title: Targeting mTORC1-mediated metabolic addiction overcomes fludarabine resistance in malignant B cells.
abstract: 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.
  
  
  CONCLUSIONS
  This study provides the first evidence for mTORC1/p70S6K-dependent regulation of pyrimidine biosynthesis in a relevant disease setting.
authors: Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio. Department of Biological, Geological and Environmental Sciences, Cleveland State University, Cleveland, Ohio.
authors: Arishya
authors: A
authors: Sharma
authors: Department of Pathobiology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio.
authors: Allison J
authors: AJ
authors: Janocha
authors: Department of Hematologic Oncology and Blood Disorders, Taussig Cancer Institute, Cleveland Clinic, Cleveland, Ohio.
authors: Brian T
authors: BT
authors: Hill
authors: Department of Hematologic Oncology and Blood Disorders, Taussig Cancer Institute, Cleveland Clinic, Cleveland, Ohio.
authors: Mitchell R
authors: MR
authors: Smith
authors: Department of Pathobiology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio.
authors: Serpil C
authors: SC
authors: Erzurum
authors: Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio. almasaa@ccf.org.
authors: Alexandru
authors: A
authors: Almasan
mesh_terms: D001402:B-Lymphocytes / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D006019:Glycolysis; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D000076222:Mechanistic Target of Rapamycin Complex 1; D046912:Multiprotein Complexes / Q000235:genetics* / Q000378:metabolism; D010085:Oxidative Phosphorylation; D011743:Pyrimidines / Q000096:biosynthesis; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics; D058570:TOR Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D014740:Vidarabine / Q000008:administration & dosage / Q000031:analogs & derivatives
chemical_list: D046912:Multiprotein Complexes; D011743:Pyrimidines; D000076222:Mechanistic Target of Rapamycin Complex 1; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D058570:TOR Serine-Threonine Kinases; C448901:ribosomal protein S6 kinase, 70kD, polypeptide 1; D014740:Vidarabine; C030986:pyrimidine; C024352:fludarabine