Dual PI3K/mTOR inhibition shows antileukemic activity in MLL-rearranged acute myeloid leukemia.

In acute myeloid leukemia (AML), several signaling pathways such as the phosphatidylinositol-3-kinase/AKT and the mammalian target of rapamycin (PI3K/AKT/mTOR) pathway are deregulated and constitutively activated as a consequence of genetic and cytogenetic abnormalities. We tested the effectiveness of PI3K/AKT/mTOR-targeting therapies and tried to identify alterations that associate with treatment sensitivity. By analyzing primary samples and cell lines, we observed a wide range of cytotoxic activity for inhibition of AKT (MK-2206), mTORC1 (rapamycin) and PI3K/mTORC1/2 (BEZ-235) with a high sensitivity of cells carrying an MLL rearrangement. In vivo PI3K/mTOR inhibition delayed tumor progression, reduced tumor load and prolonged survival in an MLL-AF9(+)/FLT3-ITD(+) xenograft mouse model. By performing targeted amplicon sequencing in 38 MLL-AF9(+) and 125 cytogenetically normal AML patient samples, we found a high additional mutation rate for genes involved in growth factor signaling in 79% of all MLL-AF9(+) samples, which could lead to a possible benefit of this cohort. PI3K/mTOR inhibition for 24 h led to the cross-activation of the ERK pathway. Further in vitro studies combining PI3K/mTOR and ERK pathway inhibition revealed highly synergistic effects in apoptosis assays. Our data implicate a possible therapeutic benefit of PI3K/mTOR inhibition in the MLL-mutated subgroup. Inhibiting rescue pathways could improve the therapeutic efficacy of PI3K-targeted therapies in AML.

Cell cycle-arrested tumor cells exhibit increased sensitivity towards TRAIL-induced apoptosis.

Resting tumor cells represent a huge challenge during anticancer therapy due to their increased treatment resistance. TNF-related apoptosis-inducing ligand (TRAIL) is a putative future anticancer drug, currently in phases I and II clinical studies. We recently showed that TRAIL is able to target leukemia stem cell surrogates. Here, we tested the ability of TRAIL to target cell cycle-arrested tumor cells. Cell cycle arrest was induced in tumor cell lines and xenografted tumor cells in G0, G1 or G2 using cytotoxic drugs, phase-specific inhibitors or RNA interference against cyclinB and E. Biochemical or molecular arrest at any point of the cell cycle increased TRAIL-induced apoptosis. Accordingly, when cell cycle arrest was disabled by addition of caffeine, the antitumor activity of TRAIL was reduced. Most important for clinical translation, tumor cells from three children with B precursor or T cell acute lymphoblastic leukemia showed increased TRAIL-induced apoptosis upon knockdown of either cyclinB or cyclinE, arresting the cell cycle in G2 or G1, respectively. Taken together and in contrast to most conventional cytotoxic drugs, TRAIL exerts enhanced antitumor activity against cell cycle-arrested tumor cells. Therefore, TRAIL might represent an interesting drug to treat static-tumor disease, for example, during minimal residual disease.

The adaptor protein FADD and the initiator caspase-8 mediate activation of NF-κB by TRAIL.

Besides inducing apoptosis, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) activates NF-κB. The apoptosis signaling pathway of TRAIL is well characterized involving TRAIL receptors, Fas-associated protein with death domain (FADD) and caspase-8. In contrast, the molecular mechanism of TRAIL signaling to NF-κB remains controversial. Here, we characterized the receptor-proximal mediators of NF-κB activation by TRAIL. Deletion of the DD of TRAIL receptors 1 and 2 revealed that it is essential in NF-κB signaling. Because FADD interacts with the TRAIL receptor DD, FADD was tested. RNAi-mediated knockdown of FADD or FADD deficiency in JURKAT T-cell leukemia cells decreased or disabled NF-κB signaling by TRAIL. In contrast, TRAIL-induced activation of NF-κB was maintained upon loss of receptor interacting protein 1 (RIP1) or knockdown of FLICE-like inhibitory protein (FLIP). Exogenous expression of FADD rescued TRAIL-induced NF-κB signaling. Loss-of-function mutations of FADD within the RHDLL motif of the death effector domain, which is required for TRAIL-induced apoptosis, abrogated FADD's ability to recruit caspase-8 and mediate NF-κB activation. Accordingly, deficiency of caspase-8 inhibited TRAIL-induced activation of NF-κB, which was rescued by wild-type caspase-8, but not by a catalytically inactive caspase-8 mutant. These data establish the mechanism of TRAIL-induced NF-κB activation involving the TRAIL receptor DD, FADD and caspase-8, but not RIP1 or FLIP. Our results show that signaling of TRAIL-induced apoptosis and NF-κB bifurcates downstream of caspase-8.

