ABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
Constitutive activation of the PI3K/AKT signaling pathway is found in ~50-70% of AML patients. The SH2-containing inositol 5-phosphatase 1 (SHIP1) is a negative regulator of PI3K/AKT signaling in hematopoietic cells. SHIP1 knockout mice develop a myeloproliferative syndrome and concomitant deletion of SHIP1 and the tumor suppressor PTEN leads to the development of lethal B-cell lymphomas. In the study presented here, we investigated the role of SHIP1 as a tumor suppressor in myeloid leukemia cells in an in vivo xenograft transplantation model. NSG Mice transplanted with UKE-1 cells derived from a secondary AML showed a significantly extended lifespan after lentiviral-mediated overexpression of SHIP1 in comparison to the vector control cohort. In contrast, the AML-derived SHIP1Y643H mutant, which has a strongly reduced enzymatic activity showed a significant reversion of the SHIP1-induced prolongation of the survival time. In addition, the analysis of 290 AML patients revealed a correlation between expression of SHIP1 and overall survival of the AML patients. These results indicate that SHIP1 can act as a tumor suppressor in acute myeloid leukemia cells and that higher SHIP1 expression is associated with prolonged overall survival in AML patients. SHIP1 may be an interesting candidate for gene therapy.
Subject(s)
Leukemia, Myeloid, Acute/therapy , Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases/genetics , Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases/metabolism , Animals , Cell Line, Tumor , Cell Proliferation , Disease Models, Animal , Gene Expression Regulation, Leukemic/genetics , Humans , Lentivirus/metabolism , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Mice , Mice, Inbred NOD , Mice, Knockout , Phosphatidylinositol 3-Kinases/metabolism , Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases/pharmacology , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction , Transplantation, Heterologous/methodsABSTRACT
We screened 136 patients with myelofibrosis and a median age of 58 years who underwent allogeneic stem cell transplantation (AHSCT) for molecular residual disease for JAKV617F (n=101), thrombopoietin receptor gene (MPL) (n=4) or calreticulin (CALR) (n=31) mutation in peripheral blood on day +100 and +180 after AHSCT. After a median follow-up of 78 months, the 5-year estimated overall survival was 60% (95% confidence interval (CI): 50-70%) and the cumulative incidence of relapse at 5 years was 26% (95% CI: 18-34%) for the entire study population. The percentage of molecular clearance on day 100 was higher in CALR-mutated patients (92%) in comparison with MPL- (75%) and JAKV617F-mutated patients (67%). Patients with detectable mutation at day +100 or at day +180 had a significant higher risk of clinical relapse at 5 years than molecular-negative patients (62% vs 10%, P<0.001) and 70% vs 10%, P<0.001, respectively) irrespectively of the underlying mutation. In a multivariate analysis, high-risk diseases status (hazard ratio (HR) 2.5; 95% CI: 1.18-5.25, P=0.016) and detectable MRD at day 180 (HR 8.36, 95% CI: 2.76-25.30, P<0.001) were significant factors for a higher risk of relapse.
Subject(s)
Calreticulin/genetics , Janus Kinase 2/genetics , Neoplasm, Residual/genetics , Pathology, Molecular/methods , Primary Myelofibrosis/diagnosis , Receptors, Thrombopoietin/genetics , Hematopoietic Stem Cell Transplantation/methods , Hematopoietic Stem Cell Transplantation/mortality , Humans , Middle Aged , Mutation , Neoplasm, Residual/diagnosis , Neoplasm, Residual/mortality , Primary Myelofibrosis/genetics , Primary Myelofibrosis/mortality , Recurrence , Survival Rate , Transplantation, HomologousABSTRACT
The hematopoietic bone marrow microenvironment is formed by proliferation and differentiation of mesenchymal stem cells (MSCs). The MSC compartment has been less studied than the hematopoietic stem cell compartment. To characterize the structure of the MSC compartment, it is necessary to trace the fate of distinct mesenchymal cells. To do so, mesenchymal progenitors need to be marked at the single-cell level. A method for individual marking of normal and cancer stem cells based on genetic "barcodes" has been developed for the last 10 years. Such approach has not yet been applied to MSCs. The aim of this study was to evaluate the possibility of using such barcoding strategy to mark MSCs and their descendants, colony-forming units of fibroblasts (CFU-Fs). Adherent cell layers (ACLs) of murine long-term bone marrow cultures (LTBMCs) were transduced with a lentiviral library with barcodes consisting of 32 + 3 degenerate nucleotides. Infected ACLs were suspended, and CFU-F derived clones were obtained. DNA was isolated from each individual colony, and barcodes were analyzed in marked CFU-F-derived colonies by means of conventional polymerase chain reaction and Sanger sequencing. Barcodes were identified in 154 marked colonies. All barcodes appeared to be unique: there were no two distinct colonies bearing the same barcode. It was shown that ACLs included CFU-Fs with different proliferative potential. MSCs are located higher in the hierarchy of mesenchymal progenitors than CFU-Fs, so the presented data indicate that MSCs proliferate rarely in LTBMCs. A method of stable individual marking and comparing the markers in mesenchymal progenitor cells has been developed in this work. We show for the first time that a barcoded library of lentiviruses is an effective tool for studying stromal progenitor cells.
