HR+/HER2- breast cancer (BC) patients often show limited response to current neoadjuvant chemotherapy, highlighting the critical need for alternative therapeutics strategies. The combination of CDK4/6 inhibitors (CDK4/6i) with endocrine therapy (ET) is the standard of care in adjuvant treatment for HR+/HER2- BC, with the potential for significantly improved outcomes in the neoadjuvant setting. However, drug resistance remains a significant challenge, with a critical need for early-stage biomarkers to predict resistance. The CARABELA phase 2 clinical trial, led by Dr. Emilio Alba (the supervisor of this proposal), provides valuable data on the efficacy and resistance patterns of prolonged CDK4/6i plus ET in neoadjuvant settings. Previous research by Dr. Chica-Parrado (the candidate) identified NF1 loss-of-function (LOF) mutations as key drivers of resistance to both ET and CDK4/6i. The CARABELA-NF1 study aims to leverage this expertise to integrate preclinical data with clinical insights from the CARABELA trial. The objectives of CARABELA-NF1 project are: (1) To perform targeted sequencing of circulating tumor DNA (ctDNA) to identify genomic alterations associated with resistance to CDK4/6i or ET and correlate these with clinical responses. (2) To develop an NF1-loss signature (NF1sig) from cell line data and validate this predictive signature using RNA from pre-treatment samples in the CARABELA trial. (3) To conduct drug screening in HR+/HER2- BC organoids derived from patients resistant to ET and CDK4/6i with NF1 mutations, focusing on the activation of pathways induced by the treatment. This project combines advanced genomic profiling with clinical trial data and drug screening to address the pressing need for effective biomarkers and novel therapeutic strategies in the neoadjuvant setting for HR+/HER2- BC.