Dissecting the structure-function relationship of the Wnt destruction complex condensate

About This Grant

Project Summary The Wnt signaling pathway has a peculiar molecular architecture: it relies on two large and highly disordered scaffold proteins, APC and Axin, that assemble into a protein machine called the Destruction Complex (DC) which regulates the interactions between pathway kinases, CK1α and GSK3β, and the transcriptional effector, β-catenin. Mutations in either of the scaffold genes drive oncogenesis across a variety of deadly cancers (e.g. colorectal, gastric, breast, melanoma, osteosarcoma) as well as diseases resulting from tissue dysbiogenesis such as Hypertrophic Cardiomyopathy (affecting 1:500 people) and Congenital Hypertrophy of the Retinal Pigment Epithelium (affecting 1-2% of populations of European ancestry). Despite the significance, we still do not grasp the answers to basic questions regarding how the DC works–how is it organized, how do mutations in these scaffolds lead to its dysregulation, and how should we treat scaffold mutations? In fact, there are currently no FDA approved drugs that target the Wnt pathway. We propose a suite of experiments to determine the structure-function relationship of the DC, taking into account our recent finding that the DC is a dynamic, biomolecular condensate. In our previous study we integrated advanced techniques for dissecting condensates, including endogenous CRISPR tagging, super resolution microscopy, optogenetic reconstitution, and partial differential equation models. These techniques enabled the detailed description of how changes in the organization of these condensates can both accelerate and inhibit Wnt signals, which suggests how changes in DC scaffolds can mechanistically lead to changes in Wnt signaling fidelity. Since then, we have used proximity labelling to identify other potential DC regulators as well as observed aberrant DC morphologies that are caused by known colorectal cancer-causing mutations in APC. Here we seek to (Aim 1) understand how DC-resident proteins affect its structure and function and (Aim 2) understand how the DC is remodeled by oncogenic mutations in APC. We employ a combination of fundamental biophysical experiments, geared towards uncovering the organizing principals of the DC, along with directly-translatable lead generating experiments, to pinpoint therapeutic targets specific to mutant APC cells. Overall, this research will significantly advance our understanding of Wnt pathway regulation and its perturbation in cancer, potentially leading to precise treatment options with fewer off-target effects that have plagued the failed attempts to drug the Wnt pathway as well as enhancing our overall comprehension of condensate biology in disease contexts.