Inhibition of Proliferation and Induction of Apoptosis in Multiple Myeloma Cell Lines by CD137 Ligand Signaling

BACKGROUND: Multiple myeloma (MM) is a malignancy of terminally-differentiated plasma cells, and the second most prevalent blood cancer. At present there is no cure for MM, and the average prognosis is only three to five years. Current treatments such as chemotherapy are able to prolong a patient's life but rarely prevent relapse of the disease. Even hematopoietic stem cell transplants and novel drug combinations are often not curative, underscoring the need for a continued search for novel therapeutics. CD137 and its ligand are members of the Tumor Necrosis Factor (TNF) receptor and TNF superfamilies, respectively. Since CD137 ligand cross-linking enhances proliferation and survival of healthy B cells we hypothesized that it would also act as a growth stimulus for B cell cancers. METHODOLOGY/PRINCIPAL FINDINGS: Proliferation and survival of B cell lymphoma cell lines were not affected or slightly enhanced by CD137 ligand agonists in vitro. But surprisingly, they had the opposite effects on MM cells, where CD137 ligand signals inhibited proliferation and induced cell death by apoptosis. Furthermore, secretion of the pro-inflammatory cytokines, IL-6 and IL-8 were also enhanced in MM but not in non-MM cell lines in response to CD137 ligand agonists. The secretion of these cytokines in response to CD137 ligand signaling was consistent with the observed activation of the classical NF-kappaB pathway. We hypothesize that the induction of this pathway results in activation-induced cell death, and that this is the underlying mechanism of CD137-induced MM cell death and growth arrest. CONCLUSIONS/SIGNIFICANCE: These data point to a hitherto unrecognized role of CD137 and CD137 ligand in MM cell biology. The selective inhibition of proliferation and induction of cell death in MM cells by CD137 ligand agonists may also warrant a closer evaluation of their therapeutic potential

CD137 ligand is expressed by B cell lymphoma and myeloma cell lines.

<p>Cells were stained by PE-conjugated monoclonal antibodies against CD137 (clone 4B4-1), or anti-CD137 ligand (clone 4B1-436), (open curves) or their isotype control (MOPC-21), (filled curve).</p

CD137 inhibits proliferation and induces cell death of MM but not of non-MM cells.

<p>Cells were cultured on plate-bound Fc or CD137-Fc protein or on uncoated plates (PBS). (A) After indicated times proliferation was determined via ³H-thymidine incorporation. (B) Cell viability was determined after 24, 48, 72 and 96 h via trypan blue staining. Depicted are means ± standard deviations of triplicate measurements. * p<0.05. This experiment is representative of three independent experiments with similar results.</p

CD137 induces apoptosis in the MM cell lines.

<p>(A) SGH-MM5 cells at a density of 10⁶ cells/ml were cultured on plate-bound Fc or CD137-Fc protein or on uncoated plates (PBS). After 24 h the cells were stained with Annexin V and 7-AAD. Similar results were obtained for the other MM cell lines. (B) Cells from (A) were stained with Acridine Orange (green) and Ethidium Bromide (red). Photographs were taken at a magnification of 40×. (C) CD137-Fc treated SGH-MM5 cells of B at a magnification of 200×. (D) Caspase 3 activity was determined 6 h after exposure of SGH-MM5 and RPMI 8226 cells to immobilized Fc or CD137-Fc protein. These experiments are representative of three independent experiments with similar results.</p

Requirement of immobilization of CD137 ligand agonists.

<p>SGH-MM5 (A and B) or SGH-MM6 (C and D) cells at a density of 10⁶ cells/ml were cultured on uncoated plates (PBS), or on plate-bound Fc, CD137-Fc, mouse IgG (MOPC21) or anti-CD137 ligand antibody (clones 5F4 and C65-485) or on uncoated plates (PBS), or to which Fc or CD137-Fc proteins were added soluble at 10 µg/ml. (A) Percentage of dead cells (left panel) and number of total live cells (right panel) were determined after 24 h via trypan blue staining. (B) Extent of apoptosis of cells in (A) was determined by Annexin V and 7-AAD staining. (C) Percentages of dead cells were determined at indicated times via trypan blue staining. (D) IL-8 concentrations in 24 h cell supernatants as determined by ELISA. Depicted are means ± standard deviations of triplicate measurements. * p<0.05.</p

CD137-induced MM cell death is not inhibited by IL-6 or IL-2.

<p>SGH-MM6 cells at a density of 1.2×10⁶ cells/ml were cultured on plate-bound Fc or CD137-Fc protein or on uncoated plates (PBS), and IL-6 (1 ng/ml) or IL-2 (100 units/ml) were added. Cell viability was determined after 24 h via trypan blue staining. Depicted are means ± standard deviations of percentages live cells from triplicate measurements. * p<0.05. This experiment is representative of three independent experiments with similar results.</p

Early activation of the classical NF-κB pathway is initiated upon CD137 ligand signaling.

<p>SGH-MM5, SGH-MM6 and RPMI 8226 cells were treated with plate-bound Fc or CD137-Fc. (A) Total protein was extracted at indicated times. NF-κB signaling proteins were detected by immunoblotting. (B) Cells were treated with CD137-Fc for indicated times and nuclear extracts were isolated and subjected to NF-κB transcription factor assay analysis. Data is represented by the average of triplicates within the experiment and is representative of two independent experiments. P values were calculated using pair wise t-test comparing time zero to time 60 min. * p<0.05. (C) Cells were treated with plate bound Fc or CD137-Fc protein for six hours following which total RNA was extracted. Real time RT-PCR was performed on both human IκBα and IL-6 transcripts. RT-PCR data is represented by fold change as calculated using the 2^ΔΔCT method where GADPH served as a reference gene, and where each data point was performed in triplicate. (D) Cells were cultured for 48 h on plates with immobilized Fc (white open curve) or CD137-Fc protein (grey filled curve), and then stained with 50 nM DiOC₆ and analyzed by flow cytometry. Cells with no DiOC₆ added were used as background control (hatched).</p