A Ligandable PNT-Domain Establishes ERG as a Directly Targetable Oncogenic Driver in Prostate Cancer

The TMPRSS2:ERG gene fusion, present in approximately 50% of prostate cancers in patients of European ancestry, drives oncogenesis through aberrant overexpression of the ERG transcription factor. Despite its role as a truncal oncogenic driver, ERG has long been considered undruggable due to the absence of enzymatic activity and an apparent lack of ligandable pockets. Here, we demonstrate continued dependency on ERG in metastatic prostate cancer and identify a druggable pocket within its structured N-terminal Pointed (PNT) domain. Using an inducible shRNA system in TMPRSS2:ERG-positive VCaP cells, we show that ERG depletion causes profound growth inhibition in vitro and in vivo. To therapeutically exploit this vulnerability, we conducted a domain-focused differential scanning fluorimetry screen targeting the ERG PNT domain, followed by structure–activity relationship optimization. This approach yielded PBITE-1 (PNT-Binding Inhibitor of the Transcription factor ERG), a lead small molecule that selectively binds the ERG PNT domain in biochemical assays. NMR chemical-shift perturbation mapping and molecular docking revealed that PBITE-1 engages a discrete, solvent-exposed surface comprising two α-helices and an adjacent flexible loop, thereby defining a coherent ligand-binding pocket within the PNT domain. In cellular models, PBITE-1 directly engaged ERG, selectively inhibited proliferation and invasion, and induced apoptosis in ERG-driven prostate and hematologic malignancies. PBITE-1 also potently suppressed growth of ERG-positive prostate organoids derived from genetically engineered mouse models, as well as patient-derived human prostate cancer organoids. Furthermore, PBITE-1 treatment significantly induced tumor cell apoptosis in vivo in VCaP xenograft models. These findings establish the ERG PNT domain as ligandable and provide the first preclinical evidence that ERG is directly targetable by small molecules, enabling future development of ERG-directed inhibitors and targeted protein degraders.