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...
The Abl-tyrosine kinase inhibitor Imatinib efficiently targets the Bcr-Abl kinase and produces major cytogenetic responses in most patients with chronic phase CML. In contrast, patients with advanced stage CML or Ph+ ALL frequently become refractory to Imatinib treatment. Resistance arises predominantly from point mutations in the Abl-kinase region, Bcr-Abl amplification or clonal evolution due to secondary genetic aberrations. To screen for genes contributing to clonal evolution, we have employed retroviral insertional mutagenesis in a murine CML/ALL model to identify potential candidate genes leading to Imatinib resistance. We found proviral insertions near the RUNX3/AML2 promoter in Imatinib resistant leukemic clones, leading to upregulation of RUNX3 mRNA expression. To analyze the effects of high RUNX3 levels on Imatinib response, we expressed RUNX3 in a Bcr-Abl-transformed murine pre-B-cell line. Significantly, whereas there was no effect on Imatinib-mediated proliferation inhibition, the cells displayed a marked reduction of apoptosis. A RUNX3R193A mutant carrying a mutation in the DNA-binding domain of RUNX3 did not protect from apoptosis, indicating that transcriptional regulation by RUNX3 was required to induce the anti-apoptotic effects. To allow for a controlled activation of RUNX transcriptional activity and to extend our analysis to other members of the RUNX family of transcription factors, we constructed 4-OH-tamoxifen (TAM) inducible RUNX3/AML2- and RUNX1/AML1-Estrogen receptor (ER) fusion proteins. These fusion proteins readily translocated from the cytoplasm into the nucleus and activated a RUNX-dependent TCRß-luciferase construct upon addition of TAM. Using these constructs, we could demonstrate that activation of RUNX3 as well as RUNX1 protected Bcr-Abl-transformed Ba/F3 cells from Imatinib-induced apoptosis. Furthermore, we found that RUNX1 mRNA levels were significantly upregulated in patients with Ph+ ALL upon resistance development. Taken together, our data indicate that elevated RUNX3 or RUNX1 levels may contribute to Imatinib resistance in Bcr-Abl expressing leukemic cells.
Interferon regulatory factors (IRF) are activating and/or repressing transcription factors induced by treatment with type I and II Interferon (IFN), other cytokines, receptor cross-linking and viral infection. In contrast to IRF-1 and IRF-2, which are widely expressed, IRF-4 and IRF-8 are tissue-restricted factors. IRF-8 is expressed mainly in cells of haematopoietic origin and has recently been shown to inhibit mitogenic activity of p210 Bcr/Abl-transformed myeloid progenitor cells by activating several genes that interfere with the c-Myc pathway. IRF-4 is most homologous with IRF-8 (approximately 70% overall homology) and its expression is highly restricted to lymphocytes of the B-cell type (pre-B, B, and plasma cells), mature T-cells and macrophages. Furthermore IRF-4 expression is significantly impaired in CML and AML patient samples predominately in T-cells. To examine a potential role of IRF-4 in Bcr/Abl mediated transformation we used a bone marrow transplant model (BMT). We transduced IRF-4 knockout (KO) bone marrow with retrovirus expressing p210 Bcr/Abl and transplanted it into lethally irradiated recipient C57/bl6 mice. For proper control we transplanted also wildtype (WT) bone marrow transduced with Bcr/Abl and mock transfected IRF-4 KO bone marrow (BM). All recipients transplanted with Bcr/Abl transduced BM (regardless of which IRF-4 KO or WT) developed rapidly a myeloproliferative disorder characterized by leukocytosis and expression of the myeloid lineage markers CD11b and Gr1. Surprisingly, IRF-4 KO Bcr/Abl infected BM recipient mice survived slightly longer than the control group transplanted with WT p210 BM (12 vs. 19 days). Histopathologic studies of the affected organs (spleen/lung) revealed extramedullary haematopoiesis in the spleens of both groups and a distinct infiltration of the tumor cells in the lung of WT Bcr/Abl transduced BM recipient mice, resulting in massive punctuated bleedings. Interestingly, preliminary analysis suggest a significantly reduced lung infiltration with almost no pulmonary bleedings in IRF-4 KO Bcr/Abl infected BM recipient mice, which we assume to be the reason for the differences in the overall survival. Taken together our data demonstrate that IRF-4 is not required for the induction of a myeloproliferative disorder by Bcr/Abl in vivo and for its ability to transform BM cells in vitro, but IRF-4 deficiency seems to have an impact on the fulminant pulmonary haemorrhage occurring in the murine CML-like disease.
