The proteasome inhibitor, bortezomib, and the histone deacetylase inhibitor, depsipeptide (FK228), up-regulate tumor death receptors. Therefore, we investigated whether pretreatment of malignant cells with these agents would potentiate natural killer (NK)–mediated tumor killing. NK cells isolated from healthy donors and patients with cancer were expanded in vitro and then tested for cytotoxicity against tumor cell lines before and after exposure to bortezomib or depsipeptide. In 11 of 13 (85%) renal cell carcinoma cell lines and in 16 of 37 (43%) other cancer cell lines, exposure to these drugs significantly increased NK cell–mediated tumor lysis compared with untreated tumor controls (P < 0.001). Furthermore, NK cells expanded from patients with metastatic renal cell carcinoma were significantly more cytotoxic against autologous tumor cells when pretreated with either bortezomib or depsipeptide compared with untreated tumors. Tumors sensitized to NK cell cytotoxicity showed a significant increase in surface expression of DR5 [tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)-R2; P < 0.05]; in contrast, surface expression of MHC class I, MIC-A/B, DR4 (TRAIL-R1), and Fas (CD95) did not change. The enhanced susceptibility to NK cell killing was completely abolished by blocking TRAIL on NK cells, and partially abolished by blocking DR5 on tumor cells. These findings show that drug-induced sensitization to TRAIL could be used as a novel strategy to potentiate the anticancer effects of adoptively infused NK cells in patients with cancer. (Cancer Res 2006; 66(14): 7317-25)

Natural killer (NK) lymphocytes have antigen-independent cytotoxicity. NK cells can shrink tumors and prevent metastases, although the mechanisms that account for these responses are uniquely different from T and B lymphocyte antigen recognition. NK cells do not rearrange genes encoding for specific antigen receptors; rather, their recognition of targets is regulated through a balance of activating and inhibitory signals (1). Even in the presence of an activating ligand, inhibitory ligands can initiate overriding signals that culminate in a net suppression of NK cell function. NK cells recognize major histocompatibility class (MHC) I and class I–like molecules through surface expression of killer immunoglobulin–like receptors (KIR; ref. 2). MHC class I molecules on tumor cells ligate NK cell–inhibitory KIR, suppressing NK cell function. The inactivation of NK cells by self-HLA molecules is a potential mechanism by which malignant cells evade host NK cell–mediated immunity. This may contribute to the failure of adoptively infused autologous NK cells to induce antitumor effects against solid tumors (3). One potential approach to bypass this escape mechanism is through the use of KIR ligand–mismatched NK cells. Allogeneic NK cells expressing KIR in which patient cells lack complimentary MHC class I ligands (so-called KIR ligand–incompatible) have enhanced cytotoxicity against acute myelogenous leukemia (AML). Ruggeri et al. and others have shown that in the setting of mismatched allogeneic hematopoietic cell transplantation with KIR incompatibility (in the graft versus host direction), KIR ligand–incompatible NK cells seem to reduce the risk of leukemia relapse compared with those who receive KIR ligand–compatible transplants (4, 5). We have previously shown that allogeneic NK cells mismatched for KIR ligands are more cytotoxic to renal cell carcinoma (RCC) and other solid tumor cells in vitro than their KIR ligand–matched autologous and allogeneic counterparts (6). However, when MHC-mismatched allogeneic NK cells are adoptively infused in patients with cancer, intense host immunosuppression must first be given before any measurable NK cell engraftment can be achieved (7). Therefore, outside the setting of an allogeneic transplant, KIR ligand–mismatched NK cells might be of limited therapeutic use as differences in MHC molecules would eventually lead to their rejection by the patient's immune system.

