Reuben J. Shaw - US grants

Studies of hereditary cancer syndromes due to inactivation of tumor suppressor genes have been some of the most informative areas of investigation in the understanding of mechanisms of tumorigenesis. Peutz-Jeghers Syndrome (PJS) is an autosomal dominantly inherited cancer syndrome which predisposes patients to intestinal hamartomatous polyps as well as a wide range of benign and malignant neoplasias. A gene responsible for PJS was recently identified on chromosome 19q13.3 and found to encode a novel serine/threonine kinase. This represents the first example of a kinase acting as a classical tumor suppressor. The goal of this study is to identify key substrates of this novel kinase which may be mediating its tumor suppressor function as well as to characterize the signaling pathways in which this kinase plays a role. Additionally, a conditional targeted disruption of the Peutz-Jeghers' gene will be created to test whether loss of function of this kinase is sufficient to predispose mice to a similar set of tumors, potentially creating a mouse model for PJS. These animals can be used to study tumor development as well as provide cells to study the effects of loss of function of this kinase in the identified signaling pathways.

DESCRIPTION (provided by applicant): Deregulation of glucose and lipid metabolism in peripheral tissues is a hallmark of type 2 diabetes. AMP- activated protein kinase (AMPK) is a master regulator of cellular and organismal metabolism controlling glucose and lipid homeostasis. AMPK is activated by low nutrients, exercise, adipokines, and by the widely used diabetes therapeutics metformin and the thiazolidinediones (TZDs). In response to these stimuli, AMPK acts in the liver to reduce gluconeogenesis and lipogenesis through poorly understood mechanisms. We, and others, identified the serine/threonine kinase LKB1 as the critical upstream kinase mediating AMPK activation. We subsequently created a genetic deletion of LKB1 in the liver of adult mice, which resulted in complete loss of hepatic AMPK activity and dramatically increased gluconeogenesis and hepatic lipid accumulation. Using these mice, we demonstrated that LKB1 was required in liver for metformin to lower blood glucose levels, the first genetic proof of a specific pathway being required for the therapeutic action of metformin. The next big question is to understand how metformin impinges on LKB1/AMPK signaling and how they in turn regulate glucose metabolism. We propose to determine the role of LKB1 and AMPK in the control of hepatic glucose metabolism and in the therapeutic action of metformin and TZDs. First, as LKB1 is known to activate 12 AMPK-related kinases in addition to AMPK, we will determine whether loss of AMPK alone mimics the effects of loss of LKB1 on glucose metabolism and the response to metformin or TZDs. To this end, we will conditionally delete LKB1 or both catalytic AMPK1 genes in the liver of adult mice. Second, using hepatocytes derived from these mice and RNAi, we will define the critical upstream and downstream components of the LKB1/AMPK pathway required for the regulation of specific metabolic processes. Finally, we will identify a number of new effectors of AMPK that control metabolism using a combination of unique proteomic approaches to purify novel AMPK substrates in addition to transcriptional profiling in our genetically defined cells following metformin or TZD treatment. These studies will better illuminate the mechanism of action of these two widely used type 2 diabetes modalities, as well as identifying many new targets for the development of future therapeutics.

DESCRIPTION (provided by applicant): Deregulation of glucose and lipid metabolism in peripheral tissues is a hallmark of type 2 diabetes. AMP- activated protein kinase (AMPK) is a master regulator of cellular and organismal metabolism which acts as sensor of cellular energy status and plays key roles in glucose and lipid homeostasis in metabolic tissues. AMPK is activated by low nutrients, exercise, adipokines such adiponectin, and by the widely used diabetes therapeutic metformin. Upon activation in liver, AMPK functions to reduce gluconeogenesis and lipogenesis through incompletely understood mechanisms. Previously, the serine/threonine kinase LKB1 was identified as the critical upstream kinase mediating AMPK activation in most mammalian tissues. Genetic deletion of LKB1 in the liver of adult mice resulted in complete loss of hepatic AMPK activity and significant increases in gluconeogenesis and hepatic lipid accumulation, while attenuating the ability of metformin to lower blood glucose. A major challenge in the field remained in decoding the molecular mechanisms through which LKB1-AMPK signaling controls metabolism. Over the past 4 years of this funding, my laboratory performed a multi-pronged screen for direct substrates of AMPK, which led to the identification and study of a number of novel AMPK substrates critical in metabolism, including Raptor, ULK1, Cry1, Srebp1, and HDACs4, 5, and 7. In this first renewal, it is proposed to further dissect the role of AMPK and related kinases in control of glucose metabolism and the therapeutic action of metformin. Given the recent advances in decoding the molecular effectors of the LKB1/AMPK signaling pathway, the relative contributions of different AMPK substrates to metabolic control and metformin's therapeutic action can now begin to be delineated. In Aim 1, the role of AMPKalpha1, AMPKalpha2, and the related SIK kinases will be compared in control of hepatic glucose metabolism using novel temporally controlled genetic mouse models. In Aim 2, we will utilize these models to define the relative requirement for LKB1 and AMPK isoforms in the therapeutic action of metformin in liver. A key aspect of this aim is a collaboration with Dr. Davi Wasserman at Vanderbilt University to define the effect of LKB1 and AMPK on metabolic flux in these models. Finally, in the Aim 3, we will expand on our recent discoveries by directly examining the relative roles of Class IIa HDAC-FOXO axis versus CRTC coactivators-CREB axis in the control of glucose homeostasis in liver. These studies disecting the role of the LKB1- AMPK pathway in liver will increase the understanding of how existing widely used diabetes modalities work, and identify critical new targets for future type 2 diabetes therapeutics.

