THERAPEUTICS FOR RARE & NEGLECTED DISEASES (TRND) PROGRAM AT NIH AND POSSIBILITIES FOR DRUG DISCOVERY IN THE RAS/MAPK DISORDERS
G. Sitta Sittampalam, Ph.D.
Therapeutics for Rare & Neglected Diseases, National Center for Advancing Translational Sciences (NCATS), National Institutes of Health, 9800 Medical Center Drive, Rockville, MD, USA.
The TRND program at the National Center for Advancing Translational Sciences (NCATSjNIH) was established in 2009 to stimulate and speed the discovery & development of new drugs for rare and neglected diseases. It is a unique collaborative model between the NIH and academic scientists, non- profit organizations and pharmaceutical and biotechnology companies.
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USE OF RAF INHIBITORS FOR TREATMENT OF MELANOMA
Chao Zhang, Gideon Bollag (on behalf of the Plexxikon team), Plexxikon Inc., Berkeley, CA.
The identification of activating BRAF mutations (primarily missense substitutions for Valine-600 or BRAFv600) in melanoma and other tumors supports a functionally important role for BRAF in the pathogenesis of these malignancies. Specific BRAF inhibitors including vemurafenib have demonstrated both objective tumor response and overall survival benefit in mutant BRAF driven melanoma. The clinical effectiveness of BRAF inhibitor-based therapy depends on complete abolition of the MAPK pathway output in tumors harboring BRAF mutations.
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USE OF ANTISENSE MORPHOLINO OLIGOMERS AS A GENE-THERAPEUTIC APPROACH TO RESTORE NORMAL SPLICING
Conxi Lazaro, Ph.D. , Hereditary Cancer Program, Genetic Diagnostics Unit, Hereditary Cancer Program, Catalan Institute of Oncology (lCO-IDIBELL), Barcelona, Spain.
A significant proportion of germ line mutations causing Neurofibromatosis type 1 affect the correct splicing of the NFl gene. Splicing is a complex mechanism by which introns from a pre-mRNA are removed. This process is finely regulated at the cellular level, and it has been shown that it can be modulated by using antisense oligonucleotides (AONs). Multiple studies have demonstrated the power of AONs in modulating abnormal splicing caused by constitutive DNA mutations, first in cultured cells, later in preclinical animal models and currently in several promising clinical trials.
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TISSUE BANKING IN PEDIATRIC ONCOLOGY
The last several decades have witnessed remarkable advances in identifying the genetic causes of many childhood syndromes, both oncologic and non-oncologic. Subsequent team efforts by clinicians, patients, their families and advocates have supported clinical trials to evaluate new therapies for those affected. In the background and often unnoticed, there are parallel team efforts by pathologists and their staff to obtain and “bank” tissue samples from children with these diseases. It is hoped that by analyzing the original tissue from the affected individuals, additional insight will be gained into disease origin and treatment possibilities.
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THERAPEUTIC APPROACHES TO HCM IN NOONAN SYNDROME WITH MULTIPLE LENTIGENES (LEOPARD SYNDROME)
Maria I. Kontaridis, Ph.D.l, 2 and Amy Roberts, M.D.3
1 Beth Israel Deaconess Medical Center, Department of Medicine, Division of Cardiology, Boston, MA
2 Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA.
3 Boston Children’s Hospital, Department of Cardiovascular Genetics, Boston, MA.
Essentially all cases of Noonan syndrome with multiple lentigenes {NS-ML; formally termed LEOPARD syndrome}, a rare autosomal dominant, multi-systemic disease, are caused by mutations in the SH2 domain-containing protein tyrosine phosphatase SHP2, encoded by PTPN11. NS-ML presents with phenotypic characteristics similar to those observed in other RASopathy disorders, including multiple lentigines, electrocardiographic conduction abnormalities, ocular hypertelorism, pulmonic stenosis, abnormal genitalia, retardation of growth, and sensorineural deafness. However, the most common cardiac manifestation in NS-ML is hypertrophic cardiomyopathy {HCM}, with an estimated prevalence of 70-80%. Currently, there is no existing treatment for LS patients with HCM, and many die early of end-stage heart failure.
STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF RAS MUTANTS
Reza Ahmadian, PhD Institute of Biochemistry & Molecular Biology II, Heinrich-Heine University Dusseldorf, Germany.
