ALBERT

Description: After single electron reduction of the dinitrogen complex [L t Bu Ni(μ-η 1 :η 1 -N 2 )NiL t Bu ] ( I ) with KC 8 , reaction of the resulting compound K[L t Bu Ni(μ-η 1 :η 1 -N 2 )NiL t Bu ] ( II ) with sodium sand yields KNa[L t Bu Ni(μ-η 1 :η 1 -N 2 )NiL t Bu ] ( 1 ), which contains two different alkali metal ions. Treatment of I with two equivalents of sodium sand leads to the symmetric complex Na 2 [L t Bu Ni(μ-η 1 :η 1 -N 2 )NiL t Bu ] ( 2 ). Complexes 1 and 2 were investigated by single crystal X-ray diffraction analysis as well as by Raman spectroscopy, and the results were compared with the data of K 2 [L t Bu Ni(μ-η 1 :η 1 -N 2 )NiL t Bu ] ( III ), which contains two K + ions. Thus, it became obvious that the nature of the alkali metal ion M in compounds M 2 [L t Bu Ni(μ-η 1 :η 1 -N 2 )NiL t Bu ] has hardly any influence on the degree of NN bond activation. Furthermore, it was shown that treatment of the dinickel(I) complex III with CO leads to the dinickel(0) compound K 2 [L t Bu Ni(CO)] 2 ( 4 ) and N 2 . Reaction of the unreduced dinickel(I) complex I with CO leads to a more simple replacement of the N 2 ligand and formation of [L t Bu Ni(CO)] ( 3 ).

Description: The hydrogenation of Citral in a fixed bed reactor using supercritical carbon dioxide was studied. A numerical model was set up using the commercial software Aspen Custom Modeler®. Applying literature experimental data, a parameter estimation for the kinetic parameters was carried out. A good fit was reached with the presented model. Furthermore, a sensitivity analysis was performed. The influence of process pressure, inlet CO2- and Hydrogen concentration and catalyst particle size on conversion, reactor length, space time yield, yield of the intermediate product 3,7-dimethyl-2-octenal and pressure drop was investigated.

Description: Linking the community structure of arbuscular mycorrhizal fungi and plants: a story of interdependence? The ISME Journal 11, 1400 (June 2017). doi:10.1038/ismej.2017.5 Authors: Sebastian Horn, Stefan Hempel, Erik Verbruggen, Matthias C Rillig & Tancredi Caruso

Description: Background: Activating mutations of the catalytic subunit of class IA phosphoinositide 3-kinase alpha (PIK3CA) are clustered in small hot-spot regions of the PIK3CA gene, including exon 9 within the helical domain and exon 11 within the kinase domain. They have been linked to several human neoplasias, including colorectal, breast and hepatocellular cancers. In acute leukemias, PIK3CA mutations have not been investigated in larger chorts and so far only been observed in a few patients. Since the PI3K/Akt/GSK3beta pathway is an important signaling cascade of receptor tyrosine kinases (e.g. Flt3R, Kit) which are frequently activated in acute leukemias we investigated the functional activity of PIK3CA mutants. Materials and methods: We transfected early hematopoietic cells (Ba/F3 cell line) with PI3KCA exon 9 and 11 mutations and investigated the cells in an in vitro factor-independent growth assay and pharmacologic inhibition experiments. Results: We demonstrate that mutations in the helical or kinase domain of PIK3CA lead to the constitutive activation of PI3Kalpha in Ba/F3 cells, inducing factor-independent growth of the IL3-dependent cells. The frequency of IL3-independent Ba/F3 cells after tranfection with exon 9 and 11 PIK3CA mutants was equivalent to the frequency confered by PIK3CA mutants containing the membrane localization signal of either src or ras. Proliferation and survival of the cells were inhibited by the PI3K inhibitors LY294002 and Quercitin or an inhibitor of the PI3K downstream target Akt. Inhibition occurred in a dose- and time-dependent manner and could be reverted by addition of IL-3. One of the major targets of PI3K/Akt signaling is GSK3beta which becomes inactivated after Akt-mediated phosphorylation. By using a GSK3beta-specific inhibitor or LiCl we could show that the inactivation of GSK3beta alone did not result in factor-independent growth of Ba/F3 cells. However, GSK3beta inhibition led to a delay in the induction of cell death after IL3-withdrawal. Conclusion: Activating mutations of PIK3CA, associated with several human neoplasias and acute leukemia are functionally active in hematopoietic cells, confer factor independency and respond to PI3K/Akt inhibition.