A t(2;5) (p23;q35) chromosomal translocation can be found in more than 50% of all cases of anaplastic large cell lymphoma (ALCL), which belong to the group of aggressive Non-Hodgkin-Lymphomas (NHL). The fusion of the nucleophosmin gene (NPM) on chromosome 5 to the anaplastic lymphoma kinase (ALK) on chromosome 2 leads to a deregulated, constitutively active kinase which has been shown to play an important role in lymphomagenesis of ALCL. Mouse models, where the NPM-ALK fusion oncogene is retrovirally expressed in whole bone marrow (BM) cells which are subsequently transplanted into lethally irrradiated recipients have shown that NPM-ALK is able to induce histiocytic and plasmacytoid malignancies. Since ALCLs are in the vast majority either of T- or ‘Null’-cell phenotype, we have now developed a murine model of T-lymphoid ALCL. By employing a retroviral vector incorporating a translational stop-cassette flanked by loxP recombination sites recognized by the Cre recombinase, we established a new method of lineage specific gene expression in-vivo. We tested the vector by infecting BM cells harvested from mice expressing Cre from different lineage specific promotors. We have infected BM cells from mice expressing Cre under the control of the lysozyme M-promotor (lysM-mice), which is active in the myeloid compartment, from mice expressing Cre from the granzyme B-promotor (GrzmB-mice), which is mainly active in T-cells, and finally we also used BM cells from C57B6 wildtype mice as a control. Mice transplanted with BM from lysM-mice succumbed to a histiocytic malignancy after 4–6 weeks, whereas mice transplanted with BM from GrzmB-mice developed a mixed T-NHL/histiocytic phenotype with a similar latency. These mice displayed infiltration of malignant, mostly CD4/CD8 DP T-cells in the thymus, lymphnodes and spleen, as well as a histiocytic infiltration in the spleen and BM. Mice transplanted with control BM from wt mice did not show signs of disease after up to 6 months. The results indicate that the hematopoetic lineage where an oncogene is expressed is paramount to the malignant phenotype The described model represents a versatile tool for the analysis of NPM-ALK signaling pathways in-vivo. With the help of this method, any gene can be rapidly expressed in a defined lineage or in a developmental stage. Thus, also more subtle phenotypes may become amenable to investigation in-vivo.
712 Transforming growth factor β (TGFβ) plays a critical role in regulating cellular processes like proliferation, extracellular matrix production, vasculogenesis and angiogenesis as well as immunomodulation. TGFβ is a pluripotent cytokine with a pronounced immunosuppressive effect by controlling proliferation, differentiation and activation of immune cells. TGFβ binding to its receptor leads to the phosphorylation of R-Smads. R-Smads again form a heteromeric complex with the cytosolic common Smad4. This Smad complex, together with additional cofactors, translocate into the nucleus, where they control the transcription of TGFβ target genes. Smif was originally identified in our lab as an interaction partner of Smad4. Functional analysis revealed a stimulatory effect in regulating TGFβ-dependent genes like the early target gene JunB. After TGFβ stimulation, Smif tranlocates, together with Smad4, into the nucleus, where Smif acts as a coactivator. To investigate the role of Smif in mammals, we generated a Smif knockout mouse. To this end exon 2 of Smif was replaced by GFP and an inverted neomycin selection cassette. Smif-deficient mice were viable but exhibit a shortened life span. On the average, these mice die at 12 month of age due to multifocal inflammatory disease. Overall pathological analysis of diseased mice revealed extensive lymphocytic infiltrates in multiple organs. Moreover, Smif-deficiency caused immune complex induced glomerulonephritis associated with proteinuria. In line with these findings, autoantibodies could be detected in the serum of Smif knockout mice. Interestingly, we identified T cells and not B-cells as the important target in Smif-deficient mice. T cells lacking Smif were spontaneously activated. In addition, TGFβ was not able to block T cell proliferation of CD4+ cells in vitro, whereas B cells isolated from Smif knockout spleens behave as wildtype. Transcription of TGFβ responsive reporter constructs was greatly reduced in Smif knockout Mefs and could be rescued by the reexpression of functional Smif. Taken all together, the observed autoimmune phenotype found in Smif-deficient mice is at least partially caused by overactivated T cells due to downregulation of the inhibitory TGFβ pathway. Disclosures: No relevant conflicts of interest to declare.
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