An FDA-Approved Antifungal, Ketoconazole, and Its Novel Derivative Suppress tGLI1-Mediated Breast Cancer Brain Metastasis by Inhibiting the DNA-Binding Activity of Brain Metastasis-Promoting Transcription Factor tGLI1

Simple Summary Breast cancer is the most commonly diagnosed cancer in American women, and metastasis accounts for the majority of breast cancer-related deaths. The common metastatic sites for breast cancer includes the bones, lungs, brain, and liver. Breast cancer brain metastasis (BCBM) patients have dismal prognoses, primarily due to the lack of understanding of the molecular mechanisms driving breast cancer cell colonization to the brain. In breast cancer, truncated glioma-associated oncogene homolog 1, tGLI1, promotes preferential metastasis to the brain through the upregulation of the cancer stem cell subpopulation and the activation of astrocytes. Whether tGLI1 is an actionable therapeutic target for any cancer type has not yet been investigated. Herein, we identified an FDA-approved antifungal, ketoconazole (KCZ), and its novel derivative, KCZ-7, to antagonize tGLI1 transcriptional activity, suppress cancer stem cells, and inhibit BCBM, rendering tGLI1, for the first time, as an actionable therapeutic target for the prevention and treatment of BCBM. The goal of this study is to identify pharmacological inhibitors that target a recently identified novel mediator of breast cancer brain metastasis (BCBM), truncated glioma-associated oncogene homolog 1 (tGLI1). Inhibitors of tGLI1 are not yet available. To identify compounds that selectively kill tGLI1-expressing breast cancer, we screened 1527 compounds using two sets of isogenic breast cancer and brain-tropic breast cancer cell lines engineered to stably express the control, GLI1, or tGLI1 vector, and identified the FDA-approved antifungal ketoconazole (KCZ) to selectively target tGLI1-positive breast cancer cells and breast cancer stem cells, but not tGLI1-negative breast cancer and normal cells. KCZ's effects are dependent on tGLI1. Two experimental mouse metastasis studies have demonstrated that systemic KCZ administration prevented the preferential brain metastasis of tGLI1-positive breast cancer and suppressed the progression of established tGLI1-positive BCBM without liver toxicities. We further developed six KCZ derivatives, two of which (KCZ-5 and KCZ-7) retained tGLI1-selectivity in vitro. KCZ-7 exhibited higher blood-brain barrier penetration than KCZ/KCZ-5 and more effectively reduced the BCBM frequency. In contrast, itraconazole, another FDA-approved antifungal, failed to suppress BCBM. The mechanistic studies suggest that KCZ and KCZ-7 inhibit tGLI1's ability to bind to DNA, activate its target stemness genes Nanog and OCT4, and promote tumor proliferation and angiogenesis. Our study establishes the rationale for using KCZ and KCZ-7 for treating and preventing BCBM and identifies their mechanism of action.