The fibroblast growth factor receptor inhibitor, derazantinib, has strong efficacy in human gastric tumor models and synergizes with paclitaxel in vivo.

Derazantinib (DZB) is an inhibitor of fibroblast growth factor receptors 1-3 (FGFR1-3), with additional activity against colony-stimulating-factor-1 receptor (CSF1R). We have profiled the activity of DZB in gastric cancer (GC) as monotherapy and combined with paclitaxel, and explored means of stratifying patients for treatment. The antiproliferative potency of DZB in vitro was quantified in 90 tumor cell lines and shown to correlate significantly with FGFR expression (<0.01) but not with FGFR DNA copy-number (CN) or FGFR mutations. In four GC cell lines in vitro , little or no synergy was observed with paclitaxel. In athymic nude mice, bearing cell-line derived xenografts (CDX) or patient-derived xenograft (PDX) GC models, DZB efficacy correlated highly significantly with FGFR gene expression ( r2 = 0.58; P = 0.0003; n = 18), but not FGFR mutations or DNA-CN. In FGFR-driven GC models, DZB had comparable efficacy to three other FGFR inhibitors and was more efficacious than paclitaxel. DZB had dose-dependent plasma pharmacokinetics but showed low brain penetration at all doses. GC models (one CDX and six PDX) were tested for sensitivity to the combination of DZB and paclitaxel and characterized by immunohistochemistry. The combination showed synergy (5) or additivity (2), and no antagonism, with synergy significantly associated ( P < 0.05) with higher levels of M2-type macrophages. The association of strong efficacy of the combination in vivo with M2 macrophages, which are known to express CSF1R, and the absence of synergy in vitro is consistent with the tumor microenvironment also being a factor in DZB efficacy and suggests additional means by which DZB could be stratified for cancer treatment in the clinic.

Microtubule-independent secretion requires functional maturation of Golgi elements.

The Golgi complex is responsible for processing and sorting of secretory cargos. Microtubules are known to accelerate the transport of proteins from the endoplasmic reticulum (ER) to the Golgi complex and from the Golgi to the plasma membrane. However, whether post-Golgi transport strictly requires microtubules is still unclear. Using the retention using selective hooks (RUSH) system to synchronize the trafficking of cargos, we show that anterograde transport of tumor necrosis factor (TNF) is strongly reduced without microtubules. We show that two populations of Golgi elements co-exist in these cells. A centrally located and giantin-positive Golgi complex that sustains trafficking, and newly formed peripheral Golgi mini-stacks that accumulate cargos in cells without microtubules. Using a genome-edited GFP-giantin cell line, we observe that the trafficking-competent Golgi population corresponds to the pre-existing population that was present before removal of microtubules. All Golgi elements support trafficking after long-term depletion of microtubules and after relocation of Golgi proteins to the ER after treatment with Brefeldin A. Our results demonstrate that functional maturation of Golgi elements is needed to ensure post-Golgi trafficking, and that microtubule-driven post-Golgi transport is not strictly required.

Muscle inactivation of mTOR causes metabolic and dystrophin defects leading to severe myopathy.

Mammalian target of rapamycin (mTOR) is a key regulator of cell growth that associates with raptor and rictor to form the mTOR complex 1 (mTORC1) and mTORC2, respectively. Raptor is required for oxidative muscle integrity, whereas rictor is dispensable. In this study, we show that muscle-specific inactivation of mTOR leads to severe myopathy, resulting in premature death. mTOR-deficient muscles display metabolic changes similar to those observed in muscles lacking raptor, including impaired oxidative metabolism, altered mitochondrial regulation, and glycogen accumulation associated with protein kinase B/Akt hyperactivation. In addition, mTOR-deficient muscles exhibit increased basal glucose uptake, whereas whole body glucose homeostasis is essentially maintained. Importantly, loss of mTOR exacerbates the myopathic features in both slow oxidative and fast glycolytic muscles. Moreover, mTOR but not raptor and rictor deficiency leads to reduced muscle dystrophin content. We provide evidence that mTOR controls dystrophin transcription in a cell-autonomous, rapamycin-resistant, and kinase-independent manner. Collectively, our results demonstrate that mTOR acts mainly via mTORC1, whereas regulation of dystrophin is raptor and rictor independent.

Differential interaction of the Pafah1b alpha subunits with the Reelin transducer Dab1.

