The histone demethylase KDM5C functions as a tumor suppressor in AML by repression of bivalently marked immature genes.

Epigenetic regulators are frequently mutated in hematological malignancies including acute myeloid leukemia (AML). Thus, the identification and characterization of novel epigenetic drivers affecting AML biology holds potential to improve our basic understanding of AML and to uncover novel options for therapeutic intervention. To identify novel tumor suppressive epigenetic regulators in AML, we performed an in vivo short hairpin RNA (shRNA) screen in the context of CEBPA mutant AML. This identified the Histone 3 Lysine 4 (H3K4) demethylase KDM5C as a tumor suppressor, and we show that reduced Kdm5c/KDM5C expression results in accelerated growth both in human and murine AML cell lines, as well as in vivo in Cebpa mutant and inv(16) AML mouse models. Mechanistically, we show that KDM5C act as a transcriptional repressor through its demethylase activity at promoters. Specifically, KDM5C knockdown results in globally increased H3K4me3 levels associated with up-regulation of bivalently marked immature genes. This is accompanied by a de-differentiation phenotype that could be reversed by modulating levels of several direct and indirect downstream mediators. Finally, the association of KDM5C levels with long-term disease-free survival of female AML patients emphasizes the clinical relevance of our findings and identifies KDM5C as a novel female-biased tumor suppressor in AML.

Transcription factor-driven coordination of cell cycle exit and lineage-specification in vivo during granulocytic differentiation : In memoriam Professor Niels Borregaard.

Differentiation of multipotent stem cells into mature cells is fundamental for development and homeostasis of mammalian tissues, and requires the coordinated induction of lineage-specific transcriptional programs and cell cycle withdrawal. To understand the underlying regulatory mechanisms of this fundamental process, we investigated how the tissue-specific transcription factors, CEBPA and CEBPE, coordinate cell cycle exit and lineage-specification in vivo during granulocytic differentiation. We demonstrate that CEBPA promotes lineage-specification by launching an enhancer-primed differentiation program and direct activation of CEBPE expression. Subsequently, CEBPE confers promoter-driven cell cycle exit by sequential repression of MYC target gene expression at the G1/S transition and E2F-meditated G2/M gene expression, as well as by the up-regulation of Cdk1/2/4 inhibitors. Following cell cycle exit, CEBPE unleashes the CEBPA-primed differentiation program to generate mature granulocytes. These findings highlight how tissue-specific transcription factors coordinate cell cycle exit with differentiation through the use of distinct gene regulatory elements.

PTBP1 promotes hematopoietic stem cell maintenance and red blood cell development by ensuring sufficient availability of ribosomal constituents.

Ribosomopathies constitute a range of disorders associated with defective protein synthesis mainly affecting hematopoietic stem cells (HSCs) and erythroid development. Here, we demonstrate that deletion of poly-pyrimidine-tract-binding protein 1 (PTBP1) in the hematopoietic compartment leads to the development of a ribosomopathy-like condition. Specifically, loss of PTBP1 is associated with decreases in HSC self-renewal, erythroid differentiation, and protein synthesis. Consistent with its function as a splicing regulator, PTBP1 deficiency results in splicing defects in hundreds of genes, and we demonstrate that the up-regulation of a specific isoform of CDC42 partly mimics the protein-synthesis defect associated with loss of PTBP1. Furthermore, PTBP1 deficiency is associated with a marked defect in ribosome biogenesis and a selective reduction in the translation of mRNAs encoding ribosomal proteins. Collectively, this work identifies PTBP1 as a key integrator of ribosomal functions and highlights the broad functional repertoire of RNA-binding proteins.

H3K9 dimethylation safeguards cancer cells against activation of the interferon pathway.

Activation of interferon genes constitutes an important anticancer pathway able to restrict proliferation of cancer cells. Here, we demonstrate that the H3K9me3 histone methyltransferase (HMT) suppressor of variegation 3-9 homolog 1 (SUV39H1) is required for the proliferation of acute myeloid leukemia (AML) and find that its loss leads to activation of the interferon pathway. Mechanistically, we show that this occurs via destabilization of a complex composed of SUV39H1 and the two H3K9me2 HMTs, G9A and GLP. Indeed, loss of H3K9me2 correlated with the activation of key interferon pathway genes, and interference with the activities of G9A/GLP largely phenocopied loss of SUV39H1. Last, we demonstrate that inhibition of G9A/GLP synergized with DNA demethylating agents and that SUV39H1 constitutes a potential biomarker for the response to hypomethylation treatment. Collectively, we uncovered a clinically relevant role for H3K9me2 in safeguarding cancer cells against activation of the interferon pathway.

The splicing factor RBM25 controls MYC activity in acute myeloid leukemia.

Cancer sequencing studies have implicated regulators of pre-mRNA splicing as important disease determinants in acute myeloid leukemia (AML), but the underlying mechanisms have remained elusive. We hypothesized that "non-mutated" splicing regulators may also play a role in AML biology and therefore conducted an in vivo shRNA screen in a mouse model of CEBPA mutant AML. This has led to the identification of the splicing regulator RBM25 as a novel tumor suppressor. In multiple human leukemic cell lines, knockdown of RBM25 promotes proliferation and decreases apoptosis. Mechanistically, we show that RBM25 controls the splicing of key genes, including those encoding the apoptotic regulator BCL-X and the MYC inhibitor BIN1. This mechanism is also operative in human AML patients where low RBM25 levels are associated with high MYC activity and poor outcome. Thus, we demonstrate that RBM25 acts as a regulator of MYC activity and sensitizes cells to increased MYC levels.

