Depleting the Action of EZH2 through PI3K–mTOR Inhibition to Overcome Metastasis and Immunotherapy Resistance in Triple-Negative Breast Cancer Open Access

Almost half of patients with triple‐negative breast cancer develop distant metastases, heralding unfavorable outcomes. Here, we provide novel insights into the contribution of the PI3K–mTOR pathway to the triple‐negative breast cancer phenotypes that promote growth, migration, metastasis, and therapy resistance. Specifically, we demonstrate that dual targeting of PI3K and mTOR but not PI3K alone inhibits cancer cell proliferation and migration in vitro. Dual PI3K–mTOR inhibition with paxalisib not only promotes a favorable mesenchymal-to-epithelial phenotype but also inhibits signatures associated with metastasis-initiating cells, including the highly aggressive cancer stem cell phenotype, persister cancer cell phenotype (p65, FOXQ1, NRF2, and NNMT), and a cancer drug resistance signature (ABCB5, SNAIL, and ALDH1). In vivo, paxalisib overcomes immunotherapy resistance to reduce primary tumor burden, circulating tumor cells, and direct and indirect indicators of metastasis with a favorable toxicity profile. Gene expression and spatial analyses show that paxalisib profoundly affects the immune microenvironment in tumors, reducing adaptive immune phenotypes associated with immunotherapy resistance (exhausted T cells and regulatory T cells) and protumor innate immune populations such as mast cells. PI3K–mTOR blockade acts upstream of EZH2, impacting both the classic repressive catalytic p85β–EZH2–H27ME3 and active EZH2–NF-κB pathways. Our data suggest that dual targeting of the PI3K–mTOR pathway disrupts both the catalytic and noncatalytic axes of EZH2 to inhibit metastasis and enhance cancer immune visibility, potentially increasing the utility of immunotherapy in resistant individuals.