CD28 Activation Induces Metabolic Adaptation in Multiple Myeloma Cells
Authors: Daniela Ventro , Utley Adam T , Lee Kelvin P 
Published: October 2019

Abstract
Alterations in cancer cell metabolism is a century old concept recently recognized as one of the hallmarks of cancer. Cancer cells, including multiple myeloma (MM), largely shift how they utilize the glucose they consume to glycolysis from that of oxidative phosphorylation (OXPHOS). This phenomenon supports the cancer cell’s large anabolic demands for continuous growth and proliferation and is reinforced by key signaling pathways. However, as a cancer of the previously non-dividing plasma cell, these metabolic adaptations are only beginning to be documented in MM.
CD28 is classically known as the T-cell co-stimulatory receptor, but is also expressed on normal plasma cells and their malignant counterparts. Previous data suggests that CD28 is required for MM cell survival, protective during stress induced conditions, and correlates with poor prognosis in the clinic. Furthermore, studies using the anti-CD28 activating monoclonal antibody (mAb) have identified phosphatidyl-inositol 3-kinase (PI3K)/Akt activation to be a key driver of its pro-survival function. Akt is also an important integrator of cellular metabolism and cell growth and proliferation signaling pathways. Therefore, its CD28 mediated activation may uncover the mechanism by which MM cells are able to metabolically sustain stress induced conditions and thrive thereafter. Herein, we show that when CD28 is activated by anti-CD28 mAb (10µg/ml) under stressful conditions (media serum reduction), Akt (T308) phosphorylation increases, resulting in an increase of total protein and cell surface expression of the glucose transporter, GLUT1. To assess if cells take in more glucose in the presence of anti-CD28 mAb they were cultured in glucose free media and glucose was then added back at concentrations of 0.5, 1 and 5mM with or without activating anti-CD28 mAb. Significant increases in glucose uptake were seen in the 5mM anti-CD28 mAb treatment group when compared to the untreated control, correlating positively with the increase in GLUT1 protein expression.
To evaluate whether or not CD28 activation induces a preferential for glycolytic breakdown of glucose, the same treatment conditions were repeated and lactate production/oxygen consumption, as a measure of glycolysis and OXPHOS respectively, were measured in a fluorometric kinetic assay. Lactate production significantly increased in MM cells treated with anti-CD28 mAb compared to untreated controls, confirming its role in enhancing glycolysis for cell growth and survival. This data is further supported by increased cell death observed in murine MM cells treated with the glycolysis inhibitor, 2-Deoxyglucose (2-DG). Interestingly, the level of oxygen consumption was comparable in all groups suggesting not only minimal effect in response to CD28 activation, but also relatively unimpaired OXPHOS in MM cells. Furthermore, analysis of mitochondrial biogenesis using the mitotracker green stain and production of reactive oxygen species by 2’,7’-difchlorofluorescin diacetate (DCFDA) oxidation in murine MM cells also revealed both processes to be intact and increased in the presence of CD28 activation. Taken together, these results suggest that CD28 signaling plays a strategic role in shifting the metabolic axis to that of increased glucose uptake and consumption via glycolysis through phosphorylation of PI3K/Akt and upregulation of GLUT1 expression. Pharmacological inhibition of CD28 is therefore an attractive avenue for therapeutic intervention in MM and we have previously shown that interfering with CD28 and its interacting ligands, CD80/CD86, using the CTLA4-Ig fusion protein, is effective in this regard.

Disclosures
No relevant conflicts of interest to declare.