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Development of metastases to central nervous system (CNS) is an increasing clinical issue following the diagnosis of advanced breast cancer. The propensity to metastasize to CNS varies by breast cancer subtype. Of the four breast cancer subtypes, triple-negative breast cancers (TNBC) have the highest rates of both parenchymal brain metastasis and leptomeningeal metastasis (LM). LM is rapidly fatal due to poor detection and limited therapeutic options. Therapy of TNBC brain metastasis and LM is challenged by multifocal brain metastasis and diffuse spread of LM, and must balance brain penetration, tumor cytotoxicity, and the avoidance of neurotoxicity. Thus, there is an urgent need for novel therapeutic options in TNBCs CNS metastasis. QBS10072S is a novel chemotherapeutic that leverages TNBC-specific defects in DNA repair and LAT1 (L-amino acid transporter type 1)-dependent transport into the brain. In our study, activity of QBS10072S was investigated in vitro with various cell lines including the human TNBC cell line MDA-MB-231 and its brain-tropic derivative MDA-MB-231-BR3. QBS10072S was preferentially toxic to TNBC cells. The efficacy of QBS10072S against brain metastasis and LM was tested using a model of brain metastasis based on the internal carotid injection of luciferase-expressing tumor cells into NuNu mice. The compound was well tolerated, delayed tumor growth and reduced leptomeningeal dissemination, resulting in significant extension of survival. Given that current treatments for LM are palliative with only few studies reporting a survival benefit, QBS10072S is planned to be investigated in clinical trials as a therapeutic for TNBC LM. Significance: TNBC brain metastasis often involves dissemination into leptomeninges. Treatment options for TNBC leptomeningeal metastasis are limited and are mostly palliative. Our study demonstrates significant efficacy of the brain-penetrating agent QBS10072S against TNBC brain metastasis and leptomeningeal spread.

PR-104 is a dinitrobenzamide mustard currently in clinical trial as a hypoxia-activated prodrug. Its major metabolite, PR-104A, is metabolized to the corresponding hydroxylamine (PR-104H) and amine (PR-104M), resulting in activation of the nitrogen mustard moiety. We characterize DNA damage responsible for cytotoxicity of PR-104A by comparing sensitivity of repair-defective hamster Chinese hamster ovary cell lines with their repair-competent counterparts. PR-104H showed a repair profile similar to the reference DNA cross-linking agents chlorambucil and mitomycin C, with marked hypersensitivity of XPF−/−, ERCC1−/−, and Rad51D−/− cells but not of XPD−/− or DNA-PKCS−/− cells. This pattern confirmed the expected dependence on the ERCC1-XPF endonuclease, implicated in unhooking DNA interstrand cross-links at blocked replication forks, and homologous recombination repair (HRR) in restarting collapsed forks. However, even under anoxia, the hypersensitivity of XPF−/−, ERCC1−/−, and Rad51D−/− cells to PR-104A itself was lower than for chlorambucil. To test whether this reflects inefficient PR-104A reduction, a soluble form of human NADPH:cytochrome P450 oxidoreductase was stably expressed in Rad51D−/− cells and their HRR-restored counterpart. This expression increased hypoxic metabolism of PR-104A to PR-104H and PR-104M as well as hypoxia-selective cytotoxicity of PR-104A and its dependence on HRR. We conclude that PR-104A cytotoxicity is primarily due to DNA interstrand cross-linking by its reduced metabolites, although under conditions of inefficient PR-104A reduction (low reductase expression or aerobic cells), a second mechanism contributes to cell killing. This study shows that hypoxia, reductase activity, and DNA interstrand cross-link repair proficiency are key variables that interact to determine PR-104A sensitivity. [Mol Cancer Ther 2009;8(6):1714–23]