The Imbalance Of Amino Acid Metabolism Promote Proliferation And Drug Resistance In Multiple Myeloma

Background:Multiple myeloma(MM)is characterized by transformed clonal plasma cells in the bone marrow(BM)microenvironment and monoclonal immunoglobulin accumulation in the blood or urine.The incidence of MM increase with the aging of our population and MM is the second most common haematological malignancy after lymphoma.As so far,MM is still an incurable malignant disease,because drug resistance or relapse cause the death of MM patients.It may provide new therapeutic options through revealing the mechanism of maintaining and promoting the malignant phenotype of MM.More and more reports suggested that metabolism plays a key role in the initiation and development of cancers.Metabolic reprogramming of cancer cells provide amount of energy to meet the rapid proliferation of cells.It is proposed to explore the metabolic features of BM microenvironment in MM patients and the mechanism of metabolites effect the proliferation and drug resistance of MM cells,which provide potential targets for the clinical treatment of MM patients.Methods:(1)The BM supernatants samples of 21 healthy doners(HDs)and 50 at newly diagnosed MM patients(MM＿ADs)were divided into training set and verification set,another peripheral blood(PB)supernatant samples of26 HDs and 31 ADs were defined PB set.Untargeted metabolomics assays of above samples were detected by time-of-flight mass spectrometry(GCTOF/MS),and the data was analysis by using SIMCA,a multivariate statistical analysis software.Other BM supernatants and PB supernatants samples of 30 HDs and 110 MM＿ADs were collected and the glycine concentration of the samples were detected by target metabolomics assay,which suggested the correlation between glycine concentration and the clinical characteristics of MM patients.(2)MM cells were cultured in glycine-free RPMI 1640 medium and5TGM1 MM mice were fed with glycine-free diet,respectively.Then,MM cells were cultured with carbon 13 isotope-labeled glycine,the isotope tracing revealed the metabolic pathway of glycine in MM cells.Meanwhile,the RNA-Seq analysis screen the key differential genes.Moreover,the inhibitors of glycine metabolism and lentiviral transfection were used to blocking glycine uptake or catabolism,which contribute to explore the mechanism of glycine promoting proliferation and drug resistance of MM cells.Finally,the indicators of bone destruction in the serum of MM patients and MM mice revealed the source of glycine in BM microenvironment.(3)The proline concentration of BM supernatants samples and CD138+ MM cells collected from 12 HDs,16 MM＿ADs and 17 sequential MM patients were detected by HPLC.In order to explore whether proline promotes proliferation of MM cells,MM cells were cultured in prolinefree medium.Then,GEP data indicated the expression of proline metabolism related genes,and the expression of PRODH in MM cells was detected by RT-PCR and western blot.Moreover,the PRODH overexpression MM cells were constructed by lentiviral transfection to explore whether PRODH effect proliferation and drug resistance in MM cells.Finally,methylation-specific PCR was used to explain why PRODH downregulated in MM cells.Results:(1)Compared with HDs,the concentrations of glycine,proline,ornithine,glutamic acid and valine were increased significantly,while the concentration of stearic acid,palmitic acid,petroselinic acid and linoleic acids were decreased significantly.Moreover,glycine concentration was closely related to the clinical characteristics of MM patients.(2)Loss of glycine inhibits proliferation of MM cells in vitro and in vivo.And lot of glycine contributes to GSH synthesis,which effect DNA damage and cell cycle.Meanwhile,RNA-Seq reveals glycine promotes GSH synthesis through GLDC.And inhibition of GLDC inhibits MM progression.Moreover,inhibition of glycine uptake enhanced the sensitive to BTZ,which inhibits proliferation of MM cells.Otherwise,the glycine concentration increased in MM patients with bone destruction and related to the indicators of bone destruction.Inhibition of the degradation of bone collagen leads to decrease of glycine concentration in MM mice serum.(3)Loss of proline inhibits proliferation of MM cells and enhances the sensitive to BTZ.The expression of PRODH downregulated gradually along with the treatment process.Moreover,overexpression of PRODH promotes proline catabolism,thereby inhibits proliferation and drug resistance in vitro and in vivo.The Cp G island hypermethylation of PRODH promoter leads to downregulation of PRODH.Conclusion:(1)The metabolic feature of BM microenvironment is the imbalance of amino acids in MM patients.(2)The amino acid increased significantly in MM patients,because MMP13 secreted by MM cells induces the degradation of collagen.(3)The abnormal increase of amino acid(glycine)promotes MM cell proliferation and drug resistance through the synthesis of GSH.(4)Hypermethylation of PRODH promoter leads to proline elevated in MM cells,which inducing drug resistance in MM.(5)Amino acid metabolism imbalance(concentration and metabolic enzyme)exhibits the potential diagnostic and therapeutic target for MM.