Rapamycin, Inhibitor of Mtor Kinase, Sensitizes Leukemia Cells to Fludarabine-Induced Apoptosis, but Protects Survival of Normal Lymphocytes.
Authors: Smolewski Piotr , Cebula Barbara , Wierzbicka Dorota , Linke Anna , Jamroziak Krzysztof , Blagosklonny Mikhail V. , Darzynkiewicz Zbigniew , Robak Tadeusz 
Published: April 2020

Abstract
Background: The mTOR kinase inhibitor, rapamycin (RAPA), inhibits cell growth by G1 phase cell cycle arrest. RAPA-induced cytotoxicity against acute lymphoblastic leukemia (ALL) cells was recently reported.
Aim: We investigated cytotoxicity of RAPA alone or in combination with fludarabine (FA), a cytostatic active in G1 phase, in ALL cells as well as in healthy lymphocytes.
Methods: NALM-6 cells (pre-B ALL-derived cell line), and phytohemaglutynin (PHA)-stimulated normal lymphocytes were treated for 24–48h with different concentrations of RAPA (0,05–500ng/ml), alone or in combination with 0.1–50.0mM FA. Some samples were initially incubated with RAPA for 24h and then FA was added for the next 24h. Untreated cultures and treated with RAPA, FA or PHA alone served as respective controls. The pro-apoptotic effect was assessed by both Annexin-V and TUNEL assays and presented as an apoptotic index (AI). Cell cycle was analyzed by DNA content distribution in propydium iodide/RN-ase stained cells. Additionally, intracellular expression of cyclin A, D3 and E was evaluated. All fluorescence measurements were performed by flow cytometry. Overall cytotoxicity was evaluated by MTT assay.
Results: RAPA was found to exert dual effect on NALM-6 cells. In lower concentrations (0.05–5ng/ml) RAPA exclusively inhibited proliferation, arresting NALM-6 cells in G1 phase of cell cycle. An increasing evidence of apoptosis, along to enhancing cytotoxicity in MTT assay was observed higher RAPA doses (10–500ng/ml). When NALM-6 were treated with RAPA+FA, the highest AIs were found for the combination of 0.5 or 1.0ng/ml RAPA with 1nM FA. Median AI induced at 24h by 1.0ng/ml RAPA+FA was 12.4%, comparing to with RAPA or FA alone (AIs 1.9% and 4.8%, respectively; both p&lt;0.0001). This effect was synergistic, with the combination index (CI) 0.68. Treatment with RAPA+FA significantly downregulated cyclin A and E expression, comparing to both untreated control and cultures treated with single agents. Importantly, 24h pretreatment of NALM-6 cells with RAPA additionally accelerated apoptosis. In PHA-stimulated lymphocytes, cytotoxicity induced by corresponding concentrations of drugs was moderately lower, than in NALM-6. In contrast to leukemic cells, 24h pretreatment of normal lymphocytes with RAPA resulted in distinct prevention of FA-induced cytotoxicity. It was accompanied by the block in PHA-induced cell cycle progression to phase S. Moreover, this blocking effect of RAPA was reversible, when after 24h of treatment lymphocytes were rinsed and placed back in fresh RAPA-free medium for the next 24h of culture.
Conclusions: In low concentration RAPA sensitizes NALM-6 cells to FA-induced apoptosis. Pretreatment with RAPA enhances cytotoxic effect on leukemia cells, but not on normal lymphocytes. RAPA administrated prior to FA blocks reversible cell cycle progression, preventing lymphocyte from FA cytotoxicity. These data provide rationale for future applying RAPA in the combination with purine nucleoside analogues for treatment of lymphoproliferative diseases. Moreover, they suggest, that the choice of optimal doses of both RAPA and the cytostatic may result in selective anti-tumor treatment, with protection of normal cells.