ABSTRACT Leukemia, a hematological malignancy resulting from genetic mutations and environmental factors, leads to uncontrolled proliferation and impaired differentiation of hematopoietic cells. Current treatment modalities face limitations such as drug resistance and off‐target toxicity, highlighting the need for novel therapeutic strategies. This study evaluates the individual and fixed‐dose combined in vitro effects of boric acid (BA) and Myeloid Ecotropic Viral Integration Site 1 Inhibitor‐2 (Meis1i‐2) in leukemia cell lines, focusing on apoptosis, cell‐cycle distribution, and selected transcript‐level changes. Three distinct leukemia cell lines (K562, RS4‐11, CCRF‐CEM) were cultured under standardized conditions and treated with varying concentrations of BA and Meis1i‐2 individually and in combination. Apoptotic cell death was evaluated using flow cytometry‐based Annexin V–FITC/PI staining, while cell cycle dynamics were assessed via Hoechst 33342 and Pyronin Y staining followed by flow cytometric analysis. Additionally, quantitative RT‐PCR (qRT‐PCR) was employed to determine the expression levels of apoptosis‐related and cell cycle regulatory genes. At the 72‐h endpoint, BA and Meis1i‐2 produced measurable changes in viability, apoptosis, and cell‐cycle distribution in a cell‐line‐ and concentration‐dependent manner. The high‐dose fixed combination of BA (2000 µM) and Meis1i‐2 (10 µM) reduced viability compared with DMSO controls in several assays; however, this condition was above the RS4‐11 IC 50 values and should be interpreted as a supra‐IC 50 exploratory condition rather than evidence of superiority over the most active single agent. Annexin V/PI and cell‐cycle analyses showed treatment‐associated apoptotic changes and redistribution of G1, S, and G2/M phases depending on the cell line. Targeted qRT‐PCR indicated transcript‐level modulation of selected apoptosis‐ and cell‐cycle‐related genes, but these changes were not uniform across all models. These findings reveal that BA and Meis1i‐2 exhibit biologically measurable antileukemic effects through apoptosis induction and cell cycle modulation. However, Chou‐Talalay analysis in RS4‐11 cells yielded CI values greater than 1 at the tested concentrations, suggesting an antagonistic rather than non‐synergistic under the tested conditions. Therefore, under the current experimental conditions, the combination demonstrates concentration‐specific antileukemic activity rather than pharmacological interaction. Future studies using expanded sub‐IC 50 dose matrices, protein‐level and functional validation, and in vivo models are required.
Meriç et al. (Mon,) studied this question.