Fig. 2: Knockout of LSD1 alters chromatin accessibility, augments H3K27Ac occupancy on PU.1 target and WNT-β-Catenin target genes, and knockout of GFI1 induces myeloid differentiation genes, LY96 and ITGAM (CD11b) and morphologic differentiation of AML cells.
From: Superior efficacy of co-targeting GFI1/KDM1A and BRD4 against AML and post-MPN secondary AML cells
A Log2 fold-change in H3K27Ac ChIP-Seq peaks (TSS + 3 kb or gene body + 10 kb) for PU.1 target genes (from the MSigDB database) in LSD1 knockout compared to Cas9-only-expressing OCI-AML5 cells. B Log2 fold-change in H3K27Ac ChIP-Seq peaks (TSS + 3 kb or gene body + 10 kb) in WNT-β-Catenin target genes (from HALLMARK datasets in the MSigDB database) in LSD1 knockout compared to Cas9-only-expressing OCI-AML5 cells. C RNA-Seq analysis was performed on OCI-AML5 LSD1 knockout (biologic triplicates) and Cas9-only-expressing (biologic duplicates) cells 8 days post-transduction. Heat map shows the number of mRNAs depleted or induced > 1.25-fold and p-value < 0.05 in the LSD1 knockout versus Cas9-only-expressing cells. D Gene set enrichment analysis was performed with RNA expression changes in OCI-AML5 LSD1-KO cells compared to GO pathways. The q-values are < 0.1 in all comparisons. E Representative immunoblot analysis of OCI-AML5 Cas9-expressing cells transduced with two lentiviral sgRNAs against GFI1 and incubated for 8 days. F Relative mRNA expression analysis of OCI-AML5 GFI1 knockout cells compared to Cas9-only-expressing cells. GAPDH expression was utilized as the normalization control. G Expression of CD11b (assessed by flow cytometry) in OCI-AML5-Cas9 and GFI1 knockout cells 8 days post-transduction. Mean of two independent experiments performed in duplicate + S.D. Significance determined by a two-tailed, unpaired t-test. H Morphologic differentiation (% myelocytes and metamyelocytes or bands by morphologic features of hematoxylin & eosin-stained cytospun cells) of OCI-AML5 GFI1 knockout cells compared to Cas9-only-expressing cells 8 days post-transduction. Mean of two independent experiments performed in duplicate + S.D. Significance determined by a two-tailed, unpaired t-test in GraphPad V8.
