46 results on '"Roberta L. Beauchamp"'
Search Results
2. Translatome analysis of tuberous sclerosis complex 1 patient-derived neural progenitor cells reveals rapamycin-dependent and independent alterations
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Inci S. Aksoylu, Pauline Martin, Francis Robert, Krzysztof J. Szkop, Nicholas E. Redmond, Srirupa Bhattacharyya, Jennifer Wang, Shan Chen, Roberta L. Beauchamp, Irene Nobeli, Jerry Pelletier, Ola Larsson, and Vijaya Ramesh
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Tuberous sclerosis complex ,TSC1 ,Neural progenitor cells ,Translatome ,Polysome profiling ,Autism spectrum disorder ,Neurology. Diseases of the nervous system ,RC346-429 - Abstract
Abstract Background Tuberous sclerosis complex (TSC) is an inherited neurocutaneous disorder caused by mutations in the TSC1 or TSC2 genes, with patients often exhibiting neurodevelopmental (ND) manifestations termed TSC-associated neuropsychiatric disorders (TAND) including autism spectrum disorder (ASD) and intellectual disability. Hamartin (TSC1) and tuberin (TSC2) proteins form a complex inhibiting mechanistic target of rapamycin complex 1 (mTORC1) signaling. Loss of TSC1 or TSC2 activates mTORC1 that, among several targets, controls protein synthesis by inhibiting translational repressor eIF4E-binding proteins. Using TSC1 patient-derived neural progenitor cells (NPCs), we recently reported early ND phenotypic changes, including increased cell proliferation and altered neurite outgrowth in TSC1-null NPCs, which were unaffected by the mTORC1 inhibitor rapamycin. Methods Here, we used polysome profiling, which quantifies changes in mRNA abundance and translational efficiencies at a transcriptome-wide level, to compare CRISPR-edited TSC1-null with CRISPR-corrected TSC1-WT NPCs generated from one TSC donor (one clone/genotype). To assess the relevance of identified gene expression alterations, we performed polysome profiling in postmortem brains from ASD donors and age-matched controls. We further compared effects on translation of a subset of transcripts and rescue of early ND phenotypes in NPCs following inhibition of mTORC1 using the allosteric inhibitor rapamycin versus a third-generation bi-steric, mTORC1-selective inhibitor RMC-6272. Results Polysome profiling of NPCs revealed numerous TSC1-associated alterations in mRNA translation that were largely recapitulated in human ASD brains. Moreover, although rapamycin treatment partially reversed the TSC1-associated alterations in mRNA translation, most genes related to neural activity/synaptic regulation or ASD were rapamycin-insensitive. In contrast, treatment with RMC-6272 inhibited rapamycin-insensitive translation and reversed TSC1-associated early ND phenotypes including proliferation and neurite outgrowth that were unaffected by rapamycin. Conclusions Our work reveals ample mRNA translation alterations in TSC1 patient-derived NPCs that recapitulate mRNA translation in ASD brain samples. Further, suppression of TSC1-associated but rapamycin-insensitive translation and ND phenotypes by RMC-6272 unveils potential implications for more efficient targeting of mTORC1 as a superior treatment strategy for TAND.
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- 2023
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3. Gene replacement therapy in a schwannoma mouse model of neurofibromatosis type 2
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Shilpa Prabhakar, Roberta L. Beauchamp, Pike See Cheah, Akiko Yoshinaga, Edwina Abou Haidar, Sevda Lule, Gayathri Mani, Katia Maalouf, Anat Stemmer-Rachamimov, David H. Jung, D. Bradley Welling, Marco Giovannini, Scott R. Plotkin, Casey A. Maguire, Vijaya Ramesh, and Xandra O. Breakefield
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neurofibromatosis type 2 ,gene therapy ,adeno-associated viral vector ,schwannoma ,Schwann cells ,Genetics ,QH426-470 ,Cytology ,QH573-671 - Abstract
Loss of function of the neurofibromatosis type 2 (NF2) tumor suppressor gene leads to the formation of schwannomas, meningiomas, and ependymomas, comprising ∼50% of all sporadic cases of primary nervous system tumors. NF2 syndrome is an autosomal dominant condition, with bi-allelic inactivation of germline and somatic alleles resulting in loss of function of the encoded protein merlin and activation of mammalian target of rapamycin (mTOR) pathway signaling in NF2-deficient cells. Here we describe a gene replacement approach through direct intratumoral injection of an adeno-associated virus vector expressing merlin in a novel human schwannoma model in nude mice. In culture, the introduction of an AAV1 vector encoding merlin into CRISPR-modified human NF2-null arachnoidal cells (ACs) or Schwann cells (SCs) was associated with decreased size and mTORC1 pathway activation consistent with restored merlin activity. In vivo, a single injection of AAV1-merlin directly into human NF2-null SC-derived tumors growing in the sciatic nerve of nude mice led to regression of tumors over a 10-week period, associated with a decrease in dividing cells and an increase in apoptosis, in comparison with vehicle. These studies establish that merlin re-expression via gene replacement in NF2-null schwannomas is sufficient to cause tumor regression, thereby potentially providing an effective treatment for NF2.
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- 2022
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4. High-content image-based analysis and proteomic profiling identifies Tau phosphorylation inhibitors in a human iPSC-derived glutamatergic neuronal model of tauopathy
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Chialin Cheng, Surya A. Reis, Emily T. Adams, Daniel M. Fass, Steven P. Angus, Timothy J. Stuhlmiller, Jared Richardson, Hailey Olafson, Eric T. Wang, Debasis Patnaik, Roberta L. Beauchamp, Danielle A. Feldman, M. Catarina Silva, Mriganka Sur, Gary L. Johnson, Vijaya Ramesh, Bruce L. Miller, Sally Temple, Kenneth S. Kosik, Bradford C. Dickerson, and Stephen J. Haggarty
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Medicine ,Science - Abstract
Abstract Mutations in MAPT (microtubule-associated protein tau) cause frontotemporal dementia (FTD). MAPT mutations are associated with abnormal tau phosphorylation levels and accumulation of misfolded tau protein that can propagate between neurons ultimately leading to cell death (tauopathy). Recently, a p.A152T tau variant was identified as a risk factor for FTD, Alzheimer's disease, and synucleinopathies. Here we used induced pluripotent stem cells (iPSC) from a patient carrying this p.A152T variant to create a robust, functional cellular assay system for probing pathophysiological tau accumulation and phosphorylation. Using stably transduced iPSC-derived neural progenitor cells engineered to enable inducible expression of the pro-neural transcription factor Neurogenin 2 (Ngn2), we generated disease-relevant, cortical-like glutamatergic neurons in a scalable, high-throughput screening compatible format. Utilizing automated confocal microscopy, and an advanced image-processing pipeline optimized for analysis of morphologically complex human neuronal cultures, we report quantitative, subcellular localization-specific effects of multiple kinase inhibitors on tau, including ones under clinical investigation not previously reported to affect tau phosphorylation. These results demonstrate the potential for using patient iPSC-derived ex vivo models of tauopathy as genetically accurate, disease-relevant systems to probe tau biochemistry and support the discovery of novel therapeutics for tauopathies.
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- 2021
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5. TSC patient-derived isogenic neural progenitor cells reveal altered early neurodevelopmental phenotypes and rapamycin-induced MNK-eIF4E signaling
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Pauline Martin, Vilas Wagh, Surya A. Reis, Serkan Erdin, Roberta L. Beauchamp, Ghalib Shaikh, Michael Talkowski, Elizabeth Thiele, Steven D. Sheridan, Stephen J. Haggarty, and Vijaya Ramesh
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Tuberous sclerosis complex ,TSC1 ,mTORC1 ,Induced pluripotent stem cells ,Neural progenitor cells ,Early neurodevelopment ,Neurology. Diseases of the nervous system ,RC346-429 - Abstract
Abstract Background Tuberous sclerosis complex (TSC) is a neurodevelopmental disorder with frequent occurrence of epilepsy, autism spectrum disorder (ASD), intellectual disability (ID), and tumors in multiple organs. The aberrant activation of mTORC1 in TSC has led to treatment with mTORC1 inhibitor rapamycin as a lifelong therapy for tumors, but TSC-associated neurocognitive manifestations remain unaffected by rapamycin. Methods Here, we generated patient-specific, induced pluripotent stem cells (iPSCs) from a TSC patient with a heterozygous, germline, nonsense mutation in exon 15 of TSC1 and established an isogenic set of heterozygous (Het), null and corrected wildtype (Corr-WT) iPSCs using CRISPR/Cas9-mediated gene editing. We differentiated these iPSCs into neural progenitor cells (NPCs) and examined neurodevelopmental phenotypes, signaling and changes in gene expression by RNA-seq. Results Differentiated NPCs revealed enlarged cell size in TSC1-Het and Null NPCs, consistent with mTORC1 activation. TSC1-Het and Null NPCs also revealed enhanced proliferation and altered neurite outgrowth in a genotype-dependent manner, which was not reversed by rapamycin. Transcriptome analyses of TSC1-NPCs revealed differentially expressed genes that display a genotype-dependent linear response, i.e., genes upregulated/downregulated in Het were further increased/decreased in Null. In particular, genes linked to ASD, epilepsy, and ID were significantly upregulated or downregulated warranting further investigation. In TSC1-Het and Null NPCs, we also observed basal activation of ERK1/2, which was further activated upon rapamycin treatment. Rapamycin also increased MNK1/2-eIF4E signaling in TSC1-deficient NPCs. Conclusion MEK-ERK and MNK-eIF4E pathways regulate protein translation, and our results suggest that aberrant translation distinct in TSC1/2-deficient NPCs could play a role in neurodevelopmental defects. Our data showing upregulation of these signaling pathways by rapamycin support a strategy to combine a MEK or a MNK inhibitor with rapamycin that may be superior for TSC-associated CNS defects. Importantly, our generation of isogenic sets of NPCs from TSC patients provides a valuable platform for translatome and large-scale drug screening studies. Overall, our studies further support the notion that early developmental events such as NPC proliferation and initial process formation, such as neurite number and length that occur prior to neuronal differentiation, represent primary events in neurogenesis critical to disease pathogenesis of neurodevelopmental disorders such as ASD.
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- 2020
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6. Comparison of Therapeutic Efficacy of Gene Therapy for Tuberous Sclerosis Type 2 with Standard of Care Everolimus in Preclinical Model (S14.002)
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Edwina Abou Haidar, Shilpa Prabhakar, Pike See Cheah, Sevda Lule, Roberta L. Beauchamp, Akiko Yoshinaga, Alexandra L. Geffrey, Anat Stemmer-Rachamimov, Vijaya Ramesh, Casey A. Maguire, and Xandra O. Breakefield
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- 2023
7. Proteasomal pathway inhibition as a potential therapy for NF2-associated meningioma and schwannoma
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Srirupa Bhattacharyya, Janet L Oblinger, Roberta L Beauchamp, Zhenzhen Yin, Serkan Erdin, Priya Koundinya, Anna D Ware, Marc Ferrer, Justin T Jordan, Scott R Plotkin, Lei Xu, Long-Sheng Chang, and Vijaya Ramesh
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Cancer Research ,Oncology ,Neurology (clinical) - Abstract
BackgroundNeurofibromatosis 2 (NF2) is an inherited disorder caused by bi-allelic inactivation of the NF2 tumor suppressor gene. NF2-associated tumors, including schwannoma and meningioma, are resistant to chemotherapy, often recurring despite surgery and/or radiation, and have generally shown cytostatic response to signal transduction pathway inhibitors, highlighting the need for improved cytotoxic therapies.MethodsLeveraging data from our previous high-throughput drug screening in NF2 preclinical models, we identified a class of compounds targeting the ubiquitin–proteasome pathway (UPP), and undertook studies using candidate UPP inhibitors, ixazomib/MLN9708, pevonedistat/MLN4924, and TAK-243/MLN7243. Employing human primary and immortalized meningioma (MN) cell lines, CRISPR-modified Schwann cells (SCs), and mouse Nf2 −/− SCs, we performed dose response testing, flow cytometry-based Annexin V and cell cycle analyses, and RNA-sequencing to identify potential underlying mechanisms of apoptosis. In vivo efficacy was also assessed in orthotopic NF2-deficient meningioma and schwannoma tumor models.ResultsTesting of three UPP inhibitors demonstrated potent reduction in cell viability and induction of apoptosis for ixazomib or TAK-243, but not pevonedistat. In vitro analyses revealed that ixazomib or TAK-243 downregulates expression of c-KIT and PDGFRα, as well as the E3 ubiquitin ligase SKP2 while upregulating genes associated with endoplasmic reticulum stress-mediated activation of the unfolded protein response (UPR). In vivo treatment of mouse models revealed delayed tumor growth, suggesting a therapeutic potential.ConclusionsThis study demonstrates the efficacy of proteasomal pathway inhibitors in meningioma and schwannoma preclinical models and lays the groundwork for use of these drugs as a promising novel treatment strategy for NF2 patients.
