Copper Sulfide Nanocomposites Enhance M6A-RNA Methylation-mediated Anti-leukemia Immunity

Acute myeloid leukemia(AML)is a malignant tumor of the hematopoietic system characterized by the uncontrolled clonal expansion of abnormally differentiated myeloid cells in Bone marrow(BM)and Peripheral blood(PB).In the past few years,although people have made good progress in the treatment of AML through chemotherapy,immunotherapy,and hematopoietic stem cell transplantation(HSCT),the majority of patients have a very high recurrence rate and poor prognosis.From the perspective of molecular mechanism,the dynamic reversible modification of Epigenetics is an important factor leading to the occurrence and development of leukemia.N6-methyladenosine(m6A)is the most abundant epigenetic modified mRNA associated with AML.It is involved in the key process of leukemia evolution by regulating the structure,function,stability and translation of RNA.Therefore,exploring m6 A modification on RNA will help to analyze cutting-edge issues of life science from the perspective of apparent transcription,reveal the pathogenesis caused by apparent transcription disorders,and provide a new entry point for the development of new drugs targeting m6 A regulatory proteins.As the earliest identified m6 A demethylase,FTO disrupts the stability of ASB2 and RARA mRNA by reducing the abundance of m6 A on the transcripts of ASB2 and RARA mRNA,thereby promoting the occurrence of leukemia and inhibiting all-trans retinoic acid(ATRA)-mediated leukemia cell differentiation.In recent years,researchers have been developing new FTO inhibitors for the treatment of leukemia.However,this single epigenetic small molecule drug therapy program is limited by problems such as poor stability,large toxicity and side effects,and easy drug resistance.Therefore,it is an urgent problem to develop a new combination therapy strategy.Inorganic nanomaterials have been widely used in biomedical fields such as drug delivery,diagnosis and imaging,screening and treatment,vaccine synthesis and development due to their high stability,good compatibility in vivo and porous structure.We designed a copper sulfide nanocomposite(PCCR)based on epigenetic modification,which consists of copper sulfide nanoparticles(CuSNP),FTO inhibitor R-2HG,chitosan CS,and targeted peptide P29.PCCR recognizes leukemia stem cells through the specific targeting of peptide 29,and then enters the cells through endocytosis.Beclin-1 binds to the autophagy related protein GAPR-1 on the surface of Golgi body and induces autophagy,thus releasing R-2HG and Cu2+ inside.R-2HG inhibited the enzyme activity of FTO protein,increased the overall m6 A modification,and inactivated related oncogenes.Cu2+ induced iron death by Fenton-like reaction.The main research contents of this paper are as follows:1)The PCCR nano apparent drug platform was designed and synthesized,and its structure,morphology,physical and chemical properties,drug loading and encapsulation rate,stability and drug release mechanism were studied.2)Confocal laser scanning microscopy(CLSM),inductively coupled plasma Atomic emission spectroscopy(ICP-AES),and small animal in vivo optical imaging system(IVIS)were used to detect the in vitro and in vivo targeting ability of PCCR.3)The mechanism of PCCR on leukemia cells was investigated by GSH/GSSG and GPX4 detection,gene-specific m6 A qPCR,LC-MS/MS,mRNA degradation,Western Blotting analysis,enzyme-linked immunosorbent assay(ELISA),flow cytometry,etc.4)The efficacy,biosafety and drug distribution of PCCR were evaluated by constructing a mouse leukemia model of C1498,and the immune activation effect of PCCR was also tested.In general,we have successfully designed and synthesized a PCCR nano epigenetic drug platform with high stability,good biocompatibility and strong targeting.This new nano epigenetic drug can reverse cell disorder caused by epigenetic network regulation,Re-modeling cancer cells or cancer stem cells,and achieve the inhibition of FTO.At the same time,it has the characteristics of multi-function,and can cooperate with multiple induced cell death modes to overcome the dependence of cell on a single mode of death,which provides a new insight for the combined use of nanomaterials and epigenetic drugs.