Loss of function mutations in STK11, encoding the Ser/Thr protein kinase, LKB1 are responsible for the familial hamartoma syndrome, Peutz Jeghers Syndrome (PJS). Importantly, recent studies have revealed sporatic mutations in STK11 in a variety of human cancers, including lung adenocarcinomas. LKB1 phosphorylates and activates AMP dependent protein kinase in response to energy stress and thus plays a key role in energy homeostasis. The hypothesis guiding this proposal is that the LKB1-AMPK signaling axis evolved to limit cell growth under conditions of energy stress and that genetic or epigenetic aberrations, as well as transcriptional and post-transcriptional events that suppress LKBI function, allow tumor cells to override metabolic control mechanisms that normally limit cell growth under energy stress. The overall goal of the proposal is to elucidate the signaling and metabolic network controlled by LKB,1-AMPK and use this information to identify targets for therapeutic intervention in tumors that lack LKB1. The specific aims are: 1) to utilize technologies developed during the previous granting period to identify druggable downstream targets in the LKB1/AMPK signaling network; 2) to utilize mass spec metabolomics to identify metabolic pathways that are altered in cancers that result from loss of LKB1 and, 3) to use genetically engineered mice that develop cancers due to loss of LKBI, in the context of other genetic aberrations observed in human lung cancers, to evaluate drugs or drug combinations, predicted on the basis of in vitro studies in Aims 1 and 2.

DESCRIPTION (provided by applicant): Cancer genetics has revealed that p53 and LKB1/STK11 are the most commonly mutated tumor suppressors in sporadic human non-small cell lung cancers (NSCLC), the leading source of annual cancer deaths in the U.S. LKB1/STK11 encodes a Ser/Thr protein kinase that directly phosphorylates the activation loop of the AMP-activated protein kinase (AMPK) as well as 12 poorly understood related kinases in the AMPK family. AMPK is a master regulator of cellular and organismal metabolism that acts as a sensor of cellular energy, arresting cell growth and reprogramming metabolism when ATP levels are low. Over the past 5 years, a number of labs including ours have decoded substrates of AMPK and related kinases that mediate downstream effects on growth and metabolism and may relate to the tumor suppressor activity of LKB1, including AMPK phosphorylation of core components in the mammalian target of rapamycin (mTOR) and autophagy pathways. In addition, the front-line type 2 diabetes drug metformin has been shown to regulate cell growth in an AMPK- and mTOR-dependent manner in some settings, suggesting it may serve as a potential anti-cancer agent. Despite these direct connections between AMPK and growth regulators, there is a great deal of overlap between the downstream functions and effectors of AMPK and its 12 related kinases, so it remains unclear which of these 14 kinases that LKB1 directly activates are the most critical for mediating its tumor suppressor function. Moreover, accumulating evidence suggests that in many settings the ability of AMPK to restore metabolic homeostasis under glucose or oxygen-poor conditions may promote survival of cancer cells. Thus, the role of AMPK in tumorigenesis may be very context dependent, and a different AMPK related kinase may be more important for the ability of LKB1 to suppress NSCLC. Finally, while epidemiological data and mouse xenograft and tobacco carcinogen models support a beneficial effect of metformin, this has not been examined in a genetically engineered mouse model of a human cancer in a manner that allows one to distinguish genotype-specific therapeutic effects. Moreover, metformin and its more potent analog phenformin are mitochondrial inhibitors that affect pathways outside of AMPK, and may selectively allow for the killing of LKB1-deficient tumors as is observed in cell culture models. The specific aims are to 1) define which of the 14 AMPK family kinases are essential for the ability of LKB1 to suppression tumorigenesis in a NSCLC xenograft model; 2) genetically define the role of AMPKa1 or AMPKa2 and related family kinases in a genetic engineered mouse model of NSCLC; and 3) examine the therapeutic efficacy and genotype selectivity of AMPK-activating biguanide compounds metformin and phenformin in multiple genetic engineered mouse models of NSCLC.

