Characteristic Amino Acid and Energy Metabolism in AML Stem Cells As Revealed By Quantitative Multiplex Proteomics

Acute Myeloid Leukemia (AML) is a rapidly progressing hematologic malignancy characterized by the accumulation of clonal myeloid progenitor cells arrested in their ability to differentiate into mature blood cells. While classic chemotherapy regimens lead to remission in the majority of patients, relapse rates are very high. Relapse and also refractoriness are caused by the hierarchical organization of AML with a minor fraction of leukemic stem cells (LSC) at the apex generating leukemic progeny, which make up the majority of leukemic cells. LSC harbor self-renewal activity, relative quiescence, resistance to apoptosis, and increased drug efflux that likely render them less susceptible to conventional therapies aimed at the bulk proliferative disease. From a clinical perspective, the leukemic stem cell model implies that in order to eradicate the disease and achieve long-term remissions, treatment courses must eliminate the LSC population.

To identify putative therapeutic target structures and/or pathways selectively aiming at LSC we performed high-resolution proteomic analyses of LSC and non-LSC populations of primary AML patient samples and healthy control subjects. To identify functional LSCs, we fractionated AML patient samples according to CD34 and CD38 surface expression by fluorescent-activated cell sorting and transplanted the resulting four populations per sample into immunocompromized NSG mice. Leukemic populations, which were able to initiate the AML in mice were labeled LSCs, while engraftment failures were considered non-LSCs. Six AML patients containing both LSC and non-LSC fractions were chosen for proteomic analysis. As healthy controls, CD34+CD38- hematopoietic stem and progenitor cells (HSPC) populations obtained from elderly patients without hematologic conditions undergoing hip replacement surgery were analyzed. We performed in-depth quantitative multiplex proteomic analysis employing tandem mass tag labeling and high-resolution mass spectrometry and identified more than 7,200 proteins. Our analysis revealed between 1,097 and 1,937 differentially expressed proteins when comparing LSC with non-LSC populations for each AML sample. Gene set enrichment analyses (GSEA) showed a significant enrichment for DNA replication, cell cycle, ribosome biogenesis, and protein translation in non-LSC populations, in agreement with a more quiescent state of LSC.

Next to Branched Chain Amino Acid degradation, which we have recently shown to control the activity of alpha-ketoglutarate dependent dioxygenases such as TET2 and EGLN1 in AML stem cells (Raffel et al., (2017). Nature 551(7680), 384-388) other metabolic processes including lipid metabolism and oxidative phosphorylation were enriched in LSC. When compared to healthy HSPCs, especially oxidative phosphorylation, RNA processing and cell adhesion were over-represented in LSC. We will present proteins differentially expressed and pathways enriched in LSCs versus non-LSCs. These may help to understand the mechanism of LSC self-renewal and function and simultaneously represent putative novel targets to eliminate leukemic stem cells in clinical settings.