Subject(s)
Lentivirus/genetics , Mesenchymal Stem Cells/metabolism , Animals , Bone Marrow Cells/cytology , Cells, Cultured , Female , Gene Library , Mesenchymal Stem Cells/cytology , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Plasmids/genetics , Plasmids/metabolism , Polymerase Chain ReactionSubject(s)
Calreticulin/genetics , Hematopoietic Stem Cell Transplantation/methods , Mutation , Primary Myelofibrosis/therapy , Adult , Aged , DNA Mutational Analysis , Humans , Middle Aged , Primary Myelofibrosis/genetics , Primary Myelofibrosis/mortality , Survival Analysis , Transplantation Conditioning/methods , Transplantation, Homologous , Treatment OutcomeSubject(s)
Calreticulin/genetics , Hematopoietic Stem Cell Transplantation , Polymerase Chain Reaction , Primary Myelofibrosis/genetics , Alleles , Calreticulin/metabolism , Humans , Immunotherapy/methods , Janus Kinase 2/genetics , Janus Kinase 2/metabolism , Mutation , Primary Myelofibrosis/diagnosis , Primary Myelofibrosis/metabolism , Recurrence , Remission Induction , Time FactorsABSTRACT
Adeno-associated viral (AAV) vectors yield high potential for clinical gene therapy but, like for other vectors systems, they frequently do not sufficiently transduce the target tissue and their unspecific tropism prevents their application for multifocal diseases such as disseminated cancer. Targeted AAV vectors have been obtained from random AAV display peptide libraries but so far, all vector variants selected from AAV libraries upon systemic administration in vivo retained some collateral tropism, frequently the heart. Here we explored, if this impediment can be overcome by microRNA-regulated transgene cassettes as the combination of library-derived capsid targeting and micro-RNA control has not been evaluated so far. We used a tumor-targeted AAV capsid variant (ESGLSQS) selected from random AAV-display peptide libraries in vivo with remaining off-target tropism toward the heart and regulated targeted transgene expression in vivo by complementary target elements for heart-specific microRNA (miRT-1d). Although this vector still maintained its strong transduction capacity for tumor target tissue after intravenous injection, transgene expression in the heart was almost completely abrogated. This strong and completely tumor-specific transgene expression was used for therapeutic gene transfer in an aggressive multifocal, transgenic, polyoma middle T-induced, murine breast cancer model. A therapeutic suicide gene, delivered systemically by this dual-targeted AAV vector to multifocal breast cancer, significantly inhibited tumor growth after one single vector administration while avoiding side effects compared with untargeted vectors.
Subject(s)
Dependovirus , Genes, Transgenic, Suicide , Genetic Therapy , Genetic Vectors , Mammary Neoplasms, Experimental/therapy , Animals , Female , Mammary Neoplasms, Experimental/genetics , Mice , MicroRNAs/administration & dosageSubject(s)
Leukemia/genetics , Pregnancy Complications, Neoplastic/genetics , Stem Cell Transplantation/methods , Adolescent , Adult , Child , Child, Preschool , Chimerism , Female , Fetus , Humans , Infant , Infant, Newborn , Male , Pregnancy , Tissue Donors , Young AdultABSTRACT
Carcinogenesis is a multistep process involving alterations in various cellular pathways. The critical genetic events driving the evolution of primary liver cancer, specifically hepatoblastoma and hepatocellular carcinoma (HCC), are still poorly understood. However, telomere stabilization is acknowledged as prerequisite for cancer progression in humans. In this project, human fetal hepatocytes were utilized as a cell culture model for untransformed, proliferating human liver cells, with telomerase activation as first oncogenic hit. To elucidate critical downstream genetic events driving further transformation of immortalized liver cells, we used retroviral insertional mutagenesis as an unbiased approach to induce genetic alterations. Following isolation of hyperproliferating, provirus-bearing cell clones, we monitored cancer-associated growth properties and characterized changes toward a malignant phenotype. Three transformed clones with the ability to form colonies in soft agar were expanded. As proof-of-principle for our experimental setup, we identified a transforming insertion on chromosome 8 within the pleiomorphic adenoma gene 1 (PLAG1), resulting in a 20-fold increase in PLAG1 expression. Upregulation of PLAG1 has already been described to promote human hepatoblastoma development. In a separate clone, a transforming insertion was detected in close proximity to the receptor-interacting serine-threonine kinase 4 (RIPK4) with an approximately eightfold suppression in RIPK4 expression. As validation for this currently unknown driver in hepatocarcinogenesis, we examined RIPK4 expression in human HCC samples and confirmed a significant suppression of RIPK4 in 80% of the samples. Furthermore, overexpression of RIPK4 in transformed human fetal hepatocytes resulted in an almost complete elimination of anchorage-independent growth. On the basis of these data, we propose RIPK4 as a novel putative tumor suppressor in human hepatocarcinogenesis.
