RUNX1-ETO Depletion in t(8;21) AML Leads to C/EBPα- and AP-1-Mediated Alterations in Enhancer-Promoter Interaction.

Acute myeloid leukemia (AML) is associated with mutations in transcriptional and epigenetic regulator genes impairing myeloid differentiation. The t(8;21)(q22;q22) translocation generates the RUNX1-ETO fusion protein, which interferes with the hematopoietic master regulator RUNX1. We previously showed that the maintenance of t(8;21) AML is dependent on RUNX1-ETO expression. Its depletion causes extensive changes in transcription factor binding, as well as gene expression, and initiates myeloid differentiation. However, how these processes are connected within a gene regulatory network is unclear. To address this question, we performed Promoter-Capture Hi-C assays, with or without RUNX1-ETO depletion and assigned interacting cis-regulatory elements to their respective genes. To construct a RUNX1-ETO-dependent gene regulatory network maintaining AML, we integrated cis-regulatory element interactions with gene expression and transcription factor binding data. This analysis shows that RUNX1-ETO participates in cis-regulatory element interactions. However, differential interactions following RUNX1-ETO depletion are driven by alterations in the binding of RUNX1-ETO-regulated transcription factors.

Ptasinska A ，Pickin A ，Assi SA ，Chin PS ，Ames L ，Avellino R ，Gröschel S ，Delwel R ，Cockerill PN ，Osborne CS ，Bonifer C ... -  《Cell Reports》

Depletion of RUNX1/ETO in t(8;21) AML cells leads to genome-wide changes in chromatin structure and transcription factor binding.

Ptasinska A ，Assi SA ，Mannari D ，James SR ，Williamson D ，Dunne J ，Hoogenkamp M ，Wu M ，Care M ，McNeill H ，Cauchy P ，Cullen M ，Tooze RM ，Tenen DG ，Young BD ，Cockerill PN ，Westhead DR ，Heidenreich O ，Bonifer C ... -  《-》

AML1/ETO trans-activates c-KIT expression through the long range interaction between promoter and intronic enhancer.

The AML1/ETO onco-fusion protein is crucial for the genesis of t(8;21) acute myeloid leukemia (AML) and is well documented as a transcriptional repressor through dominant-negative effect. However, little is known about the transactivation mechanism of AML1/ETO. Through large cohort of patient's expression level data analysis and a series of experimental validation, we report here that AML1/ETO transactivates c-KIT expression through directly binding to and mediating the long-range interaction between the promoter and intronic enhancer regions of c-KIT. Gene expression analyses verify that c-KIT expression is significantly high in t(8;21) AML. Further ChIP-seq analysis and motif scanning identify two regulatory regions located in the promoter and intronic enhancer region of c-KIT, respectively. Both regions are enriched by co-factors of AML1/ETO, such as AML1, CEBPe, c-Jun, and c-Fos. Further luciferase reporter assays show that AML1/ETO trans-activates c-KIT promoter activity through directly recognizing the AML1 motif and the co-existence of co-factors. The induction of c-KIT promoter activity is reinforced with the existence of intronic enhancer region. Furthermore, ChIP-3C-qPCR assays verify that AML1/ETO mediates the formation of DNA-looping between the c-KIT promoter and intronic enhancer region through the long-range interaction. Collectively, our data uncover a novel transcriptional activity mechanism of AML1/ETO and enrich our knowledge of the onco-fusion protein mediated transcription regulation.

Tian Y ，Wang G ，Hu Q ，Xiao X ，Chen S ... -  《-》

The Hematopoietic Transcription Factors RUNX1 and ERG Prevent AML1-ETO Oncogene Overexpression and Onset of the Apoptosis Program in t(8;21) AMLs.

The t(8;21) acute myeloid leukemia (AML)-associated oncoprotein AML1-ETO disrupts normal hematopoietic differentiation. Here, we have investigated its effects on the transcriptome and epigenome in t(8,21) patient cells. AML1-ETO binding was found at promoter regions of active genes with high levels of histone acetylation but also at distal elements characterized by low acetylation levels and binding of the hematopoietic transcription factors LYL1 and LMO2. In contrast, ERG, FLI1, TAL1, and RUNX1 bind at all AML1-ETO-occupied regulatory regions, including those of the AML1-ETO gene itself, suggesting their involvement in regulating AML1-ETO expression levels. While expression of AML1-ETO in myeloid differentiated induced pluripotent stem cells (iPSCs) induces leukemic characteristics, overexpression increases cell death. We find that expression of wild-type transcription factors RUNX1 and ERG in AML is required to prevent this oncogene overexpression. Together our results show that the interplay of the epigenome and transcription factors prevents apoptosis in t(8;21) AML cells.

Mandoli A ，Singh AA ，Prange KHM ，Tijchon E ，Oerlemans M ，Dirks R ，Ter Huurne M ，Wierenga ATJ ，Janssen-Megens EM ，Berentsen K ，Sharifi N ，Kim B ，Matarese F ，Nguyen LN ，Hubner NC ，Rao NA ，van den Akker E ，Altucci L ，Vellenga E ，Stunnenberg HG ，Martens JHA ... -  《Cell Reports》

RUNX1/ETO and mutant KIT both contribute to programming the transcriptional and chromatin landscape in t(8;21) acute myeloid leukemia.

Acute myeloid leukemia development occurs in a stepwise fashion whereby an original driver mutation is followed by additional mutations. The first type of mutations tends to be in genes encoding members of the epigenetic/transcription regulatory machinery (i.e., RUNX1, DNMT3A, TET2), while the secondary mutations often involve genes encoding members of signaling pathways that cause uncontrolled growth of such cells such as the growth factor receptors c-KIT of FLT3. Patients usually present with both types of mutations, but it is currently unclear how both mutational events shape the epigenome in developing AML cells. To this end we generated an in vitro model of t(8;21) AML by expressing its driver oncoprotein RUNX1-ETO with or without a mutated (N822K) KIT protein. Expression of N822K-c-KIT strongly increases the self-renewal capacity of RUNX1-ETO-expressing cells. Global analysis of gene expression changes and alterations in the epigenome revealed that N822K-c-KIT expression profoundly influences the open chromatin landscape and transcription factor binding. However, our experiments also revealed that double mutant cells still differ from their patient-derived counterparts, highlighting the importance of studying patient cells to obtain a true picture of how gene regulatory networks have been reprogrammed during tumorigenesis.

Chin PS ，Assi SA ，Ptasinska A ，Imperato MR ，Cockerill PN ，Bonifer C ... -  《-》