Complex I protein NDUFB9 is a metabolic vulnerability in triple negative breast cancer brain metastases.

Triple-negative breast cancer (TNBC) brain metastases (BrMs) remain a therapeutic challenge. We depict the discrepancies between primary tumors and BrMs, and examine patient-matched cerebrospinal fluid and plasma to provide detailed profiles of BrMs' metabolic microenvironment. High-throughput in vivo loss of function CRISPR screens identify NDUFB9 (NADH: Ubiquinone Oxidoreductase Subunit B9) as a brain-specific metabolic vulnerability. NDUFB9-knockout selectively inhibits the BrMs outgrowth without affecting extracranial metastases. Mechanistically, TNBC cells exhibit an imbalance between aspartate upstream supply and downstream biosynthetic demand. NDUFB9-knockout disrupts mitochondrial complex I and reduces intracellular aspartate, but this alone is insufficient to inhibit TNBC proliferation. Instead, the lower asparagine concentration in the brain microenvironment induces compensatory upregulation of asparagine synthetase, which further diverts aspartate toward asparagine biosynthesis. This dual-hit mechanism exhausts the aspartate pool and restricts nucleotide biosynthesis, thereby selectively suppressing BrM outgrowth. Our findings uncover a therapeutic strategy for TNBC BrMs.