Fig 2: In t(4;11) Cells, RUNX1 Is Highly Expressed and Has High Levels of ENL and AF9 Bound to the Locus(A) Real-time PCR quantification (see gene expression analysis in Extended Experimental Procedures) of HOXA9 (top), HOXA10 (middle), and RUNX1 (bottom) gene expression in patient cell lines. The cell lines analyzed are: RS4;11 (t-4;11), SEM (t-4;11), MV4-11 (t-4;11), THP-1 (MLL-AF9), NOMO-1 (MLL-AF9), MONO-MAC1 (MLL-AF9), KOPN-8 (MLL-ENL), ML-2 (MLL-AF6 and an MLL deletion), SHI-1 (MLL-AF6), RCH-ACV (normal MLL), CCRF-CEM (normal MLL), JURKAT (normal MLL), and K562 (normal MLL). Error bars represent the ±SD of two independent experiments. ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; N.D., not detected.(B) Western blot of RUNX1 in the cell lines as described in (A) with a short exposure (top panel) and a long exposure (middle panel).(C) Western blot of nuclear extracts in the cell lines indicated and probed with the antibodies as indicated.(D) MLL-N, AF4-C, ENL, AF9, and Cyclin T1 ChIP in RS4;11 (dark red bars), SEM (spotted red bars), MV4-11 (bright red bars), THP-1 (black bars), KOPN-8 (blue bars), and CCRF-CEM (gray bars) patient cell lines. The control primer set is from a random gene-poor region on human chromosome 8; otherwise, primer sets are as indicated in Figures 1B, 1C, and 2A. Error bars represent the ±SD of two independent experiments.See also Figure S3.

Fig 3: RUNX1 Expression Is Important for t(4;11) But Not MLL-ENL Growth, Related to Figure 5(A) Real Time PCR expression of RUNX1 in SEM (t-4;11) or KOPN-8 (MLL-ENL) cells treated with either a non-targeting control siRNA or a RUNX1 siRNA (#1). Error bars represent the ± SD of three separate PCR reactions.(B and C) Cell counts of the control (blue line) or RUNX1 siRNA (red line) treated cells from (A) in SEM (B) or KOPN-8 (C) cells over ~5 days. Error bars represent the ± SD of two independent experiments.

Fig 4: Nuclear Pfn1 interacts with the SEC(A) IP of YFP or YFP-NLS-Pfn1(WT or S137D) from nuclear extracts of MDA-MB-231 stable cells by a GFP antibody, followed by silver staining.(B) Eluates from (A) were analyzed by liquid chromatography-MS. Proteins specifically bound to NLS-Pfn1(WT) were identified using label-free quantification (LFQ) intensity and a threshold of >1.5-fold-higher intensity over those bound to YFP and NLS-Pfn1(S137D). LFQ intensities of detectable SEC components are shown.(C) Confirmation of the interaction between NLS-Pfn1(WT) and SEC by anti-GFP co-IP as in (B) using nuclear extracts of stable MCF-7 cells, followed by western blot.(D) Interaction between the SEC and endogenous Pfn1 in MCF-7 cells. ENL and cyclin T1 were pulled down followed by western blot.(E) Cyclin T1 pulldown from shLUC- versus shENL-infected MCF-7 cells, followed by western blot for endogenous Pfn1 and other proteins. In total, 50-fold-less input was used for the Pfn1 blot given the small fraction of nuclear Pfn1 (most in the cytoplasm) interacting with the SEC.(F) Size-exclusion chromatography and western blot analyses of nuclear extracts of MDA-MB-231 cells for endogenous Pfn1 and SEC components.Results in (C)–(F) were confirmed by three independent experiments. Lanes in (A), (D), and (E) were cropped and rearranged from the same blots for clarity of presentation (indicated by the black lines). See also Table S2.

Fig 5: MLL-translocation cell lines are sensitive to inhibition of pan-Cullin-dependent and CKS1-dependent protein degradation. (A) Western blots for THP-1 (MLL-AF9) and KOPN-8 (MLL-ENL) cells treated with vehicle control (DMSO), 0.1 µM MLN4924 or 1 µM C1 for 16 h and 24 h. Histone H3 was used as a loading control. Western blots are representative of 3 independent experiments. (B) Cell viability was assessed for THP-1, KOPN-8, healthy PBMC and CD34+ control cells after 3 days treatment with indicated concentrations of the pan-Cullin inhibitor (MLN4924) or the SKP2-CKS1 inhibitor (C1). Results represent the mean of 3 independent experiments with standard deviation bars. (C) Proportion of apoptotic CD34+, THP-1 and KOPN-8 cells, in response to two different concentrations of C1 or MLN4924, was measured by PI and Annexin V-FITC staining using flow cytometry. Significance was tested using the Student's t-test versus DMSO treated cells (N = 3). (D) Cell cycle status was measured for KOPN-8 cells in response to DMSO vehicle control or MLN4924 (1 µM) using the Click-iT EdU incorporation kit. Graphs are representative of 3 independent experiments.