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- 2023
8. Prospective phase II trial of the dual mTORC1/2 inhibitor vistusertib for progressive or symptomatic meningiomas in persons with neurofibromatosis 2
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Justin T Jordan, Christina C Orr, Raquel D Thalheimer, Josephine V Cambillo, Roberta L Beauchamp, Ghalib Shaikh, Alona Muzikansky, Anat Stemmer-Rachamimov, Marco Giovannini, Michel Kalamarides, Fred G Barker, Vijaya Ramesh, and Scott R Plotkin
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Oncology ,Surgery ,Neurology (clinical) - Abstract
Background Meningiomas occur in 80% of persons with neurofibromatosis 2 (NF2) and cause significant mortality and morbidity, yet there are no effective medical treatments. NF2-deficient tumors have constitutive activation of mammalian/mechanistic target of rapamycin (mTOR), and treatment with mTORC1 inhibitors results in growth arrest in a minority of tumors, with paradoxical activation of the mTORC2/AKT pathway. We studied the effect of vistusertib, a dual mTORC1/mTORC2 inhibitor, in NF2 patients with progressive or symptomatic meningiomas. Methods Vistusertib was administered orally at 125 mg twice daily for 2 consecutive days each week. The primary endpoint was the imaging response in the target meningioma, defined as a volume decrease of 20% compared with the baseline. Secondary endpoints included toxicity, imaging response of nontarget tumors, quality of life, and genetic biomarkers. Results Eighteen participants (13 female), median age of 41 (range, 18–61) years, were enrolled. In target meningiomas, the best response was partial response (PR) in 1/18 tumors (6%) and stable disease (SD) in 17/18 tumors (94%). For all measured intracranial meningiomas and vestibular schwannomas, the best imaging response was PR in 6/59 tumors (10%) and SD in 53 (90%). Treatment-related grade 3/4 adverse events occurred in 14 (78%) participants, and 9 participants discontinued treatment due to side effects. Conclusions Although the study did not meet the primary endpoint, vistusertib treatment was associated with high rates of SD in progressive NF2-related tumors. However, this dosing regimen for vistusertib was poorly tolerated. Future studies of dual mTORC inhibitors for NF2 should focus on optimizing tolerability and evaluating the relevance of tumor stability in participants.
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- 2023
9. TSC patient-derived isogenic neural progenitor cells reveal altered early neurodevelopmental phenotypes and rapamycin-induced MNK-eIF4E signaling
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Vilas Wagh, Pauline Martin, Ghalib Shaikh, Roberta L. Beauchamp, Steven D. Sheridan, Michael E. Talkowski, Stephen J. Haggarty, Elizabeth A. Thiele, Surya A. Reis, Vijaya Ramesh, and Serkan Erdin
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congenital, hereditary, and neonatal diseases and abnormalities ,Neurite ,mTORC1 ,Biology ,Early neurodevelopment ,lcsh:RC346-429 ,Transcriptome ,03 medical and health sciences ,0302 clinical medicine ,Neurodevelopmental disorder ,Developmental Neuroscience ,medicine ,Neural progenitor cells ,Induced pluripotent stem cell ,Molecular Biology ,lcsh:Neurology. Diseases of the nervous system ,030304 developmental biology ,0303 health sciences ,Neurogenesis ,medicine.disease ,Neural stem cell ,Cell biology ,TSC1 ,Psychiatry and Mental health ,Induced pluripotent stem cells ,medicine.anatomical_structure ,Tuberous sclerosis complex ,030217 neurology & neurosurgery ,Developmental Biology - Abstract
Background Tuberous sclerosis complex (TSC) is a neurodevelopmental disorder with frequent occurrence of epilepsy, autism spectrum disorder (ASD), intellectual disability (ID), and tumors in multiple organs. The aberrant activation of mTORC1 in TSC has led to treatment with mTORC1 inhibitor rapamycin as a lifelong therapy for tumors, but TSC-associated neurocognitive manifestations remain unaffected by rapamycin. Methods Here, we generated patient-specific, induced pluripotent stem cells (iPSCs) from a TSC patient with a heterozygous, germline, nonsense mutation in exon 15 of TSC1 and established an isogenic set of heterozygous (Het), null and corrected wildtype (Corr-WT) iPSCs using CRISPR/Cas9-mediated gene editing. We differentiated these iPSCs into neural progenitor cells (NPCs) and examined neurodevelopmental phenotypes, signaling and changes in gene expression by RNA-seq. Results Differentiated NPCs revealed enlarged cell size in TSC1-Het and Null NPCs, consistent with mTORC1 activation. TSC1-Het and Null NPCs also revealed enhanced proliferation and altered neurite outgrowth in a genotype-dependent manner, which was not reversed by rapamycin. Transcriptome analyses of TSC1-NPCs revealed differentially expressed genes that display a genotype-dependent linear response, i.e., genes upregulated/downregulated in Het were further increased/decreased in Null. In particular, genes linked to ASD, epilepsy, and ID were significantly upregulated or downregulated warranting further investigation. In TSC1-Het and Null NPCs, we also observed basal activation of ERK1/2, which was further activated upon rapamycin treatment. Rapamycin also increased MNK1/2-eIF4E signaling in TSC1-deficient NPCs. Conclusion MEK-ERK and MNK-eIF4E pathways regulate protein translation, and our results suggest that aberrant translation distinct in TSC1/2-deficient NPCs could play a role in neurodevelopmental defects. Our data showing upregulation of these signaling pathways by rapamycin support a strategy to combine a MEK or a MNK inhibitor with rapamycin that may be superior for TSC-associated CNS defects. Importantly, our generation of isogenic sets of NPCs from TSC patients provides a valuable platform for translatome and large-scale drug screening studies. Overall, our studies further support the notion that early developmental events such as NPC proliferation and initial process formation, such as neurite number and length that occur prior to neuronal differentiation, represent primary events in neurogenesis critical to disease pathogenesis of neurodevelopmental disorders such as ASD.
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- 2020
10. High-content image-based analysis and proteomic profiling identifies Tau phosphorylation inhibitors in a human iPSC-derived glutamatergic neuronal model of tauopathy
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Daniel M. Fass, Roberta L. Beauchamp, Steven P. Angus, Chialin Cheng, Kenneth S. Kosik, Timothy J. Stuhlmiller, Bradford C. Dickerson, Bruce L. Miller, Debasis Patnaik, Danielle A. Feldman, Jared I Richardson, Stephen J. Haggarty, Hailey Olafson, Surya A. Reis, Mriganka Sur, Gary L. Johnson, Vijaya Ramesh, Eric T. Wang, Sally Temple, Emily T Adams, and M. Catarina Silva
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Proteomics ,Aging ,Pyridines ,Image Processing ,Stem cells ,Neurodegenerative ,Alzheimer's Disease ,Computer-Assisted ,Glutamates ,Models ,Stem Cell Research - Nonembryonic - Human ,Basic Helix-Loop-Helix Transcription Factors ,Image Processing, Computer-Assisted ,2.1 Biological and endogenous factors ,Neurogenin-2 ,Aetiology ,Phosphorylation ,Induced pluripotent stem cell ,Alzheimer's Disease Related Dementias (ADRD) ,Neurons ,Multidisciplinary ,Stem Cell Research - Induced Pluripotent Stem Cell - Human ,Molecular medicine ,Drug discovery ,Chemical biology ,Neural stem cell ,Cell biology ,Frontotemporal Dementia (FTD) ,Tauopathies ,5.1 Pharmaceuticals ,Neurological ,Medicine ,Tauopathy ,Development of treatments and therapeutic interventions ,Frontotemporal dementia ,Science ,Tau protein ,Induced Pluripotent Stem Cells ,Nerve Tissue Proteins ,tau Proteins ,Biology ,Models, Biological ,Article ,Cell Line ,Small Molecule Libraries ,Medical research ,mental disorders ,medicine ,Acquired Cognitive Impairment ,Humans ,Synucleinopathies ,Stem Cell Research - Induced Pluripotent Stem Cell ,Neurosciences ,Alzheimer's Disease including Alzheimer's Disease Related Dementias (AD/ADRD) ,medicine.disease ,Biological ,Stem Cell Research ,Brain Disorders ,Pyrimidines ,biology.protein ,Dementia ,Protein Kinases ,Biomarkers ,Neuroscience - Abstract
Mutations in MAPT (microtubule-associated protein tau) cause frontotemporal dementia (FTD). MAPT mutations are associated with abnormal tau phosphorylation levels and accumulation of misfolded tau protein that can propagate between neurons ultimately leading to cell death (tauopathy). Recently, a p.A152T tau variant was identified as a risk factor for FTD, Alzheimer's disease, and synucleinopathies. Here we used induced pluripotent stem cells (iPSC) from a patient carrying this p.A152T variant to create a robust, functional cellular assay system for probing pathophysiological tau accumulation and phosphorylation. Using stably transduced iPSC-derived neural progenitor cells engineered to enable inducible expression of the pro-neural transcription factor Neurogenin 2 (Ngn2), we generated disease-relevant, cortical-like glutamatergic neurons in a scalable, high-throughput screening compatible format. Utilizing automated confocal microscopy, and an advanced image-processing pipeline optimized for analysis of morphologically complex human neuronal cultures, we report quantitative, subcellular localization-specific effects of multiple kinase inhibitors on tau, including ones under clinical investigation not previously reported to affect tau phosphorylation. These results demonstrate the potential for using patient iPSC-derived ex vivo models of tauopathy as genetically accurate, disease-relevant systems to probe tau biochemistry and support the discovery of novel therapeutics for tauopathies.
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- 2021
11. Brigatinib causes tumor shrinkage in both NF2-deficient meningioma and schwannoma through inhibition of multiple tyrosine kinases but not ALK
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Salvatore La Rosa, Helen Morrison, Jie Huang, Rajarshi Guha, Li Jiang, Yongzheng He, Gary L. Johnson, Steven P. Angus, Eric T. Hawley, Vijaya Ramesh, Justin Guinney, Sarah S. Burns, Jin Yuan, Roberta L. Beauchamp, Lars Björn Riecken, Cristina Fernandez-Valle, Marc Ferrer, Charles W. Yates, Serkan Erdin, Abbi Smith, Waylan K. Bessler, D. Bradley Welling, D. Wade Clapp, Shelley Dixon, Scott R. Plotkin, Ming Poi, Thomas S. K. Gilbert, Anat Stemmer-Rachamimov, Alejandra M. Petrilli, Craig J. Thomas, Xiaohong Li, Qingbo Lu, David R. Jones, James F. Gusella, Long-Sheng Chang, Andrea R. Masters, Xiaohu Zhang, Janet L. Oblinger, Jaishri O. Blakeley, and Stephen J. Haggarty
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0301 basic medicine ,Macroglial Cells ,Cancer Treatment ,Schwannoma ,Biochemistry ,Tyrosine Kinases ,0302 clinical medicine ,Medical Conditions ,Animal Cells ,Medicine and Health Sciences ,Meningeal Neoplasms ,Kinome ,Neurofibromatosis type 2 ,Neurological Tumors ,Neurofibromin 2 ,Multidisciplinary ,Pharmaceutics ,Animal Models ,Protein-Tyrosine Kinases ,EPH receptor A2 ,Enzymes ,Oncology ,Neurology ,Experimental Organism Systems ,Genetic Diseases ,030220 oncology & carcinogenesis ,Medicine ,Cellular Types ,Meningioma ,Tyrosine kinase ,Neurilemmoma ,Research Article ,Brigatinib ,Tumor suppressor gene ,Science ,Mouse Models ,Glial Cells ,Research and Analysis Methods ,03 medical and health sciences ,Model Organisms ,Organophosphorus Compounds ,Drug Therapy ,medicine ,otorhinolaryngologic diseases ,Humans ,neoplasms ,Cell Proliferation ,Clinical Genetics ,business.industry ,Autosomal Dominant Diseases ,Cancers and Neoplasms ,Biology and Life Sciences ,Proteins ,Cell Biology ,medicine.disease ,nervous system diseases ,030104 developmental biology ,Pyrimidines ,Neurofibromatosis Type 2 ,Mutation ,Cancer research ,Animal Studies ,Enzymology ,Schwann Cells ,business ,Protein Kinases - Abstract
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.
