A Bimetallic Metal-Organic Framework-Based Biomimetic Nanoplatform Enhances Anti-Leukemia Immunity Via Synchronizing DNA Demethylation And RNA Hypermethylation

Acute myeloid leukemia(AML)is a malignant hematologic disease characterized by abnormal accumulation and expansion of immature myeloid cells in bone marrow(BM)and peripheral blood(PB).Eventually,normal hematopoietic function fails.Although considerable progress has been made in immunotherapy for AML over the past few decades,only a small number of patients benefit from it and relapse rates are high.It was found that the reduced immunogenicity of leukemia cells(LBs)was the major reason that hindered the efficacy of immunotherapy.Abnormal epigenetic regulation was the underlying mechanism of the reduced LBs immunogenicity.Cytosine methylation at the 5’position(DNA methylation for short)plays an important role in regulating the occurrence and development of AML.The areas with high CpG sequence repetition rate(CpG island)of AML are in a state of DNA hypermethylation,which will inhibit the activity of interferon gene stimulating factor(STING),thus reducing the major histocompatibility complex(MHC),thereby reducing LBs antigen presentation and ultimately reducing LBs immunogenicity.On the other hand,N6-methyladenosine modification(m~6A-RNA methylation)on RNA regulates transcription and protein expression through shearing,export,translation,and processing.When m~6A demethylase(FTO,ALKBH5)is overexpressed in the body,decreased the stability of oncosuppressor genes in LBs and upregulated the expression of programmed cell death ligand 1(PD-L1).PD-L1 reduces the ability of CD8~+T cells to recognize and kill LBs by binding to programmed death receptor 1(PD-1)on CD8~+T cells.DNA methyltransferase inhibitor(DNMTi)and m~6A demethylation inhibitor play an important role in activating immune response as epigenetic drugs.However,the therapeutic effect of these single epigenetic drugs is not obvious,and the existing epigenetic drugs have some problems,such as weak targeting ability,severe toxic side effects and drug resistance.It is urgent to develop new combination therapy strategies and achieve targeted and controlled drug release.The metal–organic framework(MOF)has the advantages of high porosity,good stability and excellent biocompatibility,and has shown a broad application prospect in the biomedical field.Here,we construct bimetallic biomimetic nanosystem based on Mn/Fe MOF(named AFMMB),which is composed of DNA methylation inhibitor(Azacytidine,AZA),leukemia stem cell(LSC)membrane and Beclin-1 autophagotropic peptide.Due to the homing effect and immunocompatibility of LSC membranes,AFMMB can target LBs and LSCs simultaneously.Beclin-1 peptide on the surface of AFMMB can bind to Golgi associated plant pathogenesis-related protein 1(GAPR-1)and induce autophagy.This causes the AFMMB to degrade and release manganese ions,iron ions and AZA.The main research content of this study is as follows:(1)Design and synthesis of AFMMB biomimetic nano system,and the morphology,structure,stability,loading capacity,and the encapsulation rate and releasing mechanism were studied.(2)Investigate the targeting ability of AFMMB biomimetic nano system in vitro and in vivo via inductively coupled plasma atomic emission spectroscopy(ICP-MS),confocal laser scanning microscope(CLSM),small animals living optical imaging system(IVIS).(3)The molecular mechanism of AFMMB in C1498 cells was investigated by the protein immunoblot analysis(Western blotting),analysis of m~6A spot mark,enzyme linked immunosorbent assay(ELISA)and quantitative RT-PCR etc.The inhibitory effect of AFMMB on C1498 cells was analyzed by BODIPY staining and flow cytometry.(4)By building C1498 mouse models of AML,research on the AFMMB distribution,inhibiting effect,biological immune activation effect,biological safety of AML.At the same time,the 4T1 mouse breast cancer models were established to study the therapeutic effect of AFMMB on solid tumors.The results show that we have successfully synthesized AFMMB biomimetic nanosystem with high porosity,good stability and excellent targeting ability.For the mechanism,AZA and manganese ions can inhibit DNA methylation,reactivate STING pathway and up-regulate MHC-I,and iron ions can enhance modification of m~6A RNA methylation,thus reducing the transcriptional stability of PD-L1.This dual epigenetic regulation increases the immunogenicity of AML cells,thereby enhancing the immune response mediated by CD8~+T cells.Finally,we found that AFMMB also has excellent inhibitory effect on solid tumors and has wide range of therapeutic effects on cancer,providing research basis and theoretical basis for further clinical validation.