Enhancer and Transcription Factor Dynamics during Myeloid Differentiation Reveal an Early Differentiation Block in Cebpa null Progenitors.

Transcription factors PU.1 and CEBPA are required for the proper coordination of enhancer activity during granulocytic-monocytic (GM) lineage differentiation to form myeloid cells. However, precisely how these factors control the chronology of enhancer establishment during differentiation is not known. Through integrated analyses of enhancer dynamics, transcription factor binding, and proximal gene expression during successive stages of murine GM-lineage differentiation, we unravel the distinct kinetics by which PU.1 and CEBPA coordinate GM enhancer activity. We find no evidence of a pioneering function of PU.1 during late GM-lineage differentiation. Instead, we delineate a set of enhancers that gain accessibility in a CEBPA-dependent manner, suggesting a pioneering function of CEBPA. Analyses of Cebpa null bone marrow demonstrate that CEBPA controls PU.1 levels and, unexpectedly, that the loss of CEBPA results in an early differentiation block. Taken together, our data provide insights into how PU.1 and CEBPA functionally interact to drive GM-lineage differentiation.

shRNA screening identifies JMJD1C as being required for leukemia maintenance.

Epigenetic regulatory mechanisms are implicated in the pathogenesis of acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). Recent progress suggests that proteins involved in epigenetic control are amenable to drug intervention, but little is known about the cancer-specific dependency on epigenetic regulators for cell survival and proliferation. We used a mouse model of human AML induced by the MLL-AF9 fusion oncogene and an epigenetic short hairpin RNA (shRNA) library to screen for novel potential drug targets. As a counter-screen for general toxicity of shRNAs, we used normal mouse bone marrow cells. One of the best candidate drug targets identified in these screens was Jmjd1c. Depletion of Jmjd1c impairs growth and colony formation of mouse MLL-AF9 cells in vitro as well as establishment of leukemia after transplantation. Depletion of JMJD1C impairs expansion and colony formation of human leukemic cell lines, with the strongest effect observed in the MLL-rearranged ALL cell line SEM. In both mouse and human leukemic cells, the growth defect upon JMJD1C depletion appears to be primarily due to increased apoptosis, which implicates JMJD1C as a potential therapeutic target in leukemia.

C/EBPalpha in leukemogenesis: identity and origin of the leukemia-initiating cell.

The role of the transcription factor CCATT/enhancer binding protein alpha (C/EBPalpha) as a lineage instructive determinant in myelopoiesis is widely accepted. Furthermore, early mutational events ultimately leading to acute myeloid leukemia (AML) often involve abrogation of C/EBPalpha expression and/or function. The main focus of this review is the progression from a preclinical state to AML, and which preleukemic cell population(s) might-in general and in particular in patients with CEBPA mutations-be a target for the secondary genetic and epigenetic events leading to this progression.

C/EBPalpha: a tumour suppressor in multiple tissues?

The CCATT/enhancer binding protein alpha, C/EBPalpha, is a key transcription factor involved in late differentiation events of several cell types. Besides acting as a classical transcription factor, C/EBPalpha is also a well-characterized inhibitor of mitotic growth in most cell lines tested. In line with its anti-mitotic properties, C/EBPalpha has been shown to interact with, and alter the activities of, several cell cycle related proteins and a number of models as to the mechanistics of C/EBPalpha-mediated growth repression have been proposed. More recently, several reports have indicated that C/EBPalpha acts as a tumour suppressor in the hematopoietic system and that mutation within C/EBPalpha is sufficient to induce tumourigenesis. Here, we will review these data and probe the possibility that C/EBPalpha also act as a tumour suppressor in other C/EBPalpha-expressing tissues.

The proline-histidine-rich CDK2/CDK4 interaction region of C/EBPalpha is dispensable for C/EBPalpha-mediated growth regulation in vivo.

The C/EBPalpha transcription factor regulates growth and differentiation of several tissues during embryonic development. Several hypotheses as to how C/EBPalpha inhibits cellular growth in vivo have been derived, mainly from studies of tissue culture cells. In fetal liver it has been proposed that a short, centrally located, 15-amino-acid proline-histidine-rich region (PHR) of C/EBPalpha is responsible for the growth-inhibitory function of the protein through its ability to interact with CDK2 and CDK4, thereby inhibiting their activities. Homozygous Cebpa(DeltaPHR/DeltaPHR) (DeltaPHR) mice, carrying a modified cebpa allele lacking amino acids 180 to 194, were born at the Mendelian ratio, reached adulthood, and displayed no apparent adverse phenotypes. When fetal livers from the DeltaPHR mice were analyzed for their expression of cell cycle markers, bromodeoxyuridine incorporation, cyclin-dependent kinase 2 kinase activity, and global gene expression, we failed to detect any cell cycle or developmental differences between the DeltaPHR mice and their control littermates. These in vivo data demonstrate that any C/EBPalpha-mediated growth repression via the PHR as well as the basic region is dispensable for proper embryonic development of, and cell cycle control in, the liver. Surprisingly, control experiments performed in C/EBPalpha null fetal livers yielded similar results.

[Transgenic mice as disease models]. / Transgene mus som sygdomsmodeller.

Transgenic animal models have proven to be useful tools in understanding both basic biology and the events associated with disease. Recent technical advances in the area of genomic manipulation in combination with the availability of the human and murine genomic sequences now allow the precise tailoring of the mouse genome. In this review we describe a few systems in which transgenic animal models have been employed for the purpose of studying the etiology of human diseases.