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- 2021
12. Gene therapy for tuberous sclerosis complex type 2 in a mouse model by delivery of AAV9 encoding a condensed form of tuberin
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Xandra O. Breakefield, Shingo Kasamatsu, King Hwa Ling, Vijaya Ramesh, Pike See Cheah, Casey A. Maguire, Bakhos A. Tannous, Masao Kaneki, Roberta L. Beauchamp, Roderick T. Bronson, Xuan Zhang, David J. Kwiatkowski, Shilpa Prabhakar, Anat Stemmer-Rachamimov, Bence György, Elizabeth A. Thiele, and David Yellen
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congenital, hereditary, and neonatal diseases and abnormalities ,Tumor suppressor gene ,Genetic enhancement ,Diseases and Disorders ,Biology ,Virus ,03 medical and health sciences ,Tuberous sclerosis ,0302 clinical medicine ,medicine ,Recombinase ,Research Articles ,030304 developmental biology ,0303 health sciences ,Multidisciplinary ,Cell growth ,fungi ,food and beverages ,SciAdv r-articles ,Human Genetics ,medicine.disease ,nervous system diseases ,medicine.anatomical_structure ,Cancer research ,TSC1 ,TSC2 ,030217 neurology & neurosurgery ,Research Article - Abstract
Gene therapy for tuberous sclerosis type 2 proved beneficial in a mouse model of the disease, extending life span., Tuberous sclerosis complex (TSC) results from loss of a tumor suppressor gene - TSC1 or TSC2, encoding hamartin and tuberin, respectively. These proteins formed a complex to inhibit mTORC1-mediated cell growth and proliferation. Loss of either protein leads to overgrowth lesions in many vital organs. Gene therapy was evaluated in a mouse model of TSC2 using an adeno-associated virus (AAV) vector carrying the complementary for a “condensed” form of human tuberin (cTuberin). Functionality of cTuberin was verified in culture. A mouse model of TSC2 was generated by AAV-Cre recombinase disruption of Tsc2-floxed alleles at birth, leading to a shortened lifespan (mean 58 days) and brain pathology consistent with TSC. When these mice were injected intravenously on day 21 with AAV9-cTuberin, the mean survival was extended to 462 days with reduction in brain pathology. This demonstrates the potential of treating life-threatening TSC2 lesions with a single intravenous injection of AAV9-cTuberin.
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- 2021
13. mTOR kinase inhibition disrupts neuregulin 1-ERBB3 autocrine signaling and sensitizes NF2-deficient meningioma cellular models to IGF1R inhibition
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Roberta L. Beauchamp, Vijaya Ramesh, Justin T. Jordan, Serkan Erdin, Scott R. Plotkin, James F. Gusella, and Luke Witt
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0301 basic medicine ,Receptor, ErbB-3 ,insulin-like growth factor (IGF) receptor 1 ,DRC, dose–response curve ,mTORC1 ,SGK1, serum-/glucocorticoid-responsive kinase-1 ,Biochemistry ,meningioma ,Receptor, IGF Type 1 ,mTORC1/mTORC2, mTOR complex 1/mTOR complex 2 ,Cell Movement ,PI3K, Phosphoinositide 3-kinase ,Meningeal Neoplasms ,Akt PKB ,FOXO, Forkhead box protein O ,ERBB3 ,tumor suppressor gene ,CM, concentrated medium ,Benzoxazoles ,Neurofibromin 2 ,biology ,Chemistry ,Triazines ,Receptor, EphA2 ,TOR Serine-Threonine Kinases ,mammalian target of rapamycin (mTOR) ,APJ, apelin receptor ,Autocrine Communication ,MR, maximum response ,NF2, Neurofibromatosis 2 ,ERBB, V-ERB-B avian erythroblastic leukemia viral oncogene homolog ,Benzamides ,IRS, insulin receptor substrate ,PDK1, phosphoinositide-dependent kinase-1 ,RTK, receptor tyrosine kinase ,signaling ,brain tumor ,Research Article ,IC50, inhibitory concentration 50% ,Signal Transduction ,NRG1, neuregulin-1/heregulin ,Morpholines ,Neuregulin-1 ,mTOR, mechanistic/mammalian target of rapamycin ,Antibodies, Monoclonal, Humanized ,WHO, World Health Organization ,03 medical and health sciences ,Growth factor receptor ,TGFA, transforming growth factor-alpha ,Cell Line, Tumor ,otorhinolaryngologic diseases ,Humans ,Autocrine signalling ,Molecular Biology ,Mechanistic target of rapamycin ,PI3K/AKT/mTOR pathway ,dual mTORC1/mTORC2 inhibition ,Insulin-like growth factor 1 receptor ,Cell Proliferation ,Sirolimus ,Phosphoinositide 3-kinase ,030102 biochemistry & molecular biology ,qPCR, quantitative RT-PCR ,Dose-Response Relationship, Drug ,EPH receptor, erythropoietin-producing hepatocellular receptor ,Lapatinib ,Cell Biology ,EGFR, epidermal growth factor receptor ,AC, arachnoid cell ,030104 developmental biology ,Pyrimidines ,Gene Expression Regulation ,NF2 ,Cancer research ,biology.protein ,Pyrazoles ,NRG1-ERBB3 ,MN, meningioma ,Transcriptome ,IGF1R/IR, insulin-like growth factor receptor 1/insulin receptor ,Proto-Oncogene Proteins c-akt - Abstract
Meningiomas (MNs), arising from the arachnoid/meningeal layer, are nonresponsive to chemotherapies, with ∼50% showing loss of the Neurofibromatosis 2 (NF2) tumor suppressor gene. Previously, we established NF2 loss activates mechanistic target of rapamycin complex 1 (mTORC1) and mechanistic target of rapamycin complex 2 (mTORC2) signaling, leading to clinical trials for NF2 and MN. Recently our omics studies identified activated ephrin (EPH) receptor and Src family kinases upon NF2 loss. Here, we report increased expression of several ligands in NF2-null human arachnoidal cells (ACs) and the MN cell line Ben-Men-1, particularly neuregulin-1/heregulin (NRG1), and confirm increased NRG1 secretion and activation of V-ERB-B avian erythroblastic leukemia viral oncogene homolog 3 (ERBB3) receptor kinase. Conditioned-medium from NF2-null ACs or exogenous NRG1 stimulated ERBB3, EPHA2, and mTORC1/2 signaling, suggesting pathway crosstalk. NF2-null cells treated with an ERBB3-neutralizing antibody partially downregulated mTOR pathway activation but showed no effect on viability. mTORC1/2 inhibitor treatment decreased NRG1 expression and downregulated ERBB3 while re-activating pAkt T308, suggesting a mechanism independent of NRG1–ERBB3 but likely involving activation of another upstream receptor kinase. Transcriptomics after mTORC1/2 inhibition confirmed decreased ERBB3/ERBB4 while revealing increased expression of insulin-like growth factor receptor 1 (IGF1R). Drug treatment co-targeting mTORC1/2 and IGF1R/insulin receptor attenuated pAkt T308 and showed synergistic effects on viability. Our findings indicate potential autocrine signaling where NF2 loss leads to secretion/activation of NRG1-ERBB3 signaling. mTORC1/2 inhibition downregulates NRG1-ERBB3, while upregulating pAkt T308 through an adaptive response involving IGF1R/insulin receptor and co-targeting these pathways may prove effective for treatment of NF2-deficient MN.
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- 2020
14. EPH receptor signaling as a novel therapeutic target in NF2-deficient meningioma
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Patrick A. DeSouza, Timothy J. Stuhlmiller, Thomas S. K. Gilbert, Steven P. Angus, Xin Chen, James F. Gusella, Gary L. Johnson, Roberta L. Beauchamp, Vijaya Ramesh, Janet L. Oblinger, Anat Stemmer-Rachamimov, Scott R. Plotkin, Luke Witt, Stephen J. Haggarty, Justin T. Jordan, and Long-Sheng Chang
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0301 basic medicine ,Cancer Research ,Tumor suppressor gene ,Antineoplastic Agents ,Apoptosis ,Mice, SCID ,mTORC1 ,Receptor tyrosine kinase ,Mice ,03 medical and health sciences ,Mice, Inbred NOD ,Biomarkers, Tumor ,Meningeal Neoplasms ,Tumor Cells, Cultured ,otorhinolaryngologic diseases ,Animals ,Humans ,Medicine ,Kinome ,Cell Proliferation ,Receptors, Eph Family ,Neurofibromin 2 ,biology ,business.industry ,Erythropoietin-producing hepatocellular (Eph) receptor ,Xenograft Model Antitumor Assays ,Gene Expression Regulation, Neoplastic ,Dasatinib ,030104 developmental biology ,Oncology ,Tumor progression ,Basic and Translational Investigations ,biology.protein ,Cancer research ,Neurology (clinical) ,Meningioma ,business ,Proto-oncogene tyrosine-protein kinase Src ,medicine.drug - Abstract
Background Meningiomas are the most common primary brain tumor in adults, and somatic loss of the neurofibromatosis 2 (NF2) tumor suppressor gene is a frequent genetic event. There is no effective treatment for tumors that recur or continue to grow despite surgery and/or radiation. Therefore, targeted therapies that either delay tumor progression or cause tumor shrinkage are much needed. Our earlier work established mammalian target of rapamycin complex mTORC1/mTORC2 activation in NF2-deficient meningiomas. Methods High-throughput kinome analyses were performed in NF2-null human arachnoidal and meningioma cell lines to identify functional kinome changes upon NF2 loss. Immunoblotting confirmed the activation of kinases and demonstrated effectiveness of drugs to block the activation. Drugs, singly and in combination, were screened in cells for their growth inhibitory activity. Antitumor drug efficacy was tested in an orthotopic meningioma model. Results Erythropoietin-producing hepatocellular receptor tyrosine kinases (EPH RTKs), c-KIT, and Src family kinase (SFK) members, which are biological targets of dasatinib, were among the top candidates activated in NF2-null cells. Dasatinib significantly inhibited phospho-EPH receptor A2 (pEPHA2), pEPHB1, c-KIT, and Src/SFK in NF2-null cells, showing no cross-talk with mTORC1/2 signaling. Posttreatment kinome analyses showed minimal adaptive changes. While dasatinib treatment showed some activity, dual mTORC1/2 inhibitor and its combination with dasatinib elicited stronger growth inhibition in meningiomas. Conclusion Co-targeting mTORC1/2 and EPH RTK/SFK pathways could be a novel effective treatment strategy for NF2-deficient meningiomas.
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- 2018
15. CTNI-54. A SINGLE ARM PHASE II STUDY OF THE DUAL MTORC1/MTORC2 INHIBITOR VISTUSERTIB PROVIDED FOR SPORADIC PATIENTS WITH GRADE II-III MENINGIOMAS THAT RECUR OR PROGRESS AFTER SURGERY AND RADIATION
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Patrick Y. Wen, Fred G. Barker, Vijaya Ramesh, Anat Stemmer-Rachamimov, Alona Muzikansky, Miriam J. Smith, Justin T. Jordan, Roberta L. Beauchamp, Priya Kumthekar, Elizabeth R. Gerstner, and Scott R. Plotkin
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Cancer Research ,medicine.medical_specialty ,business.industry ,Surrogate endpoint ,Nausea ,Phases of clinical research ,medicine.disease ,Meningioma ,Oncology ,Troponin I ,Vomiting ,Medicine ,Neurology (clinical) ,Radiology ,Progression-free survival ,medicine.symptom ,business ,Adverse effect - Abstract
Grade II/III meningiomas have increased rates of recurrence with no approved medical therapies. The historical progression-free survival at 6 months (PFS-6) is 25% with rates >35% declared of interest for drug development. NF2 gene inactivation occurs in about half of meningiomas. Based on our studies showing mTORC1 and mTORC2/SGK1 pathway activation in NF2-deficient meningiomas and the paradoxical activation of the mTORC2/AKT pathway, we hypothesized that mTORC1/mTORC2 inhibitors would be active in meningiomas. We studied the effect of vistusertib in patients with progressive/recurrent grade II/III meningiomas (NCT03071874). Vistusertib was administered orally at 125mg twice daily on two consecutive days each week. MRIs were obtained every 56 days. Tumor size was defined as the largest cross-sectional area. Progression was defined as ≥ 25% increase in the sum of products of all measurable lesions over smallest sum observed. The primary endpoint was PFS-6. Secondary endpoints included toxicity, radiographic response, and correlative studies including immunohistochemistry for mTORC1/2 pathway activation and genetic biomarkers. Twenty-eight patients (13 female, median age 58 years, median KPS 80%) were enrolled. Median tumor size was 4.4cm; 71% were grade II and 50% harbored pathogenic NF2 variants. Four patients discontinued treatment voluntarily and 1 each withdrew for intercurrent illness and non-compliance. PFS-6 is 47% (CI, 26%-65%) and OS-12 is 72% (95%CI, 48%-86%). PFS but not OS was shorter for patients with grade 3 meningiomas; there was no difference in PFS/OS between genetic groups. Adverse events at least possibly related to vistusertib with frequency >10% include nausea, fatigue, hypophosphatemia, diarrhea, anorexia, dry mouth, hypertriglyceridemia, hypertension, vomiting, increased ALT, constipation, and weight loss. Vistusertib treatment was associated with a PFS-6 rate exceeding the target of 35% for recurrent high-grade meningioma. Adverse events were tolerable in this patient population. These data support the continued development of mTORC1/2 inhibitors in this setting.
