Mouse models in leukemia

Abstract

Human Philadelphia-positive leukemia results from a balanced chromosomal translocation, which fuses the BCR gene on chromosome 22 to the ABL proto-oncogene on chromosome 9. The understanding of Ph-positive leukemogenesis has advanced enormously over the last few decades. Although in vitro assay systems currently used, are not always relevant to human tumor biology, much can and has been learned from studies, employing cell cultures and overexpression of BCR/ABL oncogenes.Another restriction in leukemia research is the availability of primary human tumor material for study. Moreover, such tissues often represent terminally advanced stages of tumorigenesis. Therefore, the importance of in vivo models to study Philadelphia-positive leukemia is manifold. A well defined transgenic mouse model allows for tumorigenesis to be studied from its earliest stages onward and factors and mechanisms that eventually contribute to malignant progression of the leukemic cells can be uncovered. Besides an 'unlimited' provision of tumor material for analysis, more importantly, the availability of a transgenic mouse model provides a means by which cancer treatment regimes can be tested. In addition, identification of cellular components and/or pathways that contribute to the onset or progression of leukemia may eventually lead to the discovery and development of new drugs.In 1990, Heisterkamp and co-workers reported on a transgenic mouse model for Philadelphia-positive acute lymphoblastic leukemia (ALL). Since most of the transgenic animals of an earlier study had succumbed to leukemia, part of the aim of this thesis was to generate BCR/ABL P190 transgenic founder animals de novo and to derive a transgenic animal line(s) which was to be used for future studies. In order to better understand the animal model, leukemogenesis was studied in great detail in transgenic founder animals and their progeny. In the second chapter a cytogenetic study of the mouse model for acute lymphoblastic leukemia is presented. Karyotypic analysis of leukemic bone marrow of a significant number of mice shows, that leukemic cells undergo a clonal development and karyotype evolution toward a more aggressive tumor: a high frequency of aneuploidy is found in advanced leukemia, as occurs in human leukemia, with a preference for gain of chromosomes 10, 12, 14 and 17. These findings are corroborated by experiments that reveal a gain of malignancy of the cancer upon serial transplantation of leukemic bone marrow to irradiated recipient mice and by molecular analysis of lymphomas using immunoglobulin rearrangement as an indicator for tumor clonality. The results suggest that BCR/ABL has a destabilizing effect on the regulation of the proces of mitosis.In the third chapter, a correlation is described between the transcriptional status of the BCR/ABL P190 transgene and the development of leukemia: methylation of particular sequences in BCR exon-1 in the transgene is closely coupled to transgene inactivation, providing additional evidence for a direct role of BCR/ABL in leukemogenesis. A biological dissection of the oncogenic specificity of BCR/ABL is presented in the fourth chapter. Using sensitive molecular biological techniques, it is shown that, although expression of the BCR/ABL transgene is detectable in every tissue, from very early on in mouse development, no other neoplasias than of hematopoietic origin are found. The results strongly suggest that the oncogenicity of BCR/ABL is restricted to nucleated blood cells, which is very likely a reflection of cellular functions of the BCR and or ABL gene in signal transduction specific to hematopoietic lineages. The observations would also explain why the Ph-chromosome, which one would expect to arise by chance in many proliferating tissues, is found only in blood cancers.An analysis of transgenic mouse models for chronic myelogenous leukemia, using BCR/ABL P210 transgenes is presented in the fifth chapter. The clinical disease spectrum includes differentiated and undifferentiated T and B cell leukemias. The myeloid compartment is implicated only sporadically and rather late in the disease process. In some instances, the observed myelo-proliferation is a sequel to deregulation of cytokine production at advanced stages of leukemia. The course of P210 induced leukemia was acute rather than chronic, be it with an on average longer latency period than typical for ALL in BCR/ABL P190 mice. From these studies is was concluded that in the mouse, BCR/ABL P210 evokes a clinically different disease than BCR/ABL P190. Although no evidence for a chronic myeloproliferative disorder in the peripheral blood was found, an imbalance in myelopoiesis in the bone marrow suggests an effect of BCR/ABL P210 on primitive myeloid progenitors.The sixth chapter summarizes an analysis of interferon-α(IFN-α) treatment of the BCR/ABL P190 transgenic mice. (IFN-α) is currently one of the most effective drugs in the treatment of CML. Recently, (IFN-α) was tried in the treatment of ALL. No effect of (IFN-α) on animal survival or disease pattern were noted when administered to the BCR/ABL P190 mice. The conclusion was reached that, at least in a transgenic setting, (IFN-α) does not interfere with BCR/ABL P190 mediated leukemia.In order to study the normal cellular function of the BCR gene and to eventually assess its role in leukemogenesis, studies focussing on the mouse bcr gene function are presented in chapters 7 and 8. The seventh chapter describes the ablation of a functional mouse bcr gene by means of recently developed gene targeting techniques. One of two mouse bcr alleles was inactivated in a mouse embryonic stem cell line through gene interruption by insertional replacement vectors. Ibis genetically altered cell fine was then injected into developing mouse embryos. Through germline transmission of the mutated allele and subsequent breeding both bcr alleles were inactivated. Although bcr -null mutants are phenotypically normal, their neutrophils display impaired regulation of respiratory burst, which becomes apparent when these cells are activated in vivo: an overproduction of superoxide leads to significantly more oxidative tissue damage during experimental endotoxemia. The results connect Bcr in vivo with the regulation of superoxide production by the NADPH- oxidase system of leukocytes and suggest a link between the cell types affected by loss of Bcr function and the those involved in Ph -positive leukemia.Additional information on biological processes that Bcr participates in, are described. in the eighths chapter. Notwithstanding its function in hematopoietic cells, the Bcr protein is normally found in high levels in brain. The expression pattern of bcr in rodent brain was examined by means of in situ hybridization and Northern analysis. Although not directly connected with leukemogenesis, a potentially interesting role for p160Bcr in the brain is discussed as its expression pattern appears to coincide with the functional organization of particularly highly specialized structures in the brain.With the availability of well defined transgenic mouse models for BCR/ABL positive leukemia, an opportunity is created to study the nature of cellular interactions and processes that contribute to the onset and development of Ph -positive leukemia. Ultimately, such investigations are aimed at designing and testing effective therapeutic drugs to fight the disease