Authors: Mariana K. Najjar | Daniel Doheny | Sara G. Manore | Chuling Zhuang | Munazza S. Khan | Terrence L. Smalley | Hui‐Wen Lo
Source: Clinical Cancer Research (article, 2025-06-13)
Field: Chemistry — Organic Chemistry

Abstract P3-09-29: A Novel BBB-permeable Agent for Breast Cancer Brain Metastases Abstract Metastatic breast cancer is the second leading cause of cancer-related deaths among American women. The most common sites of breast cancer metastases are the bones, lungs, brain, and liver. Among the different sites, breast cancer brain metastasis (BCBM) constitutes approximately 10-30% of metastatic breast cancer and is associated with worst dismal prognoses, with a median survival time of 7.9 months. The poor outcomes are attributed to the limited understanding of the driving factors of BCBM and the scarcity of drugs that can cross the blood-brain barrier (BBB) and blood-tumor barrier (BTB). This underscores the critical need to develop new therapies for BCBM. Our lab identified truncated glioma-associated oncogene homolog 1 (tGLI1) as an alternatively spliced, gain-of-function, tumor-specific variant of GLI1. Since its discovery, tGLI1 has been reported to promote breast cancer stem cells (CSC) and breast cancer preferential metastasis to the brain. Additionally, we recently identified the FDA-approved antifungal ketoconazole to selectively target tGLI1-positive breast cancer cells in vitro. Ketoconazole was also found to prevent the preferential metastasis of tGLI1-positive breast cancer to the brain and suppress the progression of established tGLI1-positive BCBM in vivo (Doheny et al., Cancers 14:4256, 2022). Ketoconazole is a potent inhibitor of the cytochrome P450 enzyme CYP3A4 and has been associated with liver damage, adrenal insufficiency, and drug interactions. To mitigate this issue with potential toxicities, we modified the chemical moieties of ketoconazole and generated novel derivatives. We screened the compounds using isogenic brain-tropic breast cancer cell lines engineered to stably express the vector, GLI1, or tGLI1 and identified the novel derivative WF-229A to selectively target tGLI1-positive breast cancer stem cells. Foremost, our data shows that WF-229A lost the ability to inhibit CYP3A4 enzymatic activity in vitro. Additionally, WF-229A showed no toxic effects on normal brain cells (astrocytes) or the liver cells (HepG2). In efforts to evaluate the pharmacological properties of WF-229A, we conducted an in vivo maximum tolerated dose (MTD) study. Our results demonstrated that, unlike ketoconazole, increasing doses of WF-229A did not elevate serum alanine transaminase (ALT) levels, indicating no liver toxicity. Additionally, increasing doses of WF-229A did not cause an increase in serum Adrenocorticotropic hormone (ACTH) levels suggesting, no adrenal insufficiency, unlike ketoconazole. Furthermore, our experimental mouse metastasis study using intracardiac inoculation demonstrated that systemic administration of WF-229A suppressed the progression of tGLI1-positive BCBM. WF-229A can penetrates the BBB in mice. For further characterization of WF-229A, we conducted a cellular thermal shift assay (CETSA) and found that WF-229A directly binds to tGLI1 protein, but not GLI1. Additional mechanistic studies showed that WF-229A suppresses the CD44+/CD24- stem cell population, hampers migration, and induces apoptosis in tGLI1-positive BCBM cells. Our study establishes the rationale to further characterize the pharmacological properties of WF-229A for fine-tuned targeting of BCBM, and to examine WF-229A’s ability to offer additional therapeutic benefits including the prevention of BCBM in vivo. Citation Format: Mariana K. Najjar, Daniel Doheny, Sara Manore, Chuling Zhuang, Munazza Samar Khan, Terrence Smalley, Hui-Wen Lo. A Novel BBB-permeable Agent for Breast Cancer Brain Metastases [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 P3-09-29. Medicine ; Breast cancer ; Brain cancer ; Cancer ; Oncology ; Internal medicine Click Chemistry and Applications ; Histone Deacetylase Inhibitors Research ; Peptidase Inhibition and Analysis