Targeting Glucose Consumption and Autophagy in Myeloma with the Novel Nucleoside Analogue 8-Aminoadenosine

Mala Shanmugam,Samuel K Mcbrayer,Jun Qian,Kiril Raikoff,Michael J Avram,Seema Singhal,Varsha Gandhi,Paul T Schumacker,Nancy L Krett,Steven T Rosen

doi:10.1074/jbc.m109.000646

Abstract

Multiple myeloma, an incurable plasma cell malignancy, is characterized by altered cellular metabolism and resistance to apoptosis. Recent connections between glucose metabolism and resistance to apoptosis provide a compelling rationale for targeting metabolic changes in cancer. In this study, we have examined the ability of the purine analogue 8-aminoadenosine to acutely reduce glucose consumption by regulating localization and expression of key glucose transporters. Myeloma cells counteracted the metabolic stress by activating autophagy. Co-treatment with inhibitors of autophagy results in marked enhancement of cell death. Glucose consumption by drug-resistant myeloma cells was unaffected by 8-aminoadenosine, and accordingly, no activation of autophagy was observed. However, these cells can be sensitized to 8-aminoadenosine under glucose-limiting conditions. The prosurvival autophagic response of myeloma to nutrient deprivation or to nucleoside analogue treatment has not been described previously. This study establishes the potential of metabolic targeting as a broader means to kill and sensitize myeloma and identifies a compound that can achieve this goal.

Highlights

Nucleoside analogues are antimetabolites that play a pivotal role in the treatment of a spectrum of hematological malignancies
We find that 8-NH2-Ado inhibits glucose consumption that is associated with an activation of autophagy
Unique to 8-NH2-Ado—To determine if the reduction in glucose consumption is a generalized phenomenon preceding myeloma cell death or represents a unique property of 8-NH2-Ado, we examined glucose uptake in the presence of clinically relevant therapeutics that act through distinct mechanisms

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—The MM.1S cell line was developed in our laboratory. U266, ARH 77, and RPMI 8226 cell lines were from ATCC. 30 min prior to the harvest time, potentiometric dye tetramethylrhodamine ethyl ester perchlorate was added to all wells at a final concentration of 100 nM. Cells were incubated at 37 °C for 30 min, followed by PBS wash, and resuspended in PBS containing 2% fetal bovine serum and 20 nM tetramethylrhodamine ethyl ester perchlorate, and samples were analyzed by flow cytometry. The end of 5 h, cells were harvested and resuspended in 1 ml of glucose-free RPMI 1640 containing 0.2 ␮Ci/ml 2-deoxy-D-[13H]glucose (specific activity 8 Ci/mmol; GE Healthcare) and 0.5 ␮M 2-deoxyglucose, with or without cytochalasin B (5 ␮M final concentration), for 25 min. Cells were stained overnight with 1:100 dilutions of antibodies to human GLUT1, GLUT4, LC3, or Golgin-97 with detection using anti-rabbit IgG-Alexa Fluor 594 or anti-mouse IgGAlexa 488 (Invitrogen) for 60 min. Two-tailed p values were calculated using paired or unpaired t tests as indicated, with GraphPad Prism software (San Diego, CA). p values of Ͻ0.05 were considered to be statistically significant

RESULTS

Regulation of Glucose Uptake Is

DISCUSSION