The RAS-MAPK (mitogen-activated protein kinase) pathway is a kinase cascade leading to cell proliferation and differentiation. This signaling pathway has been intensively studied due to its importance in cancer development.
In the past decade a new group of genetic developmental diseases determined by germline mutations related to RAS/MAPK pathway components were identified and intensively studied. These so called RAS-MAPK-related disorders include Noonan syndrome (NS), Costello syndrome (CS), and cardiofacio-cutaneous syndrome (CFCS).
Recently, we described in detail the functional properties of a spectrum of KRAS-8 and NRAS mutations related to NS. Overall, our studies revealed several new mechanisms by which germline KRAS mutations contribute to human disease and lead to disturbed embryonic development. Two aspects are of high importance for the understanding of RAS function(s}: (i) we showed that the mild gain-of-function in the case of KRAS mutations at positions 34 and 60 is due to a mechanism counterbalancing GAP resistance by a reduced RAFl interaction; and (ii) NRAS at position 50 most likely leads to an impaired interaction between RAS and the plasma membrane.
NOONAN SYNDROME: PHENOTYPES AND THERAPEUTIC GOALS
Amy E. Roberts, MD Boston Children’s Hospital Harvard Medical School
Given the prevalence of Ras mutations (20% of malignancies) and ERK hyperactivation due to activating mutations in Ras or BRAF (30% of human cancers), the development of inhibitors of the Ras/mitogen – activated protein kinase (MAPK) pathway has been of profound interest to cancer researchers. Noonan syndrome mutations are also activating and the question has been raised if these drugs might also be useful as systemic therapies after birth. Choosing phenotypic targets requires detailed knowledge of the natural history (what is the expected disease progression so it can be determined if treatment has actually changed anything).
NEUROFIBROMATOSIS CLINICAL TRIALS CONSORTIUM
Bruce R. Korf, MD, Ph.D., Department of Genetics, University of Alabama at Birmingham.
The neurofibromatoses, including NF1, NF2, and schwannomatosis, are a set of related genetic disorders characterized by the development of benign tumors of the nerve sheath. NF1 is the most common of these disorders, affecting about 1 in 3,000 individuals. Aside from neurofibromas, patients may develop optic glioma, skeletal dysplasia, learning disabilities, and many other problems. Neurofibromas, though benign, can be disfiguring, and are at risk of transformation to malignant peripheral nerve sheath tumor. The NFl gene is a tumor suppressor that includes a GTPase activating protein domain that regulates conversion of Ras-GTP to Ras-GDP. This mechanism opens the possibility of development of specific treatments that target the Ras signaling pathway, but clinical trials are hampered by challenges in measuring outcomes and recruiting a sufficiently large number of patients to achieve a statistically valid study
MOLECULAR ANALYSIS OF NF1 AND SPRED1
Ludwine Messiaen, Ph.D. Department of Genetics, University of Alabama at Birmingham.
The NFl gene is a large and complex gene spread over 280Kb of genomic DNA on chromosome l7qll.2, comprising 57 constitutive exons and at minimum 3 alternatively spliced exons.
Mutations in the Nfl gene affect worldwide ~1/3000 individuals and are associated with Neurofibromatosis type 1. Molecular analysis can help with the clinical diagnosis, especially when atypical forms of NFl are present (such as spinal NF, NF-Noonan, Watson syndrome, segmental NF, … ).
Legius syndrome, a recently ras-o-pathy identified, is caused by mutations in the SPREDl gene on chromosome l5q13.2. Individuals with Legius syndrome present mainly with CAL-macules (CALM) and skinfold freckling, indistinguishable in size, shape and number from NFl, however, other typical NF1 associated features, such as Lisch nodules, neurofibromas, specific bone lesions, optic pathway gliomas and malignant peripheral nerve sheath tumors, are absent.
MODELING RASOPATHIES WITH HIPSC
Sonia Mulero-Navarro, The Mindich Child Health and Development Institute, lcohn School of Medicine at Mount Sinai. New York. USA.
