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Despite advances in treating acute lymphoblastic leukemia (ALL), resistance to asparaginase (ASNase) remains a major clinical hurdle. Recent evidence suggests that leukemic cells may evade ASNase-induced cytotoxicity by upregulating the glutamate/aspartate transporter EAAT1 (SLC1A3). This study aimed to evaluate whether pharmacological inhibition of EAAT1 using UCPH-101 could enhance the inhibitory effects of ASNase in T-ALL models.

Using patient-derived samples and established T-ALL cell lines (MOLT-4 and Jurkat) we assessed the effects of ASNase and UCPH-101, both alone and in combination. Combinations were designed based on IC₅₀-based fixed-ratio dosing, including sub-IC₅₀ and supra-IC₅₀ concentrations. Effects on cell viability, apoptosis, and apoptosis related gene expression were evaluated using standardized in vitro assays. Synergy was evaluated using the combination index (CI) method on the Chou-Talalay model.

Synergy analysis based on CI values revealed synergistic interactions (CI < 1) at sub and supra IC₅₀ concentrations of both agents in MOLT-4 and Jurkat cells. Dual targeting of amino acid metabolism through ASNase and EAAT1 inhibition resulted in significantly reduced cell viability and increased apoptotic cell death compared to controls. Combinatorial treatment led to significant alterations in the expression of key apoptotic regulators, suggesting a disruption of metabolic adaptation mechanisms in both leukemic cell lines.

These findings demonstrate that EAAT1 inhibition augments ASNase anti-leukemic activity in preclinical ALL models, revealing a metabolic vulnerability that can be exploited therapeutically. The mechanistic insights reported herein support further exploration of EAAT1-targeted strategies to improve outcomes in T-ALL.