Effects Of Novel Hybrid NL-101 In Combination With Traditional Chemotherapy Drugs On Acute Myeloid Leukemia And The Mechanisms

Background:Acute myeloid leukemia(AML)is a highly invasive malignant hematologic disease with high mortality and recurrence rate.Currently the pathogenesis of AML has not been fully explained.Chemotherapy is still the main treatment method for AML.Drug resistance and treatment-related toxicity are main reasons for the treatment failure in AML patients.Therefore,the research and application of novel agents are required to enhance tumor sensitivity and improve the survival and prognosis of AML patients.NL-101 is a novel hybrid,which is derived from bendamustine with the side chain replaced by the hydroxamic acid of histone deacetylase inhibitor vorinostat.Previous studies have demonstrated its efficacy in AML,but clinical efficacy and potential toxicity of NL-101 combined with other drugs is not defined.Moreover,daunorubicin and cytarabine are the most common chemotherapy drugs in clinic.In our study,we aim to demonstrate the efficacy and safety of NL-101 combined with the conventional chemotherapy drugs daunorubicin and cytarabine in the treatment of AML,and to explore the mechanisms of synergistic effect.The combination of NL-101 and daunorubicin or cytarabine may provide experimental basis for new treatment method of leukemia.Materials and Methods:(1)MTS assay was used to detect the proliferation inhibition caused by different concentrations of NL-101 on AML cell lines and primary AML cells for 48 h.(2)NL-101 was used alone or in combination with daunorubicin or cytarabine in AML cell lines and AML primary cells for 48 h.MTS assay was used to detect the proliferation inhibition and the combination index was calculated.(3)Apoptosis of MV4-11 and HL-60 cells after exposure into NL-101 in combination with daunorubicin or cytarabine for 48 h was conducted by flow cytometry.(4)AML xenograft was developed by tail vein injection of MV4-11-Luc to NSG mice and all of them would be divided into 4 groups randomly.Tumor burden was monitored by fluorescence imaging every 7 days.Mice were treated with correspondingly drug after the onset of the disease.When lower extremities were paralysis,mice would be euthanized.The bone marrow cells were collected and the expression of human-CD45 antibody was detected.The survival time should be recorded and analyzed.(5)The expression of apoptosis related proteins and DNA damage related proteins were tested by western-blot after MV4-11 and HL-60 were treated with NL-101 in combination with daunorubicin or cytarabine for 48 h.(6)Statistical analysis,independent sample t test was used to compare the single variable group,and Kaplan-Meier method was used to survival analysis.In the above statistical analysis,P<0.05 was considered to be statistically significant.Results(l)NL-101 could inhibit the growth of AML cell lines and AML primary cells in a dose-dependent manner,and different cells had different sensitivity.(2)NL-101 in combination with daunorubicin or cytarabine could synergistically inhibit the growth of AML cell lines and AML primary cells.(3)NL-101 in combination with daunorubicin or cytarabine acted on MV4-11,HL-60 cells for 48 h had apoptosis-promoting effect in dose-dependent manner.Apoptosis in combination group was significantly higher than that in monotherapy group(p<0.05).(4)NL-101 in combination with daunorubicin significantly(p<0.001)reduced tumor burden and prolonged the survival time in AML xenograft.(5)In AML cell lines MV4-11 and HL-60,a visibly increase in cleavage of Caspase-3,-7 and PARP was induced by NL-101 in combination with daunorubicin or cytarabine,and up-regulation of BAD and BIM were observed in the combination group of NL-101 and daunorubicin.(6)The DNA double strand break marker proteins y-H2AX and DNA damage related proteins P-ATM,P-ATR,P-CHK1 and P-CHK2 were obviously up-regulation in combination group.ConclusionIn this study,we found that NL-101 had a proliferation inhibitory effect on AML cell lines as well as primary cells in a concentration-dependent manner.NL-101 in combination with daunorubicin or cytarabine could synergistically inhibitor the growth and promote the apoptosis of AML cells.In vivo,our experiments confirmed that NL-101 in combination with daunorubicin could synergistically reduce the tumor burden and prolong the survival time of AML mice.Furthermore,NL-101 in combination with daunorubicin or cytarabine enhances apoptosis by promote DNA damage,thereby achieved a lethal effect on AML.Altogether,our study found NL-101 in combination with daunorubicin or cytarabine could synergistically anti-AML,and identified the mechanism of the combination regimen involving enhance DNA damage to induce the apoptosis and then kill the cells.