BAFF and APRIL protect myeloma cells from apoptosis induced by interleukin 6 deprivation and dexamethasone

Abstract

Identification of growth factors in neoplasias may be a target for future therapies by blocking either growth factor receptor interaction or the induced pathway. Using gene expression profiling, we identified overexpression of 2 receptors for a proliferation-inducing ligand (APRIL) and B-cell activating factor (BAFF) in malignant plasma cells compared with normal plasma cells. APRIL and BAFF are involved in a variety of tumor and autoimmune diseases, including B-cell malignancies. We confirmed the expression of BAFF and APRIL receptors (B-cell maturation antigen [BCMA], transmembrane activator and calcium modulator and cyclophilin ligand interactor [TACI], and BAFF-R) in a majority of 13 myeloma cell lines and in the purified primary myeloma cells of 11 patients. APRIL and BAFF were potent survival factors for exogenous cytokine-dependent myeloma cell lines and were autocrine growth factors for the RPMI8226 and L363 autonomously growing cell lines. These factors activated nuclear factor (NF)-kappaB, phosphatidylinositol-3 (PI-3) kinase/AKT, and mitogen-activated protein kinase (MAPK) kinase pathways and induced a strong up-regulation of the Mcl-1 and Bcl-2 antiapoptotic proteins in myeloma cells. BAFF or APRIL was also involved in the survival of primary myeloma cells cultured with their bone-marrow environment, and protected them from dexamethasone (DEX)-induced apoptosis. Finally, the serum levels of BAFF and APRIL were increased about 5-fold in patients with multiple myeloma (MM) as compared with healthy donors. Altogether, these data suggest that APRIL/BAFF inhibitors may be of clinical value in MM.

Figure 1. Expression of BCMA. TACI. and BAFF-R in myeloma cells and normal plasmablasts

A. Expression of BCMA, TACI, and BAFF-R mRNA was analyzed by RT-PCR in 13 HMCLs, in CD138⁺ purified primary myeloma cells from six patients with intramedullary MM (1–6) and five patients with plasma cell leukemia (7–11), and in five normal in-vitro generated polyclonal plasmablastic cells (PPC). B cells and dendritic cells (DC) were used as positive and negative controls, respectively. B. Cell surface expression of receptors for BAFF by HMCLs was determined by flow cytometry using a biotinylated human BAFF-murine CD8 fusion protein and phycoerythrin-conjugated streptavidin. C. Cell surface expression of receptors for BAFF by primary myeloma cells and PPC was determined by flow cytometry using a biotinylated human BAFF-murine CD8 fusion protein and phycoerythrin-conjugated streptavidin.

Figure 2. Expression of BAFF and APRIL in myeloma cells and normal plasmablasts

A. Expression of BAFF and APRIL mRNA was analyzed by RT-PCR in 13 HMCLs, in CD138⁺ purified primary myeloma cells from six patients with intramedullary MM (1–6) and five patients with plasma cell leukemia (7–11), and in five normal in-vitro generated polyclonal plasmablastic cells (PPC). B cells and dendritic cells (DC) were used as negative and positive controls, respectively. B. Cell surface expression of BAFF was determined by flow cytometry using an anti BAFF antibody.

Figure 3. BAFF and APRIL protect HMCLs from IL-6 deprivation-induced apoptosis

A. XG-13, XG-14 and XG-20 were IL-6 starved for 3 hours and cultured without cytokine, or in the presence of BAFF (200 ng/ml), APRIL (200 ng/ml) or IL-6 (3 ng/ml). Results are the mean values ± SD of the tritiated thymidine incorporation determined on sextuplet culture wells and are expressed as the percentage of the proliferation obtained with IL-6. Results are those of one experiment representative of five. * Indicates that the mean value is significantly different from that obtained without adding cytokine using a Student t test (P ≤ .05). B. XG-13 and XG-14 HMCLs were cultured at 10⁵ cells/ml without cytokine or in the presence of IL-6 (3 ng/ml), BAFF (200 ng/ml), or APRIL (200 ng/ml). Cells were recovered after 3 days of culture and apoptotic cells were detected by annexin V staining. Results are those of one experiment representative of five. C. XG-13 cells were IL-6 starved for 3 hours and cultured without cytokine, or in the presence of IL-6 (3 ng/ml), BAFF (200 ng/ml) or APRIL (200 ng/ml). When indicated, TACI-Fc (10 μg/ml) or anti-IL-6 MoAb (10 μg/ml) was added. Results are the mean values ± SD of the tritiated thymidine incorporation determined on sextuplet culture wells and are expressed as the percentage of the proliferation obtained with IL-6. Results are for one experiment representative of three. * Indicates that the mean value is statistically significantly different from that obtained with either BAFF, APRIL or IL-6 using a Student t test (P ≤; .05). D. XG-13 and XG-14 cells were cultured, respectively, at 2.5 × 10⁵ and at 1.5 × 10⁵ cells/ml without cytokine or in the presence of IL-6 (3 ng/ml), BAFF (200 ng/ml), or APRIL (200 ng/ml). Every 3 or 4 days, cells were counted and diluted with fresh culture medium containing the initial cytokine concentration. Results are the cumulative cell numbers obtained within 20 days of culture and are those of one experiment representative of two.

