Abstract P4-05-18: Modelling MHC-I downregulation in triple negative breast cancer to rescue resistance to ICB using diacylglycerol kinase inhibitors

Abstract Background: Immune checkpoint blockade (ICB) has recently revolutionized the treatment of triple-negative breast cancer (TNBC) and improved patient outcomes. However, most patients are intrinsically resistant and do not benefit from ICB. Moreover, no biomarkers to predict outcome have been clinically successful. Antigen presentation by MHC-I molecules is required for CD8 T cell mediated tumor death and response to immune checkpoint blockade, e.g. a-PD-1/a-PD-L1 therapy. TNBCs downregulate MHC-I to suppress anti-tumor immunity and patients with decreased antigen presentation have worse outcomes to ICB. Thus, loss of MHC-I expression may be a candidate biomarker for lack of ICB response, and therapeutic strategies to overcome MHC-I loss are an area of high translational relevance and interest. Diacylglycerol kinase α/ζ inhibitors (DGKis), which increase T cell activation, are being investigated to treat patients resistant to ICB. DGKis increase concentrations of diacylglycerol (DAG) in CD8 T cells by inhibiting conversion of DAG into phosphatidic acid, lowering the threshold for activation and amplifying TCR signaling and effector T cell function. We hypothesized that DGKis will improve response to ICB in TNBC with low MHC-I expression by improving CD8 T cell response to low antigen stimulation. However, modeling low MHC-I expression (as opposed to CRISPR-mediated KO) presents a technical challenge. Thus, we created a novel model of MHC-I downregulation, leveraging miRNA binding element mutations to titrate MHC-I expression. We assess changes in the tumor microenvironment and ICB response created by stepwise-decreased MHC-I expression and evaluate the potential of DGKis to treat MHC-I low tumors in combination with ICB. Methods: We assessed the effect of DGKi/a-PD-L1 dual therapy on an MHC-I competent, baseline model of TNBC using the murine EMT6 cell line, which is moderately responsive to a-PD-L1 therapy. To model titrated levels of MHC-I downregulation in vivo, we utilized miRNA silencing-mediated fine-tuners (misFITs). We created an MHC-I null variant of EMT6 cells through knock out of beta-2-microglobulin (B2m), a required component for MHC-I expression. To create the misFITs, we reintroduced the B2m gene into the cells with the addition of varied binding regions for the endogenously expressed miR-17. Presence of an miR-17 binding region allows for miRNA-mediated suppression of B2m and thus, MHC-I. Seven different binding regions were introduced resulting in stepwise titration of MHC-I expression. To evaluate the effects of titrated MHC-I expression on tumor growth and survival, we orthotopically inoculated mice and treated weekly with a-PD-L1. Results: In the EMT6 TNBC model, treatment with DGKi every 3 days in combination with weekly a-PD-L1 administration resulted in decreased tumor growth (Isotype control v. DGKi/a-PD-L1, p=0.0023, n=20) and doubled complete response rates from 20% in single-agent a-PD-L1 to 40% with combination therapy. Re-injection of complete responders with the original tumor cell line to assess persistence of response resulted in no tumor growth. Simultaneous bilateral injection of MHC-I null isogenic cells also resulted in significantly reduced growth, indicative of an MHC-I-independent memory response (p=0.0012, n=5-12). MisFITs representing 60%, 30%, and 10% of MHC-I levels in EMT6 cells grew at differential rates – faster with less MHC-I expression. Additionally, preliminary data demonstrates reduced a-PD-L1 benefit in mice with lower MHC-I expression. Conclusions: We determined that DGKi/a-PD-L1 treatment enhances response in the EMT6 TNBC model and establishes a memory immune response. We have created a model of MHC-I downregulation and begun to establish how MHC-I affects a-PD-L1 response and will utilize the misFITs to assess the mechanism of resistance to response and the efficacy of DGKi/a-PD-L1 treatment in improving response to ICB in MHC-I downregulated tumors. Citation Format: Jacey Marshall, Justin M. Balko. Modelling MHC-I downregulation in triple negative breast cancer to rescue resistance to ICB using diacylglycerol kinase inhibitors [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P4-05-18.