RAD001 (everolimus) induces dose-dependent changes to cell cycle regulation and modifies the cell cycle response to vincristine

Abstract

More than 50% of adults and ~20% of children with pre-B acute lymphoblastic leukemia (ALL) relapse following treatment. Dismal outcomes for patients with relapsed or refractory disease mandate novel approaches to therapy. We have previously shown that the combination of the mTOR inhibitor RAD001 (everolimus) and the chemotherapeutic agent vincristine increases the survival of non-obese diabetic/severe combined immuno-deficient (NOD/SCID) mice bearing human ALL xenografts. We have also shown that 16 μM RAD001 synergized with agents that cause DNA damage or microtubule disruption in pre-B ALL cells in vitro. Here, we demonstrate that RAD001 has dose-dependent effects on the cell cycle in ALL cells, with 1.5 μM RAD001 inhibiting pRb, Ki67 and PCNA expression and increasing G0/1 cell cycle arrest, whereas 16 μM RAD001 increases pRb, cyclin D1, Ki67 and PCNA, with no evidence of an accumulation of cells in G0/1. Transition from G2 into mitosis was promoted by 16 μM RAD001 with reduced phosphorylation of cdc2 in cells with 4 N DNA content. However, 16 μM RAD001 preferentially induced cell death in cells undergoing mitosis. When combined with vincristine, 16 μM RAD001 reduced the vincristine-induced accumulation of cells in mitosis, probably as a result of increased death in this population. Although 16 μM RAD001 weakly activated Chk1 and Chk2, it suppressed strong vincristine-induced activation of these cell cycle checkpoint regulators. We conclude that RAD001 enhances chemosensitivity at least in part through suppression of cell cycle checkpoint regulation in response to vincristine and increased progression from G2 into mitosis.

Phase I trial of the mTOR inhibitor everolimus in combination with multi‐agent chemotherapy in relapsed childhood acute lymphoblastic leukemia

“…Inhibitors of mTOR have shown promise in preclinical models of ALL through the direct inhibition of tumor cell growth and also by reversal of glucocorticoid resistance . mTOR inhibitors have also demonstrated in vitro synergy in combination with a number of other chemotherapeutic agents, including dexamethasone, doxorubicin, l ‐asparaginase, methotrexate, and etoposide …”

“…[8][9][10][11][12] mTOR inhibitors have also demonstrated in vitro synergy in combination with a number of other chemotherapeutic agents, including dexamethasone, doxorubicin, L-asparaginase, methotrexate, and etoposide. [13][14][15][16] Everolimus (Afinitor R ; Novartis), an oral mTOR inhibitor that does not require therapeutic monitoring, is approved for use in adults for a variety of indications including the treatment of breast cancer and pancreatic neuroendocrine tumors, and as an immunosuppressive agent in solid organ transplantation. In a phase I/II trial of everolimus in adults with refractory hematologic malignancies, no dose-limiting toxicities were identified at the two dose levels (DLs) tested (5 mg daily and 10 mg daily continuously).…”

New biomarkers and therapeutic strategies in acute lymphoblastic leukemias: Recent advances

“…Regarding mTOR inhibition, RAD001 has been widely investigated for its role in suppressing tumoral cell growth . This inhibitor shows a more selective activity for the mTORC1 complex, and it is crucial in inducing caspase‐independent and ‐dependent cell death . In a phase I study enrolling children and adolescents with ALL experiencing a first marrow relapse, RAD001 was tested with the natural alkaloid Vincristine and the GC Prednisone or Doxorubicin in subjects with bone marrow relapse occurring >18 months after first CR.…”

Targeting the phosphatidylinositol 3‐kinase/Akt/mechanistic target of rapamycin signaling pathway in B‐lineage acute lymphoblastic leukemia: An update

“…Also RAD001 has proven its efficacy, especially in models of Ph − pediatric B‐ALL. Importantly, RAD001 synergized with conventional chemotherapy (e.g., vincristine) or novel agents (e.g., bortezomib) both in vitro and in vivo, with increased caspase‐dependent, but p53‐independent, cell killing (Saunders, Cisterne, Weiss, Bradstock, & Bendall, ; Saunders et al, ). However, it has also been shown that RAD001 induces other types of cell death, including autophagy (Crazzolara, Bradstock, & Bendall, ) and caspase‐independent paraptosis (Baraz et al, ).…”