Ponatinib promotes a G1 cell-cycle arrest of merlin/NF2-deficient human schwann cells

Neurofibromatosis type 2 (NF2) is a genetic syndrome that predisposes individuals to multiple benign tumors of the central and peripheral nervous systems, including vestibular schwannomas. Currently, there are no FDA approved drug therapies for NF2. Loss of function of merlin encoded by the NF2 tumor suppressor gene leads to activation of multiple mitogenic signaling cascades, including platelet-derived growth factor receptor (PDGFR) and SRC in Schwann cells. The goal of this study was to determine whether ponatinib, an FDA-approved ABL/SRC inhibitor, reduced proliferation and/or survival of merlin-deficient human Schwann cells (HSC). Merlin-deficient HSC had higher levels of phosphorylated PDGFRα/β, and SRC than merlin-expressing HSC. A similar phosphorylation pattern was observed in phospho-protein arrays of human vestibular schwannoma samples compared to normal HSC. Ponatinib reduced merlin-deficient HSC viability in a dose-dependent manner by decreasing phosphorylation of PDGFRα/β, AKT, p70S6K, MEK1/2, ERK1/2 and STAT3. These changes were associated with decreased cyclin D1 and increased p27Kip1levels, leading to a G1 cell-cycle arrest as assessed by Western blotting and flow cytometry. Ponatinib did not modulate ABL, SRC, focal adhesion kinase (FAK), or paxillin phosphorylation levels. These results suggest that ponatinib is a potential therapeutic agent for NF2-associated schwannomas and warrants further in vivo investigation

Brigatinib causes tumor shrinkage in both NF2-deficient meningioma and schwannoma through inhibition of multiple tyrosine kinases but not ALK

Neurofibromatosis Type 2 (NF2) is an autosomal dominant genetic syndrome caused by mutations in the NF2 tumor suppressor gene resulting in multiple schwannomas and meningiomas. There are no FDA approved therapies for these tumors and their relentless progression results in high rates of morbidity and mortality. Through a combination of high throughput screens, preclinical in vivo modeling, and evaluation of the kinome en masse, we identified actionable drug targets and efficacious experimental therapeutics for the treatment of NF2 related schwannomas and meningiomas. These efforts identified brigatinib (ALUNBRIG®), an FDA-approved inhibitor of multiple tyrosine kinases including ALK, to be a potent inhibitor of tumor growth in established NF2 deficient xenograft meningiomas and a genetically engineered murine model of spontaneous NF2 schwannomas. Surprisingly, neither meningioma nor schwannoma cells express ALK. Instead, we demonstrate that brigatinib inhibited multiple tyrosine kinases, including EphA2, Fer and focal adhesion kinase 1 (FAK1). These data demonstrate the power of the de novo unbiased approach for drug discovery and represents a major step forward in the advancement of therapeutics for the treatment of NF2 related malignancies

Generation And Use Of Merlin-Deficient Human Schwann Cells For A High-Throughput Chemical Genomics Screening Assay

Schwannomas are benign nerve tumors that occur sporadically in the general population and in those with neurofibromatosis type 2 (NF2), a tumor predisposition genetic disorder. NF2-associated schwannomas and most sporadic schwannomas are caused by inactivating mutations in Schwann cells in the neurofibromatosis type 2 gene (NF2) that encodes the merlin tumor suppressor. Despite their benign nature, schwannomas and especially vestibular schwannomas cause considerable morbidity. The primary available therapies are surgery or radiosurgery which usually lead to loss of function of the compromised nerve. Thus, there is a need for effective chemotherapies. We established an untransformed merlin-deficient human Schwann cell line for use in drug discovery studies for NF2-associated schwannomas. We describe the generation of human Schwann cells (HSCs) with depletion of merlin and their application in high-throughput screening of chemical libraries to identify compounds that decrease their viability. This NF2-HSC model is amenable for use in independent labs and high-throughput screening (HTS) facilities