Induced pluripotent stem technology gives us the opportunity to study genetic congenital disorders such as the RASopathies, characterized by a limited cellular source. Noonan syndrome (NS) patients harboring PTPN11 mutations have a higher risk to develop juvenile myelomonocytic leukemia (JMML), and hypertrophic cardiomyopathy is observed in 90% of patients with LEOPARD syndrome (LS). To further elucidate the molecular mechanisms of hematological and cardiovascular manifestations, we generated human induced pluripotent stem cell (hiPSC}-derived myeloid and cardiomyocytes to model both syndromes. Hematopoietic cells differentiated from hiPSCs harboring NS/JMML-causing PTPNll mutations recapitulated JMML clinical features including excessive number of monocytic/granulocytic cells, granulocyte macrophage colony-stimulating factor hypersensitivity and increased fetal hemoglobin. As a new insight of this leukemia, we discovered significant up-regulation of two hematopoiesis-related micro-RNAs in hiPSC-derived NS/JMML myeloid cells and also observed reduced mRNA levels for several predicted target genes of these micro-RNAs in JMML cells from patients.
MODELING RASOPATHIES IN MOUSE AND HUMAN
Benjamin G. Neel, Princess Margaret Cancer Center, University Health Network, Toronto, ON, Canada.
We showed previously that Rafi613V/+ mice fully recapitulate the cardiac features of RAFl mutant Noonan Syndrome, including cardiac hypertrophy, enhanced contractility and chamber dilatation. Postnatal MEK inhibitor treatment normalizes all NS phenotypes in these mice, yet it remains unclear whether genetic ablation of Erk would have the same effect. It also is not known whether the cardiac phenotypes derive from distinct cell(s}-of-origin and human models for NS cardiac phenotypes remain elusive. To address these issues, we used tissue-specific Cre-expressing mice to promote inducible expression of Rafi613V/+ in various cells in the mouse heart. Mice with cardiomyocyte-specific expression of RaflL613V (Mlc2v-LV) showed enhanced contractility, but not cardiac hypertrophy.
MECHANISMS UNDERLYING COGNITIVE DEFICITS IN THE RASOPATHIES: A FAMILY WITH DISTINCT PERSONALITIES
Ype Elgersma, Ph.D., Erasmus University, Netherlands.
RASopathies are caused by mutations in the Ras/ERK signaling pathway, resulting in over-activation of this pathway. These disorders typically present with varying degrees of cognitive disability. Although the Ras/ERK pathway is ubiquitously expressed in most neurons, studies on Neurofibromatosis type 1 (NF1) which is one of the best-studied RASopathies, showed that the neuronal deficits are specific to inhibitory neurons. Here, I will present new data in which we investigated whether a gain-of function mutation in the H-Ras protein, which is negatively regulated by NF1, recapitulates this cell-specific phenotype of NF1.
We made use of a mouse mouse model of Costello syndrome (CS), which is caused by activating mutation (H_RasG12V) in the H-RAS gene. We confirm that the H- RasG12V mouse is a suitable model to study CS by showing that these mice have hyperactive Ras/ERK pathway and a profound learning deficit as assessed in the Morris water maze (MWM). Furthermore, we found that mice expressing the H-RasG12V mutation only in excitatory neurons show similar learning deficits in the MWM and a hyperactive Ras/ERK pathway, and that the plasticity deficits were not the same as the deficits observed in Nf1 mice. These results indicate that the mechanism underlying the CS phenotype is different from NFL
Yong-Seok Lee1, Dan Ehninger1+, Miou znou’, Delana Butz3, Toshiyuki Araki2, Christine I. Nam\ J. Balaji1, Aida Amln’, Corinna Burger3, Benjamin G. Neel2, and Alcino J. Silva1,*
1 Departments of Neurobiology, Psychiatry and Biobehavioral Sciences, Psychology and Brain Research Institute, Integrative Center for Learning and Memory, University of California Los Angeles, Los Angeles, CA 90095, USA.
2 Campbell Family Cancer Research Institute, Ontario Cancer Institute, Princess Margaret Hospital, University Health Network, Toronto, ON, Canada M5G1L7.
3 Department of Neurology, University of Wisconsin-Madison, Madison, WI 53706, USA
“Current address: DZNE, German Center for Neurodegenerative Diseases, Ludwig-Erhard-Allee 2, 53175 Bonn, Germany.
“Current address: Center for Neuroscience, Indian Institute of Science, Bangalore, 560012, India.
