Fig 2: AXIN1 stabilization normalizes β-catenin and partially restores cell cycle control in RUNX1-depleted cells.(a) MCF7 cells constitutively expressing shRx13′-UTR (shRx1) were treated for 36 h with either 5 μM IWR1 or its dimethyl sulphoxide vehicle followed by western blot analysis of the indicated proteins. MCF7 expressing a nonspecific shRNA (shNS) were analysed as a reference control. (b) Cells as in a were treated as indicated for 6 days and their growth rate was calculated based on MTT assays as in Fig. 2b. *P<0.05 by t-test. (c) AXIN1 and P-β-cat levels were assessed 8 h after the release of MCF7/shRx1RUNTdox cells from a G1/S double thymidine block as in Fig. 6h. Dox treatment (to silence RUNX1) initiated along with the release from the first thymidine block and IWR1 treatment (to stabilize AXIN1) initiated 17 h before harvest. (d) MCF7/shRx1RUNTdox cells were treated for 72 h with dox (to silence RUNX1) and 2 nM docetaxel for 48 h (to induce mitotic slippage) as in Fig. 6f,g and IWR1 was added for the last 24 h before FACS analysis. Data are mean±s.e.m. (n=3). *P<0.05 by t-test. (e) Working model for the tumour suppressor function of RUNX1 in ER+ breast cancer, whereby RUNX1 prevents E2-mediated AXIN1 suppression. Mechanisms linking the RUNX1/AXIN1/β–catenin axis to loss of cell cycle control in RUNX1-deficient ER+ mammary epithelial cells remain to be fully elucidated. They entail stimulation of neither LEF/TCF, nor c-MYC, nor CCND1, nor G1/S phase transition, but are associated instead with deregulated mitosis.

Fig 3: Association between RUNX1 and AXIN1 in ER+ breast cancer tumours.(a) Correlation between the RUNX1 inhibitory index and AXIN1 mRNA in the breast cancer patient cohort of TCGA (ref. 20). The RUNX1 inhibitory index was calculated for each tumour as described in the ‘Methods' section and the correlation with AXIN1 mRNA (UCSC isoform uc002cgp.1) was calculated. Bars represent the Pearson linear correlations (r) and P values were determined based on r and the sample size N. (b) Breast cancer tumour microarray TMA-1007 from Protein Biotechnologies, Inc. was immunostained for RUNX1 and AXIN1. The ER+ invasive ductal carcinomas were designated as positive or negative for RUNX1 and AXIN1. Data represent the odds ratio and the 95% confidence intervals for the association between AXIN1 status and RUNX1 status in tumours expressing ERα at either low (ERlow) or high levels (ERhigh). Association between the RUNX1 status and AXIN1 status was tested using the Pearson chi-square test for the 2 × 2 table, for ERlow and ERhigh tumours separately. Odds ratios for the ERlow and ERhigh tumours were compared using the Breslow–Day test for homogeneity of odds ratios. (c) RUNX1 and AXIN1 immunohistochemical staining of two ERhigh tumours from the TMA illustrating the association between RUNX1 and AXIN1 expression (Magnification × 20; scale bar, 50 μm; insets show the 1.1 mm cores in their entirety).

Fig 4: The HDAC Inhibitor MS275 Induces Inflammatory Signatures in SCN iPSC-Derived CD34+CD45+ Cells(A and B) GSEAs comparing CD34+CD45+CSF3R-d715 RUNX1-RHD cells from ELANE mutant SCN cells treated with DMSO or the HDAC inhibitor MS275, derived from two independent experiments (biological replicates), showing (A) induced inflammatory signaling and (B) reduced proliferative signatures in MS275-treated cells.See also Figure S5.

Fig 5: Recruitment of RUNX1 and ERα to adjacent elements in AXIN1.(a) The genome-wide response to RUNX1 was determined as described in the ‘Methods' section by comparing the mRNA profiles of MCF7/shRx13′-UTR and MCF7/shNS cells maintained in CSS±E2. The Venn diagram describes the RUNX1 responsiveness (number of genes with fold change >1.5 and P<0.05) in the presence and absence of E2. (b) RUNX1-occupied regions (R1ORs) were determined by ChIP-seq analysis of MCF7 cells and mapped with respect to the TSSs of the 599 genes that responded to RUNX1 in the presence of E2. For 111 out of these 599 genes, R1ORs were found between positions −500 kb and +500 kb of the respective TSSs, and these R1ORs (a total of 176) were enumerated in 10-kb bins. Grey area represents the results of 1,000 iterations with control random gene sets. The brown line is a density curve of R1OR occurrence generated by LOESS fitting to R1OR counts (α=0.25). Gene names are depicted in cases where R1ORs overlapped with ER-occupied regions deduced from GSE14644 (ref. 47). (c) Screen shots of RUNX1 and ERα ChIP-seq data for the AXIN1 locus, with zoom-in on ERα and RUNX1 peaks at a region co-occupied by the two transcription factors. (d) ChIP-qPCR confirmation of RUNX1 and ERα occupancy at the second intron of AXIN1. Data obtained with antibodies against RUNX1 (left) and ERα (right) are represented by brown and black bars, respectively, and data obtained with control IgG is represented by grey bars. *P<0.05 by t-test. (e) Endogenous RUNX1 in MCF7 cells was silenced by shRx13′-UTR and the indicated wild-type or mutant FLAG-tagged RUNX1 proteins were induced by dox as in Fig. 2b,d. AXIN1 expression was assessed by RT–qPCR. Data was corrected for 18S RNA and represent mean±s.e.m. from triplicate experiments. *P<0.05 by t-test. Insets represent western blot analyses of the exogenous FLAG-tagged proteins.