Lack of Association of Rare Functional Variants in TSC1/TSC2 Genes with Autism Spectrum Disorder

Background: Autism spectrum disorder (ASD) is reported in 30 to 60% of patients with tuberous sclerosis complex (TSC) but shared genetic mechanisms that exist between TSC-associated ASD and idiopathic ASD have yet to be determined. Through the small G-protein Rheb, the TSC proteins, hamartin and tuberin, negatively regulate mammalian target of rapamycin complex 1 (mTORC1) signaling. It is well established that mTORC1 plays a pivotal role in neuronal translation and connectivity, so dysregulation of mTORC1 signaling could be a common feature in many ASDs. Pam, an E3 ubiquitin ligase, binds to TSC proteins and regulates mTORC1 signaling in the CNS, and the FBXO45-Pam ubiquitin ligase complex plays an essential role in neurodevelopment by regulating synapse formation and growth. Since mounting evidence has established autism as a disorder of the synapses, we tested whether rare genetic variants in TSC1, TSC2, MYCBP2, RHEB and FBXO45, genes that regulate mTORC1 signaling and/or play a role in synapse development and function, contribute to the pathogenesis of idiopathic ASD. Methods: Exons and splice junctions of TSC1, TSC2, MYCBP2, RHEB and FBXO45 were resequenced for 300 ASD trios from the Simons Simplex Collection (SSC) using a pooled PCR amplification and next-generation sequencing strategy, targeted to the discovery of deleterious coding variation. These detected, potentially functional, variants were confirmed by Sanger sequencing of the individual samples comprising the pools in which they were identified. Results: We identified a total of 23 missense variants in MYCBP2, TSC1 and TSC2. These variants exhibited a near equal distribution between the proband and parental pools, with no statistical excess in ASD cases (P > 0.05). All proband variants were inherited. No putative deleterious variants were confirmed in RHEB and FBXO45. Three intronic variants, identified as potential splice defects in MYCBP2 did not show aberrant splicing upon RNA assay. Overall, we did not find an over-representation of ASD causal variants in the genes studied to support them as contributors to autism susceptibility. Conclusions: We did not observe an enrichment of rare functional variants in TSC1 and TSC2 genes in our sample set of 300 trios

A high-throughput kinome screen reveals serum/glucocorticoid-regulated kinase 1 as a therapeutic target for NF2-deficient meningiomas

Meningiomas are the most common primary intracranial adult tumor. All Neurofibromatosis 2 (NF2)-associated meningiomas and ~60% of sporadic meningiomas show loss of NF2 tumor suppressor protein. There are no effective medical therapies for progressive and recurrent meningiomas. Our previous work demonstrated aberrant activation of mTORC1 signaling that led to ongoing clinical trials with rapamycin analogs for NF2 and sporadic meningioma patients. Here we performed a high-throughput kinome screen to identify kinases responsible for mTORC1 pathway activation in NF2-deficient meningioma cells. Among the emerging top candidates were the mTORC2-specific target serum/glucocorticoid-regulated kinase 1 (SGK1) and p21-activated kinase 1 (PAK1). In NF2-deficient meningioma cells, inhibition of SGK1 rescues mTORC1 activation, and SGK1 activation is sensitive to dual mTORC1/2 inhibitor AZD2014, but not to rapamycin. PAK1 inhibition also leads to attenuated mTORC1 but not mTORC2 signaling, suggesting that mTORC2/SGK1 and Rac1/PAK1 pathways are independently responsible for mTORC1 activation in NF2-deficient meningiomas. Using CRISPR-Cas9 genome editing, we generated isogenic human arachnoidal cell lines (ACs), the origin cell type for meningiomas, expressing or lacking NF2. NF2-null CRISPR ACs recapitulate the signaling of NF2-deficient meningioma cells. Interestingly, we observe increased SGK1 transcription and protein expression in NF2-CRISPR ACs and in primary NF2-negative meningioma lines. Moreover, we demonstrate that the dual mTORC1/mTORC2 inhibitor, AZD2014 is superior to rapamycin and PAK inhibitor FRAX597 in blocking proliferation of meningioma cells. Importantly, AZD2014 is currently in use in several clinical trials of cancer. Therefore, we believe that AZD2014 may provide therapeutic advantage over rapalogs for recurrent and progressive meningiomas

The novel mitochondria-targeted hydrogen sulfide (H2S) donors AP123 and AP39 protect against hyperglycemic injury in microvascular endothelial cells in vitro.

