FOXA1 alterations drive endocrine therapy resistance through unique transcriptional and epigenomic programs in breast cancer

Breast cancer is histologically classified into invasive ductal carcinoma (IDC, ~70-80% of cases) and invasive lobular carcinoma (ILC, 10-15%). Both are predominantly estrogen receptor-positive (ER+) and treated with endocrine therapy, yet resistance inevitably develops, driving metastasis and poor outcomes. ILC exhibits hallmark E-cadherin loss, distinct metastatic patterns, and reduced endocrine therapy response compared with stage-matched IDC. However, treatment remains uniform across histologies, underscoring the need to define shared and subtype-specific drivers of resistance. Clinicogenomic analyses of >8,500 MSK-IMPACT tumors revealed recurrent FOXA1 mutations in ER+ disease, enriched in ILC (8% vs 4% in IDC). As a pioneer transcription factor that licenses ER chromatin binding, FOXA1 alterations may reshape ER signaling to promote therapy resistance. To investigate this, we functionally characterized recurrent FOXA1 Helix 1 (H1), Wing2 (W2), and betastrand3 (S3) mutations in IDC (MCF7, T47D), and H1, W2 variants in ILC (MDA-MB-134VI). We assessed treatment response under estrogen deprivation, fulvestrant or oral SERDs ± CDK4/6i and profiled transcriptional and chromatin changes using RNA-seq, ER CUT&RUN, ATAC-seq, and qPLEX-RIME. Across both histologies, FOXA1 mutations conferred a selective growth advantage under estrogen deprivation modeling AI therapy, while retaining sensitivity to SERDs and SERD+CDK4/6i. Mechanistically, H1 mutants were hypermorphic in IDC and ILC, increasing ER recruitment and chromatin accessibility to strengthen canonical estrogen-responsive programs. W2 variants exhibited histology-specific consequences: in IDC they increased ER chromatin binding without pioneering, consistent with enhanced EP300/NCOA3 co-activator engagement, whereas in ILC they also increased accessibility, displaying lineage-restricted pioneer function. Ductal-specific S3 mutants were neomorphic, redirecting FOXA1 to an alternative DNA motif, reprogramming accessibility, and activating proliferation, metabolic, and endocrine-resistant gene networks. Interactome profiling supported enrichment of stemness-linked chromatin remodeling networks.Together, these findings demonstrate that recurrent FOXA1 mutations establish mechanistically distinct oncogenic classes that promote endocrine resistance in ER+ breast cancer with their distribution differing by histology. ILC is enriched for hypermorphic H1 and W2 variants that enhance pioneering and chromatin opening, whereas IDC harbors W2 mutants lacking pioneer activity and neomorphic S3 variants that rewire chromatin. These insights support FOXA1 mutation status as a biomarker of aromatase inhibitor resistance and highlight the need for histology-tailored therapeutic strategies.