Subject(s)
Cell Transformation, Neoplastic/metabolism , Hepatocytes/metabolism , Protein Serine-Threonine Kinases/metabolism , Tumor Suppressor Proteins/metabolism , Animals , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Line , Cell Line, Transformed , Cell Line, Tumor , Cell Proliferation , Cell Transformation, Neoplastic/genetics , Clone Cells/metabolism , Fetus , Flow Cytometry , Gene Expression Profiling , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Mutagenesis, Insertional , Protein Serine-Threonine Kinases/genetics , Retroviridae/genetics , Reverse Transcriptase Polymerase Chain Reaction , Telomerase/genetics , Telomerase/metabolism , Tumor Suppressor Proteins/geneticsABSTRACT
Multiple myeloma is a mostly incurable malignancy characterized by the expansion of a malignant plasma cell (PC) clone in the human bone marrow (BM). Myeloma cells closely interact with the BM stroma, which secretes soluble factors that foster myeloma progression and therapy resistance. Growth arrest-specific gene 6 (Gas6) is produced by BM-derived stroma cells and can promote malignancy. However, the role of Gas6 and its receptors Axl, Tyro3 and Mer (TAM receptors) in myeloma is unknown. We therefore investigated their expression in myeloma cell lines and in the BM of myeloma patients and healthy donors. Gas6 showed increased expression in sorted BMPCs of myeloma patients compared with healthy controls. The fraction of Mer(+) BMPCs was increased in myeloma patients in comparison with healthy controls whereas Axl and Tyro3 were not expressed by BMPCs in the majority of patients. Downregulation of Gas6 and Mer inhibited the proliferation of different myeloma cell lines, whereas knocking down Axl or Tyro3 had no effect. Inhibition of the Gas6 receptor Mer or therapeutic targeting of Gas6 by warfarin reduced myeloma burden and improved survival in a systemic model of myeloma. Thus, the Gas6-Mer axis represents a novel candidate for therapeutic intervention in this incurable malignancy.
Subject(s)
Gene Expression Regulation, Neoplastic , Intercellular Signaling Peptides and Proteins/genetics , Multiple Myeloma/genetics , Plasma Cells/metabolism , Proto-Oncogene Proteins/genetics , Receptor Protein-Tyrosine Kinases/genetics , Animals , Bone Marrow Cells/metabolism , Bone Marrow Cells/pathology , Case-Control Studies , Cell Line, Tumor , Female , Humans , Intercellular Signaling Peptides and Proteins/metabolism , Mice , Mice, Inbred NOD , Multiple Myeloma/mortality , Multiple Myeloma/pathology , Multiple Myeloma/therapy , Neoplasm Transplantation , Plasma Cells/pathology , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Receptor Protein-Tyrosine Kinases/metabolism , Signal Transduction , Stromal Cells/metabolism , Stromal Cells/pathology , Survival Analysis , Warfarin/pharmacology , c-Mer Tyrosine Kinase , Axl Receptor Tyrosine KinaseSubject(s)
Antigens, Neoplasm/immunology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Immunotherapy, Adoptive/methods , Multiple Myeloma/immunology , Multiple Myeloma/therapy , Neoplasm Proteins/immunology , Antigen-Presenting Cells/immunology , Antigens, Neoplasm/administration & dosage , Epitopes/administration & dosage , Epitopes/immunology , Epitopes, B-Lymphocyte/immunology , Humans , Multiple Myeloma/blood , Neoplasm Proteins/administration & dosageABSTRACT
Adoptive immunotherapy with T lymphocytes expressing transgenic T-cell receptors (TCRs) has shown significant clinical efficacy in various malignant diseases. However, concurrent expression of endogenous and transgenic TCRs in one and the same T cell may impair efficacy and cause safety problems owing to mispairings. The most elegant approach to address these issues is the complete shutoff of the endogenous receptor chains by genome editing. To this end, we designed TCR-α and TCR-ß-specific pairs of transcription activator-like effector nucleases (TALENs). TALENs were delivered into T cells using an optimized messenger RNA-electroporation protocol. Based thereon, we obtained precise and highly efficient knockout (KO) in Jurkat (TCR-α: 59.7 ± 4.0%, TCR-ß: 37.4 ± 7.3%) as well as primary T cells (TCR-α: 58.0 ± 15.0%, TCR-ß: 41.0 ± 17.6%). Moreover, a successive KO strategy for the endogenous TCR chains combined with subsequent transduction of the respective chains of an Influenza virus-specific model TCR led to complete reprogramming of T cells with strongly improved expression and functionality of transgenic TCRs. In conclusion, we have developed novel means for the efficient genome editing in primary T lymphocytes.
