Flavin-containing monooxygenase mediated metabolism of psychoactive drugs by human brain microsomes

Flavin-containing monooxygenases (FMO) catalyze the oxidation of certain xenobiotics and drugs which contain a nucleophilic heteroatom. Here we report the first assessment of human brain flavin-containing monooxygenase from tissues obtained at autopsy from seven traffic accident victims. Human brain microsomes catalyzed the S-oxidation or N-oxidation of model substrates methimazole and N,N-dimethylaniline, respectively. The psychoactive drugs chlorpromazine, imipramine and fluoxetine, were also metabolized by human brain FMO. 'Western' immunoblot analyses revealed immunological cross-reactivity of the human brain FMO with rabbit pulmonary FMO. Immunocytochemistry further revealed the localization of the FMO predominantly in the neuronal cell bodies in the magnocellular reticular nuclei, colliculi and substantia nigra. Human brain clearly contains an active FMO system, and it is conceivable that such enzyme(s) are significantly involved in the local metabolism and modulation of pharmacological effects of psychoactive drugs

ERK inhibitor LY3214996-based treatment strategies for RAS-driven lung cancer

RAS gene mutations are the most frequent oncogenic event in lung cancer. They activate multiple RAS-centric signaling networks among them the MAPK, PI3K and RB pathways. Within the MAPK pathway ERK1/2 proteins exert a bottleneck function for transmitting mitogenic signals and activating cytoplasmic and nuclear targets. In view of disappointing anti-tumor activity and toxicity of continuously applied MEK inhibitors in patients with KRAS mutant lung cancer, research has recently focused on ERK1/2 proteins as therapeutic targets and on ERK inhibitors for their ability to prevent bypass and feedback pathway activation. Here we show that intermittent application of the novel and selective ATP-competitive ERK1/2 inhibitor LY3214996 exerts single-agent activity in patient-derived xenograft (PDX) models of RAS mutant lung cancer. Combination treatments were well tolerated and resulted in synergistic (ERKi plus PI3K/mTORi LY3023414) and additive (ERKi plus CDK4/6i abemaciclib) tumor growth inhibition in PDX models. Future clinical trials are required to investigate if intermittent ERK inhibitor-based treatment schedules can overcome toxicities observed with continuous MEK inhibition and - equally important - to identify biomarkers for patient stratification

Aurora A–Selective Inhibitor LY3295668 Leads to Dominant Mitotic Arrest, Apoptosis in Cancer Cells, and Shows Potent Preclinical Antitumor Efficacy

Although Aurora A, B, and C kinases share high sequence similarity, especially within the kinase domain, they function distinctly in cell-cycle progression. Aurora A depletion primarily leads to mitotic spindle formation defects and consequently prometaphase arrest, whereas Aurora B/C inactivation primarily induces polyploidy from cytokinesis failure. Aurora B/C inactivation phenotypes are also epistatic to those of Aurora A, such that the concomitant inactivation of Aurora A and B, or all Aurora isoforms by nonisoform–selective Aurora inhibitors, demonstrates the Aurora B/C-dominant cytokinesis failure and polyploidy phenotypes. Several Aurora inhibitors are in clinical trials for T/B-cell lymphoma, multiple myeloma, leukemia, lung, and breast cancers. Here, we describe an Aurora A–selective inhibitor, LY3295668, which potently inhibits Aurora autophosphorylation and its kinase activity in vitro and in vivo, persistently arrests cancer cells in mitosis, and induces more profound apoptosis than Aurora B or Aurora A/B dual inhibitors without Aurora B inhibition–associated cytokinesis failure and aneuploidy. LY3295668 inhibits the growth of a broad panel of cancer cell lines, including small-cell lung and breast cancer cells. It demonstrates significant efficacy in small-cell lung cancer xenograft and patient-derived tumor preclinical models as a single agent and in combination with standard-of-care agents. LY3295668, as a highly Aurora A–selective inhibitor, may represent a preferred approach to the current pan-Aurora inhibitors as a cancer therapeutic agent

Multiple forms of cytochrome P450 and associated monooxygenase activities in human brain mitochondria