To offset KIR ligand inhibition, we sought to develop a method to sensitize the patient's tumor to autologous (i.e., self) NK cells. NK cells lyse tumor targets directly via the perforin/granzyme pathway. They also express Fas ligand and tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), which directly trigger death receptor pathways inducing tumor apoptosis (810). Death receptors including Fas, TNFR1, TRAIL-R1/DR4, TRAIL-R2/DR5, DR3, and DR6 share a conserved death domain that is triggered by adaptor molecules that activate executioner caspases and initiate apoptosis (11). Recently, several agents that influence the surface expression of these death receptors, including the proteasome inhibitor bortezomib (PS-341; Velcade) and the histone deacetylase inhibitor depsipeptide (FK228) have been described (12, 13). Here, we show that pretreatment of RCC and other malignant cells in vitro with depsipeptide or bortezomib enhance NK-mediated tumor cytotoxicity by sensitizing to TRAIL-mediated apoptosis. In addition, NK cells expanded from patients with metastatic RCC were significantly more cytotoxic against the patient's own tumor cells pretreated with either bortezomib or depsipeptide compared with untreated tumors. These findings suggest that drug-induced sensitization to TRAIL could be used as a novel strategy to potentiate anticancer effects of both allogeneic and autologous adoptively infused NK cells in patients with cancer.

Generation of NK cells. NK cells were expanded from peripheral blood mononuclear cells (PBMC) of patients with metastatic cancers from whom a tumor line had been established or from healthy donors. Using the manufacturer's instructions, NK cells were isolated by negative depletion of PBMC using one of two different types of immunomagnetic beads: (a) for cancer patients, the Dynal NK cell negative isolation kit (Dynal, Carlsbad, CA) was used; (b) for healthy donors, the CliniMacs NK cell isolation kit (Miltenyi Biotec, Auburn, CA) was used. NK cell purity prior to expansion assessed by fluorescence-activated cell sorting (FACS; CD3−, CD16+, or CD56+) was between 85% and 99% and was nearly 100% devoid of CD3+ T cell contamination (CD14+ monocytes were the primary contaminating cells; data not shown). NK cells were then expanded by coculturing with irradiated (25-100 Gy) feeder cells, either EBV-transformed B cells (EBV-LCL) or PBMC at a NK/feeder cell ratio of 1:10 to 1:100. Cells were grown in either SCGM (Cambrex, East Rutherford, NJ) or X-VIVO20 (Bio Whittaker, Walkersville, MD) medium supplemented with 500 units/mL of interleukin-2 (IL-2), and either 10% AB serum (Gemini Bioproducts, Woodland, CA) or autologous plasma. Irradiated feeder cells were added every 2 weeks and the medium was supplemented weekly with 500 units/mL of IL-2. Using this protocol, NK cell expansions of >102-fold using allogeneic PBMC feeders and >104-fold using EBV-LCL feeders occurred after 2 to 4 weeks. Expanded cells assessed by FACS were typically 99% (range 97-100) pure for CD3−, CD16+, or CD56+ NK cells.

Cell lines. EBV-LCLs were generated by infecting 5 to 10 × 106 PBMC with EBV-containing supernatant and then cultured in RPMI 1640 supplemented with 10% FCS, phytohemagglutinin (1 mg/mL), and cyclosporine A (1.5 μg/mL). Tumor cell lines from RCC (JOHW, SAUJ, PORJC, and STAR), melanoma (MIRF and STEW), and pancreatic carcinoma (ZYRD and SHAW; Angelo Russo, NCI, Bethesda, MD) were established in National Heart, Lung, and Blood Institute/National Cancer Institute laboratories from surgically resected tumor specimens. Additional RCC cell lines (ORT, SNY, COL, URB, STR, MAR, BEN, WIT, and SEA) were kindly provided by members of the Surgery Branch, National Cancer Institute. Other tumor cell lines evaluated included melanoma (SK23 and 888), prostate cancer (LNCAP, PC3, and DU145), multiple myeloma (ARH77, U266B1, and RPMI 8226), lung cancer (2228, 1355, H1299, 2087, CALU6, and H466), colon cancer (HT29, DLD1, SW948, SW480, SW620, SW116, and T84), ovarian cancer (SKOV3), neuroblastoma (BE2C and SKND2), osteosarcoma (HELA), Burkitt's lymphoma (RAJI and RAMOS), AML (KG1A), acute lymphoblastic leukemia (CCL119), chronic myelogenous leukemia (K562), breast cancer (T47D, MCF7), and pancreatic cancer (PANC1), which were purchased from the American Type Culture Collection (Manassas, VA). All cell lines were maintained in RPMI 1640 or DMEM (Cellgro, Herndon, VA) supplemented with 10% FCS (HyClone, Logan, UT). Fibroblast cell lines (DEMJC and SAUJ) were established from skin punch biopsies of patients with RCC treated at the National Heart, Lung, and Blood Institute; SAUJ fibroblasts were immortalized by infecting 1-week-old cultured fibroblast cells with a retrovirus encoding the papilloma virus E6/E7 proteins and then performing limiting dilution.