Inhibition Of Sirt2 In Merlin/Nf2-Mutant Schwann Cells Triggers Necrosis

Mutations in the NF2 gene cause Neurofibromatosis Type 2 (NF2), a disorder characterized by the development of schwannomas, meningiomas and ependymomas in the nervous system. Merlin, a tumor suppressor encoded by the NF2 gene, modulates activity of many essential signaling pathways. Yet despite increasing knowledge of merlin function, there are no NF2 drug therapies. In a pilot high-throughput screen of the Library of Pharmacologically Active Compounds, we assayed for compounds capable of reducing viability of mouse Schwann cells (MSC) with Nf2 inactivation as a cellular model for human NF2 schwannomas. AGK2, a SIRT2 (sirtuin 2) inhibitor, was identified as a candidate compound. SIRT2 is one of seven mammalian sirtuins that are NAD+-dependent protein deacetylases. We show that merlin-mutant MSC have higher expression levels of SIRT2 and lower levels of overall lysine acetylation than wild-type control MSC. Pharmacological inhibition of SIRT2 decreases merlin-mutant MSC viability in a dose dependent manner without substantially reducing wild-type MSC viability. Inhibition of SIRT2 activity in merlin-mutant MSC is accompanied by release of lactate dehydrogenase and high mobility group box 1 protein into the medium in the absence of significant apoptosis, autophagy, or cell cycle arrest. These findings suggest that SIRT2 inhibition triggers necrosis of merlin-mutant MSCs and that SIRT2 is a potential NF2 drug target

Ponatinib Promotes A G1 Cell-Cycle Arrest Of Merlin/Nf2-Deficient Human Schwann Cells

Neurofibromatosis type 2 (NF2) is a genetic syndrome that predisposes individuals to multiple benign tumors of the central and peripheral nervous systems, including vestibular schwannomas. Currently, there are no FDA approved drug therapies for NF2. Loss of function of merlin encoded by the NF2 tumor suppressor gene leads to activation of multiple mitogenic signaling cascades, including platelet-derived growth factor receptor (PDGFR) and SRC in Schwann cells. The goal of this study was to determine whether ponatinib, an FDA-approved ABL/SRC inhibitor, reduced proliferation and/or survival of merlin-deficient human Schwann cells (HSC). Merlin-deficient HSC had higher levels of phosphorylated PDGFRα/β, and SRC than merlin-expressing HSC. A similar phosphorylation pattern was observed in phospho-protein arrays of human vestibular schwannoma samples compared to normal HSC. Ponatinib reduced merlin-deficient HSC viability in a dose-dependent manner by decreasing phosphorylation of PDGFRα/β, AKT, p70S6K, MEK1/2, ERK1/2 and STAT3. These changes were associated with decreased cyclin D1 and increased p27Kip1levels, leading to a G1 cell-cycle arrest as assessed by Western blotting and flow cytometry. Ponatinib did not modulate ABL, SRC, focal adhesion kinase (FAK), or paxillin phosphorylation levels. These results suggest that ponatinib is a potential therapeutic agent for NF2-associated schwannomas and warrants further in vivo investigation

Proteomics and Phosphoproteomics of Circulating Extracellular Vesicles Provide New Insights into Diabetes Pathobiology

The purpose of this study was to define the proteomic and phosphoproteomic landscape of circulating extracellular vesicles (EVs) in people with normal glucose tolerance (NGT), prediabetes (PDM), and diabetes (T2DM). Archived serum samples from 30 human subjects (n = 10 per group, ORIGINS study, NCT02226640) were used. EVs were isolated using EVtrap®. Mass spectrometry-based methods were used to detect the global EV proteome and phosphoproteome. Differentially expressed features, correlation, enriched pathways, and enriched tissue-specific protein sets were identified using custom R scripts. Phosphosite-centric analyses were conducted using directPA and PhosR software packages. A total of 2372 unique EV proteins and 716 unique EV phosphoproteins were identified among all samples. Unsupervised clustering of the differentially expressed (fold change ≥ 2, p < 0.05, FDR < 0.05) proteins and, particularly, phosphoproteins showed excellent discrimination among the three groups. CDK1 and PKCδ appear to drive key upstream phosphorylation events that define the phosphoproteomic signatures of PDM and T2DM. Circulating EVs from people with diabetes carry increased levels of specific phosphorylated kinases (i.e., AKT1, GSK3B, LYN, MAP2K2, MYLK, and PRKCD) and could potentially distribute activated kinases systemically. Among characteristic changes in the PDM and T2DM EVs, “integrin switching” appeared to be a central feature. Proteins involved in oxidative phosphorylation (OXPHOS), known to be reduced in various tissues in diabetes, were significantly increased in EVs from PDM and T2DM, which suggests that an abnormally elevated EV-mediated secretion of OXPHOS components may underlie the development of diabetes. A highly enriched signature of liver-specific markers among the downregulated EV proteins and phosphoproteins in both PDM and T2DM groups was also detected. This suggests that an alteration in liver EV composition and/or secretion may occur early in prediabetes. This study identified EV proteomic and phosphoproteomic signatures in people with prediabetes and T2DM and provides novel insight into the pathobiology of diabetes

