Abstract 1843: Androgen signaling blockade enhances NK cell-mediated killing of prostate cancer cells (PC) and promotes NK cell tumor infiltration in vivo
Authors: Schwermann Maximilian , Huntington Kelsey E. , Carlsen Lindsey , Zhou Lanlan , Srinivasan Praveen , George Andrew , Wu Laura Jinxuan , De Souza Andre , Mega Anthony E. , Safran Howard P. , Carneiro Benedito A. , El-Deiry Wafik S. 
Published: June 2023

Abstract
Background: Inhibition of the androgen pathway remains central to the treatment of prostate cancer, but most patients progress to a lethal state of metastatic castration-resistant prostate cancer (mCRPC). Immune checkpoint inhibitors have a limited role in mCRPC, although androgen receptor (AR) blockade increases CD8+ T cell function and can modulate the antitumor immune response. We investigated the effect of AR inhibitors (ARi) enzalutamide (Enza) and darolutamide (Daro) on NK cell function.Materials and Methods: We evaluated cell viability to investigate the effect of Enza and Daro on PC cells (LNCap, 22Rv1 [ARv7 mutation], DU145, PC3 [AR-]) and NK-92 cells. We performed co-culture experiments with PC and NK cells at a 1:1 ratio and analyzed immune cell-mediated tumor cell killing (ImageXpress Confocal HT). Cytokine profiling (Luminex 200) of culture supernatants was performed following treatment with ARi -/+ and anti-IFN-γ antibodies. In vivo studies were performed in NCr Nude mice harboring subcutaneous PC tumors treated orally with Daro (50mg/kg/bid) and Enza (30mg/kg/bid) for 4 weeks.
Results: Treatment of co-cultures of PC plus NK-92 cells with ARi significantly increased immune-mediated PC killing within 24 hours (control: 19%±2.7; Enza: 36%±3; Daro: 41%±4.2). Treatment of NK cells alone with ARi did not impact their viability. ARi increased the secretion of cytokines such as IL-2 (7.6 pg/ml±1.3 vs. 18 pg/ml±2.4, p=0.004), IL-1β (17.8 pg/ml±2.8 vs. 67 pg/ml±12.6, p=0.005), CXCL10 (224.2 pg/ml±16.6 vs. 504 pg/ml±42.4, p=0.017) and GDF-15 (member of the TGFβ superfamily; 8.4 ng/ml±0.43 vs. 14.3 ng/ml±3.2, p=0.042) and decreased VEGF (302.5 pg/ml±62.1 vs. 120 pg/ml±50.4, p=0.01), FGF-basic (7.4 pg/ml±1.6 vs. 3.6 pg/ml±2.1, p=0.007), M-CSF (70.9 pg/ml±14.2 vs. 25.3 pg/ml±4.5, p=0.01) and IL-8 (2645 pg/ml±201 vs. 1821 pg/ml±62.3, p=0.02). ARi increased NK-92 secretion of IFN-γ (15.2 pg/ml±5.4 and 48.2 pg/ml±8.2, p=0.01) and granzyme B (630 pg/ml±32.2 vs. 1024 pg/ml±102.4, p=0.001). IFN-γ upregulation induced by ARi was blocked with IFN-γ mAb in the co-culture experiments and inhibited the NK cell-mediated killing of PCs. Analysis of PC tumors from NCr Nude Mice treated with Daro or Enza showed increased NK cell infiltration in the tumor microenvironment.
Results: We report the discovery that ARi promotes the activation of NK cells mediated by IFN-γ, leading to increased killing of PCs. Ongoing validation of these results includes PC organoids and patient-derived samples to understand further the innate immune system response to ARi and direct novel therapeutic strategies.
Citation Format: Maximilian Schwermann, Kelsey E. Huntington, Lindsey Carlsen, Lanlan Zhou, Praveen Srinivasan, Andrew George, Laura Jinxuan Wu, Andre De Souza, Anthony E. Mega, Howard P. Safran, Benedito A. Carneiro, Wafik S. El-Deiry. Androgen signaling blockade enhances NK cell-mediated killing of prostate cancer cells (PC) and promotes NK cell tumor infiltration in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1843.