Acute Myeloid Leukemia Export Mitochondria In Extracellular Vesicles Which Induces Pro-Tumoral Changes In Bone Marrow Macrophages

It is envisioned that improved understanding of the dependency of acute myeloid leukemia (AML) on its tumour microenvironment within the bone marrow could be exploited to offer new treatment strategies and better patient outcomes. Previously we have shown that the bone marrow (BM) microenvironment supports AML growth and survival, specifically via functional mitochondrial transfer from stromal cells to AML blasts (Marlein 2017 Blood and Abdul Aziz 2019 Blood). The increase in mitochondria in AML theoretically presents a cell intrinsic problem; accumulation of excess and dysfunctional mitochondria can induce cell death via increased reactive oxygen species and apoptotic caspases (Kroemer 2007 Physiol Rev). As such, we examine the mechanisms by which AML blasts manage excess and dysfunctional mitochondria and the impact of this process on the supporting bone marrow microenvironment. Primary AML blasts and primary AML bone marrow stromal cells (BMSC) were isolated from the bone marrow of patients. Co-culture experiments show that primary AML blasts (cultured with BMSC) maintain constant levels of mitochondria despite continuous uptake of mitochondria from BMSC. Furthermore, a combination of proteomics, confocal microscopy, image cytometry and dynamic light scattering shows that AML actively export mitochondria in large extracellular vesicles (EVs), approximately 0.6micron-1.5micron in diameter, a process which maintains a steady state of mitochondrial content in the blasts. To track the recipient cell for large EV packaged AML derived mitochondria, patient derived AML were transduced with rLV.EF.mCherry-mito-9 lentivirus which fluorescently tags the mitochondria, and then transplanted into NSG mice. BM was extracted from engrafted mice and various cell populations were analysed for increases in mCherry fluorescence as evidence of AML derived mitochondria uptake. Murine F4/80+/GR1-/CD115intBM macrophages had increased mCherry fluorescence but not mouse CD45-/CD31-/Ter119-/CD105+/CD140a+BMSC, CD45-/Ter119-/CD31+endothelial cells or CD45+leukocytes. To determine the impact of AML derived mitochondria containing EVs on the function of the BM macrophages we first isolated the AML derived mitochondrial containing EVs using cell sorting for mCherry fluorescence and then BM derived macrophages (BMDM) were incubated with EVs containing mitochondria or EV with no mitochondria and assessed for changes in pro-inflammatory genes. IL-6, IL-1B, CXLC9 and CXCL10 were all induced in BMDM by EV containing mitochondria at 24 hours post incubation. However, repeated exposure of BMDM with EV containing mitochondria resulted in significantly lower levels of IL-6, IL-1B, CXLC9 and CXCL10 upregulation. Moreover, repeated exposure of BMDM to EV containing mitochondria resulted in reduced phagocytic potential compared to control BMDM, suggesting exhaustion or senescence. To determine if AML induced BM macrophage senescence we used a syngeneic model of AML, HOXA9/Meis1 or MN1 and lineage negative cells as control. Animals were sacrificed 35 days post AML injection and BM macrophages were isolated and senescent markers were analysed by real-time PCR. p16ink4a and p21 were both upregulated in BM macrophages from HOXA9/Meis1 or MN1 engrafted animals. Data indicates that mitochondrial mass in AML blasts is maintained in equipoise through export of mitochondria in large EVs. In addition, we show that AML derived EVs containing mitochondria function as a signal which changes the function of BM macrophages, towards a senescent phenotype which promotes AML blast survival and proliferation. Disclosures Bowles: Abbvie: Research Funding; Janssen: Research Funding. Rushworth:Abbvie: Research Funding; Janssen: Research Funding.