Yolanda Sanchez - US grants

The successful duplication and segregation of chromosome are essential for the maintenance of genomic integrity. Biochemical pathways, called checkpoints, provide the cell with a mechanism to sense DNA damage, and respond by arresting the cell cycle to allow DNA repair. The inability to respond by arresting the cell cycle to allow DNA repair. The inability to respond to such damage leads to increased genomic instability, which can contribute to deregulation of cellular growth and cancer Mutations in mammalian genes, such as p53 and ATM (Ataxia telangiectasia mutated), which abrogate this response, cause a genetic predisposition to cancer. At the cellular level, ATM-defective cells control are conserved among eukaryotes. In Schizosaccharomyces pombe (S. pombe), the Atm-like protein rad3 functions upstream of chk1 (checkpoint kinase 1). Chk1 function in S. pombe is required for arrest and survival following exposure to DNA-damaging agents such as ionizing radiation. We have isolated the budding yeast (Saccharomyces cerevisiae), human and murine homologues of the chk1 gene. Chk1 is required to regulate mitotic progression in response to DNA damage. The work described here will examine aspects of the DNA damage response pertaining to the Chk1 pathway that remain unanswered: 1) What is/are the pathway(s) leading to Chk1 activation? 2) Does the Chk1 pathway have a regulatory role in damage-induced DNA repair? 3) Are the transducers of the checkpoint signal associated with complexes involved in DNA replication and/or repair? 4) What are other effectors of the Chk1 pathway? Based on the conservation of CHK1 between the fission yeast and mammals, we predict that a member of the ATM family (Atm and/or related Atr) will regulate hk1 in response to DNA damage. We will undertake biochemical studies on proteins that associate with mammalian Chk1 to further elucidate the circuitry of the Chk1 pathway. Exploiting the evolutionary conservation of checkpoint regulatory components, we will identify the budding yeast homologues of hChk1 interactors and use the yeast model as a genetic tool to examine their roles in the cellular response to DNA damage. The primary goal of this work is to expand our understanding of how cells detect and respond to DNA damage. These studies will shed light on the mechanisms of the DNA damage response in mammalian cells, which may allow us to design more effective therapeutic regimens for the treatment of diseases that result from a deficiency in this capacity.

DNA damage; protein structure function; carcinogenesis; protein kinase; cell cycle; cell growth regulation; environmental stressor; alleles; gene rearrangement; phosphorylation; embryonic stem cell; fibroblasts; polymerase chain reaction; laboratory mouse; genetically modified animals; flow cytometry; electroporation; western blottings;

DNA damage; DNA repair; cell growth regulation; genetic regulation; biological signal transduction; protein protein interaction; protein localization; DNA replication; gene interaction; phosphorylation; tissue /cell culture; Schizosaccharomyces pombe; Saccharomyces cerevisiae; immunofluorescence technique; fluorescence microscopy;

DESCRIPTION (provided by applicant): Loss of function mutations in the Neurofibromatosis type 1 (NF1) gene result in an autosomal dominant disease that affects 1:3500 live births. 95% of carriers will develop neurofibromas in their lifetime. Plexiform neurofibromas can cause disfigurement and/or compression of organs, which can have devastating physical and psychological consequences. Progression of plexiform neurofibromas to malignant peripheral nerve sheath tumors (MPNST) occurs in 8-13% of patients. Currently the only treatments for neurofibromas involve surgical removal of tumor tissue and of the affected nerve with or without cancer chemotherapy. Cancer therapies frequently trigger genomic instability, thus when used in young individuals they can induce mutations that will lead to malignancies later in life. Therefore, better treatments are needed for NF1 disease. Nf1 is a GTPase-activating protein (GAP) for Ras proteins and loss of NF1 results in increased levels of Ras-GTP, the activated form of Ras, which can lead to many of the phenotypes observed in NF1 patients. Loss of human NF1 leads to activation of PKA and MAPK pathways in Schwann cells contributing to the aberrant migration and proliferation of these cells. The budding yeast Saccharomyces cerevisiae has two NF1-like genes called IRA1 and IRA2 that when mutated lead to phenotypes that are reminiscent of Schwann cells with mutations in NF1. Expression of the catalytic domain of human NF1 can suppress the phenotypes of ira yeast mutants. The conservation of these pathways makes the genetically amenable yeast an excellent model system to use to identify drug targets for NF1. The work proposed here addresses two hurdles in the studies of NF1 and can only be achieved using a system such as yeast. The first hurdle is the lack of a system that allows identification of therapies specific to cells lacking NF1 and the second is the need for a resource amenable to high throughput analyses with which to carry out chemical screens. We propose to take a two-pronged approach by using two models, human cells and yeast for the identification of drug targets for NF1. There is no better model than the yeast for these types of screens, thus by combining these two models we present a powerful strategy for screening compounds to identify and validate potential drug targets for NF1. PUBLIC HEALTH RELEVANCE: NF1 is an inherited disease that affects 1:3500 live births in which 95% of carriers develop neurofibromas that include plexiform neurofibromas. Plexiform neurofibromas can cause disfigurement and/or compression of organs with devastating consequences. Despite the fact that NF1 is one of the most frequently inherited genetic disorders and that the gene involved in the disease is known, currently there is no effective pharmacological therapy for NF1. This proposal addresses two hurdles in the identification of therapies for NF1. The first hurdle is the lack of a system that allows identification of therapies specific to cells with NF1 mutations and the second is the need for a resource amenable to high throughput drug screens. The work proposed here takes a two-pronged approach using human cells and a genetic model system, the yeast for the identification of drug targets for NF1. There is no better model than the yeast for these types of screens, thus by combining these two models we present a powerful approach for screening compounds to identify and validate potential drug targets for NF1.

