Fig. 2: Compound DW0254 inhibits RAC activation and shows anti-leukemic activity in vitro in leukemia cell lines.

A Chemical structure, physicochemical properties, and biological activities cell line for compounds 1–4 and NSC23766 (structure not shown), a known inhibitor of RAC. IC₅₀ values represent the dose at which 50% cell viability was achieved on P12-ICHIKAWA cells. B GTP-RAC activity inhibition in P12-ICHIKAWA cells treated with different doses of DW0254 for 3 h. GST pulldown assays were conducted by incubating lysates with PAK1-PBD beads. Cell lysates to detect total RAC and proteins eluted from the PAK1-PBD beads to detect GTP-RAC were subjected to Western blotting using anti-RAC (610651, BD Transduction laboratories, San Jose, CA) and anti-beta ACTIN (A5441, Sigma-Aldrich) antibodies. Data are representative of three individual experiments. C Representative peaks of Far Red CellTrace staining of P12-ICHIKAWA cells treated with different doses of DW0254 and examined by FACS on three consecutive days. Peaks 1–4 represent the number of times the cells in each peak have divided; data shown from one of the three independent experiments. D Bar graph showing percentage of apoptosis by AnnV/PI staining of P12-ICHIKAWA cells treated for 3 days with different doses of DW0254, data represent mean ± SD of two independent experiments with n = 3 samples for each condition. Live: AnnV⁻/PI⁻; Early apoptosis: AnnV⁺/PI⁻; Late apoptosis: AnnV⁺/PI⁺; and Dead: AnnV⁻/PI⁺. E Drug dosage curve showing live cell viability assay after 3 days of DW0254 treatment of human ALL and F AML cell lines with diverse backgrounds and RAS status as described, n = 4 at each dosage, data show mean ± SD, one of three individual experiments showing the consistent results. Color code: WT RAS green, G12 mutant RAS blue, Q61 mutant RAS burgundy, other RAS mutations yellow. G GTP-RAC activity inhibition in a panel of T-ALL and AML cell lines treated with 50 µM DW0254 for 1 h.