X-linked SCID (SCID-X1) is amenable to correction by gene therapy using conventional gammaretroviral vectors. Here, we describe the occurrence of clonal T cell acute lymphoblastic leukemia (T-ALL) promoted by insertional mutagenesis in a completed gene therapy trial of 10 SCID-X1 patients. Integration of the vector in an antisense orientation 35 kb upstream of the protooncogene LIM domain only 2 (LMO2) caused overexpression of LMO2 in the leukemic clone. However, leukemogenesis was likely precipitated by the acquisition of other genetic abnormalities unrelated to vector insertion, including a gain-of-function mutation in NOTCH1, deletion of the tumor suppressor gene locus cyclin-dependent kinase 2A (CDKN2A), and translocation of the TCR-β region to the STIL-TAL1 locus. These findings highlight a general toxicity of endogenous gammaretroviral enhancer elements and also identify a combinatorial process during leukemic evolution that will be important for risk stratification and for future protocol design.

Type 1 diabetes is associated with reduced vascular repair, as indicated by impaired wound healing and reduced collateral formation in ischemia. Recently, endothelial progenitor cells (EPCs) have been identified as important regulators of these processes. We therefore explored the concept that EPCs are dysfunctional in diabetes. The number of EPCs obtained from type 1 diabetic patients in culture was 44% lower compared with age-and sex-matched control subjects (P < 0.001). This reduction was inversely related to levels of HbA 1c (R ‫؍‬ ؊0.68, P ‫؍‬ 0.01). In addition, we demonstrated that patient EPCs were also impaired in function using an in vitro angiogenesis assay. Conditioned media from patient EPCs were significantly reduced in their capacity to support endothelial tube formation in comparison to control EPCs. Therefore, despite culturing the EPCs under normoglycemic conditions, functional differences between patient and control EPCs were maintained. Our findings demonstrate that adverse metabolic stress factors in type 1 diabetes are associated with reduced EPC numbers and angiogenicity. We hypothesize that EPC dysfunction contributes to the pathogenesis of vascular complications in type 1 diabetes. Diabetes 53: 195

WNT proteins are secreted morphogens that are required for basic developmental processes, such as cell-fate specification, progenitor-cell proliferation and the control of asymmetric cell division, in many different species and organs. In blood and immune cells, WNT signalling controls the proliferation of progenitor cells and might also affect the cell-fate decisions of stem cells. Recent studies indicate that WNT proteins also regulate effector T-cell development, regulatory T-cell activation and dendritic-cell maturation. WNT signalling seems to function as a universal mechanism in leukocytes to establish a pool of undifferentiated cells for further selection, effector-cell maturation and terminal differentiation. WNT signalling is therefore subject to strict molecular control, and dysregulated WNT signalling is implicated in the development of haematological malignancies.

The activation of nuclear factor cB (NF-KB) has been implicated in the regulation of transcription of a variety of genes and has been shown to be essential for the expression of genes controlled by the long terminal repeat of human immunodeficiency virus (HIV LTR). We show here that intracellular thiol levels play a key role in regulating this process. That is, stimulation with tumor necrosis factor a and/or phorbol 12-myristate 13-acetate activates NF-cB and markedly decreases intracellular thiols; N-acetyl-L-cysteine, an efficient thiol source, prevents this thiol decrease and blocks the activation of NF-KB; and the lack of activated NF-icB prevents the activation of the HIV LTR and the transcription of genes under its control. These rmdings reveal a previously unrecognized genetic regulatory mechanism in which cytokine-induced shifts in intracellular thiol levels are crucial in the control of NF-KB activity and thereby influence the spectrum of genes expressed by cytokine-stimulated cells.Fauci and colleagues (1) have demonstrated that tumor necrosis factor a (TNF-a) stimulates human immunodeficiency virus (HIV) transcription via activation of the transcription factor nuclear factor KB (NF-KcB). We have shown (2) that this stimulation is inhibited by N-acetyl-L-cysteine (NAC), a nontoxic drug that enters cells readily and serves both as a scavenger for reactive oxidative intermediates (ROI) and as a precursor for glutathione (y-glutamylcysteinylglycine, GSH), the major intracellular thiol and ROI scavenger present in all eukaryotic forms of life (3-5).GSH is a cofactor for many glycolytic enzymes (6) and for enzymes involved in amino acid catabolism and conversion (7). It plays a key role in the accurate formation of protein disulfide bonds. In addition, by maintaining the redox potential within cells, it protects against oxidative damage that would otherwise be caused by free radicals and ROI produced during cell metabolism or as the result of drug overdose (e.g., acetaminophen) (8). NAC, which replenishes the intracellular cysteine required to produce GSH, also reacts directly with ROI and thereby provides further protection against cell damage caused by conditions that induce oxidative stress.

To identify oncogenic pathways in T cell acute lymphoblastic leukemia (T-ALL), we combined expression profiling of 117 pediatric patient samples and detailed molecular-cytogenetic analyses including the Chromosome Conformation Capture on Chip (4C) method. Two T-ALL subtypes were identified that lacked rearrangements of known oncogenes. One subtype associated with cortical arrest, expression of cell cycle genes, and ectopic NKX2-1 or NKX2-2 expression for which rearrangements were identified. The second subtype associated with immature T cell development and high expression of the MEF2C transcription factor as consequence of rearrangements of MEF2C, transcription factors that target MEF2C, or MEF2C-associated cofactors. We propose NKX2-1, NKX2-2, and MEF2C as T-ALL oncogenes that are activated by various rearrangements.