A t(2;5) (p23;q35) chromosomal translocation can be found in a high percentage of anaplastic large-cell lymphomas (ALCL). This genetic abnormality leads to the expression of the NPM-ALK fusion protein, which encodes a constitutively active tyrosine kinase that plays a causative role in lymphomagenesis. Employing a modified infection/transplantation protocol utilizing an MSCVbased vector, we were able to reproducibly induce two phenotypically different lymphoma-like diseases dependent on the retroviral titers used. The first phenotype presented as a polyclonal histiocytic malignancy of myeloid/macrophage origin with a short latency period of 3-4 weeks. Clinically, the diseased mice showed rapidly progressive wasting, lymphadenopathy and pancytopenia. Mice displaying the second phenotype developed monoclonal B-lymphoid tumors with a longer latency of approximately 12-16 weeks, primarily involving the spleen and the bone marrow, with less extensive lymph node but also histologically evident extranodal organ infiltration by large immature plasmoblastic cells. The described retroviral mouse model will be useful to analyse the role of NPM-ALK in lymphomagenesis in vivo and may contribute to the development of new treatment options for NPM-ALK induced malignancies.

Mutations in the Bcr-Abl kinase domain are a frequent cause of imatinib resistance in patients with advanced CML or Ph þ ALL. The impact of these mutations on the overall oncogenic potential of Bcr-Abl and on the clinical course of the disease in the absence of imatinib is presently unclear. In this study, we analyzed the effects of the Bcr-Abl P-loop mutation Y253H and the highly imatinib resistant T315I mutation on kinase activity in vitro and transforming efficiency of Bcr-Abl in vitro and in vivo. Immunoprecipitated Bcr-Abl Y253H and Bcr-Abl T315I proteins displayed similar kinase activities and substrate phosphorylation patterns as Bcr-Abl wildtype. We directly compared the proliferative capacity of mutant to wildtype Bcr-Abl in primary BM cells in vitro and in a murine transplantation model of CML by using a competitive repopulation assay. The results implicate that in the absence of imatinib, there is no growth advantage for cells carrying Bcr-Abl T315I or Bcr-Abl Y253H compared to Bcr-Abl wt , whereas imatinib treatment clearly selects for leukemic cells expressing mutant Bcr-Abl both in vitro and in vivo. Thus, the analysed Bcr-Abl mutants confer imatinib resistance, but do not induce a growth advantage in the absence of imatinib.

The kinase inhibitor imatinib mesylate targeting the oncoprotein Bcr-Abl has revolutionized the treatment of chronic myeloid leukemia (CML). However, even though imatinib successfully controls the leukemia in chronic phase, it seems not to be able to cure the disease, potentially necessitating lifelong treatment with the inhibitor under constant risk of relapse. On a molecular level, the cause of disease persistence is not well understood. Initial studies implied that innate features of primitive progenitor cancer stem cells may be responsible for the phenomenon. Here, we describe an assay using retroviral insertional mutagenesis (RIM) to identify genes contributing to disease persistence in vivo.

The Bcr-Abl fusion protein arising through the t(9;22)(q34;q11) reciprocal translocation is the causative agent in chronic myeloid leukemia and a subset of acute lymphocytic leukemia. Imatinib mesylate is a specific inhibitor of the Bcr-Abl kinase and has shown promising results in clinical studies. The structural relation between the Bcr-Abl oncogene and the tyrosine kinase inhibitor imatinib has recently been elucidated by an elegant crystal structure analysis, emphasizing the importance of dephosphorylated tyrosine 393 (Tyr393) in BcrAbl for access of the inhibitor to the kinase domain. By mutating this tyrosine to phenylalanine and thereby mimicking a constitutively dephosphorylated state, we now show that Ba/ F3 cells transformed by this mutant demonstrate an increased sensitivity towards imatinib in vivo. This effect is not due to an impaired kinase activity of Bcr-Abl Y393F, since a synthetic substrate is phosphorylated with similar kinetics. Treatment of Ba/F3 cells transfected with Bcr-Abl wild type with a phosphatase inhibitor diminished the effect of imatinib, but did not influence the growth of Ba/F3 cells transfected with BcrAblY393F. The results support the findings of the crystal structure and indicate that Tyr393 indeed plays a significant role for the sensitivity of Bcr-Abl towards imatinib in vivo. These data implicate the regulation of Tyr393 phosphorylation as a potential mechanism of imatinib resistance. Leukemia (2003) IntroductionThe tyrosine kinase inhibitor imatinib has recently emerged as an important new treatment option in patients with chronic myeloid leukemia (CML) and Philadelphia chromosome-positive (Ph + ) acute lymphocytic leukemias (ALL) carrying the t(9;22) translocation. 1,2 Unfortunately, many patients with advanced CML and nearly all patients with ALL quickly develop resistance to imatinib treatment. [3][4][5] Imatinib has been shown to bind specifically to the nucleotide-binding pocket in the catalytic domain of Bcr-Abl. 6 Tyrosine 393 (Tyr393) is located in the activation loop of the Bcr-Abl kinase domain and has been proposed to stabilize the activation loop in the open formation when phosphorylated, thereby restricting the access of imatinib to the catalytic region and compromising its inhibitory function. Thus, phosphorylation of Tyr393 may function as a switch, regulating accessibility of imatinib to its binding site in the Bcr-Abl protein. Therefore, other molecules that are able to 'flick the switch' may influence imatinib sensitivity in a Bcr-Abl tranformed cell, and overexpression of kinases or downregulation of phosphatases targeting Tyr393 may induce imatinib resistance. As the importance of this amino acid for imatinib binding has been shown in vitro, we aimed at examining the relevance of phosphorylation of Tyr393 in vivo. We demonstrate here that a mutant Bcr-Abl imitating a constitutively dephosphorylated Tyr393 renders Ba/F3 cells more sensitive towards imatinib inhibition. Materials and methods DNA constructs, cells and transfectionsThe mutation of Tyr3...