B-ALL is characterized by the accumulation of lymphoblasts with clonal origin in the bone marrow and peripheral blood (Cobaleda and Sanchez-Garcia, 2009; Onciu, 2009). Lymphoblasts are immature cells, which would normally differentiate into leukocytes, but instead proliferate in an uncontrolled manner in the bone marrow and aggressively infiltrate other organs. This form of leukemia is found mainly in children from 2-5 years and then peaks again in elderly people (>50 years; Borkhardt et al., 1997). Because these immature cells proliferate quickly, patients require treatment within weeks after diagnosis (McGregor et al., 2012). Today, the overall cure rate with combined radiation- and multi chemotherapy is about 80% in children and 50% in adults (Gokbuget and Hoelzer, 2009; Yeoh et al., 2002). The diagnosis is based on a complete blood cell count and a blood smear. A bone marrow biopsy is a final proof.
Introduction 20
The analysis of chromosomal translocations is routinely done in B-ALL diagnosis and serves as prognostic marker to identify patients who require more intense therapy (Harrison, 2009). The TEL-AML1 fusion t(12;21) is the most commonly detected chromosomal abnormality and found in 25% of patients (Zelent et al., 2004). The TEL gene and the AML1 gene both encode transcription factors, which are essential for early hematopoietic development/stem cell maintenance (Ford et al., 2009). The TEL-AML1 translocation links the helix-loop-helix domain of TEL to the DNA binding and transactivation domains of AML1. Detection of this fusion is a favorable prognostic marker (Zelent et al., 2004).
As part of an epidemiologic study, it was shown that the TEL-AML1 translocation could rarely be detected in neonatal blood spots (Greaves and Wiemels, 2003). Still, it took 5-10 years until leukemia was diagnosed in these children. Recurrent chromosomal translocations are a hallmark of B-ALL and are commonly found in TEL-AML1 leukemias, indicating that B-ALL develops from an immature cell accumulating additional mutations until it can drive leukemogenesis (Armstrong and Look, 2005).
Fusion of the Abelson (ABL1) tyrosine kinase to the BCR (breakpoint cluster region) gene is a commonly found chromosomal translocation t(9;22)(q34;q11) in B-ALL (Nowell and Hungerford, 1960). The resulting Philadelphia chromosome encodes the constitutively active BCR-ABL tyrosine kinase, which drives tumorigenesis by increased cell division, inhibiting DNA repair mechanisms and providing anti apoptotic factors via constitutive STAT5 (Signal transducer and activator of transcription) signaling (Malin et al., 2010). The BCR-ABL fusion is found in 40% of adult patients and is associated with a poor prognosis (Gleissner et al., 2002).
To this day, cancer stem cells have not been identified in B-ALL by a certain CD phenotype (Buss and Ho, 2011; Cox et al., 2009). However, their existence was functionally proven and their frequency can be defined using mouse models (Cox et al., 2004). To determine their frequency, patient derived cells are transplanted at limiting dilutions into immunocompromised mice. The CSC frequency is then calculated from the number of engrafted mice for each cell dose injected, using ELDA software (Hu and Smyth, 2009).
Aim of the study 21
3
Aim of the study
Hematopoietic stem cells (HSCs) have the unique ability to undergo self-renewing divisions. Thereby they maintain steady levels of hematopoiesis without depleting the stem cell pool. The balance between quiescence, proliferation and differentiation is regulated by intrinsic and extrinsic factors, provided by specialized microenvironments in the bone marrow. While HSCs themselves are well defined, the molecular and cellular composition of the niche they reside in is poorly understood. The transcription factor Ebf2 is expressed in stromal cells of the bone marrow and has been shown to regulate HSC homeostasis. Still, the underlying mechanisms and the contribution of Ebf2 on a cellular level remain undefined. The aim of this study was to examine whether all Ebf2-expressing bone marrow stromal cells contribute to Ebf2-mediated niche function for HSCs or whether this potential lies in a certain cell lineage. Therefore, mice carrying a conditional allele of Ebf2 were generated and combined with Cre lines to disrupt Ebf2-expression specifically in mesenchymal stem cells, adipocytes or osteoblasts.
HSCs and leukemic Cancer Stem Cells (CSC) share certain characteristics. CSCs have the unique ability to propagate a heterogeneous disease upon transplantation of a single cell. Therefore, CSCs like HSCs are multipotent and can undergo self-renewing divisions. As IEO (immature Ebf2-expressing osteoblastic) cells support HSCs in vitro in an Ebf2-dependent manner, we addressed the question whether CSCs depend on the same Ebf2-mediated bone marrow niches. Therefore, co-culture experiments of human B-ALL and B-CLL leukemic cells with IEO cells and stromal cell lines were carried out. Thereby, the influence of Ebf2 on proliferation, survival and maintenance of CSCs was investigated.
Finally, the role of Ebf2 in T cell development was studied. Ebf2-deficient mice show a strong reduction in thymic cellularity. In the thymus, Ebf2 is expressed by a small population of stromal cells at the boundary between the outer cortex and the inner medulla. Applying FACS, the nature of this cell population was characterized and we investigated whether different T cell types are equally affected by the loss of Ebf2 in thymic stromal cells. Furthermore, thymic embryonic lobes were transplanted into wild type recipients to determine whether the observed phenotype originates in the thymus or on the level of a bone marrow-derived T cell progenitor.
Results 22