Tumor cells require a constant supply of macromolecular precursors, and interrupting this supply has been proposed as a therapeutic strategy in
cancer. Precursors for
lipids,
nucleic acids, and
proteins are generated in the
tricarboxylic acid (TCA) cycle and removed from the mitochondria to participate in biosynthetic reactions. Refilling the pool of precursor molecules (anaplerosis) is therefore crucial to maintain cell growth. Many
tumor cells use
glutamine to feed anaplerosis. Here we studied how "
glutamine-addicted" cells react to interruptions of
glutamine metabolism. Silencing of
glutaminase (GLS), which catalyzes the first step in
glutamine-dependent anaplerosis, suppressed but did not eliminate the growth of
glioblastoma cells in culture and in vivo. Profiling metabolic fluxes in GLS-suppressed cells revealed induction of a compensatory anaplerotic mechanism catalyzed by
pyruvate carboxylase (PC), allowing the cells to use
glucose-derived
pyruvate rather than
glutamine for anaplerosis. Although PC was dispensable when
glutamine was available, forcing cells to adapt to low-
glutamine conditions rendered them absolutely dependent on PC for growth. Furthermore, in other cell lines, measuring PC activity in nutrient-replete conditions predicted dependence on specific anaplerotic
enzymes. Cells with high PC activity were resistant to GLS silencing and did not require
glutamine for survival or growth, but displayed suppressed growth when PC was silenced. Thus, PC-mediated,
glucose-dependent anaplerosis allows cells to achieve
glutamine independence. Induction of PC during chronic suppression of
glutamine metabolism may prove to be a mechanism of resistance to
therapies targeting glutaminolysis.