Description: The phosphoinositide 3-kinase (PI3K)-AKT pathway is a relevant signal transduction axis which regulates survival, growth and proliferation of hematopoietic cells through a variety of downstream targets. Constitutive activation of the PI3K-AKT pathway is observed in up to 70% of acute myelogenous leukemia (AML), primarily due to activating mutations in receptor tyrosine kinases (e.g. c-KIT) or RAS and is also present in T-cell-non-Hodgkins-lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, Hodgkins Lymphoma and myelodysplastic syndrome (MDS). We analyzed the effect of activating point mutations in the catalytic subunit p110alpha of class IA PI3K (PIK3CA) in hematopoietic cells. We transfected early hematopoietic, IL-3 dependent cells (Ba/F3 cell line) with point mutations in the helical (exon 9, E542K, E545A) and kinase domain (exon 20, H1047R) of the p110alpha catalytic subunit and determined the effect of PI3K-AKT pathway activation on cellular proliferation, survival and leukemogenic expansion in vitro and in vivo. We demonstrate that the p110alpha point mutations constitutively activated the PI3K-AKT pathway and resulted in factor-independent growth of Ba/F3 cells. Proliferation and survival of the cells were inhibited in a time- and dose-dependent manner using inhibitors of either PI3K or AKT. The mammalian target of rapamycin (mTOR) was demonstrated to be important for mitogenic, but not anti-apoptotic signaling of mutant PIK3CA. In a syngenic mouse model, hematopoietic cells expressing mutant p110alpha induced a leukemia-like disease. FACS analysis demonstrated a median chimerism of 68% in the bone marrow and 35% in peripheral blood of diseased mice, which were characterized by anemia, leukemic infiltration of hematopoietic organs, and 90% mortality within 5 weeks. No substantial differences were observed between E542K, E545A and H1047R. Mice, which were transplanted with an activating c-KIT point mutation (D814V), showed a significantly reduced survival and 100% died within 9 days. In conclusion our data show, that PIK3CA point mutations, by activating the PI3K-AKT pathway, confer factor-independence to hematopoietic cells in vitro and induce leukemogenic activity in vivo. As mutant c-KIT (D814V) showed enhanced leukemogenic potential, signaling through c-KIT may involve additional downstream pathways which cooperate with activated PI3K in leukemia progression. Our model is useful for the differential investigation of the leukemogenic relevance of the PI3K-AKT pathway and pharmacological inhibitor studies.

Description: Purpose: Signal transduction pathways, such as the PI3K/AKT cascade, are frequently activated in acute myeloid leukemia and stimulate the proliferation and survival of leukemic cells. Mutations in upstream genes such as Class III receptor tyrosine kinases are frequent but not exclusive causes of that activation. An important downstream target of PI3K/AKT is the key regulator GSK-3β. It controls anti-apoptotic genes such as NF-kappaB and Cyclin D1, is involved in wnt-pathway activation and drug resistance of leukemic cells. Deregulated signaling by GSK-3β occurs through inhibition by AKT-mediated phosphorylation. We have observed that constitutive phosphorylation of GSK-3β occurred in hematopoietic cells with pro-leukemogenic PI3K-mutations. We wanted to evaluate the relevance of GSK-3β inactivation in the transformation process of hematopoietic cells. Methods and Results: We used an in vitro factor-independent growth assay, GSK-3b inhibitors (Lithium, BIO) and established a second hit model using retroviral gene transfer of the weak oncogene Bcl XL. Signaling cascades were analysed by western blot. We demonstrate that inactivation of GSK-3b alone was not sufficient to induce factor-independent growth in IL-3 dependent early hematopoietic cells (Ba/F3). Induction of apoptosis upon growth factor withdrawal was reduced, but not prevented, in the presence of GSK-3b inhibitors, leading to a delayed Caspase 3 activation, PARP cleavage and DNA fragmentation. Overexpression of Bcl-XL also did not result in a prevention of apoptosis. GSK-3b inhibition in synergy with Bcl-XL overexpression resulted in the establishement of several growth factor-independent cell lines, which were characterized by the activation of multiple signaling cascades including AKT, MAPK, STAT5, but not STAT3. Also Cyclin D1 was overexpressed in contrast to other cyclines (D2, D3) which are no substrates of GSK-3b. Conclusions: Our data show that GSK-3β is part of the apoptotic response to growth factor withdrawal and suggest that GSK-3β is causaly involved in the transformation process of hematopoietic cells and seems to have an synergistic role in addition to other pro-leukemic mutations.

