Cancer stem cells synthesize proline to attenuate oxidative stress.

Cancers reprogram their metabolism to provide anabolic needs without driving excessive oxidative stress. Attention has focused on glucose metabolism, yet amino acid synthesis and degradation also promote tumor cell states and growth. Here, we assessed amino acids that maintain cancer stem cells in glioblastoma and found increased proline levels relative to differentiated tumor progeny through increased proline synthesis. Cancer stem cells preferentially expressed the signaling molecule FAM3C induced by the stem cell transcription factor SOX2 to drive expression of proline synthesis enzymes. FAM3C classically mediated cellular responses as a secreted protein but gained intracellular functions in cancer stem cells through binding the histone reader spindlin 1 (SPIN1), thereby preventing its lysosomal degradation, assisting its nuclear localization, and promoting epigenetic regulation of proline synthesis. Proline synthesis depleted ROS, and genetic targeting of FAM3C attenuated ROS scavenging, whereas SPIN1 OE restored ROS levels. Molecular docking identified tucatinib as a brain-penetrant pharmacologic disruptor of FAM3C-SPIN1 interactions, promoting SPIN1 degradation and reducing intracellular proline levels. Thus, cancer stem cells induced a favorable metabolic state through proline synthesis and ROS depletion, revealing potential therapeutic dependencies.