Abstract Metastasis is the major cause of mortality for breast cancer. Common metastatic sites include bones, lungs, brain, and liver; with breast cancer brain metastasis (BCBM) accounting for 20-40% of the metastatic cases. HER2-enriched and triple-negative breast cancer (TNBC) subtypes are more likely to develop BCBM compared to others. BCBM is associated with poor prognoses, with a median survival time of 7.9 months. The dismal outcomes are due to limited understanding of the molecular mechanisms driving BCBM and the lack of effective drugs capable of penetrating the blood-brain barrier (BBB). This underscores the critical need to identify new druggable targets and therapeutics for HER2 and TNBC BCBM. To address this urgent need, in earlier work our group identified a tumor-specific, alternatively spliced variant of the transcription factor GLI1 (glioma-associated oncogene homolog 1), termed truncated GLI1 (tGLI1). tGLI1 represents a novel therapeutic target for BCBM because of its selective overexpression in BCBM and the promotion of breast cancer stem cells (CSC), BCBM progression, and tumor microenvironment remodeling. In prior studies, screening of commercial drug libraries for tGLI1-selective agents revealed that the FDA-approved antifungal ketoconazole (KCZ) selectively inhibited tGLI1-positive breast cancer cells in vitro and suppressed tGLI1-driven brain metastases in vivo. However, KCZ’s potent inhibition of the CYP3A4 enzyme raises safety concerns, including liver damage, adrenal insufficiency, and drug-drug interactions. To overcome these issues with toxicities, we modified the chemical structure of KCZ and identified the novel derivative KCZ-229A as a tGLI1 inhibitor candidate. We found that KCZ-229A retains selective inhibition of tGLI1-positive breast CSCs but loses the ability to inhibit CYP3A4 enzymatic activity in vitro. Additionally, KCZ-229A demonstrates no toxicity to normal brain, liver, or mammary epithelial cells in vitro, while in vivo studies confirmed no liver toxicity (normal serum ALT), no adrenal insufficiency (normal serum ACTH), no hematologic abnormalities, and no adverse effects on major organs. Moreover, mass spectrometry analysis of serum showed that KCZ-229A has enhanced bioavailability and BBB permeability. Mechanistic studies revealed that KCZ-229A binds directly and specifically to tGLI1 protein, induces apoptosis, suppresses the CD44+/CD24- stem cell population, and hampers migration and invasion of tGLI1-positive BCBM cells in vitro. An experimental mouse metastasis study using intracardiac inoculation demonstrated that systemic administration of KCZ-229A suppressed the progression of tGLI1-positive BCBM. These findings establish KCZ-229A as a brain-penetrant, and safe tGLI1-targeting compound, supporting its further preclinical development to optimize its pharmacological profile and assess its potential to both treat and prevent BCBM. Citation Format: Mariana Najjar, Daniel Doheny, Sara Manore, Joshua Cha, Phi-Long Tran, Elissa Bloom, Hui-Wen Lo. Targeting tGLI1 in breast cancer brain metastases with a novel ketoconazole derivative 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 424.
Najjar et al. (Fri,) studied this question.