John Blenis - US grants

The long-term objective of this research is to understand the molecular basis of how oncogene products interfere with the normal regulation of intracellular signal transduction pathways. These studies focus on the mechanism of regulating 40S ribosomal protein S6 protein kinase activity in cells incubated with growth factors or phorbol esters or in cells expressing the Rous sarcoma virus transforming gene product, pp60v-src. The key to this understanding will require continued purification of the S6 kinase for characterization as well as production of antibodies. Classical biochemical approaches for enzyme purification will continue to be used. Strategies for producing polyclonal and monoclonal antibodies with limited quantities of antigen will be modified from existing protocols. Antibodies will be used to immunoprecipitate biosynthetically-labeled S6 kinase from culture cells, to identify src- and growth-regulated post- translational modifications, to screen expression libraries for S6 kinase cDNA, to perform immuno-cytological studies and to improve purification protocols if required. In parallel with S6 kinase purification, pharmacological agents will be used to study possible interactions of S6 protein kinase with other potential signal transducers such as protein kinase C, cAMP-dependent protein kinase, Ca2+/calmodulin-dependent protein kinases and G- proteins at a biochemical and cell biological level. Additional S6 protein kinase substrates will be identified by a combination of in vitro protein phosphorylation and two-dimensional gel analysis of phosphoproteins in 32Pi-labeled cells in which S6 protein kinase activity has been stimulated. The pharmacological studies and cell growth variants will be particularly useful in the latter approach as the S6 kinase can be stimulated under conditions not affecting the activity of other signal transducing proteins. Finally, the growth-stimulated phosphorylation of nuclear proteins and the enzymes responsible for these modifications will be studied. Interest will focus on nuclear matrix or DNA-binding phosphoproteins potentially involved in regulating gene expression. Gel retardation studies will be used to identify 32-Pi- labeled proteins which bind to the control elements of growth- regulated genes. These studies will strengthen our understanding of the molecular basis of communication within cells and will provide a biochemical means for elucidating the molecular mechanism of src-induced oncogenesis.

The long-term goals of this proposal are to define the mechanism of regulation of the 70kD-S6 protein kinases (pp70S6k) in response to a variety of growth modulators including platelet-derived growth factor (PDGF), epidermal growth factor (EGF), nerve growth factor (NGF), insulin, interleukin2 (IL-2) and tumor promoting phorbol esters. These growth modulators have been previously shown to regulate pp70S6k activity,, but how this occurs and what role the signalling pathway that regulates pp70S6k plays in the physiological response of cells to these factors in not known. Our immediate goals are to generate and express by various means, different forms of pp70S6k (wild type, constitutively-activated and dominant-interfering) in cultured cells to help define the roll of this protein kinase in cell physiological processes; to further define how pp7OS6k is regulated by phosphorylation; to identify and characterize the pp70S6k-protein kinases; and to identify and characterize other components of this important signalling system. Defining the components that regulate pp70S6k activity and the further characterization of the enzyme itself may lead not only to a better general understanding of cell growth control and its obvious relevance to the cancer problem, but also may be particularly relevant with regards to our understanding of the immune system and insulin responsiveness. Recent genetic studies in Drosophila indicate that the 40S ribosomal protein S6 (the only known target of pp70S6k) may act as a tumor suppressor in the immune system. This observation may be related to the inability of unphosphorylated S6, within the 40S subunit, to translate specific mRNA with unique 5' untranslated regions. The hypothesis is that S6 phosphorylation or removal of the protein would relieve this inhibition of translation. Additionally, the ability of the T cell immunosuppressant rapamycin to potently inhibit IL-2-dependent T cell proliferation and very specifically, pp70S6k activity, suggests an important role for pp70S6k in T cell proliferation. One of the apparent defects in some insulin-resistant humans is the inability of a normal insulin receptor to activate pp70S6k as seen in insulin-responsive humans. Additionally, specific inhibitors of the growth-regulated lipid kinase phosphatidylinositol 3-kinase, not only antagonize IL-2-, insulin-, PDGF- and NGF-mediated activation of pp70S6k, but also interfere with insulin- regulated translocation of the glucose transporter GLUT4, PDGF-stimulated actin cytoskeleton reorganization and IgE-mediated histamine release in different cell systems. Thus it is clear that defining this signalling pathway will improve our understanding of how pp70S6k is regulated and its potential downstream effects on translation, transcription and in general growth control, as well as improve our understanding of processes regulating protein sorting/secretion and cell shape.

