High-resolution genomic profiling of childhood T-ALL reveals frequent copy-number alterations affecting the TGF-beta and PI3K-AKT pathways and deletions at 6q15-16.1 as a genomic marker for unfavorable early treatment response.

Precursor T-cell acute lymphoblastic leukemia (T-ALL) in children represents a clinical challenge, because relapses are usually fatal. It is thus necessary to identify high-risk patients as early as possible to effectively individualize treatment. We aimed to define novel molecular risk markers in T-ALL and performed array-based comparative genomic hybridization (array-CGH) and expression analyses in 73 patients. We show that DNA copy-number changes are common in T-ALL and affect 70 of 73 (96%) patients. Notably, genomic imbalances predicted to down-regulate the TGF-beta or up-regulate the PI3K-AKT pathways are identified in 25 of 73 (34%) and 21 of 73 (29%) patients, suggesting that these pathways play key roles in T-ALL leukemogenesis. Furthermore, we identified a deletion at 6q15-16.1 in 9 of 73 (12%) of the patients, which predicts poor early treatment response. This deletion includes the CASP8AP2 gene, whose expression is shown to be down-regulated. The interaction of CASP8AP2 with CASP8 plays a crucial role in apoptotic regulation, suggesting a functional link between the clinical effect of the deletion and the molecular mode of action. The data presented here implicate the TGF-beta and PI3K-AKT pathways in T-ALL leukemogenesis and identify a subgroup of patients with CASP8AP2 deletions and poor early treatment response.

Downregulation of Notch signaling by gamma-secretase inhibition can abrogate chemotherapy-induced apoptosis in T-ALL cell lines.

Activation of Notch1 signaling plays an important role in the pathogenesis of precursor T-cell lymphoblastic leukemia (T-ALL). The Notch1 receptor is cleaved and activated via the gamma-secretase complex. Downregulation of Notch1 signaling by gamma-secretase inhibitors (GSIs) thus represents a potential novel therapeutic approach. In this study, we analyzed the response of four T-ALL cell lines to compound E, a potent gamma-secretase inhibitor, and to the combination of compound E with vincristine, daunorubicin, L-asparaginase (L-ASP), and dexamethasone (DEX). We identified two distinct types of responses: In type 1 cell lines, represented by TALL1 and HSB2, GSI-induced apoptosis followed cell cycle arrest and enhanced the induction of apoptosis caused by DEX and L-ASP. In type 2 cell lines, represented by CEM and Jurkat J6, GSI caused neither cell cycle block nor cell death. Notably, the combination of GSI with chemotherapy-induced resistance by decreasing apoptosis. In type 2 cells, GSI induced the upregulation of Bcl-xl mRNA and protein, which was thus identified as a candidate mechanism for the inhibition of apoptosis. In conclusion, the data presented here caution against clinical use of a combination treatment of GSI and chemotherapy in T-ALL.

The abundance of RNPS1, a protein component of the exon junction complex, can determine the variability in efficiency of the Nonsense Mediated Decay pathway.

Nonsense-mediated mRNA decay (NMD) is a molecular pathway of mRNA surveillance that ensures rapid degradation of mRNAs containing premature translation termination codons (PTCs) in eukaryotes. NMD has been shown to also regulate normal gene expression and thus emerged as one of the key post-transcriptional mechanisms of gene regulation. Recently, NMD efficiency has been shown to vary between cell types and individuals thus implicating NMD as a modulator of genetic disease severity. We have now specifically analysed the molecular mechanism of variable NMD efficiency and first established an assay system for the quantification of NMD efficiency, which is based on carefully validated cellular NMD target transcripts. In a HeLa cell model system, NMD efficiency is shown to be remarkably variable and to represent a stable characteristic of different strains. In one of these strains, low NMD efficiency is shown to be functionally related to the reduced abundance of the exon junction component RNPS1. Furthermore, restoration of functional RNPS1 expression, but not of NMD-inactive mutant proteins, also restores efficient NMD in this model. We conclude that cellular concentrations of RNPS1 can modify NMD efficiency and propose that cell type specific co-factor availability represents a novel principle that controls NMD.

The uORF-containing thrombopoietin mRNA escapes nonsense-mediated decay (NMD).

Platelet production is induced by the cytokine thrombopoietin (TPO). It is physiologically critical that TPO expression is tightly regulated, because lack of TPO causes life-threatening thrombocytopenia while an excess of TPO results in thrombocytosis. The plasma concentration of TPO is controlled by a negative feedback loop involving receptor-mediated uptake of TPO by platelets. Furthermore, TPO biosynthesis is limited by upstream open reading frames (uORFs) that curtail the translation of the TPO mRNA. uORFs are suggested to activate RNA degradation by nonsense-mediated decay (NMD) in a number of physiological transcripts. Here, we determine whether NMD affects TPO expression. We show that reporter mRNAs bearing the seventh TPO uORF escape NMD. Importantly, endogenously expressed TPO mRNA from HuH7 cells is unaffected by abrogation of NMD by RNAi. Thus, regulation of TPO expression is independent of NMD, implying that mRNAs bearing uORFs cannot generally be considered to represent NMD targets.

