Stromal Cell Weakening And Salinomycin Enhance The Effect And Mechanism Of BTK Inhibitor Against MCL

[Objective](1)To study the effect of conditioned medium(CM)of bone marrow stromal cell line HS-5 cells on the anti-mantle cell lymphoma(MCL)effect of first-generation Bruton's Tyrosine Kinase(BTK)inhibitor ibrutinib and second-generation BTK inhibitor acalabrutinib,and its molecular mechanism was revealed by high-throughput transcriptome sequencing technique.(2)To evaluate whether salinomycin(Sal)can enhance the anti-MCL effect of acalabrutinib,a second-generation BTK inhibitor,and reveal its potential molecular mechanism[Methods](1)In this study,mantle cell lymphoma cell line Mino cells cultured in conventional medium without conditioned medium and drugs were used as control group,and CM group,ibrutinib/acalabrutinib group and ibrutinib/acalabrutinib+CM group were divided into three groups.Serum-free CM,was obtained by culturing HS-5 cells,and its effect on the proliferation of Mino cells and the effect of ibrutinib/acalabrutinib on the proliferation of Mino cells enhanced by CM were detected by Alamar Blue method.With the help of high-throughput transcriptome sequencing,the differentially expressed mRNA,among groups were compared and analyzed by bioinformatics.The analysis methods mainly included differential expression pattern clustering analysis,Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment classification analysis,Pathway enrichment factor analysis and so on,so as to reveal the possible functions of differential genes and the signal pathways involved HMAs treatment group(D14),but Tregs content in HMAs treatment group(D28)was lower than that in HMAs treatment group(D14).1.4 in AML patients,the Tregs content in HMAs treatment group(D28)was significantly lower than that in non-HMAs treatment group(P=0.036),and there was no significant difference compared with newly diagnosed group and HMAs treatment group(D14)(P=0,586).1.5 In the MDS patients,there was no significant difference in Tregs relative content among newly diagnosed group,HMAs treatment group(D14)and HMAs treatment group(D28).In patients with AML and MDS,the relative content of Tregs in the control group was not significantly different from that in the HMAs group(P=0.662),but significantly lower than that in the non-HMAs group(P=0.001),and in the patients with AML and MDS,there was no significant difference in the relative content of Tregs between the D AC treatment group and the AZA treatment group(P=0.405).1.8 there was no significant difference in the relative content of Tregs between the remission group after HMAs treatment,the non-remission group after HMAs treatment and the non-HMAs treatment group(P=0.01).2.Th cells:2.1 There was no significant difference in the relative contents of Thl and Th2 cells and the ratio of Th1/Th2 cells between the newly diagnosed AML and MDS groups and the control group(P=0.224).The Th2 cells in the newly diagnosed AML and MDS groups were higher than those in the control group,while the Th1/Th2 cells in the AML and MDS groups were lower than those in the control group.In patients with AML,there was no significant difference in the relative content of Thl and Th2 cells and the ratio of Th1/Th2 cells in peripheral blood between the newly diagnosed group and the control group.However,there was a downward trend of Th1 cells in the peripheral blood of the newly diagnosed patients with AML compared with the control group.2.2 Patients with AML and MDS,there was no significant difference in the relative content of Th1,Th2 cells and the ratio of Thl/Th2 cells in peripheral blood among newly diagnosed group,HMAs treatment group and non-HMAs treatment group(P=0.05).However,it was observed that Th1 cells in HMAs group were higher than those in non-HMAs treatment group.3.NKT cells:the content of NKT cells in control group was higher than that in AML and MDS groups(P=0.01).3.2 in patients with AML and MDS,the of Bcl-2 and cyclinDl protein and increased the expression level of cleaved-PARP and cleaved-caspase3 protein in Mino cells treated with acalabrutinib alone.[Conclusion](1)HS-5 conditioned medium not only enhanced the proliferative activity of Mino cells,but also weakened the cytotoxicity of ibrutinib and acalabrutinib to Mino cells.It was confirmed that stromal cells played an important role in the occurrence and development of MCL and its resistance to BTK inhibitors.With the help of high-throughput sequencing results,it was found that the main enriched signal pathways of differentially expressed genes supporting the growth of Mino cells in conditioned medium included B cell signal transduction,apoptosis,cell adhesion/migration and cytokine signals in the immune system,in which some genes could be regulated by ibrutinib.However,there are still some genes that can not be regulated by ibrutinib,which provides an idea for finding new therapeutic targets for MCL and finding new targets to cooperate with BTK inhibitors to overcome their drug resistance.(2)The combination of salinomycin and acalabrutinib can synergistically inhibit the survival of Mino cells,enhance apoptosis and block the cell cycle.