Homoharringtonine inhibits the NOTCH/MYC pathway and exhibits antitumor effects in T-cell acute lymphoblastic leukemia

Abstract

We report on the antileukemic activity of homoharringtonine (HHT) in T-cell acute lymphoblastic leukemia (T-ALL). We showed that HHT inhibited the NOTCH/MYC pathway and induced significantly longer survival in mouse and patient-derived T-ALL xenograft models, supporting HHT as a promising agent for T-ALL.

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. Jurkat^Luc+ 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 2.

HHT exhibits antileukemic activity by inhibiting the NOTCH1/MYC pathway. (A) RNA sequencing analysis was performed on Jurkat cells treated with PBS or HHT (10 or 15 ng/mL) for 24 hours. Normalized enrichment scores (NES) of the top 6 gene sets downregulated by HHT treatment were identified through integrated analysis. (B) Jurkat cells (left) and blasts from patient with T-ALL (right, T-ALL 003) were treated with increasing concentrations of HHT for 24 hours, followed by western blot analysis for NOTCH1, c-MYC, MCL1, and BCL-2 levels with β-actin as the loading control. (C-K) Experimental design and results of the Notch1–induced T-ALL model. (C) Schematic experimental design. BM cells (2 × 10⁵ cells per mouse) from the Notch1–induced T-ALL mice (B6-Ly5.2) were transplanted into congenic recipient mice (B6-Ly5.1, n = 7 mice per group), and 10 days after transplantation these mice were treated with vehicle (PBS) or HHT (1 mg/kg body weight). (D) White blood cell (WBC) counts; (E) percentages of leukemic blasts in PB and representative PB smears; (F) spleen size, weight, and percentages of leukemic blasts; (G) representative BM smears and femur hematoxylin and eosin (H&E) staining; (H) percentages of blasts in BM; (I) mRNA expression levels of Notch1 in BM cells; (J) western blot analysis of NOTCH1; c-MYC, and MCL1 in BM cells, with β-actin as the loading control; and (K) survival of the HHT-treated (n = 9) vs PBS-treated (n = 10) mice 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 ± SEM. Significance values: ns, not significant.