Description: Acute myelogenous leukemia (AML) is often associated with mutations in transcription factors that are essential for normal hematopoietic development and differentiation. The basic leucine zipper (bZIP) protein C/EBPalpha is mutated in 7–10% of AML. Two different classes of C/EBPalpha mutations are found in AML: 1) mutations that introduce a stop codon between the primary translational initiation site and a downstream ATG, resulting in translation of an N-terminally truncated protein (p30) 2) mutations that disrupt the basic region of the bZIP domain in the C-terminus, abolishing DNA binding. Of note, biallellic mutations are often but not always observed. It has been proposed that p30 is dominant-negative over wt C/EBPalpha (p42), blocking its transcriptional activity. We have previously shown that p30 blocks erythrocytic commitment and inhibits terminal granulocytic differentiation when expressed in human but not murine primary hematopoietic cells. To identify the molecular mechanism by which the p30 inhibits differentiation and to evaluate the role of homo- and heterodimerization between p30 and p42 in leukemogenesis, we created a p30 that carries point mutations in bZIP (p30-L12V) that disrupt dimerization. Expression of p30-L12V had no effect on the differentiation of human CD34+ cells, arguing for the requirement of dimerization. To inhibit potential heterodimerization between p30 and p42 but permit p30 homodimerization, we generated a p30-GZ, in which the leucine zipper was replaced by an artificial dimerization domain. Expression of p30-GZ induced a similar differentiation block as the normal p30, arguing against a dominant-negative function of p30 over p42 mediated by dimerization. In a second approach to understand the mechanism by which p30 inhibits differentiation, we wanted to identify gene targets of p30. For this purpose, p30 was fused to the modified ligand-binding domain of the estrogen receptor (p30-ERtm). Expression of p30-ERtm in CD34+ cells in the presence of tamoxifen showed a phenotype very similar to the one observed after the expression of p30, while in the absence of tamoxifen no effect was observed. We used this system of inducible p30 expression in human CD34+ cells to evaluate gene expression patterns. If p30 is dominant-negative and interferes with the transcriptional activation of p42, reciprocal expression patterns of target genes should be observed for wt C/EBPalpha and p30. Our data show that homodimerization of p30 is necessary and sufficient to block erythrocytic commitment and terminal granulocytic differentiation of human CD34+ cells and to induce known C/EBPalpha target genes. These data argue that the formation of p30/p42 heterodimers is not required for the effect of C/EBPalpha p30, questioning the proposed dominant-negative function.

Description: The RUNX1 gene encodes an alpha subunit of the core-binding factor (CBF), an important heterodimeric transcription factor in hematopoietic ontogeny and development, and is one of the most frequently disrupted genes in acute leukemia. In addition to its involvement in several translocations, the RUNX1 gene is often subject to deletions or point mutations in acute myelogenous leukemia (AML). Interestingly, in addition to complete loss-of-function mutations, many of the alterations involve missense point mutations within the Runt domain that disrupt DNA binding activity (DB-mutants). In vitro assays have suggested that these DB mutants have a dominant-negative (DN) activity, presumably due to their ability to bind and sequester CBF beta but inability to bind DNA. A strict correlation between the type of mutation and its monoallelic or biallelic incidence is not apparent even though a DN mutant should only affect one allele while a loss of function mutation should affect both alleles. It has been hypothesized that loss of one allele (haploinsufficiency) is sufficient for loss of tumor suppressor activity but the relative high incidence of specific DB mutations suggests a more complex scenario. We thus sought to determine if expression of DB mutants in murine bone marrow (BM) resulted in a similar phenotype as the loss of Runx1, or if these mutations are associated with a gain-of-function. Two RUNX1-DB mutants were thus evaluated using the established retroviral transduction/transplantation mouse model. Between 3 and 6 months after transplantation, peripheral blood, spleen and BM cells were analyzed. Long-term repopulating cells expressing RUNX1 DB-mutants were able to contribute normally to both myeloid and lymphoid compartments, although a disproportionate increase in the B-cell compartment was observed in 3 out of 10 mice. Surprisingly, and inconsistent with a DN activity, disruption of normal T-cell or megakaryocytic development was not observed in the mice, in contrast to Runx1−/+ mice. Significantly, however, replating assays in vitro demonstrated that RUNX1-DB mutants lead to a significant increase in self-renewal activity, in contrast to BM cells of floxed Runx1 mice expressing the Cre recombinase, which showed a less dramatic effect on self-renewal. Colonies derived from CFU-Cs expressing RUNX1-DB mutants were composed of dysplastic granulocytic and monocytic cells, with an increasing number of immature blasts after multiple replatings (〉7), whereas residual colonies from Runx1fl/− BM receiving CRE showed a different morphology with more mature cells. Thus our data suggest that RUNX1-DB mutants do not act in a dominant negative fashion to inhibit normal RUNX1 function, but impart a gain-of-function that results in impaired myeloid differentiation and increased self-renewal potential, consistent with its association with AML.