Figure 3.

Disrupting the MLL-AF9 and DOT1L interaction has the same consequences on leukemogenesis as enzymatic inactivation of DOT1L. (A) H3K79me2 for Days 1–3 of the proliferation assay showing a slower decrease in protein-protein inhibition cell lines than enzymatically inactivated or empty vector controls (representative data from two or more independent experiments). (B) ChIP-qPCR for H3K79me2 at the HoxA9 and Meis1 promoters plotted relative to H3 for each cell line confirmed the decrease in H3K79me2 in the MigR1, Δ 10, I867A and RCR cells in comparison to WT (representative data from two independent experiments with technical triplicates). (C) The abundance of MLL target genes Meis1 and (D) HoxA9 mRNA is significantly reduced in 4-OHT-treated cells in comparison to EtOH-treated controls in the MigR1, Δ10, I867A, and RCR-transduced cells. Expression of these genes was not decreased in the WT control cell line when comparing treated and non-treated cells (n = 3). (E) Wright-Giemsa stain of cells after 4-OHT treatment Wright-Giemsa stain of cells after 4-OHT treatment (Olympus IX83 Inverted Microscope; Original magnification ×400) and corresponding flow cytometry for cell differentiation marker CD14 and progenitor marker c-Kit, showing cell differentiation in MigR1, Δ10, I867A, and RCR cells while WT cells retain their blast-like morphology and cell surface markers (representative data from four independent experiments).Significance key calculated using a t-test in Prism 6 software (ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001).