Targeting Glycolysis and Compensatory Mitochondrial Metabolism In An In Vivo Xenograft Model Of Multiple Myeloma With FDA Approved Ritonavir and Metformin

Multiple Myeloma (MM) is an incurable plasma cell malignancy accounting for 11,000 deaths annually in the US and 20% of deaths from all hematological malignancies. MM is one of myriad malignancies exhibiting enhanced glucose consumption associated with an aerobic glycolytic phenotype (i.e. the Warburg effect). We have previously published a study defining key glucose transporters responsible for facilitating glucose entry in myeloma and observed that MM cells rely on constitutively cell surface-localized GLUT4 for basal glucose consumption. MM cells cultured in the absence of glucose or upon GLUT4 suppression exhibit either overt apoptosis (sensitive phenotype) or growth arrest (resistant phenotype). To further demonstrate the clinical utility of targeting GLUT4 for MM therapy we tested a HIV protease inhibitor ritonavir that has an off-target inhibitory effect on GLUT4. Treatment of MM cells with physiologically achievable concentrations of ritonavir blocks glucose entry resulting in MM cell death or growth suppression. To determine ritonavir’s specificity in targeting GLUT4 we generated KMS11 myeloma cell lines over-expressing GLUT1. As anticipated, in in vitro studies GLUT1 overexpressing cells were found to be resistant to ritonavir. MM cells that were resistant to glucose-deprivation were further sensitized when co-treated with metformin, that we believe targets compensatory mitochondrial complex I activity. The objective of this study was to determine the in vivo efficacy of combinatorial ritonavir, metformin or the combination using the KMS11-GLUT1 or GFP (green fluorescent protein, as a negative control) expressing cells in a xenograft model of MM.

KMS11 myeloma cells expressing GFP or GLUT1 were generated as previously described (Multiple myeloma exhibits novel dependence on GLUT4, GLUT8, and GLUT11: implications for glucose transporter-directed therapy. McBrayer SK, Cheng JC, Singhal S, Krett NL, Rosen ST, Shanmugam M. Blood. 2012 May 17;119(20)). NOD SCID mice were injected subcutaneously with KMS11-GFP or GLUT1 cells on the left or right side, respectively. When tumors were approximately 125 mm³, ritonavir 50 mgs/kg by oral gavage or metformin (125 mgs/kg) by i.p. injection or the combination were administered daily. Tumor volumes, weight, survival, glucose uptake and analysis of specific proteins in tumor lysates were performed.

We first evaluated the impact of ritonavir/metformin or the combination on the viability of KMS11-GFP or GLUT1 expressing cells. Ritonavir elicits apoptosis in KMS11-GFP cells and less so in GLUT1 over-expressing cells, as anticipated (McBrayer S et.al Blood 2012). In vivo, however GLUT1 expressing cells were found to be highly sensitive to ritonavir while the parental GFP expressing cells were resistant. We have previously determined that cells resistant to glucose deprivation-elicited apoptosis have increased oxygen consumption and mitochondrial metabolism that can be targeted with metformin. Therefore, in order to target potential upregulation of mitochondrial OXPHOS we tested the complex 1 inhibitor metformin. We hypothesized that the KMS11-GFP cells in vivo were insensitive to ritonavir due to increased engagement of mitochondrial OXPHOS. In vitro studies with ritonavir and metformin demonstrate potent elicitation of apoptosis correlating with a loss of oxygen consumption and coupled respiration. Indeed, the administration of ritonavir with metformin elicits potent toxicity in both cell types in vivo. An investigation of cellular signaling pathways engaged in vivo indicates that ritonavir in combination with metformin suppresses AKT and AMPK activity and suppresses expression of pro-survival BCL-2 family member MCL-1. We have also demonstrated the efficacy of this treatment regimen in CLL primary patient samples and DLBCL and mantle cell lymphoma and other MM cell lines. HIV patients chronically treated with ritonavir who exhibit diabetic symptoms have been treated with metformin indicating this combination treatment is well tolerated in humans (Kohli, R., et.al 2007. A randomized placebo-controlled trial of metformin for the treatment of HIV lipodystrophy. HIV Med 8:420-426). Our studies reveal a potent combinatorial regimen involving repurposed, FDA approved, ritonavir and metformin for the treatment of MM and potentially other glucose-driven malignancies.

No relevant conflicts of interest to declare.