Projects

Jongen-Lavrencic lab

We aim to identify microRNAs that have a function in pathogenesis of AML and may be used to improve diagnostics, prognostics and potentially therapy of heterogeneous AML.

Our approach to identify candidate microRNAs is based on microRNA expression profiling (Q-RT-PCR) of large number of AML samples as well as cell subsets of normal human differentiation (in vitro and in vivo models).

 

General Introduction:

AML

Acute myeloid leukemia (AML) is a heterogeneous group of blood cancer, which is characterized by increased, uncontrolled proliferation of hematopoietic progenitors with lost ability to properly differentiate. The pathogenesis of AML is a multi-step process affecting cell differentiation, proliferation and apoptosis that ultimately lead to malignant transformation of hematopoietic progenitors. The pathogenetic notion is that at least two, probably in most cases multiple, hits are required for leukemic transformation. One category of genetic abnormalities (class 1) results in constitutive activation of proliferative signaling and involves signaling molecules such as RAS, FLT3 and c-KIT. A second type of lesions (class 2) such as formation of fusion genes, as a result of the chromosomal lesions t(8;21) or inv(16) involving transcription factors result in a block of myeloid differentiation. The prognosis of AML is correlated with different prognostic factors, such as age and known cytogenetic abnormalities namely t(15;17), t(8;21), inv(16), 11q23, 3q26 abnormalities, -5/5q, -7/7q. Novel genome-wide approaches to molecularly analyze extensive groups of well-characterized AML patients, have revealed new insights in the pathogenesis of AML and disclosed novel distinct entities of this type of leukemia.

microRNAs

MicroRNAs are small non-protein-coding RNAs that posttranscriptionally regulate gene expression by forming base pairing with sequences in the 3’ untranslated region (3’UTR) of protein coding genes and thereby repressing translation or inducing degradation of target transcript. MicroRNAs are single-stranded RNAs of 18-25 nucleotides and are generated from precursor molecules. There is a significant body of evidence suggesting that microRNAs play important roles in cellular processes such as proliferation, differentiation and apoptosis.

microRNAs in myelopoiesis

Chen and the colleagues [30] were the first who cloned microRNAs from normal mouse bone marrow and found microRNA-223, -142 and -181 preferentially expressed in hematopoietic tissue. MicroRNA-233 is expressed at low levels in CD34+ hematopoietic progenitors (HPCs) and this expression increase during myeloid differentiation towards granulocytes. Using a loss-of-function allele in mice it was demonstrated that the microRNA-223 negatively regulates progenitor proliferation and granulocyte differentiation by targeting myocyte enhancer factor 2C (Mef2c), a transcription factor that promotes myeloid progenitor proliferation. In an acute pro-myelocytic leukemia cell-line, an auto-regulation circuit of microRNA-223, CEBPA and neurofibromin 1A (NF1A) has been propose. Furthermore, PU.1 was found to activate microRNA-223 transcription in mice.

Besides results obtained from cell lines only limited data is available on microRNA expression profiles during normal myeloid differentiation of primary human cells.

Projects:

1. microRNAs expression profiling in AML and prognostic value of microRNAs in AML

The aim of this project is a) to determine microRNA signatures and their possible association with disease development and prognosis in AML and b) to identify candidate oncomirs and tumor suppressor microRNAs in AML

 microRNAs in AML

Our and others recent studies have shown an association between microRNA expression signatures and (cyto)genetic subsets of AML. We used quantitative real-time RT-PCR to study the expression of 260 known human microRNAs. Using an approach of unsupervised ordering (i.e. not taking account of morphologic subtype, cytogenetics, molecular abnormalities or other external information) 215 cases of AML were classified into the subgroups with similar expression patterns of microRNAs. Unsupervised analysis of microRNA expression revealed signatures that correlate with particular cytogenetic and molecular subgroups of AML. Furthermore in our study, several established oncogenic and tumor suppressive microRNAs, such as microRNA-155, microRNA-21, let-7 appear to be associated with specific subtypes of AML.

Others demonstrated that a microRNA expression signature containing microRNA-223, -128a -128b and let7b could accurately distinguish AML from ALL and that increased expression of microRNA-191 and -199a was associated with poor survival in one AML study. A microRNA signature of cytogenetically normal AML was identified that is predictive for adverse prognosis. These data suggest that microRNAs are implicated in the pathogenesis of AML and may have a useful role in future diagnostics.

Our approach:

We study aberrant expression of microRNAs in human AML in relationship to treatment outcome and hematological features using clinically and molecularly fully characterized biobank of clinical specimens (500 cases of AML). Potential cooperation with other molecular abnormalities in AML is investigated by integrating our data of transcript profiling, single nucleotide polymorphism data and hypermethylation profiling (collaboration with P.Valk and R.Delwel)

2. Functional studies on candidate microRNAs in AML

The aim of this project is to study the function and identify the targets of candidate microRNAs in myeloid differentiation and proliferation and their role in pathogenesis of AML.

There is limited knowledge about function of aberrant microRNA expression in AML. MicroRNA-125b overexpression, that is directly linked to t(2;11) abrogates myeloid cell differentiation in vitro indicating a function of this microRNA in leukemogenesis. Overexpression of microRNA-155 in mouse bone marrow expands the granulocyte/monocyte population with pathological features characteristic of myeloid neoplasia. In AML, upregulation of microRNA-155 is associated with mutations in the gene of the kinase receptor FLT3, so called FLT3-ITD. In a recent study increased expression of microRNA-196b by mixed-lineage leukemia (MLL) fusion proteins has been suggested to contribute to development of AML via increased cell proliferation capacity and survival as well as partial block in differentiation in vitro.

Our approach:

Based on our microRNA expression profiling in human AML, we selected several candidate microRNAs for a series of experiments that are intended to clarify the role of these microRNA at various levels in myelopoiesis and AML:

(I)

We use a) Luciferase assay en Western blotting in cell lines and primary cells to experimentally validate selected number of in silico predicted candidate microRNA targets involved in stress response, cell cycle, apoptosis and differentiation using and b) Ago2-RISC-mRNA immunoprecipitation approach to identify new candidate microRNA targets in relevant cell type using

(II)

32D murine cell line model and primary murine bone marrow including sorted cell subsets (LSK, CMP, GMPs) as well as primary human CD34+ cells are examined as regards their maturation, proliferative, cell cycle and apoptosis abilities following forced expression of candidate microRNA using retroviral delivery system. Furthermore, the transcriptome analysis is performed to study the effect of candidate microRNA on known transcriptional programs involved in myeloid differentiation (such as CEBPA differentiation pathway, retinoic acid signaling pathway), and pathways involved in stress response (such as RB tumor suppressor pathway.

(III)

We study the transforming abilities of candidate microRNAs in vivo in a hematopoietic stem cell transplantation model in mice. Hematopoietic effects are assessed with pathology/cytology/immunophenotyping and transfer experiments (stem cell renewal).