Effects Of M6A Methylation Modification Of IRF8 And Targeted Nanoparticles Coloaded With Gamma-secretase Inhibitor And Dexamethasone On T-cell Acute Lymphoblastic Leukemia

PART Ⅰ Mechanism of m6A methylation modification of IRF8 mediated the development of T cell acute lymphoblastic leukemiaBackgroundT-cell acute lymphoblastic leukemia(T-ALL)is a malignant clonal disease which originated from hematopoietic stem cells,with a poor prognosis and long-term survival rate of only 25%-50%.To deeply explore the molecular mechanism of T-ALL,find new therapeutic targets,develop targeted strategies and prolong patient survival are not only the top issue of clinical and pathological research of T-ALL,but also have important scientific significance and potential clinical application value.Interferon regulatory factor 8(IRF8)is a transcription factor mainly expressed in hematopoietic cells,which plays an important role in regulating hematopoietic lineage differentiation.It plays a crucial regulatory role in lymphocyte differentiation.IRF8 can promote the transformation of naive CD8 T cells into effector cells by integrating T cell receptor(TCR)and cytokine-related pathways.In addition,it is also a negative regulator of Th17 cell differentiation and inhibits the formation and function of Th17 cells by transcriptionally inhibiting the expression of the solitary nuclear receptor RORC.By mining public database information,we found that the expression of IRF8 in T-ALL decreased significantly,suggesting that it may be involved in T-ALL.However,the role and mechanism of IRF8 in T-ALL are not clear.m6A methylation is an internal chemical modification that is the most prevelant in mRNA and has the greatest impact on its dynamic regulation.FTO is the first identified m6A demethylase,which is responsible for "erasing" the m6A modification of RNA,thus making the m6A modification of cells a reversible process.FTO-mediated m6A methylation regulate the pathogenesis of multiple malignancies,including breast cancer,respiratory system tumor,and gastrointestinal cancer.In AML with MLL rearrangement,FTO is highly activated,which regulates gene expression by inhibiting the m6A modification level of ASB2 and RARA,so as to enhance the survival and drug resistance of AML cells.We analyzed the gene data of T-ALL patients in the GEO database and found that the expression of FTO was remarkably elavated in T-ALL.However,whether FTO-mediated m6A modification of IRF8 can affect the development of T-ALL remains to be further studied.Objectives1.We sought to confirm the important anti-cancer effect of IRF8 in T-ALL through clinical specimen analysis,in vivo and in vitro experiments,clarify that IRF8 negatively regulates PI3K/AKT pathway through transcriptional inhibition of PIK3R5,2.We sought to uncover the underlying mechanism of FTO-mediated m6A methylation modification regulating IRF8 expression,and study the feasibility of targeting IRF8 by FTO inhibitor in the treatment of T-ALL in vivo and in vitro.Methods1.Differential genes were screened in the GEO database,and IRF8 expression was verified in patient samples.2.Lentivirus was used to up-regulate the expression of IRF8 in T-ALL cell lines Molt4 and Jurkat.CCK8 assays were applied to detect cell proliferation,EdU experiments were performed to detect DNA replication rate.Cell cycle assays were used to detect cell cycle distribution.Semi-solid culture assays were applied to determine cell clone formation ability,and Transwell chamber culture assays were performed to evaluate cell invasion and migration ability.3.Irf8 knockout mice were applied to construct Irf8 knockout and Irf8 wild-type T-ALL bone marrow transplantation mouse models by overexpression of Notch1 in bone marrow hematopoietic stem/progenitor cells(Lin-cells).Wright’s-Giemsa staining of Bone marrow and blood,H&E staining of liver and spleen,peripheral blood cell count and flow cytometry were accessed to detect the burden of leukemia cells.The survival of the two groups of mice was observed.4.The downstream molecules and pathways of IRF8 were screened by RNA-seq and verified by Western blot(WB)and immunohistochemistry.ChIP assays and dual-luciferase experiments were applied to verify the regulation mechanism and binding sites of IRF8 as a transcription factor.5.Patient gene data were analyzed