Fig 2: Cellular assays showing Cpd-1 inhibited the PPIs between AF9/ENL and DOT1L, AF4 or AFF4. (A) Co-immunoprecipitation using an antibody against the N-terminal MLL shows treatment of Molm-13 cells with Cpd-1 for 2 days dose-dependently disrupted the PPIs between MLL-AF9 and DOT1L or AF4; Upon incubation for 3 days, (B) Cpd-1 reduced the nuclear levels of DOT1L, H3K79me1 and me2, ENL and AFF4 in Molm-13 cells, while inactive Cpd-3 did not. A DOT1L inhibitor EPZ4777 only reduced those of H3K79me1 and me2, but not other proteins; (C) Cpd-1 reduced the nuclear protein levels of H3K79me1 and me2 and ENL in MV4;11 cells.

Fig 3: Trithorax homology domain 2 (THD2)-mediated functions promote MLL-fusion-dependent leukemic transformation.(A) Structure/function analysis of MLL-ELL. Various MLL-ELL constructs were examined for the transformation of myeloid progenitors. HA-tag (h: indicated as a red triangle) was fused to MTM and MTMT constructs. A schema of myeloid progenitor transformation assay is shown on top. Hoxa9 expression normalized to Gapdh in first-round and second-round colonies (left) is shown as the relative value of MLL-ELL (arbitrarily set at 100%) with error bars (mean ± SD of PCR triplicates). Colony-forming ability at third- and fourth-round passages (right) is shown with error bars (mean ± SD of =3 biological replicates). IBD: integrase-binding domain; hMBM: high-affinity MENIN-binding motif; LBD: LEDGF-binding domain; THD1/2: trithorax homology domains 1 and 2; SET: Suver3-9/enhancer-of-zeste/trithorax motif; TED: transcription elongation domain; OHD: occludin homology domain. (B) Requirement of THD2 for various MLL fusion proteins in leukemic transformation. Various MLL fusion constructs were examined for the transformation of myeloid progenitors as in (A). RA1/2: RAS association domains 1/2; PDZ: PSD-95/Dlg/ZO-1 domain; OM: octapeptide motif; LZ: leucine zipper. MISD: minimum interaction site of DOT1L: YEATS: Yaf9 ENL AF9 Taf14 Sas5 domain; AHD: ANC1 homology domain. (C) Colony morphologies of MTMTh or MTMh-ENL' transformed cells. The colonies on day 5 of fourth passage are shown with a scale bar. The ratio of compact colonies (=100 total colonies were counted in each experiment) is shown on the left (mean ± SD of six biological replicates). Representative images are shown on the right.

Fig 4: Ser5-P, Ser2-P, CDK9, negative elongation factor (NELF), and super-elongation complex (SEC) subunits on the Ad5 genome.(a, top) Global Run-On sequencing in cells expressing wt E1A; (bottom) ChIP-seq of C-terminal domain (CTD) Ser5-P, CTD Ser2-P, CDK9, and NELF. (b) CDK9, AF9, ENL, and BRD4 ChIP-seq in cells expressing wt or DM E1A. AF9 and ENL ChIP-seq data are plotted with two different y-axes. (c) Average plots of AF9 and ENL binding near transcription start sites on the human genome.

Fig 5: A-485-sensitive promoter H3K18/27ac and high super-elongation complex association at 2XPI genes.(a) Average H3K18ac, H3K27ac, and H3K9ac near transcription start sites (TSSs) for all active genes, 2XPI genes, and 2Xin genes after DMSO (dimethyl sulfoxide) or A-485 treatment. (b) Metagene plots of AF9 and ENL ChIP-seq counts across all active genes and 2XPI genes after DMSO (dimethyl sulfoxide) or A-485 treatment. (c) Average BRD4 near TSSs for all active genes and 2XPI genes after DMSO (dimethyl sulfoxide) or A-485 treatment.