Combined Radiation and PI3Kγδ Inhibitor Enhances Anti-Tumor Effect of PD-1 Blockade in Immune-competent Syngeneic Breast Cancer Model

Abstract

Breast cancer is generally viewed as immunologically ‘cold’, imposing an immune-suppressive tumor microenvironment (TME) and responding poorly to lone immune checkpoint blockade (ICB). As an adjunct to ICB, radiation therapy (RT) holds promise in terms of in situ tumor vaccination effect, although it is known to promote immune suppression, increasing regulatory T cells (Treg), myeloid-derived suppressor cells (MDSCs), and M2 tumor-associated macrophages (TAMs). It was our contention that combined use of RT and a PI3Kγδ inhibitor to combat immune suppression might enhance the efficacy of ICB. Murine breast cancer cells (4T1) were grown in both immune-competent and -deficient BALB/c mice, and tumors were irradiated by 3 fractions of 24 Gy. A PD-1 blockade (10 mg/kg) and a PI3Kγδ inhibitor (IPI145; 15 mg/kg) were then administered every other day for 2 weeks. Fluorescence-activated cell sorting (FACS) and immunohistochemistry served to monitor subsequent changes in immune cell repertoire. We used mRNA-sequencing data of patient-derived breast cancer xenografts in a humanized NSG mouse model to analyze differentially expressed genes. Transcriptomic and clinical data were acquired from The Cancer Genome Atlas (TCGA) pan-cancer cohort, and a deconvolution algorithm (xCell) was used to profile immune cellular distributions at certain levels (high vs. low) of PI3Kγδ expression. In the immune-competent syngeneic 4T1 murine tumor model, PD-1 blockade alone led to tumor hyperprogression, whereas a three-pronged strategy of PI3Kγδ inhibitor, RT, and PD-1 blockade significantly delayed primary tumor growth, boosted abscopal effect, and improved animal survival by comparison. The immune-deficient syngeneic 4T1 murine tumor model failed to show this synergism in delaying tumor growth. According to FACS analysis, RT significantly increased not only CD8+cytotoxic T-cell fractions but also immune-suppressive Treg cells, MDSCs, and M2 TAMs. However, PI3Kγδ inhibitor significantly lowered proportions of Treg, MDSCs, and M2 TAMs, achieving dramatic gains in splenic, nodal, and tumor CD8+ T-cell populations after triple combination therapy. There was significantly decreased tumor expression of p-AKT, PD-L1, and HIF1α by PI3Kγδ inhibition. The analyses of RNAseq data of humanized PDX showed triple combination increased immune response pathways and increased CD8 T cell and decreased Treg and M2 macrophage shown by the Xcell deconvolution. In the TCGA pan-cancer cohort, higher tumor purity-adjusted Treg/CD8+T-cell and M2/M1 TAM ratios and worse overall patient survival were associated with high PIK3CG (PI3Kγ) or PIK3CD (PI3Kδ) expression. These findings collectively indicate that PI3Kγ and PI3Kδ are clinically relevant targets in an immunosuppressive TME. Combining PI3Kγδ inhibitor, RT, and PD-1 blockade may thus be a viable approach, helping to overcome the therapeutic resistance of immunologically cold tumors such as breast cancer.