Reagents and antibodies. The purity of NK cell cultures was evaluated by flow cytometry (FACSCalibur; BD PharMingen, Franklin Lakes, NJ) after staining with CD3, CD14, CD16, and CD56 monoclonal antibodies (BD PharMingen). Depsipeptide (FK228; Fujisawa Pharmaceuticals, Osaka, Japan) and bortezomib (Velcade, PS-341; Millennium Pharmaceuticals, Cambridge, MA) were purchased from the NIH, Division of Veterinary Resources pharmacy. The pan-caspase inhibitor Z-VAD (R&D Systems, Minneapolis, MN) was used at a concentration of 20 μmol/L. Mouse anti-human Fas (ZB4; Abcam, Cambridge, MA) was added to assays at a concentration of 10 μg/mL. MHC class I antibody (W6/32; Dako, Carpinteria, CA) was added to cytotoxicity assays at a concentration of 10 μg/mL. To inhibit NK cell cytotoxicity via TRAIL, mouse anti-human TRAIL antibody (Biolegend, San Diego, CA) was added at a concentration of 10 μg/mL. DR4, DR5 (Biolegend), MIC-A/B, HLA-ABC (PharMingen) antibodies were added at concentrations recommended by the manufacturers. Secondary goat anti-mouse PE-labeled antibody was added at a concentration of 5 μg/mL. To inhibit NK cell cytotoxicity via perforin, concanamycin A (Sigma, St. Louis, MO) was added to assays at a concentration of 10 μmol/L. To induce tumor killing via the TRAIL pathway, recombinant TRAIL (Peprotech, Rocky Hill, NJ) was added to target cells at a concentration between 37.5 and 300 ng/mL.

Apoptosis, cytotoxicity, and proliferation assays. Apoptosis was measured by flow cytometry staining for Annexin-V (BD) and 7-AAD (Beckman Coulter, Fullerton, CA) following the manufacturer's instructions. All flow cytometry assays were acquired on a FACSCalibur and analyzed by FCS express (De Novo Software, Thornhill, Canada). Chromium-51 (Amersham Biosciences, Inc., Piscataway, NJ) cytotoxicity assays were done by plating 4,000 51Cr-labeled target cells/well (96-well plate) with varying amounts of unlabeled NK cell effectors. After 4 or 16 hours of incubation, the supernatants were harvested onto Luma plates (Perkin-Elmer, Wellesley, MA) and analyzed using a MicroBeta scintillation counter (Perkin-Elmer). The effects of bortezomib and depsipeptide on cell proliferation were analyzed by incubating tumor or NK cells (10,000 or 50,000 cells, respectively, per well in 96-well plate) in culture media for 2 to 5 days. 3H-thymidine (1 μCi) was then added, and after 16 hours, the cells were harvested onto solid filters (MicroBeta FilterMate 96 Harvester; Perkin-Elmer) and analyzed with scintillation counting (MicroBeta, Perkin-Elmer).

