Fig 1: 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.
Fig 2: CRISPR/Cas9-mediated correction of the ASXL1 mutation leads to increased myeloid differentiation.A The ASXL1 gene is made up of 12 exons with the ASXL1G710X mutation in KBM5 cells located in exon 12. RNA-sequencing demonstrates CRISPR/Cas9-mediated correction of the ASXL1G710X mutation in the leukemic KBM5 cell line. For the ASXL1 mutant cells, the red bar demonstrates the presence of the ASXL1 mutation at amino acid location 710, exon 12, chromosome 20. For the corrected cells, the red bar is absent at amino acid location 710, exon 12, chromosome 20, demonstrating correction of the ASXL1 mutation. In addition, the different colored bars in the corrected trace demonstrate silent nucleotide changes that were introduced to avoid undesired Cas9 activity in the mutation-corrected cells. B RNA-sequencing demonstrates that correction of the ASXL1G710X mutation in the KBM5 cells by CRISPR/Cas9 gene-editing results in decreased expression of the HOXA9 gene. Increased expression of ITGAM/CD11b is demonstrated in the corrected isogenic cell line, suggesting increased myeloid differentiation. C? Flow cytometry demonstrates increased expression of three myeloid surface markers, CD11b, CD14, and CD15, in the corrected cell line compared to the ASXL1 mutant cells, demonstrating increased myeloid differentiation with correction of the ASXL1G710X mutation. Representative plots are shown. D Representative images of Giemsa stained ASXL1 mutant cells and those with homozygous correction of the ASXL1G710X mutation. Red arrows identify cells with decreased nucleus to cytoplasm ratio and condensed nuclei, consistent with myeloid maturation/differentiation.
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