Study Of TGF-β1 Regulating Bone Marrow Microenvironment Mediated Drug Resistance In FLT3-ITD Mutated AML

BackgroundAcute myeloid leukemia(AML)is a common hematological malignancy.About 30%of newly diagnosed AML patients have FMS like tyrosine kinase 3(FLT3)mutations,including the internal tandem repeat mutation(1TD)of FLT3 proximal membrane region and the point mutation(TKD)at the "activation ring".At present,chemotherapy is the main treatment of AML,induction chemotherapy can make about 80%of the initial patients get complete remission(CR).However,most of the patients relapsed after remission and eventually developed into refractory leukemia.The reason may be closely related to AML drug resistance.AML drug resistance is not only related to the biological characteristics of leukemia cells,but also related to the external environment of leukemia cells.It is found that FLT3-ITD mutation is not only involved in the development of leukemia,but also mediates chemotherapy resistance by inducing RUNX3.The traditional regimen for the treatment of FLT3-ITD mutation-positive(FLT3-ITD+)AML has low overall survival(OS)and high recurrence rate.High-intensity chemotherapy cannot benefit the patients with this subtype,and allogeneic hematopoietic stem cell transplantation(allo-HSCT)is also difficult to improve the prognosis of some patients.Midostaurin,a new type of FLT3 tyrosine kinase inhibitor(TKI),was approved by the US Food and Drug Administration(FDA)in 2017 for the treatment of FLT3 positive AML.Compared with the traditional chemotherapy,Mdostaurin combined chemotherapy can improve the OS of FLT3+AML patients for 4 years(44.3%to 51.4%),but it can not improve the CR and the OS of ITD(high or low)subgroup,and the effect is still limited.TKI combined with chemotherapy can clear FLT3-ITD+AML sensitive clone,but the emergence of drug-resistant clones under the selective pressure of chemotherapy drugs,the bypass activation of signaling pathways,the chemotherapy protection of bone marrow microenvironment and the survival of leukemia stem cells(LSC)are closely related to the drug resistance of leukemia recurrence.At present,it is believed that FLT3-ITD+AML resistance induced by bone marrow microenvironment and LSC protection are the root causes of MRD failure to be completely eliminated and leukemia recurrence.The mechanism is not completely clear.Therefore,there is no effective method to overcome microenvironment-mediated drug resistance.To study the mechanism of FLT3-ITD+ AML resistance regulated by bone marrow microenvironment,and to lay a theoretical foundation for targeted intervention to improve the efficacy.Transforming growth factor-β1(TGF-β1)is one of the important cytokines in bone marrow microenvironment,which plays an important role in the regulation of AML cells and LSC self-renewal and cell cycle.In many solid tumors,the abnormal expression of TGF-(β1 is closely related to the occurrence,metastasis and drug resistance of tumors.However,the role and mechanism of TGF-β1 in AML resistance are not clear.Research objective1.To detect the molecular biological changes of bone marrow-derived mesenchymal stem cells(MSC)from AML patients and analyze the correlation with gene changes in AML patients.2.To detect the expression of TGF-β1 in bone marrow fluid of healthy donors,primary AML and remission AML,analyze the differences of different stages and subtypes of AML,and explore the correlation with clinical efficacy.3.To study the role and mechanism of TGF-β1 in the induction of AML resistance by bone marrow microenvironment.Contents and Methods1.MSC culture and identification were performed in 30 healthy donors and 80 AML patients after initial diagnosis and remission2.Molecular biological changes of bone marrow MSC in 17 AML patients were detected,and their correlation with genetic changes in AML patients was analyzed3.The expression and subtype distribution of TGF-β1 in bone marrow fluid of healthy donors,primary patients and remission AML were detected by ELISA,and the relationship between clinical characteristics and chemotherapy effect of AML patients with TGF-β1 expression in bone marrow was analyzed by SPSS software4.The effect of bone marrow microenvironment on sorafenib sensitivity of FLT3-ITD positive acute