The mTOR Inhibitor Rapamycin Inhibits Drug Transport in Multidrug Resistant Cell Lines and in Acute Myeloid Leukemia (AML) Cells.
Authors: Pawarode Attaphol , O’Loughlin Kieran L. , Cuviello Nicholas W. , Greco William R. , Ford Laurie A. , Minderman Hans , Baer Maria R. 
Published: April 2020

Abstract
Mechanisms of drug resistance in acute myeloid leukemia (AML) include cellular drug efflux mediated by the multidrug resistance (MDR)-associated ATP-binding cassette (ABC) proteins P-glycoprotein (Pgp), multidrug resistance protein-1 (MRP-1) and breast cancer resistance protein (BCRP) and impaired cytoplasmic-nuclear drug transport mediated by the major vault protein lung resistance protein (LRP), as well as aberrant signal transduction due to activation of the mammalian target of rapamycin (mTOR) and other signaling molecules. Rapamycin has antiproliferative effects in AML by virtue of inhibition of mTOR and its downstream signaling pathways, and has clinical activity in this disease. Rapamycin is also known to block drug transport mediated by Pgp, and we sought to determine its effects on transport mediated by MRP-1, BCRP and LRP and its effects on cytotoxicity of MDR substrate drugs. Rapamycin effects on cellular uptake and efflux of mitoxantrone, a substrate for Pgp, MRP-1 and BCRP, were studied by flow cytometry in cell lines with MDR mediated by these proteins, and effects on nuclear-cytoplasmic distribution of doxorubicin were studied by confocal microscopy in cell lines with MDR mediated by LRP. Rapamycin cytotoxicity and effects on cytotoxicity of MDR protein substrate drugs were assessed in 96-well microculture survival assays. Additionally, effects on cellular drug transport were studied by flow cytometry in blasts from 30 untreated AML patients. Rapamycin, at ≥0.5 μM, had concentration-dependent inhibitory effects on drug transport mediated by Pgp, MRP-1 and BCRP and, at ≥2.5 μM, by LRP in cell lines with MDR mediated by these proteins. The magnitude of rapamycin effects was similar to those of the Pgp modulator PSC-833, the MRP-1 modulator MK-571 and the broad-spectrum MDR modulator cyclosporine A (CsA) in Pgp+ and MRP-1+ cell lines, and approximately half those of the BCRP modulator fumitremorgin C (FTC) and of CsA in BCRP+ cells. Rapamycin as a single agent was cytotoxic toward all cell lines tested and cytotoxicity was not affected by the presence of MDR proteins. Concurrent treatment with rapamycin and mitoxantrone resulted in a synergistic drug interaction in HL60/VCR and HL60/ADR cells, with 20-fold and 40-fold increases in potency compared with the expected effects of additivity, but synergy was not seen in parental HL60 cells. Finally, rapamycin increased mitoxantrone uptake and reduced efflux in 25 of 30 AML samples, and its effects on uptake were greater than those of CsA (p= 0.05; 2-tailed Wilcoxon signed-ranks test). Thus, in addition to its known effects on signal transduction, rapamycin also modulates drug transport in cell lines expressing Pgp, MRP-1, BCRP and LRP, synergizes with MDR protein substrate chemotherapy drugs in these cell lines, and enhances uptake and decreases efflux of MDR substrate drugs in AML cells. Effects of rapamycin on transport of chemotherapy drugs, as well as on signal transduction, should be considered in the design of combination regimens in AML.