HHT showed in vitro and in vivo antileukemic activity in T-ALL. (A) T-ALL cell lines, including the ETP-ALL cell line Loucy, were treated with HHT for 24 hours. Cell viability was assessed using the CellTiter-Glo, and IC50 at 24 hours was calculated (left). The values were normalized to the average of the untreated samples for each cell line. Western blot analysis was conducted to assess NOTCH1, c-MYC, MCL1, and BCL-2 levels in T-ALL cell lines with β-actin as the loading control (right). (B) Cell viability of primary blasts of patients with T-ALL treated with HHT for 24 hours was assessed using CellTiter-Glo. The IC50 at 24 hours was calculated (left). Western blot analysis of NOTCH1, c-MYC, MCL1, and BCL-2 levels was performed in samples of patients with T-ALL with β-actin as the loading control (right). (C-G) Experimental design and results of the cell line-derived xenograft (CDX) model. (C) Schematic experimental design. JurkatLuc+ T-ALL cells (2 × 106 cells per mouse) were IV injected into NSG mice. Eight days later, these mice (n = 7 for each group) were treated with vehicle (PBS) or HHT (1 mg/kg body weight) for 10 days. (D-E) Leukemic burden was determined by luminescence imaging on day 8 (before treatment), day 14 (5 days after treatment), and day 19 (10 days after treatment). (F-G) Percentages of blasts in peripheral blood (PB) (F) and survival (G) of the above 2 groups of mice are shown. (H-J) Experimental design and results of the PDX models. (H) Schematic experimental design of the PDX models. Two PDXs were generated by transplanting blasts from 1 patient with T-ALL or from 1 patient with ETP-ALL (1 × 106 cells per mouse) IV into NSG mice. Two weeks later, these mice were treated with vehicle (PBS) or HHT (1 mg/kg body weight) for 10 to 12 days. (I-J) The percentage of blasts in the PB and survival of the T-ALL PDX (I; n = 9 mice for each group) and ETP-ALL PDX (J; n = 10 mice for each group) models are shown. For the western blot (WB) analysis in panels A-B, 1 of 3 independent experiments with similar results are shown. Statistical analysis was conducted using the 2-tailed, unpaired Student t test. Survival was analyzed using the log-rank test. Results shown represent the mean ± standard error of mean (SEM). Significance values: ns, not significant.
Figure 1.

HHT showed in vitro and in vivo antileukemic activity in T-ALL. (A) T-ALL cell lines, including the ETP-ALL cell line Loucy, were treated with HHT for 24 hours. Cell viability was assessed using the CellTiter-Glo, and IC50 at 24 hours was calculated (left). The values were normalized to the average of the untreated samples for each cell line. Western blot analysis was conducted to assess NOTCH1, c-MYC, MCL1, and BCL-2 levels in T-ALL cell lines with β-actin as the loading control (right). (B) Cell viability of primary blasts of patients with T-ALL treated with HHT for 24 hours was assessed using CellTiter-Glo. The IC50 at 24 hours was calculated (left). Western blot analysis of NOTCH1, c-MYC, MCL1, and BCL-2 levels was performed in samples of patients with T-ALL with β-actin as the loading control (right). (C-G) Experimental design and results of the cell line-derived xenograft (CDX) model. (C) Schematic experimental design. JurkatLuc+ T-ALL cells (2 × 106 cells per mouse) were IV injected into NSG mice. Eight days later, these mice (n = 7 for each group) were treated with vehicle (PBS) or HHT (1 mg/kg body weight) for 10 days. (D-E) Leukemic burden was determined by luminescence imaging on day 8 (before treatment), day 14 (5 days after treatment), and day 19 (10 days after treatment). (F-G) Percentages of blasts in peripheral blood (PB) (F) and survival (G) of the above 2 groups of mice are shown. (H-J) Experimental design and results of the PDX models. (H) Schematic experimental design of the PDX models. Two PDXs were generated by transplanting blasts from 1 patient with T-ALL or from 1 patient with ETP-ALL (1 × 106 cells per mouse) IV into NSG mice. Two weeks later, these mice were treated with vehicle (PBS) or HHT (1 mg/kg body weight) for 10 to 12 days. (I-J) The percentage of blasts in the PB and survival of the T-ALL PDX (I; n = 9 mice for each group) and ETP-ALL PDX (J; n = 10 mice for each group) models are shown. For the western blot (WB) analysis in panels A-B, 1 of 3 independent experiments with similar results are shown. Statistical analysis was conducted using the 2-tailed, unpaired Student t test. Survival was analyzed using the log-rank test. Results shown represent the mean ± standard error of mean (SEM). Significance values: ns, not significant.

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