RESUMO
Activating mutations in FMS-like tyrosine kinase receptor-3 (FLT3) and Nucleophosmin-1 (NPM1) are most frequent alterations in acute myeloid leukemia (AML), and are often coincidental. The mutational status of NPM1 has strong prognostic relevance to patients with point mutations of the FLT3 tyrosine kinase domain (TKD), but the biological mechanism underlying this effect remains unclear. In the present study, we investigated the effect of the coincidence of NPM1c and FLT3-TKD. Although expression of FLT3-TKD is not sufficient to induce a disease in mice, coexpression with NPM1c rapidly leads to an aggressive myeloproliferative disease in mice with a latency of 31.5 days. Mechanistically, we could show that FLT3-TKD is able to activate the downstream effector molecule signal transducer and activator of transcription 5 (STAT5) exclusively in the presence of mutated NPM1c. Moreover, NPM1c alters the cellular localization of FLT3-TKD from the cell surface to the endoplasmic reticulum, which might thereby lead to the aberrant STAT5 activation. Importantly, aberrant STAT5 activation occurs not only in primary murine cells but also in patients with AML with combined FLT3-TKD and NPM1c mutations. Thus, our data indicate a new mechanism, how NPM1c mislocalizes FLT3-TKD and changes its signal transduction ability.
Assuntos
Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Mutação , Proteínas Nucleares/genética , Transdução de Sinais , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/metabolismo , Substituição de Aminoácidos , Animais , Modelos Animais de Doenças , Retículo Endoplasmático/metabolismo , Duplicação Gênica , Regulação Leucêmica da Expressão Gênica , Humanos , Camundongos , Proteínas Nucleares/metabolismo , Nucleofosmina , Transporte Proteico , Fator de Transcrição STAT5/metabolismo , Sequências de Repetição em TandemRESUMO
The NPM-ALK fusion kinase is expressed in 60% of systemic anaplastic large-cell lymphomas (ALCL). A Nuclear Interaction Partner of ALK (NIPA) was identified as a binding partner of NPM-ALK. To identify the precise role of NIPA for NPM-ALK-driven lymphomagenesis, we investigated various NPM-ALK+ cell lines and mouse models. Nipa deletion in primary mouse embryonic fibroblasts resulted in reduced transformation ability and colony formation upon NPM-ALK expression. Downregulating NIPA in murine NPM-ALK+ Ba/F3 and human ALCL cells decreased their proliferation ability and demonstrated synergistic effects of ALK inhibition and NIPA knockdown. Comprehensive in vivo analyses using short- and long-latency transplantation mouse models with NPM-ALK+ bone marrow (BM) revealed that Nipa deletion inhibited NPM-ALK-induced tumorigenesis with prolonged survival and reduced spleen colonies. To avoid off-target effects, we combined Nipa deletion and NPM-ALK expression exclusively in T cells using a lineage-restricted murine ALCL-like model resembling human disease: control mice died from neoplastic T-cell infiltration, whereas mice transplanted with Lck-CreTG/wtNipaflox/flox NPM-ALK+ BM showed significantly prolonged survival. Immunophenotypic analyses indicated a characteristic ALCL-like phenotype in all recipients but revealed fewer "stem-cell-like" features of Nipa-deficient lymphomas compared to controls. Our results identify NIPA as a crucial player in effective NPM-ALK-driven ALCL-like disease in clinically relevant murine and cell-based models.
RESUMO
While cancer stem cells are well established in certain hematologic and solid malignancies, their existence in T cell lymphoma is unclear and the origin of disease is not fully understood. To examine the existence of lymphoma stem cells, we utilized a mouse model of anaplastic large cell lymphoma. Established NPM-ALK+ lymphomas contained heterogeneous cell populations ranging from mature T cells to undifferentiated hematopoietic stem cells. Interestingly, CD4-/CD8- double negative (DN) lymphoma cells aberrantly expressed the T cell receptor α/ß chain. Serial transplantation of sorted CD4/CD8 and DN lymphoma subpopulations identified lymphoma stem cells within the DN3/DN4 T cell population, whereas all other subpopulations failed to establish serial lymphomas. Moreover, transplanted lymphoma DN3/DN4 T cells were able to differentiate and gave rise to mature lymphoma T cells. Gene expression analyses unmasked stem-cell-like transcriptional regulation of the identified lymphoma stem cell population. Furthermore, these lymphoma stem cells are characterized by low CD30 expression levels, which might contribute to limited long-term therapeutic success in patients treated with anti-CD30-targeted therapies. In summary, our results highlight the existence of a lymphoma stem cell population in a NPM-ALK-driven CD30+ mouse model, thereby giving the opportunity to test innovative treatment strategies developed to eradicate the origin of disease.
Assuntos
Linfoma Anaplásico de Células Grandes/imunologia , Linfoma de Células T/imunologia , Células-Tronco/imunologia , Quinase do Linfoma Anaplásico/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular , Modelos Animais de Doenças , Feminino , Expressão Gênica/imunologia , Antígeno Ki-1/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Translocação Genética/imunologiaRESUMO
Targeted expression of transgenes is essential for the accurate representation of human disease in in vivo models. Current approaches to generate conditional transgenic mouse models are cumbersome and not amenable to high-throughput analysis since they require de novo generation and characterization of genetically modified mice. Here we describe a new system for lineage-restricted expression of transgenes based on a retroviral vector incorporating a translational stop cassette flanked by loxP recombination sites. Conditional transgene expression in chimeric mice is achieved by retroviral infection and transplantation of hematopoietic stem cells (HSC) derived from transgenic mice expressing Cre-recombinase from a lineage-specific promoter. For validation, we directed expression of NPM-ALK, the fusion oncogene driving a subset of anaplastic large cell lymphoma (ALCL), to T-cells by infecting hematopoietic stem cells from Lck-Cre-transgenic mice with a retroviral construct containing the NPM-ALK cDNA preceded by a translational stop cassette. These mice developed T-cell lymphomas within 12-16 weeks, featuring increased expression of the ALCL hallmark antigen CD30 as well as other cytotoxic T-cell markers, similar to the human disease. The new model represents a versatile tool for the rapid analysis of gene function in a defined lineage or in a developmental stage in vivo.