Figure 3. BAFF and APRIL protect HMCLs from IL-6 deprivation-induced apoptosis

A. XG-13, XG-14 and XG-20 were IL-6 starved for 3 hours and cultured without cytokine, or in the presence of BAFF (200 ng/ml), APRIL (200 ng/ml) or IL-6 (3 ng/ml). Results are the mean values ± SD of the tritiated thymidine incorporation determined on sextuplet culture wells and are expressed as the percentage of the proliferation obtained with IL-6. Results are those of one experiment representative of five. * Indicates that the mean value is significantly different from that obtained without adding cytokine using a Student t test (P ≤ .05). B. XG-13 and XG-14 HMCLs were cultured at 10⁵ cells/ml without cytokine or in the presence of IL-6 (3 ng/ml), BAFF (200 ng/ml), or APRIL (200 ng/ml). Cells were recovered after 3 days of culture and apoptotic cells were detected by annexin V staining. Results are those of one experiment representative of five. C. XG-13 cells were IL-6 starved for 3 hours and cultured without cytokine, or in the presence of IL-6 (3 ng/ml), BAFF (200 ng/ml) or APRIL (200 ng/ml). When indicated, TACI-Fc (10 μg/ml) or anti-IL-6 MoAb (10 μg/ml) was added. Results are the mean values ± SD of the tritiated thymidine incorporation determined on sextuplet culture wells and are expressed as the percentage of the proliferation obtained with IL-6. Results are for one experiment representative of three. * Indicates that the mean value is statistically significantly different from that obtained with either BAFF, APRIL or IL-6 using a Student t test (P ≤; .05). D. XG-13 and XG-14 cells were cultured, respectively, at 2.5 × 10⁵ and at 1.5 × 10⁵ cells/ml without cytokine or in the presence of IL-6 (3 ng/ml), BAFF (200 ng/ml), or APRIL (200 ng/ml). Every 3 or 4 days, cells were counted and diluted with fresh culture medium containing the initial cytokine concentration. Results are the cumulative cell numbers obtained within 20 days of culture and are those of one experiment representative of two.

Figure 4. Autocrine BAFF or APRIL are involved in the growth of autonomously growing HMCLs

L363 and RPM18226 cells were starved for 3 hours and cultured without cytokine, or in the presence of TACI-Fc (10 μg/ml) or TACI-Fc (10 μg/ml) and BAFF/APRIL (200 ng/ml BAFF and 200 ng/ml APRIL). Results are the mean values ± SD of the tritiated thymidine incorporation determined on sextuplet culture wells and are expressed as the percentage of the proliferation obtained without cytokine. * Indicates that the mean value is statistically significantly different from that obtained without adding TACI-Fc using a Student t test (P ≤.05).

Figure 5. BAFF and APRIL rescue myeloma cells from dexamethasone-induced apoptosis

A. RPMI 8226 and L363 myeloma cells were cultured in the presence of DEX (10⁻⁶ M) with or without IL-6 (3 ng/ml), BAFF (200 ng/ml) or APRIL (200 ng/ml). Cells were recovered after 3 days of culture and apoptotic cells were detected by annexin V staining. Results are those of one experiment out of five. B. Mononuclear cells from eight patients with MM were cultured for 4 days in the presence of IL-6 (1 ng/ml) with or without DEX (10⁻⁶ M), BAFF (200 ng/ml) or APRIL (200 ng/ml). At day 4 of culture, the viability and total cell counts were assessed and the percentage of CD138⁺ viable plasma cells was determined by flow cytometry. Results are median values of the numbers of myeloma cells in the culture wells. The values were compared with a Wilcoxon test for pairs.

Figure 6. TACI-Fc induces apoptosis of primary myeloma cells

Mononuclear cells from tumor samples of six patients with MM were cultured for 4 days in the presence of IL-6 (1 ng/ml) with or without DEX (10⁻⁶ M), TACI-Fc (10 ng/ml) or B-E8 (10 ng/ml). At day 4 of culture, the cell count and viability were determined and the percentage of CD138⁺ viable plasma cells was determined by flow cytometry. The power of an inhibitor is given as the percentage of reduction of the median value of viable myeloma cell count with the inhibitor compared to the median value of viable myeloma cell count without inhibitor.

