Ablation of CD28-86 Signaling Results in Induction of Both Caspase-Dependent and Caspase-Independent Cell Death in Myeloma Cells

Multiple myeloma is a disease of long-lived plasma cells (LLPCs), and is the 2^nd most common hematologic malignancy. In recent years, pharmacologic advances have led to an increase in median and overall survival rates. However, the disease remains incurable for the majority of patients, and research on the underlying survival mechanisms of myeloma cells is relevant for discovering new therapeutic approaches that could eventually lead to a cure.

Myeloma cells retain most of the physiological characteristics of their normal counterpart – the LLPC. They secrete antibodies, express CD138, and home and reside in the bone marrow, where they are heavily reliant on growth and survival signals from the stromal microenvironment. Recent reports have shown that the CD28-86 costimulation pathway is important for the generation and survival of LLPCs. Consistent with a pro-survival function, previous studies have demonstrated that CD28 and CD86 high expression are poor prognostic indicators for myeloma patients. Additionally we have shown that CD28 signaling mediates resistance to different chemotherapeutic agents. To better understand the role of CD28 and CD86 in myeloma we have been studying the effects of loss of expression or blockade in myeloma cell lines and patient samples. We have shown that myeloma cells also require CD28-86 signaling for their survival, as knockdown of either CD28 or CD86 via shRNAs, or blockade with CTLA4Ig (Abatacept), led to cell death in 5 myeloma cell lines and 1 patient sample. We have also shown that CD28-86 signaling regulates expression of integrins (β7, β1) that play important roles in cell-cell or cell-matrix interactions that facilitate cell growth and survival. Taken together, our previous work indicates that the CD28-86 signaling pathway plays an important role in maintaining myeloma cell viability. Interestingly, our data indicate that CD86 relays a survival signal that is different from its function as a CD28 ligand. Overexpression of an shRNA-resistant CD86 (CD86FLm) protected against CD86 silencing, while overexpression of CD86TLm (where the intracellular domain of CD86 has been deleted) does not, indicating that the cytoplasmic tail of CD86 plays a role in myeloma cell survival.

In order to determine the survival mechanisms mediated by this signaling pair, we investigated different pathways known to protect myeloma cells from pro-apoptotic signals.We first demonstrated that exogenous IL-6, a myeloma growth and survival factor, cannot protect against cell death from CD28 or CD86 silencing, suggesting that the CD28-CD86 pathway is distinct from IL-6 signaling and provides survival signals that are complementary to IL6 receptor signaling. In contrast, overexpression of pro-survival Bcl-2 family members protects against cell death induced by silencing of CD28 and CD86. However, when we performed expression analyses (RNA-seq, pRT-PCR and Western blot), no consistent significant changes were observed in any of the Bcl-2 family members following CD28 or CD86 knockdown. Since Bcl-2 proteins can inhibit both apoptotic and non-apoptotic forms of cell death (e.g. autophagy), we determined if the cell death was caspase dependent. Caspase-3 is activated by CD28 or CD86 silencing or CTLA4Ig treatment. However pan-caspase inhibitors Boc-D-FMK or QVD-Oph can only partially protect against this cell death despite demonstrating a complete blockade of caspase-3 cleavage. Overall, our data show that cell death induced upon ablation of CD28-86 signaling is pleiotropic, as it appears to be both caspase-dependent and caspase-independent. We will present data on the mechanism of non-apoptotic death (autophagy or necrosis). Preliminary data indicates that autophagy is activated by CD28/CD86 silencing.

Our data suggest that blocking the CD28-86 pathway may be a viable therapeutic addition to current regimens since it induces myeloma cell death through multiple mechanisms and therefore may not be susceptible to drug resistance that is associated with relapsed/refractory disease.