Cytoplasmic and receptor tyrosine kinases, Ras small G proteins, heterotrimeric G proteins, and tumor promoting phorbol esters are among the major regulators of various signaling systems that modulate cell growth, differentiation and development. Inappropriate regulation can result in a variety of diseases due to improper differentiation/development or malignant transformation. One of these signaling systems, often referred to as the MAP kinase pathway, is composed of a regulated serine/threonine kinase module, downstream of the c-Ras protooncogene. Presented simply, this module consists of the protooncogene c-Raf - greater than the dual-specificity kinase MEK - greater than the mitogen-activated kinase, ERK. An ERK-activated kinase, RSK, lies downstream of the ERKs. ERK and RSK translocate to the nucleus in response to mitogens, providing a mechanism for signal transduction from the cytoplasm to nucleus. The research proposed in this application addresses issues regarding the regulation of ERK/RSK activation and downstream signaling. The first objective is to characterize novel inputs into the ERK pathway, downstream of Ras, Raf and MEK, that play an important role in the activation kinetics of ERK and RSK, and subsequent nuclear signaling. An activity that is inactivated upon mitogen stimulation and that negatively regulates the ERKs has been previously identified. Experiments are proposed to biochemically or molecularly identify ERK phosphatases or noncatalytic inhibitors that exhibit the properties of this inhibitor. Candidate molecules will also be examined. The second objective of this proposal is to thoroughly characterize the regulation and biological function of RSK, about which little is known. Experiments proposed to characterize RSK include: structure/function of analysis, identification and mutational analysis of novel, ERK-mediated, and auto phosphorylation sites, characterization of dominant-negative RSK alleles and either effects of downstream targets, the mechanism of RSK nuclear translocation, the regulation of gene expression by RSK, and the role of RSK in regulating cell proliferation. The third major objective of this proposal characterizes nuclear signal transduction by ERK and RSK. The focus of this aim will be on the characterization of c-Fos phosphorylation by ERK and RSK, and the subsequent recruitment of a novel Fos kinase activity. Experiments described are aimed at identifying the Fos kinase using biochemical or molecular approaches. Candidate molecules will also be examined.

DESCRIPTION (provided by applicant): Many human diseases result from improper regulation of cell growth (an increase in cell mass and size), proliferation, migration, survival and death. These processes are critically regulated by a complex containing the mammalian target of rapamycin (mTOR), Lst8 and Raptor, called the mTORC1 complex. The S6 protein kinases (S6K) are major effectors of mTORC1. The mTORC1/S6K signaling system is the cell's central integration point for multiple homeostatic inputs, sensing growth factor availability, energy levels, and amino acid sufficiency. Hyperactivation of mTORC1/S6K signaling is a common feature of nearly all human cancers. mTORC1 inhibitors, such as rapamycin and its analogs, are currently being clinically evaluated for the treatment of cancer. While the inhibitors have exhibited some promise, rapamycin- insensitive mTOR signaling also influences tumorigenesis, and feedback loops exist that up- regulate survival pathways following rapamycin treatment. Thus, additional therapeutic agents targeting other components of this pathway are needed. By taking a systems-wide approach towards defining mTORC1/S6K pathway regulation and the mechanisms by which this signaling system modulates various biological processes, we hope to provide insights that will lead to the identification of novel therapeutic strategies for the treatment of mTORC1/S6K- dependent cancers and other metabolic disorders. Aim 1 will focus on the connection between S6K1 signaling, gene expression and cell growth control through an S6K1-specific interacting protein SKAR. Approaches are described that investigate the role of SKAR and S6K1 in the regulation of mRNA biogenesis and protein translation. This aim also sets the foundation for how we will approach all mRNA binding proteins linked to the mTORC1/S6K signaling system identified in Aim 2. The approach outlined in Aim 2 combines a variety of biochemical purification approaches with mass spectrometry analysis to identify and validate a common set of proximal upstream regulators and downstream effectors of the S6K signaling system. The use of multiple converging lines of investigation will focus our efforts on the most critical components of the pathway, allowing us to dissect how S6K regulates so many disparate cellular processes. Aim 3 utilizes RNAi-based genetic approaches to elucidate the mechanism of homeostatic regulation of the mTORC1/S6K pathway. To this end, we have developed a sensitive, high- throughput, image-based screening strategy for monitoring S6K activity in vivo. We propose to utilize this unique assay to broadly interrogate mitogen- and nutrient-regulated inputs into the mTORC1/S6K pathway. PUBLIC HEALTH RELEVANCE: We hope that the studies outlined in this proposal will deepen our understanding of the defects in mTORC1/S6K signaling that are responsible for cancer progression and cell growth-associated diseases, such as the childhood cancer predisposition syndrome Tuberous Sclerosis. These studies will also impact our understanding of other metabolic diseases linked to S6K, such as diabetes and obesity. We believe such mechanistic insight will open the door to the identification of novel therapeutic strategies for inhibiting the growth factor and/or amino acid sensing arms of the mTORC1/S6K signaling network.