- Published
- 2021
16. Combination therapy with mTOR kinase inhibitor and dasatinib as a novel therapeutic strategy for vestibular schwannoma
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Konstantina M. Stankovic, Yanling Zhang, Wenjianlong Zhou, Limeng Wu, Sasa Vasilijic, Patrick A. DeSouza, Lei Xu, Roberta L. Beauchamp, Jessica E. Sagers, Vijaya Ramesh, and Richard Seist
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Male ,Dasatinib ,lcsh:Medicine ,mTORC1 ,Tyrosine-kinase inhibitor ,Receptor tyrosine kinase ,Mice ,0302 clinical medicine ,lcsh:Science ,Tumour-suppressor proteins ,Mice, Knockout ,0303 health sciences ,Neurofibromin 2 ,Multidisciplinary ,biology ,Receptor, EphA1 ,TOR Serine-Threonine Kinases ,Neuroma, Acoustic ,3. Good health ,030220 oncology & carcinogenesis ,Benzamides ,Drug Therapy, Combination ,Female ,biological phenomena, cell phenomena, and immunity ,medicine.drug ,Combination therapy ,medicine.drug_class ,Morpholines ,Article ,03 medical and health sciences ,In vivo ,medicine ,otorhinolaryngologic diseases ,Animals ,Humans ,Protein Kinase Inhibitors ,PI3K/AKT/mTOR pathway ,030304 developmental biology ,Pharmacology ,business.industry ,lcsh:R ,Erythropoietin-producing hepatocellular (Eph) receptor ,Mice, Inbred C57BL ,Disease Models, Animal ,Pyrimidines ,Cancer research ,biology.protein ,lcsh:Q ,business - Abstract
Neurofibromatosis type 2 (NF2) is an inherited disorder characterized by bilateral vestibular schwannomas (VS) that arise from neoplastic Schwann cells (SCs). NF2-associated VSs are often accompanied by meningioma (MN), and the majority of NF2 patients show loss of the NF2 tumor suppressor. mTORC1 and mTORC2-specific serum/glucocorticoid-regulated kinase 1 (SGK1) are constitutively activated in MN with loss of NF2. In a recent high-throughput kinome screen in NF2-null human arachnoidal and meningioma cells, we showed activation of EPH RTKs, c-KIT, and SFK members independent of mTORC1/2 activation. Subsequently, we demonstrated in vitro and in vivo efficacy of combination therapy with the dual mTORC1/2 inhibitor AZD2014 and the multi-kinase inhibitor dasatinib. For these reasons, we investigated activated mTORC1/2 and EPH receptor-mediated signaling in sporadic and NF2-associated VS. Using primary human VS cells and a mouse allograft model of schwannoma, we evaluated the dual mTORC1/2 inhibitor AZD2014 and the tyrosine kinase inhibitor dasatinib as monotherapies and in combination. Escalating dose-response experiments on primary VS cells grown from 15 human tumors show that combination therapy with AZD2014 and dasatinib is more effective at reducing metabolic activity than either drug alone and exhibits a therapeutic effect at a physiologically reasonable concentration (~0.1 µM). In vivo, while AZD2014 and dasatinib each inhibit tumor growth alone, the effect of combination therapy exceeds that of either drug. Co-targeting the mTOR and EPH receptor pathways with these or similar compounds may constitute a novel therapeutic strategy for VS, a condition for which there is no FDA-approved pharmacotherapy.
- Published
- 2019
17. Multi-center, single arm phase II study of the dual mTORC1/mTORC2 inhibitor vistusertib for patients with recurrent or progressive grade II-III meningiomas
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Justin T. Jordan, Vijaya Ramesh, Scott R. Plotkin, Fred G. Barker, Alona Muzikansky, Anat Stemmer-Rachamimov, Patrick Y. Wen, Priya Kumthekar, and Roberta L. Beauchamp
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Cancer Research ,medicine.medical_specialty ,Oncology ,business.industry ,VISTUSERTIB ,Medicine ,Phases of clinical research ,Center (algebra and category theory) ,Radiology ,business - Abstract
2024 Background: Grade II/III meningiomas represent about 20% of tumors and have increased rates of recurrence with no approved medical therapies. Historically, the progression-free survival at 6 months (PFS-6) for these tumors is 25%. The Response Assessment in Neuro-Oncology (RANO) group identified a PFS-6 rate of > 35% to be of interest for trials of grade II/III meningioma. Methods : NF2 gene inactivation occurs in the majority of meningiomas and is associated with mTORC1 activation. Human studies of everolimus for neurofibromatosis 2 patients documented growth arrest in only a minority of tumors. Based on our studies showing mTORC2/SGK1 pathway activation in NF2-deficient meningiomas and the known paradoxical activation of the mTORC2/AKT pathway in meningiomas, we hypothesized that dual inhibition of mTORC1/2 would be superior in meningiomas. Treatment of primary meningioma cells with vistusertib led to decreased cell proliferation and showed greater efficacy than rapamycin, regardless of NF2 expression. We studied the effect of vistusertib in patients with progressive or recurrent grade II/III meningiomas (NCT03071874). Vistusertib was administered orally at 125mg twice daily on two consecutive days each week. MRIs were obtained every 2 cycles (1 cycle = 28 days). Tumor size was defined as the largest cross-sectional area. Progression was defined as ≥25% increase in the sum of products of all measurable lesions over smallest sum observed. The primary endpoint was PFS-6. Secondary endpoints included toxicity, radiographic response, and correlative studies including immunohistochemistry for mTORC1/2 pathway activation and genetic biomarkers. Results: Twenty-eight patients (13 female), with a median age of 58 years (range, 32 to 77 years), were enrolled in this multicenter study. The median Karnofsky performance status was 80. Twenty-five patients have been followed to six months or to tumor progression. The median duration of treatment was 6.5 month (range, 1-18 months). Four patients chose to discontinue treatment, 1 withdrew to intercurrent illness, and 1 was withdrawn due to non-compliance. PFS-6 is 51.5% (CI, 29.3% - 70.0%). Adverse events at least possibly related to vistusertib with frequency > 10% include nausea (54%); fatigue (36%); hypophosphatemia (29%); diarrhea, anorexia, dry mouth, and hypertriglyceridemia (all 14%); hypertension, vomiting, increased ALT, constipation, and weight loss (all 11%). Conclusions: Vistusertib treatment was associated with a PFS-6 rate that exceeds the RANO target of 35% for recurrent high-grade meningioma. The follow-up data continue to mature. Adverse events were tolerable in this patient population. Correlative studies to identify biological factors that correlate with response are under way. These data support the initiation of larger randomized studies of vistusertib in this setting. Clinical trial information: NCT03071874.
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- 2021
18. CSIG-42. HIGH THROUGHPUT KINOME AND TRANSCRIPTOME ANALYSES REVEAL NOVEL THERAPEUTIC TARGETS IN NF2-DEFICIENT MENINGIOMA
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Stephen J. Haggarty, James F. Gusella, Gary L. Johnson, Janet L. Oblinger, Steven P. Angus, Vijaya Ramesh, Roberta L. Beauchamp, Justin T. Jordan, Long-Sheng Chang, Timothy J. Stuhlmiller, Serkan Erdin, and Scott R. Plotkin
- Subjects
Transcriptome ,Cancer Research ,Abstracts ,Text mining ,Oncology ,business.industry ,otorhinolaryngologic diseases ,Kinome ,Neurology (clinical) ,Computational biology ,Biology ,business ,Throughput (business) - Abstract
Meningiomas (MN), the most common adult primary intracranial tumor, arise from the arachnoid/meninges and are non-responsive to chemotherapies with a high recurrence rate despite surgery, necessitating effective non-invasive therapies. Our previous work showed that NF2 loss activates mechanistic target of rapamycin complex 1 (mTORC1) and mTORC2 signaling, which led to past NF2 clinical trials using rapalogs (RAD001/everolimus), and current meningioma clinical trials with dual mTORC1/mTORC2 inhibitor (mTORi) AZD2014. To understand additional dysregulated, potentially druggable pathways, we undertook an ‘omics approach of large-scale kinomics and RNA-sequencing employing CRISPR-modified human arachnoidal cells (ACs), NF2-expressing vs NF2-null. In NF2-null ACs, several kinases were elevated including erythropoietin-producing hepatocellular (EPH)-receptor tyrosine kinase (RTK) family members, Src family kinase (SFK) members, and c-KIT, all targets of dasatinib. In vitro treatment of MN cells using mTORi (AZD2014 or INK128) and dasatinib enhanced growth inhibition upon combination mTORi+dasatinib. In vivo treatment of an orthotopic mouse MN model showed moderate response to dasatinib with stronger response using INK128 or INK128+dasatinib (e-published in Neuro-Oncology). Our transcriptomic data also revealed increased expression of several ligands/growth factors, particularly NRG1/neuregulin. Expanding these results, we have confirmed increased expression of NRG1 in human NF2-null ACs. We also find NF2-null ACs secrete NRG1, and in conditioned-media experiments we observe stimulation of ErbB3, EPHA2 and mTOR pathways, suggesting an autocrine signaling mechanism. NF2-null AC or MN cells, when stimulated with exogenous NRG1, show enhanced activation of mTOR and EPH pathways besides ErbB3 signaling. Further, lapatinib (multi-ErbB inhibitor) but not erlotinib (EGFR inhibitor) attenuates the NRG1-stimulated activation of ErbB3, EPHA2 and mTOR, suggesting that NRG1-induced activation is EGFR-independent. Taken together, our results support a mechanistic link where NF2 loss increases NRG1/ErbB signaling to EPH/SFK and mTOR pathways, which may be a critical driver of tumorigenesis, thus providing a therapeutic opportunity to co-target these pathways in NF2-deficient meningiomas.
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- 2018
19. A high-throughput kinome screen reveals serum/glucocorticoid-regulated kinase 1 as a therapeutic target for NF2-deficient meningiomas
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Vilas Wagh, Patrick A. DeSouza, Anat Stemmer-Rachamimov, Justin T. Jordan, Scott R. Plotkin, Wen-Ning Zhao, Stephen J. Haggarty, James F. Gusella, Roberta L. Beauchamp, Marianne James, and Vijaya Ramesh
- Subjects
Neurofibromatosis 2 ,Morpholines ,Immunoblotting ,Antineoplastic Agents ,mTORC1 ,Biology ,Protein Serine-Threonine Kinases ,mTORC2 ,meningioma ,Polymerase Chain Reaction ,Immediate early protein ,Immediate-Early Proteins ,Meningioma ,PAK1 ,AZD2014 ,Cell Line, Tumor ,medicine ,otorhinolaryngologic diseases ,Meningeal Neoplasms ,Humans ,Kinome ,SGK1 ,neoplasms ,Kinase ,High-Throughput Nucleotide Sequencing ,medicine.disease ,Molecular biology ,nervous system diseases ,Pyrimidines ,Oncology ,NF2 ,Gene Knockdown Techniques ,Benzamides ,Cancer research ,mTOR signaling ,Signal transduction ,Priority Research Paper ,Signal Transduction - Abstract
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.
