Homoharringtonine Exhibits Anti-Tumor Effect in T-Cell Acute Lymphoblastic Leukemia By Targeting Notch1/Myc Pathway

Abstract

T cell acute lymphoblastic leukemia (T-ALL) is a highly proliferative hematologic malignancy, mainly mediated by NOTCH/MYC signaling. Therefore, new therapies targeting NOTCH/MYC pathway are urgently required. Homoharringtonine (HHT), a potent inhibitor of translation elongation, has been used in China for many years for the treatment of myeloid leukemia and was approved in 2012 by the US Food and Drug Administration for the treatment of chronic myeloid leukemia with resistance to tyrosine kinase inhibitors. However, little is known about its efficacy in T-ALL. Herein, we examined the therapeutic role of HHT in T-ALL using mouse model and patient cells. First, we treated T-ALL cell lines (i.e., Jurkat, Molt4 and CCRF-CEM) and patient samples with HHT in vitro. After 24-hour(h) treatment, we observed very low IC50 values (5-20ng/mL) in all three T-ALL cell lines, suggesting highly sensitive to HHT treatment. HHT dramatically induced apoptosis and cell cycle arrest in G0/G1 phase in all three T-ALL cell lines. We observed similar results in human T-ALL cells treated with HHT for 24h, with much lower IC50 values (2-5ng/mL vs 30-110 ng/mL) in all three T-ALL patient samples compared to mononuclear cells from healthy donors, with increased apoptosis rates (89.2% vs 29.6%, p<0.001). Collectively, these results showed that T-ALL cell lines and patient samples exhibit high sensitivity to HHT in vitro. To evaluate the in vivo efficacy of HHT in T-ALL, we established a cohort of T-ALL mice by transplanting Luci+ Jurkat cells into NSG mice and then treated these mice with HHT (1mg/kg, ip) or vehicle (PBS) for 10 days, starting on day 9 post transplantation. Leukemia burden was compared by measuring Bioluminescence and engraftment at day 14 and day 19 post transplantation in HTT-treated and PBS-treated groups. We observed significantly decreased leukemia burden in HHT-treated mice compared to PBS-treated controls (median total flux: 796.3 vs 3701*106 at day 19, P=0.0001). HHT significantly prolonged the survival of the T-ALL mice as compared to PBS treatment (median survival: 29 vs 25 days, P=0.0003). T-ALL patient-derived-xenograft (PDX) model was also constructed and treated with HHT or PBS for 10 days. We observed significantly reduced human T-ALL engraftment rates (14.4-25.3% vs 37.8-65.3% at day 10 post transplantation, p<0.0001) and prolonged survival (median survival: 54 vs 45 days, P=0.0098) in the HHT-treated PDX compared to PBS-treated PDX, suggesting an in vivo anti-leukemia effect of HHT in T-ALL. Next, we performed RNA-seq of T-ALL cells with or without HHT treatment to identify downstream pathways that are regulated by HHT. Through gene set enrichment analysis (GSEA), we found that Myc pathway and Notch pathway were among the top 6 upregulated gene sets. Further, using western-blot analysis, we showed that all the expression levels of Notch1, c-MYC and MCL-1 were decreasing in T-ALL cell lines and patient samples after exposing to increased concentrations of HHT, suggesting that they were all down-regulated by HHT. Interestingly, we observed that a much lower concentration of HHT is needed to down-regulate Notch 1 (4ng/mL) than that needed to down-regulate MCL1(8-16ng/mL). Similar results were found in T-ALL human samples. Taken together, these results suggest that HHT could attenuate NOTCH 1 and MYC expression in T-ALL cells. Further, we used the Notch-1 induced T-ALL mouse model to verify the effect of HHT on Notch pathway and test its in vivo anti-ALL efficacy. Strikingly, we found that HHT treatment significantly reduced white blood cell (WBC) counts (0.82-1.68 *109/L vs 9.73-17.46*109/L, p<0.0001) and T-ALL burden (0.0-0.8% vs 7.9-57.1%, p=0.0003) and significantly prolonged survival (median survival: not reached vs 31 days, p=0.0007) as compared to PBS treatment. Interestingly, 60% (six of ten) HHT-treated T-ALL mice versus 0% of PBS-treated mice, obtained long-term survival, free of T-ALL after monitoring for 75 days, suggesting that these mice likely are cured by HHT monotherapy. QPCR and western-blot analysis using bone marrow cells of the treated mice showed that HHT dramatically reduced both the mRNA and protein levels of NOTCH1 compared to the control group. In summary, we showed here that HHT, approved by the FDA for CML treatment, exhibited in vivo therapeutic efficacy in T-ALL, by inhibiting the NOTCH-MYC pathway. Our data support to explore HHT as a novel therapy for T-ALL in the clinic.