Untargeted Metabolomics Identifies Glutamine Metabolism As a Driver of Chemoresistance in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is one of the most challenging cancers to treat. Induction chemotherapy (iCT) remains the standard of care, but the incidence of refractory and relapsed AML is high. Unraveling the molecular regulation of this chemoresistance is critical to provide new treatment options for patients. Cellular metabolism plays a central role in the control of cell fate, and a dysregulated metabolism is now widely accepted as a hallmark of many cancers. We hypothesized that chemoresistance in AML arises at the time of maximal iCT response, with the residual cells manifesting distinctive metabolic features that enable their survival under the extreme stress of chemotherapy.

To test this hypothesis, we used a mouse model of aggressive, MLL-AF9-driven AML that allows real-time monitoring of leukemic burden through bioluminescence imaging, and therefore identification of the moment of maximal response to iCT. We isolated GFP-expressing AML cells from bone marrow of mice receiving treatment with vehicle or iCT (cytarabine for 5 days and doxorubicin for the first 3 days), both at the moment of maximal response (3 days after last dose of iCT) and after relapse (10 days after last dose). We then developed a platform for untargeted metabolomics analysis of freshly sorted cells, which allows us to measure the levels of more than 670 metabolites using 500,000 cells per sample. Amongst the top hits were multiple metabolites involved in glutamine metabolism, including glutamine, glutamate, aspartate and pyroglutamate. All of these metabolites were low in vehicle-treated cells, very high in the residual cells at the moment of maximal response to iCT, and low again in cells after relapse, suggesting a dynamic role for glutamine metabolism in the immediate stress response to iCT. Analysis of the transcriptomic profile of the cells (RNAseq) showed that expression of the majority of genes encoding for enzymes involved in glutamine metabolism did not differ between groups. In contrast, mRNA levels of several glutamine transporters were increased in chemoresistant AML cells. To confirm that glutamine metabolism plays a functional role in protecting AML cells from chemotherapy, we treated AML cells in culture with iCT in combination with 6-diazo-5-oxo-L-norleucine (DON), a glutamine analog and antagonist that inhibits all glutamine-dependent enzymes. At several concentrations synergism between DON and iCT was observed, proving that activation of glutamine metabolism confers chemoresistance in AML cells.

These results highlight the power of using untargeted metabolomics to uncover novel chemoprotective metabolic pathways, and underscore the uniqueness of our approach as glutamine metabolism would not have been picked up through transcriptomics analysis alone. In addition, we identified several currently unknown metabolites, of which levels differed significantly in chemoresistant AML cells, that will be further investigated in future studies. Taken together, our findings provide insight into the metabolic programs that determine chemoresistance in vivo and indicate that targeting glutamine metabolism may provide a basis for overcoming chemoresistance in AML.