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- 2015
20. Traditional and systems biology based drug discovery for the rare tumor syndrome neurofibromatosis type 2
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Jaishri O. Blakeley, Anat Stemmer-Rachamimov, Wen-Ning Zhao, Noah Sciaky, Sarah S. Burns, Cristina Fernandez-Valle, Marga Bott, Long-Sheng Chang, Gary L. Johnson, Vijaya Ramesh, Justin Guinney, Michael E. Talkowski, Steve Angus, Serkan Erdin, Annemarie Carlstedt, Alejandra M. Petrilli, Roberta L. Beauchamp, Charles W. Yates, Xin Chen, Stephen J. Haggarty, Patrick A. DeSouza, Tim J. Stuhlmiller, Abhishek Pratap, D. Bradley Welling, Jon S. Zawistowski, D. Wade Clapp, Scott R. Plotkin, Salvatore La Rosa, James F. Gusella, Helen Morrison, and Robert J. Allaway
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0301 basic medicine ,Neurofibromatosis 2 ,Carcinogenesis ,Cell Survival ,Morpholines ,lcsh:Medicine ,Schwannoma ,Biology ,medicine.disease_cause ,Meningioma ,Mice ,03 medical and health sciences ,chemistry.chemical_compound ,Cell Line, Tumor ,Panobinostat ,Meningeal Neoplasms ,otorhinolaryngologic diseases ,medicine ,Animals ,Humans ,Kinome ,Neurofibromatosis ,Neurofibromatosis type 2 ,lcsh:Science ,neoplasms ,Neurofibromin 2 ,Sulfonamides ,Multidisciplinary ,Systems Biology ,lcsh:R ,medicine.disease ,nervous system diseases ,Gene Expression Regulation, Neoplastic ,Pyridazines ,Merlin (protein) ,Pyrimidines ,030104 developmental biology ,chemistry ,Quinolines ,Cancer research ,lcsh:Q ,Transcriptome ,Neurilemmoma - Abstract
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.
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- 2018
21. Kinome Screen Reveals SGK1 as a Therapeutic Target for NF2: Inhibition of mTORC1/2 is More Effective than Rapamycin
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Anat Stemmer-Rachamimov, Roberta L. Beauchamp, Patrick A. DeSouza, Vijaya Ramesh, Scott R. Plotkin, Vilas Wagh, James F. Gusella, Marianne James, Stephen J. Haggarty, and Wen-Ning Zhao
- Subjects
Chemistry ,Kinase ,mTORC1 ,medicine.disease ,Biochemistry ,mTORC2 ,law.invention ,PAK1 ,law ,otorhinolaryngologic diseases ,Genetics ,SGK1 ,medicine ,Cancer research ,Suppressor ,Kinome ,biological phenomena, cell phenomena, and immunity ,Neurofibromatosis type 2 ,Molecular Biology ,Biotechnology - Abstract
Neurofibromatosis type 2 (NF2) is characterized by vestibular schwannomas and meningiomas (MN) due to bi-allelic NF2 inactivation with loss of NF2 tumor suppressor protein. Besides NF2-associated MNs, ~60% of sporadic MNs show loss of NF2, and these tumors are non-responsive to chemotherapeutic intervention. We previously showed activation of mammalian/mechanistic target of rapamycin complex 1 (mTORC1) signaling upon NF2 loss, leading to clinical trials with rapalogs. Here we carried out a high throughput kinome screen to identify kinases responsible for mTORC1 activation in NF2-null MN cells. Among the top candidates were the mTORC2-specific target serum/glucocorticoid-regulated kinase 1 (SGK1) and p21-activated kinase 1 (PAK1). In NF2-null MNs, SGK1 inhibition rescues mTORC1 activation, and SGK1 activation is sensitive to dual mTORC1/2 inhibitor AZD2014, but not to rapamycin. PAK1 inhibition also rescues mTORC1 activation, but in a manner independent of mTORC2/SGK1 signaling. Using clustered regularly i...
- Published
- 2015
22. Mediator Subunit Med28 Is Essential for Mouse Peri-Implantation Development and Pluripotency
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Yuh-Shin Chang, Vilas Wagh, Roberta L. Beauchamp, Ryan M. Walsh, Marianne James, James F. Gusella, Konrad Hochedlinger, Lin Li, and Vijaya Ramesh
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Homeobox protein NANOG ,Cellular differentiation ,Rex1 ,Induced Pluripotent Stem Cells ,lcsh:Medicine ,Biology ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Mediator ,Animals ,Embryo Implantation ,Induced pluripotent stem cell ,lcsh:Science ,030304 developmental biology ,Mice, Knockout ,0303 health sciences ,Multidisciplinary ,Mediator Complex ,lcsh:R ,Gene Expression Regulation, Developmental ,Cell Differentiation ,MED28 ,Cellular Reprogramming ,Embryonic stem cell ,Molecular biology ,Cell biology ,Mice, Inbred C57BL ,030220 oncology & carcinogenesis ,Embryo Loss ,lcsh:Q ,Reprogramming ,Gene Deletion ,Research Article - Abstract
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 in vitro studies have been performed on MED28, its in vivo 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 Oct4 and Nanog. Further, overexpression of Med28 in mouse embryonic fibroblasts enhances the efficiency of their reprogramming to pluripotency. Cre-mediated inactivation of Med28 in induced pluripotent stem cells shows that Med28 is required for their survival. Intriguingly, heterozygous loss of Med28 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.
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- 2015
23. A NHERF binding site links the βPDGFR to the cytoskeleton and regulates cell spreading and migration
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Roberta L. Beauchamp, Nitasha Manchanda, Marianne James, Andrius Kazlauskas, and Vijaya Ramesh
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Sodium-Hydrogen Exchangers ,Moesin ,macromolecular substances ,Biology ,Cell Line ,Receptor, Platelet-Derived Growth Factor beta ,Cell membrane ,Focal adhesion ,Mice ,Ezrin ,Cell Movement ,Radixin ,medicine ,Animals ,Humans ,Phosphorylation ,Cytoskeleton ,Cell Shape ,Platelet-Derived Growth Factor ,Focal Adhesions ,Neurofibromin 2 ,Binding Sites ,Cell Membrane ,Cortical actin cytoskeleton ,Cell Biology ,Phosphoproteins ,Actin cytoskeleton ,Actins ,Cell biology ,medicine.anatomical_structure ,Mutation ,Tyrosine - Abstract
The Na(+)/H(+) exchanger regulatory factor, NHERF, is a multifunctional adapter protein involved in a wide range of physiological activities. NHERF associates with merlin and the ezrin/radixin/moesin (MERM) family of membrane-actin cytoskeletal linker proteins through its C-terminus and is capable of interacting via its PDZ1 domain to the betaPDGF receptor (betaPDGFR). Thus, NHERF, potentially links the betaPDGFR to the actin cytoskeleton through its interaction with MERM proteins. In the present study, we have examined whether abolishing the interaction of betaPDGFR with NHERF results in actin cytoskeletal rearrangements. We have stably expressed a wild-type betaPDGFR, a mutant betaPDGFR (L1106A) that is incapable of interacting with NHERF, as well as a kinase defective mutant receptor (K634R), in PDGFR-deficient mouse embryonic fibroblasts. Our observations indicate that cells expressing betaPDGFR (L1106A) were impaired in their ability to spread and migrate on fibronectin compared with wild-type and K634R cells. L1106A mutant cells also revealed an increased number of focal adhesions, a condensed F-actin ring at the cell periphery and a decrease in total focal adhesion kinase (FAK) tyrosine phosphorylation. Further, we show that NHERF and MERM proteins could act as intermediary bridging proteins between betaPDGFR and FAK. Thus, the interaction of betaPDGFR with NHERF may provide an essential link between the cell membrane and the cortical actin cytoskeleton independent of receptor activity.
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- 2004
24. TorsinB - perinuclear location and association with torsinA
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Christoph Kamm, Roberta L. Beauchamp, Laurie J. Ozelius, Phyllis I. Hanson, Jeffrey W. Hewett, Xandra O. Breakefield, Heather Boston, Vijaya Ramesh, and Teri Naismith
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Glycosylation ,Nuclear Envelope ,Cytoplasmic inclusion ,Immunoprecipitation ,Blotting, Western ,Mutant ,Endoplasmic Reticulum ,Kidney ,Biochemistry ,Mice ,Neuroblastoma ,Cellular and Molecular Neuroscience ,Antibody Specificity ,Gene expression ,Animals ,Humans ,Nuclear protein ,Gene ,Brain Chemistry ,biology ,Endoplasmic reticulum ,Brain ,Immunohistochemistry ,Precipitin Tests ,Cell Compartmentation ,Cell biology ,Chaperone (protein) ,biology.protein ,Carrier Proteins ,Neuroscience ,Molecular Chaperones - Abstract
The torsins comprise a four-member family of AAA+ chaperone proteins, including torsinA, torsinB, torp2A and torp3A in humans. Mutations in torsinA underlie early onset torsion dystonia, an autosomal dominant, neurologically based movement disorder. TorsinB is highly homologous to torsinA with its gene adjacent to that for torsinA on human chromosome 9q34. Antibodies have been generated which can distinguish torsinA and torsinB from each other, and from the torps in human and rodent cells. TorsinB (approximately MW 38 kDa), like torsinA ( approximately MW 37 kDa), is an N-glycosylated protein and both reside primarily in the endoplasmic reticulum (ER) and nuclear envelope in cultured cells. Immunoprecipitation studies in cultured cells and human brain tissue indicate that torsinA and torsinB are associated with each other in cells. Overexpression of both wild-type torsinB and mutant torsinA lead to enrichment of the protein in the nuclear envelope and formation of large cytoplasmic inclusions. We conclude that torsinB and torsinA are localized in overlapping cell compartments within the same protein complex, and thus may carry out related functions in vivo.
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- 2004
25. Developmental expression of the tuberous sclerosis proteins tuberin and hamartin
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Vanishree Murthy, Andrea N. Cutone, Roberta L. Beauchamp, Anat Stemmer-Rachamimov, Vijaya Ramesh, David N. Louis, Nicole A. Smith, Luciana A. Haddad, and Jennifer E. Roy
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Pathology ,medicine.medical_specialty ,Biology ,Epithelium ,Tuberous Sclerosis Complex 1 Protein ,Pathology and Forensic Medicine ,Cellular and Molecular Neuroscience ,Tuberous sclerosis ,Fetus ,Western blot ,Pregnancy ,Tuberous Sclerosis ,Tuberous Sclerosis Complex 2 Protein ,medicine ,Animals ,Humans ,Rats, Wistar ,medicine.diagnostic_test ,Tumor Suppressor Proteins ,Age Factors ,Gene Expression Regulation, Developmental ,Proteins ,Embryo, Mammalian ,medicine.disease ,Immunohistochemistry ,Rats ,Repressor Proteins ,Blot ,Viscera ,medicine.anatomical_structure ,Female ,Choroid plexus ,Neurology (clinical) ,TSC1 ,TSC2 ,Immunostaining - Abstract
Tuberous sclerosis complex is an autosomal dominant multisystem disorder, characterized by the development of hamartomas in multiple organs, primarily the skin, heart, kidney, and brain. The tuberous sclerosis genes, TSC1 and TSC2, encode hamartin and tuberin, respectively. Employing specific antibodies for hamartin and tuberin, we analyzed the expression of these two proteins by Western blot analyses in normal developing human and rat tissues. Both proteins are expressed ubiquitously in human fetal tissues and placenta, but are expressed at relatively low levels in human adult tissues, except brain. Similarly, high expression of these two proteins is observed in rat embryonic tissues, with a progressive decline after birth. To better characterize the developmental expression of tuberin and hamartin, we conducted a detailed study in rat tissues from embryonic day 13 to adult by Western blot analysis and immunohistochemistry. Immunohistochemical staining of rat tissues for tuberin and hamartin revealed tissue-specific expression patterns throughout development. Both tuberin and hamartin are expressed in epithelia, muscle (smooth, cardiac and skeletal muscle) and the nervous system (neurons, glia, choroid plexus and arachnoid). Except for the central nervous system, immunostaining intensity declines with age, confirming the protein blot analysis. These results indicate that tuberin and hamartin may play a critical role in development, and thus provide a framework for understanding the developmental and hamartomatous manifestations of tuberous sclerosis. These findings also suggest that tuberin and hamartin have additional functions in the adult brain, consistent with the marked neurological problems that afflict many patients with tuberous sclerosis.
