KSR2 is a novel oncogene in TAL1 subtype of T-cell acute lymphoblastic leukemia with potential clinical and prognostic implications - Supplementary Methods

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Supplementary Materials and Methods for "KSR2 is a novel oncogene in TAL1 subtype of T-cell acute lymphoblastic leukemia with potential clinical and prognostic implications" accepted for publication in Haematologica (2026) The published article includes an abbreviated Methods section due to journal word limits. This document provides the full methodological details required for transparency and reproducibility and accompanies the associated publication. T-cell acute lymphoblastic leukemia (T-ALL) is a rare hematological malignancy characterized by substantial molecular heterogeneity and aggressiveness manifested by relatively high incidence of relapse and drug resistance. Despite the growing understanding of T-ALL biology and increasing cure rates, novel oncogenes with biomarker and therapeutic potential are needed to recognize high-risk patients and improve therapy effectiveness. We demonstrate that KSR2 is overexpressed in a subgroup of T-ALL patients and that its expression is linked to TAL1 molecular subtype of this malignancy. We show that the ectopic KSR2 expression results from demethylation of its regulatory region followed by direct binding of TAL1 transcription factor. We demonstrate that the KSR2 expression is a potential marker of unfavorable outcome in T-ALL. We show that KSR2 provides growth advantage in T-ALL in vitro, indicating its oncogenic activity. In addition, we observe a dose-dependent cytotoxic effect of APS-2-79, a small molecule KSR2 inhibitor, in T-ALL cell lines expressing KSR2. Finally, we show that KSR2 is involved in metabolic processes, such as glycolysis and biosynthesis of lipids and proteins and that this effect is mediated by supporting AMPK signaling. Therefore, we propose KSR2 as a novel oncogene with prognostic biomarker potential and druggable vulnerability in T-ALL.