Subject(s)
Gene Transfer Techniques , Lentivirus/genetics , Receptors, Antigen, T-Cell, alpha-beta/genetics , T-Lymphocytes/physiology , Deoxyribonucleases, Type II Site-Specific/genetics , Electroporation/methods , Gene Knockout Techniques , Genetic Vectors , Humans , Jurkat Cells , Plasmids , RNA, Messenger , Receptors, Antigen, T-Cell, alpha-beta/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Transfection/methodsSubject(s)
Calreticulin/genetics , Mutation , Primary Myelofibrosis/genetics , Primary Myelofibrosis/mortality , Graft vs Host Disease/etiology , Hematopoietic Stem Cell Transplantation/adverse effects , Humans , Primary Myelofibrosis/therapy , Prognosis , Transplantation, Homologous , Treatment OutcomeABSTRACT
Here we describe a system based on recombinant lentiviral vectors for the safe screening of potential anti-HIV drugs. The system allows to evaluate the sensitivity of HIVl-1 reverse transcriptase and integrase (wild-type as well as mutant forms of these enzymes detected in drug-resistant virus isolates) towards different drugs and substances, but also to screen inhibitors of other stages of HIV-1 life cycle.
Subject(s)
Anti-HIV Agents/pharmacology , HIV Integrase Inhibitors/pharmacology , HIV-1/drug effects , High-Throughput Screening Assays , Virus Replication/drug effects , Drug Resistance, Viral , Flow Cytometry , Gene Expression , Genes, Reporter , Genetic Vectors , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , HIV Integrase/genetics , HIV Integrase/metabolism , HIV Reverse Transcriptase/antagonists & inhibitors , HIV Reverse Transcriptase/genetics , HIV Reverse Transcriptase/metabolism , HIV-1/enzymology , HIV-1/genetics , Humans , Lentivirus/genetics , Transduction, Genetic , Virion/drug effects , Virion/growth & developmentABSTRACT
Six p53 wild-type cancer cell lines from infrequently p53-mutated entities (neuroblastoma, rhabdomyosarcoma, and melanoma) were continuously exposed to increasing concentrations of the murine double minute 2 inhibitor nutlin-3, resulting in the emergence of nutlin-3-resistant, p53-mutated sublines displaying a multi-drug resistance phenotype. Only 2 out of 28 sublines adapted to various cytotoxic drugs harboured p53 mutations. Nutlin-3-adapted UKF-NB-3 cells (UKF-NB-3(r)Nutlin(10 µM), harbouring a G245C mutation) were also radiation resistant. Analysis of UKF-NB-3 and UKF-NB-3(r)Nutlin(10 µM) cells by RNA interference experiments and lentiviral transduction of wild-type p53 into p53-mutated UKF-NB-3(r)Nutlin(10 µM) cells revealed that the loss of p53 function contributes to the multi-drug resistance of UKF-NB-3(r)Nutlin(10 µM) cells. Bioinformatics PANTHER pathway analysis based on microarray measurements of mRNA abundance indicated a substantial overlap in the signalling pathways differentially regulated between UKF-NB-3(r)Nutlin(10 µM) and UKF-NB-3 and between UKF-NB-3 and its cisplatin-, doxorubicin-, or vincristine-resistant sublines. Repeated nutlin-3 adaptation of neuroblastoma cells resulted in sublines harbouring various p53 mutations with high frequency. A p53 wild-type single cell-derived UKF-NB-3 clone was adapted to nutlin-3 in independent experiments. Eight out of ten resulting sublines were p53-mutated harbouring six different p53 mutations. This indicates that nutlin-3 induces de novo p53 mutations not initially present in the original cell population. Therefore, nutlin-3-treated cancer patients should be carefully monitored for the emergence of p53-mutated, multi-drug-resistant cells.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Resistance, Neoplasm/genetics , Imidazoles/pharmacology , Piperazines/pharmacology , Proto-Oncogene Proteins c-mdm2/antagonists & inhibitors , Adaptation, Biological/drug effects , Caspase 3/metabolism , Caspase 7/metabolism , Humans , Mutation , Proto-Oncogene Proteins c-mdm2/metabolism , RNA Interference , Tumor Cells, Cultured , Tumor Suppressor Protein p53/metabolismABSTRACT
