Immunomodulatory drugs Revlimid (lenalidomide) and CC-4047 induce apoptosis of both hematological and solid tumor cells through NK cell activation

Abstract

Revlimid (Lenalidomide, CC-5013) and CC-4047 are IMiDs immunomodulatory drugs that have been described as having immunomodulatory properties and anti-tumor activity. Here we report proapoptotic effects of CC-5013 and CC-4047 on tumor cells in a co-culture model of PBMC and tumor cells. CC-5013 and CC-4047 enhanced PBMC activity leading to tumor cell apoptosis in K562/PBMC co-culture model. We also demonstrate that the natural killer (NK) cell population of PBMC was essential in inducing K562 apoptosis. Increases of NK and natural killer T (NKT) cell populations by CC-5013 and CC-4047 was observed along with modulation of NK cell CD56 adhesion marker. In addition, our data indicate that NK activation by CC-4047 was dependent on other cell types of PBMC. We expanded the application of K562/PBMC co-culture model to other hematological and solid tumors. In Raji/PBMC co-culture model, CC-5013 and CC-4047 dose-dependently augmented tumor cell apoptosis. Pre-treatment of Raji cells with Rituximab further enhanced apoptosis induced by CC-5013 or CC-4047-treated PBMC. Moreover, CC-5013 and CC-4047 significantly increased PC-3 prostate cancer cell apoptosis in PC-3/PBMC co-culture, either as single agent or in combination with Docetaxel. Together, the results reveal that co-culture models are suitable cellular systems to assess anti-tumor activities of these compounds. Our findings support clinical evaluation of CC-5013 and CC-4047 in relapsed NHL with Rituximab and in prostate cancer with Docetaxel.

Fig. 1

CC-4047 enhances PBMC activity in inducing K562 apoptosis with or without IL-2 pretreatment. a PBMC were treated with or without 20 units/ml of IL-2 for 72 h followed by 10 μM CC-4047 for 72 h. K562 cells were labeled with PKH26 dye prior to co-culture with PBMC at 15:1 effector-to-target ratio. After co-culture, the mixture of K562 cells and PBMC was labeled with Annexin V-FITC. This experiment was performed with PBMC from three different donors. Representative density plots of K562/PBMC co-culture with or without CC-4047 treatment are shown here. K562 cells can be distinguished from PBMC by gating PKH26 positive population (y-axis). Double positive cell population represents apoptotic K562 cells as shown in the upper right-hand box of each plot. CC-4047 increased apoptosis of K562 cells to 61.9% (no IL-2) or 80.1% (with IL-2) compared to DMSO-treated groups (22.1 or 59.5%, respectively). b CC-4047 enhances PBMC-mediated apoptosis of K562 cells at various effector-to-target ratios with or without IL-2 treatment. PBMC pretreated with CC-4047 were mixed with K562 cells at the indicated effector-to-target ratios for 3 h. Cells were then analyzed as described in Materials and methods. Results shown are representative of three independent experiments with different PBMC donors

Fig. 2

CC-4047 and CC-5013 increase PBMC-mediated apoptosis of K562 cells in the absence of IL-2. Various doses of CC-4047 and CC-5013 were evaluated in the co-culture model without IL-2 pretreatment of PBMC. Fold increase in K562 apoptosis in CC-4047 and CC-5013 treated group versus DMSO treated group was calculated. Results shown are mean fold increase of apoptosis (±SD) from three independent experiments using different PBMC donors

Fig. 3

NK cells play important roles in inducing K562 apoptosis enhanced by CC-4047. a After three days in culture with CC-4047 or CC-5013, increased CD56bright NK cells and NKT cells (CD56+CD3+ cells) numbers were detected in PBMC from six tested donors. Plots show representative results. b Histograms of a representative experiment comparing regular PBMC and NK-depleted PBMC in the co-culture model. NK cells were depleted from PBMC and tested in the co-culture model with K562 cells as described in Materials and methods. Non-depleted regular PBMC from the same donor are shown on the left panel and NK-depleted PBMC on the right. For each double-peaked sample, the right peak indicates the apoptotic population of K562. The level of apoptosis is listed above each histogram. This experiment was repeated three times using PBMC from different donors. c Comparison of CC-4047 effects on regular non-depleted PBMC (PBMC), NK-depleted PBMC (PBMC-NK), and purified NK cells (NK). NK cells were isolated from PBMC and treated with IL-2 followed by DMSO or 1 μM CC-4047 as described in Materials and methods. As a control, regular PBMC and NK-depleted PBMC from the same donor were treated under the same conditions. Data show representative results of four independent experiments with PBMC from different donors. d Effect of 1 μM CC-4047 on purified NK-mediated K562 apoptosis at different NK-to-target ratios. Different effector-to-target ratios were tested for isolated NK-mediated K562 apoptosis. Ratio of 1:1 matches the NK cell-to-K562 ratio in non-depleted K562/PBMC co-culture. Data in c and d show representative results of four independent experiments with PBMC from different donors

Fig. 4

CC-4047 and CC-5013 show no additive effect in combination with Rituximab and complement in Raji mono-cell culture but enhance Raji apoptosis in co-culture model as single agent or in combination with Rituximab. a Raji cells were cultured with Rituximab (10 μg/ml) and increasing concentrations of complement in the absence or presence of CC-4047 and CC-5013. Rituximab plus complement inhibited cell proliferation in a dose dependent manner. CC-4047 and CC-5013 had a very modest effect on Raji cell proliferation and showed no additive effect with Rituximab plus complement in this mono-cell culture assay. b Enhancement of PBMC-mediated Raji apoptosis by CC-4047 and CC-5013 is dose-dependent. PBMC were treated with CC-4047 or CC-5013 for 72 h prior to co-culture. These PBMC were then co-cultured with pre-labeled Raji cells at 30:1 or 15:1 ratio for 3 h, followed by labeling with AnnexinV-APC and analysis with FACSArray Bioanalyzer. Each data point represents the average of triplicate results. Shown is the result from 30:1 ratio. Similar trend of apoptosis induction was obtained with 15:1 ratio (data not shown). c Combination studies of Rituximab and CC-4047 and CC-5013 in Raji/PBMC co-culture model. Raji cells were treated with or without various doses of Rituximab for 20 min. PBMC were treated with CC-4047 (10 μM) and CC-5013 (10 μM) for 72 h prior to the co-culture with Raji cells. Each data point represents the average of triplicate results (* Synergistic and ♣ additive as determined by fractional product method.)

Fig. 5

CC-4047 and CC-5013 enhance PC-3 cell apoptosis in co-culture model, in the presence or absence of Docetaxel pretreatment. a Enhancement of PBMC-mediated apoptosis by CC-4047 and CC-5013 is dose-dependent. PBMC were treated with CC-4047 or CC-5013 for 72 h. These PBMC were then co-cultured with pre-labeled PC-3 cells at 10:1 or 5:1 ratio for 18 h, followed by labeling with AnnexinV-APC and analysis with FACSArray Bioanalyzer. Each data point represents the average of triplicate results. Shown above is the result from 10:1 ratio. A similar dose response was obtained with the 5:1 ratio. Combination studies of Docetaxel and CC-4047 (b) or CC-5013 (c) in PC-3/PBMC co-culture model. PKH26 pre-labeled PC-3 cells were treated with or without various doses of Docetaxel for 6 h. PBMC were treated with CC-4047 or CC-5013 at various doses for 72 h prior to co-culture with PC-3 cells. Each data point represents the average of triplicate results (* Synergistic and ♣ additive as determined by fractional product method.)