Homoharringtonine Synergizes Strongly with Venetoclax Against the Early T-Cell Progenitor Acute Lymphoblastic Leukemia：from Bench to Bed

Early T cell precursor acute lymphoblastic leukemia (ETP-ALL) is a distinct subtype of T-ALL with poor outcome and novel therapies are urgently needed. Venetoclax (VEN), a BCL2 inhibitor, has been approved by FDA for AML. Combination of VEN and demethylating drugs or targeted therapies such as MCL1 inhibitor showed promising effect in AML. Loucy, an ETP-ALL cell line, was also reported high expression of BCL2. But little is known about VEN combination treatment in ETP-ALL. Our data showed that homoharringtonine (HHT), a plant alkaloid, downregulated MCL1 in T-ALL. Herein, we examined the treatment effect of VEN combined with HHT on ETP-ALL using murine model and patient samples. First, we compared BCL2 levels and treatment effect of VEN alone on ETP-ALL (i.e., Loucy) and T-ALL (i.e., Jurkat, Molt4, and CCRF-CEM) cell lines. We observed higher levels of BCL2 and lower IC50 values (IC50 at 24h: 0.093 uM vs.>1uM) in Loucy vs T-ALL cells. Similar results were found in human samples, with IC50 of 0.539uM in ETP-ALL sample vs 3.165-23.25uM in T-ALL samples treated with VEN for 24h. Next, to determine whether the combination of VEN and HHT shows a synergistic effect on ETP-ALL, Loucy cells were treated with a serial concentration of VEN (range: 0.025-0.1uM) and HHT (range:10-40ng/mL) alone and in combination for 24h. VEN and HHT combination showed significant synergism in reducing cell viability, with a mean combination index (CI) of < 0.1. We observed similar results in patient samples, with mean CI lower than 0.01 indicating highly synergistic effect. To assess this in vivo, a cohort of ETP-ALL mice generated by transplanting Loucy cells into NSG mice were randomly divided into 4 groups and treated with vehicle, HHT (1 mg/kg, ip), VEN (25 mg/kg, oral gavage) or HHT+VEN combination. HHT and VEN combination significantly prolonged survival of the ETP-ALL mice compared to either drug alone or vehicle (median survival of combination vs VEN vs HHT vs control: 113.5 vs 95.5 vs 85.5 vs 69.0 days, p<0.01), suggesting a synergistic effect of HHT and VEN in vivo. To identify the mechanical basis for the synergy of VEN and HHT in ETP-ALL, we performed single-cell RNA-seq using primary patient ETP-ALL cells treated with vehicle, HHT, VEN or their combination for 24h. Based on the gene expression profile, we identified a cluster of ETP-ALL cells sensitive to HHT , and a cluster only sensitive to combination of VEN and HHT which showed higher levels of Bcl2 and Mcl1 and upregulated anti-apoptosis signal by gene set enrichment analysis (GSEA), suggesting intra-tumoral heterogeneity of ETP-ALL blasts. These results suggest that HHT or VEN alone is not sufficient to induce apoptosis in the resistant cells and combination of HHT and VEN could induce apoptosis by reducing MCL1 expression in these cells which was confirmed by western blot. Further, with the promising results from bench work and given that both HHT and VEN are approved anti-leukemia drugs, we translated these findings into clinic. The HAG (HHT, low dose Cytarabine, G-CSF priming) regimen has been widely used in China for the treatment of AML for 15 years and proved to be effective and safer than intensive chemotherapy. Based on above findings, with informed consent from patients, we applied venetoclax plus HAG(V-HAG) regimen to 3 relapsed/refractory(R/R) ETP-ALL patients and 4 newly diagnosed (ND) patients, including an 82-year-old refractory patient. CR/CRi rates after the first cycle treatment were 100%, which was significantly higher than the current induction therapy (CR rate ranging from 33.3-55.9% after the first induction cycle). Except for the 82-year-old patient who refused post-remission treatment, all the remaining 6 patients remained in remission until today. Of note, no clinical manifestations of tumor lysis syndrome were observed. The median time to absolute neutrophil count recovery of >500/μL and >1000/μL was 13 (range: 0-22) and 16 (range: 0-24) days, and to platelet count recovery of > 20,000/ μL was 16 (range: 0-21) days, indicating this regimen was relatively safe and well tolerated. In conclusion, our findings demonstrate that HHT and VEN showed synergistic effect on ETP-ALL and HHT/VEN based therapy is a promising and safe regimen in newly diagnosed and R/R ETP-ALL patients. With these promising results from bench to bedside, 2 multi-center phase Ib/II clinical trials using V-HAG in patients with R/R ETP-ALL and ND-ETP have been initiated at our institution.