Key Points• Microdeletions represent an additional inactivation mechanism for PTEN in human T-cell acute lymphoblastic leukemia.• PTEN microdeletions are RAG-mediated aberrations.Phosphatase and tensin homolog (PTEN)-inactivating mutations and/or deletions are an independent risk factor for relapse of T-cell acute lymphoblastic leukemia (T-ALL) patients treated on Dutch Childhood Oncology Group or German Cooperative Study Group for Childhood Acute Lymphoblastic Leukemia protocols. Some monoallelic mutated or PTEN wild-type patients lack PTEN protein, implying that additional PTEN inactivation mechanisms exist. We show that PTEN is inactivated by small deletions affecting a few exons in 8% of pediatric T-ALL patients. These microdeletions were clonal in 3% and subclonal in 5% of patients. Conserved deletion breakpoints are flanked by cryptic recombination signal sequences (cRSSs) and frequently have non-template-derived nucleotides inserted in between breakpoints, pointing to an illegitimate RAG recombination-driven activity. Identified cRSSs drive RAG-dependent recombination in a reporter system as efficiently as bona fide RSSs that flank gene segments of the T-cell receptor locus. Remarkably, equivalent microdeletions were detected in thymocytes of healthy individuals. Microdeletions strongly associate with the TALLMO subtype characterized by TAL1 or LMO2 rearrangements. Primary and secondary xenotransplantation of TAL1-rearranged leukemia allowed development of leukemic subclones with newly acquired PTEN microdeletions. Ongoing RAG activity may therefore actively contribute to the acquisition of preleukemic hits, clonal diversification, and disease progression. (Blood. 2014;124(4):567-578)

Mesenchymal stem cells (MSCs) hold a great promise for application in several therapies due to their unique biological characteristics. In order to harness their full potential in cell-or gene-based therapies it might be advantageous to enhance some of their features through gene delivery strategies. Accordingly, we are interested in developing an efficient and safe methodology to genetically engineer human bone marrow MSC (BM MSC), enhancing their therapeutic efficacy in Regenerative Medicine. The plasmid DNA delivery was optimized using a cationic liposome-based reagent. Transfection efficiencies ranged from ~2% to ~35%, resulting from using a Lipid/DNA ratio of 1.25 with a transgene expression of 7 days. Importantly, the number of plasmid copies in different cell passages was quantified for the first time and ~20,000 plasmid copies/cell were obtained independently of cell passage. As transfected MSC have shown high viabilities (>90%) and recoveries (>52%) while maintaining their multipotency, this might be an advantageous transfection strategy when the goal is to express a therapeutic gene in a safe and transient way.

Human T-cell development is less well studied than its murine counterpart due to the lack of genetic tools and the difficulty of obtaining cells and tissues. Here, we report the transcriptional landscape of 11 immature, consecutive human T-cell developmental stages. The changes in gene expression of cultured stem cells on OP9-DL1 match those of ex vivo isolated murine and human thymocytes. These analyses led us to define evolutionary conserved gene signatures that represent pre- and post-αβ T-cell commitment stages. We found that loss of dim expression of CD44 marks human T-cell commitment in early CD7+CD5+CD45dim cells, before the acquisition of CD1a surface expression. The CD44−CD1a− post-committed thymocytes have initiated in frame T-cell receptor rearrangements that are accompanied by loss of capacity to differentiate toward myeloid, B- and NK-lineages, unlike uncommitted CD44dimCD1a− thymocytes. Therefore, loss of CD44 represents a previously unrecognized human thymocyte stage that defines the earliest committed T-cell population in the thymus.

