Rapamycin Causes Reversible Cell Cycle Slowing, Rather Than Cell Cycle Arrest, in Myeloid and Lymphoid Cell Lines and in Acute Leukemia Cells

The mammalian target of rapamycin (mTOR), a serine/threonine protein kinase that regulates cell survival and proliferation, is constitutively activated in diverse malignancies, and constitutive mTOR activation has been implicated in both malignant transformation and resistance to chemotherapy. The mTOR inhibitor rapamycin, which inhibits mTOR signaling and has immunosuppressive effects at nanomolar concentrations, also has antineoplastic activity, but its reported effects on cell cycle and on apoptosis have been variable. Several studies evaluated lymphoid and myeloid cell lines after 24 or 48 hours of exposure to rapamycin and reported cell cycle arrest in G1, while others reported apoptosis. Rapamycin is being incorporated into treatment regimens in acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and other hematologic malignancies, and knowledge of its growth inhibitory and cytotoxic effects will be useful in designing combination regimens. To this end, the concentration-dependent effects of rapamycin on survival, proliferation, cell cycle and apoptosis of myeloid and lymphoid cell lines and acute leukemia cells were studied. Assays were performed at serial time points extending to 96 hours. Cells studied included HL60 myeloid and Jurkat T-lymphoblastic leukemia, 8226 myeloma and U937 histiocytic lymphoma cells and ML-1 myeloid leukemia cells, which have intact p53, as well as multidrug resistant HL60/VCR, HL60/ADR and 8226/MR20 cells overexpressing the ATP-binding cassette (ABC) proteins ABCB1, ABCC1 and ABCG2, respectively. Pretreatment blasts from 9 patients with AML and 4 with ALL were also studied. Cell survival was measured following 96-hour (cell lines) or 48-hour (patient samples) culture with rapamycin at picomolar to micromolar concentrations using the WST-1 colorimetric assay. Cells grown in suspension culture without rapamycin and with 1 nanomolar to 100 micromolar rapamycin were counted at serial time points. Cell cycle was analyzed by propidium iodide labeling and bromodeoxyuridine incorporation, cell division by carboxyfluorescein succinimidyl ester (CFSE) staining and apoptosis by Annexin V labeling, all measured by flow cytometry. Concentration-dependent effects of rapamycin on cell lines and acute leukemia cells in the WST-1 assay were biphasic, occurring at nanomolar and again at micromolar concentrations, with a plateau from approximately 10 nanomolar to 10 micromolar. At nanomolar concentrations rapamycin prolonged cell doubling times by 20–40%, as evidenced by CFSE staining at serial time points, but it did not cause cell cycle arrest. Examined at serial time points, cells continuously exposed to rapamycin at nanomolar concentrations did not accumulate in any single phase of the cell cycle, nor did they become apoptotic. Doubling times increased, resulting in decreased cell numbers at each time point in relation to control cultures without rapamycin. When cells cultured with rapamycin were transferred to rapamycin-free medium, doubling times reverted to those in rapamycin-free cultures, demonstrating that cell cycle slowing resulted from continuous exposure to rapamycin and was fully reversible in the absence of rapamycin. In contrast to rapamycin at nanomolar concentrations, rapamycin at micromolar concentrations caused apoptosis of cell lines and acute leukemia cells. Thus rapamycin at nanomolar concentrations increases the doubling time of myeloid and lymphoid cell lines and acute leukemia cells, while rapamycin at micromolar concentrations causes apoptosis. Rapamycin doses used in immunosuppressive regimens are well tolerated and yield nanomolar plasma concentrations. Our data suggest that continuous oral administration of rapamycin or its analogs at the doses used in immunosuppressive regimens may slow growth of leukemia cells and might have utility as maintenance therapy in the minimal residual disease setting, given the known lack of effect on normal cells. Higher doses of rapamycin are being studied in phase I clinical trials. Finally, increased understanding of rapamycin’s effect on leukemia cell growth will help direct its incorporation into combination therapy.