MTOR complex-1(mTORC1) activation occurs frequently in
cancers, yet clinical efficacy of
rapalogs is limited because of the associated activation of upstream survival pathways. An alternative approach is to inhibit downstream of
mTORC1; therefore, acquired resistance to
fludarabine (Flu), a
purine analogue and
antimetabolite chemotherapy, active agent for
chronic lymphocytic leukemia (CLL) was investigated. Elevated phospho-p70S6K, also known as RPS6KB1 (
ribosomal protein S6 kinase, 70kDa,
polypeptide 1) (T389), an
mTORC1 activation marker, predicted Flu resistance in a panel of B-cell lines, isogenic Flu-resistant (FluR) derivatives, and primary human CLL cells. Consistent with the anabolic role of
mTORC1, FluR cells had higher rates of glycolysis and oxidative phosphorylation than Flu-sensitive (FluS) cells.
Rapalogs (
everolimus and
rapamycin) induced moderate cell death in FluR and primary CLL cells, and
everolimus significantly inhibited glycolysis and oxidative phosphorylation in FluR cells. Strikingly, the higher oxidative phosphorylation in FluR cells was not coupled to higher
ATP synthesis. Instead, it contributed primarily to an essential,
dihydroorotate dehydrogenase catalyzed, step in de novo
pyrimidine biosynthesis.
mTORC1 promotes
pyrimidine biosynthesis by p70S6
kinase-mediated phosphorylation of CAD (
carbamoyl-phosphate synthetase 2,
aspartate transcarbamylase, and
dihydroorotase; Ser1859) and favors S-phase cell-cycle progression. We found increased phospho-CAD (S1859) and higher S-phase population in FluR cells. Pharmacological inhibition of de novo
pyrimidine biosynthesis using
N-phosphonacetyl-l-aspartate and
leflunomide, RNAi-mediated knockdown of
p70S6K, and inhibition of mitochondrial respiration were selectively cytotoxic to FluR, but not FluS, cells. These results reveal a novel link between mTORC1-mediated metabolic reprogramming and Flu resistance identifying mitochondrial respiration and de novo
pyrimidine biosynthesis as potential therapeutic targets.
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