The development of diabetic vascular complications is initiated, at least in part, by mitochondrial reactive oxygen species (ROS) production in endothelial cells. Hyperglycemia induces superoxide production in the mitochondria and initiates changes in the mitochondrial membrane potential that leads to mitochondrial dysfunction. Hydrogen sulfide (H2S) supplementation has been shown to reduce the mitochondrial oxidant production and shows efficacy against diabetic vascular damage in vivo. However, the half-life of H2S is very short and it is not specific for the mitochondria. We have therefore evaluated two novel mitochondria-targeted anethole dithiolethione and hydroxythiobenzamide H2S donors (AP39 and AP123 respectively) at preventing hyperglycemia-induced oxidative stress and metabolic changes in microvascular endothelial cells in vitro. Hyperglycemia (HG) induced significant increase in the activity of the citric acid cycle and led to elevated mitochondrial membrane potential. Mitochondrial oxidant production was increased and the mitochondrial electron transport decreased in hyperglycemic cells. AP39 and AP123 (30-300nM) decreased HG-induced hyperpolarisation of the mitochondrial membrane and inhibited the mitochondrial oxidant production. Both H2S donors (30-300nM) increased the electron transport at respiratory complex III and improved the cellular metabolism. Targeting H2S to mitochondria retained the cytoprotective effect of H2S against glucose-induced damage in endothelial cells suggesting that the molecular target of H2S action is within the mitochondria. Mitochondrial targeting of H2S also induced >1000-fold increase in the potency of H2S against hyperglycemia-induced injury. The high potency and long-lasting effect elicited by these H2S donors strongly suggests that these compounds could be useful against diabetic vascular complications

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

Traditional And Systems Biology Based Drug Discovery For The Rare Tumor Syndrome Neurofibromatosis Type 2

Neurofibromatosis 2 (NF2) is a rare tumor suppressor syndrome that manifests with multiple schwannomas and meningiomas. There are no effective drug therapies for these benign tumors and conventional therapies have limited efficacy. Various model systems have been created and several drug targets have been implicated in NF2-driven tumorigenesis based on known effects of the absence of merlin, the product of the NF2 gene. We tested priority compounds based on known biology with traditional dose-concentration studies in meningioma and schwann cell systems. Concurrently, we studied functional kinome and gene expression in these cells pre- and post-treatment to determine merlin deficient molecular phenotypes. Cell viability results showed that three agents (GSK2126458, Panobinostat, CUDC-907) had the greatest activity across schwannoma and meningioma cell systems, but merlin status did not significantly influence response. In vivo, drug effect was tumor specific with meningioma, but not schwannoma, showing response to GSK2126458 and Panobinostat. In culture, changes in both the transcriptome and kinome in response to treatment clustered predominantly based on tumor type. However, there were differences in both gene expression and functional kinome at baseline between meningioma and schwannoma cell systems that may form the basis for future selective therapies. This work has created an openly accessible resource (www.synapse.org/SynodosNF2) of fully characterized isogenic schwannoma and meningioma cell systems as well as a rich data source of kinome and transcriptome data from these assay systems before and after treatment that enables single and combination drug discovery based on molecular phenotype

Mediator Subunit Med28 Is Essential for Mouse Peri-Implantation Development and Pluripotency

