or without 1 μM DEX in technical triplicate for 24 hours. DP cells could not be analyzed (NA) because of a lack of viable cells remaining after DEX exposure. (G) MFI of BCL-2 in murine thymocytes treated ex vivo in the presence of 100 pg/mL IL-7 with or without 1 μM DEX in technical triplicate for 24 hours. DP cells could not be analyzed (NA) because of a lack of viable cells remaining after DEX exposure. (H) MFI of BCL-2 in thymocytes isolated from mice treated with vehicle control (n = 3) or DEX (n = 3) at a dose of 2 mg/kg/day for 3 days. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 2-sample t test (A, F, G, and H) or 1-way ANOVA with Tukey’s method for multiple comparisons adjustment (B). All data are representative of 3 independent experiments.

Article Snippet: Consistent with their presence on both of these gene lists, targeted analysis of the RNA-Seq data revealed that these genes were induced by the combination of DEX and IL-7 relative to treatment with DEX or IL-7 alone only in the scrambled control clones but not in the STAT5A/B-KO clones ( ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 3 caption a7 caption a8 STAT5B, but not STAT5A, mediates the upregulation of BCL-2 expression in cells exposed to the combination of DEX and IL-7. ( A ) Evaluation of STAT5A and STAT5B expression by Western blotting in independent scrambled control (S1–S4) and STAT5 single-KO (A1–A4 or B1–B4) and double-KO (AB1–AB4) CCRF-CEM cell clones ( n = 4 per genotype). ( B ) Viability of independent scrambled control and STAT5-KO CCRF-CEM cell clones ( n = 4 per genotype) treated with 100 nM DEX with or without 25 ng/mL IL-7 for 72 hours. ( C ) Venn diagram depicting the overlap between the top differentially expressed genes among scrambled control CCRF-CEM cell clones ( n = 4) treated with DEX versus DEX plus IL-7 and STAT5B target genes. ( D and E ) Fold change in the fragments per kilobase per million mapped reads (FPKM) values for ( D ) BCL2 transcript and ( E ) ARHGEF3 transcript levels as determined by RNA-Seq analysis of scrambled control cells ( n = 4) and STAT5A/B double-KO ( n = 4) CCRF-CEM cell clones treated in the absence or presence of 100 ng/mL IL-7 and/or 1 μM DEX for 16 hours. ( F ) ΔMFI of BCL-2 protein expression in scrambled control ( n = 4) and STAT5-KO ( n = 4) CCRF-CEM cell clones treated with 100 ng/mL IL-7 and 1 μM DEX relative to 100 ng/mL IL-7 alone for 48 hours. ( G ) BCL2L11 and BCL2 transcript expression in CCRF-CEM cells cultured in the absence or presence of 1 μM DEX, 100 ng/mL IL-7, and/or 10 μg/mL CHX for 16 hours as determined by qPCR performed in technical triplicate.

Techniques: Isolation, Control, Expressing, Ex Vivo

Journal: The Journal of Clinical Investigation

Article Title: Glucocorticoids paradoxically facilitate steroid resistance in T cell acute lymphoblastic leukemias and thymocytes

doi: 10.1172/JCI130189

Figure Lengend Snippet: (A) Heatmap depicting the clustering of 76 primary T-ALL samples by the expression of genes that are upregulated in early developing thymocytes relative to later developing thymocytes. (B) MFI of cell-surface IL-7Rα in 15 early and 12 late T-ALL PDX samples following exposure to 1 μM DEX for 24 hours in technical triplicate. (C) Viability relative to vehicle control of 15 early and 12 late T-ALL PDX samples treated with 1 μM DEX in the absence or presence of 25 ng/mL IL-7 for 48 hours in technical triplicate. (D) MFI of BCL-2 protein expression in 10 early T-ALL PDX samples following exposure to 100 ng/mL IL-7 with or without 1 μM DEX and/or 500 nM RUX for 16 hours in technical triplicate. Some samples were not analyzed because of limited cell numbers. (E) Viability relative to vehicle control of 15 early T-ALL samples exposed to 25 ng/mL IL-7 with or without 1 μM DEX and/or 500 nM RUX or 1 μM ABT-199 for 48 hours in technical triplicate. (F) Kaplan-Meier survival analysis of mice transplanted with early T-ALL T24 and treated with vehicle control (n = 5), DEX (n = 5), RUX (n = 5), or a combination of DEX and RUX (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by paired t test (B and C), 1-way ANOVA with Tukey’s method for multiple comparisons adjustment (D and E), or a log-rank test (F).

Article Snippet: Consistent with their presence on both of these gene lists, targeted analysis of the RNA-Seq data revealed that these genes were induced by the combination of DEX and IL-7 relative to treatment with DEX or IL-7 alone only in the scrambled control clones but not in the STAT5A/B-KO clones ( ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 3 caption a7 caption a8 STAT5B, but not STAT5A, mediates the upregulation of BCL-2 expression in cells exposed to the combination of DEX and IL-7. ( A ) Evaluation of STAT5A and STAT5B expression by Western blotting in independent scrambled control (S1–S4) and STAT5 single-KO (A1–A4 or B1–B4) and double-KO (AB1–AB4) CCRF-CEM cell clones ( n = 4 per genotype). ( B ) Viability of independent scrambled control and STAT5-KO CCRF-CEM cell clones ( n = 4 per genotype) treated with 100 nM DEX with or without 25 ng/mL IL-7 for 72 hours. ( C ) Venn diagram depicting the overlap between the top differentially expressed genes among scrambled control CCRF-CEM cell clones ( n = 4) treated with DEX versus DEX plus IL-7 and STAT5B target genes. ( D and E ) Fold change in the fragments per kilobase per million mapped reads (FPKM) values for ( D ) BCL2 transcript and ( E ) ARHGEF3 transcript levels as determined by RNA-Seq analysis of scrambled control cells ( n = 4) and STAT5A/B double-KO ( n = 4) CCRF-CEM cell clones treated in the absence or presence of 100 ng/mL IL-7 and/or 1 μM DEX for 16 hours. ( F ) ΔMFI of BCL-2 protein expression in scrambled control ( n = 4) and STAT5-KO ( n = 4) CCRF-CEM cell clones treated with 100 ng/mL IL-7 and 1 μM DEX relative to 100 ng/mL IL-7 alone for 48 hours. ( G ) BCL2L11 and BCL2 transcript expression in CCRF-CEM cells cultured in the absence or presence of 1 μM DEX, 100 ng/mL IL-7, and/or 10 μg/mL CHX for 16 hours as determined by qPCR performed in technical triplicate.

Techniques: Expressing, Control