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Cellular biology of mature B-cell lymphomas (Master thesis)

Σμαροπούλου, Μαρία/ Smaropoulou, Maria/ Μεταφραστική Έρευνα στη Μοριακή Βιολογία και Γενετική

Chronic lymphocyEc leukemia (CLL) is a mature B-cell malignancy characterized by pronounced clinical and biological heterogeneity. A defining feature of CLL is the expression of “stereotyped” B-cell receptors (BcR), which classify paEents into subsets with shared immunogeneEc, molecular and clinical characterisEcs. While differences in signaling responses between these subsets have been extensively documented, the contribuEon of metabolic reprogramming to their disEnct biological behavior remains largely unexplored. In this study, we invesEgated the metabolic and signaling profiles of three major CLL stereotyped subsets, namely #1, #2 and #4, which represent diverse clinical phenotypes. Peripheral blood mononuclear cells (PBMCs) were isolated from 20 CLL paEents and sEmulated in vitro with anE-IgM and/or CpG. Flow cytometry was used to assess the acEvaEon markers CD25 and CD86, and Western blo ng was performed to analyze the expression of key metabolic and signaling proteins, including HIF-1α, EGLN3, BCAT1, LAT1, p70S6K, and phospho-p70S6K. In addiEon, intracellular metabolites, such as glutamine, glutamate, glucose, lactate, branched-chain amino acids (BCAAs) and D-2-hydroxyglutarate (D-2-HG) were quanEfied through targeted biochemical assays and high-performance liquid chromatography (HPLC). Subset #1 displayed a pronounced anabolic profile characterized by elevated LAT1 and BCAT1 expression and increased mTORC1 acEvity, without a corresponding increase in glucose consumpEon or lactate producEon, indicaEng coordinated acEvaEon of amino acid metabolism and upregulaEon of oxidaEve phosphorylaEon. However, subset #1 showed a tendency toward higher glucose uptake compared with the other subsets. Subset #2 exhibited moderate metabolic acEvaEon with intermediate levels of mTOR signaling, while no glucose uptake and lactate producEon were detected, supporEng a predominantly non-glycolyEc metabolic program. Subset #4 displayed a metabolically quiescent state marked by high glutamine retenEon and low responsiveness to sEmulaEon, consistent with B-cell anergy. Notably, subset #1 demonstrated elevated HIF-1α despite higher EGLN3, which suggests funcEonal inhibiEon of the prolyl-hydroxylase pathway, consistent with a pseudohypoxic state. Together, these findings reveal subset-specific metabolic hierarchies that integrate amino acid transport, mTOR acEvaEon, and hypoxia-associated signaling. This work provides new insights into the metabolic basis of CLL heterogeneity and idenEfies potenEal therapeuEc vulnerabiliEes within metabolically acEve and clinicallyc aggressive CLL subsets.