The Role Of TIFAB In Mll-AF9-induced Acute Myeloid Leukemia

Objective:Leukemia is a malignant tumor of the human blood system.Leukemia can be classified into acute leukemia and chronic leukemia according to the severity of the disease and the maturity of the cells.Due to its rapid onset and rapid progress,acute leukemia has not yet had clinically effective therapeutic drugs.Therefore,the research on the pathogenic mechanism of acute leukemia has attracted wide attention from scholars.In the bone marrow of acute leukemia patients,abnormal blasts and immature cells proliferate indefinitely under the action of various carcinogenic factors,replace the original normal hematopoietic cells,inhibit normal hematopoiesis in the human body,and infiltrate the lymph nodes,liver,spleen,testes and the central nervous system(including the brain and spinal cord),manifested by anemia(fatigue),thrombocytopenia(bleeding),and neutropenia(frequent infection).Acute leukemia can be further classified into acute lymphoblastic leukemia and acute myeloid leukemia(AML)according to the cell type.AML is one of the most common types of leukemia in adults,and it usually affects elderly patients.The incidence of AML is low before the age of 45,and with the increase of age,the higher the incidence,the worse the prognosis.According to reports,the average age of patients first diagnosed with AML is 68 years.The treatment of leukemia represented by AML is still a clinical problem.As we all know,leukemia involving mixed-lineage leukemia(MLL)gene rearrangement on chromosome 11q23 is the most aggressive and drug-resistant leukemia.It has the characteristics of high malignancy,poor chemotherapy effect,and poor prognosis.When the World Health Organization released the revision of the leukemia classification in 2011,the 11q23/MLL gene rearrangement leukemia was classified as a separate category.MLL gene rearrangement is very common in AML.At present,more than 60 different fusion genes and 100 rearrangements have been found,of which the most common is the MLL fusion gene AF9,which accounts for2%to 5%of all AML patients.The canonical NF-κB signaling pathway is activated in AML stem cells to maintain the stemness of LSC.In previous studies of our group,it was found that activation of non-canonical NF-κB-inducible kinase(NIK)can inhibit MLL-AF9-induced AML,suggesting that the roles of non-canonical NF-κB signaling pathway and the canonical NF-κB signaling pathway are opposite in AML.However,the specific mechanism of non-canonical NF-κB pathway in AML and its downstream targets still need to be further explored to clarify the pathogenesis of AML and explore potential therapeutic targets for AML.In previous studies,we found that after activating the expression of NIK,the expression of TRAF-interacting protein with a forkhead-associated Domain B(TIFAB)in AML cells was significantly down-regulated.According to reports,TIFAB is related to the loss of 5q chromosome in myeloid malignancies;TIFAB can significantly affect the ratio of hematopoietic stem/progenitor cells and bone marrow differentiation.Therefore,in this study,we focused on the role of TIFAB in MLL-AF9-induced AML,aiming to further explore the specific mechanism of the non-canonical NF-κB pathway in AML and how TIFAB affects the MLL-AF9-induced AML through HOXA9.Methods:1.Cell line research model:MLL-AF9-induced AML cells,human renal epithelial cell293T.2.Gene mutant mice,CD45.1 receptor mice.3.Molecular biology experiments:use q PCR and western blot experiments to detect molecular expression;CHIP-q PCR is used to determine the interaction of intracellular DNA and protein;use cell counting,Brd U,Ki67,colony formation experiments to detect cell proliferation;use Caspase 3 staining experiment to detect cell apoptosis ability Mito-tracker staining experiment was used to detect mitochondrial quality;Mito Sox staining experiment was used to detect mitochondrial reactive oxygen level;2-NBDG staining experiment to detect cellular glucose uptake;mouse AML cell transplantation experiment was used for phenotype verification.4.Database information:The data used for relevant bioinformatics analysis was obtained by database or online bioinformatic tools such as GEO(Gene Expression