Background: In prostate cancer, androgen receptor (AR)–targeting agents are very effective in various stages of the disease. However, therapy resistance inevitably occurs, and understanding how prostate tumor cells adapt to bypass AR suppression is critical for identifying novel drug targets to improve the clinical management of treatment-resistant disease.

Results: Here, we performed integrative multi-omics analyses on tissues isolated before and after 3 months of AR-targeting enzalutamide monotherapy from patients with high-risk prostate cancer enrolled in a neoadjuvant clinical trial. Transcriptomic analyses demonstrated that AR inhibition drove practically all tumors toward a neuroendocrine-like disease state. Additionally, epigenomic profiling revealed massive enzalutamide-induced reprogramming of pioneer factor FOXA1 from inactive chromatin sites toward active cis-regulatory elements that dictate prosurvival signals. Notably, these treatment-induced FOXA1 sites were enriched for the circadian clock component ARNTL, whose expression was elevated upon hormone therapy in our patient cohort. In addition, high posttreatment ARNTL levels were associated with poor clinical outcomes, and ARNTL knockout could restore enzalutamide sensitivity in treatment-resistant cell line and xenograft models.

Conclusions: Taken together, our data highlight a remarkable cistromic plasticity of FOXA1 following AR-targeted therapy and revealed an unexpected biological interplay between hormonal resistance and circadian rhythm regulator ARNTL, a novel candidate therapeutic target.