Meritxell Alberich-Jorda

DNMT1-interacting RNAs block gene-specific DNA methylation

DNA methylation was first described almost a century ago; however, the rules governing its establishment and maintenance remain elusive. Here we present data demonstrating that active transcription regulates levels of genomic methylation. We identify a novel RNA arising from the CEBPA gene locus that is critical in regulating the local DNA methylation profile. This RNA binds to DNMT1 and prevents CEBPA gene locus methylation. Deep sequencing of transcripts associated with DNMT1 combined with genome-scale methylation and expression profiling extend the generality of this finding to numerous gene loci. Collectively, these results delineate the nature of DNMT1–RNA interactions and suggest strategies for gene-selective demethylation of therapeutic targets in human diseases.

C/EBPa controls acquisition and maintenance of adult haematopoietic stem cell quiescence

In blood, the transcription factor C/EBPa is essential for myeloid differentiation and has been implicated in regulating self-renewal of fetal liver haematopoietic stem cells (HSCs). However, its function in adult HSCs has remained unknown. Here, using an inducible knockout model we found that C/EBPa-deficient adult HSCs underwent a pronounced increase in number with enhanced proliferation, characteristics resembling fetal liver HSCs. Consistently, transcription profiling of C/EBPa-deficient HSCs revealed a gene expression program similar to fetal liver HSCs. Moreover, we observed that age-specific Cebpa expression correlated with its inhibitory effect on the HSC cell cycle. Mechanistically we identified N-Myc as a downstream target of C/EBPa, and loss of C/EBPa resulted in de-repression of N-Myc. Our data establish C/EBPa as a central determinant in the switch from fetal to adult HSCs.

Sustained PU.1 Levels Balance Cell-Cycle Regulators to Prevent Exhaustion of Adult Hematopoietic Stem Cells

To provide a lifelong supply of blood cells, hematopoietic stem cells (HSCs) need to carefully balance both self-renewing cell divisions and quiescence. Although several regulators that control this mechanism have been identified, we demonstrate that the transcription factor PU.1 acts upstream of these regulators. So far, attempts to uncover PU.1s role in HSC biology have failed because of the technical limitations of complete loss-of-function models. With the use of hypomorphic mice with decreased PU.1 levels specifically in phenotypic HSCs, we found reduced HSC long-term repopulation potential that could be rescued completely by restoring PU.1 levels. PU.1 prevented excessive HSC division and exhaustion by controlling the transcription of multiple cell-cycle regulators. Levels of PU.1 were sustained through autoregulatory PU.1 binding to an upstream enhancer that formed an active looped chromosome architecture in HSCs. These results establish that PU.1 mediates chromosome looping and functions as a master regulator of HSC proliferation.

Sox4 Is a Key Oncogenic Target in C/EBPα Mutant Acute Myeloid Leukemia

Mutation or epigenetic silencing of the transcription factor C/EBPα is observed in ∼10% of patients with acute myeloid leukemia (AML). In both cases, a common global gene expression profile is observed, but downstream targets relevant for leukemogenesis are not known. Here, we identify Sox4 as a direct target of C/EBPα whereby its expression is inversely correlated with C/EBPα activity. Downregulation of Sox4 abrogated increased self-renewal of leukemic cells and restored their differentiation. Gene expression profiles of leukemia-initiating cells (LICs) from both Sox4 overexpression and murine C/EBPα mutant AML models clustered together but differed from other types of AML. Our data demonstrate that Sox4 overexpression resulting from C/EBPα inactivation contributes to the development of leukemia with a distinct LIC phenotype.

Targeting CDK1 promotes FLT3-activated acute myeloid leukemia differentiation through C/EBPα

Mutations that activate the fms-like tyrosine kinase 3 (FLT3) receptor are among the most prevalent mutations in acute myeloid leukemias. The oncogenic role of FLT3 mutants has been attributed to the abnormal activation of several downstream signaling pathways, such as STAT3, STAT5, ERK1/2, and AKT. Here, we discovered that the cyclin-dependent kinase 1 (CDK1) pathway is also affected by internal tandem duplication mutations in FLT3. Moreover, we also identified C/EBPα, a granulopoiesis-promoting transcription factor, as a substrate for CDK1. We further demonstrated that CDK1 phosphorylates C/EBPα on serine 21, which inhibits its differentiation-inducing function. Importantly, we found that inhibition of CDK1 activity relieves the differentiation block in cell lines with mutated FLT3 as well as in primary patient-derived peripheral blood samples. Clinical trials with CDK1 inhibitors are currently under way for various malignancies. Our data strongly suggest that targeting the CDK1 pathway might be applied in the treatment of FLT3ITD mutant leukemias, especially those resistant to FLT3 inhibitor therapies.

C/EBPγ deregulation results in differentiation arrest in acute myeloid leukemia.

C/EBPs are a family of transcription factors that regulate growth control and differentiation of various tissues. We found that C/EBPγ is highly upregulated in a subset of acute myeloid leukemia (AML) samples characterized by C/EBPα hypermethylation/silencing. Similarly, C/EBPγ was upregulated in murine hematopoietic stem/progenitor cells lacking C/EBPα, as C/EBPα mediates C/EBPγ 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/EBPα-C/EBPγ balance and upregulated the expression of myeloid differentiation markers. Our results indicate that C/EBPγ mediates the myeloid differentiation arrest induced by C/EBPα deficiency and that targeting the C/EBPα-C/EBPγ axis rescues neutrophilic differentiation in this unique subset of AMLs.

Cannabinoid receptor 2 and its agonists mediate hematopoiesis and hematopoietic stem and progenitor cell mobilization.

