Models of chronic myeloid leukemia (CML) have proven invaluable for furthering our understanding of the molecular pathophysiology of this disease. Xenotransplantation of primary human CML cells into immunodeficient mice allows investigation into the nature of the most primitive repopulating cells in this leukemia, but the system is limited by variability and difficulty with experimental manipulation. Accordingly, a large effort has been invested in developing models of CML through expression of the BCR/ABL oncogene in the hematopoietic system of laboratory mice. Despite numerous attempts, an accurate transgenic mouse model of CML has not been produced, possibly because of the toxicity of BCR/ABL. Conditional transgenic mice are a promising new approach to this problem. A more successful strategy is retroviral transduction of BCR/ABL into mouse bone marrow in vitro, followed by transplantation into syngeneic or immunodeficient recipient mice. Recipients of marrow transduced with p210 BCR/ABL develop a fatal myeloproliferative illness that closely resembles human CML. This model is being used to define the signaling pathways required for leukemogenesis by BCR/ABL, and for developing new therapeutic approaches.

Several methods to model human Ph+ leukemia in laboratory mice are available, including propagation of BCR/ABL-expressing cells in mice, xenotransplantation of primary Ph+ leukemia cells into immunodeficient mice, BCR/ABL transgenic mice, and BCR/ABL retroviral bone marrow transduction and transplantation. Recent studies in these different model systems have yielded important advances in our knowledge of the pathogenesis and therapy of human chronic myeloid leukemia and Ph+ B-lymphoblastic leukemia, and are the subject of this review.

c-Abl, the product of the cellular homologue of the transforming gene of Abelson murine leukaemia virus, has been a protein in search of a purpose for over two decades. Because c-Abl is implicated in the pathogenesis of several human leukaemias, understanding the functions of Abl is an important goal. Recently, biochemical and genetic approaches have converged to shed new light on the mechanism of regulation of c-Abl kinase activity and the multiple roles of c-Abl in cellular physiology. This review summarizes our current understanding of the many facets of c-Abl biology, emphasizing recent studies on Drosophila and mammalian Abl.

There are two commonly used approaches to modeling human leukemia in mice: generation of mutant mice by traditional transgenic or knock-out/knock-in methods and retroviral bone marrow transduction and transplantation. For modeling leukemia, the retroviral model system has some distinct advantages over transgenic mice. Testing different forms and mutants of a given oncogene is much easier with the retroviral system and avoids the potential deleterious effects of expression of a transgene in nonhematopoietic tissues and during development. The retroviral provirus serves as a clonal marker of a transduced cell, facilitating analysis of clonality and transplantability of the malignancy. Finally, the retroviral system allows the assessment of the action of an oncogene in different subsets of hematopoietic precursor cells in the bone marrow, which is difficult or impossible with transgenic models. This article summarizes recent progress in modeling human Philadelphia-positive leukemia in mice with the retroviral bone marrow transduction/transplantation system and emphasizes the advantages and limitations of this approach with examples from the BCR-ABL leukemogenesis literature.

The subcellular localization of the mouse type IV c-abl protein was determined by indirect immunofluorescence of nontransformed NIH 3T3 fibroblasts that overexpress the protein. Unlike the viral transforming protein p160gag/v-abl, which has cytoplasmic and plasma membrane localization, a large fraction of the c-abl (IV) protein is nuclear, with the remainder in the cytoplasm and plasma membrane. Deletion of a small N-terminal regulatory region of the c-abl (IV) protein, sufficient to activate its transforming potential fully, changes the distribution of the protein from the nucleus to the cytoplasm. Mapping of an amino acid sequence responsible for the nuclear localization of the c-abl (IV) protein reveals a nuclear localization signal similar to that of SV40 large T antigen.

Using the specific Abl tyrosine kinase inhibitor STI 571, we purified unphosphorylated murine type IV c-Abl and measured the kinetic parameters of c-Abl tyrosine kinase activity in a solution with a peptide-based assay. Unphosphorylated c-Abl exhibited substantial peptide kinase activity with K(m) of 204 microm and V(max) of 33 pmol min(-1). Contrary to previous observations using immune complex kinase assays, we found that a transforming c-Abl mutant with a Src homology 3 domain point mutation (P131L) had significantly (about 6-fold) higher intrinsic kinase activity than wild-type c-Abl (K(m) = 91 microm, V(max) = 112 pmol min(-1)). Autophosphorylation stimulated the activity of wild-type c-Abl about 18-fold and c-Abl P131L about 3.6-fold, resulting in highly active kinases with similar catalytic rates. The autophosphorylation rate was dependent on Abl protein concentration consistent with an intermolecular reaction. A tyrosine to phenylalanine mutation (Y412F) at the c-Abl residue homologous to the c-Src catalytic domain autophosphorylation site impaired the activation of wild-type c-Abl by 90% but reduced activation of c-Abl P131L by only 45%. Mutation of a tyrosine (Tyr-245) in the linker region between the Src homology 2 and catalytic domains that is conserved among the Abl family inhibited the autophosphorylation-induced activation of wild-type c-Abl by 50%, whereas the c-Abl Y245F/Y412F double mutant was minimally activated by autophosphorylation. These results support a model where c-Abl is inhibited in part through an intramolecular Src homology 3-linker interaction and stimulated to full catalytic activity by sequential phosphorylation at Tyr-412 and Tyr-245.

Src-homology region 2 (SH2) domains, by binding to tyrosine-phosphorylated sequences, mediate specific protein-protein interactions important in diverse signal transduction pathways. Previous studies have shown that activated forms of the Abl tyrosine kinase, including P210BCR/ABL of human chronic myelogenous leukemia, require the SH2 domain for the transformation of fibroblasts. To determine whether SH2 is also required for Bcr/Abl to transform hematopoietic cells, we have studied two SH2 domain mutations in P210BCR/ABL: a point mutation in the conserved FLVRES motif (P210/R1033K), which interferes with phosphotyrosine-binding by SH2, and a complete deletion of SH2 (P210/delta SH2). Despite a negative effect on intrinsic Abl kinase activity, both P210 SH2 mutants were still able to transform the hematopoietic factor-dependent cell lines Ba/F3 and FDC-P1 to growth factor independence. Unexpectedly, both mutants showed greater transforming activity than wild-type P210 in a quantitative transformation assay, probably as a consequence of increased stability of the SH2 mutant proteins in vivo. Cells transformed by both P210 SH2 mutants were leukemogenic in synaptic mice and P210/r1053K mice exhibited a distinct disease phenotype, reminiscent of that induced by v-Abl. These results demonstrate that while the Abl SH2 domain is essential for BCR/ABL transformation of fibroblasts, it is dispensable for the transformation of hematopoietic factor-dependent cell lines.