Sub theme 2.1.3
Malignant transformation of hematopoietic stem cells

Goals of research: general outline
Scientific achievements
Future plans: special goals and approach
Running projects
Associated staff

Goals of research: general outline

The research program aims to elucidate key regulatory abnormalities of leukemogenesis. In the previous report period, major emphasis of this program was on growth factor receptor and signal transduction derangements and perturbations of transcription and epigenetic control determining functional abnormalities of survival, proliferative, cell cycle, and maturation fates of hematopoietic stem cells (von Lindern, Delwel, Touw). We have now expanded this theme with studies aimed at epigenetic modifications by histone and DNA modifications, specifically methylation (Delwel, Touw) and by the regulatory actions of microRNA’s (Erkeland).  Epigenetic control of gene expression has been suggested to play a pivotal role in determining the biological behavior of cells. One such epigenetic mechanism is DNA cytosine methylation, which can alter gene expression by creating new binding sites for methylation dependent repressor proteins or by disrupting the ability of transcription factors to bind to their target sequences. Disruption of normal DNA methylation distribution is a hallmark of cancer and can play critical roles in initiation, progression and maintenance of the malignant phenotype. Based on these data we hypothesize that DNA methylation distributes into specific patterns in cancer, and that these methylation profiles impose and reflect critical biological differences with practical clinical and therapeutic implications. In order to test this hypothesis we aim at large-scale DNA methylation profiling in humans, AML on a well-characterized cohort of ~350 patients with AML.

Scientific achievements
  • EVI1 encodes a nuclear DNA binding zinc-finger protein that is frequently aberrantly expressed in murine and human myeloid leukemia. The protein transforms myeloid precursors via a unique mechanism. We identified several partner proteins which all suggest that the protein may be involved in epigenetic gene-silencing and chromatin remodeling. We demonstrated that EVI1 transformed AML expresses aberrant promoter hypermethylation as well.
  • We identified an AML subtype with a gene expression signature that was highly identical to that of CEBPA-mutant AML. Using mouse models combined with patient sample analysis we demonstrated that promoter hypermethylation of CEBPA is the driving force of the phenotype and gene expression signature of this AML subtype.
  • Applying a methylation profiling strategy, we demonstrated that CEBPA-silenced AML carry a hypermethylation phenotype, which means that CEBPA is one out of hundreds of genes that are abnormally methylated. These findings suggest that epigenetic defects are involved in the pathogenesis of these leukemias.
  • We found that mouse leukemia genes identified by provirus tagging are differentially expressed in subgroups of adult and pediatric AML. Genes directly flanking a virus integration site but not more distantly located genes contributed significantly to patient classification, suggesting an involvement of the virus-flanking genes in the pathogenesis of AML (refs 6 and 7).
  • During his postdoctoral training in Boston, Stefan Erkeland studied the role for miRNAs in cancer. He developed retroviral and lentiviral vectors to express miRNAs in mammalian cells. To investigate the functions of miRNAs and tumor suppressor genes in cancer, a doxycyclin (DOX)-regulated lentiviral expression vector that highly induces miRNAs expression was developed (ref 10). Both in vitro and allo-transplantation experiments in nude mice showed DOX-dependent tumor suppressing activity of the miRNA Let7g (ref 8). To investigate the role of miRNAs in cancer a miRNA expression library containing over 200 single miRNAs was generated and clusters introduced in an MSCV-based retrovirus vector. This library will be used to study functions of miRNAs in hematopoiesis and leukemia.
  • Retroviral integration mutagenesis in mice has been proven a powerful tool to identify new genes relevant for human AML. By using methylated DNA immunoprecipitation (MeDIP) in combination with inverse PCR, we developed a new approach of this technique in which we can specifically identify (new) candidate TSG in murine AML. The strength of this approach lies in the fact that identification of these potential TSG gives us the opportunity to investigate large human AML datasets using a candidate gene approach.

Future plans: special goals and approach


  • An in vivo model system has been developed, such that we will be able to relatively easily generate conditional and inducible mutant mice. We will specifically focus on the role of Evi1 in epigenetic regulation in leukemic transformation. Using the in vivo mouse models in combination with inducible in vitro differentiation assays, we wish to study how Evi1 may interact with chromatin remodeling complexes, interferes with epigenetic gene-silencing and how these functions play a role in leukemic transformation. These studies will also involve promoter methylation and histone modification profiling. Large scale analysis of AML samples will be carried out to elucidate the role EVI1 in these epigenetic modifications in human AML.
  • We will define EVI1 target genes in models and in patient samples and study whether and how the expression of these targets is affected by EVI1. Genes aberrantly expressed by EVI1 will be further analyzed using the in vivo and in vitro models.


  • We will study using inducible in vitro models how mutant CEBPalpha affects gene expression of CEBPalpha wild type target genes. Proteomic approaches will be applied to study whether the protein complexes have been affected by mutations in the basic-zipper region of the protein. These studies are meant to elucidate how mutant CEBPalpha affects transcriptional control and alter myeloid differentiation.