T-cell acute lymphoblastic leukemia (T-ALL) is a cancer of developing T cells in the thymus. T-ALL is characterized by chromosomal rearrangements. These rearrangements can lead to the aberrant activation of oncogenic transcription factors by placing their genes under the control of promoters and/or enhancers of Tcell receptor genes, the BCL11B gene, or other genes; occasionally, these rearrangements can give rise to oncogenic fusion proteins. The activated oncogenic transcription factors include TAL1 and LMO2 (and related family members), TLX1, TLX3, NKX2-1, HOXA, and MEF2C; in addition, certain oncogenic fusion proteins can directly activate the HOXA or MEF2C genes.1,2 Oncogenic proteins facilitate the developmental arrest of pre-leukemic immature T cells. We previously proposed that these chromosomal rearrangements should be classified as type A aberrations, as they are generally considered to be the driving oncogenic event associated with unique expression profiles.2 Based upon their gene expression signatures, T-ALL can be classified into the following four major subtypes: ETP-ALL, TLX, proliferative, and TALLMO. [3][4][5] Maturation arrest induces a pre-leukemic condition in which additional mutations can give rise to T-ALL.1,2 These secondary mutations are not necessarily clonal events and are often selected during disease progression or post-treatment T he tumor suppressor phosphatase and tensin homolog (PTEN) negatively regulates phosphatidylinositol 3-kinase (PI3K)-AKT signaling and is often inactivated by mutations (including deletions) in a variety of cancer types, including T-cell acute lymphoblastic leukemia. Here we review mutation-associated mechanisms that inactivate PTEN together with other molecular mechanisms that activate AKT and contribute to T-cell leukemogenesis. In addition, we discuss how Pten mutations in mouse models affect the efficacy of gamma-secretase inhibitors to block NOTCH1 signaling through activation of AKT. Based on these models and on observations in primary diagnostic samples from patients with T-cell acute lymphoblastic leukemia, we speculate that PTEN-deficient cells employ an intrinsic homeostatic mechanism in which PI3K-AKT signaling is dampened over time. As a result of this reduced PI3K-AKT signaling, the level of AKT activation may be insufficient to compensate for NOTCH1 inhibition, resulting in responsiveness to gamma-secretase inhibitors. On the other hand, de novo acquired PTEN-inactivating events in NOTCH1-dependent leukemia could result in temporary, strong activation of PI3K-AKT signaling, increased glycoly sis and glutaminolysis, and consequently gamma-secretase inhibitor resistance. Due to the central role of PTEN-AKT signaling and in the resistance to NOTCH1 inhibition, AKT inhibitors may be a promising addition to current treatment protocols for T-cell acute lymphoblastic leukemia.

Contemporary biomedical research increasingly depends on techniques to induce or to inhibit expression of genes in hematopoietic stem cells (HSCs) or other primary cells to assess their roles on cellular processes including differentiation, apoptosis and migration. Surprisingly little information is available to optimize lentiviral transduction of HSCs. We have therefore carefully optimized transduction of murine and human HSCs by optimizing vector design, serum-free virus production and virus quantitation. We conclude that the viral RNA length, even in relatively small vectors, is an important factor affecting the lentiviral gene transfer on the level of both the virus production and the cellular transduction efficiency. Efficient transfer of large gene sequences into difficult-to-transduce primary cells will benefit from reducing the lentiviral construct size.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-016-2118-z) contains supplementary material, which is available to authorized users.

Genetic modification of human mesenchymal stem cells (MSC) is a powerful tool to improve the therapeutic utility of these cells and to increase the knowledge on their regulation mechanisms. In this context, strong efforts have been made recently to develop efficient nonviral gene delivery systems. Although several studies addressed this question most of them use the end product of a reporter gene instead of the DNA uptake quantification to test the transfection efficiency. In this study, we established a method based on quantitative real-time PCR (RT-PCR) to determine the intracellular plasmid DNA copy number in human MSC after lipofection. The procedure requires neither specific cell lysis nor DNA purification. The influence of cell number on the RT-PCR sensitivity was evaluated. The method showed good reproducibility, high sensitivity, and a wide linear range of 75-2.5 x 10⁶ plasmid DNA copies per cell. RT-PCR results were then compared with the percentage of transfected cells assessed by flow cytometry analysis, which showed that flow cytometry-based results are not always proportional to plasmid cellular uptake determined by RT-PCR. This work contributed for the establishment of a rapid quantitative assay to determine intracellular plasmid DNA in stem cells, which will be extremely beneficial for the optimization of gene delivery strategies.

