FOXA1 is an evolutionary conserved pioneer factor that binds to condensed chromatin allowing the recruitment of other transcription factors to the DNA. Although mutations in FOXA1 are a hallmark of estrogen receptor-positive (ER+) breast cancers, it is currently unknown whether and how these mutations affect breast cancer development and response to endocrine therapies. In this work, we studied how the genome-wide chromatin recruitment, accessibility and transcriptional outcomes of recurrent FOXA1 mutations can affect therapeutic response in ER+ breast cancer. By examining the landscape of FOXA1 mutations in a cohort of 4,952 breast cancer patients, we identified several hotspot mutations, some of them present also in other malignancies and some others specific to breast cancer. In particular, we characterized three mutations in the Wing2 region and a breast-cancer specific third β strand mutation, namely SY242CS. We also showed that FOXA1 mutations are enriched in metastatic tumors and mutually exclusive with ESR1 mutations, well-known drivers of resistance to endocrine therapy. Using a clinico-genomicaly curated cohort of patients, together with in vitro and in vivo breast models, we associated FOXA1 missense mutations with a lower response to endocrine therapy. Mechanistically, by means of ChIP-seq, ATAC-seq and RNA-seq analyses, we found that FOXA1 mutations in the Wing2 loop display increased chromatin binding affinity at ER loci upon estrogen stimulation, and an enhanced ER-mediated transcription without changes in chromatin accessibility, decoupling FOXA1 Wing2 mutant binding from their pioneering function. These data correlated with the highly organized 3D conformation conferred by FOXA1 Wing2 mutations we predicted by structural modeling. Thus. the enhanced chromatin binding affinity gained by FOXA1 Wing2 mutations might be a mechanism to sustain active estrogen response even in the presence of therapeutic pressure. In contrast, breast specific SY242CS mutant shows neomorphic properties including the ability to open novel chromatin regions, and activate an alternative cistrome and transcriptome. Using an engineered luciferase reporter system, we validated that a non-canonical motif, shared by both gained accessibility and binding sites, is SY242CS-specific. Structural modeling of the binding of WT or SY242CS FOXA1 to this new motif revealed that SY242CS undergoes conformational change that results in a tight interaction with the new DNA motif, not observed in WT FOXA1. Taken together, our findings provide mechanistic insights into how FOXA1 mutations modulate its function in breast cancer to dictate malignant progression and response to endocrine therapy. More broadly, these results position FOXA1 mutations as potential biomarkers of response and potential targets for the treatment of metastatic ER+ breast cancer.

Citation Format: Amaia Arruabarrena-Aristorena, Jesper LV Maag, Srushti Kittane, Yanyan Cai, Jane Park, Pedram Razavi, Lorenzo Ferrando, Pier Selenica, Wouter R. Karthaus, Srinivasaraghavan Kannan, Emiliano Cocco, Sik Y. Ho, Daisylyn Senna Tan, Mirna Sallaku, Charles L. Sawyers, Jorge S. Reis-Fihlo, Chandra S. Verma, Ralf Jauch, Richard Koche, José Baselga, Eneda Toska, Maurizio Scaltriti. FOXA1 mutations reveal distinct chromatin profiles and influence therapeutic response in breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3439.