Figure 5.

Cooperative gene induction through promoter-enhancer activation. A, Heat map of RNA-seq data depicting candidate genes preferentially upregulated by GSK-LSD1+5Aza treatment (log₂ FC ≥1.5, RPKM ≥5) that were not passing the threshold by monotherapy treatment alone in patient-derived TET2^mut AML1566 cells at day 8 (n=3 per treatment). Gene expression changes relative to total average were ranked by hypo-DMCs at promoters. B, Competitive proliferation experiment in TET2^mut AML cells with overexpression of candidate genes (DLX2, FUCA1, GATA2, GFI1, LSP1, and TYROBP) and GFP control. Shown is the ratio of candidate gene-GFP⁺ fraction compared to empty-GFP⁺ relative to the initial measurement. Error bars represent means ± s.d. from three replicate plates. C, Shown are TET2^mut+DNMT3A^wt AML samples (red) vs non-responder samples (black) that exhibited less additional impairment after combination therapy compared to GSK-LSD1 monotherapy. D, Log₂ fold change (FC) of gene expression of GATA2, GFI1 and DLX2 in TET2^mut+DNMT3A^wt (red, n=3) and non-responder AMLs (black, n=3) after treatment rel. to vehicle condition. qRT-PCR analysis normalized to COX6B1. Asterisk declares combination therapy as significant for the target gene compared to both monotherapies (*P≤0.05, Student’s t-test, means of triplicate measurements ± s.d.). E, Integrative scheme of ChIP-seq and ERRBS at the GATA2 locus in patient-derived TET2^mut AML cells. Upper panel showing CpG methylation changes after treatment (rel. to vehicle) as well as DMCs after combinatorial treatment vs. GSK-LSD1 monotherapy. Lower panel showing ChIP-seq tracks for H3K4me1, H3K27ac, and LSD1 at the distal GATA2 enhancer (DG2E). F, G, QChIP for H3K4me1 (left panel), H3K4me2 (middle panel) and H3K27ac (right panel) at a non-target (F) and LSD1-bound DG2E (F) site. Results of each treatment at day 8 presented as percentage of its input from three independent experiments (Student’s t-test; *P<0.05, ** P<0.01). H, Q3C performed to detect chromatin-chromatin interactions of the GATA2 promoter with DG2E as well as with down- and upstream intergenic regions. Q3C results from two independent experiments performed in TET2^mut AML cells after treatment with vehicle condition set as 1 on day 8 (Student’s t-test; *P<0.05, ** P<0.01). I, QPCR results for GATA2 after combination therapy on day 8 in CRISPRi-transduced TET2^mut AML cells containing two sgRNA against DG2E and a non-target ctrl sgRNA (means of n=3 with s.d.; Student’s t-test; **P<0.01). J, Proposed concept of the dual targeting in AML responsive to combinatorial treatment. Removal of 5mC promoter methylation by 5Aza treatment combined with LSD1 inhibition (GSK-LSD1) facilitates stronger interactions of the LSD1-occupied enhancers with the target promoter resulting in greater induction of target genes such as GATA2.