Yasmath Ahmed - US grants

The Wnt signal transduction pathway directs fundamental cellular processes during development and tissue homeostasis. Deregulation of Wnt signaling results in numerous developmental defects and triggers 90% of colorectal cancers, the vast majority of which result from inactivation of the tumor suppressor, Adenomatous polyposis coli (APC). Although we have known for more than two decades that APC plays a central role in Wnt signaling, the mechanistic basis underlying APC function has remained elusive. Our long-term goal is to understand the mechanisms by which APC regulates Wnt signaling, as this knowledge is critical for devising new strategies that target Wnt-driven diseases. APC and its binding partner, Axin, establish a multiprotein ?destruction complex? that inhibits Wnt signaling by promoting turnover of the transcriptional coactivator, beta- catenin, the key mediator of Wnt pathway activation. Axin plays key roles not only in the destruction complex, but also in assembly of the ?signalosome?, a complex that activates signaling following Wnt stimulation. In the prevailing model for Wnt signaling, the role of APC is limited to promoting beta-catenin degradation and, thus, pathway inhibition in the unstimulated state. However, our unexpected findings in the current funding period force revision of this model, as we have discovered completely novel aspects of APC function. Our new findings indicate that the primary role of APC is to control both of Axin's essential roles: to inhibit signaling in the unstimulated state, and to activate signaling following Wnt stimulation. We found that APC regulates two post-translational modifications critical for Axin function in both the inhibition and the activation of Wnt signaling. In the forthcoming funding period, we propose to elucidate the novel mechanisms by which APC regulates Axin, and thereby controls assembly of both the destruction complex and the signalosome. We have developed innovative experimental systems for in vivo Wnt pathway dissection in Drosophila and cultured mammalian cells as well as in vitro pathway reconstitution with Xenopus egg extracts and purified proteins. We propose to combine our unique genetic and biochemical approaches to interrogate the functions of APC in Wnt pathway regulation. The knowledge gained from these studies on the fundamental mechanisms that underlie APC function will be invaluable in the development of future treatments for Wnt-driven diseases.

The Wnt signal transduction pathway directs essential steps in embryonic development. Inappropriate activation of Wnt signaling triggers the development of several cancers, including the vast majority of colorectal cancers. Thus, understanding the basic mechanisms that underlie Wnt pathway activation will facilitate the design of innovative strategies for the treatment of a large number of diseases. A major goal of our research is to elucidate the regulation of two distinct multiprotein complexes - termed the ?beta-catenin destruction complex? and the ?signalosome?- that are fundamental for the control of Wnt signaling in the ?off? and ?on? states, respectively. Axin, a concentration-limiting scaffold protein, plays important roles in the assembly of both complexes. How these distinct roles of Axin are coordinated remains a mystery. Regulators of Axin, including the ADP-ribose polymerase Tankyrase (Tnks) have recently emerged as promising therapeutic targets. In the current model, the sole role of Tnks is to target Axin for proteasomal degradation, and thereby to control steady state Axin levels in the unstimulated state. However, we have found, unexpectedly, that ADP- ribosylation of Axin by Tnks also promotes Axin's critical role in activation of the pathway following Wnt stimulation. Our findings force major revision of the prevailing model for Tnks function in Wnt signaling and may underlie the effectiveness of small molecule Tnks inhibitors, which are among the most promising anti- Wnt pathway agents under development. Thus, in contrast with the prevailing model, we hypothesize that Tnks plays key roles in controlling Axin activity both in the destruction complex (in the unstimulated state) and in the signalosome (following Wnt stimulation). We propose to test this hypothesis by analyzing differences in the state of Axin ADP-ribosylation under basal and Wnt-stimulated conditions. We will determine how Axin ADP- ribosylation regulates the composition and activity of the ?-catenin destruction complex and signalosome. We will address the physiological roles of ADP-ribosylation on Axin activity in the unstimulated and Wnt-stimulated states in vivo. These studies are driven by an ongoing collaboration between two scientists who have a shared interest in Wnt signaling: Yashi Ahmed, an experienced Drosophila geneticist and cell biologist, and Ethan Lee, an expert in using in vivo approaches in Xenopus and in vitro pathway reconstitution. We will combine our innovative genetic, cell biological, and biochemical approaches in vertebrate and invertebrate models to provide insight into the regulation of Wnt signaling. The proposed research has significance for the development of new therapeutic strategies for Wnt-driven diseases.

