Gene expression profiling of acute myeloid leukemia (AML) allows the discovery of previously unrecognized molecular entities. Here, we identified a specific subgroup of AML, defined by an expression ...profile resembling that of AMLs with mutations in the myeloid transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα), while lacking such mutations. We found that in these leukemias, the CEBPA gene was silenced, which was associated with frequent promoter hypermethylation. The leukemias phenotypically showed aberrant expression of T-cell genes, of which CD7 was most consistent. We identified 2 mechanisms that may contribute to this phenotype. First, absence of Cebpa led to up-regulation of specific T-cell transcripts (ie, Cd7 and Lck) in hematopoietic stem cells isolated from conditional Cebpa knockout mice. Second, the enhanced expression of TRIB2, which we identify here as a direct target of the T-cell commitment factor NOTCH1, suggested aberrantly activated Notch signaling. Putatively activating NOTCH1 mutations were found in several specimens of the newly identified subgroup, while a large set of control AMLs was mutation negative. A gene expression prediction signature allowed the detection of similar cases of leukemia in independent series of AML.
Hematopoietic stem cells (HSCs) ensure blood cell production during the life-time of an organism, and to do so they need to balance self-renewal, proliferation, differentiation, and migration in a ...steady state as well as in response to stress or injury. Importantly, aberrant proliferation of HSCs leads to hematological malignancies, and thus, tight regulation by various tumor suppressor pathways, including p53, is essential. Protein phosphatase magnesium-dependent 1 delta (PPM1D) is a negative regulator of p53 and promotes cell survival upon induction of genotoxic stress. Truncating mutations in the last exon of PPM1D lead to the production of a stable, enzymatically active protein and are commonly associated with clonal hematopoiesis. Using a transgenic mouse model, we demonstrate that truncated PPM1D reduces self-renewal of HSCs in basal conditions but promotes the development of aggressive AML after exposure to ionizing radiation. Inhibition of PPM1D suppressed the colony growth of leukemic stem and progenitor cells carrying the truncated PPM1D, and remarkably, it provided protection against irradiation-induced cell growth. Altogether, we demonstrate that truncated PPM1D affects HSC maintenance, disrupts normal hematopoiesis, and that its inhibition could be beneficial in the context of therapy-induced AML.
•CMO mice (suffering from sterile chronic inflammation) succumb to MLL-AF9 AML faster than WT mice•Hyperactivation of IL-6/Jak/Stat3 in CMO mice contributes to leukemic expansion•CMO Tp53+/− mice ...show accelerated tumor development compared with that in Tp53+/− mice•CMO Tp53+/− mice exhibit reduced survival and increased risk of AML compared with those in Tp53+/− mice
Acute myeloid leukemia (AML) is a malignant neoplasia of the hematopoietic system characterized by the accumulation of immature and nonfunctional leukemic blasts in the bone marrow and peripheral tissues. Mechanistically, the development of AML is explained by the “two-hit” theory, which is based on the accumulation of driver mutations that will cooperate to induce transformation. However, a significant percentage of patients with AML exhibit only one driver mutation, and thus, how leukemic transformation occurs in these cases is unclear. Accumulating evidence suggests that nongenetic factors, such as chronic inflammation, might influence AML development, and accordingly, clinical data have reported that patients with chronic inflammatory disorders have an increased risk of developing hematological malignancies. Here, using a mouse model of chronic inflammation, we demonstrate that systemic elevated levels of cytokines and chemokines and hyperactivation of the Jak/Stat3 signaling pathway may substitute “second hit” mutations and accelerate tumorigenesis. Altogether, our data highlight chronic inflammation as an additional factor in the development of AML, providing additional understanding of the mechanisms of transformation and opening new avenues for the treatment of this disease.
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Cb2 is a novel protooncogene encoding the peripheral cannabinoid receptor. Previous studies demonstrated that 2 distinct noncoding first exons exist: exon-1A and exon-1B, which both splice to ...protein-coding exon-2. We demonstrate that in retrovirally induced murine myeloid leukemia cells with proviral insertion inCb2, exon-1B/exon-2 Cb2messenger RNA levels have been increased, resulting in high receptor numbers. In myeloid leukemia cells without virus insertion in this locus, low levels of only exon-1A/exon-2 Cb2transcripts were present and receptors could not be detected. To elucidate the function of Cb2 in myeloid leukemia cells, a set of in vitro experiments was carried out using 32D/G-CSF-R (granulocyte colony-stimulating factor receptor) cells transfected with exon-1B/exon-2 Cb2complementary DNA and a myeloid cell line carrying a virus insertion in Cb2(ie, NFS 78). We demonstrate that a major function of the Cb2 receptor is stimulation of migration as determined in a transwell assay. Exposure of Cb2-expressing cells to different cannabinoids showed that the true ligand for Cb2 is 2-arachidonoylglycerol (2-AG), which may act as chemoatractant and as a chemokinetic agent. Furthermore, we observed a significant synergistic activity between 2-AG and interleukin-3 or G-CSF, suggesting cross-talk between the different receptor systems. Radioactive-ligand binding studies revealed significant numbers of Cb2 receptors in normal spleen. Transwell experiments carried out with normal mouse spleen cells showed 2-AG–induced migration of B220-, CD19-, immunoglobulin M–, and immunoglobulin D–expressing B lymphocytes. Our study demonstrates that a major function of Cb2 receptor expressed on myeloid leukemia cells or normal splenocytes is stimulation of migration.
