Leukaemia Cells Induced Metabolic Alterations in AML Associated Mesenchymal Stem Cells Via Notch Signalling

Abstract

Introduction: Acute myeloid leukaemia (AML) is a haematological malignancy with a high relapse rate and poor prognosis. Leukaemia cell proliferation is dependent on its interaction with the bone marrow (BM) microenvironment. AML associated mesenchymal stem cells (AML-MSCs) supported the proliferation of leukaemia cells and contributed to disease progression. Stromal microenvironment promoted a metabolic switch but precise underlying molecular mechanisms are poorly understood. Previous studies have demonstrated transfer of functional mitochondria from AML-MSCs to AML blasts facilitating energy requirements. To further improve our understanding of the crosstalk between leukaemia and AML-MSCs, we sought to determine contribution of AML-MSCs and signalling cascades regulating metabolic processes.Methods: Sorted MSCs from non-leukaemic and MLL-AF9 leukaemic mice were isolated, and gene expression profiling was performed using RNA microarray. Additionally sorted MSCs from long-term cultures were cultured alone or with MLL-AF9 leukaemia cells and analysed by RNA-sequencing. Gene set enrichment analysis (GSEA) was used to identify the hallmark gene sets overrepresented in AML-MSCs. We further cocultured murine wild type BM-MSCs alone or together with murine AML cells (C1498 and MLL-AF9) or the control lineage negative cells (Lin -). Metabolic alterations, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were analysed by Agilent Seahorse XFe96 analyser. Additionally, glucose consumption, lactate secretion and mitochondrial DNA copy number were measured.Results: Microarray analysis in sorted MSCs from leukaemic and non-leukaemic mice have identified hallmark oxidative phosphorylation (p<0.01, NES=-1.6) and glycolysis (p<0.01, NES=-1.3) gene sets to be negatively enriched in AML-MSCs. Interestingly, both the gene sets were also negatively enriched in sorted AML-MSCs when cocultured with leukaemia but not control cells. To validate these findings, we analysed OCR and EACR in WT-MSCs in an identical setting. The oxidative phosphorylation was significantly decreased in MSCs cocultured with C1498 (p<0.0001) and MLL-AF9 (p<0.005) but not with Lin - cells. Interestingly, glycolysis rate, glucose consumption, lactate secretion were significantly decreased in MSCs cocultured with leukaemia cells. Mitochondrial DNA copy number were significantly decreased in MSCs cocultured with C1498 (p<0.001) or MLL-AF9 (p<0.005) but not with control cells.Recent evidence from the lab has demonstrated an essential role for Notch signalling in the leukaemia and AML-MSCs interaction. To functionally determine the crosstalk of leukaemia-MSC interaction and subsequent Notch signalling, we ectopically expressed the Notch intracellular domain (Notch-ICN1) to mimic Notch activation in a murine stromal cell line, MS-5. Confirming Notch activation, Hes1 mRNA expression (encoding a transcriptional target of Notch signalling) was significantly increased in these cells. Underscoring a role for Notch signalling and activation, Notch-ICN1 overexpression in MS-5 cells demonstrated less oxidative phosphorylation and glycolysis rates as compared to MS-5 cells transduced with empty vector.Conclusion: In line with our microarray and GSEA analysis, our findings confirmed that leukaemia cells indeed induced metabolic alterations decreasing oxidative phosphorylation and glycolysis, and thereby potentially altering AML-MSCs function. At the molecular level, Notch signalling (via upregulated Notch1 and 2 expressions and Notch-ICN) in AML-MSCs contributed to metabolic alterations. Therefore, therapeutically interfering this pathway could target the bidirectional interaction between leukaemia and AML-MSCs improving therapeutic efficacy of AML. DisclosuresKhandanpour: GSK: Honoraria; Takeda: Honoraria; Janssen: Honoraria; AstraZeneca: Honoraria, Research Funding; Pfizer: Honoraria; Sanofi: Honoraria, Research Funding; BMS/Celgene: Honoraria.