DESCRIPTION (provided by applicant): The goal of the signal transduction field is to identify and characterize the biochemical and molecular basis of communication from one molecule to another as part of a signaling pathway. Over the last 30 years, this knowledge has provided a vastly improved understanding of how kinases and phosphorylation events control all known cellular processes associated with normal growth and development. Unfortunately, these pathways are also deregulated through an assortment of mechanisms, and when this occurs a variety of human developmental disorders and diseases arise. There are over 500 protein kinases in the human genome with the potential to regulate protein phosphorylation, but relatively few are well characterized. Of those that are, many are now known to possess important functions in normal development and disease. Because of the central role these enzymes play in a variety of biological processes and in many human diseases, and because protein kinases are potential targets for therapeutic intervention, a tremendous effort has been made at determining the molecular basis for information transmission and the biological processes affected. The characterization of new kinases and their functions will likely reveal even more novel therapeutic targets. The study of molecular cancer research has often been intertwined with advances in the study of kinases and phosphorylation, as it is now known that alterations in kinases are amongst the most common mutations found across nearly all known forms of human cancer. As a result, kinase inhibitors have emerged as the most prevalent form of anti-cancer agents in the past 50 years, with hundreds of new kinase inhibitors entering clinical trials every year for the past few years. These are truly exciting times in the protein kinase and protein phosphorylation arena. Thus, it is critical that the exchange of information and ideas between the leading scientists in the field occur in order for this scientific discovery o proceed. Since its inception in 1983, the FASEB summer research conference on Protein Kinases and Protein Phosphorylation (held biennially) has consistently been one of the preeminent meetings in the field of signal transduction, providing a premier venue for the communication of research findings from both academia and industry. The goal of the proposed conference is to continue this tradition of exciting and enthusiastic scientific exchange.

Salk Cancer Center ? Overall Project Summary / Abstract The Salk Institute Cancer Center is a premier cancer research center that uses innovative and collaborative approaches to tackle some of the most pressing challenges facing cancer biologists today. Researchers in the Salk Cancer Center are employing cutting edge genomic, epigenomic, transcriptome, and metabolomic approaches to molecularly characterize cancer. The goals of this research are to develop new diagnostic tools and to also define cancer subtypes so that clinicians will be able to select the most effective therapeutic approaches, while also identifying new molecular targets. Salk Cancer Center researchers are at the forefront of developing and applying these approaches; discoveries like reprogramming tumor metabolism and tumor microenvironment have the power to revolutionize diagnostics and are already demonstrating tremendous potential in the clinic. The Salk Cancer Center is building upon its outstanding foundation in basic discovery science to identify and develop new therapeutic targets for multiple tumor types. Cancer Center researchers have discovered new cancer genes, identified potential therapeutic targets, and defined new drug resistance mechanisms. During this past funding period, multiple basic discoveries from a handful of labs at the Salk Cancer Center have led to early phase clinical trials, greatly expanding translational efforts at Salk. Over the next five years, as part of the Conquering Cancer Initiative, the Salk Cancer Center will bring more of its basic research discoveries to preclinical and clinical settings. Funding from the National Cancer Institute provides the Salk Cancer Center with the organizational and financial support needed to make a significant impact on cancer diagnosis and treatment. The two reorganized research programs (Genetic, Epigenetic, and Immune Circuits in Cancer; Animal Models of Cancer and Therapeutics) provide opportunities for members to come together, maximizing collaboration and communication. The seven Shared Resources provide access to technologies, services, and expertise to enhance productivity and promote multidisciplinary interactions. Essential Developmental Funds enable the Center to remain at the forefront of cancer research. Together, these elements of the Salk Cancer Center create an environment that supports the level of scientific discovery required to make real progress in improving cancer diagnosis and treatment.