<div><p>The multi-subunit mammalian Mediator complex acts as an integrator of transcriptional regulation by RNA Polymerase II, and has emerged as a master coordinator of development and cell fate determination. We previously identified the Mediator subunit, MED28, as a cytosolic binding partner of merlin, the Neurofibromatosis 2 (NF2) tumor suppressor, and thus MED28 is distinct in having a cytosolic role as an NF2 interacting protein as well as a nuclear role as a Mediator complex subunit. Although limited <i>in vitro</i> studies have been performed on MED28, its <i>in vivo</i> function remains unknown. Employing a knockout mouse model, we describe for the first time the requirement for Med28 in the developing mouse embryo. Med28-deficiency causes peri-implantation lethality resulting from the loss of pluripotency of the inner cell mass accompanied by reduced expression of key pluripotency transcription factors <i>Oct4</i> and <i>Nanog</i>. Further, overexpression of Med28 in mouse embryonic fibroblasts enhances the efficiency of their reprogramming to pluripotency. Cre-mediated inactivation of <i>Med28</i> in induced pluripotent stem cells shows that Med28 is required for their survival. Intriguingly, heterozygous loss of <i>Med28</i> results in differentiation of induced pluripotent stem cells into extraembryonic trophectoderm and primitive endoderm lineages. Our findings document the essential role of Med28 in the developing embryo as well as in acquisition and maintenance of pluripotency during reprogramming.</p></div

Loss of Med28 in MEFs results in lack of reprogramming.

<p><b>(A)</b> Western blot analysis shows that Med28 expression is much higher in ES cells compared to MEFs. <b>(B)</b> Western blot shows reduced Med28 expression in <i>Med28</i>^<i>fl/fl</i> MEFs at 6 days post-infection with Ad-Cre compared to Ad-empty control adenovirus. <b>(C)</b> Immunofluorescent staining of <i>Med28</i>^<i>fl/fl</i> MEFs demonstrates significant reduction of Med28 (red) in Ad-Cre-infected cells compared to Ad-empty control. DAPI staining (blue) of cell nuclei is shown. <b>(D)</b> Quantitation of number of iPSC colonies (triplicate experiments) from <i>Med28</i>^<i>fl/fl</i> primary MEFs infected with OKSM and rtTA followed by infection with Ad-empty (OKSM+Ad-empty) or Ad-Cre (OKSM+Ad-Cre) one day after Dox treatment. Note that infection with OKSM+Ad-Cre produced no colonies. Data are presented as mean +/- SD (*<i>p</i><0.05).</p

Targeted disruption of <i>Med28</i>.

<p><b>(A)</b> Schematic representation of the targeted allele and null allele. Targeted <i>Med28</i> allele in mouse ES cells with neo cassette in the reverse orientation (top, Med28(fl-frt), targeted <i>Med28</i> allele without neo cassette after transient transfection with Flp (middle, Med28(fl)) and Med28 null allele with Exon 1 and 2 deletions after breeding with Protamine-Cre mouse (bottom, Med28(-)). Shaded boxes with numbers represent the exons. Open triangle: loxP site; closed triangle: Frt site; B: BspH1 restriction site; S: SpHI restriction site. Numbered arrows represent PCR primers used for genotyping. Short solid bar represents 5’ probe outside the homologous region used for Southern blot analysis. <b>(B)</b> Representative Southern blot analysis used to confirm correct targeting. DNA was digested with either BspHI or SpH1 restrict enzymes and the 5’Probe (shown in <b>A</b>) outside the homologous region was used to detect wildtype (+), floxed (fl) and null (-) alleles at expected sizes for WT (+) allele, 14.6 kb (BspHI) or 15.2 kb (SpHI); floxed allele without neo (fl), 15 kb (BspHI) or 12 kb (SpHI); and null (-) allele, 12.1 kb (BspHI) or 12.7 kb (SpHI). <b>(C)</b> Representative PCR analysis using primers 1 and 2 (shown in A) confirm the presence of 5’loxP site (top panel, 324 bp) and WT allele (top panel, 290 bp). PCR analysis using primers 1 and 3 (shown in <b>A</b>) confirm presence of only the null (-) allele (bottom panel, 310 bp). <b>(D)</b> Histological analysis of 6.5-dpc (E6.5) presumed to be Med28 mutant embryos shows disorganized extraembryonic tissue with no discernible epiblast. Sections from whole decidual swellings were stained with hematoxylin and eosin. Arrow, epiblast; arrowhead, trophoblast giant cell.</p