myeloid leukemia cell line Molm13:in vitro,AML patient derived bone marrow MSC was co-cultured with Molm13 cells to simulate the bone marrow microenvironment in vitro.CCK8 was used to detect the effects of sorafenib on the proliferation of Molm13 cells.Flow cytometry was used to detect the apoptosis of Molm13 cells induced by sorafenib.To study the effect of bone marrow microenvironment on the sensitivity of FLT3-ITD positive AML cell to sorafenib.5.The effect of TGF-β1 on the sensitivity of sorafenib inFLT3-ITD positive AML cell Molm13 cells:TGF-β1 and TGF-β1 receptor inhibitor LY2109761 were added into the co-culture system and non co-culture system respectively.The proliferation was detected by CCK8,and apoptosis was detected by flow cytometry,and and to analyze the effect of TGF-β1 on sorafenib sensitivity of Molm13 cells.6.Statistical analysis:SPSS 23.0 and GraphPad Prism 8.0 software were used for data analysis.T-test was used for the comparison of measurement data between the two groups,and one-way analysis of variance(ANOVA)was used for the comparison of multi-group mean.P<0.05 was considered as statistically significant.Results:1.Bone marrow derived MSC was detected in 17 patients with primary AML.It was found that MSC had no AML specific molecular biological changes.2.TGF-β1 protein expression was detected in the normal control and AML patients:TGF-β1 level was significantly lower than that of the normal control group(P=0.0027).TGF-β1 level in remission AML patients was significantly higher than that in primary AML patients(P<0.0001).The expression of TGF-β1 protein in bone marrow fluid of patients with AML at low risk was significantly higher than that of patients with AML at high risk(P=0.0038).However,there were no statistical differences among subgroups with different clinical biological characteristics,such as gender,age,chromosomal karyotype,leukocyte count,difficulty in treating diseases,presence of FLT3-ITD mutation,presence of CEBPA mutation,and remission(The P values are 0.966;0.156;0.081;0.872;0.593;0.199;0.707;0.347).The expression level of TGF-β1 mRNA of MSC was detected by Q-PCR,and there was no statistical difference between the patients with initial AML and remission AML and the normal donors(P=0.6225).3.CCK-8 was used to detect the effect of sorafenib on the proliferation of Molm13 cells.The results showed that different concentrations of sorafenib could inhibit the growth of Molm13 cells.With the increase of drug dosage,the inhibition rate of cell proliferation increased,showing a significant dose-dependent relationship.Molm13 cells were treated with different concentrations of sorafenib for 24h,48h and 72h.Compared with non-co-culture,the sensitivity of Molm13 cells to sorafenib was decreased in the co-culture system(P=0.0013;P=0.0008;P=0.003).4.Effect of rhTGF-β1 on the drug sensitivity of sorafenib in Molm13 cells:The effect of rhTGF-β1 on the sorafenib sensitivity of Molm13 cells was detected by adding rhTGF-β1 to the non-co-culture and co-culture systems.It was found that rhTGF-β1 could increase the inhibitory effect of sorafenib on the proliferation of Molm13 cells(P=0.0305),the addition of rhTGF-β1 receptor antagonist LY2109761 can reverse the sensitization effect of TGF-β1(P=0.0444).Compared with non-co-culture,the increase of rhTGF-β1 sorafenib in the co-culture system had a more obvious inhibitory effect on the proliferation of Molm13(P=0.0104),and LY2109761 could also reverse this effect(P=0.0071).Flow cytometry showed that in non-co-culture and co-culture systems,rhTGF-β1 could improve the apoptotic effect of sorafenib on Molm 13 cells(P=0.0031).Conclusion1.AML patients with bone marrow-derived MSC do not inherit AML specific molecular biological changes2.The expression level of TGF-β1 protein in bone marrow of patients with initial AML was significantly lower than that of normal donors and remission AML patients.3.Sorafenib significantly proliferation inhibition of Molm13 cells.In vitro MSC co-culture simulated the bone marrow microenvironment,and the sensitivity of Molm13 cells to sorafenib decreased after co-culture.4.TGF-β1 can significantly increase the sensitivity of Molm13 cells to sorafenib,and its antagonist can reduce the sensitization of TGF-β1 to Molm13 cells.