A few of our ongoing projects are detailed below.
Identifying Therapeutic Targets for Wnt-driven Disease.
A major focus of our group’s current work is the identification of novel Wnt pathway components that may serve as therapeutic targets for Wnt-driven diseases. We have identified previously unknown Wnt pathway activators through genetic and cell culture-based screens in collaboration with Dr. Ethan Lee at Vanderbilt University and Dr. David Robbins at The University of Miami. We are harnessing the rigor of Drosophila genetics to test the in vivo roles of novel enzymes that either work with beta-catenin to promote Wnt target gene regulation or regulate the activity of the Wnt receptor complex, and we are investigating their promise as therapeutic targets for Wnt-driven diseases.
Function of the Adenomatous polyposis coli Tumor Suppressor in Wnt signaling.
In the classical model for Wnt signaling, the primary role of APC is to act, together with the concentration-limiting scaffold protein Axin, in a “destruction complex” that directs the phosphorylation and consequent proteasomal degradation of the transcriptional activator beta-catenin, thereby preventing signaling in the Wnt-off state. Following Wnt stimulation, Axin is recruited to a multiprotein “signalosome” required for pathway activation.
However, an alternative model in which APC has additional roles is supported by the fact that mutations in APC in nearly all colorectal carcinomas retain the APC amino-terminus while eliminating the carboxyl terminus. Despite intense effort over two decades, the molecular mechanisms by which APC destabilizes beta-catenin remain a mystery.
Our lab developed a powerful in vivo Drosophila model to elucidate APC function in Wnt signaling. We have made three major discoveries that have elucidated APC function. First, we found that APC has dual negative and positive roles that are essential for Wnt signal transduction. Second, building on our finding that APC not only inhibits, but also promotes Wnt signaling in vivo, we discovered that APC is essential for the phosphorylation of Axin by GSK3, a critical step in Axin regulation that is required for both the unstimulated and Wnt-stimulated states. Third, we demonstrated that APC is essential not only in the destruction complex, but also for the rapid transition in Axin that occurs after Wnt stimulation and the resulting association of Axin with the Wnt co-receptor LRP6, one of the earliest steps in pathway activation. Therefore, this requirement for APC in Axin regulation through phosphorylation both prevents signaling in the Wnt-off state and promotes signaling immediately following Wnt stimulation. These unanticipated findings indicate that the functions of the essential tumor suppressor APC are much broader than previously known, transform the classical model of Wnt signaling, and are relevant for the development of novel therapeutics for Wnt-driven disease.
Tankyrase: A Therapeutic Target in Wnt-driven Disease.
Axin regulation is essential for Wnt/beta-catenin signaling and thus relevant for therapeutic intervention; however, our understanding of this process has been impeded by inability to detect endogenous Axin in vivo, presenting a major obstacle in the Wnt signaling field. To overcome this gap, my lab developed a unique in vivo system for detecting epitope-tagged functional Axin expressed at physiological levels. This assay overcame a long-standing barrier in the field, as all previous in vivo studies relied on Axin overexpression, which disrupts physiological regulation. For the first time in any experimental system, our model allowed visualization of the rapid changes in Axin that are induced immediately following Wnt stimulation in vivo. In addition, we developed sensitive Axin antibodies that allow unprecedented detection of endogenous Drosophila Axin by immunostaining and immunoblotting.
We investigated the function of one of the most promising therapeutic targets in Wnt signaling, the ADP-ribose polymerase Tankyrase, with our new experimental system. Groundbreaking work by investigators at Novartis Pharmaceuticals had revealed that ADP-ribosylation mediated by Tankyrase targets Axin for degradation, thereby stabilizing beta-catenin and promoting Wnt signaling. Small molecule inhibitors of Tankyrase thus impede the Wnt-dependent proliferation of cancer cells, raising the exciting possibility that these agents hold significant clinical promise. However, due to functional redundancy in vertebrate Tankyrase homologs, the physiological role of Tankyrase in Wnt signaling was unknown. Our studies in Drosophila provided in vivo evidence that supports the Novartis conclusions. In addition, and to our surprise, we also found that the levels of ADP-ribosylated Axin increase rapidly in response to Wnt stimulation in both Drosophila and human cells, and that ADP-ribosylation enhances the association of Axin with the Wnt co-receptor LRP6 following Wnt stimulation, a step important for signalosome formation. Our findings support a new model in which Tankyrase not only targets Axin for proteolysis under basal conditions, but also enhances the role of Axin in the activation of signaling following Wnt exposure. In addition to controlling Axin levels, Tankyrase-dependent ADP-ribosylation promotes the reprogramming of Axin following Wnt stimulation. Therefore, Tankyrase inhibition blocks Wnt signaling not only by increasing destruction complex activity, but also by impeding signalosome assembly. This discovery forces revision of our understanding of Axin regulation, Tankyrase function in the Wnt pathway, and the mechanistic basis for the clinical effectiveness of small molecule Tankyrase inhibitors.
Wnt Gradients Regulate Intestinal Development and Homeostasis.
In the classical model, secretion and spreading of Wingless to cells distant from its source of synthesis is essential for long-range signaling. However, this widely-held tenet was upended recently by a surprising finding: replacement of wild-type Drosophila Wingless with a membrane-tethered form produces viable adults with normal external morphology, supporting the opposing model that Wingless spreading is dispensable for tissue patterning.
However, whether this dispensability of Wingless spreading holds true broadly, in particular for the formation of internal organs is not known. To tackle this fundamental issue, we are using a powerful new model that we developed in the adult Drosophila intestine. The Drosophila gut, like its mammalian counterpart, is subdivided into compartments with distinct functional roles, anatomy, physiology, cell types, and gene expression patterns. We discovered that the Wingless/Wnt ligand is expressed at the boundaries between all major compartments in the adult intestine. We also found that Wingless has essential roles during development and homeostasis of the intestine, non-autonomously controlling stem cell proliferation inside compartments, and autonomously specifying cell fate near compartment boundaries. Since Wingless gradients in the gut persist throughout the lifetime of the organism, despite the weekly turnover of the entire intestinal epithelium, and the injury that can result from environmental assaults including toxins and infection, we are currently testing whether Wingless spreading is indeed dispensable for cell specification in this adult gut model. To learn more, click below for Yashi’s plenary talk at the 2018 Annual Drosophila Research Conference, courtesy of the Genetics Society of America: