Page 64 - JCTR-11-5

Journal of Clinical and
Translational Research Metabolism of healthy and leukemic stem cells

progression after chemotherapy-based treatment in mouse concentrations, including glycolytic, FAO, or TCA cycle
models of AML. 112,113 Thus, it remains unclear if HIF-1 is a intermediates, as well as amino acids, nucleotides, lipids,
feasible therapeutic target. and small molecules or drugs, at specific time points.
Other genomic alterations contribute to Techniques such as liquid chromatography–MS (LC-
chemoresistance through well-established mechanisms. MS) and gas chromatography–MS (GC-MS) are commonly
Activating mutations in metabolic enzymes such as used for metabolite separation and detection 115,116
isocitrate dehydrogenase (IDH) 1 and IDH2 lead to the (Figure 3). LC-MS separates metabolites using a liquid
accumulation of the oncometabolite 2-hydroxyglutarate mobile phase and a stationary phase based on properties
(2-HG). The presence of 2-HG induces hypermethylation such as charge, polarity, or hydrophilicity, whereas
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of specific genes, preventing cellular differentiation and GC-MS uses a gas mobile phase to separate compounds
triggering widespread epigenetic changes. In addition, according to volatility and boiling point. These targeted
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2-HG upregulates B-cell lymphoma 2 (BCL-2), conferring MS-based methods are especially valuable for examining
resistance to apoptosis and increasing sensitivity to BCL-2 the metabolic profiles of quiescent and active HSCs or
inhibitors. Overall, elevated BCL-2 expression is crucial LSCs, where subtle changes in glycolysis, OXPHOS,
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for LSC survival, supporting a resting, low-ROS state that FAO, or amino acid metabolism can influence stemness,
depends on OXPHOS for energy production. 112 activation, proliferation, or therapeutic resistance.
4. Metabolomic approaches However, a major limitation of these approaches is the
low abundance of HSC and LSC populations within the
Recent advances in metabolomic technologies have BM. 123,124 This rarity makes it difficult to obtain sufficient
enabled more precise and comprehensive studies of cellular material for MS, often necessitating strategies
metabolism in HSCs and LSCs. Metabolomics allows such as pooling samples from multiple donors to achieve
for highly precise measurements of small molecules and the required input for metabolomic analyses or
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metabolites that reflect the metabolic state at the cellular extensive cell sorting for metabolic flux measurements.
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or tissue level. 15,16,114 These measurements provide critical Consequently, the feasibility of single-sample untargeted
insights into processes that regulate energy production, workflows in these rare cell populations remains a
biosynthesis, redox balance, and metabolism, thereby significant challenge.
enabling thorough characterization of the metabolic states Untargeted metabolomics is a bottom-up approach that
of quiescent and active HSCs as well as LSCs. This is
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particularly important because both HSCs and LSCs rely focuses on identifying and quantifying potentially 100 or
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on distinct metabolic patterns and pathways (Table 1). 1000 metabolites along with novel metabolic features.
This technique allows researchers to identify and analyze
In addition, metabolic profiling can be used to shifts in the metabolic profile and metabolic pathways,
identify diagnostic biomarkers, reveal alterations in as well as the effects of different treatments on the entire
metabolic pathways associated with treatment, monitor system. This technique enabled the identification of
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therapeutic responses, and uncover vulnerabilities that chemotherapy-induced metabolic shifts in osteosarcoma
may be exploited for the development of novel therapies stem cells using untargeted metabolomics by LC-MS that
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(Figure 3). To better understand the metabolic profile of would not have been detected by targeted metabolomics.
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HSCs and LSCs, researchers employ technologies such A prominent example is the identification of 2-HG,
as mass spectrometry (MS), single-cell and multi-omics which accumulates in IDH1/2-mutant AML and serves
approaches, and live-cell metabolic assays. as both a key biomarker and a direct therapeutic target.
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Similarly, analyses of CML stem cells have identified
4.1. Targeted versus untargeted metabolomics
distinct metabolic enzyme signatures compared to normal
One widely used technology to study metabolism is MS, HSCs, highlighting novel vulnerabilities for therapeutic
an analytical technique that ionizes chemical compounds exploitation. It should be noted that choosing between
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and measures their mass-to-charge (m/z) ratios with high targeted and untargeted approaches ultimately relies on
sensitivity and throughput. Metabolomic analysis using the biological question, as both are suitable for testing
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MS typically employs two main approaches: targeted and hypotheses. In the context of exploring the metabolic
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untargeted metabolomics. Targeted metabolomics focuses profile of HSCs and LSCs, either method can be used to
on quantifying a specific set of metabolites using known map metabolic changes associated with HSC activation
standards for each compound under investigation, thereby or oncogenic transformation. Furthermore, these
providing a quantitative snapshot of the metabolome. 121,122 technologies provide foundational data for enhancing our
This approach enables profiling of metabolite understanding of HSC and LSC metabolism.

Volume 11 Issue 5 (2025) 58 doi: 10.36922/JCTR025320053