We have investigated cytochrome P450 (P450) and associated monooxygenase activities in human brain mitochondria isolated from eight regions of four human brain samples obtained at autopsy. P450-associated monooxygenase activities including aminopyrine N-demethylase (APD), 7-ethoxycoumarin O-deethylase (ECD), p-nitrophenol hydroxylase (PNPH), and N-nitrosodimethylamine N-demethylase (NDMAD) were detectable in the mitochondria from human brain regions. Immunoblot experiments using antisera to purified rat liver microsomal P450, namely P4502B½, P4501A½, and P4502E1, revealed immunoreactive bands in isolated mitochondria from different regions of the human brain. The antibody to P4502B½ and P4501A½ inhibited the human brain mitochondrial APD and ECD activities, respectively. The addition of antiserum to microsomal NADPH cytochrome P450 reductase did not affect the mitochondrial P450-associated monooxygenase activities, although it completely inhibited the corresponding activities in brain microsomes. Overall, the present study demonstrates, in human brain mitochondria, the presence of multiple forms of P450 belonging to the 1A, 2B, and 2E subfamilies that are involved in xenobiotic metabolism

Cerebral metabolism of imipramine and a purified flavin-containing monooxygenase from human brain

Flavin-containing monooxygenase (FMO), previously reported both from hepatic and extrahepatic tissues, including brain, catalyze the oxidation of certain xenobiotics and drugs that contain a nucleophilic heteroatom. Psychoactive drugs, including the antidepressant imipramine, are substrates for the brain FMO. Since FMO-mediated metabolism of these drugs might contribute to local pharmacodynamic modulation within the human brain, the metabolism of imipramine by human brain FMO was studied in further detail. In the present study, the FMO activity was determined in human brain microsomes by estimating the actual amount of imipramine N-oxide formed. It was then compared with the corresponding activity measured using substrate (imipramine)-stimulated rates of nicotinamide adenine dinucleotide phosphate (NADPH) oxidation, which was significantly higher than the activity estimated as the amount of N-oxide assayed using high-pressure liquid chromatography (HPLC). The brain FMO activity was measurable only in the presence of detergents (sodium cholate or Lubrol PX) or in microsomes that were freeze-thawed several times. The activity was inhibited by an antibody to rabbit pulmonary FMO, but an antiserum to the rat liver NADPH cytochrome P-450 reductase had no effect indicating that cytochrome P-450 was not involved in the above metabolic pathway. The optimum pH for N-oxidation of imipramine was found to be 8.5; thermolability experiments indicated that the FMO activity was completely lost only after the incubation of brain microsomes at 45°C for 20 minutes. An FMO purified to apparent homogeneity from a human brain had a molecular weight of 71,000 Da. The purified enzyme cross-reacted with the antibody to rabbit pulmonary FMO and efficiently catalyzed the metabolism of imipramine to its N-oxide. The human brain clearly contains an active FMO system, and it is conceivable that such enzymes are significantly involved in the local metabolism and modulation of pharmacological and/or toxic effects of certain xenobiotics, including psychoactive drugs

Identification of druggable cancer driver genes amplified across TCGA datasets.

The Cancer Genome Atlas (TCGA) projects have advanced our understanding of the driver mutations, genetic backgrounds, and key pathways activated across cancer types. Analysis of TCGA datasets have mostly focused on somatic mutations and translocations, with less emphasis placed on gene amplifications. Here we describe a bioinformatics screening strategy to identify putative cancer driver genes amplified across TCGA datasets. We carried out GISTIC2 analysis of TCGA datasets spanning 16 cancer subtypes and identified 486 genes that were amplified in two or more datasets. The list was narrowed to 75 cancer-associated genes with potential "druggable" properties. The majority of the genes were localized to 14 amplicons spread across the genome. To identify potential cancer driver genes, we analyzed gene copy number and mRNA expression data from individual patient samples and identified 42 putative cancer driver genes linked to diverse oncogenic processes. Oncogenic activity was further validated by siRNA/shRNA knockdown and by referencing the Project Achilles datasets. The amplified genes represented a number of gene families, including epigenetic regulators, cell cycle-associated genes, DNA damage response/repair genes, metabolic regulators, and genes linked to the Wnt, Notch, Hedgehog, JAK/STAT, NF-KB and MAPK signaling pathways. Among the 42 putative driver genes were known driver genes, such as EGFR, ERBB2 and PIK3CA. Wild-type KRAS was amplified in several cancer types, and KRAS-amplified cancer cell lines were most sensitive to KRAS shRNA, suggesting that KRAS amplification was an independent oncogenic event. A number of MAP kinase adapters were co-amplified with their receptor tyrosine kinases, such as the FGFR adapter FRS2 and the EGFR family adapters GRB2 and GRB7. The ubiquitin-like ligase DCUN1D1 and the histone methyltransferase NSD3 were also identified as novel putative cancer driver genes. We discuss the patient tailoring implications for existing cancer drug targets and we further discuss potential novel opportunities for drug discovery efforts

Epigenetic regulatory genes as putative cancer amplified driver genes.