Endocannabinoids are arachidonic acid derivatives and part of a novel bioactive lipid signaling system, along with their G-coupled cannabinoid receptors (CB₁ and CB₂) and the enzymes involved in their biosynthesis and degradation. However, their roles in hematopoiesis and hematopoietic stem and progenitor cell (HSPC) functions are not well characterized. Here, we show that bone marrow stromal cells express endocannabinoids (anandamide and 2-arachidonylglycerol), whereas CB₂ receptors are expressed in human and murine HSPCs. On ligand stimulation with CB₂ agonists, CB₂ receptors induced chemotaxis, migration, and enhanced colony formation of bone marrow cells, which were mediated via ERK, PI3-kinase, and Gαi-Rac1 pathways. In vivo, the CB₂ agonist AM1241 induced mobilization of murine HSPCs with short- and long-term repopulating abilities. In addition, granulocyte colony-stimulating factor -induced mobilization of HSPCs was significantly decreased by specific CB₂ antagonists and was impaired in Cnr2(-/-) cannabinoid type 2 receptor knockout mice. Taken together, these results demonstrate that the endocannabinoid system is involved in hematopoiesis and that CB₂/CB₂ agonist axis mediates repopulation of hematopoiesis and mobilization of HSPCs. Thus, CB₂ agonists may be therapeutically applied in clinical conditions, such as bone marrow transplantation.

RUNX1 regulates the CD34 gene in haematopoietic stem cells by mediating interactions with a distal regulatory element

The transcription factor RUNX1 is essential to establish the haematopoietic gene expression programme; however, the mechanism of how it activates transcription of haematopoietic stem cell (HSC) genes is still elusive. Here, we obtained novel insights into RUNX1 function by studying regulation of the human CD34 gene, which is expressed in HSCs. Using transgenic mice carrying human CD34 PAC constructs, we identified a novel downstream regulatory element (DRE), which is bound by RUNX1 and is necessary for human CD34 expression in long‐term (LT)‐HSCs. Conditional deletion of Runx1 in mice harbouring human CD34 promoter–DRE constructs abrogates human CD34 expression. We demonstrate by chromosome conformation capture assays in LT‐HSCs that the DRE physically interacts with the human CD34 promoter. Targeted mutagenesis of RUNX binding sites leads to perturbation of this interaction and decreased human CD34 expression in LT‐HSCs. Overall, our in vivo data provide novel evidence about the role of RUNX1 in mediating interactions between distal and proximal elements of the HSC gene CD34.

Targeted BMI1 inhibition impairs tumor growth in lung adenocarcinomas with low CEBPα expression

In lung cancers with low expression of C/EBPα, BMI1 expression correlates with worse prognosis but can be targeted with a drug. The right drug for the right tumor The expression of a tumor suppressor called C/EBPα is often lost in non–small cell lung cancer, as well as in other cancer types. Yong et al. discovered that lung tumors deficient in C/EBPα often overexpress a particular oncogenic protein, BMI1, and that higher expression of BMI1 correlates with worse prognosis in this group of patients. The authors characterized the role of these two proteins and their interaction in lung cancer development, then used cell lines and a genetic mouse model to test a therapeutic approach, showing that a pharmaceutical inhibitor of BMI1 is effective against non–small cell lung cancer with low C/EBPα and high BMI1. Lung cancer is the most common cause of cancer deaths. The expression of the transcription factor C/EBPα (CCAAT/enhancer binding protein α) is frequently lost in non–small cell lung cancer, but the mechanisms by which C/EBPα suppresses tumor formation are not fully understood. In addition, no pharmacological therapy is available to specifically target C/EBPα expression. We discovered a subset of pulmonary adenocarcinoma patients in whom negative/low C/EBPα expression and positive expression of the oncogenic protein BMI1 (B lymphoma Mo-MLV insertion region 1 homolog) have prognostic value. We also generated a lung-specific mouse model of C/EBPα deletion that develops lung adenocarcinomas, which are prevented by Bmi1 haploinsufficiency. BMI1 activity is required for both tumor initiation and maintenance in the C/EBPα-null background, and pharmacological inhibition of BMI1 exhibits antitumor effects in both murine and human adenocarcinoma lines. Overall, we show that C/EBPα is a tumor suppressor in lung cancer and that BMI1 is required for the oncogenic process downstream of C/EBPα loss. Therefore, anti-BMI1 pharmacological inhibition may offer a therapeutic benefit for lung cancer patients with low expression of C/EBPα and high BMI1.

Prolyl Isomerase Pin1 Regulates Axon Guidance by Stabilizing CRMP2A Selectively in Distal Axons

Axon guidance relies on precise translation of extracellular signal gradients into local changes in cytoskeletal dynamics, but the molecular mechanisms regulating dose-dependent responses of growth cones are still poorly understood. Here, we show that during embryonic development in growing axons, a low level of Semaphorin3A stimulation is buffered by the prolyl isomerase Pin1. We demonstrate that Pin1 stabilizes CDK5-phosphorylated CRMP2A, the major isoform of CRMP2 in distal axons. Consequently, Pin1 knockdown or knockout reduces CRMP2A levels specifically in distal axons and inhibits axon growth, which can be fully rescued by Pin1 or CRMP2A expression. Moreover, Pin1 knockdown or knockout increases sensitivity to Sema3A-induced growth cone collapse in vitro and in vivo, leading to developmental abnormalities in axon guidance. These results identify an important isoform-specific function and regulation of CRMP2A in controlling axon growth and uncover Pin1-catalyzed prolyl isomerization as a regulatory mechanism in axon guidance.