Evaluation of metabolic vulnerabilities of Early T-cell Progenitor Acute Lymphoblastic Leukaemia (ETP ALL) cells

T-cell acute lymphoblastic leukaemia (T-ALL) is a rare and aggressive tumour originating from the malignant transformation of T-lineage progenitor cells. Among its subtypes, Early T-cell Precursor (ETP) ALL is distinguished by an immature immunophenotype, a transcriptional program closer to that of hematopoietic stem cells, and a poor clinical prognosis due to therapy resistance and high relapse rates. Unlike typical T-ALL, ETP ALL harbours a distinct genetic landscape, with frequent alterations in genes involved in hematopoiesis and myeloid signaling, and fewer classic T-ALL mutations such as those in NOTCH1 and CDKN2A/B. In light of the distinct biological and clinical characteristics of ETP ALL, the aim of this thesis was to explore the possible metabolic differences between ETP ALL and non-ETP ALL subtypes. To this end, a previously generated metabolomic dataset was analysed using the MetaboAnalyst platform, incorporating the Kyoto Encyclopedia of Genes and Genomes (KEGG) database to identify metabolites and metabolic pathways differentially utilized between the two groups. Among the most significantly altered pathways were glutathione, taurine, and cysteine metabolism—each crucial in counteracting oxidative stress. These findings indicate extensive metabolic reprogramming in ETP ALL cells, particularly involving redox homeostasis, and suggest a potential metabolic vulnerability in this aggressive T-ALL subset. Given the close link between glutathione metabolism and ferroptosis—a form of regulated cell death driven by oxidative stress—we evaluated the expression of ferroptosis-related genes in ETP (CUTLL3, LOUCY, and PEER) versus non-ETP (e.g., MOLT-4, JURKAT E6, DND41, HPB-ALL, HSB-2, KOPTK1, RPMI 8402, P12 ICHIKAWA, PF382, CCRF-CEM, and CUTLL1) T-ALL cell lines. Flow cytometric analyses quantified key redox-related parameters, including oxidized lipids, cytoplasmic and mitochondrial reactive oxygen species (ROS), reduced glutathione (GSH), and intracellular ferrous iron. ETP ALL cell lines showed elevated lipid peroxidation and a trend toward increased mitochondrial ROS levels, suggesting a heightened redox vulnerability, particularly affecting membranes and mitochondrial function. Western blot analyses further revealed elevated expression of oxidative stress and ferroptosis-related proteins (e.g., xCT/SLC7A11, NRF2, HO-1, and GPX4) in ETP ALL cells, supporting the presence of an altered oxidative state. Subsequently, we assessed the effects of ferroptosis-inducing agents—Buthionine sulfoximine (BSO), RSL3, and Erastin—on cell viability. While both ETP and non-ETP T-ALL cells were resistant to BSO, ETP ALL cells showed significantly greater sensitivity to RSL3 and Erastin, consistent with the protein expression profiles. These findings highlight a functional correlation between ferroptosis-related gene expression and susceptibility to oxidative stress inducers in ETP ALL, uncovering a potential therapeutic vulnerability in this high-risk T-ALL subgroup.