The Clinical Significance Of PARP-1 And The Research Of PARP-1 Inhibitor In Acute Myeloid Leukemia

BackgroundAcute myeloid leukemia(AML)is a group of highly heterogeneous hematopoietic stem and progenitor cells caused by acquired and incidental genetic alterations,accounting for approximately 70%of adult acute leukemia.With the in-depth development of basic research work and the improvement of clinical treatment methods,the prognosis of AML has been significantly improved.However,the five-year survival rate is still only 10%-30%.At present,the pathogenesis of AML is not completely clear,and the treatment for patients with refractory recurrence and elderly patients still needs to be continuously explored and improved.DNA damage response(DDR)and repair in hematological malignancies is not fully understood.Poly(ADP-ribose)polymerase 1(PARP-1)is one of the most important genes in DNA damage repair.It is reported that PARP-1 is highly expressed in AML,and another important gene of DNA damage repair,BRCA1,is underexpressed in AML.When a therapeutic drug causes DNA damage in leukemia cells,loss of repair function leads to accumulation of genomic changes,which ultimately leads to cell death.Therefore,PARP-1 inhibitors may be a promising and effective therapeutic drug for AML.ObjectiveThe bone marrow samples from healthy people and cytogenetically normal AML(CN-AML)patients were collected.The expression of PARP-1 mRNA was detected by q-PCR.The significance of PARP-1 on clinical prognosis was analyzed according to the expression of PARP-1.To investigate the inhibitory effect of PARP-1 inhibitor BMN673 and BMN673 combined with SAHA-bendamustine hybrid NL101 on AML cells,and explore the mechanism of action of the combination in vitro.The safety and effectiveness of the combination treatment through in vivo study was explored.In addition,the effect and mechanism of PARP-1 inhibitor BMN673 combined with HHT on FLT3-ITD mutant AML was also explored.Methods1.Bone marrow blood from AML patients and healthy people was collceded and mononuclear cells were isolated.RNA was extracted to detect PARP-1 mRNA expression.The clinical data of the patients were collected including the time of initial diagnosis of the disease,gender,age,white blood cell(WBC)and platelet(PLT)count,hemoglobin(HB),proportion of bone marrow(BM)blasts,karyotype,type of gene mutation,treatment method,therapeutic effect,survival time,etc.Patients were grouped according to the expression level of PARP-1,and the survival time curves were plotted and compared using the Kaplan-Meier method and the log-rank test.A multivariate Cox proportional hazards model was used to identify independent outcome predictors after adjusting for confounding factors.2.PARP-1 inhibitor BMN673 and SAHA-bendamustine hybrid NL101 were treated alone or in combination in AML cell lines and patient primary cells to detect growth inhibition,calculate the combination index(CI)values,and detect apoptosis and cell cycle by flow cytometry.The cells treated with single and combination group were collected,and the protein levels of apoptosis pathway,cell cycle and DNA damage response pathway were verified by q-PCR,Western blot and immunofluorescence.3.In vivo study of BMN673 in combination with NL101 on AML animal models.Injection of MV4-11-luc or MOLM-13 cells into NSG mice via the tail vein.Cell engraftment was assessed by intraperitoneal injection of luciferin followed by imaging using IVIS Lumina LT system.Mice were treated with either BMN673,NL101,in combination at indicated concentrations,or vehicle.The tumor burden in mice was measured by fluorescence imaging every week,and the body weight of the mice was recorded every three day.The mice were sacrificed when the mice were paralyzed in both lower limbs.Bone marrow blood cells were collected for detection of huamn-CD45 expression,and the survival time of each group was recorded for survival analysis.HE staining was used to detect the degree of spleen tumor cell infiltration in mice.4.BMN673 combined with HHT alone or in combination treated on FLT3-ITD mutant and non-mutant acute myeloid leukemia cell lines and primary patient cells,detection of growth inhibition,calculation the combination index(CI)values and detection apoptosis by flow cytometry.The cells treated with the single and the combination group were collected,and the apoptosis-related protein,DNA damage-related pathway and FLT3 kinase-related protein were detected by Western blot and immunofluorescence.Results1.The expression level of PARP-1 in 339 CN-AML patients and AML cell lines were higher than that in the normal group.The overall survival(OS)and event free survival(EFS)of patients with high PARP-1 expression were shorter than those of the low expression group(Median survival OS:646 vs 374;EFS:425 vs 240).High expression of PARP-1 is an independent risk factor for prognosis of AML(HR:OS:1.949(1.384,2.745),P<0.001;EFS:1.822(1.323,2.509),P<0.001).2.The peripheral WBC(17.00×109/L[3.60×109-87.20×109]vs 9.65×109/L[2.42×109-38.60×109],P=0.008)and the bone marrow blast cells(72.00%[51.50-85.00]vs 63.00%[37.00-78.38],P=0.003)in the high PARP-1 expression group were higher than those in the low expression group.The frequency of FLT3-ITD mutations(28.2%vs 17.3%,P=0.031)in the PARP-1 high expression group was higher than in the low expression group.3.PARP-1 inhibitor BMN673 combined with NL101 significantly inhibited the growth of leukemia cells.The combination group significantly caused cell cycle arrest at G2/M phase,and up-regulated the CDK inhibitor protein P21 and increased G2/M regulatory molecules,cyclin B1 and p-CDC2(Tyr-15).Furthermore,the combination resulted in a significant increase of apoptosis and an increase in the active form of Caspase-3(Cleaved Caspase3)and inactive form of PARP-1(Cleaved PARP-1)compare to BMN673 or NL101.4.BMN673 combined with NL101 significantly increased the expression of y-H2AX in leukemia cells,increased levels of the DNA damage markers P-ATM,P-CHK1,P-CHK2,and inhibited the expression of DNA repair protein poly(ADP)ribosylation(PAR).5.BMN673 combined with NL101 significantly reduced the burden of leukemia cells in AML mice,prolonged the survival time,reduced hCD45 in mouse bone marrow and the infiltration of leukemia cells in mouse spleen.6.BMN673 combined with HHT synergistically inhibited the growth of FLT3-ITD mutant AML cells,promoted apoptosis,increased DNA damage and inhibited FLT3 and P-FLT3 kinase.ConclusionIn this study,we found PARP-1 expression was significantly increased in CN-AML patients.Patients with high PARP-1 expression had higher levels of blast cells in bone marrow and WBC in peripheral blood,and was associated with a more frequent of FLT3-ITD mutation.The OS and EFS of the high expression group were significantly shorter than those in the low expression group.High PARP-1 expression predicted poor survival in CN-AML patients.BMN673 combined with NL101 had a strong synergistic effect in treating AML.The combination significantly induced cell apoptosis and arrested cell cycle in G2/M phase.Mechanistically,BMN673 and NL101 combinatorial treatment promoted DNA damage.In vivo,the combination effectively delayed the development of AML and prolonged survival.In addition,we also found that BMN673 combined with HHT synergistically inhibited FLT3-ITD mutant AML.