Abstract 5025: Antifungal ketoconazole inhibits tumor-specific transcription factor tGLI1 leading to suppression of breast cancer stem cells and brain metastasis

Breast cancer is the second leading cause of brain metastases in women; patients with breast cancer brain metastasis (BCBM) survive only 6-18 months following diagnosis. Current standard of care options for BCBM include stereotactic radiosurgery, whole-brain radiotherapy, and surgical resection. Cancer stem cells are thought to be one of the driving forces behind not only distant metastasis, but also late-stage recurrence. The hedgehog pathway has been identified as an important mediator of stem cells, however, FDA-approved therapies targeting this pathway have demonstrated limited efficacy in breast cancer clinical trials. Despite our current knowledge of breast cancer stem cells (BCSC), there remains a significant challenge in managing patients with metastatic breast cancer, underscoring the need to identify novel, actionable targets. Our laboratory discovered an alternative splice variant of glioma-associated oncogene homolog 1 (GLI1), termed truncated GLI1 (tGLI1), that is a tumor-specific gain-of-function transcription factor and terminal effector of the hedgehog pathway that is preferentially expressed in most BCBM samples and recurrent gliomas. Recent results established that tGLI1 promotes BCSC and is associated with preferential metastasis to the brain and radioresistance, justifying tGLI1 as an ideal therapeutic target for BCBM patients. To identify tGLI1-targeting agents, we screened 1,520 compounds across three commercial drug libraries and found ketoconazole (KCZ), an FDA-approved imidazole antifungal and component of previously studied anti-neoplastic regimens, selectively killed tGLI1-expressing breast cancer cells with increased efficacy against BCSC in vitro. tGLI1 knockdown abolished the ability of KCZ to target BCSC, indicating that KCZ-mediated suppression of BCSC is dependent on tGLI1. Intracardiac mouse studies showed KCZ selectively inhibited circulating tGLI1-positive breast cancer cells from developing into brain metastases and suppressed the progression of existing brain metastases. Mass spectrometry demonstrated KCZ effectively penetrated the blood-brain barrier (BBB) and blood-tumor barrier (BTB). Mechanistic studies suggest that KCZ-dependent cell kill is, in part, mediated through disruption of the functional tGLI1-STAT3 interaction. Furthermore, modification of KCZ side chains produced derivative compounds that retained tGLI1-selectivity in both in vitro models of BCSC and in vivo models of BCBM with increased BBB penetrance. Collectively, our preclinical results demonstrate that KCZ is an effective inhibitor of BCSC and brain metastasis of tGLI1-positive breast cancer. Based on these promising preclinical data, we opened a window-of-opportunity study in patients with BCBM and recurrent gliomas to determine if KCZ treatment alters tGLI1 signaling in humans (NCT03796273). Citation Format: Daniel L. Doheny, Sherona R. Sirkisoon, Tadas Rimkus, Dongqin Zhu, Noah R. Aguayo, Alexandria Harrison, Marlyn Anguelov, Sara Manore, Fei Xing, Linda J. Metheny-Barlow, Kounosuke Watabe, Alexandra Thomas, Adrianna Henson Masters, Roy Strowd, Hui-Wen Lo. Antifungal ketoconazole inhibits tumor-specific transcription factor tGLI1 leading to suppression of breast cancer stem cells and brain metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5025.