Targeting mTORC1–Mediated Metabolic Addiction Overcomes Fludarabine Resistance in Malignant B Cells

Abstract

mTORC1 activation occurs frequently in cancers, yet clinical efficacy of rapalogs is limited due to the associated activation of upstream survival pathways. An alternative approach is to inhibit downstream of mTORC1; therefore, acquired resistance to fludarabine (Flu), a purine analog and anti-metabolite chemotherapy, active agent for chronic lymphocytic leukemia (CLL) was investigated. Elevated phospho-p70S6K (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, 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 (DHODH) catalyzed, step in de novo pyrimidine biosynthesis. mTORC1 promotes pyrimidine biosynthesis by p70S6 kinase-mediated phosphorylation of CAD (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 (PALA) 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. Implications This study provides the first evidence for mTORC1/p70S6K-dependent regulation of pyrimidine biosynthesis in a relevant disease setting.

MCL-1 and BCL-xL-dependent resistance to the BCL-2 inhibitor ABT-199 can be overcome by preventing PI3K/AKT/mTOR activation in lymphoid malignancies

“…Given that the dependency of ABT-199-R cells on MCL-1 is regulated by the PI3K/mTOR pathway, we next asked whether these cells could be sensitized to ABT-199 by targeting the PI3K or mTOR pathways. We used the PI3K δ and mTORC1 inhibitors, GS-1101 and RAD001, respectively, as before, 32 , 33 in combination with ABT-199. Cell viability analyses with Annexin V-PI indicated that GS-1101 in combination with ABT-199 sensitized SU-DHL-6 ABT199-R cells more than the RAD001+ABT-199 combination ( Figures 6a and d ).…”

mTOR Regulation of Metabolism in Hematologic Malignancies

“…In addition, both metformin, a respiratory chain complex I inhibitor, and rapamycin similarly synergized with dasatinib in inducing apoptosis in the sensitive subset [104]. Sharma et al focused on the metabolic response to chemotherapeutic agent fludarabine: CLL cells, namely MEC-1 and 2 cell lines and primary samples showed an entirely similar profile to fludarabine resistant cells, a profound mTOR activation that caused an overall increase in glycolysis and OXPHOS rates, combined with an upregulation of purine biosynthesis [105]. An interesting observation was made by Siska et al: chronic and acute B leukemia cells can induce metabolic changes in T lymphocytes by reducing their signaling through mTOR, thus slowing down their GLUT1-mediated glucose import and glycolytic rates, ultimately impairing their anti-leukemic action [106].…”

Dual TORK/DNA-PK inhibition blocks critical signaling pathways in chronic lymphocytic leukemia

“…23 Complete deletion of the TORK kinase (TORK) gene in mouse B cells resulted in impaired germinal center formation. 24 Inhibition of mTORC1 by rapamycin in CLL cells results in increased fludarabine sensitivity 25 and inhibition of cytosine guanine dinucleotide-induced proliferation of CLL cells. 26 ATM, ATR, and DNA-PK are critical regulators of the DNA damage repair (DDR) pathway.…”