PROJECT SUMMARY The evolutionarily-conserved Wnt signal transduction pathway directs cell proliferation and differentiation during animal development and tissue homeostasis. Despite the fact that deregulation of Wnt signaling underlies numerous developmental disorders and cancers, including nearly all colorectal cancers, many of these mechanisms remain poorly understood. Thus, a deeper understanding of the mechanisms that activate this pathway will guide the development of new therapeutic strategies to combat Wnt-driven diseases. The long-term goal of the PI?s research program is to elucidate the mechanisms that activate Wnt signaling during animal development and to use this knowledge to identify control points in the pathway susceptible to therapeutic targeting in Wnt-driven diseases. In support of this effort, the PI and her laboratory group have established innovative in vivo models in Drosophila that have revealed unanticipated functions of three core Wnt pathway effectors: the tumor suppressor, APC; the scaffold protein, Axin; and the ADP-ribose polymerase, Tankyrase. Building on these findings and enabled by genome-wide screens designed to uncover new Wnt signaling regulators, the current project will address three major unsolved questions: 1) how the membrane- associated receptor activation complex is assembled and activated; 2) how the negative regulatory cytosolic beta-catenin destruction complex is inhibited upon Wnt stimulation; and 3) how the nuclear beta-catenin-TCF transcription complex is activated. To elucidate the role of previously unknown Wnt pathway activators, this project will couple genetic, cell biological and biochemical approaches with in vivo assays previously developed by the PI to monitor pathway activation within Wnt signaling gradients. It will apply an innovative focus centered on three components: a deubiquitinating complex essential for Wnt receptor stability, a ubiquitin ligase essential for signaling activation, and a kinase that activates the beta-catenin-TCF transcription complex. This work is complemented by productive collaborations with investigators who have expertise in the biochemical reconstitution of Wnt signaling, Wnt pathway analysis in vertebrate models, and in the identification of Wnt-dependent post-translational protein modifications using mass spectrometry analysis. The successful completion of this work will provide an understanding of: 1) the control of Wnt receptor activity; 2) the control of the beta-catenin destruction complex; 3) the activation of the Wnt transcription complex; and 4) novel therapeutic strategies to target Wnt-driven diseases.

PROJECT SUMMARY The evolutionarily-conserved Wnt signal transduction pathway directs cell proliferation and differentiation during animal development and tissue homeostasis. Despite the fact that deregulation of Wnt signaling underlies numerous developmental disorders and cancers, including nearly all colorectal cancers, many of these mechanisms remain poorly understood. Thus, a deeper understanding of the mechanisms that activate this pathway will guide the development of new therapeutic strategies to combat Wnt-driven diseases. The long-term goal of the PI?s research program is to elucidate the mechanisms that activate Wnt signaling during animal development and to use this knowledge to identify control points in the pathway susceptible to therapeutic targeting in Wnt-driven diseases. In support of this effort, the PI and her laboratory group have established innovative in vivo models in Drosophila that have revealed unanticipated functions of three core Wnt pathway effectors: the tumor suppressor, APC; the scaffold protein, Axin; and the ADP-ribose polymerase, Tankyrase. Building on these findings and enabled by genome-wide screens designed to uncover new Wnt signaling regulators, the current project will address three major unsolved questions: 1) how the membrane- associated receptor activation complex is assembled and activated; 2) how the negative regulatory cytosolic beta-catenin destruction complex is inhibited upon Wnt stimulation; and 3) how the nuclear beta-catenin-TCF transcription complex is activated. To elucidate the role of previously unknown Wnt pathway activators, this project will couple genetic, cell biological and biochemical approaches with in vivo assays previously developed by the PI to monitor pathway activation within Wnt signaling gradients. It will apply an innovative focus centered on three components: a deubiquitinating complex essential for Wnt receptor stability, a ubiquitin ligase essential for signaling activation, and a kinase that activates the beta-catenin-TCF transcription complex. This work is complemented by productive collaborations with investigators who have expertise in the biochemical reconstitution of Wnt signaling, Wnt pathway analysis in vertebrate models, and in the identification of Wnt-dependent post-translational protein modifications using mass spectrometry analysis. The successful completion of this work will provide an understanding of: 1) the control of Wnt receptor activity; 2) the control of the beta-catenin destruction complex; 3) the activation of the Wnt transcription complex; and 4) novel therapeutic strategies to target Wnt-driven diseases.

Project Summary Inhibition of Wnt Receptor Activation by the Tumor Suppressor Adenomatous Polyposis Coli The long-term objective of this study is to investigate how the tumor suppressor Adenomatous polyposis coli (APC) inhibits the Wnt signal transduction pathway by regulating the Wnt receptor complex (signalosome) and to demonstrate how this can be exploited to target APC mutant colorectal cancers (CRCs). Wnt signaling is essential for intestinal stem cell maintenance, whereas aberrant activation of this pathway, which occurs most frequently through mutational inactivation of APC, triggers the development of the vast majority of CRCs. In the classical model for Wnt signaling, the sole role of APC is to destabilize the key transcriptional activator in the Wnt pathway, beta-catenin. However, our recently published findings reveal an additional and entirely new function ? APC prevents the internalization and consequent activation of the signalosome, a novel role that is evolutionarily conserved. We have shown that: 1) inducible loss of APC is rapidly followed by ligand-independent signalosome activation; 2) depletion or antibody-mediated inhibition of LRP6 (a signalosome component) inhibits the stabilization of beta-catenin, the transcriptional activation of Wnt target genes, and the proliferation of APC mutant cells; and 3) in APC mutant cells, endocytosis of Wnt receptors is required for the aberrant activation of Wnt signaling. The goal of this project is to use in vitro, ex vivo, and in vivo approaches to gain a better understanding of how APC inhibits signalosome activation under physiological conditions and to determine how aberrant activation of the signalosome underlies the consequences of APC inactivation in tumors. The three specific aims are to: 1) elucidate the mechanism by which APC loss promotes signalosome assembly in CRC cells; 2) identify the APC mutant CRC cells most susceptible to LRP6 inactivation; and 3) test the efficacy of LRP6 inactivation on CRC tumorigenicity in vivo. Because the molecular mechanisms by which APC prevents the aberrant activation of Wnt signaling are important for our understanding of colorectal carcinogenesis, the knowledge gained from this study will aid in the development of new therapeutic strategies for the treatment of CRC and other Wnt-driven cancers.