Validação pré-clínica da eficácia da inibição combinada de EGFR e STAT3 em carcinomas da próstata com rearranjos ETS

The primary objective of this project is to validate the clinical utility of repurposing Erlotinib, an anti-EGFR inhibitor, in combination with a specific STAT3 inhibitor (TTI-101), for the treatment of prostate carcinomas characterized by ETV1 overexpression, collectively accounting for 8-10% of all prostate carcinomas. To achieve this objective, the project is structured in four distinct and complementary goals encompassing six tasks: 1 – Validate the synergistic efficacy and ETS-specificity of combined inhibition of EGFR and STAT3 in complex in vitro 3D models mimicking human tumours; We will take advantage of the different prostate cell models previously developed in the group with modulation of the expression of different ETS transcription factors (ERG, ETV1 or ETV4), leveraged by our groups’ expertise in 3D cell models and fluorescence-based high content screening, to assess the effectiveness of Erlotinib and TTI-101 combination therapy in co-cultures integrating PCa cells with diverse ETS backgrounds and prostate-derived myofibroblasts or cancer-associated fibroblasts (CAFs). This approach, delineated in Task 1, will widen our understanding of the paracrine mechanisms potentially governing therapy response, offering critical insights into stromal involvement in oncogenic EGFR/STAT3 signalling. 2) Validate whether combined inhibition of EGFR and STAT3 limits tumour growth and metastization of cell line-derived xenografts (CDXs) recapitulating human disease; To properly evaluate potential clinical relevance, we will evaluate tumour growth in an environment faithfully mimicking human androgen-independent disease. We will mimic CRPC disease by subcutaneous implantation of fluorescently-stable cell line spheroids in a sophisticated humanized mouse model, and evaluate response to the combination therapy post-castration, using immunofluorescence imaging. With this strategy (Task 2), we will be able to validate the ETS-specificity of the therapeutic response across primary and metastatic sites, obviating the need for predefined endpoints. Detailed analysis of tumours and metastatic lesions will clarify how well these models replicate human metastatic disease (Task 3). 3) Investigate the complex interplay between the tumour profile and the immune system in TME remodelling in response to the combination therapy; To gain insights into the intricate mechanisms underlying drug efficacy in a specific tumour subtype under an active immune system, we will dissect the cellular players and the molecular changes occurring in CDXs exposed to the combination therapy, using single-cell RNA sequencing (scRNA-seq; Task 4). This approach will highlight critical players and shed light on the unknown mechanisms underlying oncogenic ETS transcription factor signalling, with potential implications in other ETS-driven carcinomas. 4) Development of a preclinical model for timely assessment of patient eligibility to the combination therapy; To establish a straight line with the potential clinical translatability of the proposed combination therapy inhibiting EGFR and STAT3 signalling, we will develop an in vivo platform for drug testing in metastatic prostate cancer cells using the chick embryo chorioallantoic membrane (CAM) model (Task 5). Subsequently, we will leverage the CAM platform to identify patients that could benefit from the combination therapy by assessing therapeutic efficacy in CAM xenografts derived from patients’ circulating tumour cells.