Stem/progenitor cells hold a great promise for application in several therapies due to their unique biological characteristics. With the purpose of harnessing these cells full potential in cell-or gene-based therapies it might be advantageous to enhance some of their features through gene delivery strategies. Accordingly, we are interested in developing efficient and safe methodologies to genetically engineer stem cells, boosting their therapeutic efficacy in Regenerative Medicine. In our work, delivery of plasmid DNA to human Bone Marrow Mesenchymal Stem Cells (BM-MSC) was optimized by lipofection and by a recently available microporation technique and no effect was observed in their immunophenotypic characteristics or differentiative potential. After lipofection similar number of plasmid copies was determined at different cell passages. Importantly, cell proliferation kinetics slowed down due to the presence of plasmid. Overall, we believe our findings are extremely useful towards the maximization of gene delivery to human MSC, without compromising cell function and viability.

Background: T-cell development in the thymus is a complex process that depends on sequential transcriptional and epigenetic events that induce T-cell lineage commitment and simultaneously suppress alternative cell fates. In T-cell acute lymphoblastic leukemia (T-ALL), aberrantly expressed oncogenes result in the arrest of developing thymocytes, which can lead to the acquisition of secondary mutations, uncontrolled proliferation and disease progression. MEF2C is often expressed as a result of chromosomal rearrangements in immature, early T-cell progenitor ALL (ETP-ALL), but is also expressed in normal thymocyte progenitors before T-cell commitment (in the ETP stage). As the only hematopoietic lineage, thymocytes that have passed the T-cell commitment checkpoint (as well as mature T-cells) do no longer express MEF2C. Aims: We aimed to investigate the effect of constitutive MEF2C expression on early T-cell development. OP9-DL1 co-cultures have been most useful for mimicking in vitro T-cell development starting with hematopoietic stem cells (HSCs) derived from human cord blood or bone marrow. We also aimed to investigate the impact of MEF2C in comparison to LYL1 and LMO2; two T-ALL oncogenes also highly expressed at the ETP stage. Methods: We have utilized the OP9-DL1 in vitro co-culture system to gradually differentiate CD34+ HSCs from umbilical cord blood into the T-cell lineage. HSCs in this co-culture will recapitulate in vivo T-cell development as measured by incremental acquisition of surface markers CD7, CD5, CD1a, and reach the CD4, CD8 double-positive (DP) stage. We generated gene expression profiles of 11 subsequent in vitro stages of differentiation to help us match them to in vivo development stages. We investigated in vitro T-cell differentiation of HSCs after lentiviral transduction with MEF2C or control vectors, as well as with other transcriptional regulators LYL1 and LMO2 that are expressed at the ETP stage. Results: The major change in gene expression of subsequent early T-cell differentiation stages defines two distinct T-cell differentiation clusters that correlate with in vivo pre- and post-T-cell commitment profiles. We found that T-cell commitment occurs in CD7+ CD5+ cells before the acquisition of CD1a surface expression. Expression of control vectors in HSCs does not affect the in vitro T-cell differentiation, but MEF2C expression blocks differentiation into the direction of T-cells as measured by the failure of most cells to acquire CD7 as the first marker. Instead, with increased passage number cells gradually lose CD34 expression and eventually disappear from the co-culture. Similar effects were observed for the expression of LYL1 and LMO2; LYL1 expression arrests the cells at the most immature CD7+ ETP stage and prevents the transition towards CD7+ CD5+ cells, whereas LMO2 expressing cells reach the CD7+ CD5+ stage but fail to acquire CD1a as a marker of T-cell commitment. Summary/Conclusion: The gene expression profiles of 11 human in vitro T-cell differentiation subsets has enabled us to pinpoint T-cell commitment to a stage in which cells have acquired CD7 and CD5, just prior to the acquisition of CD1a. MEF2C, LYL1, and LMO2, expressed in ETP-ALL as well as in normal thymocyte progenitors, do not allow the transition to T-cell commitment when constitutively expressed. These proteins each result in the arrest of in vitro differentiating T-cells at different ETP stages, all before the T-cell commitment as marked by CD1a expression. Constitutive expression of MEF2C, LYL1, or LMO2 in very early thymocyte progenitors is incompatible with development into and beyond the T-cell commitment checkpoint and these proteins could therefore play important roles in the pathogenesis of ETP-ALL. Disclosures No relevant conflicts of interest to declare.