CD28-Mediated Pro-Survival Signaling in Multiple Myeloma

Abstract 2921

Multiple myeloma (MM) is a plasma cell neoplasm which is resident in the bone marrow. It has been observed that MM cells are critically dependent upon the bone marrow (BM) microenvironment for survival, which likely plays a role in therapeutic resistance. Thus, a better understanding of the critical interactions of MM with the BM microenvironment may yield new therapeutic targets. One approach to elucidate these survival mechanisms is to characterize cell surface receptors and their stromal ligands. In particular, it has been observed that MM cells express CD28, a molecule best understood as the prototypic T-cell costimulatory receptor. It has since been demonstrated that expression of CD28 correlates with increasing disease burden and that expression at the time of diagnosis correlated with worse prognosis, which is consistent with CD28's function as a potential pro-survival molecule in MM. CD86, one of the ligands for CD28, has also been identified on MM cell lines and in patient biopsies and correlates with worse prognosis than patient biopsies which are CD86 negative. Together, these data suggest that CD28 is a pro-survival molecule in MM. However, the molecular mechanisms by which CD28 may be mediating a pro-survival response in MM remain largely uncharacterized.

The ligands for CD28, CD80 and CD86, are expressed on APCs, particularly dendritic cells (DC), which are found in the BM microenvironment. We and others have observed that DCs infiltrate myelomatous portions of patient BM biopsies and murine plasmacytomas. Therefore, we hypothesize that DC in the microenvironment are providing the pro-survival signal to the MM via a CD28 :CD80/86 interaction. To test this in vitro, we have co-cultured MM cells and DC + 2 μM melphalan. CD28 signaling was then blocked either on the CD28 side with an anti-CD28 blocking antibody (CD28.6) or the CD80/CD86 side with the fusion protein CTLA4-Ig. In melphalan alone, MM cells have markedly reduced survival. With the addition of DC, MM cell survival significantly increases. However, this pro-survival signal is abrogated by blockade of CD28 signaling via CD28.6 or CTLA4-Ig.

To better understand the mechanisms by which CD28 signaling may be increasing MM cell survival, we first examined expression of apoptotic factors. In particular, it has been shown that diminished levels of the pro-apoptotic molecule Bim plays a critical role in the survival of MM cells. Consistent with these observations, we have data which demonstrate that activation of CD28 decreases Bim protein expression (correlated with increased survival of the MM cell), and blockade of CD28 signaling increase Bim protein expression (correlated with decreasing survival). This mechanism is at least in part transcriptionally regulated; we have demonstrated via RT-PCR that chemotherapy alone increases Bim expression levels, and CD28 activation decreases Bim expression levels without chemotherapy and maintains these lower levels in the presence of chemotherapy.

Additionally, MM cells must be highly metabolically efficient due to their forced production of high levels of immunoglobulin (explaining one mechanism by which proteasome inhibitors may be functioning). In the T cell field, it has been shown that CD28 activation increases expression of the glucose transporter Glut1. Similarly, CD28 activation in MM cells also increases Glut1 protein expression, especially in the absence of glucose. These data demonstrate that CD28 may increase metabolic efficiency in low nutrient conditions. Moreover, we have data which demonstrate that MM cells increase Glut1 protein levels in response to CD28 activation, particularly in the presence of chemotherapies.

Taken together, these data demonstrate that cells in the microenvironment which provide the CD28 ligands (such as DC) are critical for MM cell survival. Moreover, our data show that this CD28-mediated protection of MM cells occurs via at least two separate pathways and affects both apoptosis and metabolism. These data demonstrate that the CD28 receptor and its downstream signaling component represent novel therapeutic targets for the treatment of multiple myeloma.