Activating NOTCH1 mutations predict favorable early treatment response and long-term outcome in childhood precursor T-cell lymphoblastic leukemia.

Activating mutations of the transmembrane receptor NOTCH1 are common in precursor T-cell lymphoblastic leukemia (T-ALL). We systematically analyzed the impact of activating NOTCH1 mutations on early treatment response and long-term outcome in 157 patients with T-ALL of the pediatric ALL-Berlin-Frankfurt-Munster (BFM) 2000 study. We confirm previous results that NOTCH1 mutations occur in more than 50% of T-ALL in children. In 82 patients (82/157; 52.2%), activating NOTCH1 mutations were identified either in the heterodimerization (55/82; 67.1%), in the PEST (13/82; 15.9%), or in both domains (14/82; 17.0%). The presence of NOTCH1 mutations was significantly correlated with a good prednisone response and favorable minimal residual disease (MRD) kinetics, which was independent from sex, age, white blood cell count, and T-cell immunophenotype at the time of diagnosis. Furthermore, activating NOTCH1 mutations specified a large subgroup of patients with an excellent prognosis. These findings indicate that in the context of the ALL-BFM 2000 treatment strategy, NOTCH1 mutations predict a more rapid early treatment response and a favorable long-term outcome in children with T-ALL.

Exon-junction complex components specify distinct routes of nonsense-mediated mRNA decay with differential cofactor requirements.

Messenger RNAs (mRNAs) bearing premature translation termination codons (PTCs) are degraded by nonsense-mediated mRNA decay (NMD). For mammalian NMD, current models propose a linear pathway that involves the splicing-dependent deposition of exon-junction complexes (EJCs) and the sequential action of the NMD factors UPF3, UPF2, and UPF1. We show here that different EJC proteins serve as entry points for the formation of distinguishable NMD-activating mRNPs. Specifically, Y14, MAGOH, and eIF4A3 can activate NMD in an UPF2-independent manner, whereas RNPS1-induced NMD requires UPF2. We identify the relevant regions of RNPS1, eIF4A3, Y14, and MAGOH, which are essential for NMD and provide insights into the formation of complexes, that classify alternative NMD pathways. These results are integrated into a nonlinear model for mammalian NMD involving alternative routes of entry that converge at a common requirement of UPF1.

Impact of pre-analytical handling on bone marrow mRNA gene expression.

Large clinical trials on leukaemia, require the transport of bone marrow (BM) from participating clinics to central diagnostic laboratories. We have investigated the impact of RNA extraction protocols and time delays between sample aspiration and RNA extraction on RNA quality and gene expression profiles. Intact RNA can be extracted from BM samples stored at room temperature for up to 48 h. Gene expression analyses using Affymetrix U95Av2 GeneChips and a custom-designed cDNA array in parallel showed that even short-term storage of BM has dramatic effects on mRNA expression of individual transcripts. Many probe sets/genes showed either reproducible deregulation (18.8%, analysis of variance <0.05), or inconsistent expression that differed from patient to patient (38.4%). Moderate alterations were observed in 42.8% genes, with a maximum fold change <2.0 in all experiments and at all time points. These profound effects complicate the use of unstabilized, shipped BM samples for gene expression analyses. The comparison of a variety of RNA stabilization reagents (e.g. PAXgene) resulted in partial conservation of the mRNA expression patterns. Immediate density centrifugation or erythrocyte lysis and freezing at -80 degrees C represent simple procedures that reliably preserved mRNA gene expression patterns in BM.

Profiling and functional annotation of mRNA gene expression in pediatric rhabdomyosarcoma and Ewing's sarcoma.