Omnibus),GEPIA2(Gene Expression Profiling Interactive Analysis 2),TCGA(The Cancer Genome Atlas).5.Statistical analysis:Whether there are differences were analyzed by using independent sample t test.Graph Pad Prism 7,R 3.6.1 and other software were used to complete the above related analysis.The two-tailed P value less than 0.05 was defined as the standard for the difference of statistical significance.Result:1.NIK inhibits the progression of MLL-AF9-induced AML through RELB.RELB directly regulates TIFAB and RELB inhibits the expression of TIFAB.The result of mouse bone marrow transplantation showed that activation of NIK can prolong the survival of AML mice,and knocking out RELB can reverse the inhibitory effect of NIK on AML.Data analysis of GSE97389 showed that activation of NIK in MLL-AF9-induced AML cells can inhibit the expression of TIFAB at the m RNA level,which was verified by q PCR experiment.The result of western blot experiment showed that NIK upregulated the expression of RELB and downregulated the expression of RELA in MLL-AF9-induced AML cells;NIK inhibited the expression of TIFAB,and knocking out RELB could reverse the inhibitory effect of NIK on TIFAB.CHIP-q PCR results showed that RELB directly regulates TIFAB.The result of western blot showed that overexpression of RELB in MLL-AF9-induced AML cells inhibited the expression of TIFAB,while overexpression of RELA had no such effect.2.TIFAB promotes the progression of MLL-AF9-induced AML.GEPIA2 database test result showed that the expression level of TIFAB m RNA in AML patients is higher than that of normal controls.TCGA database analysis result showed that AML patients with high TIFAB expression have a poorer prognosis.When TIFAB is overexpressed in MLL-AF9-induced AML cells after transfection,the result of cell counting experiments,Brd U and Ki67 staining experiments showed that TIFAB promoted the proliferation of c-Kit~+AML cells.Caspase3 staining experiments showed that TIFAB inhibited c-Kit~+AML cells apoptosis.Mouse bone marrow transplantation experiments showed that TIFAB significantly shortened the survival of AML mice.The leukemia stem cells(LSC)with TIFAB overexpression and its control were sorted by flow cytometry,and then RNA-seq sequencing was performed on them.The result showed that TIFAB upregulated the HOXA family and other genes involved in maintaining the expression of LSC characteristic.GSEA analysis of RNA-seq data showed that TIFAB affects the myeloid differentiation,stemness and cell energy metabolism of LSC cells.Mito-tracker staining result showed that TIFAB promoted the increase of mitochondrial mass in c-Kit~+AML cells.Mito Sox staining experiment show that TIFAB promoted the increase of mitochondrial reactive oxygen species in c-Kit~+AML cells.The results of 2-NBDG staining experiment showed that TIFAB promoted the absorption of glucose by c-Kit~+AML cells.The result of western blot showed that TIFAB upregulated the expression of UQCRC2 and MTCO1 proteins in AML cells.3.TIFAB accelerates MLL-AF9-induced AML progression through HOXA9.Western blot experiments showed that TIFAB inhibited RELB and RELA protein expression.The result of q PCR experiment showed that HOXA2,HOXA3,HOXA5,HOXA9 and HOXA10 were upregulated by TIFAB on m RNA expression level.Western blot experiment showed that TIFAB upregulated the HOXA9 protein expression.Mouse bone marrow transplantation experiment and cell colony formation experiment showed that NIK can significantly prolong the survival of AML mice and inhibit AML colony formation,and both TIFAB and HOXA9 could reverse the inhibitory effect of NIK on AML.The result of western blot experiments showed that the RELB protein expression in AML cells was upregulated after the activation of NIK,and the RELA and TIFAB protein expression were downregulated;HOXA9 could reverse the upregulation of RELB after NIK activation and HOXA9 could restore the expression of RELA and TIFAB inhibited by NIK.Conclusions:1.NIK inhibits the progression of MLL-AF9-induced AML through RELB.2.TIFAB is the direct target of RELB,and RELB inhibits the expression of TIFAB.3.TIFAB promotes the progression of MLL-AF9-induced AML.4.TIFAB accelerates MLL-AF9-induced AML progression through HOXA9.