to screen out the key molecule FTO in T-ALL.Patients’ samples were used to verify the expression of FTO.Lentivirus was used to down-regulate FTO in T-ALL cells,and FTO inhibitor was applied to inhibit FTO demethylase activity.Dot blot assays were used to detect the level of m6A.Cell proliferation function and IRF8 expression levels were accessed.Recovery experiment verified whether the regulation of FTO on cell proliferation depended on the change of IRF8 expression level.6.RNA-seq and m6A database analyzed the role and potential binding sites of FTO in regulating IRF8 expression through m6A methylation modification,which was verified by MeRIP and dual-luciferase experiments.The up-regulation mechanism of IRF8 mRNA was detected by RNA stability assays.7.Intravenous injection of FTO inhibitor in Irf8 knockout and Irf8 wild-type T-ALL bone marrow transplantation mice,and exploring the feasibility of up-regulating IRF8 expression by FTO inhibitor in the treatment of T-ALL.Results1.IRF8 was identified as a common DEG through analysis of multiple T-ALL-related datasets.Patients’ samples verified that the expression of IRF8 was aberrantly inhibited in T-ALL.2.Upregulation of IRF8 in T-ALL cell lines Molt4 and Jurkat significantly inhibited cell proliferation and slowed down the rate of DNA replication,so as to block cell cycle in GO/G1 phase.At the same time,the ability of cell colony formation,invasion and migration were inhibited.3.Compared with Irf8 wild-type T-ALL mice,Irf8 knockout T-ALL mice showed more severe leukemia infiltration in bone marrow,peripheral blood,liver and spleen.The leukemia burden was significantly increased,the proliferation of leukemia cells was promoted,and the median survival time of mice was significantly shortened.4.RNA-seq analysis screened out the downstream molecule PIK3R5 and the enriched pathway PI3K/AKT.WB and immunohistochemistry experiments confirmed that IRF8 could negatively regulate PIK3R5 and PI3K/Akt signaling pathways in vivo and in vitro.ChIP assays and dual-luciferase assays verified that IRF8,as a transcription factor,can directly bind to the binding site of PIK3R5 promoter region and inhibit gene transcription.We also determined the specific binding site.5.GSEA analysis and patient gene data analysis screened out the key molecule FTO that related to the pathogenesis of T-ALL,and verified the aberrant high expression of FTO with clinical specimens.Using lentivirus to down-regulate FTO in T-ALL cells,the expression of IRF8 increased significantly and cell proliferation was inhibited.We used FTO inhibitor to inhibit FTO demethylase activity.Dot blot assays showed that m6A level was increased,IRF8 expression was increased and cell proliferation was inhibited.The recovery experiment showed that downregulation of IRF8 in Molt4 cells treated with FTO inhibitor could restore the cell proliferation ability.6.RNA-seq showed that FTO negatively regulated the expression of IRF8 mRNA.m6A database analysis showed two potential m6A modification sites at the 3’ UTR end of IRF8 mRNA.MeRIP and dual-luciferase assays confirmed that FTO negatively regulated the expression of IRF8 mRNA by regulating the m6A modification level of the two m6A sites at the 3’ UTR end of IRF8 mRNA.RNA stability experiments confirmed that FTO inhibitors could prolong the half-life of IRF8 mRNA in Molt4 cells.7.Irf8 knockout and Irf8 wild-type T-ALL bone marrow transplantation mice were injected intravenously with FTO inhibitors.It was found that the leukemia burden in bone marrow,peripheral blood and spleen of Irf8 wild-type T-ALL mice were decreased significantly,Besides,cell proliferation was slowed down,and survival time was prolonged after treatment with FTO inhibitors.To explore the feasibility of FTO inhibitor up-regulating the expression of IRF8 in the treatment of T-ALL.The therapeutic effect of FTO inhibitor on Irf8 knockout T-ALL mice was not obvious.Dot blot assays showed that FTO inhibitor significantly increased the m6A level of T-ALL cells in Irf8 wild-type T-ALL mice.WB and immunohistochemical experiments confirmed that FTO inhibitor increased the expression of IRF8 and inhibited the downstream PIK3R5/PI3K/AKT pathway.Conclusions1.IRF8 is aberrantly silenced in T-ALL.Through inhibition of PIK3R5 transcription and PI3K/AKT pathway activation,IRF8 