Using Affymetrix oligonucleotide microarrays, we analyzed mRNA gene expression patterns of 12 primary pediatric rhabdomyosarcomas (RMS) and 11 Ewing's sarcomas (EWS), which belong to the small round blue cell tumors (SRBCTs). Diagnostic classification of these cancers is frequently complicated by the highly similar appearance in routine histology, and additional molecular markers could significantly improve tumor classification. A combination of three independent statistical approaches (t-test, SAM, k-nearest neighborhood analysis) resulted in 101 highly significant probe sets that clearly discriminate between EWS and RMS. We identified novel marker transcripts that have not been previously associated with either RMS or EWS yet, including CITED2, glypican 3 (GPC3), and cyclin D1 (CCND1). Expression levels for selected candidate genes were validated by quantitative real-time reverse-transcription PCR. Furthermore, to identify biologically meaningful trends, functional annotations were assigned to 946 genes differentially expressed between EWS and RMS (t-test). Genes involved in protein biosynthesis (n = 28) and complex assembly (n = 9), lipid metabolism (n = 23), energy generation (n = 22), and mRNA processing (n = 11) were expressed significantly higher in EWS. Thus, functional annotation of tumor-specific genes reveals detailed insights into tumor biology and differentiation-specific expression patterns and gives important clues related to the possible cellular origin of these pediatric tumors. Supplementary material for this article is available at the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020-7136/suppmat/index.html.

Cartilage-like gene expression in differentiated human stem cell spheroids: a comparison of bone marrow-derived and adipose tissue-derived stromal cells.

OBJECTIVE: To compare the chondrogenic potential of human bone marrow-derived mesenchymal stem cells (BMSC) and adipose tissue-derived stromal cells (ATSC), because the availability of an unlimited cell source replacing human chondrocytes could be strongly beneficial for cell therapy, tissue engineering, in vitro drug screening, and development of new therapeutic options to enhance the regenerative capacity of human cartilage. METHODS: Quantitative gene expression of common cartilage and cell interaction molecules was analyzed using complementary DNA array technology and reverse transcription-polymerase chain reaction during optimization of cell differentiation, in order to achieve a molecular phenotype similar to that of chondrocytes in cartilage. RESULTS: The multilineage potential of BMSC and ATSC was similar according to cell morphology and histology, but minor differences in marker gene expression occurred in diverse differentiation pathways. Although chondrogenic differentiation of BMSC and ATSC was indistinguishable in monolayer and remained partial, only BMSC responded (with improved chondrogenesis) to a shift to high-density 3-dimensional cell culture, and reached a gene expression profile highly homologous to that of osteoarthritic (OA) cartilage. CONCLUSION: Hypertrophy of chondrocytes and high matrix-remodeling activity in differentiated BMSC spheroids and in OA cartilage may be the basis for the strong similarities in gene expression profiles between these samples. Differentiated stem cell spheroids represent an attractive tool for use in drug development and identification of drug targets in OA cartilage-like tissue outside the human body. However, optimization of differentiation protocols to achieve the phenotype of healthy chondrocytes is desired for cell therapy and tissue engineering approaches.

Enhanced expression of the human chitinase 3-like 2 gene (YKL-39) but not chitinase 3-like 1 gene (YKL-40) in osteoarthritic cartilage.

The knowledge of molecular alterations in osteoarthritic cartilage is important to identify novel therapeutic targets or to develop new diagnostic tools. We aimed to characterize the molecular response to cartilage degeneration by identification of differentially expressed genes in human osteoarthritic versus normal cartilage. Gene fragments selectively amplified in osteoarthritic cartilage by cDNA representational difference analysis included YKL-39 and the oesophageal-cancer-related-gene-4 (ECRG4). YKL-39 expression was significantly upregulated in cartilage from patients with osteoarthritis (n=14) versus normal subjects (n=8) according to real-time PCR (19-fold, p=0.009) and cDNA array analysis (mean 15-fold, p<0.001) and correlated with collagen 2 up-regulation. In contrast, the homologous cousin molecule YKL-40 (chitinase 3-like 1), which is elevated in serum and synovial fluid of patients with arthritis, showed no significant regulation in OA cartilage. Enhanced levels of YKL-40 may, therefore, be derived from synovial cells while modulation of YKL-39 and collagen 2 expression reflected the cartilage metabolism in response to degradation.

Molecular analysis of expansion, differentiation, and growth factor treatment of human chondrocytes identifies differentiation markers and growth-related genes.

This study is intended to optimise expansion and differentiation of cultured human chondrocytes by growth factor application and to identify molecular markers to monitor their differentiation state. We dissected the molecular consequences of matrix release, monolayer, and 3D-alginate culture, growth factor optimised expansion, and re-differentiation protocols by gene expression analysis. Among 19 common cartilage molecules assessed by cDNA array, six proved best to monitor differentiation. Instant down-regulation at release of cells from the matrix was strongest for COL 2A1, fibromodulin, and PRELP while LUM, CHI3L1, and CHI3L2 were expansion-related. Both gene sets reflected the physiologic effects of the most potent growth-inducing (PDGF-BB) and proteoglycan-inducing (BMP-4) factors. Only CRTAC1 expression correlated with 2D/3D switches while the molecular phenotype of native chondrocytes was not restored. The markers and optimised protocols we suggest can help to improve cell therapy of cartilage defects and chondrocyte differentiation from stem cell sources.