Effects of depsipeptide and bortezomib on cell viability and proliferation. We first assessed the effect of exposing tumor cells, NK cells, and fibroblasts to depsipeptide and bortezomib. Human tumor cells, NK cells, and fibroblasts were incubated with depsipeptide (25 ng/mL), bortezomib (10 nmol/L), or the combination of both drugs for 24 hours. These concentrations were chosen as they induce apoptosis in myeloma (bortezomib) and AML (depsipeptide) cell lines in vitro but are >10-fold lower than the mean plasma concentration achieved with either drug given at the maximum tolerated dose in humans (14, 15). At these concentrations, the level of apoptosis induced in myeloma cells (ARH-77) was higher with depsipeptide (62 ± 3%) compared with bortezomib (19 ± 0%; P = 0.002; Fig. 1A). Similar levels of apoptosis with each drug were observed in other myeloma cell lines (RPMI 8226 and U266B1; data not shown). A small percentage (5.6%) of NK cells was induced into apoptosis following exposure to depsipeptide; in contrast, bortezomib did not cause NK cell apoptosis. When given alone, depsipeptide and bortezomib did not induce significant apoptosis of either RCC cells or primary fibroblast cultures. A similar level of apoptosis, comparable to that observed with RCC cells, was observed in a panel of tumor cell lines including prostate cancer, lung cancer, and melanoma following exposure to depsipeptide or bortezomib (data not shown). In contrast to either agent alone, the combination of both drugs caused substantial levels of apoptosis in RCC cells (Fig. 1A). Increasing the length of exposure of RCC cells to individual agents did not significantly increase apoptosis (Fig. 1B). Both agents, even at low concentrations, reduced the proliferation of RCC cells (Fig. 1C). Similar to RCC, inhibition of NK cell proliferation was observed at the same dose range (data not shown).

Figure 1.

Viability and proliferation of human multiple myeloma, RCC, NK cells, and fibroblasts after exposure to bortezomib and/or depsipeptide. Human multiple myeloma cell line (ARH-77), human RCC cell line (SAUJ), NK cells expanded from a healthy donor, and human skin-derived fibroblasts were treated with 10 nmol/L of bortezomib and/or 25 ng/mL of depsipeptide for 24 hours in complete medium. Cells were harvested and stained with Annexin-V and 7-AAD and analyzed by flow cytometry (A). RCC cell lines (JOHW and SAUJ) were treated with 25 ng/mL of depsipeptide or 10 nmol/L of bortezomib for varying time periods (up to 40 hours) then analyzed by flow cytometry for viability by Annexin-V and 7-AAD staining (B). RCC cells were exposed to varying concentrations of depsipeptide (▪, JOHW; □, SAUJ) or bortezomib (▴, JOHW; △, SAUJ) for 72 hours and analyzed for proliferation by 3H uptake (C). Values for proliferation of untreated cells ranged between 40,000 and 120,000 cpm. Values were normalized to 100% viability and proliferation for untreated cells. One of three representative experiments is shown. n.d., not done.

Figure 1.

Viability and proliferation of human multiple myeloma, RCC, NK cells, and fibroblasts after exposure to bortezomib and/or depsipeptide. Human multiple myeloma cell line (ARH-77), human RCC cell line (SAUJ), NK cells expanded from a healthy donor, and human skin-derived fibroblasts were treated with 10 nmol/L of bortezomib and/or 25 ng/mL of depsipeptide for 24 hours in complete medium. Cells were harvested and stained with Annexin-V and 7-AAD and analyzed by flow cytometry (A). RCC cell lines (JOHW and SAUJ) were treated with 25 ng/mL of depsipeptide or 10 nmol/L of bortezomib for varying time periods (up to 40 hours) then analyzed by flow cytometry for viability by Annexin-V and 7-AAD staining (B). RCC cells were exposed to varying concentrations of depsipeptide (▪, JOHW; □, SAUJ) or bortezomib (▴, JOHW; △, SAUJ) for 72 hours and analyzed for proliferation by 3H uptake (C). Values for proliferation of untreated cells ranged between 40,000 and 120,000 cpm. Values were normalized to 100% viability and proliferation for untreated cells. One of three representative experiments is shown. n.d., not done.

