A bedside-to-bench translational analysis of NF1 alterations and CDK4/6 inhibitor resistance in hormone receptor-positive metastatic breast cancer

Background CDK4/6 inhibitors (CDK4/6i) are used for management of hormone receptor-positive (HR+) metastatic breast cancer (MBC), and activation of the RAS/MAPK and PI3K/AKT signalling pathways has been implicated in resistance to these agents. Pathogenic NF1 mutations (pNF1m) dysregulate RAS signalling, but NF1 has not been linked to CDK4/6i resistance. We analysed multi-institutional data, real-world evidence, and preclinical models to characterise the impact of pNF1m on CDK4/6i sensitivity. Methods A retrospective cohort of patients with pNF1m tumours were identified from 4 institutions between 2/2015-5/2023 and evaluated for progression-free survival and intrinsic/acquired resistance on CDK4/6i. Real-world clinical-genomic data from GuardantINFORM between 6/2014 and 3/2023 was analysed for associations between pNF1m and time-to-next-treatment or overall survival following CDK4/6i, adjusted using propensity score weighting. We used CRISPR/Cas9 to delete NF1 in MCF7 and T47D breast cancer cells in vitro. NF1-knockout (NF1-KO) and-wild-type (WT) cells were analysed with respect to CDK4/6i sensitivity, MAPK and PI3K pathway activation, and sensitivity to MAPK and PI3K pathway inhibitors. In parallel, we assessed treatment response in a patient-derived organoid (PDO) harbouring NF1 loss, established from an HR+/HER2-breast tumor following progression on a CDK4/6i. Findings Among 1962 multicentre patients, we identified 38 with HR+/HER2-MBC, pNF1m, and exposure to CDK4/6i. NF1-associated intrinsic or acquired resistance to CDK4/6i was observed in a majority of tumours, and in those with baseline pNF1m on first-line CDK4/6i, a median progression-free survival of 6.2 months was much less than expected in routine practice. Real-world weighted analysis of 1161 patients comparing 28 pNF1m to 1133 NF1 non-altered tumours demonstrated shorter time-to-next-treatment on CDK4/6i regimens (4.2 vs. 12.4 months, hazard ratio 3.14, 95% confidence interval 2.01-4.93) and overall survival (15.8 vs. 45.2 months, hazard ratio 2.04, 95% confidence interval 1.09-3.82). NF1-deleted cells exhibited reduced sensitivity to CDK4/6i with or without oestrogen suppression, which was accompanied by induction of both MAPK and PI3K pathways, the latter of which was exacerbated by CDK4/6i. Blockade of RAS or AKT, but not MEK or ERK, reversed CDK4/6i resistance mediated by NF1 loss in cell lines and the PDO. Interpretation NF1 mutations are associated with shorter therapy duration on CDK4/6i in MBC. A causal link between NF1 loss and CDK4/6i resistance was supported by experiments in HR + breast cancer cells. NF1 deletion was accompanied by activation of ERK and AKT, and blockade of RAS or AKT combined with CDK4/6i was effective in NF1-deleted cells and an NF1-mutant PDO. Copyright (c) 2025 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).