Noonan syndrome (NS) is an autosomal dominant genetic disorder with an incidence of ~l in 2,500 live births characterized by facial abnormalities, short stature, motor delay and cardiac defects. Importantly, 30% to 50% of NS patients show cognitive deficits. Mutations in the PTPN11 gene, which up-regulate Ras-ERK signaling, account for ~50% of NS. We will report that heterozygous knock-in mice expressing common NS-associated gain-of-function Ptpn11 mutations(Ptpn11D61G/+ and Ptpn11N308D/+) show hippocampal-dependent spatial learning impairments caused by deficits in hippocampal long-term potentiation (LTP). First, we show that the learning and the LTP phenotypes associated with the more severe Ptpn11D61G/+ mutation in mice are more profound than the LTP and learning phenotypes associated with milder Ptpn11N308D/+ mutation. Second, viral overexpression of the PTPN11D61Ggene, specifically in adult CA fields of the hippocampus, results in increased basal ERK signaling, deficits in hippocampal CA1 LTP and consequently in spatial learning impairments, demonstrating that altered Ptpn11 function and associated LTP deficits specifically in adult CA fields are sufficient to cause spatial learning deficits. Third, a MEK inhibitor (SL327), that targets the hyperactive ERK signaling pathway, can reverse the LTP and learning deficits caused by the PTPN11D61Gmutation.
Scott R. Plotkin, Massachusetts General Hospital; Boston, MA
Neurofibromatosis 1 (Nfl) is a complex neurogenetic disorder with features of a tumor-suppressor syndrome and a rasopathy. The condition is characterized by a predisposition to multiple tumor types, learning disability, bony lesions, and other disease manifestations, which often result in functional disability, reduced quality of life, pain, and in some cases malignancy. With increasing knowledge of the biology and pathogenesis of NFl, clinical trials with targeted agents directed at NFl-related complications have become available. Most clinical trials for patients with NF have used designs and endpoints similar to oncology trials. However, differences in the disease manifestations and natural history of NFl (compared to cancers) require the development of new designs and endpoints to perform meaningful clinical trials.
GENETIC SYNDROMES ASSOCIATED WITH THE RAS/MAPK PATHWAY AND THE IDENTIFICATION OF MUTATIONS IN A NEW GENE, RIT1, FOR NOONAN SYNDROME
Yoko Aoki, Tetsuya Niihori, Shin-Ichi Inoue, Yoichi Matsubara, Department of Medical Genetics, Tohoku University School of Medicine, Sendai, Japan.
Recent studies have shown that a group of genetic disorders results from dysregulation of the Ras/MAPK cascade. These disorders include:
1) Noonan syndrome caused by mutations in PTPN11, SOS1, RAF1, KRAS, BRAF, and NRAS;
2) LEOPARD (multiple lentigines, electrocardiographic conduction abnormalities, ocular hypertelorism, pulmonary stenosis, abnormal genitalia, retardation of growth and sensorineural deafness) syndrome caused by mutations in PTPN11 and RAF1;
3) Costello syndrome caused by activating mutations in HRAS;
4) cardio-facio-cutaneous (CFC) syndrome caused by mutations in BRAF, MAP2K1/2 and KRAS;
5) Noonan-like syndrome caused by mutations in SHOC2 or CBL;
6) neurofibromatosis type I caused by haploinsufficiency of neurofibromin;
7) NF-llike syndrome caused by haploinsufficiency of SPRED1; 7) hereditary gingival fibromatosis caused by a mutation in SOS1;
8) capillary malformation-arteriovenous malformation caused by haploinsufficiency of RASA1 (p120 GAP).
It has been suggested that these syndromes be comprehensively termed Ras/MAPK pathway syndromes or RASopathies.
DRUG SCREENING FOR NOONAN SYNDROME WITH DROSOPHILA
Bruce D. Gelb. Mindich Child Health and Development Institute, /cahn School of Medicine at Mount Sinai, New York, NY.
Noonan syndrome (NS) has pleimorphic features of varying severity, which can not currently be prevented or addressed at their root cause. The elucidation of the genetic abnormalities underlying NS and subsequent mechanistic studies, particularly animal modeling, have provided the potential to develop therapies directly addressing the altered signal transduction. Currently, the two leading clinicap aspects to target are hypertrophic cardiomyopathy (HCM) and intellectual and developmental delays. While strategies relying on small molecules directly inhibiting the canonical RAS-MAPK signaling, which might leverage current efforts primarily targeting cancer, might prove efficacious, there is risk that the safety profile of such therapies will not be acceptable for more aspects of NS.
