The Molecular Mechanism Of FGFR3 Negative Regulation On MLL-AF9-induced Acute Leukemia Cells Reprogramming Into Leukemia Stem Cells

Background and Objective:Leukemia is a malignant clonal disease of hematopoietic stem cells.Leukemic stem cells?LSCs?are the source of drug resistance and relapse in acute myeloid leukemia?AML?.It is important to clarify the biological characteristics and mechanism of LSCs resistance in order to remove LSCs and cure AML.In this study,the MLL-AF9 MA-induced leukemic cell line?MA-KO LC?induced by the deletion of fibroblast factor receptor 3?FGFR3?gene was used as the research object.It was found that knockout of FGFR3 in MA-induced mouse acute leukemia cell lines significantly increased the percentage of LSCs CD117.However,the transplantation experiment showed that the survival time of leukemia mice was significantly prolonged in vivo.Therefore,we demonstrated the molecular mechanism of reprogramming MA cells into LSCs by activating FGFR1-ERG-CD117 signal pathway after deletion of FGFR3 by sequencing results of transcription group.By analyzing the in vivo implantation of leukemic cells and the changes of bone marrow microenvironment,it was demonstrated that FGFR3 could influence the implantation of leukemic cells and prolong the survival time of mice by regulating the expression of chemokine CCLs gene.It provides a new idea for drug development and clinical treatment of LSCs-targeted AML.Methods:1.The MA-KO LC cells of FGFR3-deficient mice?MA-WT LC cells as control?were used to detect the proliferation of MA-KO LC cells by cloning,proliferation count in vitro and Brdu labeling method.2.Signal pathway inhibitors were used to verify how FGFR1 could up-regulate CD117 expression by activating PI3K/AKT and/or IKK-NF-?B signals.By using receptor inhibitors or RNA interference techniques to down-regulate genes such as FGFR1 and ERG respectively,it is clear whether these genes or signals are the root cause of LSCs reprogramming.The over-expression of virus or knock-down of ERG,in MA-WT LC cells was used to verify whether LSCs could also be enriched or decreased.Chip-qPCR and double-luciferase reporter gene methods were used to verify the direct regulation of CD117 transcriptional activity by ERG.3.Leukemic cell transplantation and enzyme linked immunosorbent assay?Elisa?were used to determine the expression level of chemokine CCLs gene to verify the survival of mice and the implantation of leukemic cells.Results:1.The clone ability and proliferation ability of MA-WT LC cells in vitro were 1² times as much as that of MA-KO LC.At three concentrations of cytarabine?Ara-c?,the cell death rate of MA-WT LC was 2³ times higher than that of MA-KO LC,indicating that MA cells with FGFR3 deletion showed strong drug tolerance.In vitro migration assay showed that the migration ability of MA-WT LC cells was 1.5 times higher than that of MA-KO LC;2.The percentage of LSCs(CD117⁺CD11b^low)in MA-KO LC was 27 times higher than that in control group,and the percentage of LICs?CD117⁺CD11b⁺?in MA-KO LC was 8 times higher than that in control group;3.FGFR1-PI3K/AKT and FGFR1-IKK/NF-?B signals were involved in the production of LSCs and LICs;4.After FGFR3 knockout,the expression of FGFR1 was up-regulated by 1.5 times;the expression of ERG was up-regulated about 10 times;the expression of ERG gene was regulated by FGFR1signal;ERG can directly bind to the promoter region of CD117 to regulate the expression of CD117;5.The survival time of the mice transplanted with MA-KO PreLC and MA-KO LC cells was nearly 2 times longer than that of the control mice.The deletion of FGFR3 did not affect the homing ability of MA cells in BM,SP and PB.However,the implanted ability of MA-KO LC cells was significantly decreased by 2.5 times and 1.5 times,respectively.The expression levels of CCL3 and CCL4in bone marrow supernatant of leukemia mice transplanted with MA-WT LC cells were 3 and 5 times higher than those of FGFR3-deficient leukemia mice,respectively.Conclusion:After FGFR3 deletion,the expression of CD117 is regulated at the transcriptional level by activating the FGFR1-ERG signal,and MA cells are reprogrammed into CD117⁺LSCs or LICs cells.FGFR3 deletion prolongs the survival time of MA mice.