[unreadable] DESCRIPTION (provided by applicant): Mammalian target of rapamycin (mTOR) is a critical regulator of cell growth and proliferation, serving as the central integration point for multiple homeostatic inputs, including growth factor availability, energy levels and amino-acid sufficiency. Constitutive, unregulated mTOR kinase activity is a nearly universal feature of cancer cells, and defects in the mTOR signaling network have also been implicated in metabolic disorders and benign tumor syndromes. For these reasons there has been intense interest in the clinical application of derivatives of the natural product rapamycin which inhibits some aspects of mTOR-mediated signal transduction. While rapamycin has shown promise as an anti-cancer agent in clinical trials, the potential contribution of rapamycin-insensitive aspects of mTOR signaling to cancer progression, including the recently identified feedback loop involving the direct activation of Akt by mTOR, points to the critical need for additional therapeutic avenues in the treatment of diseases associated with deregulation of the mTOR signaling network. In order facilitate the identification of additional small molecules targeting mTOR, we will develop a high throughput, cell-based assay for the quantitative detection of rpS6 phosphorylation as a measure of the activation state of the mTOR pathway. Using this assay, we will perform a series of pilot studies, starting with known inhibitors of mTOR signaling, such as rapamycin and wortmannin, and culminating in pilot screens of a library comprising 200 commercially available kinase inhibitors, as well as a larger library of chemically diverse bioactive compounds. In addition, we will develop a series of secondary assays, combining high content imaging, biochemical assays, and cell based functional readouts, to evaluate targets identified in our high throughput assay for their ability to modulate mTOR signaling. Particular emphasis will be placed on identifying compounds that are able to act as 'broad spectrum' mTOR inhibitors that block the activity of both mTORC1 and mTORC2. Specific inhibitors identified through this work will not only serve as valuable research tools but may advance the treatment of diseases involving hyperactive mTOR signaling. In many cancers, the defective regulation of a key protein in the cell known as mTOR directly contributes to the uncontrolled growth of malignant cells. Rapamycin is a drug that blocks some, but not all, cellular functions of mTOR and has shown some promise in cancer treatment, but it is not always effective. Our work is aimed at finding new drugs to specifically inhibit all aspects of mTOR activity, with the goal of improving treatments for cancers involving mis-regulation of mTOR signaling. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Pulmonary Lymphangioleiomyomatosis (LAM) is a slowly progressing disease characterized by cystic destruction of the lungs and eventual respiratory failure, and affects 3.4-7.8 per million women. Biochemically, LAM is caused by loss of function of the Tuberous Sclerosis Complex (TSC) and appears to require activation of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway. Despite encouraging clinical trial results from the use of mTORC1 inhibitors to treat LAM, these drugs are limited for two reasons: first, rapamycin-based treatments are not curative as tumors regrow following cessation of treatment; and second, long-term rapamycin usage has tremendous side effects including immune disorders and diabetes. To resolve this currently, unmet clinical need, this project will investigate three unresolved mechanistic questions, with the long-term goal of developing long-lasting therapies for LAM. The first aim will determine the contribution of glutamine (Gln) metabolism in AML and LAM cells and its importance in cellular survival as inability of mTORC1-targeted therapies to induce toxicity remains an unaddressed hurdle. This aim will investigate the mechanism of Gln metabolism in these cells as well as new approaches to target the growth and survival of AML and LAM cells via pharmacologic and shRNA methods, both in vitro and in vivo. The second aim will garner a greater understanding of how the mTORC1 pathway regulates the alternative splicing of key genes involved in the benign metastasis phenotype seen in LAM. While mRNA splicing is highly regulated and pervasive (occurring in ~95% of human genes and frequently deregulated in cancer), the mechanisms causing deregulation are unknown. Using novel hits from a phospho-proteomic screen we recently completed, this aim will investigate how the mTORC1 pathway regulates the splicing of key genes involved in the benign metastasis phenotype of LAM. Finally, the third aim will identify key mechanistic insights into how estrogen contributes to the pathogenesis of LAM, which occurs almost exclusively in females during childbearing years. The aim will investigate the role of estrogen receptor signaling via the ERK-Fra1-ZEB pathway, in the context of mTORC1 hyperactivation, in regulating LAM cell survival, migration and invasion. In addition to addressing basic questions of LAM biology, each of these aims has clinical implications, and in two of the aims animal studies have been proposed to translate the cell biology discoveries into relevant in vivo models. In conclusion, there's a great need for greater understanding of the biology of LAM, and the expectations are that successful completion of the proposed work will impact LAM treatment through identification of new biomarkers as well as novel drugs that can specifically eliminate abnormal cell growth, migration and tumor formation in TSC and LAM patients.

