Prostate cancer is one of the most common cancers and a leading cause of cancer death in men. Tumor cell plasticity is a key driver of prostate cancer progression, metastasis, and resistance to androgen receptor pathway inhibitors (ARPIs), yet no approved therapies directly target this process. ESE3/EHF (EHF) is an epithelial-specific ETS transcription factor highly expressed in normal prostate epithelium and frequently downregulated in aggressive primary tumors, castration-resistant (CRPC) and neuroendocrine (NEPC) prostate cancers. Reduced EHF expression is associated with attenuated androgen receptor (AR) signaling, lineage plasticity and treatment resistance. In human cell lines and murine models with prostate-specific EHF knockout, loss of EHF expression is sufficient to disrupt luminal identity and induce phenotypic plasticity and multilineage transitions, favoring tumor progression. These findings position EHF as a master regulator of epithelial cell identity and a tractable target for therapeutic cell reprogramming. To test this approach, we have developed mRNA- and DNA-based EHF delivery systems formulated in lipid-polymeric nanoparticles (LPNP) for cell cultures and in vivo administration. The activity of EHF-based constructs was evaluated across a translational preclinical platform including human and murine cell lines, cell line- and patient-derived organoids and xenografts, syngeneic murine allografts, and genetically engineered mouse models. Functional assays and drug combination studies were performed to assess cell differentiation and sensitivity to ARPIs. Multiple molecular, phenotypic, and pharmacodynamic endpoints were analyzed. mRNA- and DNA-based EHF delivery led to efficient and sustained EHF expression in human and murine models and reprogrammed tumor cells toward a luminal differentiation state, restoring canonical AR signaling and responsiveness to AR-directed therapies while suppressing phenotypic plasticity and stem-like features. Functionally, EHF delivery in vitro inhibited proliferation, tumor organoid growth, and tumor sphere formation, without affecting normal epithelial cells. Systemic delivery of LPNP-formulated EHF-encoding mRNA by intravenous injection achieved efficient, sustained, and selective intratumoral EHF expression, resulting in significant tumor growth inhibition in subcutaneous human xenografts and murine allograft models without observable toxicity. Intravenous delivery in mice also enabled efficient target engagement in metastatic lesions in bone and visceral organs, leading to a substantial reduction in metastatic burden. These data establish cell lineage reprogramming via EHF restoration as a novel modality to counteract tumor cell plasticity in prostate cancer. With a defined biomarker framework (low EHF expression, plasticity signatures) and compatibility with various delivery platforms, this approach provides a clear path toward clinical translation. This strategy represents a first-in-class opportunity to target tumor plasticity and overcome resistance in advanced prostate cancer.
Domenico Albino, Carola Musumeci, Elisa Storelli, Atik Balla, Elisa Federici, Gianluca Civenni, Daniela Impellizzieri, Giada Andrea Cassanmagnago, Marco Bolis, Steve Pascolo, Carlo V. Catapano, Giuseppina MR. Carbone. Therapeutic Reversal of Tumor Cell Plasticity by EHF mRNA Delivery in Prostate Cancer [abstract]. In: Proceedings of AACR Drug Discovery and Development (AACR D3) Conference; 2026 Jul 21-24; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(14_Suppl):Abstract nr B014.
D. Albino, C. Musumeci, Elisa Storelli et al.· Clinical Cancer Research· 0 citations
Advanced prostate cancer is a leading cause of cancer-related deaths with limited effective targeted therapies for the different molecularly defined subtypes. About 50% of prostate cancers harbor ERG gene fusions, driving sustained expression of this oncogenic transcription factor associated with tumorigenesis and cancer progression. Despite its clinical relevance, ERG remains undruggable, highlighting a significant unmet need for novel therapeutic modalities. ERG mono-methylation at lysine 362 (K362) is a unique post-translational modification catalyzed by the protein methyltransferase EZH2, frequently upregulated and co-expressed in advanced ERG-fusion positive tumors (Zoma et al., 2021). Modifying the protein secondary structure and disrupting an auto-inhibitory domain, K362 methylation promotes ERG transcriptional and oncogenic activity in cancer cells. K362 methylation also provides an accessible, tumor-specific epitope for drug discovery. To exploit this unique vulnerability, we engineered a single-chain variable fragment (scFv) mini-antibody (mERG mini-body) that recognizes mono-methylated K362 ERG with remarkable affinity and selectivity, enabling specific targeting of ERG active oncogenic state. To perform proof-of-principle studies, the mERG mini-body was delivered intracellularly via DNA and mRNA expression systems formulated with lipid-polymeric nanoparticles (LPNP). Delivery efficiency, efficacy and mechanism of action were evaluated in multiple ERG-fusion positive models, including human and murine cell lines, cell line- and patient-derived organoids, murine allograft models derived from Pb-Cre4; Pten flox/flox ; Rosa26 ERG/ERG (ERG/PTEN) transgenic mice. DNA- and mRNA-based delivery in vitro demonstrated robust intracellular expression and nuclear localization of mERG mini-body, with effective and selective engagement of methylated ERG. K362 mutations in ERG abolished target recognition and binding, confirming the mERG mini-body specificity. Target engagement resulted in inhibition of ERG-driven transcriptional programs and suppression of oncogenic phenotypes, including reduced cell proliferation, organoid and tumor-sphere growth. Pharmacodynamic responses were durable and consistent across delivery systems, supporting platform flexibility. Systemic administration of LPNP-formulated mRNA by intravenous injection to tumor bearing mice achieved efficient and selective intratumoral delivery with sustained mERG mini-body expression, target engagement, and marked tumor growth inhibition in murine allografts, without evidence of systemic toxicity. These data establish the feasibility and efficacy of this novel mini-body-based strategy as a viable therapeutic modality for targeting an oncogenic transcription factor previously considered undruggable. With a defined biomarker strategy (ERG gene fusion and K362 methylation) and leveraging on validated mRNA delivery technologies, this work highlights the potential for this first-in-class therapeutic and supports further translational development for targeting ERG-driven prostate cancer.
Carola Musumeci, Concetta Guerra, Domenico Albino, Elisa Storelli, Jacopo Sgrignani, Daniela Impellizieri, Simone Moro, Elisa Federici, Andrea Cavalli, Giuseppina MR. Carbone, Carlo V. Catapano. Suppressing ERG-driven oncogenesis by mRNA-based delivery of an intracellular mini-body targeting a unique protein methylation site in ERG fusion-positive prostate cancer [abstract]. In: Proceedings of AACR Drug Discovery and Development (AACR D3) Conference; 2026 Jul 21-24; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(14_Suppl):Abstract nr B094.
C. Musumeci, Concetta Guerra, D. Albino et al.· Clinical Cancer Research· 0 citations
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