CHAF1A phosphorylation by PLK1 promotes AR-driven enzalutamide resistance in prostate cancer

Prostate cancer (PCa) remains a major global health challenge, ranking as the second most frequently diagnosed malignancy and the fifth leading cause of cancer-related mortality among men. Although androgen deprivation therapy and next-generation androgen receptor (AR) pathway inhibitors, such as enzalutamide, have improved clinical outcomes, the inevitable emergence of castration-resistant prostate cancer (CRPC) and treatment resistance underscores the need to elucidate the underlying molecular mechanisms. Polo-like kinase 1 (PLK1), a conserved serine/threonine kinase essential for mitosis, has been implicated in promoting enzalutamide resistance; however, the mechanistic basis of this phenomenon remains insufficiently understood. In this study, we identify Chromatin Assembly Factor 1 subunit A (CHAF1A) as a previously unrecognized modulator of AR signaling and a critical contributor to enzalutamide resistance. Analysis of the clinical database revealed that CHAF1A is significantly overexpressed in PCa compared with benign prostate tissues, and elevated CHAF1A levels correlate with poor patient prognosis. Functional assays demonstrated that CHAF1A knockdown in C4-2 and 22Rv1 cells suppresses cell proliferation, decreases PSA expression, and restores sensitivity to enzalutamide. Conversely, CHAF1A overexpression in C4-2 and LNCaP cells enhances proliferation and upregulates AR downstream effectors. Mechanistic studies revealed that CHAF1A is a potential substrate of PLK1 and undergoes phosphorylation at threonine 591 (T591). Introduction of a phosphorylation-deficient mutant, CHAF1A-T591A, into C4-2 cells attenuated AR signaling and significantly increased their responsiveness to enzalutamide. RNA sequencing further demonstrated pronounced downregulation of AR signaling in CHAF1A-T591A-expressing cells compared with wild-type controls. Using ChIP-qPCR, we show that CHAF1A enhances AR binding to promoter regions of key downstream targets, thereby elevating AR transcriptional activity. Importantly, the T591A mutation markedly diminished AR chromatin occupancy, indicating that PLK1-mediated phosphorylation at T591 is required for CHAF1A-dependent AR activation. Collectively, these findings identify CHAF1A as a novel enhancer of AR chromatin binding and transcriptional output, acting through a PLK1-dependent phosphorylation mechanism. We propose that elevated CHAF1A expression drives enzalutamide resistance by sustaining AR pathway activation, and that disruption of PLK1-mediated CHAF1A phosphorylation represents a promising therapeutic strategy to overcome resistance in CRPC.