Fig. 5: Cellular activity of ASH1L inhibitor AS-99.

a, b Titration curves from the MTT cell viability assay performed after 7 days of treatment of human MLL1 rearranged (MLL1-r) leukemia cell lines (MV4;11, MOLM13, KOPN8, RS4;11) and control leukemia cell lines, non–MLL1-r (K562 and SET2) with AS-99 (a) or AS-nc (b); mean ± SD, n = 4 biological replicates. Representative graphs are shown from 2–3 independent experiments. GI₅₀ values correspond to AS-99 concentrations required to achieve 50% inhibition of cell proliferation. c Colony counts and representative images of colonies from the colony-forming assay performed with normal human hematopoietic CD34⁺ cells isolated from cord blood treated for 7 days with DMSO or AS-99; mean ± SD, n = 2. The experiment was performed twice. Representative data is shown. d Quantification of CD11B expression in human leukemia cells treated for 7 days with AS-99, detected by flow cytometry; mean ± SD, n = 3 biological replicates. P values (MV4;11: 6 µM: 0.0021, 4.5 µM: 0.026, 3 µM: 0.028, 1.5 µM: 0.73); KOPN8 (4 µM <0.0001, 3 µM: 0.0012, 2 µM: <0.0001, 1 µM: 0.055); K562 (8 µM: 0.78, 6 µM: 0.070, 4 µM: 0.055, 2 µM: 0.081) were calculated using unpaired 2-tailed t test. Two independent experiments were performed for each cell line in triplicates. Representative graphs are shown. Gating strategy is presented in Supplementary Fig. 8c. e Wright-Giemsa–stained cytospins for MV4;11 and KOPN8 cells after 7 days of treatment with DMSO or AS-99: in MV4;11 at 6 µM and in KOPN8 at 4 µM. f Flow cytometry analysis of apoptosis induced by AS-99 in MV4;11, KOPN8, and K562 cells after 7 days of treatment. Mean ± SD, n = 3 biological replicates. P values (MV4;11: 6 µM: 0.026, 4.5 µM: 0.049, 3 µM: 0.65, 1.5 µM: 0.70; KOPN8: 4 µM: < 0.0001, 3 µM: 0.012, 2 µM: 0.010, 1 µM: 0.24; K562: 8 µM: 0.31, 6 µM: 0.004, 4 µM: 0.006, 2 µM: 0.39) were calculated using unpaired 2-tailed t test. Two independent experiments were performed in triplicates. Representative graphs are shown. *P < 0.05; **P < 0.01; ****P < 0.0001; NS not significant. Gating strategy is presented in Supplementary Fig. 8d. g CUT&RUN experiment in MV4;11 cells treated with AS-99 (5.5 µM) or DMSO showing H3K36me2 peaks. The rows show the RPKM (Reads Per Kilobase Million) values on the peaks (normalized to 1 kb) and 4 kb regions flanking the peaks. Peaks were sorted by total normalized signals of DMSO-treated cells within each category. Violin plots show the RPKM values of each peak in DMSO and AS-99 treated cells. The boxes represent 25th (minima) and 75th (maxima) percentile and the lines represent the median. The high and low whisker ends are the largest values within 1.5-times interquartile range above 75th and the smallest value within 1.5-times interquartile range below the 25th percentile, respectively. Mann–Whitney two-sided U test was used for statistical analysis to compare the difference of signals between DMSO- and AS-99-treated cells.