Increased Release Of Microvesicles Containing Mitochondria Is Associated With The Myeloid Differentiation Of AML-M5 Leukaemia Cells

Acute myeloid leukaemia(AML)is a heterogeneous clonal disease characterized by immature myeloid blasts that are blocked in early stages of differentiation,resulting in malignant proliferation in bone marrow.The standard treatment for AML is the combination medication of cytarabine and anthracycline,intending to induce remission,but this regimen bring strong side effects at the meantime.Thus,effective therapy strategies against poor outcome in AML patients need to be identified.Acute monocytic leukaemia(AML-M5)is a common subtype,accounting for up to 20% of AML cases,and is differentially characterized by the presence of monocytic-lineage maturation features in at least 80% of the blast cells,which show monoblasts,promonocytes,and monocytes.Additionally,patients with AML-M5 show a worse prognosis than those with the non-monocytic subtypes.The pathogenesis of AML-M5 is closely related to the disruption of monocyte-macrophage differentiation.Therefore,by exploring the mechanism of monocyte-macrophage differentiation,we try to identify the factors that would be of immense research and therapeutic significance for AML-M5 patients.Recently,striking differences in mitochondrial characteristics between normal hematopoietic stem cells(HSCs)and leukaemia stem cells(LSCs)have been revealed.AML leukaemia cells and patient samples show increased mitochondrial biogenesis compared with normal hematopoietic progenitors.Recent reports have emphasized that leukaemia blasts rely primarily on mitochondrial function for their survival,and evidence has shown that AML is both sensitive to the repression of mitochondrial DNA replication and protein synthesis.Furthermore,AML leukaemia blasts show increased reliance on oxidative phosphorylation(OXPHOS).And it is reported that mitochondrial gene knockout HL60 cells show preferential differentiation into monocytes/macrophages.Based on current discoveries,we asked whether mitochondria may act as a crucial player during leukaemia blast overcoming the differentiation blockade.The biological and pathological significance for MVs is beginning to be gradually recognized in recent years,such as participating in the maintenance of normal physiology and stimulating tumour progression.The various cargos of MVs include m RNA,mi RNA,non-coding RNAs,cytoplasmic proteins and membrane proteins,which may play important roles in normal physiology and disease pathogenesis.Interestingly,some human cells package mitochondria into extracellular MVs.Bone marrow mesenchymal stem cells have been found to target mitochondria to the cellular membrane through arrestin domain-mediated MVs.Our data demonstrated the role of mitochondria as a metabolic regulator of differentiation and point to its inhibition as a strategy for overcoming differentiation blockade in AML.Our work stresses the importance of MVs containing mitochondria in malignant AML-M5 leukaemia blast biology.(1)The mitochondrial mass and ATP levels were decreased during myeloid differentiation in AML-M5 cells.Primarily,we established a pro-differentiation model in vitro with a positive control drug PMA,and next explored the relationship of mitochondrial mass and energy metabolism with myeloid differentiation.Myeloid differentiation was confirmed by analysing the cell-surface CD11 b and CD14 expression levels and the uptake of fluorescent-labelled latex beads using FACS.For the mitochondrial mass,we chose to use Tom20 staining and Mitotracker Green staining.(2)The release of MVs containing mitochondria is increased during myeloid differentiation in AML.Firstly,we used differential ultracentrifugation,which is recognized as a common conventional approach to isolate and enrich MVs.Then,dynamic light scattering(DLS)and WB were used to define its size and phenotypic characteristics.Finally,FACS,PCR and WB were used to confirm the presence of mitochondria in MVs.(3)The MVs formation inhibitor Y-27632 restrains myeloid differentiation.Targeting the formation of MVs using a specific inhibitor(Y-27632)restrained myeloid differentiation,suggesting that the increasing release of MVs plays an important role in regulating myeloid differentiation.(4)Oxidative phosphorylation inhibition leads to myeloid differentiation.An OXPHOS inhibitor(rotenone)used to inhibit the respiratory chain and ATP production showed a strong myeloid differentiation effect on monocytic leukaemia cells,indicating that disturbing mitochondrial energy metabolism might break the blockade of myeloid differentiation.Here,we identified that the disruption of the mitochondrial mass and energy metabolism promotes leukaemia cellular myeloid differentiation.In this study,we showed that AML-M5 cells package mitochondria in MVs when MVs shed from membranes.Additionally,during myeloid differentiation,we report for the first time that differentiated leukaemia cells release more MVs than monocytic leukaemia cells.Targeting the formation of MVs using a specific inhibitor(Y-27632)restrained myeloid differentiation,suggesting that the increased release level of MVs plays an important role in regulating myeloid differentiation.Furthermore,the intracellular mitochondria and ATP levels were decreased after leukaemia cells overcame the differentiation blockade.Moreover,rotenone,which is used to inhibit the respiratory chain and ATP production,had a strong effect on myeloid differentiation in monocytic leukaemia cells.Collectively,these studies uncovered the relationship between mitochondrial function and myeloid differentiation and may provide more insight into the diagnosis and treatment of AML.