NMDA and D1 receptors regulate the phosphorylation of CREB and the induction of c-fos in striatal neurons in primary culture.

Numerous in vivo studies have demonstrated that psychostimulant drugs such as amphetamine and cocaine can induce the expression of the immediate early gene c-fos in striatal neurons via the activation of D1 dopamine receptors. NMDA receptor activation is also known to induce c-fos in the striatum. In the present study we have used a primary striatal neuronal culture preparation to examine the mechanisms whereby these stimuli lead to changes in gene expression. Direct application of NMDA to striatal cells in culture caused a rapid increase in the expression of c-fos as well as an increase in the phosphorylation of the transcription factor CRE binding protein (CREB). This was prevented by NMDA receptor antagonists, and required extracellular calcium, but did not involve L-type calcium channels. The induction of c-fos and CREB phosphorylation following NMDA were unaffected by inhibition of protein kinase C; tyrosine kinases or nitric oxide synthase. However, the response to NMDA was blocked by KN62, a selective inhibitor of calcium/calmodulin-dependent protein kinase. Application of the D1 agonist SKF 38393, or direct stimulation of adenylyl cyclase with forskolin, also resulted in the phosphorylation of CREB and the induction of c-fos in striatal neurons. These effects were blocked by the protein kinase A inhibitor H89. These observations are consistent with the hypothesis that calcium/calmodulin-dependent phosphorylation of CREB induced by NMDA, or cAMP-dependent phosphorylation of CREB induced by D1 agonists, underlie the induction of c-fos seen following activation of these receptors in striatal neurons.

Structural and dielectric properties of synthetic glycolipids in mixtures with water.

Three synthetically produced glycolipids, N-(beta-D-glucopyranosyl)-N-octadecyl-stearoylamide (OSGA), N-(beta-D-glucopyranosyl-N-octadecyl-oleoylamide (OOGA), N-(beta-D-galactopyranosyl)-N-octadecyl-lauroylamide (OLGA) have been studied in different mixtures with water by x-ray diffraction and dielectric measurements with microwaves at 9.4 GHz. The measurements were performed in the temperature range -50-70 degrees C. X-Ray diffraction revealed a direct L(beta') --> H( parallel) transition at 20 degrees C, 60 degrees C, and 45 degrees C depending on the glycolipid species but nearly not on the water content. The hexagonal phases are saturated at a water content of approximately 20 wt%. The lamellar phase absorbs even less water (< 10 wt%). The dielectric data show that in the H( parallel) phase the binding of water is stronger than in the L(beta') phase. In the temperature range below 0 degrees C, OSGA and OOGA show a "subzero transition" due to the freeze-out of water in a separate ice phase. This transition can be seen in an abrupt decrease of the dielectric function because the dielectric response of ice is much smaller at microwave frequencies. OLGA does not show the subzero transition but an additional transition, hexagonal --> distorted hexagonal at 60 degrees C.

[Proteins of bacterial membranes. H+-adenosinetriphosphatase from Acholeplasma laidlawii cells]. / Belki bakterial'nykh membran. H+-adenozintrifosfataza kletok Acholeplasma laidlawii.

The cytoplasmic membrane of micoplasmic cells, in particular of A. laidlawii cells, contains a proton-carrier Mg2+ -activated ATPase. A whole H+ -ATPase complex (F0-F1) was isolated from these cells and characterized. The isolation procedure included solubilization of the enzyme with Triton X-100 followed by ion-exchange chromatography on DEAE-cellulose and gel filtration on Sepharose 6B. The enzyme was inhibited by dicyclohexylcarbodiimide (10(-4) M). The Km value for ATP hydrolysis and Ki for ADP hydrolysis were determined. The order of the constants did not differ from those measured earlier for factor F1 of the complex. The purified enzyme, similar to its hydrophylic moiety is sensitive to the action of bivalent cations. The subunit composition of the whole complex and of its water-soluble part was investigated. The complex was found to contain 11 polypeptides, five of which belong to factor F1. The molecular weights of these polypeptides were determined.