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Potentiation of allogeneic NK cell–mediated cytotoxicity against tumor cells treated with depsipeptide or bortezomib. We next evaluated whether treatment of malignant cells with depsipeptide or bortezomib would potentiate NK-mediated tumor killing. RCC cells were first treated with depsipeptide or bortezomib and then tested in vitro for their susceptibility to lysis by allogeneic NK cells. In the majority (11 of 13) of RCC cell lines tested, a significantly higher percentage of tumor cells exposed to depsipeptide or bortezomib for 18 hours were lysed by NK cells compared with NK cell lysis of untreated RCC controls (P < 0.001). This potentiation in NK cell killing was evident as early as 8 hours following exposure of tumor cells to either agent (Fig. 2A). Moreover, tumor cells of multiple cellular origins, including AML, acute lymphoblastic leukemia, melanoma, and carcinomas of the prostate, pancreas, lung, and colon showed enhanced susceptibility to NK cell cytotoxicity following pretreatment with depsipeptide or bortezomib (Table 1). Overall, 54% (27 of 50) of tumors tested were killed at significantly higher levels by allogeneic NK cells after exposure to either drug; the fold median increase in cytotoxicity after depsipeptide and bortezomib treatment compared with untreated controls was 2.3 (P < 0.001) and 1.9 (P < 0.001), respectively. In 11 of 50 tumor cell lines tested, exposure to depsipeptide or bortezomib augmented NK cell cytotoxicity >3-fold compared with untreated controls (Table 2). Some tumor cells were sensitized to NK cell cytotoxicity with both drugs, whereas others were selectively sensitized to only one of the two drugs. No increase in NK cell–mediated killing of fibroblasts occurred following exposure to either agent.

Figure 2.

Potentiation of autologous and allogeneic MHC class I-identical NK cells against depsipeptide- and bortezomib-treated RCC. RCC cells (JOHW) were treated for 2 to 40 hours with depsipeptide (gray columns, 25 ng/mL) or bortezomib (black columns, 10 nmol/L) and evaluated for NK cell lysis in a 4-hour 51Cr release assay at an effector to target (E/T) ratio of 4:1 (A). Human RCC cell lines (JOHW, STAR, and SAUJ) were cultured in the presence of 10 nmol/L of bortezomib or 25 ng/mL of depsipeptide for 14 to 20 hours. Autologous human NK cells isolated and expanded from RCC patients (JOHW and STAR; B) or from RCC patient (SAUJ) prior to allogeneic transplantation (autologous in origin) and post–HLA-matched allogeneic transplant (C; 100% allogeneic donor in origin) were tested against untreated (•), bortezomib-treated (▴), or depsipeptide-treated (▪) 51Cr-labeled tumor cells in a 4- (B) and 16-hour (C) assay. One of two (for A and B) and one of four (for C) representative experiments is shown. *, P < 0.05, statistically significant difference (by paired Student's t test) between untreated and treated cells.

Figure 2.

Potentiation of autologous and allogeneic MHC class I-identical NK cells against depsipeptide- and bortezomib-treated RCC. RCC cells (JOHW) were treated for 2 to 40 hours with depsipeptide (gray columns, 25 ng/mL) or bortezomib (black columns, 10 nmol/L) and evaluated for NK cell lysis in a 4-hour 51Cr release assay at an effector to target (E/T) ratio of 4:1 (A). Human RCC cell lines (JOHW, STAR, and SAUJ) were cultured in the presence of 10 nmol/L of bortezomib or 25 ng/mL of depsipeptide for 14 to 20 hours. Autologous human NK cells isolated and expanded from RCC patients (JOHW and STAR; B) or from RCC patient (SAUJ) prior to allogeneic transplantation (autologous in origin) and post–HLA-matched allogeneic transplant (C; 100% allogeneic donor in origin) were tested against untreated (•), bortezomib-treated (▴), or depsipeptide-treated (▪) 51Cr-labeled tumor cells in a 4- (B) and 16-hour (C) assay. One of two (for A and B) and one of four (for C) representative experiments is shown. *, P < 0.05, statistically significant difference (by paired Student's t test) between untreated and treated cells.

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Table 1.