inhibits the proliferation of T-ALL cells,blocks the cell cycle in G0/G1 phase,and inhibits the ability of colony formation,invasion and migration.Irf8 knockout promotes the pathogenesis of T-ALL in vivo and shortens the survival time of T-ALL mice.2.FTO can promote the degradation of IRF8 mRNA and inhibit the expression of IRF8 by regulating the m6A modification of m6A site on IRF8 mRNA.The use of FTO inhibitors in vivo can up-regulate the expression of IRF8 and delay the development of T-ALL.PART Ⅱ DT7 peptide-modified lecithin nanoparticles co-loaded with y-secretase inhibitor and dexamethasone efficiently inhibit T-cell acute lymphoblastic leukemiaBackgroundThe prognosis of acute T-lymphoblastic leukemia(T-ALL)is poor,and the long-term survival rate is less than 50%.About 40%of adult T-ALL patients relapse in the course of the disease.Relapsed T-ALL patients often have a poor prognosis and a significantly increased risk of death.The five-year survival rate is less than 7%,which seriously affects the survival of patients.Glucocorticoid resistance is an important risk factor for the relapse of T-ALL.Therefore,there is still an urgent clinical need to develop targeted strategies to increase the sensitivity of T-ALL to glucocorticoid,improve the therapeutic effect and prolong the survival of patients.It was found that NOTCH1 signaling pathway may be involved in dexamethasone resistance in T-ALL.γ-Secretase inhibitor(GSI)is an efficient and selective small-molecule inhibitor of NOTCH1 signaling pathway.It can effectively inhibit the activation of NOTCH1 signaling pathway and enhance the expression of glucocorticoid receptor(GR)on the surface of T-ALL cells,so as to restore the sensitivity of cells to glucocorticoids.Systemic application of GSI can easily lead to more serious gastrointestinal adverse reactions.However,dexamethasone(DEX)can significantly inhibit goblet cell metaplasia in the intestine,so as to alleviate the adverse gastrointestinal reactions caused by GSI.Therefore,the combination of DEX and GSI not only has a synergistic anti-leukemia effect,but also can reduce adverse drug reactions.However,the traditional drug delivery methods have low drug accumulation efficiency in bone marrow and serious drug side effects.It is urgent to develop new targeted drug delivery methods.In this study,we designed and prepared D-form T7(DT7)peptide-modified nanoparticles(TLnp)to enhance the bone marrow targeted delivery of GSI and DEX.The basic component of TLnp is lecithin,which has good cell affinity and histocompatibility and is easier to transport to bone marrow.Secondly,the particle size of TLnp is below 40nm,which can be more effectively transported to bone marrow through passive targeting.More importantly,the surface of TLnp is modified with DT7 peptides,which can target the transferrin receptor(TFR)on the surface of T-ALL cells and promote the active absorbance of drugs.Objectives1.We sought to prepare and characterize DT7 peptide-modified lecithin nanoparticles(TLnp/D&G)loaded with DEX and GSI,clarify that TLnp/D&G enhances drug accumulation in bone marrow T-ALL cells.2.We sought to determine the anti-leukemia efficacy and molecular mechanism of TLnp/D&G in vivo and in vitro,and explore the safety and gastrointestinal toxicity of TLnp/D&G.Methods1.The optimal molar ratio of γ-secretase inhibitor and dexamethasone was calculated.DT7-modified lecithin nanoparticles coloaded with DEX and GSI were prepared by thin-film hydration methods.The coupling of DT7 was characterized by 1H NMR.The drug morphology was observed by transmission electron microscope.The particle size,potential,drug loading and entrapment efficiency of nanoparticles were detected.Dynamic membrane dialysis was further performed to investigate the release behavior of TLnp/D&G nanoparticles.2.The real-time in vivo fluorescence imaging in mice were used to investigate the active targeting of TLnp in vivo and its accumulation ability in tumor cells.TfR-APC-Cy7 antibody was used to measure the expression level of TfR on the surface of T-ALL cell lines and primary cells by flow cytometry.The ability of drug uptake was detected by HUVEC and CEM co-culture model in vitro.The co-delivery efficiency of two drugs into T-ALL cells was evaluated by in vitro cell uptake assay.3.CCK8 cytotoxicity test and apoptosis