The antiangiogenic activity of thalidomide (Thal), coupled with an increase in bone marrow angiogenesis in multiple myeloma (MM), provided the rationale for the use of Thal in MM. Previously, the direct anti-MM activity of Thal and its analogues (immunomodulatory drugs, IMiDs) on MM cells was demonstrated, suggesting multiple mechanisms of action. In this study, the potential immunomodulatory effects of Thal/IMiDs in MM were examined. It was demonstrated that Thal/IMiDs do not induce T-cell proliferation alone but act as costimulators to trigger proliferation of anti-CD3–stimulated T cells from patients with MM, accompanied by an increase in interferon-γ and IL-2 secretion. However, an increase in autologous T-cell killing of patient MM cells could not be demonstrated. A role for natural killer (NK)- and LAK-cell–mediated killing is suggested because IL-2–primed peripheral blood mononuclear cells (PBMCs) treated with Thal/IMiDs demonstrated significantly increased lysis of MM cell lines. Cold target inhibition assays suggested NK- rather than LAK-cell–mediated killing. Furthermore, this killing was not major histocompatibility complex-class restricted, and the depletion of CD56⁺ cells blocked the drug-induced MM cell lysis. It was significant that increased killing of patient MM cells by autologous PBMCs treated with Thal/IMiDs was also observed. Although the in vivo relevance of NK-cell–mediated MM cell killing is unknown, phenotypic analysis performed in MM patients receiving Thal therapy demonstrated an increase in CD3⁻CD56⁺cells in patients responding to therapy. Thus in vitro and in vivo data support the hypothesis that Thal may mediate its anti-MM effect, at least in part, by modulating NK cell number and function.

Selinexor, a selective inhibitor of nuclear export compound that blocks exportin 1 (XPO1) and forces nuclear accumulation and activation of tumor suppressor proteins, inhibits nuclear factor κB, and reduces oncoprotein messenger RNA translation, is a potential novel treatment for myeloma that is refractory to current therapeutic options.

We have recently shown that proteasome inhibitor PS-341 induces apoptosis in drug-resistant multiple myeloma (MM) cells, inhibits binding of MM cells in the bone marrow microenvironment, and inhibits cytokines mediating MM cell growth, survival, drug resistance, and migration in vitro. PS-341 also inhibits human MM cell growth and prolongs survival in a SCID mouse model. Importantly, PS-341 has achieved remarkable clinical responses in patients with refractory relapsed MM. We here demonstrate molecular mechanisms whereby PS-341 mediates anti-MM activity by inducing p53 and MDM2 protein expression; inducing the phosphorylation (Ser15) of p53 protein; activating c-Jun NH₂-terminal kinase (JNK), caspase-8, and caspase-3; and cleaving the DNA protein kinase catalytic subunit, ATM, and MDM2. Inhibition of JNK activity abrogates PS-341–induced MM cell death. These studies identify molecular targets of PS-341 and provide the rationale for the development of second-generation, more targeted therapies.

Increased angiogenesis has recently been recognized in active multiple myeloma (MM). Since vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are two key mediators of angiogenesis, we characterized the production of VEGF, b-FGF and interleukin-6 (IL-6) (a MM growth and survival factor) in MM cell lines and Epstein–Barr virus (EBV) transformed B cell lines from MM patients, patient MM cells, as well as bone marrow stromal cells (BMSCs) from normal healthy donors and MM patients. We detected secretion of VEGF, but no bFGF and IL-6, in MM cell lines (MM.1S, RPMI 8226 and U266); EBV transformed B cell lines from MM patients (IM-9, HS-Sultan and ARH77); MM cell lines resistant to doxorubicin (RPMI-DOX40), mitoxantrone (RPMI-MR20), melphalan (RPMI-LR5) and dexamethasone (MM.1R); and patient MM cells (MM1 and MM2). BMSCs from MM patients and normal donors secreted VEGF, b-FGF and IL-6. Importantly, when MM cells were adhered to BMSCs, there was a significant increase in VEGF (1.5- to 3.1-fold) and IL-6 (1.9- to 56-fold) secretion. In contrast, the bFGF decreased in co-cultures of BMSCs and MM cells. Paraformaldehyde fixation of BMSCs or MM cells prior to adhesion revealed that VEGF was produced both from BMSCs and MM cells, though it may come primarily from BMSCs in some cultures. IL-6 was produced exclusively in BMSCs, rather than MM cells. Moreover, when MM cells were placed in Transwell insert chambers to allow their juxtaposition to BMSCs without cell to cell contact, induction of VEGF and IL-6 secretion persisted, suggesting the importance of humoral factors. Addition of exogenous IL-6 (10 ng/ml) increased VEGF secretion by BMSCs. Conversely, VEGF (100 ng/ml) significantly increased IL-6 secretion by BMSCs. Moreover, anti-human VEGF (1 μg/ml) and anti-human IL-6 (10 μg/ml) neutralizing antibodies reduced IL-6 and VEGF secretion, respectively, in cultures of BMSCs alone and co-cultures of BMSCs and MM cells. Finally, thalidomide (100 μM) and its immunomodulatory analog IMiD1-CC4047 (1 μM) decreased the upregulation of IL-6 and VEGF secretion in cultures of BMSCs, MM cells and co-cultures of BMSCs with MM cells. These data demonstrate the importance of stromal–MM cell interactions in regulating VEGF and IL-6 secretion, and suggest additional mechanisms whereby thalidomide and IMiD1-CC4047 act against MM cells in the BM millieu.