Androgen receptor signaling blockade enhances NK cell-mediated killing of prostate cancer cells and sensitivity to NK cell checkpoint blockade

BackgroundThe blockade of the androgen receptor (AR) pathway is an effective treatment for prostate cancer (PCa), but many patients progress to metastatic castration-resistant prostate cancer (mCRPC). Therapies for mCRPC include AR inhibitors (ARi), chemotherapy, PARP inhibitors, and radioligands. Checkpoint inhibitor activity is limited to a small subset of MSI-H mCRPC. AR signaling modulates CD8+ T cell function, but its impact on NK cell (NKc) cytotoxicity is unknown. We investigated the effect of ARi on NKc activation, cytokine secretion, expression of inhibitory receptor NKG2A, and killing of PCa cells in vitro. MethodsPCa cell lines (LNCaP, 22Rv1 [ARv7 mutation], DU145[AR-], PC3 [AR-]) were co-cultured with NK-92 cells and treated with ARi (enzalutamide [enza] and darolutamide [daro]) or in combination with anti-NKG2A antibody monalizumab. Immune cell-mediated tumor cell killing assays and multiplexed cytokine profiling were performed. NKc expression of NKG2A and PCa cells expression of HLA-E were investigated by flow cytometry. The AR-negative cell lines (PC3 and DU145) were stably transduced with a functional AR pathway to evaluate the modulation of HLA-E by AR. The activation status of peripheral blood NKc isolated from patients with PCa before and post-initiation of androgen deprivation therapy (ADT) was investigated by flow cytometry. ResultsARi significantly increased immune-mediated NK-92 cell killing of PCa cells independent of their sensitivity to androgen signaling. Cytokine analysis revealed that ARi-induced NKc activation is mediated by IFN-{gamma} and TRAIL, as confirmed by blocking antibodies. ARi increased NKG2A expression in NK cells. Immune killing of PCa cells was enhanced with the combination of ARi and monalizumab. ARi also increased the expression of HLA-E, the ligand of the inhibitory NKG2A receptor, on PCa cell lines. Using AR-negative cell lines (PC3 and DU145) and stable transduction of AR, we demonstrate that androgen signaling regulates HLA-E expression. HDAC inhibitors (vorinostat and panobinostat) did not alter the androgen-induced expression of HLA-E in PCa cells. Mirroring the results from NK-92 cells, ADT also activated peripheral blood NK cells isolated from patients with metastatic PCa. ConclusionsARi activates NK cells through upregulating IFN-{gamma} and TRAIL and promotes the killing of PCa cells. This enhanced cytotoxic killing of PCa cells is augmented by monalizumab. ARi upregulates PCa cells expression of HLA-E, suggesting a mechanism suppressing the innate immune response against PCa. These results support novel therapeutic strategies for PCa targeting NK activation with the combination of ARi and monalizumab. Graphical AbstractAndrogen receptor signaling blockade enhances NK cell-mediated killing of prostate cancer cells and sensitivity to NK cell checkpoint blockade. Nonetheless, ARi can potentially upregulate an NK cell inhibitor ligand (HLA-E), thus suppressing NK cell killing of PCa. This regulation is dependent on a functional AR signal on tumor cell lines. Adding an anti-NKG2a-HLA-E mAb with ARi further enhances the NK cell-mediated killing of PCa. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/567201v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@1571a67org.highwire.dtl.DTLVardef@186942borg.highwire.dtl.DTLVardef@69b08eorg.highwire.dtl.DTLVardef@c6547d_HPS_FORMAT_FIGEXP M_FIG C_FIG