CTF THERAPEUTIC DEVELOPMENT PIPELINE
Annette Bakker, Salvatore La Rosa, Marco Nievo
Children’s Tumor Foundation.
The Children’s Tumor Foundation is the world‘s leading non-government funder of NF research to find effective treatments for neurofibromatosis. Foundation-funded research was critical in isolating the genes that cause NF and is now focused on finding treatments to improve the lives of those living with the disorder. This includes the foundation’s active participation in drug discovery research and clinical trials. We sponsor a network of 44 NF Clinics throughout the country to improve access to quality care for those with NF, and ensure best practices in treating the disorder.
The key research goals for the Foundation are to attract pharmaceutical industry and biotech to the NF field via a collaborative business model, actively bridging academic science to industry to patients.
COSTELLO SYNDROME PHENOTYPES AND THERAPEUTIC GOALS
Bronwyn Kerr, MBBS
Manchester Centre for Genomic Medicine, Central Manchester University Hospitals NHS Foundation Trust, Manchester Academic Health Sciences Centre (MAHSC), Manchester M13 9WL, UK.
Costello syndrome was first described in the 1970s by Jack Costello, a New Zealand Paediatrician, who recognised the key characteristics in 2 patients. There were no further publications until 1991, when der Kaloustian published a third case, suggested the name “Costello syndrome” and noted the resemblance to Noonan and Cardio-facio-cutaneous syndromes. Over the next decade, a number of reports expanded the clinical features, and a high risk of malignancy, particularly embryonal rhabdomyosarcoma, emerged.
CLINICAL TRIALS FOR FRAGILE X SYNDROME
Reymundo Lozano MD.
Department of Pediatrics, UC California, Davis, Health System. Fragile X Research and Treatment Center, UC Davis, MIND Institute, Sacramento, CA.
Fragile X syndrome (FXS) is the most prevalent and well-understood monogenetic cause of intellectual disability (ID) and autism spectrum disorder (ASD); its high penetrance and the seminal importance of FMRP in synaptic plasticity make FXS an ideal model for the study of neurodevelopmental disorders in general. In fact recent studies suggest that there is functional convergence of a number of genes that are implicated in ID and ASD, indicating that an understanding of the cellular and biochemical dysfunction that occurs in monogenic forms of ID are likely to reveal common targeted treatments.
CARDIO-FACIO-CUTANEOUS SYNDROME
Emma M. M. Burkitt Wright 1,2
Wellcome Trust Clinical Research Training Fellow, Manchester Centre for Genomic Medicine1,2,
lnstitute of Human Development, Faculty of Medical and Human Sciences, University of Manchester;2St. Mary’s Hospital, Central Manchester University Hospitals NHS Foundation Trust, Manchester Academic Health Science Centre, Manchester M13 9WL, UK.
Cardio-facio-cutaneous syndrome (CFe} is a multisystem disorder due to germline mutations that cause dysregulation of the Ras-MAPK pathway, and frequently has severe manifestations. Patients demonstrate a wide variety of clinical problems, including neurological features such as learning disability and epilepsy, congenital heart disease and cardiomyopathy, and a wide variety of cutaneous manifestations. Genetic and phenotypic similarities to Noonan syndrome (NS) are present, and as in NS, extensive genetic heterogeneity is demonstrated, and a proportion of patients remain without a molecular diagnosis. These barriers to comprehensive molecular confirmation, combined with the wide variety and variability of clinical manifestations, make it challenging to characterise this disorder fully.
A CLINICAL TRIAL OF MEK INHIBITION IN NOONAN SYNDROME WITH HYPERTROPHIC CARDIOMYOPATHY
Calum A. MacRae1, William J. McKenna2, Craig T. Besson’, Eric Svensson’, Denise Yates’, Jessie Gu3•
1, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA;
2, The Heart Hospital and University College London, UK;
3 Novartis Institute of Biomedical Research, Cambridge, MA
The purpose of the study is to provide proof-of-concept to determine whether the ability of MEK162 to antagonize MEK activation in Noonan Syndrome HCM patients, who usually have upstream mutations in the Ras-Raf-Mek-Erk pathway that lead to MEK activation, would be beneficial over a 6 month treatment period in hypertrophy regression. The study is designed as an open label study to assess safety, tolerability, pharmacokinetics and pharmacodynamics of MEK162 in Noonan Syndrome Hypertrophic Cardiomyopathy.