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- 2001
26. Analysis of bothTSC1 andTSC2 for germline mutations in 126 unrelated patients with tuberous sclerosis
- Author
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Roberta L. Beauchamp, Erica Hammer, Ashleigh Banwell, Laurie J. Ozelius, Yo Niida, Vijaya Ramesh, Janine Lewis, Katherine B. Sims, and Nicole Lawrence-Smith
- Subjects
Genetics ,congenital, hereditary, and neonatal diseases and abnormalities ,education.field_of_study ,Population ,Single-strand conformation polymorphism ,Biology ,Bioinformatics ,medicine.disease ,nervous system diseases ,Tuberous sclerosis protein ,Tuberous sclerosis ,Germline mutation ,medicine.anatomical_structure ,medicine ,Missense mutation ,TSC1 ,TSC2 ,education ,Genetics (clinical) - Abstract
Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by the development of multiple hamartomas involving many organs. About two-thirds of the cases are sporadic and appear to represent new mutations. With the cloning of two causative genes, TSC1 and TSC2 it is now possible to analyze both genes in TSC patients and identify germline mutations. Here we report the mutational analysis of the entire coding region of both TSC1 and TSC2 genes in 126 unrelated TSC patients, including 40 familial and 86 sporadic cases, by single-stranded conformational polymorphism (SSCP) analysis followed by direct sequencing. Mutations were identified in a total of 74 (59%) cases, including 16 TSC1 mutations (5 sporadic and 11 familial cases) and 58 TSC2 mutations (42 sporadic and 16 familial cases). Overall, significantly more TSC2 mutations were found in our population, with a relatively equal distribution of mutations between TSC1 and TSC2 among the familial cases, but a marked underrepresentation of TSC1 mutations among the sporadic cases (P = 0.0035, Fisher's exact test). All TSC1 mutations were predicted to be protein truncating. However, in TSC2 13 missense mutations were found, five clustering in the GAP-related domain and three others occurring in exon 16. Upon comparison of clinical manifestations, including the incidence of intellectual disability, we could not find any observable differences between TSC1 and TSC2 patients. Our data help define the distribution and spectrum of mutations associated with the TSC loci and will be useful for both understanding the function of these genes as well as genetic counseling in patients with the disease. Hum Mutat 14:412–422, 1999. © 1999 Wiley-Liss, Inc.
- Published
- 1999
27. Exon scanning of the entire TSC2 gene for germline mutations in 40 unrelated patients with tuberous sclerosis
- Author
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Hope Northrup, Matthew Jacobsen, Roberta L. Beauchamp, Katherine B. Sims, Vijaya Ramesh, Priscilla Short, Erica Higgins, Patrick J. McNamara, Laurie J. Ozelius, and Ashleigh Banwell
- Subjects
Adult ,Male ,congenital, hereditary, and neonatal diseases and abnormalities ,Adolescent ,DNA Mutational Analysis ,Molecular Sequence Data ,Biology ,Tuberous sclerosis ,Exon ,Germline mutation ,Tuberous Sclerosis ,Tuberous Sclerosis Complex 2 Protein ,medicine ,Genetics ,Humans ,Missense mutation ,Amino Acid Sequence ,Child ,Germ-Line Mutation ,Genetics (clinical) ,Polymorphism, Genetic ,Base Sequence ,Genetic heterogeneity ,Tumor Suppressor Proteins ,Infant ,Exons ,Middle Aged ,medicine.disease ,Pedigree ,Repressor Proteins ,Tuberous sclerosis protein ,medicine.anatomical_structure ,Child, Preschool ,Female ,TSC1 ,TSC2 - Abstract
Tuberous sclerosis complex (TSC) is a dominantly inherited multisystem disorder resulting in the development of hamartomatous growths in many organs. Genetic heterogeneity has been demonstrated linking the familial cases to either TSC1 at 9q34.3, or TSC2 at 16p13.3. About two-thirds of the TSC cases are sporadic and appear to represent new mutations. While both genes are thought to account for all familial cases, with each representing approximately 50% of the mutations, the proportion of sporadic cases with mutations in TSC1 and TSC2 is yet to be determined. We have examined the entire coding sequence of the TSC2 gene in 20 familial and 20 sporadic cases and identified a total of twenty-one mutations representing 50% and 55% of familial and sporadic cases respectively. Our rate of mutation detection is significantly higher than other published reports. Twenty out of 21 mutations are novel and include 6 missense, 6 nonsense, 5 frameshifts, 2 splice alterations, a 34 bp deletion resulting in abnormal splicing, and an 18 bp deletion which maintains the reading frame. The mutations are distributed throughout the coding sequence with no specific hot spots. There is no apparent correlation between mutation type and clinical severity of the disease. Our results document that at least 50% of sporadic cases arise from mutations in the TSC2 gene. The location of the mutations described here, particularly the missense events, should be valuable for further functional analysis of this tumor suppressor protein.
- Published
- 1998
28. Analysis of Molecular Domains of Epitope-Tagged Merlin Isoforms in Cos-7 Cells and Primary Rat Schwann Cells
- Author
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Vijaya Ramesh, Christopher Sterner, Charo Gonzalez-Agosti, Denise Pinney, Lin Xu, and Roberta L. Beauchamp
- Subjects
Moesin ,Biology ,Transfection ,Gene product ,Ezrin ,Radixin ,Genes, Neurofibromatosis 2 ,Animals ,Rats, Wistar ,Cytoskeleton ,Cells, Cultured ,Sequence Tagged Sites ,Neurofibromin 2 ,Membrane Proteins ,Cell Biology ,Recombinant Proteins ,Rats ,Cell biology ,Merlin (protein) ,Animals, Newborn ,COS Cells ,Schwann Cells ,Peptides ,Oligopeptides ,Filopodia ,Cell Division ,Subcellular Fractions - Abstract
The Neurofibromatosis 2 gene product, merlin, has striking similarity to ezrin, radixin, and moesin (ERM), members of the protein 4.1 family which have been demonstrated to connect proteins in the plasma membrane to the cytoskeletal components. The recent localization of merlin to the motile regions in cultured cells such as membrane ruffles further supports the notion that merlin represents a new class of tumor suppressors. Here we describe the localization of full-length and truncated polypeptides of merlin expressed as Flag-tagged proteins in transfected cells. Similar to endogenous merlin, the epitope-tagged full-length merlin localizes to the membrane ruffles in transfected Cos-7 cells and rat Schwann cells. In addition, the overexpressed merlin localizes to other actin-rich cortical structures, such as microvilli and filopodia. The amino-terminal half of merlin is seen dispersed throughout the cells and in membrane ruffles. Compared to the amino-terminal half of merlin, its carboxy-terminal half localizes more distinctly to membrane ruffles. The full-length and the carboxy-terminal portion of merlin co-localize with F-actin at the membrane ruffles. However, distinct from the ERM proteins, the carboxy-terminal-truncated merlin and F-actin do not co-localize with each other at the stress fibers. Our results suggest that both the amino- and the carboxy-terminal domains of merlin contribute to its membrane ruffle localization.
- Published
- 1998
29. Lack of association of rare functional variants in TSC1/TSC2 genes with autism spectrum disorder
- Author
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Roberta L. Beauchamp, Mark J. Daly, James F. Gusella, Samira Bahl, Vijaya Ramesh, Colby Chiang, Michael E. Talkowski, and Benjamin M. Neale
- Subjects
Genetics ,Proband ,congenital, hereditary, and neonatal diseases and abnormalities ,Mammalian target of rapamycin ,biology ,Research ,Rare variants ,Ubiquitin ligase ,Psychiatry and Mental health ,Exon ,medicine.anatomical_structure ,Developmental Neuroscience ,Tuberous sclerosis complex ,Ubiquitin ligase complex ,Next-generation sequencing ,biology.protein ,medicine ,TSC1 ,Autism spectrum disorder ,TSC2 ,Molecular Biology ,Gene ,Developmental Biology ,RHEB - Abstract
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.
- Published
- 2013
30. p53 gene analysis of ovarian borderline tumors and stage I carcinomas*1
- Author
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Dino Dimeo, Roberta L. Beauchamp, Debra A. Bell, David W. Yandell, Jolanta Kupryjanczyk, and Ann D. Thor
- Subjects
Pathology ,medicine.medical_specialty ,Tumor suppressor gene ,Epithelioma ,Single-strand conformation polymorphism ,Biology ,medicine.disease ,Pathology and Forensic Medicine ,Malignant transformation ,Exon ,Carcinoma ,medicine ,Cancer research ,Ovarian cancer ,Gene - Abstract
Mutations of the p53 gene are common in human ovarian carcinomas; however, their role in the early development of ovarian cancer is unclear. Twelve ovarian borderline tumors (BTs; eight of them p53 immunopositive) and 10 stage I carcinomas (four of them p53 immunopositive) were studied for genetic alterations in the p53 gene. The study was based on single-strand conformation polymorphism (SSCP) analysis and DNA sequencing of exons 2 through 11 of the p53 gene using DNA preparations from microdissected tumors. Mutations were found in 40% of the carcinomas (including a borderline component adjacent to carcinoma in one lesion) but in none of the pure BTs. These findings suggest that p53 mutations may not be commonly associated with the borderline phenotype of ovarian epithelial tumors but may occur during malignant transformation.
- Published
- 1995
31. The E3 ubiquitin ligase protein associated with Myc (Pam) regulates mammalian/mechanistic target of rapamycin complex 1 (mTORC1) signaling in vivo through N- and C-terminal domains
- Author
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Sangyeul Han, Lin Li, Pradeep G. Bhide, Marianne James, Aaron DiAntonio, Vijaya Ramesh, Roberta L. Beauchamp, Clara F. Burande, Samira Bahl, Nicole A. Smith, and Sun Kim
- Subjects
Cell signaling ,Ubiquitin-Protein Ligases ,Nerve Tissue Proteins ,mTORC1 ,Mechanistic Target of Rapamycin Complex 1 ,Biochemistry ,Corpus Callosum ,Mice ,Ubiquitin ,stomatognathic system ,parasitic diseases ,Tuberous Sclerosis Complex 2 Protein ,Animals ,Humans ,Caenorhabditis elegans ,Molecular Biology ,Mechanistic target of rapamycin ,PI3K/AKT/mTOR pathway ,reproductive and urinary physiology ,biology ,F-Box Proteins ,TOR Serine-Threonine Kinases ,Tumor Suppressor Proteins ,Ubiquitination ,Proteins ,Cell Biology ,Molecular biology ,Axons ,Mice, Mutant Strains ,Ubiquitin ligase ,Cell biology ,Protein Structure, Tertiary ,HEK293 Cells ,nervous system ,Ubiquitin ligase complex ,Multiprotein Complexes ,embryonic structures ,Synapses ,biology.protein ,Drosophila ,Signal transduction ,Carrier Proteins ,Signal Transduction - Abstract
Pam and its homologs (the PHR protein family) are large E3 ubiquitin ligases that function to regulate synapse formation and growth in mammals, zebrafish, Drosophila, and Caenorhabditis elegans. Phr1-deficient mouse models (Phr1(Δ8,9) and Phr1(Magellan), with deletions in the N-terminal putative guanine exchange factor region and the C-terminal ubiquitin ligase region, respectively) exhibit axon guidance/outgrowth defects and striking defects of major axon tracts in the CNS. Our earlier studies identified Pam to be associated with tuberous sclerosis complex (TSC) proteins, ubiquitinating TSC2 and regulating mammalian/mechanistic target of rapamycin (mTOR) signaling. Here, we examine the potential involvement of the TSC/mTOR complex 1(mTORC1) signaling pathway in Phr1-deficient mouse models. We observed attenuation of mTORC1 signaling in the brains of both Phr1(Δ8,9) and Phr1(Magellan) mouse models. Our results establish that Pam regulates TSC/mTOR signaling in vitro and in vivo through two distinct domains. To further address whether Pam regulates mTORC1 through two functionally independent domains, we undertook heterozygous mutant crossing between Phr1(Δ8,9) and Phr1(Magellan) mice to generate a compound heterozygous model to determine whether these two domains can complement each other. mTORC1 signaling was not attenuated in the brains of double mutants (Phr1(Δ8,9/Mag)), confirming that Pam displays dual regulation of the mTORC1 pathway through two functional domains. Our results also suggest that although dysregulation of mTORC1 signaling may be responsible for the corpus callosum defects, other neurodevelopmental defects observed with Phr1 deficiency are independent of mTORC1 signaling. The ubiquitin ligase complex containing Pam-Fbxo45 likely targets additional synaptic and axonal proteins, which may explain the overlapping neurodevelopmental defects observed in Phr1 and Fbxo45 deficiency.
