Fig. 6: BRD9 inhibition exhibits anti-proliferative and pro-apoptotic effects in SF3B1-mutated cell lines.

A Dose-response analysis of I-BRD9, PROTAC BRD9 Degrader-1, and dBRD9 treatments in 3 SF3B1^WT cell lines, MEC1, PGA1, and HG3 (all CLL), and 3 SF3B1^MUT cell lines, CII (CLL), HNT34 (AML), and MEL202 (UVM). The cell lines were treated with drug concentrations ranging from 0.001 to 50 µM for 3 days, and cell viability was determined by CellTiter-Glo 2.0. Complete cell killing was exclusively observed with I-BRD9 treatment, allowing for the determination of corresponding IC₅₀ values for each cell line. The HNT34 cell line exhibited sensitivity to all three drugs, thereby allowing for the determination of IC₅₀ values for all conditions. Dose-response curves are shown with 95% confidence intervals, while individual dots display the mean values of triplicates, with error bars representing the standard deviation. B I-BRD9 sensitivity profile in 958 cancer cell lines from GDSC [39]. C Assessment of proliferation in an SF3B1^WT cell line, PGA1, and SF3B1^MUT cell lines, CII and HNT34, upon treatment with 10 µM I-BRD9, 25 µM PROTAC BRD9 Degrader-1, or 25 µM dBRD9. Cell lines were treated for 3 days with a 5-hour exposure to BrdU at the end, and proliferation was quantified by flow cytometry, measured as BrdU+ cells. Vehicle (DMSO) was used as the negative control for drug treatment, while cells not exposed to BrdU served as the negative control to assess the specificity of the anti-BrdU antibody. The upper left quadrants (BrdU+ cells) in the density plots represent the percentages of proliferating cells. D Bar plot displaying differences in the percentages of proliferating cells compared to negative controls with corresponding P values (one-way ANOVA). The bar plot displays the mean values from two repeated experiments, with error bars representing the standard deviation. E Assessment of apoptosis in the same cell lines and the identical samples as in (C). Treatment with 5 µM Camptothecin and vehicle (DMSO) were used as the positive and negative controls, respectively. Apoptosis was evaluated by Annexin V/PI staining and subsequent flow cytometry analysis. The lower left (Annexin V-/PI- cells), upper left (Annexin V+/PI- cells), and upper right (Annexin V+/PI+ cells) quadrants in the density plots represent the percentages of viable, early apoptotic, and late apoptotic cells, respectively. F Stacked bar plot displaying differences in the percentages of viable, early apoptotic, and late apoptotic cells compared to negative controls with corresponding P values (one-way ANOVA). The bar plot displays the mean values from two repeated experiments, with error bars representing the standard deviation. G Bar plot showing cell viability differences as determined by CellTiter-Glo 2.0 in the same cell lines under the same experimental conditions as in panels (C, E) with corresponding P values (one-way ANOVA). The bar plot displays the mean values from two repeated experiments in triplicates, with error bars representing the standard deviation. WT: wildtype; MUT: mutated; IC₅₀: half-maximal inhibitory concentration; RLU: relative luminescence unit.