Hyperactivation of mTORC1 and mTORC2 by multiple oncogenic events causes addiction to eIF4E-dependent mRNA translation in T-cell leukemia.

High activation of the PI3K-AKT-mTOR pathway is characteristic for T-cell acute lymphoblastic leukemia (T-ALL). The activity of the master regulator of this pathway, PTEN, is often impaired in T-ALL. However, experimental evidence suggests that input from receptor tyrosine kinases (RTKs) is required for sustained mTOR activation, even in the absence of PTEN. We previously reported the expression of Neurotrophin receptor tyrosine kinases (TRKs) and their respective ligands in primary human leukemia samples. In the present study we aimed to dissect the downstream signaling cascades of TRK-induced T-ALL in a murine model and show that T-ALLs induced by deregulated receptor tyrosine kinase signaling acquire activating mutations in Notch1 and lose PTEN during clonal evolution. Some clones additionally lost one allele of the homeodomain transcription factor Cux1. All events independently led to a gradual hyperactivation of both mTORC1 and mTORC2 signaling. We dissected the role of the individual mTOR complexes by shRNA knockdown and found that the separate depletion of mTORC1 or mTORC2 reduced the growth of T-ALL blasts, but was not sufficient to induce apoptosis. In contrast, knockdown of the mTOR downstream effector eIF4E caused a striking cytotoxic effect, demonstrating a critical addiction to cap-dependent mRNA-translation. Although high mTORC2-AKT activation is commonly associated with drug-resistance, we demonstrate that T-ALL displaying a strong mTORC2-AKT activation were specifically susceptible to 4EGI-1, an inhibitor of the eIF4E-eIF4G interaction. To decipher the mechanism of 4EGI-1, we performed a genome-wide analysis of mRNAs that are translationally regulated by 4EGI-1 in T-ALL. 4EGI-1 effectively reduced the ribosomal occupancy of mRNAs that were strongly upregulated in T-ALL blasts compared with normal thymocytes including transcripts important for translation, mitochondria and cell cycle progression, such as cyclins and ribosomal proteins. These data suggest that disrupting the eIF4E-eIF4G interaction constitutes a promising therapy strategy in mTOR-deregulated T-cell leukemia.

Activation of Evi1 inhibits cell cycle progression and differentiation of hematopoietic progenitor cells.

The transcription factor Evi1 has an outstanding role in the formation and transformation of hematopoietic cells. Its activation by chromosomal rearrangement induces a myelodysplastic syndrome with progression to acute myeloid leukemia of poor prognosis. Similarly, retroviral insertion-mediated upregulation confers a competitive advantage to transplanted hematopoietic cells, triggering clonal dominance or even leukemia. To study the molecular and functional response of primary murine hematopoietic progenitor cells to the activation of Evi1, we established an inducible lentiviral expression system. EVI1 had a biphasic effect with initial growth inhibition and retarded myeloid differentiation linked to enhanced survival of myeloblasts in long-term cultures. Gene expression microarray analysis revealed that within 24 h EVI1 upregulated 'stemness' genes characteristic for long-term hematopoietic stem cells (Aldh1a1, Abca1, Cdkn1b, Cdkn1c, Epcam, among others) but downregulated genes involved in DNA replication (Cyclins and their kinases, among others) and DNA repair (including Brca1, Brca2, Rad51). Cell cycle analysis demonstrated EVI1's anti-proliferative effect to be strictly dose-dependent with accumulation of cells in G0/G1, but preservation of a small fraction of long-term proliferating cells. Although confined to cultured cells, our study contributes to new hypotheses addressing the mechanisms and molecular targets involved in preleukemic clonal dominance or leukemic transformation by Evi1.

Wnt signaling strength regulates normal hematopoiesis and its deregulation is involved in leukemia development.

A strict balance between self-renewal and differentiation of hematopoietic stem cells (HSCs) is required in order to maintain homeostasis, as well as to efficiently respond to injury and infections. Numbers and fate decisions made by progenitors derived from HSC must also be carefully regulated to sustain large-scale production of blood cells. The complex Wnt family of molecules generally is thought to be important to these processes, delivering critical signals to HSC and progenitors as they reside in specialized niches. Wnt proteins have also been extensively studied in connection with malignancies and are causatively involved in the development of several types of leukemias. However, studies with experimental animal models have produced contradictory findings regarding the importance of Wnt signals for normal hematopoiesis and lymphopoiesis. Here, we will argue that dose dependency of signaling via particular Wnt pathways accounts for much, if not all of this controversy. We conclude that there seems little doubt that Wnt proteins are required to sustain normal hematopoiesis, but are likely to be presented in carefully controlled gradients in a tissue-specific manner.

Lentiviral vectors for induction of self-differentiation and conditional ablation of dendritic cells.