The Norris Cotton Cancer Center (NCCC) Molecular Biology Shared Resource Laboratory (MBSR) has been operating since 1995 as a core facility. This core initially was developed within the Cancer Center, but was quickly expanded to provide services to other laboratories with the addition of other sources of support. It has operated as a university cost center since 1997, but remains a central and important shared resource for NCCC investigators, who represent a major proportion of its user base. In 2000, proteomics services were added to this shared resource. In 2007 these were split off as the new Proteomics Shared Resource (PRSR) described separately elsewhere. The MBSR provides state-of-the-art molecular biology support including DMA and RNA oligonucleotide synthesis, DMA sequencing, fragment analysis, traditional and real-time PCR services, and various imaging support services for the NCCC membership. The MBSR also collaborates with the Proteomics, Bioinformatics, Genomics, and Biostatistics Shared Resources in providing seamless support for the investigator who is applying these tools for genomic level analyses as part of the Integrative Biology Group (IBG). The MBSR currently provides 54 NCCC members and their laboratories with services which totaled $131 K in chargebacks for FY 2007 ($273K total chargebacks). The NCCC users represented 47% of the total laboratories using this facility at Dartmouth (116 labs total), and their usage constituted 48% of the total usage based on chargeback fees and 44% based on units of use. The total chargebacks for this core were $273 K for FY 2007, and the total operating budget was $597K, requiring $324K in subvention which was derived from the NCCC Core Grant ($57K) and other institutional resources ($267K). This shared resource is requesting a budget of $52,787 from the NCCC Core Grant for the first year of this renewal, representing a level comparable to the current year's support and representing approximately 14% of its total operating budget for FY 2009 of approximately $378K (note that the decrease in subvention support and total budget is due to the splitting of MBSR and PRSR and concomitant reduction in services, costs, and staffing for MBSR). The MBSR continues to add and upgrade instrumentation, including recent upgrades to its DNA sequencing services. It plans to add the new Solexa technology from Agilent in the near future, which will facilitate important laboratory research and allow the NCCC investigators to comprehensively analyze the genetic polymorphisms for the entire genomes of individual cancer patients and thereby provide a means of customizing individual diagnosis and treatment. The overall goal of the MBSR is to continue to provide NCCC researchers with state-of-the-art technical support and services for their cancer research at as low a rate as possible so that they can continue to successfully meet their research objectives individually and collectively.

? DESCRIPTION (provided by applicant): Genomic information can allow investigators to devise precision therapies that target molecular lesions specific to a patient's cancer. One of the molecular lesions present in many malignant tumors is loss of the NF1 tumor suppressor, which is a driver in neurofibromatosis type 1 (NF1), one of the most frequently inherited genetic disorders. NF1 exhibits a broad clinical spectrum including benign nervous system tumors called neurofibromas, low-grade astrocytomas, pheochromocytoma, and juvenile myelomonocytic leukemia. Plexiform neurofibromas (PN) occur in deep nerves and can degenerate into malignant peripheral nerve sheath tumors (MPNSTs), chemo- and radiation-resistant sarcomas with a dismal 20% five-year survival rate. In NF1 carriers, the lifetime risk is 30% for PN and 8-15% for MPNST. NF1 mutations are also found in sporadic tumors including glioblastoma multiforme (GBM), melanoma, pheochromocytoma, ovarian, uterine, and lung cancers. Furthermore, in animal models and human tumor lines NF1 loss has been shown to drive GBM. A targeted molecular therapy designed to inhibit tumor-initiating and -promoting cells would substantially advance our ability to treat tumors that develop as a result of NF1 loss. Using complementary screening platforms, we identified small molecules that selectively killed or stopped the growth of cells carrying a mutation in NF1 as well as their molecular targets. In this application we focus on our top small molecule leads and the power of our model systems to test the hypothesis that aggressive neurological cancers that are known to be driven by NF1 loss and for which no cure exists, including PN, MPNST and GBM, will respond to molecules that we identified as synthetic lethal with NF1 loss. For this, we will determine pharmacokinetic properties of our top small molecule leads and, where needed, conduct structure-activity relationship studies to improve kinetics and/or reduce toxicity in order to test their efficacy in pre-clinical models of PN and GBM. We will leverage our model systems to define the mechanisms of action of our small molecule leads and add to our pipeline drugs in clinical trials that share the same targets. In addition to mutational inactivation, some GBM tumors exhibit down-regulation of the NF1 protein. Our lead compounds also stopped the growth of human GBM and neuroblastoma cells with low NF1 protein in vitro, supporting the broad application of the small molecule leads that we have identified. To capture tumors that have lost NF1 by any mechanism, we constructed an RNA-based classifier, which is capable of identifying downstream transcriptomic effects that indicate NF1 inactivation in GBM, using machine learning. We will apply the RNA-based classifier, along with targeted sequencing of NF1, to identify additional patient derived xenograft (PDX) GBM tumors that have an inactivating mutation of NF1 or molecular signatures of NF1 loss and test their response to our lead compounds in pre-clinical models. We expect that this work will provide new targets and therapeutic leads for aggressive neurological cancers driven by NF1 loss.