Role Of Homoharringtonine Combined With Cladribine And Aclarubicin In Acute Myeloid Leukemia

Objective: To investigate the clinical efficacy and mechanism of homoharringtonine combined with cladribine and aclarubicin(HCA)regimen in the treatment of children with acute myeloid leukemia(AML).Methods:1.The clinical data of 5 children with refractory AML treated with HCA regimen from June 2019 to December 2019 were collected to analyze the complete remission rate,adverse reactions and disease-free survival of children.2.CCK8 was used to determine the effects of cladribine,homoharringtonine and aclarubicin,alone or in combination,on the proliferation of HL60 and THP1 cells.3.CCK8 was used to determine the effects of HCA,alone or in combination with apoptosis inhibitor,necroptosis inhibitor,ferroptosis inhibitor or autophagy inhibitor,on the proliferation of HL60 and THP1 cells,and screen for possible death pathways that HCA-mediated in AML cells.4.The pathway of HCA-mediated in AML cells was further verified by Hoechst/PI staining,flow cytometry and Western blot.Results:1.Five children with refractory AML were treated with the HCA regimen for two cycles,4 of 5 achieved a complete remission(CR),and another achieved a CR with incomplete hematological recovery(CRi).Adverse reactions mainly included myelosuppression,febrile neutropenia,pneumonia,sepsis,intestinal infection and no treatment-related deaths.After CR,3 children underwent hematopoietic stem cell transplantation,and 2 only received consolidation therapy.As of the latest follow-up,all5 patients were in disease-free survival(DFS),ranging from 23 to 28 months.2.The proliferation of HL60 and THP1 cells treated with homoharringtonine,cladribine and aclarubicin alone for 24 h or 48 h could be inhibited in a time-and dose-dependent manner.Homoharringtonine,cladribine combined with aclarubicin had higher growth inhibiton rate to single or both drugs in combination.When drugs were combined,the combination index(CI)value was < 1,suggerting a synergistic inhibitory effect between drugs.3.The inhibitors of apoptosis and necroptosis,but not ferroptosis and autophagy,could suppress cell death induced by HCA,suggesting ferroptosis and autophagy independent mechanism of cell death.4.Hoechst/PI staining and flow cytometry results showed that apoptosis inhibitor itself did no effect on apoptosis.The number of apoptotic cells was significantly higher in the HCA group as compared with control.Furthermore,the number of apoptotic cells was decreased when apoptosis inhibitor was added.Western blot results showed that compared with the control group,PARP,Caspase-3 and Caspase-8proteins were activated and cleaved in the HCA group,while the expression of Bax was up-regulate and Bcl-2 was down-regulated.The expressions of apoptosis related proteins could be reversed by apoptosis inhibition.The results suggested that HCA could induce apoptosis in AML cells.5.Western blot results showed that compared with the control group,the expressions of necroptosis related proteins RIP1,RIP3 and MLKL were down-regulated in the HCA group,but were not phosphorylated.Compared with the HCA group,the apoptosis inhibitor showed no significant change in the expression of necroptosis related proteins,while the necroptosis inhibitor could increase the expression of RIP1,and had no significant changes in RIP3 and MLKL,and none of them was phosphorylated.These results suggested that HCA did not promote AML cell death through necroptosis.Conclusions:1.HCA as a new regimen of conventional drugs were a high safety and efficacy treatment in refractory AML.2.Homoharringtonine combined with cladribine and aclarubicin exhibit synergistic growth inhibition of AML cells and induce cell death mainly through apoptosis.