The Reelin signaling pathway controls radial neuronal migration and maturation in the developing brain. The platelet activating factor (PAF) acetyl hydrolase 1b (Pafah1b) complex is also involved in multiple aspects of brain development. We previously showed that the Reelin pathway and the Pafah1b complex interact genetically and biochemically. Lis1, the regulatory subunit of Pafah1b interacts with phosphoDab1, an essential mediator of Reelin signaling. Compound mutants carrying mutations in both, the Reelin pathway and Lis1 exhibit hydrocephalus, a phenotype that is suppressed by mutations in the gene encoding the Alpha2 subunit of Pafah1b. This subunit, like the Alpha1 catalytic subunit of Pafah1b also binds the Reelin receptor VLDLR. Here we investigated the molecular interactions of the Pafah1b catalytic subunits with Dab1. We found that Alpha2 coprecipitates with Dab1 from brain extracts of normal and reeler mutant mice lacking Reelin, and from cell-free extracts containing normal or a phosphorylation mutant form of Dab1, suggesting that Dab1 phosphorylation is not necessary for binding to Alpha2. This interaction is specific for Alpha2 and not Alpha1, and depends on a unique tyrosine residue of Alpha2. Biochemical assays using mutant mice lacking Alpha2 further demonstrated that this subunit is not required for Reelin-induced Dab1 phosphorylation. However, increasing amounts of Alpha2 in a cell-free system disrupted the formation of Dab1-Lis1 complexes without affecting the association of Dab1 with VLDLR. Our data suggest that the Alpha2 subunit may play a modulatory role in the formation of protein complexes that affect brain development and hydrocephalus.

S6 kinase inactivation impairs growth and translational target phosphorylation in muscle cells maintaining proper regulation of protein turnover.

A defect in protein turnover underlies multiple forms of cell atrophy. Since S6 kinase (S6K)-deficient cells are small and display a blunted response to nutrient and growth factor availability, we have hypothesized that mutant cell atrophy may be triggered by a change in global protein synthesis. By using mouse genetics and pharmacological inhibitors targeting the mammalian target of rapamycin (mTOR)/S6K pathway, here we evaluate the control of translational target phosphorylation and protein turnover by the mTOR/S6K pathway in skeletal muscle and liver tissues. The phosphorylation of ribosomal protein S6 (rpS6), eukaryotic initiation factor-4B (eIF4B), and eukaryotic elongation factor-2 (eEF2) is predominantly regulated by mTOR in muscle cells. Conversely, in liver, the MAPK and phosphatidylinositol 3-kinase pathways also play an important role, suggesting a tissue-specific control. S6K deletion in muscle mimics the effect of the mTOR inhibitor rapamycin on rpS6 and eIF4B phosphorylation without affecting eEF2 phosphorylation. To gain insight on the functional consequences of these modifications, methionine incorporation and polysomal distribution were assessed in muscle cells. Rates and rapamycin sensitivity of global translation initiation are not altered in S6K-deficient muscle cells. In addition, two major pathways of protein degradation, autophagy and expression of the muscle-specific atrophy-related E3 ubiquitin ligases, are not affected by S6K deletion. Our results do not support a role for global translational control in the growth defect due to S6K deletion, suggesting specific modes of growth control and translational target regulation downstream of mTOR.

Postnatal repeated maternal deprivation produces age-dependent changes of brain-derived neurotrophic factor expression in selected rat brain regions.

BACKGROUND: Adverse life events occurring early in development may alter the correct program of brain maturation and render the organism more vulnerable to psychiatric disorders. Identification of persistent changes associated with these events is crucial for the development of novel therapeutic strategies. METHODS: We used postnatal repeated maternal deprivation (MD) from postnatal day (PND) 2-14 to investigate changes in brain-derived neurotrophic factor (BDNF) levels. RNase protection assay and enzyme linked immunosorbent assay were employed to determine the anatomic profile of neurotrophin expression at different ages following MD. RESULTS: We found that MD produces a short-term up-regulation of neurotrophin expression in hippocampus and prefrontal cortex, as measured on PND 17, whereas at adulthood, a selective reduction of BDNF expression was observed in prefrontal cortex. When adult animals were challenged with a chronic swim stress paradigm, both a reduced expression of BDNF in prefrontal cortex and a significant reduction in striatal protein levels were found only in control subjects, whereas levels in the MD group were not further decreased. CONCLUSIONS: Our data suggest that MD produces a significant reduction of BDNF expression within prefrontal cortex and striatum, which may render these structures less plastic and more vulnerable under challenging conditions.

Effect of antipsychotic drugs on brain-derived neurotrophic factor expression under reduced N-methyl-D-aspartate receptor activity.

Brain-derived neurotrophic factor (BDNF) promotes a variety of neuromodulatory processes during development as well as in adulthood. This neurotrophin has been associated with synaptic plasticity, suggesting that its regulation may represent one of the mechanisms through which psychotropic drugs alter brain function. Because reduced glutamatergic function represents a major feature of schizophrenia, we investigated the effects of the concomitant administration of haloperidol or olanzapine with the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 on BDNF expression. MK-801 reduces the hippocampal expression of the neurotrophin; this effect was exacerbated by haloperidol, but it was normalized by olanzapine. Our data reveal a fine tuning of BDNF biosynthesis and a differential modulation by antipsychotic drugs when NMDA-mediated transmission is reduced, suggesting that haloperidol and olanzapine can produce different effects on brain plasticity through the modulation of BDNF expression.