Study On The Mechanism Of Tocilizumab Reversing The Drug Resistance Of AML Cells Induced In The Bone Marrow Microenvironment

Background: Acute myeloid leukemia(AML)is a highly heterogeneous malignant hematological disease that originates from hematopoietic stem cells(HSCs).It is characterized by myeloid cells’ delayed differentiation,blocked apoptosis and abnormal proliferation,which is the most common type of adult acute leukemia.Its recurrence and drug resistance are always the main obstacles affecting its prognosis.The recurrence and drug resistance are closely related to the bone marrow microenvironment.Preliminary studies have found that AML cells remaining in the bone marrow microenvironment have abnormal activation of PI3K/AKT,JAK/STAT3,MEK/ERK signaling pathways,and this abnormality is involved in microenvironment-induced drug resistance;on the other hand,it is found that compared with normal donors,untreated and relapsed/refractory AML patients secreted more IL-6,and the co-culture of AML cells with human bone marrow stromal cells HS-5 significantly increased the expression level of IL-6.Objective: To study the role of tocilizumab in reversing the microenvironment-induced drug resistance of AML cells through blocking IL-6-mediated JAK-STAT3 signaling pathway.Methods: 1.Used bioinformatics technology to explore the role of IL-6 in the microenvironment-induced drug resistance of AML cells;2.Established a co-culture system with AML cell lines(U937,HL-60)adhered to human bone marrow stromal cells(HS-5)to simulate the bone marrow microenvironment that leukemia cells depend on;3.Built HS-5 cell line with IL-6 gene knockdown;4.The CCK-8 method and Annexin V-APC/7-AAD double staining method were used to detect the drug resistance of before and after HS-5 cells’ IL-6 gene knockout,and before and after tocilizumab treatment of co-cultured AML cells;5.Western Blot method to detect the key role of IL-6-JAK-STAT3 signaling pathway in AML cells before and after co-culture,and before and after tocilizumab treatment;6.Construct a subcutaneous tumor-bearing model of human AML mice,and explore the mechanism of tocilizumab(TCZ)assisting cytarabine(Ara-C)to eliminate leukemia cells in vivo.Results:1.The results of bioinformatics analysis suggest that the differentially expressed genes in poor prognosis AML patients with FLT3-ITD gene mutations are enriched in the IL-6 signaling pathway and cancer pathway,and the IL-6 has potential interactions between differentially expressed genes of AML patients with FLT3-ITD gene mutations.2.TCZ alone had no obvious inhibitory effect on AML cells before and after co-culture;after adhesion co-culture,the signal transducer and activator of transcription 3 increased and increased with time.3.Adhesion co-culture can significantly reduce the chemotherapeutic sensitivity of AML cell lines(U937,HL-60)to Ara-C,DNR and HHT.Combined with TCZ,the chemotherapy effect has been significantly improved,and inhibited the increase of p-STAT3、p-AKT、p-ERK1/2 in AML cells after adhesion co-culture.4.Adhered with HS-5 which IL-6 gene knock-down enhances the chemotherapy sensitivity of AML cells.5.In vivo studies,the data have shown that TCZ can cooperate with Ara-C to eliminate HL-60 leukemia cells,significantly inhibit the growth of tumor volume in mice,and significantly reduce tumor burden;and it is detected by immunohistochemistry that IL-6-JAK-STAT3 axis key protein molecules STAT3,p-STAT3,p-AKT and p-ERK1/2 were reduced in expression under the action of TCZ.Conclusion: IL-6-JAK-STAT3 axis participates in TCZ to assist Ara-C to kill AML cells,and inhibiting the activation of this axis is beneficial to enhance the chemotherapy sensitivity of AML cells.