CD28-Mediated Pro-Survival Signaling: A Novel Therapeutic Target for the Treatment of Multiple Myeloma
Murray, Megan Elizabeth
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Multiple myeloma (MM) is the neoplasm of plasma cells (PC); until late in disease progression, it is dependent upon the bone marrow microenvironment for survival, similar to its normal PC counterparts. Although the last decade has seen the addition of novel therapeutics for myeloma treatment, the disease is still considered to be almost uniformly fatal. In particular, chemotherapeutic resistance after relapse remains the significant clinical challenge in myeloma therapy and much of this resistance can be attributed to interactions with the microenvironment. Here, we have shown that the prototypic T cell costimulatory receptor CD28 is expressed on MM cells and directly contributes to chemotherapeutic resistance. The ligands for CD28, CD80 and CD86, are expressed on antigen presenting cells, including dendritic cells (DC). We show that DC-mediated protection of MM cells from chemotherapy in vitro is completely dependent upon the CD28:CD80/CD86 interaction; if this interaction is blocked with the FDA approved chimeric fusion protein CTLA4-Ig (which binds to CD80 and CD86), MM cells are re-sensitized to chemotherapy. Consistent with these results, treatment of two different murine models of MM with CTLA4-Ig sensitize the disease to a sub-therapeutic dose of melphalan, suggesting that the CD28:CD80/CD86 interaction is critical for maintenance of MM tumors in vivo. We believe these findings establish a key role for CD28 in MM resistance to chemotherapy and targeting this interaction is a novel approach to resensitize MM. Since these data have demonstrated that CD28 is essential for the maintenance of MM cells both in vitro and in vivo, we have begun to examine the signaling pathways downstream of CD28 which contribute to survival. We have previously shown that CD28 activation induces the phosphorylation of phosphatidylinositol-3-kinase (PI3K), a molecule that induces phosphorylation of the kinase Akt. Here we demonstrate that both PI3K and Akt are necessary for CD28-mediated pro-survival signaling--consistent with what is observed downstream of other pro-survival receptors (such as insulin-like growth factor-1 (IGF-1)) in myeloma. Akt is a central signaling molecule in MM cells with many downstream targets so we examined targets that regulate apoptotic factors in MM. One such molecule is the transcription factor forkhead box O3a (FoxO3a) which is phosphorylated and therefore inactivated by Akt; this subsequently leads to a downregulation in the FoxO3a transcriptional target Bim, a pro-apoptotic molecule whose balance is critical for MM cell survival or apoptosis. Here we have shown that CD28 activation induces phosphorylation of FoxO3a and decreases Bim expression. Conversely, CD28 signaling blockade induces Bim expression and apoptosis of MM cells. Knockdown of Bim resulted in partial resistance to CD28-blockade induced apoptosis, pointing to a direct role for Bim in CD28 signaling in MM cells. Together, these data demonstrate a CD28[arrow right]PI3K[arrow right]Akt[arrow right]pFoxO3a[arrow right]Bim pathway which modulates cell survival in myeloma. However, knockdown of Bim does not completely abrogate CD28 blockade-induced apoptosis which suggests that other molecules downstream of PI3K/Akt also contribute to CD28-dependent survival. In T cells, CD28 directly regulates cellular metabolism by upregulating the glucose transporter Glut1. Additionally, we have recently observed that PC also regulate their metabolic efficiency by increasing total Glut1 expression. We found that this pathway is also conserved in MM cells, and CD28 activation increases Glut1 in metabolic stresses (like serum or glucose starvation) as well as in chemotherapy. This increase is Akt dependent and directly correlates with survival. Finally, we examined the role that interferon regulatory factor 4 (IRF4) may have downstream of CD28; it has been shown that MM cells are ritically dependent on IRF4 and it is regulated in an Akt-dependent fashion. Here we show that CD28[arrow right]Akt increases both IRF4 and IRF4's transcriptional target, c-myc (a transcription factor whose disregulation has been shown to be a critical early initiating event in many myelomas). Moreover, CD28 signaling blockade with CTLA4-Ig decreases both IRF4 and c-myc, suggesting another mechanism by which CD28 blockade may sensitize myeloma cells to stresses. Finally, knockdown of c-myc abrogates MM cell survival and sensitizes MM cells to CTLA4-Ig treatment, suggesting that myc is a key downstream component of the CD28 signaling pathway. In summary, our findings suggest a model of CD28-mediated pro-survival signaling in multiple myeloma that is PI3K/Akt-dependent and has a number of downstream targets that impact apoptosis and metabolism. We have also shown that CD28 signaling is essential for MM cell survival both in vitro and in vivo, and this is the first report that demonstrates that targeting CD28 can sensitize myeloma to chemotherapeutics in vivo. These findings suggest that CD28 in the MM/PC compartment is therapeutically targetable for not only MM, but also for auto-immune PC and allergy-induced PC in human disease.