Summary / Abstract Research over the past decade has begun to reveal several direct linkages between genes mutated in human cancer and genes that control cell metabolism. The LKB1 tumor suppressor is a serine/threonine kinase mutationally inactivated in the familial cancer disease Peutz-Jeghers Syndrome, as well as in ~25% of non-small cell lung cancers, making it the third most frequent gene altered in this cancer type, which is responsible for the most deaths by cancer each year. Thirteen years ago, the Shaw lab and others discovered that LKB1 directly phosphorylates the activation loop of the AMP-activated protein kinase (AMPK) and 12 related kinases. AMPK is a serine/threonine kinase that is activated by LKB1 under conditions of low cellular energy, such as those that accompany loss of nutrients, in particular glucose and oxygen. AMPK plays a highly conserved role as an energy sensor and acts to restore metabolic homeostasis on a cellular and ultimately organismal level by downregulating anabolic biosynthetic ATP- consuming processes (like protein and lipid biosynthesis), and upregulating catabolic ATP-restoring processes (like autophagy and fatty acid oxidation). Studies by the Shaw lab over the past decade have sought to: 1) understand the mechanistic basis for how AMPK reprograms growth and metabolism by decoding direct substrates of AMPK that mediate its downstream effects, and 2) identify new cancer therapy approaches based on their understanding of the rate-limiting nodes of metabolism and growth that AMPK endogenously utilizes under low energy conditions. The Shaw lab has used a number of genetically engineered mouse models of non-small cell lung cancer to perform preclinical studies with novel cancer metabolism drugs, and this grant builds upon their expertise accumulated over the past decade. Three lines of research are proposed. First, advances in proteomics and genetic technologies will be used by the Shaw lab to conduct phospho-proteome screens in primary tumors that are with or without intact LKB1 to identify relevant targets required for tumor suppression in lung. These events will be rapidly modeled genetically in cell lines and ultimately in murine cancer models using CRISPR. Second, based on their understanding of how AMPK inhibits growth, the Shaw lab has explored the use of direct inhibitors of the lipogenesis enzyme Acetyl-CoA carboxylase (ACC) and found broad anti-cancer activity in genetic models of lung cancer. This proposal seeks to examine whether other fatty acid synthesis enzymes may offer therapeutic windows in lung cancer. Third, this proposal will explore the role of AMPK and its target the autophagy kinase ULK1 in promoting tumor cell survival, particularly in the context of therapeutic response. Altogether, these studies emphasize the need to gain a deep understanding of the molecular wiring of this signaling network and how it interfaces with key cellular processes in order to reveal novel vulnerabilities that can be exploited to selectively kill cancer cells.

Abstract Tumor cells arise upon escape from two distinct and critical barriers that limit proliferation of human cells, replicative senescence and crisis. Cells in replicative senescence arrest permanently while continuing to metabolize, triggered by short telomeres. Senescence entry however, is avoided by impairment of the main cell cycle checkpoints controlled by the p53 and Rb tumor suppressive pathways. Following senescence bypass and continued proliferation, cells undergo crisis, which is a phase highlighted by substantial telomere deprotection and widespread cell death. Crisis is a stringent tumor-suppressive barrier, as it removes the vast majority of cells that avoid senescence. However, rarely cells overcome this barrier and become neoplastic. The molecular mechanisms and pathways underlying cell death in crisis and spontaneous crisis evasion are not understood. Here, it is proposed to investigate the molecular mechanisms underlying the escape from crisis and crisis bypass, with the expectation that the resulting discoveries will have a strong impact on our understanding of the early steps in cancer development. The preliminary data presented here suggest a novel concept for replicative crisis that implicates autophagy as a major regulator of cell death. Autophagy suppression allowed cells to bypass crisis and continue to proliferate, while accumulating multiple genomic aberrations. This discovery is of profound significance for understanding how genome instability evolves during the early steps of cancer development. Furthermore, the finding suggests that autophagy inhibitors might have counterproductive effects and promote the establishment of neoplastic cells instead of eliminating them. In three specific aims it is proposed to decipher the exact signaling pathways that lead from dysfunctional telomeres to the activation of autophagy-controlled cell death (Aim 1), to determine the consequences of telomere-driven autophagy and of autophagy inhibition during crisis (Aim 2), and to understand the role of autophagy-driven cell death in crisis on tumor development in vivo (Aim 3). In summary, this grant proposal focuses on the mechanisms underlying cell death during replicative crisis, the mechanism of how autophagy is activated and regulated in response to replicative crisis, and how inhibition of autophagy during crisis enables cells with an unstable genome to escape this final barrier against tumor cell establishment and drive malignancy. We will thereby explore our novel hypothesis, in which temporary or permanent resistance to autophagic cell death is the initial event required for the emergence of post-crisis cells and an abrupt rise in genome instability, leading to the establishment of neoplastic cells.