Introduction E-twenty-six (ETS) specific transcription factors are a family of Ͼ 20 helix-loop-helix domain transcription factors that have been implicated in a myriad of cellular processes, including hematopoiesis. 1 The hallmark ETS factor protein involved in hematopoietic development is SPI1 (Spleen focus forming virus Proviral Integration site 1; PU.1), which activates gene expression during myeloid and B-lymphoid cell development. Other ETS factors include the 2 closely related transcriptional activator proteins ERG (Ets Related Gene) and FLI1 (Friend Leukemia virus Integration site 1), which both play crucial roles in hematopoietic development 2,3 and multiple forms of cancer. 4,5 Recently, SPI1 was identified as a binding partner of the PML-RAR-␣ oncofusion protein complex in an inducible overexpression model. 6 The PML-RAR-␣ oncofusion protein is the result of a translocation t(15;17)(q22;q21) involving the promyelocytic leukemia (PML) gene on chromosome 15 and the retinoic acid receptor-␣ (RAR-␣) on chromosome 17. 7,8 Another translocation, t(8;21)(q22;q22), is present in ϳ 10% of all de novo acute myeloid leukemia (AML) cases, and results in the expression of the AML1-ETO (RUNX1-RUNX1T1) oncofusion protein. Expression of the AML1-ETO oncofusion protein in hematopoietic cells results in a stage-specific arrest of maturation and increased cell survival, predisposing cells to develop leukemia. 9 At the molecular level RUNX1 (Runt-related transcription factor 1; AML1, CBFA2) represents a DNA-binding transcriptional activator factor required for hematopoiesis, 10,11 while ETO (eight-twenty-one; MTG8, RUNX1T1) acts as a corepressor molecule. 12 The t(8;21) translocation replaces the transactivation domain of RUNX1/AML1 with the almost complete ETO protein, thereby converting an essential transcriptional activator into a strong repressor. 13,14 Here we extend genome-wide AML1-ETO studies 15,16 and reveal that a subset of AML1-ETO binding sites are bound by CBF-␤ (core binding factor-␤), whereas nearly all are bound by HEB (HeLa E-box-binding factor), RUNX1/AML1 as well as by the ETS factors ERG and FLI1. Subsequent analysis in t(8;21) cells revealed cell type specific ETS factor binding and preferential AML1-ETO binding to the cell type specific ETS factor binding sites, suggesting that these proteins facilitate oncofusion protein binding. In addition, we uncovered that binding of the ETS factors correlates with the "active" histone acetylation mark. Together, our results suggest that ETS factors demarcate hematopoietic regulatory sites that provide a target for (aberrant) epigenetic regulation by oncofusion proteins. Methods ChIPChromatin was harvested as described. 17 ChIPs were performed using specific antibodies to ETO, HEB, ERG, FLI1 (Santa Cruz Biotechnology), H3K9K14ac, AML1-ETO, ETO, CBF-␤, RNAPII (Diagenode), RUNX1, and FLI1 (Abcam), and H4panAc (Millipore) and analyzed by quantitative PCR or ChIP-seq. Primers for quantitative PCR are described in supplemental Methods (available on the Blood Web site...

Different mechanisms for CBFb-MYH11 function in acute myeloid leukemia with inv(16) have been proposed such as tethering of RUNX1 outside the nucleus, interference with transcription factor complex assembly and recruitment of histone deacetylases, all resulting in transcriptional repression of RUNX1 target genes. Here, through genome-wide CBFb-MYH11-binding site analysis and quantitative interaction proteomics, we found that CBFb-MYH11 localizes to RUNX1 occupied promoters, where it interacts with TAL1, FLI1 and TBP-associated factors (TAFs) in the context of the hematopoietic transcription factors ERG, GATA2 and PU.1/SPI1 and the coregulators EP300 and HDAC1. Transcriptional analysis revealed that upon fusion protein knockdown, a small subset of the CBFb-MYH11 target genes show increased expression, confirming a role in transcriptional repression. However, the majority of CBFb-MYH11 target genes, including genes implicated in hematopoietic stem cell self-renewal such as ID1, LMO1 and JAG1, are actively transcribed and repressed upon fusion protein knockdown. Together these results suggest an essential role for CBFb-MYH11 in regulating the expression of genes involved in maintaining a stem cell phenotype.

Despite the fact that loss of E-cadherin is causal to the development and progression of invasive lobular carcinoma (ILC), options to treat this major breast cancer subtype are limited if tumours develop resistance to anti-oestrogen treatment regimens. This study aimed to identify clinically targetable pathways that are aberrantly active downstream of E-cadherin loss in ILC. Using a combination of reverse-phase protein array (RPPA) analyses, mRNA sequencing, conditioned medium growth assays and CRISPR/Cas9-based knock-out experiments, we demonstrate that E-cadherin loss causes increased responsiveness to autocrine growth factor receptor (GFR)-dependent activation of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt signalling. Autocrine activation of GFR signalling and its downstream PI3K/Akt hub was independent of oncogenic mutations in PIK3CA, AKT1 or PTEN. Analyses of human ILC samples confirmed growth factor production and pathway activity. Pharmacological inhibition of Akt using AZD5363 or MK2206 resulted in robust inhibition of cell growth and survival of ILC cells, and impeded tumour growth in a mouse ILC model. Because E-cadherin loss evokes hypersensitisation of PI3K/Akt activation independent of oncogenic mutations in this pathway, we propose clinical intervention of PI3K/Akt in ILC based on functional E-cadherin inactivation, irrespective of activating pathway mutations.