Cd28 Mediates Pro-Survival Auto-Phagy In Multiple Myeloma

Multiple myeloma (MM) is incurable by current therapies, primarily due to acquired chemotherapy resistance and subsequent relapse. Defining the pro-survival responses in MM is critical to discover novel targets and develop new therapies. Our lab previously reported that CD28, the prototypic T cell costimulatory molecule, is also expressed on myeloma cells and is essential to support MM stress adaptation, survival and chemotherapy resistance. However, how CD28 mediates pro-survival responses has been largely undetermined. Here, we report that CD28 mediates a pro-survival response in MM through autophagy. Direct CD28 activation on MM cells by anti-CD28 mAb or co-culture with dendritic cells expressing CD28 ligands CD80 and CD86 protected MM cells from chemotherapy (melphalan) or serum starvation induced death, improving MM viability more than 2 folds. Inhibition of autophagy by the pharmacologic reagents bafilomycin A1 (Baf) or 3-methyladenine (3MA), or knockdown ATG5, a critical autophagy regulator, abrogated CD28's pro-survival effects. CD28 activation resulted in a 2-fold increase in the autophagy marker LC3II as well as elevated autophagosome numbers assessed by CytoID. Conversely, knockdown of CD28 decreased these effects. By using the MM cell lines that expressed tandem mCherry-GFP tagged LC3, which GFP is quenched by autophagic degradation, we confirmed that CD28 engagement induced autophagic flux. Additionally, we found CD28 activation upregulated ATG5 protein expression 2 folds, which has not been previously reported in any cell type. Inhibition of autophagy by Baf or 3MA failed to inhibit upregulation of ATG5 by CD28 signaling, which indicates CD28 signaling upregulates ATG5. Activation of CD28 did not robustly increase the expression of the ATG5 gene as measured by mRNA expression. Analysis of gene expression databases of CD138+ cells from healthy donors, MGUS, smoldering myeloma and MM patients (GSE5900, GSE4581 ) demonstrated that CD28 expression did not correlate ATG5 mRNA expression. Blocking translation with cycloheximide failed to inhibit upregulation of ATG5 by CD28 activation. However, blocking protein degradation using the proteasome inhibitor MG132 completely abolished ATG5 upregulation, suggesting CD28 signaling induces ATG5 protein expression by preventing ATG5 degradation through a novel mechanism. To answer how CD28-mediated autophagy improves MM survival, we found inhibition of autophagy by 3MA or shATG5 or shATG7 minimizes CD28-mediated induction of oxidative phosphorylation in MM. Active mitochondrial respiration requires fatty acids fueled and released by breakdown of lipid droplet. We found lipid droplets in MM decreased upon CD28 activation. Blocking lysosomal lipase activity by lalistat1&2 decreased CD28-mediated pro-survival effects. These findings suggest a novel mechanism where CD28-mediated autophagy is fueling fatty acid oxidation and mitochondrial respiration to maintain metabolic fitness and improve survival.