The development and usage of safe cell systems for testing agents which possess anti-HIV activity is a very important factor in the design of new drugs. We have described in detail a system we designed that is based on lentiviral vectors (Prokofjeva et. al.,Antiviral Therapy,in print) for swift and completely safe screening of potential HIV-1 replication inhibitors. The system enables one to test the efficiency of the inhibitory activity of compounds whose action is directed towards either wild-type HIV-1 reverse transcriptase or integrase, or mutant enzymes corresponding to the drug-resistant virus form. Testing results of a number of already known drugs, which correlate well with published data as well as data on newly synthesized compounds, were obtained. Application of this system substantially broadens the possibilities of preclinical anti-HIV drugs testing.
ABSTRACT
Monitoring of minimal residual disease (MRD) after allogeneic (allo)-SCT for myelofibrosis (MF) allows recognizing the depth of remission and thus guides application of appropriate therapeutic interventions. MPL W515L/K mutations, which are detected in 5-10% of JAK2V617F-negative patients, may be useful for this purpose. Using a highly sensitive quantitative PCR method, we tested 90 patients with MF who underwent allo-SCT for the presence of MPL W515L/K mutations. Two patients with primary MF were found to harbor MPLW515L while no patient was positive for MPLW515K mutation. Both patients were JAK2V617F negative and cleared the mutation rapidly after allo-SCT and remained negative for a median follow-up of 19 months. The results of molecular monitoring correlated well with other remission parameters such as normalization of peripheral blood counts and morphology and complete donor chimerism. We conclude that MPLW515L can be cleared after allo-SCT and hence may be used as an MRD marker in a proportion of JAK2V617F-negative MF patients.
Subject(s)
Genetic Testing/methods , Hematopoietic Stem Cell Transplantation , Primary Myelofibrosis/genetics , Primary Myelofibrosis/therapy , Receptors, Thrombopoietin/genetics , Aged , Female , Follow-Up Studies , Genetic Markers/genetics , Genetic Testing/standards , Humans , Janus Kinase 2/genetics , Neoplasm, Residual/genetics , Neoplasm, Residual/therapy , Reproducibility of Results , Retrospective Studies , Reverse Transcriptase Polymerase Chain Reaction , Transplantation, HomologousABSTRACT
We investigated efficacy and toxicity of lenalidomide in 24 heavily pretreated myeloma patients with a median age of 59 years (range: 37-70) and relapse after allo-SCT. Lenalidomide was given at a dose of 15 mg (n=4), or 25 mg (n=20), orally once daily on day 1 to day 1 every 28 days, with (n=20) or without (n=4) DHAP. The median number of lenalidomide cycles was five (range: 2-17). Major side effects were leukopenia (grade 4: 4%, grade 3: 21% and grade 2: 17%) and thrombocytopenia (grade 3: 17% and grade 2: 29%); infectious complications were observed in 50%. Non-hematological toxicity consisted of muscle cramps (n=9), fatigue (n=5) and constipation (n=2). Mild grade I-II GVHD was seen in three patients. Response was achieved in 66%: CR in 8%, VGPR in 8%, PR in 50% and SD in 13%. The median time to progression was 9.7 months (95% confidence interval (CI): 7.5-11.9), and median OS was 19.9 months (95% CI: 17.3-22.5). Immunomonitoring after lenalidomide showed significant increase of activated NK (NKp44(+)) and T (HLA-DR(+)) cells, as well as regulatory T cells (CD4(+), CD25(+), CD127(lo)), supporting an immunomodulating anti-myeloma effect of lenalidomide.