Development of lentiviral vectors (LVs) in the field of immunotherapy and immune regeneration will strongly rely on biosafety of the gene transfer. We demonstrated previously the feasibility of ex vivo genetic programming of mouse bone marrow precursors with LVs encoding granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), which induced autonomous differentiation of long-lived dendritic cells (DCs), referred to as self-differentiated myeloid-derived antigen-presenting-cells reactive against tumors (SMART-DCs). Here, LV biosafety was enhanced by using a DC-restricted and physiological promoter, the major histocompatibility complex (MHC) II promoter, and including co-expression of the herpes simplex virus-thymidine kinase (sr39HSV-TK) conditional suicide gene. Tricistronic vectors co-expressing sr39HSV-TK, GM-CSF and IL-4 transcriptionally regulated by the MHCII promoter or the ubiquitous cytomegalovirus (CMV) promoter were compared. Despite the different gene transfer effects, such as the kinetics, levels of transgene expression and persistency of integrated vector copies, both vectors induced highly viable SMART-DCs, which persisted for at least 70 days in vivo and could be ablated with the pro-drug Ganciclovir (GCV). SMART-DCs co-expressing the tyrosine-related protein 2 melanoma antigen administered subcutaneously generated antigen-specific, anti-melanoma protective and therapeutic responses in the mouse B16 melanoma model. GCV administration after immunotherapy did not abrogate DC vaccination efficacy. This demonstrates proof-of-principle of genetically programmed DCs that can be ablated pharmacologically.

Correction of B-cell development in Btk-deficient mice using lentiviral vectors with codon-optimized human BTK.

X-linked agammaglobulinemia (XLA) is the most common primary immunodeficiency (PID) in man and caused by mutations in the Bruton's tyrosine kinase (BTK) gene. XLA is characterized by a B-cell differentiation arrest in bone marrow, absence of mature B cells and immunoglobulins (Igs), and recurrent bacterial infections. We used self-inactivating lentiviral vectors expressing codon-optimized human BTK under the control of three different ubiquitous or B cell-specific promoters. Btk-/- mice engrafted with transduced cells showed correction of both precursor B-cell and peripheral B-cell development. Lentiviral vectors containing the wildtype BTK sequence did not correct the phenotype. All treated mice with codon-optimized BTK exhibited the recovery of B1 cells in the peritoneal cavity, and of serum IgM and IgG3 levels. Calcium mobilization responses upon B-cell receptor stimulation as well as in vivo responses to T cell-independent antigens were restored. Viral promoters overexpressing BTK >100-fold above normal resulted in erythro-myeloid proliferations independent of insertional mutagenesis. However, transplantation into secondary Btk-/- recipients using cellular promoters resulted in functional restoration of peripheral B cells and IgM levels, without any adverse effects. In conclusion, transduction of human BTK corrects B-cell development and antigen-specific antibody responses in Btk-/- mice, thus indicating the feasibility of lentiviral gene therapy for XLA, provided that BTK expression does not vastly exceed normal levels.

Mechanisms controlling titer and expression of bidirectional lentiviral and gammaretroviral vectors.

Bidirectional lentiviral vectors mediate expression of two or more cDNAs from a single internal promoter. In this study, we examined mechanisms that control titer and expression properties of this vector system. To address whether the bidirectional design depends on lentiviral (LV) backbone components, especially the Rev/Rev responsive element (RRE) system, we constructed similar expression cassettes for LV and gammaretroviral (GV) vectors. Bidirectional expression levels could be adjusted by the use of different internal promoters. Furthermore, removal of the constitutive RNA transport element of Mason-Pfizer monkey virus, used in first generation bidirectional LV vectors, improved gene expression. Titers of bidirectional vectors were approximately 10-fold reduced in comparison to unidirectional vectors, independent of the Rev/RRE interaction. We reasoned that titer reductions were due to the formation of interfering double-stranded RNA in packaging cells. Indeed, cotransfection of Nodamuravirus B2 protein, an RNA interference suppressor, increased bidirectional vector titers at least fivefold. We validated the potential of high titer bidirectional vectors by coexpressing a fluorescent marker with O(6)-methylguanine-DNA methyltransferase from integrating, or with Cre recombinase from integrating and non-integrating GV and LV backbones. This allowed for the tracking of chemoprotected and recombined cells by fluorescence marker expression.

Leukemia induction after a single retroviral vector insertion in Evi1 or Prdm16.

Insertional activation of cellular proto-oncogenes by replication-defective retroviral vectors can trigger clonal dominance and leukemogenesis in animal models and clinical trials. Here, we addressed the leukemogenic potential of vectors expressing interleukin-2 receptor common gamma-chain (IL2RG), the coding sequence required for correction of X-linked severe combined immunodeficiency. Similar to conventional gamma-retroviral vectors, self-inactivating (SIN) vectors with strong internal enhancers also triggered profound clonal imbalance, yet with a characteristic insertion preference for a window located downstream of the transcriptional start site. Controls including lentivirally transduced cells revealed that ectopic IL2RG expression was not sufficient to trigger leukemia. After serial bone marrow transplantation involving 106 C57Bl6/J mice monitored for up to 18 months, we observed leukemic progression of six distinct clones harboring gamma-retroviral long terminal repeat (LTR) or SIN vector insertions in Evi1 or Prdm16, two functionally related genes. Three leukemic clones had single vector integrations, and identical clones manifested with a remarkably similar latency and phenotype in independent recipients. We conclude that upregulation of Evi1 or Prdm16 was sufficient to initiate a leukemogenic cascade with consistent intrinsic dynamics. Our study also shows that insertional mutagenesis is required for leukemia induction by IL2RG vectors, a risk to be addressed by improved vector design.

