Fig. 5. Azacytidine leads to increased differentiation in ASXL1 mutant cells.

A Due to the increased methylation in the ASXL1 mutant cells, we treated the isogenic KBM5 cells with the DNMT inhibitor 5-azacytidine. Proliferation assays of the ASXL1 mutant cells and homozygous corrected cells treated with 5-Azacytidine revealed significantly decreased cell viability of the ASXL1 mutant cells, particularly when treated with 1 μM at 72 and 96 h. B Flow cytometric analysis of the isogenic KBM5 cells treated with 1 μM 5-Azacytidine for 96 h demonstrated increased differentiation with increased expression of CD11b and CD14 in the treated vs. untreated ASXL1 mutant cells. Furthermore, there is increased expression of CD11b and CD14 in the treated ASXL1 mutant cells compared to the untreated homozygous corrected cells. C Representative images of Giemsa stained untreated and 5-Azacytidine treated ASXL1 mutant cells and untreated and 5-Azacytidine treated homozygous corrected cells. Red arrows identify cells with decreased nucleus to cytoplasm ratio and condensed nuclei, consistent with myeloid maturation/differentiation. D Proliferation assays of the ASXL1 mutant and homozygous corrected cells treated with the combination of Venetoclax and 5-Azacytidine revealed significantly decreased cell viability of the ASXL1 mutant cells.