target menu
... / ... / ... / Researchers / Ruud Delwel
 

Ruud Delwel

About the research

In acute myeloid leukemia (AML) immature myeloid precursor cells accumulate in the marrow and blood. The disease is the result of acquired mutations in genes critical in development towards neutrophils. By applying a variety of molecular approaches, e.g. retroviral insertional mutagenesis, nucleotide sequencing, Q-PCR and GeneChip analysis, we identified several genes that may be involved the development of AML. We currently focus on three of them: i.e. EVI1, C/EBPA and Cb2.  

Aberrant EVI1 expression in mouse and human AML.
The EVI1 gene, which was first identified as a common virus integration site in murine leukemias, encodes a nuclear DNA binding zincfinger protein. The gene is aberrantly expressed in human AML carrying 3q26 abnormalities, the locus where the gene resides. Moreover, even in 5 –10% of AML patients without 3q26 aberrations, high EVI1 expression has been observed, although the mechanism of this overexpression is unknown. AML patients expressing EVI1 respond poorly to anti-leukemic treatment. Gene expression analysis of 285 cases of human AML and subsequent unsupervised cluster analysis (See Valk-lab), revealed one subgroup of AML patients of which approximately 50% were EVI1 positive. This entire subgroup of AML patients responded poor to treament. To study the role of EVI1 in transformation of hematopoietic precursor cells we generated an inducible EVI1 model. In this model we cloned the EVI1 gene into a VAV-promoter construct with a loxp-flanked transcriptional stop box (LSL) 5’of the EVI1 gene. We studied the immediate in vivo effects of enforced EVI1 expression on erythroid development by crossing Vav1-loxP-Stop-loxP-Evi1 transgenic lines, which conditionally express EVI1 in the hematopoietic system, with the erythroid-lineage specific pEV-Cre line. Day 12.5 double transgenic fetal livers were small, pale, contained less CFU-E and had decreased overall cell numbers compared to livers from single transgenic or wild type littermates. Moreover, a block in erythroid differentiation and erythroid dysplasia was observed, which are both characteristic for MDS. Our data convincingly demonstrate that Evi1 interferes with fetal erythropoiesis and supports the notion that overexpression of this gene may be a key event causing erythroid defects in AML/MDS patients carrying 3q26 abnormalities. We are currently studying the effects of other Cre expressing lines, with the Cre gene under the control of various hematopoietic promoter constructs. Retroviral infection of these animals may reveal genes that may cooperate with EVI1 in full myeloid transformation.

 

C/EBPA abnormalities in AML.
The CCAAT/Enhancer Binding Protein a (CEBPA) is a 37kDa transcription factor essential for granulocytic differentiation. The C-terminal part is composed of a basic region that binds to DNA and a leucine zipper that mediates dimerization (bZIP). Recent studies reported N and C-terminal mutations in approximately 5 – 10% of patients with acute myeloid leukemia (AML). In case a C-terminal mutation is observed in one CEBPA allele, the other allele most frequently harbors an N-terminal mutation (See Figure). N-terminal mutations cause the expression of a 30kDa protein, whereas C-terminal mutations usually represent in-frame insertions or deletions in the bZIP-domain resulting in deficient DNA-binding of CEBPA. Gene array analysis carried out on 285 AML cases, revealed a group of AML patients with a unique gene expression profile, which contained most of AML cases with biallelic, CEBPA mutations (See Valk-lab). Interestingly, among this cluster 50% of the cases did not harbor any CEBPA mutation, while presenting exactly the same gene expression profile. Instead of carrying CEBPA mutations, these particular cases did not express CEBPA mRNA at all. Moreover, the patients with low CEBPA expression showed a poor response to anti leukemic treatment, whereas the CEBPA mutant AML cases respond favorably to therapy. To study the mechanism by which CEBPA expression might have been down regulated, methylation-specific PCR was applied using the bisulfite method. This analysis revealed that in most of the cases with low CEBPA mRNA levels, the CEBPA promoter was silenced by hypermethylation. None of the other AML cases that we analyzed showed CEBPA hyper methylation. Intriguingly, the majority of the genes within this profile are strongly down regulated, suggesting that they may represent CEBPA target genes whose expression is negatively affected by the absence of wt-CEBPA or the presence of mutated CEBPA. The gene that was most significantly down regulated in AML cases with either CEBPA mutations or CEBPA methylation is alpha-catenin (CTNNA1). We suggest that down regulation of CTNNA1 in CEBPA defective AML may be a critical step in leukemic transformation. The role of CTNNA1 in CEBPA defective AML is currently under investgation.


Cb2, a gene encoding the peripheral cannabinoid receptor.
By retroviral insertional mutagenesis we identified Cb2, the gene encoding Gai protein coupled receptor (GaiPCR) as a transforming gene involved in the development of mouse AML. We previously demonstrated that this receptor is highly expressed in approximately 50% of the patients with AML, whereas no receptor expression has been observed on normal bone marrow myeloid precursor cells. To study the mechanism by which this receptor may be involved in defective myeloid development we introduced it into mouse normal bone marrow precursors and into the mouse myeloid differentiation model 32D-G-CSFR. Stimulation of this receptor with its ligand CP55,940 completely abolished G-CSF induced neutrophilic differentiation. This block of differentiation could be reversed by the CB2 receptor specific antagonist but not by a CB1 (CB2 homologue) specific antagonist. Addition of pertussis toxin, an agent that interferes with G-protein signaling of GaiPCRs, could also prohibit the block of differentiation. We are currently studying 1) the interaction of Cb2 with other membrane receptors and signaling molecules and 2) how it may be involved in myeloid transformation in human AML.