Figure 7. Signal transduction induced by BAFF or APRIL in myeloma cells

A. XG-13, XG-14 and RPMI8226 cells were starved overnight and cultured without cytokine, or with either IL-6 (30 ng/ml), BAFF (800 ng/ml) or APRIL (800 ng/ml) for 10 and 30 min at 37°C. Cell lysates were analyzed by Western blotting with antisera against phospho-STAT3 (pSTAT3), phospho-ERK1/2 (pMAPK) and phospho-AKT (pAKT). Immunoblotting for STAT3, MAPK and AKT confirmed equal protein loading. Western blots are of one representative experiment out of three. B. XG-13, XG-14 and RPMI8226 cells were starved overnight and cultured without cytokine, or with either IL-6 (3 ng/ml), TNF-α (20 ng/ml), BAFF (200 ng/ml), or APRIL (200 ng/ml) for 30 min at 37°C. NF-κB activity was detected by ELISA according to the manufacturer’s instructions. C. XG-13 cells were IL-6 starved for 3 hours and cultured without cytokine or in the presence of BAFF (200 ng/ml) or APRIL (200 ng/ml). When indicated, Ly 294002 (25 μM), SN50 (100 μg/ml), or the SN50 inactive peptide (100 μg/ml) was added. Results are the mean values ± SD of the tritiated thymidine incorporation determined on sextuplet culture wells and are expressed as the percentage of the proliferation obtained with APRIL and SN50 inactive peptide. * Indicates that the mean value is statistically significantly different from that obtained with BAFF or APRIL using a Student t test (P ≤ .05).

Figure 7. Signal transduction induced by BAFF or APRIL in myeloma cells

A. XG-13, XG-14 and RPMI8226 cells were starved overnight and cultured without cytokine, or with either IL-6 (30 ng/ml), BAFF (800 ng/ml) or APRIL (800 ng/ml) for 10 and 30 min at 37°C. Cell lysates were analyzed by Western blotting with antisera against phospho-STAT3 (pSTAT3), phospho-ERK1/2 (pMAPK) and phospho-AKT (pAKT). Immunoblotting for STAT3, MAPK and AKT confirmed equal protein loading. Western blots are of one representative experiment out of three. B. XG-13, XG-14 and RPMI8226 cells were starved overnight and cultured without cytokine, or with either IL-6 (3 ng/ml), TNF-α (20 ng/ml), BAFF (200 ng/ml), or APRIL (200 ng/ml) for 30 min at 37°C. NF-κB activity was detected by ELISA according to the manufacturer’s instructions. C. XG-13 cells were IL-6 starved for 3 hours and cultured without cytokine or in the presence of BAFF (200 ng/ml) or APRIL (200 ng/ml). When indicated, Ly 294002 (25 μM), SN50 (100 μg/ml), or the SN50 inactive peptide (100 μg/ml) was added. Results are the mean values ± SD of the tritiated thymidine incorporation determined on sextuplet culture wells and are expressed as the percentage of the proliferation obtained with APRIL and SN50 inactive peptide. * Indicates that the mean value is statistically significantly different from that obtained with BAFF or APRIL using a Student t test (P ≤ .05).

Figure 8. Regulation of Bcl-2 family antiapoptotic proteins by BAFF and APRIL

A. XG-13, XG-14 and RPMI8226 cells were starved overnight before culture with no cytokine, or with IL-6 (30 ng/ml), BAFF (800 ng/ml) or APRIL (800 ng/ml) for 6 hours in RPMI-1% BSA. At the end of the culture, cells were immediately lysed and assayed for Bcl-2 family antiapoptotic protein expression using Western blot analysis. In this experiment, STAT3 expression was used as loading protein control. B. Blots of three independent experiments were scanned and the values were normalized using STAT3-band intensities as internal standards. Results are the median values for the three main Bcl-2-family members expressed in the XG-13 HMCLs starved of IL-6 and cultured for 6 hours with no cytokine, or with IL-6 (30 ng/ml), BAFF (800 ng/ml) or APRIL (800 ng/ml). * Indicates a significant increase in expression with a Student t test for pairs (P < .05).

Figure 9. Serum level of circulating BAFF and APRIL in myeloma patients

Serum levels of BAFF and APRIL were determined by ELISA in the sera from 36 patients with myeloma and nine age-related healthy individuals.