DataSheet_1_Coordinated regulation of gene expression and microRNA changes in adipose tissue and circulating extracellular vesicles in response to pioglitazone treatment in humans with type 2 diabetes.docx

Pioglitazone, a PPARγ agonist, is used to treat type 2 diabetes (T2D). PPARγ is highly expressed in adipose tissue (AT), however the effects of pioglitazone to improve insulin sensitivity are also evident in other tissues and PPARγ agonism has been shown to alter cancer derived extracellular vesicle (EV)-miRNAs. We hypothesized that pioglitazone modifies the cargo of circulating AT-derived EVs to alter interorgan crosstalk in people with diabetes. We tested our hypothesis in a 3-month trial in which 24 subjects with T2D were randomized to treatment with either pioglitazone 45 mg/day or placebo (NCT00656864). Levels of 42 adipocyte-derived EV-miRNAs were measured in plasma EVs using low density TaqMan arrays. Levels of differentially expressed EV-miRNAs and their most relevant target genes were also measure in adipose tissue from the same participants, using individual TaqMan assays. Levels of 5 miRNAs (i.e., miR-7-5p, miR-20a-5p, miR-92a-3p, miR-195-5p, and miR-374b-5p) were significantly downregulated in EVs in response to pioglitazone treatment relative to placebo. The opposite occurred for miR-195-5p in subcutaneous AT. Changes in miRNA expression in EVs and AT correlated with changes in suppression of lipolysis and improved insulin sensitivity, among others. DICER was downregulated and exosomal miRNA sorting-related genes YBX1 and hnRNPA2B1 displayed a downregulation trend in AT. Furthermore, analysis of EV-miRNA targeted genes identified a network of transcripts that changed in a coordinated manner in AT. Collectively, our results suggest that some beneficial pharmacologic effects of pioglitazone are mediated by adipose-specific miRNA regulation and exosomal/EV trafficking.Clinical Trial RegistrationClinicalTrials.gov, identifier NCT00656864.</p

A chemical biology approach identified PI3K as a potential therapeutic target for neurofibromatosis type 2

Mutations in the merlin tumor suppressor gene cause Neurofibromatosis type 2 (NF2), which is a disease characterized by development of multiple benign tumors in the nervous system. The current standard of care for NF2 calls for surgical resection of the characteristic tumors, often with devastating neurological consequences. There are currently no approved non-surgical therapies for NF2. In an attempt to identify much needed targets and therapeutically active compounds for NF2 treatment, we employed a chemical biology approach using ultra-highthroughput screening. To support this goal, we created a merlin-null mouse Schwann cell (MSC) line to screen for compounds that selectively decrease their viability and proliferation. We optimized conditions for 384-well plate assays and executed a proof-of-concept screen of the Library of Pharmacologically Active Compounds. Further confirmatory and selectivity assays identified phosphatidylinositol 3-kinase (PI3K) as a potential NF2 drug target. Notably, loss of merlin function is associated with activation of the PI3K/Akt pathway in human schwannomas. We report that AS605240, a PI3K inhibitor, decreased merlin-null MSC viability in a dose-dependent manner without significantly decreasing viability of control Schwann cells. AS605240 exerted its action on merlin-null MSCs by promoting caspase-dependent apoptosis and inducing autophagy. Additional PI3K inhibitors tested also decreased viability of merlin-null MSCs in a dose-dependent manner. In summary, our chemical genomic screen and subsequent hit validation studies have identified PI3K as potential target for NF2 therapy