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Members of the TALE (three-amino-acid loop extension) family of atypical homeodomain-containing transcription factors are important downstream effectors of oncogenic fusion proteins involving the mixed lineage leukemia (MLL) gene. A well-characterized member of this protein family is MEIS1, which orchestrates a transcriptional program required for the maintenance of MLL-rearranged acute myeloid leukemia (AML). TGIF1/TGIF2 are relatively uncharacterized TALE transcription factors, which, in contrast to the remaining family, have been shown to act as transcriptional repressors. Given the general importance of this family in malignant hematopoiesis, we therefore tested the potential function of TGIF1 in the maintenance of MLL-rearranged AML. Gene expression analysis of MLL-rearranged patient blasts demonstrated reduced TGIF1 levels, and, in accordance, we find that forced expression of TGIF1 in MLL-AF9-transformed cells promoted differentiation and cell cycle exit in vitro, and delayed leukemic onset in vivo. Mechanistically, we show that TGIF1 interferes with a MEIS1-dependent transcriptional program by associating with MEIS1-bound regions in a competitive manner and that the MEIS1:TGIF1 ratio influence the clinical outcome. Collectively, these findings demonstrate that TALE family members can act both positively and negatively on transcriptional programs responsible for leukemic maintenance and provide novel insights into the regulatory gene expression circuitries in MLL-rearranged AML.

C/EBPα collaborates with MLL-ENL to activate a group of genes that, together with Hoxa9 and Meis1, are responsible for the early events that transforms normal hematopoietic cells into leukemic cells, MLL-fusion proteins are potent inducers of oncogenic transformation, and their expression is considered to be the main oncogenic driving force in ∼10% of human acute myeloid leukemia (AML) patients. These oncogenic fusion proteins are responsible for the initiation of a downstream transcriptional program leading to the expression of factors such as MEIS1 and HOXA9, which in turn can replace MLL-fusion proteins in overexpression experiments. To what extent MLL fusion proteins act on their own during tumor initiation, or if they collaborate with other transcriptional regulators, is unclear. Here, we have compared gene expression profiles from human MLL-rearranged AML to normal progenitors and identified the myeloid tumor suppressor C/EBPα as a putative collaborator in MLL-rearranged AML. Interestingly, we find that deletion of Cebpa rendered murine hematopoietic progenitors completely resistant to MLL-ENL–induced leukemic transformation, whereas C/EBPα was dispensable in already established AMLs. Furthermore, we show that Cebpa-deficient granulocytic-monocytic progenitors were equally resistant to transformation and that C/EBPα collaborates with MLL-ENL in the induction of a transcriptional program, which is also apparent in human AML. Thus, our studies demonstrate a key role of C/EBPα in MLL fusion–driven transformation and find that it sharply demarcates tumor initiation and maintenance.

The process of blood formation, haematopoiesis, depends upon a small number of haematopoietic stem cells (HSCs) that reside in the bone marrow. Differentiation of HSCs is characterised by decreased expression of genes associated with self-renewal accompanied by a stepwise activation of genes promoting differentiation. Lineage branching is further directed by groups of cooperating and counteracting genes forming complex networks of lineage-specific transcription factors. Imbalances in such networks can result in blockage of differentiation, lineage reprogramming and malignant transformation. CCAAT/enhancer-binding protein-α (C/EBPα) was originally identified 30 years ago as a transcription factor that binds both promoter and enhancer regions. Most of the early work focused on the role of C/EBPα in regulating transcriptional processes as well as on its functions in key differentiation processes during liver, adipogenic and haematopoietic development. Specifically, C/EBPα was shown to control differentiation by its ability to coordinate transcriptional output with cell cycle progression. Later, its role as an important tumour suppressor, mainly in acute myeloid leukaemia (AML), was recognised and has been the focus of intense studies by a number of investigators. More recent work has revisited the role of C/EBPα in normal haematopoiesis, especially its function in HSCs, and also started to provide more mechanistic insights into its role in normal and malignant haematopoiesis. In particular, the differential actions of C/EBPα isoforms, as well as its importance in chromatin remodelling and cellular reprogramming, are beginning to be elucidated. Finally, recent work has also shed light on the dichotomous function of C/EBPα in AML by demonstrating its ability to act as both a tumour suppressor and promoter. In the present review, we will summarise the current knowledge on the functions of C/EBPα during normal and malignant haematopoiesis with special emphasis on the recent work.

The balance between self-renewal and differentiation is crucial for the maintenance of hematopoietic stem cells (HSCs). Whereas numerous gene regulatory factors have been shown to control HSC self-renewal or drive their differentiation, we have relatively few insights into transcription factors that serve to restrict HSC differentiation. In the present work, we identify ETS (E-twenty-six)-related gene (ERG) as a critical factor protecting HSCs from differentiation. Specifically, loss of Erg accelerates HSC differentiation by >20-fold, thus leading to rapid depletion of immunophenotypic and functional HSCs. Molecularly, we could demonstrate that ERG, in addition to promoting the expression of HSC self-renewal genes, also represses a group of MYC targets, thereby explaining why Erg loss closely mimics Myc overexpression. Consistently, the BET domain inhibitor CPI-203, known to repress Myc expression, confers a partial phenotypic rescue. In summary, ERG plays a critical role in coordinating the balance between self-renewal and differentiation of HSCs.

C/EBPalpha (42 kDa) is commonly dysregulated in acute myeloid leukaemia (AML) by mutation or via the action of AML oncogenes, which can increase the 30 kDa protein oncogenic form of C/EBPalpha (p30). The Trib2 oncogene is associated with dysregulated C/EBPalpha in human AML and leads to the degradation of p42, leaving p30 intact. Indeed there exists a novel paradigm whereby an E2F1-p30-Trib2 positive regulatory loop and an E2F1-p42-Trib2 negative feedback loop plays a key role in Trib2 expression, essential for AML cell proliferation and survival. Thus, it is important to understand the Trib2-C/EBPalpha relationship in the context of p42 degradation and/or C/EBPalpha mutation in AML. Using mouse genetics our data reveals that in the absence of C/EBPalpha (-/-) Trib2 was unable to induce AML whereas in the presence of p42 and mutant p30 (L/+), Trib2 and p30 cooperate to decrease the latency of AML disease. In the situation where p30 is expressed in the absence of p42, which alone can lead to AML (L/L), Trib2 does not lead to a more aggressive disease than that caused by p30 itself. These data show that the modulation of p42 rather than the expression of p30 is a key driver in Trib2 AML. Peptide array analysis showed the site-specific direct interaction between Trib2 and p42, and mutational analysis showed that these site-specific interactions were required for the K48-specific ubiquitin-dependent proteasomal degradation of p42. A C-terminal lysine residue of C/EBPalpha commonly found duplicated in AML patients was critical for Trib2-mediated p42 ubiquitination and proteasomal degradation. Proteasome inhibition using bortezomib induced cell death in Trib2 positive AML cell lines and AML patient samples in vitro and in vivo. Knockdown and overexpression of Trib2 in AML cells followed by transplantation in NSG mice and treated with bortezomib selectively killed Trib2 positive AML. Our data show the mechanism required for oncogene function centres on p42 degradation.