Basic Biology I
Tissue repair and regeneration critically depend on the recruitment and function of stem cells that reside within adult tissues. For example, neural stem cells are localized in distinct locations in the adult brain, the so called neurogenic niches. They are able to reproduce (self-renew) throughout life and to differentiate into functional cell types of the brain such as neurons, astrocytes and oligodendrocytes. Therefore, it may be possible to pharmacologically mobilize these stem cells to compensate for damage in the central nervous system, for example after stroke. Moreover, functional similarities exist between normal stem cells and so called cancer stem cells that can initiate aggressive brain tumors and fuel tumor growth. However, little is known about signals and factors controlling the balance between stem cell self-renewal and differentiation, and how normal stem cells can develop into a source of cancer. Taking the biological complexity of mechanisms regulating stem cells into account, unbiased large-scale screens are a promising tool for revealing key mechanisms controlling cell fate decisions in normal and cancerous brain stem cells in culture and ultimately in animal models. To probe these interrelated aspects of brain stem cell biology, we will use unbiased cell-based screens that identify small molecule modulators and genes which control the balance between human adult neural progenitor replication, growth and differentiation, and that induce differentiation or programmed cell death of patient-derived brain cancer stem cells. We will investigate the effects of newly identified lead compounds or genes on stem cell behavior and we will elucidate their specific mechanisms of action. Chemically optimized molecules will also be studied in animal models for example upon transplantation of human cancer stem cells into the brains of rodents or upon pharmacological treatment of mice showing neurological symptoms. This approach should provide new insights into those factors that control stem cell biology in normal and disease states, and may accelerate the development of novel and more effective therapeutic options for the treatment of aggressive brain tumors, as well as of neurodegenerative diseases.
Statement of Benefit to California:
In the United States approximately 1.2 million people aged 18 years and older are diagnosed annually with adult onset brain disease or disorders including brain tumors (~35,000 people diagnosed annually), epilepsy (~135,000 people diagnosed annually), Huntington's disease (~30,000 people are currently living with the disorder), stroke (~600,000 people diagnosed annually), and traumatic brain injury (~80,000 people diagnosed annually). An estimated 13% - 16% of the United States and California households may currently be dealing with the burden of long-term care (medical and non-medical care) to a family member suffering from a brain disease. Hence, there is a pressing need for non-invasive treatment strategies and one such example is regenerative medicine in which new cells such as neurons and oligodendrocytes are generated to replace tissues lost to degenerative diseases or aging. So far, the pharmaceutical industry has been reluctant to invest into this approach and without government funding of basic research in regenerative medicine, progress is likely to be hindered. A regenerative therapy approach requires both an understanding of the genes and pathways that control stem cell biology and the identification and chemical optimization of drug-like small molecules promoting the mobilization of tissue stem cells, for example neural stem cells residing in the adult brain. To this end, we will use unbiased cell-based screens that provide a powerful strategy for identifying small molecule modulators and genes which control stem cell fate decisions such as self-renewal and differentiation. Given the overlapping developmental pathways shared by normal and cancer-initiating stem cells, we will pursue screens of both in parallel as they share common pathways, screening strategies and methodologies. The chemical and biological screens also complement each other well, are synergistic and have historically often provided distinct insights. Thus, our proposed strategy may shed new light on the link between stem cell biology, tumorigenesis, and chemical strategies for intervention in disease. By using biological and chemical approaches synergistically, we hope to accelerate the development of novel and more effective therapeutic options for treatment of brain diseases such as brain cancer and neurodegenerative disorders.
The goal of this proposal is to elucidate the signals that regulate self-renewal and differentiation in adult neural progenitor cells (NPCs) and brain tumor stem cells (BTSCs). By gaining a mechanistic understanding of these processes, the applicants hope to accelerate the development of novel and more effective therapeutic options for treatment of brain tumors and neurodegenerative diseases. In the first aim, the applicants propose to expand and characterize NPCs and BTSCs in vitro while developing unbiased screens for factors that control neural proliferation and plasticity. Next, the applicants propose to utilize high throughput chemical and functional genomics screens to identify small molecule modulators and genes that control the balance between self-renewal and differentiation in NPCs, as well as differentiation and apoptosis in BTSCs. For the third Aim, the applicants will explore the effects of the newly identified hit molecules or gene knockdowns on cellular processes in order to elucidate their mechanisms of action. Finally, the most promising of these candidates will be selected and tested for efficacy in one or more models of brain cancer or neurodegenerative disease. Although the reviewers appreciated the timely and important topic of this application, the sweeping scope and lack of mechanistic focus were thought to significantly limit its potential for impact. Their enthusiasm was further diminished by the fairly uninnovative approaches that were proposed as well as what was perceived as a serious methodological flaw: As the applicants had not demonstrated an ability to propagate cells without reliance on growth factors and mitogens, the reviewers were not convinced that any data obtained would be meaningful, as the presence of these agents would almost certainly obfuscate and confound interpretation of the behaviors to be analyzed. In general, the feasibility of this effort was significantly criticized, particularly as each Aim was contingent on the success of the one preceding. One reviewer highlighted weaknesses in the preliminary data, which were described as neither novel nor hypothesis-generating. Another reviewer was not convinced that the described cell culture conditions would permit the expansion and self-renewal of brain tumor stem cells in sufficient quantities to screen the number of compounds proposed. Furthermore, it was not clear whether the low incidence of cancer stem cells in the tumor cell populations would preclude the identification of compounds that could select for their growth. Several reviewers indicated that the proposed assays seemed ill-considered and were described in a very cursory manner, with little attention given to the number of cells that would be needed per well, the number of replicates required, etc. They noted that the budget and scope were excessive and suggested that a more targeted approach would be far less costly and present a shorter and more efficient route to a mechanistic understanding. The PI was described as a superb chemist with a highly regarded and productive track record. The research environment was also found to be exceptional. Reviewers were concerned, however, that two postdoctoral fellows who would perform the day-to-day work have not been named, whereas the designated associate has limited experience with brain cancer research. The addition of a stem cell expert and/or a senior developmental neurobiologist or cancer biologist to the team would have alleviated these concerns considerably. In summary, the reviewers felt that this application addressed an important topic but was limited in impact and feasibility due to methodological flaws, an overly broad and unfocussed approach, and a lack of vital expertise within the applicant team.