Fig. 4: SF3B1 mutation sensitizes K562 cells to UBA1 inhibition.

A Dose-response curves of SF3B1^WT and SF3B1^K700E K562 cells treated with TAK-243 or DMSO for 72 h (n = 3). Chemical structure of the small-molecule UBA1 inhibitor TAK-243 (MLN7243) displayed under the curve. Data points represent mean ± SEM live cell fractions, normalized to control-treated samples. Interpolated sigmoidal, four parameter logistic (4PL), standard curves of mean (solid line) and 95% confidence interval (dotted line). Live cells were defined as Aqua/Apotracker Green double negative singlets, assessed by flow cytometry (see supplementary Fig. 5A for gating strategy). IC₅₀ of TAK-243 are quantified in B. Mean ± SEM nM TAK-243. Unpaired t-test. C Immunoblot analysis and D quantification of UBA1a/b protein levels in SF3B1^WT and SF3B1^K700E K562 cells 72 h after transfection with negative control siRNA (control) or UBA1 siRNA (siUBA1). Actin was used as a loading control and results were normalized to the highest relative signal. Mean ± SEM relative UBA1a/b signals. Two-way ANOVA with Tukey’s multiple comparisons test. E Dose-response curves of SF3B1^WT and SF3B1^K700E K562 cells 72 h after transfection with negative control siRNA (control) or UBA1 siRNA (siUBA1), treated with TAK-243 or DMSO for 72 h (n = 3). Data points represent mean ± SEM live cell fractions, normalized to control-treated samples. Interpolated sigmoidal, 4PL, standard curves of mean (solid line) and 95% confidence interval (dotted line). Live cells were defined as Aqua/Apotracker Green double negative singlets, assessed by flow cytometry. IC₅₀ of TAK-243 are quantified in F. Mean ± SEM nM TAK-243. Two-way ANOVA with Tukey’s multiple comparisons test. G Experimental strategy to assess the effect of targeted UBA1 inhibition by TAK-243 treatment on SF3B1^WT and SF3B1^K700E cells. H SF3B1^K700E VAF in co-cultures of 1:1 SF3B1^WT and SF3B1^K700E K562 cells after 72 h of treatment with 50 nM TAK-243 or DMSO, as determined by ddPCR (n = 3). Mean ± SEM. Unpaired t-test. I CFU counts per 1000 seeded SF3B1^WT and SF3B1^K700E K562 cells treated with indicated concentrations of TAK-243 for 7 days (n^DMSO = 7, n^{15 nM} = 5, n^{32 nM} = 3; n^{50 nM} = 4). Mean ± SEM. Two-way ANOVA with Dunnett’s multiple comparisons test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ns not significant.