ABSTRACT The past decade, an explosion of research and technological achievements in molecular oncology and genomics have offered an unprecedented opportunity to transform patient care. This shift in the traditional cancer research paradigm, demands for more sophisticated skills to be acquired by cancer scientists. We are requesting support via a new T32 training grant for the research training of four postdoctoral fellows in Molecular and Translational Oncology Research (MTOR) at Weill Cornell Medicine (WCM). WCM will provide funding for an additional fifth training slot. The objective of this program is to recruit the best possible candidates with a solid foundation in basic science and train them in molecular and translational oncology so that they can apply their knowledge to address important clinically unmet needs in the prevention, diagnosis, and treatment of cancer patients. MTOR trainees will receive program support for two years during which they will follow a structured and rigorous postdoctoral training path. The MTOR program is a joint effort of 17 outstanding WCM preceptors and co-mentors, and a dense network of clinical collaborators, who are international leaders in their respective fields. Trainees will further profit from the leveraging of institutional resources such as the Sandra and Edward Meyer Cancer Center and Weill Cornell's Clinical and Translational Science Center (CTSC). MTOR's highly personalized training plan will include 1) individual development plans and customized workplans; 2) rigorous translationally focused research training; 3) hands-on experience in cutting-edge methodologies; and 4) a comprehensive curriculum of innovative core and ancillary skill acquisition workshops, including writing, leadership, communication, and entrepreneurial perspectives. We anticipate a pool of at least 300 candidates. We have assembled an outstanding roster of external and internal advisory committee members with the mandate to evaluate the program and provide specific recommendations to improve its efficiency. MTOR has a strong diversity focus and through its rigorous, structured and highly personalized curriculum, seeks to train the future leaders in translational cancer research in the USA.

PROJECT SUMMARY/ ABSTRACT The current understanding of how primary tumor cells acquire the capacity to metastasize is still very limited. This becomes a major problem to find effective cures for all types of cancer, as metastatic disease accounts for the majority of cancer-related mortality. Therefore, a deep understanding of the changes that occur in the primary tumor cells that lead them to acquire the ability to escape, migrate, invade, colonize and survive other niches is necessary. Chromatin remodeling is at the intersection of signaling pathways and their ability to elicit cellular changes, such as increased survival, proliferation and metastatic properties. However, the role of chromatin remodeling factors, although studied in primary tumors, remains largely unknown in the metastatic process. Here we propose that chromatin remodeling is mediated by histone chaperones, particularly by suppression of the chromatin assembly factor 1 (CAF-1) complex. Our preliminary data shows that suppression of this complex is sufficient to induce metastatic potential in cells in culture and that CAF-1 suppression is regulated by ERK. We hypothesize that ERK2-mediated CAF-1 suppression leads to a decline in the incorporation of the canonical histone H3.1/H3.2 variants in the chromatin and an increase of H3.3 variant at the promoter of genes that drive the acquisition of metastatic properties. If true, this opens a new line of research in the metastasis field and yields novel potential therapeutic targets to be considered for cancer treatment.