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.
