Abstract Introduction: Triple-negative breast cancer (TNBC) has higher tumor immunogenicity than other subtypes, and immune checkpoint inhibitors (ICIs) targeting the PD-1/PD-L1 axis have been applied in its treatment. However, responses to immune checkpoint blockade (ICB) remain limited by intrinsic and acquired resistance, highlighting the need for predictive biomarkers and new combination strategies to improve anti-PD-L1 efficacy.The aryl hydrocarbon receptor (AHR) regulates immune responses and PD-L1 expression, promoting an immunosuppressive tumor microenvironment (TME). AHR signaling may also interfere with interferon-mediated immune activation and affect ICB response. This study aimed to examine how AHR activation contributes to anti-PD-L1 resistance in PD-L1 overexpressing TNBC and whether AHR blockade enhances therapeutic efficacy. Methods: PD-L1-overexpressing (PD-L1 OE) and control (Ctl) 4T1 cells were generated. The AHR antagonist (CH-223191) and anti-PD-L1 antibody were applied in vitro and in vivo. Flow cytometry, western blotting, cell proliferation assay, quantitative RT-PCR, immunohistochemistry (IHC), and co-culture of macrophages and cancer cells were performed. An orthotopic syngeneic mouse model was established by injecting 4T1 Ctl or PD-L1 OE cells into the mammary fat pad. Mice were treated with CH-223191 (5 mg/kg, i.p., four times weekly) and anti-PD-L1 antibody (150 µg, i.p., twice weekly), alone or in combination. Results: PD-L1 OE tumors showed reduced responsiveness to anti-PD-L1 therapy compared with control in vivo, confirming intrinsic resistance to ICB. AHR expression was elevated at both RNA and protein levels in PD-L1 OE tumors after anti-PD-L1 treatment. Combination therapy with CH-223191 and anti-PD-L1 antibody tended to suppress tumor growth compared with monotherapy, suggesting an ability to overcome PD-L1-driven resistance in vivo. In vitro, CH-223191 reduced 4T1 viability in a dose-dependent manner, suggesting a potential tumor-intrinsic role of AHR signaling. Co-culture of PD-L1 OE 4T1 cells with macrophages promoted M2-like polarization, supporting an immunosuppressive microenvironment. Collectively these findings suggest that AHR activation contributes to anti-PD-L1 resistance by promoting an immunosuppressive TME, and that AHR blockade could enhance anti-PD-L1 efficacy in TNBC. Conclusions: AHR activation was linked to PD-L1-driven anti-PD-L1 resistance, and AHR inhibition enhanced responsiveness in vivo. These findings suggest that AHR mediates PD-L1 axis-driven resistance and that its inhibition may serve as a promising combination strategy in TNBC. Further studies should elucidate the mechanistic link between AHR signaling and PD-L1-mediated immune regulation and validate its therapeutic potential. Citation Format: Minkyoung Choi, Ju Hee Kim, Han-Byoel Lee, Wonshik Han. Targeting AHR overcomes PD-L1-driven resistance to immune checkpoint blockade in triple-negative breast cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2813.
Choi et al. (Fri,) studied this question.