ALBERT

Description: Leukemia caused by retroviral insertional mutagenesis after stem cell gene transfer has been reported in several experimental animals and in patients treated for X-linked severe combined immunodeficiency. Here, we analyzed whether gene transfer into mature T cells bears the same genotoxic risk. To address this issue in an experimental “worst case scenario,” we transduced mature T cells and hematopoietic progenitor cells from C57BL/6 (Ly5.1) donor mice with high copy numbers of gamma retroviral vectors encoding the potent T-cell oncogenes LMO2, TCL1, or ΔTrkA, a constitutively active mutant of TrkA. After transplantation into RAG-1–deficient recipients (Ly5.2), animals that received stem cell transplants developed T-cell lymphoma/leukemia for all investigated oncogenes with a characteristic phenotype and after characteristic latency periods. Ligation-mediated polymerase chain reaction analysis revealed monoclonality or oligoclonality of the malignancies. In striking contrast, none of the mice that received T-cell transplants transduced with the same vectors developed leukemia/lymphoma despite persistence of gene-modified cells. Thus, our data provide direct evidence that mature T cells are less prone to transformation than hematopoietic progenitor cells.

Description: After the report of two cases of leukaemia caused by insertional mutagenesis of a retroviral vector in children with SCID, it became clear that safety issues of therapeutic gene transfer must be addressed more thoroughly. We analysed whether gene transfer into mature T cells and haematopoietic stem cells bear the same risk of generating T cell leukaemia through activation of specific T cell oncogenes, such as LMO2, TCL1 and ΔTrkA. To address this issue, we used the Rag-1 mouse model, which allows long term analysis of transplanted T cells and haematopoietic stem cells. We were able to transduce mature T cells and haematopoietic stem cells of C57BL/6 (Ly5.1) donor mice with oncoretroviral vectors expressing LMO2, TCL1 and ΔTrkA. Transduction efficacies of up to 70% were achieved for mature T cells and approximately 90% for haematopoietic stem cells. After transplantation into Rag-1-deficient recipients, stem cell transplanted animals developed T cell lymphomas/leukemia for all investigated oncogenes after characteristic incubation times, mostly of a CD8+CD4+ double positive phenotype. T cell lymphomas were characterised by gross thymic mass, splenomegaly and heavily enlarged lymph nodes, although none of the control- vector- transduced mice developed lymphoma/leukaemia. LM PCR analysis revealed mono- or oligoclonality of the tumours. T cell transplanted animals showed no signs of leukaemia development so far. However, after several attempts, one immortalized T cell progenitor clone could be generated after transduction with LMO2. Our results so far indicate that mature T cells are less susceptible to transformation by known T cell proto-oncogenes, but the studies are still ongoing.

Description: Acute lymphoblastic leukemia (ALL) is known to consist of several clones that might have different chromosomal, genetic or epigenetic aberrations. However, little is known about functional diversity in these different clones. In some patients, cells cannot be eradicated by standard therapy regimens, and aggressive or otherwise unfavorable clones might survive, eventually resulting in relapse and a poor prognosis of the patients. Here, we asked whether genetically distinct clones of ALL from a single patient would show a functionally distinct response towards drug treatment in vivo. As technical approach, we genetically engineered primary patients' ALL cells growing in immuno-compromized NSG mice as patient derived xenograft (PDX) cells by lentiviral transduction. ALL PDX cells were red-green-blue (RGB) color marked in order to discriminate several differently colored cell populations in the same mouse in functional in vivo experiments. ALL PDX cells further expressed luciferase for bioluminescence in vivo imaging (BLI) for sensitive and reliable monitoring of disease burden. Limiting dilution transplantation of RGB marked PDX cells transplanted into groups of mice allowed generating individually marked single cell clones which were discriminated by flow cytometry. Populations expressing a distinct color were sorted and analyzed by ligation mediated PCR to verify distinct integration of lentiviral inserts to prove single cell clone (SCC) origin of the population. In sum, eight distinct SCCs could be generated and were used for functional and -OMICs approaches. Targeted resequencing of the eight SCCs and the bulk cells revealed that all samples had mutations in CSMD1 and HERC1 with variant allele frequencies (VAF) of 0.5, indicating that these mutations represent the founding clone. However, we also found mutations that were only present in single samples: FAT1 and STAG2 mutations were found in SCC 3, whereas CSMD1 and USP6 mutations were found in SCC 6. Whole exome sequencing revealed SCC specific patterns, identifying SCC 6 being the clone furthest away from the bulk population. As the patient showed a high hyperdiploidy (+6,+13,+14,+17,+18,+21,+22,+X), we tested SCC and bulk cells by fluorescence in situ hybridization (FISH) and found that both the bulk sample and the SCCs consisted mainly of cells harboring three X chromosomes and to a minor proportion (between 2% and 20%) of cells harboring two X chromosomes. Only SCC 6 consisted exclusively of cells harboring two X chromosomes. Additionally, this SCC showed a distinct DNA-methylation pattern analyzed by 450K arrays (illumina). To analyze if the chromosomal, genetic and epigenetic differences also resulted in functional diversity, we first performed a competitive transplantation assay, injecting a mixture of five SCCs in the same ratio (20% each) into single mice. After 42 days when overt leukemia had established in the mice, cells were re-isolated and proportion of SCCs reanalyzed according to their specific color. Interestingly, SCC 5 (25%) and 7 (36%) had a clear growth advantage over SCCs 1 (14%), 6 (13%) and 8 (12%). The same pattern could be overserved if only SCC 5 (50% in, 92% out) and SCC 6 (50% in, 8% out) were transplanted. Next, response towards chemotherapeutic drugs was assessed. In vitro, SCC 6 was much more resistant towards the glucocorticoids prednisolon and dexamethasone (Dexa) compared to all other SCCs and bulk cells. Cells of SCC 5 and SCC 6 were mixed in equal amounts and transplanted into mice. Four days after transplantation, mice were randomized and treated with PBS or Dexa (2 or 8 mg/kg i.p., 5 days a week, 5 weeks). BLI showed a clear response towards therapy of the entire tumor. After 61 days, control treated mice showed again an outgrowth of SCC 5 (83% vs. 17% SCC 6), while Dexa treated animals showed the opposite pattern (Dexa 2 mg/kg: SCC 6 35%; Dexa 8 mg/kg: SCC 6 59%) indicating that SCC 6 was more resistant towards Dexa treatment in vivo. Taken together, our results clearly show that within a single ALL patient, genetically and functionally distinct subpopulations exist. Combining PDX model with genetic marking of the cells enables us to in-depth analyze SCCs of a single patient sample and eventually identify adverse prognostic markers. Disclosures No relevant conflicts of interest to declare.