Ectopic retroviral expression of LMO2, but not IL2Rgamma, blocks human T-cell development from CD34+ cells: implications for leukemogenesis in gene therapy.

The occurrence of leukemia in a gene therapy trial for SCID-X1 has highlighted insertional mutagenesis as an adverse effect. Although retroviral integration near the T-cell acute lymphoblastic leukemia (T-ALL) oncogene LIM-only protein 2 (LMO2) appears to be a common event, it is unclear why LMO2 was preferentially targeted. We show that of classical T-ALL oncogenes, LMO2 is most highly transcribed in CD34+ progenitor cells. Upon stimulation with growth factors typically used in gene therapy protocols transcription of LMO2, LYL1, TAL1 and TAN1 is most prominent. Therefore, these oncogenes may be susceptible to viral integration. The interleukin-2 receptor gamma chain (IL2Rgamma), which is mutated in SCID-X1, has been proposed as a cooperating oncogene to LMO2. However, we found that overexpressing IL2Rgamma had no effect on T-cell development. In contrast, retroviral overexpression of LMO2 in CD34+ cells caused severe abnormalities in T-cell development, but B-cell and myeloid development remained unaffected. Our data help explain why LMO2 was preferentially targeted over many of the other known T-ALL oncogenes. Furthermore, during T-cell development retrovirus-mediated expression of IL2Rgamma may not be directly oncogenic. Instead, restoration of normal IL7-receptor signaling may allow progression of T-cell development to stages where ectopic LMO2 expression causes aberrant thymocyte growth.

Monitoreo de los glaciares del Popocatépetl

Los glaciares existentes en volcanes activos como el Popocatépetl, representan un peligro adicional a los peligros volcánicos comunes cuando se verifican erupciones explosivas. En caso de presentarse éstas, existe la posibilidad de generación de flujos de lodo a la mezcla de material piroclástico y agua proveniente de la fusión del glaciar, que viajarían por los cauces que son drenados actualmente por los glaciares. Esta clase de eventos han sucedido en el pasado geólogico del volcán. De verificarse este tipo de fenómenos en la actualidad, éstos plantearían un problema serio ya que existen numerosos asentamientos humanos a lo largo de los mencionados cauces. Por razones de protección civil, es importante estudiar los glaciares de volcanes activos para evitar que se repitan desgracias como la de Armero, Colombia en noviembre de 1985. Además, el estudio de los glaciares implica conocer más acerca de otros peligros geológicos de largo como la desertificación (AU)

Influence of enzyme induction and inhibition on the oxidation of nifedipine, sparteine, mephenytoin and antipyrine in humans as assessed by a "cocktail" study design.

Nifedipine (NF), sparteine (SP), mephenytoin (MP) and antipyrine (AP) were administered simultaneously ("cocktail" design) to 15 healthy subjects, including 4 poor metabolizers (PM) of SP and 4 PMs of MP, on three different occasions: without pretreatment, after pentobarbital (PB) pretreatment and together with cimetidine (cim). Concentrations of AP, NF, its pyridine metabolite 2,6-dimethyl-4-(2-nitrophenyl)-pyridine-3,5-dicarboxylate-methylester (M-O), SP, dehydrosparteine (DHS) were determined in plasma; 2,6-dimethyl-4-(2-nitrophenyl)-pyridine-3,5-dicarboxylate-monomethyle ster (which is ester hydrolyzed M-O), SP, DHS, 4-hydroxymephenytoin, AP and metabolites 3-hydroxymethylantipyrine, norantipyrine and 4-hydroxyantipyrine were measured in urine. Clearance of NF had increased 270% after PB and decreased 32% with cim. Area under the plasma-concentration time curve of M-O and urinary excretion of 2,6-dimethyl-4-(2-nitrophenyl)-pyridine-3,5-dicarboxylate-monoethyles ter had also decreased after PB. Neither extensive metabolizers nor PMs of SP and MP were sensitive to PB treatment, but sparteine clearance was reduced 55% by cim in extensive metabolizers and 59% in PMs. Ratio SP/DHS in urine was hardly influenced by cim. AP oxidation was significantly induced and inhibited by PB and cim, respectively. The cocktail study design seems to be suitable to assess induction and inhibition of the metabolism of the applied model substrates, which are metabolized by at least partly independent isozymes of the cytochrome P-450 system, simultaneously.

Glacier surge mechanism: 1982-1983 surge of variegated glacier, alaska.

The hundredfold speedup in glacier motion in a surge of the kind the kind that took place in Variegated Glacier in 1982-1983 is caused by the buildup of high water pressure in the basal passageway system, which is made possible by a fundamental and pervasive change in the geometry and water-transport characteristics of this system. The behavior of the glacier in surge has many remarkable features, which can provide clues to a detailed theory of the surging process. The surge mechanism is akin to a proposed mechanism of overthrust faulting.