- Published
- 2012
32. p53 gene mutations and protein accumulation in human ovarian cancer
- Author
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Debra A. Bell, David W. Yandell, Susan M. Edgerton, Jolanta Kupryjanczyk, Ann D. Thor, Roberta L. Beauchamp, and Victor Merritt
- Subjects
Nonsense mutation ,Biology ,Gene mutation ,medicine.disease_cause ,Polymerase Chain Reaction ,medicine ,Humans ,Point Mutation ,Amino Acid Sequence ,Neoplasm Metastasis ,Codon ,Gene ,Neoplasm Staging ,Ovarian Neoplasms ,Mutation ,Multidisciplinary ,Base Sequence ,Point mutation ,Cancer ,DNA, Neoplasm ,Exons ,Genes, p53 ,medicine.disease ,Immunohistochemistry ,Primary tumor ,Cancer research ,Female ,Tumor Suppressor Protein p53 ,Ovarian cancer ,Research Article ,Chromosomes, Human, Pair 17 - Abstract
Mutations of the p53 gene on chromosome 17p are a common genetic change in the malignant progression of many cancers. We have analyzed 38 malignant tumors of ovarian or peritoneal müllerian type for evidence of p53 variations at either the DNA or protein levels. Genetic studies were based on single-strand conformation polymorphism analysis and DNA sequencing of exons 2 through 11 of the p53 gene; mutations were detected in 79% of the tumors. These data show a statistically significant association between mutations at C.G pairs and a history of estrogen therapy. Two of 20 patients whose normal tissue could be studied carried germ-line mutations of p53. Immunohistochemical analysis of the p53 protein was carried out using monoclonal antibody PAb1801. Ninety-six percent of the missense mutations were associated with abnormal accumulation of p53 protein, but nonsense mutations, a splicing mutation, and most deletions did not result in p53 protein accumulation. A statistically significant association between p53 protein accumulation in poorly differentiated stage III serous carcinomas and small primary tumor size at diagnosis was found, perhaps suggesting that p53 protein accumulation accelerates the metastatic spread from a primary tumor. Overall, our findings indicate that alterations of p53 play a major role in ovarian cancer, including predisposition to the disease in some patients, and suggest a possible mechanism for somatic mutations leading to this cancer.
- Published
- 1993
33. Mediator subunit MED28 (Magicin) is a repressor of smooth muscle cell differentiation
- Author
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Kim S. Beyer, Anders M. Näär, James F. Gusella, Ming-Fen Lee, Vijaya Ramesh, and Roberta L. Beauchamp
- Subjects
Regulation of gene expression ,Mediator Complex ,Protein subunit ,Smooth muscle cell differentiation ,Myocytes, Smooth Muscle ,Intracellular Signaling Peptides and Proteins ,Repressor ,Cell Biology ,MED28 ,MED6 ,Biology ,Fibroblasts ,Biochemistry ,Molecular biology ,MED1 ,Up-Regulation ,Cytoskeletal Proteins ,Mice ,Mediator ,Cell Transdifferentiation ,NIH 3T3 Cells ,Animals ,Humans ,RNA Interference ,Molecular Biology - Abstract
Magicin, a protein that we isolated earlier as an interactor of the neurofibromatosis 2 protein merlin, was independently identified as MED28, a subunit of the mammalian Mediator complex. Mediator complex is an evolutionarily conserved transcriptional cofactor, which plays an essential role in positive and negative gene regulation. Distinct Mediator subunit composition is thought to contribute to gene regulation specificity based on the interaction of specific subunits with subsets of transcription factors. Here we report that down-regulation of Med28 expression in NIH3T3 cells results in a significant induction of several genes associated with smooth muscle cell (SMC) differentiation. Conversely, overexpression of MED28 represses expression of SMC genes, in concordance with our knockdown data. More importantly, multipotent mesenchymal-derived murine precursors can transdifferentiate into SMCs when Med28 is down-regulated. Our data also show that Med28 functions as a negative regulator of SMC differentiation in concert with other Mediator subunits including Med6, Med8, and Med18 within the Mediator head module. Our results provide strong evidence that MED28 may function as a scaffolding protein by maintaining the stability of a submodule within the head module and that components of this submodule act together in a gene regulatory program to suppress SMC differentiation. The results presented here demonstrate for the first time that the mammalian Mediator subunit MED28 functions as a repressor of SMC differentiation, which could have implications for disorders associated with abnormalities in SMC growth and differentiation, including atherosclerosis, asthma, hypertension, and smooth muscle tumors.
- Published
- 2007
34. Magicin associates with the Src-family kinases and is phosphorylated upon CD3 stimulation
- Author
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Roberta L. Beauchamp, Kim S. Beyer, James F. Gusella, Vijaya Ramesh, and Ming-Fen Lee
- Subjects
CD3 Complex ,Biophysics ,macromolecular substances ,Biology ,Proto-Oncogene Proteins c-fyn ,Biochemistry ,Jurkat cells ,SH3 domain ,Cell Line ,Jurkat Cells ,Mice ,FYN ,Animals ,Humans ,Src family kinase ,Phosphorylation ,Molecular Biology ,Cytoskeleton ,Cell Nucleus ,Tyrosine-protein kinase CSK ,Mediator Complex ,Kinase ,Intracellular Signaling Peptides and Proteins ,hemic and immune systems ,Cell Biology ,Cell biology ,Cytoskeletal Proteins ,src-Family Kinases ,biology.protein ,Mutagenesis, Site-Directed ,GRB2 ,biological phenomena, cell phenomena, and immunity ,Proto-oncogene tyrosine-protein kinase Src ,Signal Transduction - Abstract
We recently identified a novel actin cytoskeleton-associated protein magicin, for merlin and Grb2 interacting cytoskeletal protein. To unravel the cellular functions of magicin, we used a yeast two-hybrid system and identified Fyn tyrosine kinase as a specific binding partner for magicin. Fyn phosphorylates magicin in vitro. In addition to Fyn, Src and Lck also interact with magicin. Upon stimulation with anti-CD3 antibody, magicin is phosphorylated in the T lymphocyte leukemia Jurkat cell line. Magicin phosphorylation is not observed in an Lck-deficient line, J.CaM1.6, indicating that Lck is the major Src family kinase for phosphorylating magicin in Jurkat cells. Employing site-directed mutagenesis along with in vitro kinase assays, we found that Y64 of magicin is phosphorylated by Lck creating a SH2-Grb2 binding motif. Magicin has also been identified as a Mediator subunit (MED28) in the nucleus involved in transcriptional regulation, therefore we propose that magicin may serve as a multi-faceted adaptor/scaffold to relay cellular signaling to the cytoskeleton and from the cytoskeleton to the nucleus.
- Published
- 2006
35. Magicin (MED28), a Potential Adaptor Protein
- Author
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Ming-Fen Lee, James F. Gusella, Vijaya Ramesh, Kim S. Beyer, and Roberta L. Beauchamp
- Subjects
Chemistry ,Genetics ,Signal transducing adaptor protein ,MED28 ,Molecular Biology ,Biochemistry ,Biotechnology ,Cell biology - Published
- 2006
36. Alternative splicing in protein associated with Myc (Pam) influences its binding to c-Myc
- Author
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Mark Bowser, Vanishree Murthy, Pradeep G. Bhide, Túlio M. Santos, Vijaya Ramesh, Karen Sazani, Sangyeul Han, and Roberta L. Beauchamp
- Subjects
Gene isoform ,Transcriptional Activation ,Blotting, Western ,Molecular Sequence Data ,Gene Expression ,Biology ,Kidney ,Mixed Function Oxygenases ,Proto-Oncogene Proteins c-myc ,Cellular and Molecular Neuroscience ,Exon ,Mice ,Multienzyme Complexes ,Animals ,Humans ,Protein Isoforms ,Amino Acid Sequence ,RNA, Messenger ,Cloning, Molecular ,Peptide sequence ,Gene ,Lung ,Reverse Transcriptase Polymerase Chain Reaction ,Alternative splicing ,RNA ,Kidney metabolism ,Brain ,Exons ,Blotting, Northern ,Molecular biology ,Immunohistochemistry ,Rats ,Alternative Splicing ,RNA splicing ,Protein Binding - Abstract
We recently identified Pam (for protein associated with c-Myc), as a binding partner for the tuberous sclerosis complex (TSC) protein tuberin in brain. The highly conserved Pam homologs in Drosophila and C. elegans are neuron-specific proteins that regulate synaptic growth. The Pam gene contains 83 exons and encodes a 4,641-amino-acid polypeptide with a predicted molecular weight of approximately 510 kDa. In a previous study, we demonstrated that Pam is expressed as two forms, approximately 450 kDa in rat embryonic and a approximately 350 kDa in rat adult brain. Here we have extended that work to show the approximately 450 kDa form is expressed in rat embryonic kidney, heart, and lung and in rat cell lines, and the approximately 350 kDa form is expressed in adult rat tissues as well as in human and mouse brain and human and mouse cell lines. To understand the size difference, we investigated alternative splicing of Pam in brain and detected six isoforms in the Myc-binding region resulting from splicing of exon 53, and three new exons, 52A, 56, and 56A. We also demonstrate that the presence of exon 52A in Pam significantly enhances binding to Myc, suggesting functional importance of this alternative splicing. The presence of Pam in many cellular compartments, its spliced variants, as well as its multiple binding partners, including tuberin, make it a complex, yet intriguing protein in the nervous system.
- Published
- 2005
37. Inactivation patterns of NF2 and DAL-1/4.1B (EPB41L3) in sporadic meningioma
- Author
-
Fabio P. Nunes, Vijaya Ramesh, Yo Niida, Mia MacCollin, Anat Stemmer-Rachamimov, Yiping Shen, Roberta L. Beauchamp, and James F. Gusella
- Subjects
Cancer Research ,Monosomy ,Chromosomes, Human, Pair 22 ,Loss of Heterozygosity ,Locus (genetics) ,Biology ,Meningioma ,Loss of heterozygosity ,Genes, Neurofibromatosis 2 ,otorhinolaryngologic diseases ,Genetics ,medicine ,Meningeal Neoplasms ,Humans ,Meningeal Neoplasm ,Genes, Tumor Suppressor ,Neurofibromatosis ,neoplasms ,Molecular Biology ,Tumor Suppressor Proteins ,Microfilament Proteins ,Membrane Proteins ,medicine.disease ,Tumor progression ,Cancer research ,Chromosomes, Human, Pair 18 ,Comparative genomic hybridization ,Microsatellite Repeats - Abstract
The molecular basis of tumorigenesis and tumor progression in meningiomas is not fully understood. The neurofibromatosis 2 (NF2) locus is inactivated in 50-60% of sporadic meningiomas, but the genetic basis of sporadic meningiomas not inactivated at the NF2 locus remains unclear. Specifically, there is conflicting data regarding the role of the tumor suppressor gene DAL-1/4.1B. Using microsatellite markers, we studied 63 sporadic meningiomas to determine loss of heterozygosity (LOH) at the NF2 and DAL-1/4.1B loci. Array comparative genomic hybridization analysis of 52 of these tumors was performed to determine copy number changes on chromosomes 18 and 22. Forty-one of 62 informative tumors showed LOH at the NF2 locus (66%) while only 12 of 62 informative tumors (19%) showed LOH of DAL-1/4.1B. Eleven of 12 (92%) tumors with DAL-1/4.1B LOH also had NF2 LOH. Monosomy or large deletions of chromosomes 18 and 22 were the main mechanism for LOH in these tumors. These studies implicate the DAL-1/4.1B locus in sporadic meningiomas less commonly than reported previously, and suggest that it is a progression rather than an initiation locus. Furthermore, we found the majority of meningiomas developed monosomy rather than isodisomy at the NF2 and DAL-1/4.1B loci as the mechanism for LOH.
- Published
- 2005
38. Neurofibromatosis 2 gene in human colorectal cancer
- Author
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Denise Pinney, Vijaya Ramesh, Christopher Sterner, Stephen D. Schmidt, Anil K. Rustgi, Lin Xu, Roberta L. Beauchamp, and James F. Gusella
- Subjects
Genetics ,Cancer Research ,Base Sequence ,Tumor suppressor gene ,Colorectal cancer ,Chromosomes, Human, Pair 22 ,Molecular Sequence Data ,Single-strand conformation polymorphism ,Biology ,medicine.disease ,Candidate Tumor Suppressor Gene ,Merlin (protein) ,Exon ,Genes, Neurofibromatosis 2 ,otorhinolaryngologic diseases ,medicine ,Cancer research ,Humans ,Chromosome Deletion ,Neurofibromatosis ,Colorectal Neoplasms ,Molecular Biology ,Gene - Abstract
Colon cancers commonly have allelic losses of chromosome 22q, which suggests the presence of a tumor suppressor gene on 22q. The candidate tumor suppressor gene on 22q is the neurofibromatosis 2 (NF2) gene. Using single strand conformation polymorphism (SSCP) analysis, we screened 24 pairs of colorectal cancer and adjacent normal mucosa, as well as 10 colon cancer cell lines from non-NF2 patients, for mutations in the coding sequence of the NF2 gene. Two SSCP variants, one in exon 14 and another one in exon 16, were detected in two of the sporadic colorectal cancers, but not in adjacent normal mucosa samples. Sequencing of these variants in one tumor detected an A-to-G transition in bp 1459 of the NF2 cDNA, resulting in the change of Ile to Val at codon 487 of merlin, the NF2 protein product. The other tumor showed a 2-bp (CT) deletion in the intronic sequence of the alternatively spliced exon 16. These results suggest that the NF2 gene is probably involved in some colorectal tumors, but is not the critical chromosome 22q tumor suppressor gene involved in colon tumorigenesis.