Description: The mechanism of cell transformation by Fms-like tyrosine kinase 3 (FLT3) in acute myeloid leukemia (AML) is incompletely understood. The most prevalent activated mutant FLT3 ITD exhibits an altered signaling quality, including strong activation of the STAT5 transcription factor. FLT3 ITD has also been found partially retained as a high-mannose precursor in an intracellular compartment. To analyze the role of intracellular retention of FLT3 for transformation, we have generated FLT3 versions that are anchored in the perinuclear endoplasmic reticulum (ER) by appending an ER retention sequence containing a RRR (R3) motif. ER retention of R3, but not of corresponding A3 FLT3 versions, is shown by biochemical, fluorescence-activated cell sorting, and immunocytochemical analyses. ER anchoring reduced global autophosphorylation and diminished constitutive activation of ERK1/2 and AKT of the constitutively active FLT3 versions. ER anchoring was, however, associated with elevated signaling to STAT3. Transforming activity of the FLT3 D835Y mutant was suppressed by ER anchoring. In contrast, ER-anchored FLT3 ITD retained STAT5-activating capacity and was transforming in vitro and in vivo. The findings highlight another aspect of the different signaling quality of FLT3 ITD: It can transform cells from an intracellular location.

Description: Leukemia-initiating cells reside within the bone marrow (BM) in specialized niches where they undergo complex interactions with their surrounding stromal cells. In order to identify genes being implicated in the interaction of acute myeloid leukemia (AML) cells and stromal cells, we performed co-cultures of primary AML cells with primary endothelial cells and osteoblasts. The gene expression of co-cultured AML blasts was compared to AML cells grown without adherent cells using microarray analysis. Amongst those genes being dysregulated upon co-culture was the actin binding protein plastin-3 (PLS3). Further RT-qPCR analysis revealed an endogenous PLS3 expression in about 50% of BM samples from AML patients (n=25). In contrast, expression of PLS3 was only detected in 2 of 12 analyzed AML cell lines with Kasumi-1 showing strong and THP-1 showing only weak expression. Therefore, functional analysis of PLS3 in AML was studied using shRNA knockdown and overexpression of PLS3 in Kasumi-1 cells. We could show that PLS3 has an impact on the colony formation capacity of AML cells in vitro as the knockdown resulted in significantly reduced colony numbers while increased colony growth was observed in the Kasumi-1 cells overexpressing PLS3 (p

Description: Evidence from model organisms and clinical trials reveals that the random insertion of retrovirus-based vectors in the genome of long-term repopulating hematopoietic cells may increase self-renewal or initiate malignant transformation. Clonal dominance of nonmalignant cells is a particularly interesting phenotype as it may be caused by the dysregulation of genes that affect self-renewal and competitive fitness. We have accumulated 280 retrovirus vector insertion sites (RVISs) from murine long-term studies resulting in benign or malignant clonal dominance. RVISs (22.5%) are located in or near (up to 100 kb [kilobase]) to known proto-oncogenes, 49.6% in signaling genes, and 27.9% in other or unknown genes. The resulting insertional dominance database (IDDb) shows substantial overlaps with the transcriptome of hematopoietic stem/progenitor cells and the retrovirus-tagged cancer gene database (RTCGD). RVISs preferentially marked genes with high expression in hematopoietic stem/progenitor cells, and Gene Ontology revealed an overrepresentation of genes associated with cell-cycle control, apoptosis signaling, and transcriptional regulation, including major “stemness” pathways. The IDDb forms a powerful resource for the identification of genes that stimulate or transform hematopoietic stem/progenitor cells and is an important reference for vector biosafety studies in human gene therapy.