Figure 2.
Constitutively active STAT5 promotes T-ALL progression and maintenance. (A) Schematic overview of the conditional and inducible STAT5B1∗6–targeted ROSA26 locus. Cre-mediated removal of the conditional NeoR STOP cassette leads to ROSA26-based reverse tetracycline-transactivator 3 (rtTA3) expression and availability of the tetracycline response element (TRE). Doxycycline (DOX) administration leads to inducible expression of the FLAG-tagged STAT5B1∗6 oncogene, which can be monitored by mCherry reporter expression. (B) Representative flow cytometric analysis of mCherry expression in DN3a thymocytes from 6-week-old CD2-rtTA3-STAT5B1∗6 (Control) and CD2-rtTA3-STAT5B1∗6; Lmo2Tg (Lmo2Tg) mice maintained or not on doxycycline-enriched diet (DOX; red). MFI ± SEM, 2-way ANOVA with Tukey correction test; ∗∗P < .01, ∗∗∗P < .001 compared with untreated (gray). (C) Kaplan-Meier curves of the time to leukemia for WT, CD2-rtTA3-STAT5B1∗6, Lmo2Tg, and CD2-rtTA3-STAT5B1∗6; Lmo2Tg mice maintained on DOX-enriched diet (). Log-rank (Mantel-Cox) test; ∗∗P < .01 as compared with Lmo2Tg littermates. All malignant thymic tumors were diagnosed at necropsy median time to leukemia (days) is indicated. All malignant thymic tumors were diagnosed at necropsy. (D-E) Levels of the FLAG-tagged STAT5B1∗6 oncoprotein, phosphorylated STAT5 (pSTAT5), and total STAT5 (D), as well as proportion of T-cell populations (%; E), in T-ALL cells harvested from the thymus of Lmo2Tg and CD2-rtTA3-STAT5B1∗6; Lmo2Tg mice at the onset of disease. Actin was used as a loading control. Immature CD4−CD8− (DN), CD4+CD8+ DP, and mature CD4+CD8− and CD4−CD8+ SP populations are indicated. Mean ± SEM, 2-way ANOVA with Tukey correction test; ∗P < .05 compared with mice maintained on standard diet. (F) Flow cytometry analysis and schematic representation of the transplantation strategy of primary STAT5B1∗6–expressing Lmo2Tg (Cd45.2+) leukemia cells into sublethally irradiated Cd45.1+ recipients maintained or not on DOX-enriched diet. (G) Expression of the FLAG-tagged STAT5B1∗6 oncoprotein and resulting pSTAT5 in leukemic cells from the thymus of 3 recipients per cohort was confirmed by western blot. Actin was used as a loading control. (H) Kaplan-Meier curves of the time to leukemia for recipients transplanted with primary CD2-rtTA3-STAT5B1∗6; Lmo2Tg T-ALL 580, with leukemia growth delay (LGD; days) indicated between cohorts of mice maintained (maintenance; dark red) or not (withdrawal; light red) on DOX-enriched feed. Log-rank (Mantel-Cox) test (N = 6 per cohort); ∗∗P < .01 as compared with maintenance recipients. IRES: internal ribosome entry site; LUC: luciferase; PURO: puromycin resistance sequence; T2A: Thosea asigna virus 2A self-cleaving peptide sequence.

Constitutively active STAT5 promotes T-ALL progression and maintenance. (A) Schematic overview of the conditional and inducible STAT5B1∗6–targeted ROSA26 locus. Cre-mediated removal of the conditional NeoR STOP cassette leads to ROSA26-based reverse tetracycline-transactivator 3 (rtTA3) expression and availability of the tetracycline response element (TRE). Doxycycline (DOX) administration leads to inducible expression of the FLAG-tagged STAT5B1∗6 oncogene, which can be monitored by mCherry reporter expression. (B) Representative flow cytometric analysis of mCherry expression in DN3a thymocytes from 6-week-old CD2-rtTA3-STAT5B1∗6 (Control) and CD2-rtTA3-STAT5B1∗6; Lmo2Tg (Lmo2Tg) mice maintained or not on doxycycline-enriched diet (DOX; red). MFI ± SEM, 2-way ANOVA with Tukey correction test; ∗∗P < .01, ∗∗∗P < .001 compared with untreated (gray). (C) Kaplan-Meier curves of the time to leukemia for WT, CD2-rtTA3-STAT5B1∗6, Lmo2Tg, and CD2-rtTA3-STAT5B1∗6; Lmo2Tg mice maintained on DOX-enriched diet (). Log-rank (Mantel-Cox) test; ∗∗P < .01 as compared with Lmo2Tg littermates. All malignant thymic tumors were diagnosed at necropsy median time to leukemia (days) is indicated. All malignant thymic tumors were diagnosed at necropsy. (D-E) Levels of the FLAG-tagged STAT5B1∗6 oncoprotein, phosphorylated STAT5 (pSTAT5), and total STAT5 (D), as well as proportion of T-cell populations (%; E), in T-ALL cells harvested from the thymus of Lmo2Tg and CD2-rtTA3-STAT5B1∗6; Lmo2Tg mice at the onset of disease. Actin was used as a loading control. Immature CD4−CD8− (DN), CD4+CD8+ DP, and mature CD4+CD8− and CD4−CD8+ SP populations are indicated. Mean ± SEM, 2-way ANOVA with Tukey correction test; ∗P < .05 compared with mice maintained on standard diet. (F) Flow cytometry analysis and schematic representation of the transplantation strategy of primary STAT5B1∗6–expressing Lmo2Tg (Cd45.2+) leukemia cells into sublethally irradiated Cd45.1+ recipients maintained or not on DOX-enriched diet. (G) Expression of the FLAG-tagged STAT5B1∗6 oncoprotein and resulting pSTAT5 in leukemic cells from the thymus of 3 recipients per cohort was confirmed by western blot. Actin was used as a loading control. (H) Kaplan-Meier curves of the time to leukemia for recipients transplanted with primary CD2-rtTA3-STAT5B1∗6; Lmo2Tg T-ALL 580, with leukemia growth delay (LGD; days) indicated between cohorts of mice maintained (maintenance; dark red) or not (withdrawal; light red) on DOX-enriched feed. Log-rank (Mantel-Cox) test (N = 6 per cohort); ∗∗P < .01 as compared with maintenance recipients. IRES: internal ribosome entry site; LUC: luciferase; PURO: puromycin resistance sequence; T2A: Thosea asigna virus 2A self-cleaving peptide sequence.

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