Enhancers play a central role in the spatiotemporal control of gene expression and tend to work in a cell-type-specific manner. In addition, they are suggested to be major contributors to phenotypic ...variation, evolution and disease. There is growing evidence that enhancer dysfunction due to genetic, structural or epigenetic mechanisms contributes to a broad range of human diseases referred to as enhanceropathies. Such mechanisms often underlie the susceptibility to common diseases, but can also play a direct causal role in cancer or Mendelian diseases. Despite the recent gain of insights into enhancer biology and function, we still have a limited ability to predict how enhancer dysfunction impacts gene expression. Here we discuss the major challenges that need to be overcome when studying the role of enhancers in disease etiology and highlight opportunities and directions for future studies, aiming to disentangle the molecular basis of enhanceropathies.
The canonical Wnt signaling pathway is mediated by interaction of β-catenin with the T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) transcription factors and subsequent transcription ...activation of Wnt-target genes. In the hematopoietic system, the function of the pathway has been mainly investigated by rather unspecific genetic manipulations of β-catenin that yielded contradictory results. Here, we used a mouse expressing a truncated dominant negative form of the human TCF4 transcription factor (dnTCF4) that specifically abrogates β-catenin-TCF/LEF interaction. Disruption of the β-catenin-TCF/LEF interaction resulted in the accumulation of immature cells and reduced granulocytic differentiation. Mechanistically, dnTCF4 progenitors exhibited downregulation of the Csf3r gene, reduced granulocyte colony-stimulating factor (G-CSF) receptor levels, attenuation of downstream Stat3 phosphorylation after G-CSF treatment, and impaired G-CSF-mediated differentiation. Chromatin immunoprecipitation assays confirmed direct binding of TCF/LEF factors to the promoter and putative enhancer regions of CSF3R. Inhibition of β-catenin signaling compromised activation of the emergency granulopoiesis program, which requires maintenance and expansion of myeloid progenitors. Consequently, dnTCF4 mice were more susceptible to Candida albicans infection and more sensitive to 5-fluorouracil-induced granulocytic regeneration. Importantly, genetic and chemical inhibition of β-catenin-TCF/LEF signaling in human CD34+ cells reduced granulocytic differentiation, whereas its activation enhanced myelopoiesis. Altogether, our data indicate that the β-catenin-TCF/LEF complex directly regulates G-CSF receptor levels, and consequently controls proper differentiation of myeloid progenitors into granulocytes in steady-state and emergency granulopoiesis. Our results uncover a role for the β-catenin signaling pathway in fine tuning the granulocytic production, opening venues for clinical intervention that require enhanced or reduced production of neutrophils.
•Disruption of the β-catenin-TCF/LEF interaction compromises steady-state and emergency granulopoiesis.•TCF/LEF factors regulate G-CSF receptor expression by directly interacting with CSF3R promoter and enhancer regions.
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C/EPBα proteins, encoded by the CCAAT-enhancer-binding protein α gene, play a crucial role in granulocytic development, and defects in this transcription factor have been reported in acute myeloid ...leukemia. Here, we defined the C/EBPα signature characterized by a set of genes up-regulated upon C/EBPα activation. We analyzed expression of the C/EBPα signature in a cohort of 525 patients with acute myeloid leukemia and identified a subset characterized by low expression of this signature. We referred to this group of patients as the C/EBPα dysfunctional subset. Remarkably, a large percentage of samples harboring C/EBPα biallelic mutations clustered within this subset. We hypothesize that re-activation of the C/EBPα signature in the C/EBPα dysfunctional subset could have therapeutic potential. In search for small molecules able to reverse the low expression of the C/EBPα signature we applied the connectivity map. This analysis predicted positive connectivity between the C/EBPα activation signature and histone deacetylase inhibitors. We showed that these inhibitors reactivate expression of the C/EBPα signature and promote granulocytic differentiation of primary samples from the C/EBPα dysfunctional subset harboring biallelic C/EBPα mutations. Altogether, our study identifies histone deacetylase inhibitors as potential candidates for the treatment of certain leukemias characterized by down-regulation of the C/EBPα signature.
C/EBPs are a family of transcription factors that regulate growth control and differentiation of various tissues. We found that C/EBPgamma is highly upregulated in a subset of acute myeloid leukemia ...(AML) samples characterized by C/EBPalpha hypermethylation/silencing. Similarly, C/EBPgamma was upregulated in murine hematopoietic stem/progenitor cells lacking C/EBPalpha, as C/EBPalpha mediates C/EBPgamma suppression. Studies in myeloid cells demonstrated that CEBPG overexpression blocked neutrophilic differentiation. Further, downregulation of Cebpg in murine Cebpa-deficient stem/progenitor cells or in human CEBPA-silenced AML samples restored granulocytic differentiation. In addition, treatment of these leukemias with demethylating agents restored the C/EBPalpha-C/EBPgamma balance and upregulated the expression of myeloid differentiation markers. Our results indicate that C/EBPgamma mediates the myeloid differentiation arrest induced by C/EBPalpha deficiency and that targeting the C/EBPalpha-C/EBPgamma axis rescues neutrophilic differentiation in this unique subset of AMLs.
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