STAT5 transcription factors are frequently activated in hematopoietic neoplasms and are targets of various tyrosine kinase oncogenes. Evidences for a crosstalk between STAT5 and reactive oxygen species (ROS) metabolism have recently emerged but mechanisms involved in STAT5-mediated regulation of ROS still remain elusive. We demonstrate that sustained activation of STAT5 induced by Bcr-Abl in chronic myeloid leukemia (CML) cells promotes ROS production by repressing expression of two antioxidant enzymes, catalase and glutaredoxin-1(Glrx1). Downregulation of catalase and Glrx1 expression was also observed in primary cells from CML patients. Catalase was shown not only to reduce ROS levels but also, to induce quiescence in Bcr-Abl-positive leukemia cells. Furthermore, reduction of STAT5 phosphorylation and upregulation of catalase and Glrx1 were also evidenced in leukemia cells co-cultured with bone marrow stromal cells to mimic a leukemic niche. This caused downregulation of ROS levels and enhancement of leukemic cell quiescence. These data support a role of persistent STAT5 signaling in the regulation of ROS production in myeloid leukemias and highlight the repression of antioxidant defenses as an important regulatory mechanism.

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Nowadays, nonviral gene transfer is currently of great importance for introducing exogenous genes into genomes and for ensuring that transgene expression is suitable for therapeutic and bioproduction purposes. The piggyBac transposon-based system is particularly interesting since it is easy to engineer and has a large cargo capacity, up to 100 kb. In its setup, the system requires only the piggyBac transposase protein and the transgene delineated by the two piggyBac-specific inverted terminal repeats. Usually the source of transposase is carried by a DNA plasmid. However, the principal drawback of this method is the lasting presence of the transposase, due to episomal persistence or possible integration of the transposase gene vector into the cell's genome. This can lead to genotoxic effects such as multiple genomic integration events and remobilization of the transposon vector once it has been integrated. One alternative to improve the safety of the system is to deliver the transposase as in vitro-synthesized messenger RNA in order to define a very narrow expression window during which a one-shot transposition process would occur. Issues that can be encountered when working on mRNA cell transfer are related to the quality of the synthetic mRNA, the system used to introduce mRNA into the cells and the bioavailability of the mRNA molecules. Here we describe a method to produce mRNA, verify its quality, determine which transfecting reagents can be used and how this mRNA is available to promote the transposition process in HeLa cells. Additionally, we illustrate this method in stromal mesenchymal cell lines in order to support hematopoiesis.

Introduction The contribution of the stromal cell compartment to leukemogenesis remains poorly understood. Several studies have described abnormalities involving this compartment in acute myeloid leukemias (AML) and myelodysplastic syndromes (MDS) including proliferative defect of mesenchymal stem/stromal cells (MSCs) as reported by our group in AML patients (Domenech et al., Haematologica 2012, EHA meeting, abstr. 37). Recently, evidence of an involvement of MSCs in the leukemic process has been provided in murine models (Walkley et al., Cell 2007; Raaijmakers et al. Nature 2010). In the present study, we investigated potential modifications of human AML blast cell biology induced in vitro by MSCs from healthy individuals and AML patients. Methods Bone marrow MSCs from 6 AML patients (3 M0, 3 M1 [FAB classification]) were compared to those from 6 normal individuals. All the MSCs were analyzed at the end of the second passage of culture. Capacity of MSCs to influence leukemic cell behavior was assessed by co-culture with immature leukemia cells from KG1a line and with heterologous or autologous primary blast cells from the AML patients. Apoptotic cell frequencies, cell cycle phase distributions, and presence of DNA double-strand breaks of leukemia cells with or without MSCs were performed by flow cytometry. Results In co-cultures, no difference in leukemia cell adhesion were found on AML and normal MSCs. The presence of MSCs reduced apoptosis of primary blast cells (-30%, p=0.002) although no effect was observed on campthotecin-induced apoptosis of KG1a cells, as compared to MSC-free culture conditions. Cell cycle analysis revealed that G0/G1 ratios of KG1a cells were strongly increased by the presence of MSCs considering the whole cells (+128%, p=0.005) as well as the cell fraction adherent to MSCs (+138%, p=0.006). Comparable results were obtained with primary blast cells considering all the cells (+51%, p=0.117), especially within the adherent cell fraction (+229%, p=0.004). In addition, direct contact with MSCs was associated with a slight decrease in the proportion of blast cells with DNA double-strand breaks compared to MSC-free cultures (-10%, p= 0.034). No significant differences were found for all these results when co-cultures with AML and normal MSCs were compared. Likewise, no differences existed between co-cultures with M0 and M1 AML MSCs or between co-cultures of autologous and heterologous primary blast cells with AML MSCs. Conclusions This study shows that MSCs influence leukemic cell behavior, irrespective of their healthy or leukemic origin. In particular, they protect blast cells from apoptosis and induce their quiescence, (mainly by direct contact) which could contribute to decreased yields of DNA double-strand breaks, a source of genetic instability. Further experiments are in progress to evaluate potential changes in the capacity of AML MSCs to support normal hematopoiesis. Disclosures: Gyan: FRESENIUS KABI: Consultancy, Research Funding. Domenech:Celgene Corporation: Research Funding.