Lentiviral marking of patient-derived acute lymphoblastic leukaemic cells allows in vivo tracking of disease progression
1] Newcastle Cancer Centre, Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne, UK  Department of Paediatric and Adolescent Haematology and Oncology, Great North Children's Hospital, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK. Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K
(Impact Factor: 10.43).
08/2013; 27(8):1792. DOI: 10.1038/leu.2013.113
Leukemia is one of the leading journals in hematology and oncology. It is published monthly and covers all aspects of the research and treatment of leukemia and allied diseases. Studies of normal hemopoiesis are covered because of their comparative relevance.
Available from: Abhishek Sharma
- "A protocol described previously  to transduce lymphoblastic leukaemic blasts was adapted to transduce Ewing-sarcoma cells with a lentiviral vector (pSLIEW) encoding both enhanced green fluorescent protein (EGFP) and firefly Luciferase (fLuc), allowing in vitro analysis and cell sorting (EGFP) and in vivo bioluminescent imaging (fLuc). The SLIEW virus was produced by transfection of HEK293T cells with equimolar amounts of a packaging vector (pCMVΔ8.91), "
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ABSTRACT: Ewing sarcoma and osteosarcoma represent the two most common primary bone tumours in childhood and adolescence, with bone metastases being the most adverse prognostic factor. In prostate cancer, osseous metastasis poses a major clinical challenge. We developed a preclinical orthotopic model of Ewing sarcoma, reflecting the biology of the tumour-bone interactions in human disease and allowing in vivo monitoring of disease progression, and compared this with models of osteosarcoma and prostate carcinoma. Human tumour cell lines were transplanted into non-obese diabetic/severe combined immunodeficient (NSG) and Rag2(-/-/)γc(-/-) mice by intrafemoral injection. For Ewing sarcoma, minimal cell numbers (1000-5000) injected in small volumes were able to induce orthotopic tumour growth. Tumour progression was studied using positron emission tomography, computed tomography, magnetic resonance imaging and bioluminescent imaging. Tumours and their interactions with bones were examined by histology. Each tumour induced bone destruction and outgrowth of extramedullary tumour masses, together with characteristic changes in bone that were well visualised by computed tomography, which correlated with post-mortem histology. Ewing sarcoma and, to a lesser extent, osteosarcoma cells induced prominent reactive new bone formation. Osteosarcoma cells produced osteoid and mineralised "malignant" bone within the tumour mass itself. Injection of prostate carcinoma cells led to osteoclast-driven osteolytic lesions. Bioluminescent imaging of Ewing sarcoma xenografts allowed easy and rapid monitoring of tumour growth and detection of tumour dissemination to lungs, liver and bone. Magnetic resonance imaging proved useful for monitoring soft tissue tumour growth and volume. Positron emission tomography proved to be of limited use in this model. Overall, we have developed an orthotopic in vivo model for Ewing sarcoma and other primary and secondary human bone malignancies, which resemble the human disease. We have shown the utility of small animal bioimaging for tracking disease progression, making this model a useful assay for preclinical drug testing.
Available from: Arnold Ganser
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ABSTRACT: Despite advances in allogeneic stem cell transplantation, BCR-ABL positive acute lymphoblastic leukemia (ALL) remains a high-risk disease, necessitating the development of novel treatment strategies. As the known oncomir, mir-17~92, is regulated by BCR-ABL fusion in chronic myeloid leukaemia, we investigated its role in BCR-ABL translocated ALL. miR-17~92-encoded miRNAs were significantly less abundant in BCR-ABL-positive as compared to -negative ALL-cells and over-expression of miR-17~19b triggered apoptosis in a BCR-ABL-dependent manner. Stable isotope labeling of amino acids in culture (SILAC) followed by liquid chromatography and mass spectroscopy (LC-MS) identified several apoptosis-related proteins including Bcl2 as potential targets of miR-17~19b. We validated Bcl2 as a direct target of this miRNA cluster in mice and humans, and, similar to miR-17~19b over-expression, Bcl2 specific RNAi strongly induced apoptosis in BCR-ABL-positive cells. Furthermore, BCR-ABL- positive human ALL-cell lines were more sensitive to pharmacological BCL2-inhibition than negative ones. Finally, in a xenograft model using patient-derived leukaemic blasts, real time, in vivo imaging confirmed pharmacological inhibition of BCL2 as a new therapeutic strategy in BCR-ABL-positive ALL. These data demonstrate the role of miR-17~92 in regulation of apoptosis, and identify BCL2 as a therapeutic target of particular relevance in BCR-ABL positive ALL.Leukemia accepted article preview online, 27 November 2013. doi:10.1038/leu.2013.361.
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ABSTRACT: The oncogenic fusion protein AML1-ETO, also known as RUNX1-RUNX1T1 is generated by the t(8;21)(q22;q22) translocation, one of the most frequent chromosomal rearrangements in acute myeloid leukemia (AML). Identifying the genes that cooperate with or are required for the oncogenic activity of this chimeric transcription factor remains a major challenge. Our previous studies showed that Drosophila provides a genuine model to study how AML1-ETO promotes leukemia. Here, using an in vivo RNAi screen for suppressors of AML1-ETO activity, we identified pontin/RUVBL1 as a gene required for AML1-ETO-induced lethality and blood cell proliferation in Drosophila. We further show that PONTIN inhibition strongly impaired the growth of human t(8;21)(+) or AML1-ETO-expressing leukemic blood cells. Interestingly, AML1-ETO promoted the transcription of PONTIN. Moreover transcriptome analysis in Kasumi-1 cells revealed a strong correlation between PONTIN and AML1-ETO gene signatures and demonstrated that PONTIN chiefly regulated the expression of genes implicated in cell cycle progression. Concordantly, PONTIN depletion inhibited leukemic self-renewal and caused cell cycle arrest. All together our data suggest that the up-regulation of PONTIN by AML1-ETO participate in the oncogenic growth of t(8;21) cells.Leukemia accepted article preview online, 17 December 2013. doi:10.1038/leu.2013.376.
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