Assuntos
Antígeno Ki-1/metabolismo , Linfoma Anaplásico de Células Grandes/patologia , Linfoma de Células T/patologia , Processamento de Proteína Pós-Traducional , Proteínas Tirosina Quinases/metabolismo , Animais , Apoptose , Proliferação de Células , Feminino , Humanos , Linfoma Anaplásico de Células Grandes/genética , Linfoma Anaplásico de Células Grandes/metabolismo , Linfoma de Células T/genética , Linfoma de Células T/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Tirosina Quinases/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Inherited bone marrow failure syndromes (IBMFSs) are a heterogeneous group of disorders characterized by defective hematopoiesis, impaired stem cell function, and cancer susceptibility. Diagnosis of IBMFS presents a major challenge due to the large variety of associated phenotypes, and novel, clinically relevant biomarkers are urgently needed. Our study identified nuclear interaction partner of ALK (NIPA) as an IBMFS gene, as it is significantly downregulated in a distinct subset of myelodysplastic syndrome-type (MDS-type) refractory cytopenia in children. Mechanistically, we showed that NIPA is major player in the Fanconi anemia (FA) pathway, which binds FANCD2 and regulates its nuclear abundance, making it essential for a functional DNA repair/FA/BRCA pathway. In a knockout mouse model, Nipa deficiency led to major cell-intrinsic defects, including a premature aging phenotype, with accumulation of DNA damage in hematopoietic stem cells (HSCs). Induction of replication stress triggered a reduction in and functional decline of murine HSCs, resulting in complete bone marrow failure and death of the knockout mice with 100% penetrance. Taken together, the results of our study add NIPA to the short list of FA-associated proteins, thereby highlighting its potential as a diagnostic marker and/or possible target in diseases characterized by hematopoietic failure.
Assuntos
Síndrome Congênita de Insuficiência da Medula Óssea , Proteína do Grupo de Complementação D2 da Anemia de Fanconi , Células-Tronco Hematopoéticas/metabolismo , Proteínas Nucleares , Animais , Síndrome Congênita de Insuficiência da Medula Óssea/genética , Síndrome Congênita de Insuficiência da Medula Óssea/metabolismo , Síndrome Congênita de Insuficiência da Medula Óssea/patologia , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Células-Tronco Hematopoéticas/patologia , Camundongos , Camundongos Knockout , Proteínas Nucleares/deficiência , Proteínas Nucleares/metabolismo , Ligação ProteicaRESUMO
UNLABELLED: The CD30-specific antibody-drug conjugate, brentuximab vedotin, is approved for the treatment of relapsed, refractory Hodgkin lymphomas and systemic anaplastic large T-cell lymphomas. Multiple ongoing clinical trials are investigating brentuximab vedotin efficacy in other CD30-positive hematologic malignancies. Because CD30 expression varies among different types of lymphoma and can also change during the course of treatment, companion diagnostic imaging of CD30 could be a valuable tool in optimizing patient-specific brentuximab vedotin treatment regimens. METHODS: The mouse antihuman CD30 antibody AC-10 was radiolabeled with the positron-emitting radionuclide (89)Zr. The stability and specificity of (89)Zr-desferrioxamine (DFO)-labeled CD30-specific AC-10 antibody ((89)Zr-DFO-AC-10) was evaluated in vitro. The pharmacokinetics of (89)Zr-DFO-AC-10 was studied in BALB/c nude mice bearing subcutaneous human Karpas 299 tumors (CD30-positive model) or A-431 tumors (CD30-negative model) using PET/CT imaging, biodistribution studies, and autoradiography. RESULTS: AC-10 was conjugated with a DFO B chelator and radiolabeled with (89)Zr to give formulated (89)Zr-DFO-AC-10 with a radiochemical yield of 80%, radiochemical purity greater than 99%, and specific activity of 111-148 MBq/mg. (89)Zr-DFO-AC-10 was stable in mouse and human sera and preserved the immunoreactivity toward CD30. Biodistribution data showed the highest tissue accumulation of (89)Zr-DFO-AC-10 in CD30-positive tumors, with 37.9% ± 8.2% injected activity per gram of tissue at 72 h after injection, whereas uptake in CD30-negative tumors was 11.0% ± 0.4%. The specificity of (89)Zr-DFO-AC-10 binding to CD30 in vivo was confirmed by blocking studies. Time-activity curves showed that between 24 and 144 h after injection, tumor-to-muscle ratios increased from 18.9 to 51.8 in the CD30-positive model and from 4.8 to 8.7 in the CD30-negative model. Tumor-to-blood ratios also increased, from 3.2 to 13.6 and from 1 to 2 in the CD30-positive and -negative models, respectively. CONCLUSION: Our results demonstrate that for measuring CD30 expression, (89)Zr-DFO-AC-10 is a sensitive PET agent with high tumor-to-normal-tissue contrast. (89)Zr-DFO-AC-10 is a promising CD30-imaging radiotracer for clinical translation in patients with various lymphomas and other diseases.