SIRT5 induces a metabolic switch to fuel nucleotide pools and chemoresistance in triple-negative breast cancer

Chemoresistance is a primary cause of relapse and mortality in triple-negative breast cancer (TNBC). To reveal the metabolic characteristics that contribute to de novo chemoresistance in human TNBC, we analyzed primary tumor biopsies prior to preoperative chemotherapy, employing quantitative mass spectrometry-based proteomics and metabolomics. Remarkably, our findings reveal that chemoresistant TNBCs exhibit a significant enrichment in metabolic traits associated with oxidative phosphorylation (OXPHOS) and altered nucleotide metabolism, which converge on the overexpression of SIRT5, a master regulator of mitochondrial metabolism. Notably, SIRT5 is frequently overexpressed in breast cancer due to copy number gains and amplifications. Through gain- and loss-of-function studies, we confirm that SIRT5 mediates chemoresistance through its catalytic activity. Using metabolomics and stable isotope tracing, we further demonstrate that SIRT5 induces a metabolic switch that redirects glycolysis to the pentose phosphate pathway (PPP), thereby replenishing nucleotide pools while enhancing glutaminolysis to support the tricarboxylic acid (TCA) cycle. Specifically, we show that SIRT5 catalyzes the conversion of 6-phospho-D-gluconate to ribulose-5-phosphate (R-5-P) by demalonylating the lysine residue (K59) on 6-phosphogluconate dehydrogenase (6-PGD). Furthermore, we reveal that SIRT5 drives cellular dependence on glutamine as a bioenergetic substrate through activation of oncogenic MYC. Dependency analysis reveals a significant genetic codependence between SIRT5 expression and ATR replication stress checkpoint activation. We find that the combination of ATR inhibitors and chemotherapeutic agents shows significant synergistic effects in reversing chemoresistance in TNBC. In summary, our findings illustrate that elevated SIRT5 orchestrates a coordinated metabolic switch to sustain the PPP and alter nucleotide pools, leading to replication stress and ATR checkpoint dependence. Simultaneously, it activates glutaminolysis to fuel the TCA cycle for bioenergetic demands. Thus, targeting ATR represents a crucial and selective metabolic vulnerability of SIRT5-overexpressing TNBC.