Targeting Undruggable SMAD4 HyperPPI with Myhre iPSC Biosensors and Organoids

About This Grant

SUMMARY Adaptor proteins that function exclusively through protein–protein interactions (PPIs) comprise one of the largest classes of undruggable targets. SMAD4, the central adaptor in the canonical TGF-β signaling pathway, exemplifies this challenge. Germline mutations in SMAD4 cause Myhre Syndrome (MS), a rare autosomal dominant multisystem disorder characterized by severe fibrosis, vascular stenosis, restrictive cardiomyopathy, and respiratory complications. Our recent studies identified SMAD4-I500V as a gain-of-function mutation that drives a hypermorphic SMAD4–SMAD3 interaction (hyperPPI), leading to excessive TGF-β signaling output. This discovery establishes a new paradigm in Myhre biology, in which hyperPPIs can function as pathogenic drivers and potential therapeutic targets. However, no physiologically relevant platforms currently exist to interrogate SMAD4-I500V hyperPPI in the patient context or to discover small-molecule modulators that could normalize this signaling axis. This proposal will establish two complementary, patient-derived experimental systems that together address this critical gap. In Aim 1, we will develop the Myhre iPSC hyperPPI Biosensor System (MiBS) by CRISPR-engineering Myhre patient–derived and sibling control induced pluripotent stem cells (iPSCs) with live-cell biosensors for allele-specific monitoring of SMAD4-I500V/SMAD3 hyperPPI. MiBS will enable multiplexed, quantitative, and high-throughput detection of pathogenic SMAD4 interactions in their native genomic and cellular context, providing an unprecedented platform for chemical screening and mechanistic dissection. In Aim 2, we will establish the Cardiac Organoid Phenotyping System (CoPS) by differentiating Myhre and control iPSCs into multicellular 3D cardiac organoids that recapitulate cardiomyocyte, fibroblast, and endothelial interplay. CoPS will be used to define functional disease phenotypes—including contractility, calcium handling, and electrophysiology—as well as molecular signatures of fibrosis, vascular integrity, and cardiomyocyte maturation. Single-cell transcriptomic profiling will further resolve lineage-specific alterations and disrupted intercellular communication networks. By integrating MiBS and CoPS, this project will generate the first physiologically and mechanistically relevant platforms to model SMAD4-I500V hyperPPI in Myhre Syndrome. These tools will illuminate how undruggable adaptor proteins drive rare disease, while establishing scalable, HTS-compatible systems for future therapeutic discovery. More broadly, the approaches developed here are generalizable to other adaptor proteins and PPI-driven pathologies, offering a conceptual and experimental framework for expanding the target landscape to previously intractable biology.