Fig 2: MLL-AF4 Directly Regulates RUNX1 and Other Target Genes by Stabilizing AF9 and ENL Binding(A) MLL, MLL-AF4, HOXA9, and RUNX1 real-time PCR expression in scrambled control siRNA-treated cells (black bars), MLL-AF4 siRNA-treated SEM (gray bars), and RS4;11 (white bars) cells. Data are the mean ± SD (error bars) of three independent knockdown experiments. In each individual experiment, control values were set to 1.(B and C) Western blots as indicated in SEM cells (B) or RS4;11 cells (C) treated with the siRNAs as indicated. Proteins were detected using the antibodies indicated except MLL-AF4, which was detected with an AF4-C antibody.(D) A summary of AF4 protein interactions.(E) MLL-N, AF4-C, AFF4, ENL, AF9, and Cyclin T1 ChIP + real-time PCR with scrambled control versus MLL-AF4 siRNA-treated SEM cells from (A). Values and error bars represent the mean ± SD of at least two independent ChIP experiments. Primer sets are as in Figure 1B, 1C, and 2A.

Fig 3: PKM2 controls RUNX1 stability.A Gene set enrichment analysis (GSEA) of proteins the in PKM2-depleted U937 and NB4 cells measured by MS. B Heatmaps of levels of the indicated transcription factors in NB4 or U937 cells in which endogenous PKM2 was stably knocked out and WT PKM2 (WT) or K270R mutant (KR) were expressed. C, D NB4, U937, and 32DBCR-ABL rescue cell lines were subjected to western blots and the indicated proteins were detected. E The mRNA of RUNX1 was detected by quantitative real-time PCR in NB4, U937, and 32DBCR-ABL rescue cell lines. F Western blot analysis of RUNX1 in U937 cells transfected with shRNA targeting PKM2 and treated with MG132 (2 mM for 4 h) as indicated. G Western blot analysis of whole cell lysates derived from U937 cells stably expressing shRNA empty vector (EV) or shPKM2 and treated with cycloheximide (CHX, 20 mg/mL) for the indicated time points. ß-actin was analyzed as an internal loading control. H U937 rescue cell lines were treated with MG132 (2 mM for 4 h) and IP assay were performed using antibody against RUNX1 followed by western blot analysis.

Fig 4: The transcriptional function of CBFB is critical for breast tumor suppression. a A Venn diagram showing genes (identified by RNAseq) co-regulated by CBFB and RUNX1. b Kaplan-Meier curve showing 10-gene signature upregulated by CBFB and RUNX1 predicts disease-free survival of breast cancer patients. c IB showing the upregulation of Notch intracellular domain (NICD) of NOTCH3 in CBFB_KO and RUNX1_KO cells. d ChIPseq of RUNX1 showing the binding of RUNX1 on the NOTCH3 locus. e Anchorage independent growth assays of double knockout (DKO) of CBFB (C) and NOTCH3 (N) or DKO of RUNX1(R) and NOTCH3(N). n = 3 (biological); two asterisks, p value < 0.01 (single KO vs. double KO comparisons, two-tailed t test). f Representative IHC images showing the protein levels of CBFB, RUNX1, and NOTCH3 in human breast tumors and normal tissues. g IB showing the effect of exogenous expression of CBFB on RUNX1 and NOTCH3 expression in MCF7 and BT474 cells. h Weight and (i) volume of orthotopically transplanted tumors from MCF7 cells that were transduced with an empty vector (EV) or a lentiviral vector expressing CBFB (CBFB). Tumor incidence is shown on the top

Fig 5: CBFB and hnRNPK regulate translation. a Flow chart showing the in vitro translation assay to test the effect of a translation element (RNA) and a protein. In vitro transcribed RNA was used in the in vitro translation assays using HeLa cells lysate. b In vitro translation assay using the 1-Step Human coupled IVT kit showing the effect of CBFB and hnRNPK binding sites (T14) on RUNX1 translation. c In vitro translation assays using the 1-Step Human coupled IVT showing effect of recombinant CBFB (rCBFB) and hnRNPK (rhnRNPK) on RUNX1 translation. d In vitro translation assay using WT and CBFB_KO HEK 293 T cells lysate. See “Methods” section for details of preparation of 293 T cell lysate. e In vitro translation assay. Un-transfected (control) and hnRNPK knockdown (KD) HEK 293T cells lysate replaced the HeLa cell lysate in the 1-Step Human coupled IVT kit with or without the supplement of rhnRNPK. f IB showing the effect of CBFB deletion on the proteins encoded by bound transcripts. g IB showing the effect of hnRNPK KD on the proteins encoded by bound transcripts