Endothelial cell-derived mitochondrial transfer to Acute Myeloid Leukemia alters global gene expression to confer chemoresistance

Abstract Acute myeloid leukemia (AML) exhibits high relapse rates, suggesting the existence of robust mechanisms that protect leukemic cells from therapy, leading to measurable residual disease (MRD). Our previous research demonstrated that endothelial cell (EC) interactions with AML cells trigger EC activation, promoting AML adherence, quiescence, and chemoresistance. We further identified that this interaction also increases EC secretion of interleukin-8 as a key mediator enhancing AML proliferation and therapy resistance. Mitochondrial transfer has been implicated in chemoresistance across various cancers. In AML, mitochondrial metabolic function is known to affect therapy response, and furthermore, stromal cells have been shown to transfer mitochondria to AML cells in the presence of chemotherapy. However, the potential role of EC-to-AML mitochondrial transfer remains unexplored, despite the critical interface between these cell populations in patients. We hypothesized that ECs transfer mitochondria to AML cells, fundamentally altering their activity and inducing chemoresistance. Using a co-culture system incorporating both established AML cell lines and primary patient-derived samples with ECs, we tracked mitochondrial transfer through fluorescent labeling and flow cytometry. Our studies revealed mitochondrial transfer occurs through both contact-dependent and independent mechanisms, including microvesicle-mediated transfer. When AML cells with and without EC-derived mitochondria were isolated using FACS sorting and exposed to cytarabine or idarubicin, those with EC-derived mitochondria were significantly more chemoresistant, even with concentrations of chemotherapy that were 10x greater than used clinically. To elucidate the mechanism behind the EC mitochondria transfer induced chemoresistance, we performed single cell RNAseq (scRNAseq) of AML cells co-cultured with ECs. The results demonstrated unique transcript signatures of the co-cultured AML cells in comparison to AML cells not exposed to ECs. To identify candidate genes that could be responsible for the observed chemoresistance, we analyzed the scRNAseq data and found that hepatocyte growth factor (HGF) gene expression was highly upregulated in AML cells exposed to ECs compared to AML monocultures. In subsequent studies using isolated AML cells containing EC-derived mitochondria, RT-PCR and qPCR demonstrated elevated HGF gene expression, and ELISA analysis confirmed significantly higher production of HGF when compared to AML cells without mitochondrial transfer. Moreover, treatment with capmatinib, a HGF receptor (MET) inhibitor, effectively reversed the chemoresistant phenotype of the AML cells containing EC-derived mitochondria, restoring sensitivity to levels comparable to control AML cells.This research elucidates a novel mechanism whereby EC-AML interactions promote chemoresistance through altered AML gene expression that occurs following mitochondrial transfer from EC cells. The specific identification of EC mitochondrial transfer and the genetic changes that occur as a result should help identify key mediators of resistance (e.g., HGF) and help identify promising therapeutic targets. These findings contribute to our still growing understanding of AML pathology and suggest potential strategies to address the persistent challenge of MRD and disease relapse.