Identification and functional investigation of miRNAs in AML

·     We will determine the effects of global down-regulation of miRNA expression for AML progression.

  • We will identify critical tumor suppressing and oncogenic miRNAs involved in AML progression using conditional pre-leukemia models and newly developed miRNA-expressing and knock-down models.
  • We will investigate the critical miRNA-regulated pathways for AML development using a newly developed miRNA expression library.

Identification of (new) tumor suppressor genes in human acute myeloid leukemia (AML)  

  • We have developed a strategy based on genetic screens in mouse leukemias (see above) to identify candidate tumor suppressor or haplo-insufficient genes that are currently being further explored for their significance for human AML. We have identified a number of candidates that have been shown to be affected by LOH or hypermethylation in certain cases of human AML or MDS. The prognostic value of these findings will be analyzed and some of the genes will be selected for future functional studies.  

Most recent publications
  1. Bas J. Wouters, Meritxell Alberich Jordà, Karen Keeshan, Irene Louwers, Claudia A.J. Erpelinck-Verschueren, Dennis Tielemans, Anton W. Langerak, Yiping He, Pu Zhang, Christopher J. Hetherington, Roel G.W. Verhaak, Peter J.M. Valk, Bob Löwenberg, Daniel G. Tenen, Warren S. Pear, and Ruud Delwel: Distinct gene expression profiles of acute myeloid/T-lymphoid leukemia with silenced CEBPA and mutations in NOTCH1. Blood. 2007 Nov 15;110(10):3706-14.
  2. Maria Figueroa, Bas Wouters, Lucy Skrabanek. Jacob Glass, Yushan Li, Claudia Erpelinck, Ton Langerak, B Lowenberg, M Fazzari, JM Greally, P Valk.  Ari Melnick and Ruud Delwel:  Integrated epigenetic analyses delineates a biologically distinct form of acute myeloid leukemia with T-cell lineage infidelity.  Blood, Mar 2009; 113: 2795 - 2804.
  3. Ulrich Steidl, Frank Rosenbauer, Roel G W Verhaak, Xuesong Gu, Alexander Ebralidze, Hasan H Otu, Steffen Klippel, Christian Steidl, Ingmar Bruns, Daniel B Costa, Katharina Wagner, Manuel Aivado, Guido Kobbe, Peter J M Valk, Emmanuelle Passegué, Towia A Libermann, Ruud Delwel & Daniel G Tenen: Essential role of Jun family transcription factors in PU.1 knockdown–induced leukemic stem cells. Nat.Gen. 2006 Nov;38(11):1269-77.
  4. Karen Keeshan, Yiping He, Bas J. Wouters, Olga Shestova, Hong Sai, Carlos G. Rodriguez, Ivan Maillard, John W. Tobias, Peter Valk, Martin Carroll, Jon C. Aster, Ruud Delwel, and Warren S. Pear: Tribbles homolog 2 (Trib2) inactivates C/EBP alpha and causes acute myelogenous leukemia. Cancer Cell. 2006 Nov;10(5):40
  5. Peter J.M. Valk, Roel G.W. Verhaak, M. Antoinette Beijen, Claudia A.J. Erpelinck, Sahar Barjesteh van Waalwijk van Doorn - Khosrovani, Judith M. Boer, H. Berna Beverloo, Michael J. Moorhouse, Peter J. van der Spek, Bob Löwenberg, Ruud Delwel:  Prognostically Useful Gene Expression Profiles in Acute Myeloid Leukemia . N Engl J Med. 2004 Apr 15;350(16):1617-28.
  6. Erkeland SJ, Verhaak RG, Valk PJ, Delwel R, Lowenberg B, Touw IP. Significance of murine retroviral mutagenesis for identification of disease genes in human acute myeloid leukemia. Cancer Res. 2006 Jan 15;66(2):622-6.
  7. Touw IP, Erkeland SJ,. Retroviral insertion mutagenesis in mice as a comparative oncogenomics tool to identify disease genes in human leukemia. Mol Ther. 2007 Jan;15(1):13-9.
  8. Erkeland SJ, Madhu S. Kumar, Ryan E. Pester, Cindy Y. Chen, Phillip A. Sharp, Tyler Jacks. Suppression of non-small cell lung tumor development by the let-7 microRNA family. Proc Natl Acad Sci U S A. 2008 Mar 11;105(10):3903-8.
  9. Ventura A, Young AG, Winslow M, Lintault L, Meissner A, Erkeland SJ, Newman J, Bronson RT, Crowley D, Stone JR, Jaenisch R, Sharp PA, Jacks T. Targeted deletion reveals essential and overlapping functions of the miR-17 through 92 family of miRNA clusters. Cell. 2008 Mar 7;132(5):875-86.
  10. Stern P, Astrof S, Erkeland SJ, Schustak J, Sharp PA, Hynes RO.A system for Cre-regulated RNA interference in vivo. Proc Natl Acad Sci U S A. 2008 Sep 16;105(37):13895-900.