- Published
- 1995
39. Pam and its ortholog highwire interact with and may negatively regulate the TSC1.TSC2 complex
- Author
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Roberta L. Beauchamp, Naoto Ito, Vijaya Ramesh, Sangyeul Han, Luciana A. Haddad, Nicole A. Smith, and Vanishree Murthy
- Subjects
Central Nervous System ,Amino Acid Motifs ,Biochemistry ,PC12 Cells ,Tuberous Sclerosis Complex 1 Protein ,Mixed Function Oxygenases ,Tuberous sclerosis ,Ubiquitin ,Drosophila Proteins ,Caenorhabditis elegans ,Conserved Sequence ,Glutathione Transferase ,Zinc finger ,Genetics ,Neurons ,TSC1-TSC2 complex ,biology ,Brain ,Zinc Fingers ,Immunohistochemistry ,Ubiquitin ligase ,Cell biology ,medicine.anatomical_structure ,Phenotype ,Drosophila ,Protein Binding ,congenital, hereditary, and neonatal diseases and abnormalities ,DNA, Complementary ,Genotype ,Recombinant Fusion Proteins ,Ubiquitin-Protein Ligases ,Nerve Tissue Proteins ,Transfection ,Models, Biological ,Cell Line ,Proto-Oncogene Proteins c-myc ,parasitic diseases ,Tuberous Sclerosis Complex 2 Protein ,medicine ,Animals ,Humans ,Molecular Biology ,Adaptor Proteins, Signal Transducing ,Models, Genetic ,Tumor Suppressor Proteins ,Proteins ,Cell Biology ,biology.organism_classification ,medicine.disease ,Precipitin Tests ,Protein Structure, Tertiary ,Rats ,Repressor Proteins ,Mutation ,biology.protein ,TSC1 ,TSC2 ,Carrier Proteins - Abstract
Tuberous Sclerosis Complex (TSC) is an autosomal dominant disorder associated with mutations in TSC1, which codes for hamartin, or TSC2, which codes for tuberin. The brain is one of the most severely affected organs, and CNS lesions include cortical tubers and subependymal giant cell astrocytomas, resulting in mental retardation and seizures. Tuberin and hamartin function together as a complex in mammals and Drosophila. We report here the association of Pam, a protein identified as an interactor of Myc, with the tuberin-hamartin complex in the brain. The C terminus of Pam containing the RING zinc finger motif binds to tuberin. Pam is expressed in embryonic and adult brain as well as in cultured neurons. Pam has two forms in the rat CNS, an approximately 450-kDa form expressed in early embryonic stages and an approximately 350-kDa form observed in the postnatal period. In cortical neurons, Pam co-localizes with tuberin and hamartin in neurites and growth cones. Although Pam function(s) are yet to be defined, the highly conserved Pam homologs, HIW (Drosophila) and RPM-1 (Caenorhabditis elegans), are neuron-specific proteins that regulate synaptic growth. Here we show that HIW can genetically interact with the Tsc1.Tsc2 complex in Drosophila and could negatively regulate Tsc1.Tsc2 activity. Based on genetic studies, HIW has been implicated in ubiquitination, possibly functioning as an E3 ubiquitin ligase through the RING zinc finger domain. Therefore, we hypothesize that Pam, through its interaction with tuberin, could regulate the ubiquitination and proteasomal degradation of the tuberin-hamartin complex particularly in the CNS.
- Published
- 2003
40. Ki-ras and p53 mutations in pancreatic ductal adenocarcinoma
- Author
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Christopher J. N. Rall, Stephen P. Povoski, David W. Yandell, Yu-Xin Yan, Anil K. Rustgi, Roberta L. Beauchamp, and Fiona Graeme-Cook
- Subjects
Adult ,Male ,Pancreatic disease ,Endocrinology, Diabetes and Metabolism ,Nonsense mutation ,Biology ,Adenocarcinoma ,medicine.disease_cause ,Endocrinology ,Pancreatic cancer ,Gene duplication ,Internal Medicine ,medicine ,Humans ,Aged ,Hepatology ,Point mutation ,Exons ,Middle Aged ,medicine.disease ,Molecular biology ,Pancreatic Neoplasms ,medicine.anatomical_structure ,Genes, ras ,Mutation ,Cancer research ,Female ,Tumor Suppressor Protein p53 ,Pancreas ,Carcinogenesis - Abstract
Pancreatic adenocarcinoma involves activation of the Ki-ras oncogene, inactivation of the p53 tumor suppressor gene, and dysregulation of growth factors and perhaps metastasis genes. Ki-ras oncogene point mutations are known to be involved in pancreatic oncogenesis. The p53 tumor suppressor gene product plays a critical role in cell cycle regulation and also functions as a nuclear transcription factor. Point mutations in the p53 gene have been observed in a variety of malignancies. We determined the frequency of p53 protein overexpression and p53 point mutations in the conserved and nonconserved domains in pancreatic cancers as well as the coincidence of Ki-ras mutation in pancreatic ductal adenocarcinoma. Genomic DNA was isolated from 20 frozen pancreatic adenocarcinomas (14 primary, six metastases) along with six specimens of control pancreatic tissue and screened by single-strand conformation polymorphism (SSCP) analysis followed by direct genomic sequencing of SSCP variants. SSCP analysis was accomplished by incorporating 32P-dCTP in 12 separate polymerase chain (PCR) amplifications covering the p53 coding exons 2-11. All mobility shifts on SSCP were subjected to direct genomic sequencing by the modified dideoxy method. Immunoperoxidase (IP) staining was also done with a p53 monoclonal antibody. Ki-ras codon 12 mutational analysis was accomplished by incorporating 32P-dCTP by polymerase chain reaction amplification utilizing mismatched primers, which create a BstN1 restriction endonuclease site spanning codon 12; the products were digested by BstN1. Polyacrylamide gel electrophoresis allowed distinction between wild-type and mutant Ki-ras. p53 mutations were found in 5 of 20 pancreatic cancers (three of 14 primary tumors, two of six metastatic tumors). Point mutations were observed in three of 14 primary tumors, and one of six metastases, while a 2-base pair duplication resulting in a premature stop codon in exon 5 was found in one metastatic tumor. Point mutations were noted in conserved domains (exons 4, 5, 8) and in the nonconserved domain (exon 10). IP staining revealed that eight of 14 of the primary tumors and two of six metastases exhibited moderate to strong nuclear staining (> 30%), while no nuclear staining was evident in the controls. Ki-ras codon 12 mutations were found in 14 of 20 (70%) pancreatic cancers (nine of 14 primary tumors, five of six metastatic tumors) and none of the six controls. Fifty percent of the primary pancreatic tumors demonstrated moderate to strong nuclear staining. Extensive genetic analysis demonstrated mutations in 30% of the pancreatic cancers. One cancer had a nonsense mutation not detected by IP. Seven of 19 (37%) pancreatic cancers exhibited both Ki-ras point mutation and p53 protein overexpression or mutation. Both genetic analysis and IP are required to characterize all p53 mutations in pancreatic cancer. Ki-ras codon 12 mutations and p53 protein overexpression are important steps in pancreatic oncogenesis.
- Published
- 1996
41. Detection of Heterozygous Mutation in the Retinoblastoma Gene in a Human Pituitary Adenoma Using PCR-SSCP Analysis and Direct Sequencing
- Author
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Takashi Yoshimoto, Hidetoshi Ikeda, Roberta L. Beauchamp, and David W. Yandell
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Silent mutation ,Mutation ,Tumor suppressor gene ,Retinoblastoma ,Endocrinology, Diabetes and Metabolism ,Point mutation ,Single-strand conformation polymorphism ,General Medicine ,Biology ,medicine.disease ,medicine.disease_cause ,Molecular biology ,Pathology and Forensic Medicine ,Exon ,Endocrinology ,Pituitary adenoma ,Cancer research ,medicine - Abstract
Genomic deoxyribonucleic acid from surgical specimens of 25 pituitary adenomas was screened for the presence of mutations in the tumor suppressor gene, retinoblastoma gene, using polymerase chain reaction and single-strand conformation polymorphism analysis, followed by direct deoxyribonucleic acid sequencing. Mutation causing an amino acid change was found in one of the 25 pituitary adenomas. The mutation site was in exon 19 (codon 621) of the retinoblastoma gene. In addition, there were three types of silent mutations in introns of the gene. The patient in whom the retinoblastoma mutation was identified had a tumor with high clinical malignancy, a high percentage of c-myc protein-labeled cells, and a diagnosis of plurihormonal pituitary adenoma based on the presence of cells immunoreactive for five pituitary hormones. This article suggests that point mutation of retinoblastoma gene is rare in human pituitary adenomas but may provide a marker for aggressive pituitary adenoma.
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- 1995
42. Genetic alteratations in treatment-induced sarcomas
- Author
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David W. Yandell, Roberta L. Beauchamp, Herman D. Suit, Ira J. Piro, and Andrew E. Rosenberg
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Oncology ,Cancer Research ,medicine.medical_specialty ,Radiation ,business.industry ,Internal medicine ,medicine ,Radiology, Nuclear Medicine and imaging ,business - Published
- 1992
43. Alternative splicing in protein associated with Myc (Pam) influences its binding to c‐Myc.
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Túlio M. Santos, Sangyeul Han, Mark Bowser, Karen Sazani, Roberta L. Beauchamp, Vanishree Murthy, Pradeep G. Bhide, and Vijaya Ramesh
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- 2006
- Full Text
- View/download PDF
44. Brigatinib causes tumor shrinkage in both NF2-deficient meningioma and schwannoma through inhibition of multiple tyrosine kinases but not ALK.
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Long-Sheng Chang, Janet L Oblinger, Abbi E Smith, Marc Ferrer, Steven P Angus, Eric Hawley, Alejandra M Petrilli, Roberta L Beauchamp, Lars Björn Riecken, Serkan Erdin, Ming Poi, Jie Huang, Waylan K Bessler, Xiaohu Zhang, Rajarshi Guha, Craig Thomas, Sarah S Burns, Thomas S K Gilbert, Li Jiang, Xiaohong Li, Qingbo Lu, Jin Yuan, Yongzheng He, Shelley A H Dixon, Andrea Masters, David R Jones, Charles W Yates, Stephen J Haggarty, Salvatore La Rosa, D Bradley Welling, Anat O Stemmer-Rachamimov, Scott R Plotkin, James F Gusella, Justin Guinney, Helen Morrison, Vijaya Ramesh, Cristina Fernandez-Valle, Gary L Johnson, Jaishri O Blakeley, D Wade Clapp, and Synodos for NF2 Consortium
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Medicine ,Science - Abstract
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.
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- 2021
- Full Text
- View/download PDF
45. Traditional and systems biology based drug discovery for the rare tumor syndrome neurofibromatosis type 2.
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Synodos for NF2 Consortium, Robert Allaway, Steve P Angus, Roberta L Beauchamp, Jaishri O Blakeley, Marga Bott, Sarah S Burns, Annemarie Carlstedt, Long-Sheng Chang, Xin Chen, D Wade Clapp, Patrick A Desouza, Serkan Erdin, Cristina Fernandez-Valle, Justin Guinney, James F Gusella, Stephen J Haggarty, Gary L Johnson, Salvatore La Rosa, Helen Morrison, Alejandra M Petrilli, Scott R Plotkin, Abhishek Pratap, Vijaya Ramesh, Noah Sciaky, Anat Stemmer-Rachamimov, Tim J Stuhlmiller, Michael E Talkowski, D Bradley Welling, Charles W Yates, Jon S Zawistowski, and Wen-Ning Zhao
- Subjects
Medicine ,Science - Abstract
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.
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- 2018
- Full Text
- View/download PDF
46. Mediator Subunit Med28 Is Essential for Mouse Peri-Implantation Development and Pluripotency.
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Lin Li, Ryan M Walsh, Vilas Wagh, Marianne F James, Roberta L Beauchamp, Yuh-Shin Chang, James F Gusella, Konrad Hochedlinger, and Vijaya Ramesh
- Subjects
Medicine ,Science - Abstract
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 in vitro studies have been performed on MED28, its in vivo 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 Oct4 and Nanog. Further, overexpression of Med28 in mouse embryonic fibroblasts enhances the efficiency of their reprogramming to pluripotency. Cre-mediated inactivation of Med28 in induced pluripotent stem cells shows that Med28 is required for their survival. Intriguingly, heterozygous loss of Med28 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.
- Published
- 2015
- Full Text
- View/download PDF
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