2013 Research Grant Program Winning Abstract. Neuroblastoma-Causing
Mutations in Human Neural Crest Stem Cells. By Stephen Roberts.
Neuroblastoma ...
2013 Research Grant Program Winning Abstract Neuroblastoma-Causing Mutations in Human Neural Crest Stem Cells By Stephen Roberts
Neuroblastoma (NB) is the most common extra-cranial tumor of childhood and annually accounts for 15% of all pediatric cancer deaths. It shows strikingly variable clinical behavior from spontaneous regression without therapy to aggressive, therapy-resistant disease. Currently, our understanding of the underlying molecular pathways that lead to this diversity of clinical behavior is lacking. A better knowledge of the pathogenesis of neuroblastoma is critically needed to help develop more effective therapies. Recently, both germline and somatic mutations in the anaplastic lymphoma kinase (ALK) gene were reported in NB. Importantly, these mutations were found throughout all stages of disease, suggesting that ALK mutation may be an early step in NB pathogenesis. In addition to ALK mutations, amplification of the MYCN proto-oncogene has long been associated with a significantly worse prognosis. However, the precise role these genetic aberrations play in NB pathogenesis remains unknown. Neuroblastoma arises from neural crest stem cells (NCSCs). Because the neural crest is a transient embryologic structure that undergoes differentiation in utero, pathogenesis studies using human NCSCs have not been possible until recently. Over the past several years, however, derivation of NCSCs in vitro has become feasible. The objective of the proposed studies is to determine the role that ALK mutations with and without MYCN over-expression play in the earliest stages of neuroblastomagenesis. Our long-range goal is to develop effective new therapies for patients with relapsed or refractory high-risk neuroblastoma. The objective of this application is to explore the role of known NB-specific mutations in the earliest stages of neuroblastoma pathogenesis in a relevant human model system. Our central hypothesis is that ALK mutations are an early event in the malignant transformation of NCSCs and that understanding the effects of these mutations will provide critical insights into the biological variability of this tumor that can ultimately be exploited in new therapies. To achieve the objective of this application, we will pursue the following two specific aims: 1. Determine the effects of ALK mutations with and without MYCN over-expression on the growth and differentiation of human neural crest stem cells. To perform these studies we will use previously derived induced pluripotent stem cells. These cells will be differentiated into NCSCs following established protocols. The critical step in the isolation of the final NCSCs involves using the BD FACSAria™ cell sorter to sort for the appropriate population of CD56/CD273 +/+ cells using BD antibodies. After sorting, we will infect these NCSCs with Gateway-modified lentiviral (pLenti6.3/V5 DEST) vectors containing ALK mutations, as well as with a tetracycline-inducible MYCN plasmid. Importantly, these constructs are already made and have been used successfully in NIH 3T3 cells (manuscript in preparation). We will measure the effects of these mutations on cell growth and proliferation, apoptosis, transformation, and terminal differentiation. Successful completion of these experiments will provide valuable information on the effects of ALK mutations and MYCN expression on the earliest stages of neuroblastoma
development, as well as validate the use of these human NCSCs as a powerful in vitro model for the study of neuroblastoma pathogenesis. 2. Identify downstream genetic pathways affected by ALK mutations. To achieve this aim, we will perform flow cytometry analysis using BD reagents as well as gene expression profiling of normal and mutant NCSCs to identify down-stream pathways that are activated by ALK mutation and/or MYCN over-expression. Using our BD FACSCalibur™ cell analyzer as well as the core facility FACSAria cell sorter and BD embryonic and neural stem cell specific antibodies, we will measure the changes in stem cell marker expression in cells before and after introduction of mutations. Briefly, we will use Illumina expression arrays to detect expression changes induced by ALK mutations and/or MYCN expression. These will be compared to previously performed gene expression profiling of neuroblastoma tumors. Significant changes will be validated by real-time qRT-PCR. Successful completion of this aim will provide the critical preliminary data for further studies investigating the role of discovered genetic pathways in neuroblastoma development. Successfully completing these two aims and identifying the sequence of critical steps that shape the development of neuroblastoma from its earliest lineage is the critical next step towards achieving our long-range goal of developing effective new treatments for high-risk neuroblastoma. Our expectations are that successful completion of this research will provide us with a unique and validated in vivo neuroblastoma stem cell model, along with the necessary preliminary data to pursue NIH funding for further studies of the novel genetic pathways that have been identified. As the genomic landscape of neuroblastoma becomes increasingly well characterized, each of the genetic aberrations found will need to be tested, singly and in combination, not in cell lines which are far too late in the oncogenic pathway, but in stem cells that were previously out of our reach. This reverse genetics approach reenacts the natural pathway of pediatric developmental tumors, and we expect that it will shed new light on its causes and ultimately offer novel treatment opportunities. BD flow cytometry reagents are critical to the success of the proposed research and the support from BD will be instrumental in advancing our understanding of this devastating childhood cancer. The BD Biosciences Research Grant Program aims to reward and enable important research by providing vital funding for scientists pursuing innovative experiments to advance the scientific understanding of disease. Visit bdbiosciences.com/grant to learn more and apply online.