<p>(A) Copy number (x-axis) and mRNA expression (y-axis) for <i>NSD3</i> and <i>SETD1</i> in breast cancers and melanomas, respectively. Correlation coefficient for copy number and mRNA expression are listed in the top right (r value). (B) <i>BRD4</i> and <i>YEATS4</i> shRNA activity in a panel of cancer cell lines (Project Achilles). shRNA score denotes the log2 based decrease in the representative shRNA compared to pooled shRNA in cancer cell lines after several rounds of proliferation post-shRNA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098293#pone.0098293-Cheung1" target="_blank">[11]</a>. Yellow bars indicate cell lines with <i>BRD4</i> or <i>YEATS4</i> copy number >4 and black bars indicate cell lines with <i>BRD4</i> or <i>YEATS4</i> copy number <4. (C) Frequency of amplification (red bar), mutation (green bar), and deletion (blue bar) for <i>NSD3</i>, <i>SETDB1</i>, <i>YEATS4</i>, and <i>BRD4</i> in various cancers. The percentages shown reflect the overall rate of gene amplification, mutation and/or deletion in each cancer type. Vertical aligned bars reflect samples from the same patient. (D) Relative NSD3 protein level (y-axis, normalized to b-actin protein levels) compared with <i>NSD3</i> copy number (x-axis) in SW48, H1581, SW837, and H1703 cells. (E) Relative proliferation (y-axis) and (F) relative apoptosis levels of cancer cell lines H1581, H1703, SW48, and SW837 cells 3 days after transfection with <i>NSD3</i> siRNA, as measured by Cell Titer Glo and Caspase Glo assays, respectively. (G) Cell cycle profile of H1703 cells 24 or 48 hours after transfection with <i>NSD3</i> siRNA compared to non-transfected controls. (H) Relative changes of cells in apoptosis, G1 or G2 phases (y-axis) in cell lines 48 hours-post <i>NSD3</i> siRNA transfection compared to uninfected controls.</p

Combined CDK4/6 and ERK1/2 inhibition enhances anti-tumor activity in NF1-associated plexiform neurofibroma

Purpose: Plexiform neurofibromas (PNF) are peripheral nerve sheath tumors that cause significant morbidity in persons with neurofibromatosis type 1 (NF1), yet treatment options remain limited. To identify novel therapeutic targets for PNF, we applied an integrated multi-omic approach to quantitatively profile kinome enrichment in a mouse model that has predicted therapeutic responses in clinical trials for NF1-associated PNF with high fidelity. Experimental design: Utilizing RNA sequencing combined with chemical proteomic profiling of the functionally enriched kinome using multiplexed inhibitor beads coupled with mass spectrometry, we identified molecular signatures predictive of response to CDK4/6 and RAS/MAPK pathway inhibition in PNF. Informed by these results, we evaluated the efficacy of the CDK4/6 inhibitor, abemaciclib, and the ERK1/2 inhibitor, LY3214996, alone and in combination in reducing PNF tumor burden in Nf1flox/flox;PostnCre mice. Results: Converging signatures of CDK4/6 and RAS/MAPK pathway activation were identified within the transcriptome and kinome that were conserved in both murine and human PNF. We observed robust additivity of the CDK4/6 inhibitor, abemaciclib, in combination with the ERK1/2 inhibitor, LY3214996, in murine and human NF1(Nf1) mutant Schwann cells. Consistent with these findings, the combination of abemaciclib (CDK4/6i) and LY3214996 (ERK1/2i) synergized to suppress molecular signatures of MAPK activation and exhibited enhanced antitumor activity in Nf1flox/flox;PostnCre mice in vivo. Conclusions: These findings provide rationale for the clinical translation of CDK4/6 inhibitors alone and in combination with therapies targeting the RAS/MAPK pathway for the treatment of PNF and other peripheral nerve sheath tumors in persons with NF1