Glutamine Catabolism Maintains MCL-1 Expression in Glucose-Deprived Multiple Myeloma

Multiple myeloma (MM) is a plasma cell malignancy accounting for 11,000 deaths annually in the US with the majority succumbing to disease due to the development of resistance. A key component to the development of resistance lies in ineffective engagement of the BCL-2 family of apoptosis regulators. We and others have demonstrated that glucose maintains levels of a key resistance-promoting BCL-2 family member, myeloid cell leukemia factor 1 (MCL-1). Expression of MCL-1 promotes resistance of MM to a wide range of therapeutics. MM, like other cancers, exhibits elevated glucose consumption associated with manifestation of the Warburg effect. We previously reported that myeloma cells maintain the glycolytic phenotype by achieving constitutive plasma membrane localization of GLUT4, and GLUT4 inhibition or glucose deprivation suppressed MCL-1 expression. In this study, we further investigated the mechanism underlying the suppression of MCL-1 expression upon glucose deprivation.

L363 and KMS18 cell lines are sensitive to glucose deprivation, exhibiting significant cytotoxicity upon glucose deprivation and demonstrating rapid reduction of MCL-1 protein in contrast to JJN3 and KMS11 cells that are resistant to glucose deprivation. Knockdown of MCL-1 in JJN3 dramatically induced apoptosis while overexpression of MCL-1 in L363 significantly reversed cell death upon glucose deprivation, supporting a role for MCL-1 suppression in mediating glucose-deprivation elicited cell death. We next proceeded to investigate the metabolic pathways contributing to the persistence of MCL-1 in the glucose-insensitive lines. Since glutamine is an important carbon source that can compensate for glucose deprivation, we examined glutamine uptake in L363 and JJN3 cells. Evaluation of ³H-glutamine uptake revealed that JJN3 cells exhibit higher glutamine uptake than L363 cells. In addition, we performed ¹³C-based metabolic flux analysis in order to identify metabolites that may be contributing to the maintenance of MCL-1 in glucose-deprived cells. Interestingly the level of glutathione (GSH) was much higher in JJN3 than in L363 that was maintained even upon glucose deprivation. Since GSH is a major cellular reactive oxygen species (ROS) scavenger, we evaluated levels of ROS using a cyto-roGFP construct and found that it was significantly elevated upon glucose withdrawal in L363 but not in JJN3. In addition, treating glucose deprived L363 with cell-permeant GSH monoethyl ester (GSH-MEE) rescued MCL-1 expression, further supporting a role for ROS in suppressing MCL-1 expression.

Glutamine uptake evaluation revealed that glutamine consumption was maintained upon glucose withdrawal in both JJN3 and L363, so we were then interested in the TCA cycle-related differences in glutamine metabolism between JJN3 and L363. The level of pyruvate dehydrogenase alpha 1 (PDHA1) phosphorylation decreased upon glucose deprivation only in JJN3 cells, which activates PDHA1 and allows pyruvate to flow into the TCA cycle. In accordance with this, treatment of JJN3 with dichloroacetate (DCA), which inhibits pyruvate dehydrogenase kinase and therefore activates PDHA1, restored MCL-1 expression and prevented cell death of L363 during glucose deprivation. To study how TCA cycle helps maintain the GSH level, glutamate dehydrogenase (GDH) knockdown cells were generated because GDH catalyzes the reversible inter-conversion of glutamate to α-ketoglutarate and glutamate is the precursor for GSH. Interestingly, in GDH knockdown L363 cells, treatment of DCA was not able to reverse MCL-1 protein level upon glucose deprivation. Taken together, our observations suggest that glucose-deprived resistant cells sustain higher levels of GSH via greater glutamine uptake, thereby scavenging ROS to sustain cell viability; while sensitive cells cannot make GSH effectively, leading to increased ROS level, decreased MCL-1 expression, and ultimately, apoptosis.

In sum, we have discovered that targeting glucose entry elicits apoptosis via an induction of ROS leading to suppression of MCL-1. These observations provide further rationale for the development of GLUT4-specific biologics to target aberrant glucose metabolism in myeloma and the need to further understand how glutamine metabolism and the TCA cycle contribute to maintaining MCL-1 expression.