test were used to investigate the killing ability of TLnp/D&G on T-ALL cell line and primary cells compared with other non-targeted and non-combined preparations in vitro.The protein levels of NOTCH1 intracellular domain(NICD),p-AKT,GR and BCL2 were detected by Western blot to explore the molecular mechanism of synergistic enhancement of anti-leukemia effect.4.Different preparations were injected intravenously into CEM-bearing mice and NOTCH1-induced T-ALL mice to detect the leukemia burden and survival of mice.Ki67 and Tunel immunofluorescence staining were used to investigate the anti-leukemia ability of TLnp/D&G in vivo.NICD,p-AKT,GR and BCL2 immunofluorescence assays were performed to verify the molecular mechanism of synergistic enhancement in anti-leukemic effect of TLnp/D&G.5.The reduced effect gastrointestinal toxicity of TLnp/D&G on were explored by H&E staining,PAS staining,KLF4 immunohistochemical staining,and bodyweight of mice were observed.The preliminary safety of TLnp/D&G was investigated by H&E staining of main organs and hemolysis assays.Results1.When the molar ratio of DEX:GSI=1:1,the combination index of the two drugs is the lowest as 0.52±0.04,indicating that the combination of the two drugs achieves synergy effect.NMR confirmed that the targeted ligand coupling was successful.TLnp/D&G has a small particle size,with an average diameter of 32.36±2.15 nm.An obvious sustained-release effect,high drug load and good dispersion was demonstrated,which can fulfill the requirement of in vitro and in vivo experiments.2.In CEM-bearing mice,the fluorescence of TLnp group at the tumor site was obviously stronger than that of Lnp group,while the free drug group was the weakest.24-hour fluorescence observation showed that the preparation had a good active targeting effect on tumor tissue,long circulation effect and sustained-release effect.In NOTCH1-induced systemic T-ALL mice,the fluorescence of femurs in TLnp group was also significantly higher than that in Lnp group and free drug group,and the fluorescence of bone marrow cells was significantly increased.3.The in vitro uptake experiment of cells in a co-culture system showed that the fluorescence of CEM cells in TLnp group was significantly higher than that in Lnp group.In vitro cell uptake experiments showed that the co-delivery ability of TLnp was significantly better than that of Lnp group and free preparation group.4.CCK8 cytotoxicity assays and cell apoptosis assays in vitro showed that TLnp/D&G exhibited a better inhibitory effect on the survival and stronger ability to induce apoptosis than Lnp/D&G and other groups.TLnp/D&G also showed the strongest ability to induce apoptosis of primary T-ALL cells.WB experiment showed that TLnp/D&G synergistically inhibited the activation of Notch1 and the expression of p-Akt,increased the expression of glucocorticoid receptor(GR),thus synergistically inhibiting the expression of BCL2.5.In vivo anti-leukemia experiment showed that TLnp/D&G had the strongest inhibitory effect on tumor growth in CEM-bearing mice.TLnp/D&G remarkably inhibited the expression of Ki67 and increased the positive rate of TUNEL in tumor sections.Immunofluorescence showed that the expression of activated Notch1(NICD)and p-AKT were down-regulated,the expression of GR was increased,and the level of BCL2 was significantly inhibited.In Notch1-induced T-ALL mice,TLnp/D&G remarkably reduced leukemia burden in BM,blood and spleen,and prolonged mouse survival time.Immunofluorescence further confirmed the above molecular mechanism.6.TLnp/D&G significantly inhibited the expression of KLF4 and the metaplasia of goblet cells in the small intestine.The structure of main organs was normal,and the hemolysis test was negative.Conclusions1.DT7 peptide-modified lecithin nanoparticles coloaded with DEX and GSI(TLnp/D&G)were successfully synthesized.TLnp/D&G significantly enhances the efficiency of drug co-delivery to bone marrow T-ALL cells.2.TLnp/D&G exhibited excellent anti-leukemia effects in vivo and in vitro.By inhibiting the activation of NOTCH1 and the expression of p-AKT,TLnp/D&G can increase the expression of GR,synergistically inhibit BCL2,promote apoptosis and reverse dexamethasone resistance.3.TLnp/D&G significantly reduces gastrointestinal adverse reactions and shows excellent preliminary safety.