Enhanced cytotoxicity of NK cells against depsipeptide or bortezomib-treated tumor cells of different origin

Number of cell lines testedNumber of cell lines sensitized to NK cells after treatment with
Depsipeptide*BortezomibDepsipeptide or Bortezomib
Renal cell cancer 13 11 11 
Melanoma 
Prostate cancer 
Neuroblastoma 
Multiple myeloma 
Breast cancer 
Pancreatic cancer 
Lung cancer 
Colon cancer 
Ovarian cancer 
Osteosarcoma 
Burkitt's lymphoma 
Leukemias 
Fibroblasts 
Total 52 20 25 27 
Number of cell lines testedNumber of cell lines sensitized to NK cells after treatment with
Depsipeptide*BortezomibDepsipeptide or Bortezomib
Renal cell cancer 13 11 11 
Melanoma 
Prostate cancer 
Neuroblastoma 
Multiple myeloma 
Breast cancer 
Pancreatic cancer 
Lung cancer 
Colon cancer 
Ovarian cancer 
Osteosarcoma 
Burkitt's lymphoma 
Leukemias 
Fibroblasts 
Total 52 20 25 27 

NOTE: Allogeneic NK cells were evaluated for their lytic activity against different tumor cell lines after 18 hours of treatment with depsipeptide (25 ng/mL) or bortezomib (10 nmol/L) by 51Cr release assay at an E/T ratio between 1:1 and 10:1. This assay was done at least twice for all cell lines tested. Data are presented as the number of cell lines of each tumor type where the level of lysis by NK cells was significantly higher (P < 0.05) compared with untreated tumor controls.

*

Number of cell lines that were sensitized to depsipeptide only.

Number of cell lines that were sensitized to bortezomib only.

Number of cell lines that were sensitized to depsipeptide or bortezomib.

Table 2.

NK cell cytotoxicity against tumor cells of different origin

Untreated
Depsipeptide
Bortezomib
UntreatedDepsipeptideBortezomib
Lysis ± SD (%)Lysis ± SD (%)Fold change*Lysis ± SD (%)Fold change*Lysis ± SD (%)Lysis ± SD (%)Fold change*Lysis ± SD (%)Fold change*
Renal cell carcinoma JOHW 7 ± 0 22 ± 3 3.1 41 ± 3 5.7 Melanoma STEW 8 ± 4 23 ± 6 2.9 33 ± 9 4.1 
 SAUJ 13 ± 1 27 ± 4 2.1 24 ± 1 1.8  SK23 2 ± 2 36 ± 0 7.2 34 ± 1 6.8 
 PORJC 41 ± 2 46 ± 2 1.1 48 ± 2 1.1  888 38 ± 1 39 ± 3 1.0 44 ± 2 1.2 
 STAR 9 ± 1 27 ± 2 3.0 21 ± 1 2.3  MIRF 8 ± 4 31 ± 2 3.9 15 ± 2 1.9 
 ORT 15 ± 3 34 ± 4 2.3 39 ± 2 2.6 Lung carcinoma 2228 12 ± 2 44 ± 5 3.7 36 ± 3 3.0 
 SNY 27 ± 1 47 ± 1 1.7 50 ± 1 1.9  1355 14 ± 2 16 ± 1 1.1 13 ± 2 0.9 
 COL 22 ± 1 36 ± 6 1.6 55 ± 6 2.5  H1299 14 ± 3 23 ± 0 1.9 23 ± 4 1.6 
 URB 42 ± 3 45 ± 3 1.1 59 ± 3 1.4  2087 23 ± 0 42 ± 2 1.8 44 ± 0 1.7 
 STR 23 ± 5 31 ± 5 1.3 46 ± 2 2.0  CALU-6 59 ± 7 64 ± 9 1.1 66 ± 9 1.1 
 WIT 32 ± 3 47 ± 1 1.5 48 ± 0 1.5  H466 37 ± 5 44 ± 4 1.2 30 ± 2 0.8 
 MAR 31 ± 3 23 ± 1 0.7 33 ± 2 1.1 Leukemias KG1a 11 ± 0 13 ± 2 1.2 39 ± 3 3.5 
 BEN 33 ± 4 50 ± 8 1.5 46 ± 5 1.4  CCL119 50 ± 7 54 ± 5 1.1 71 ± 2 1.4 
 SEA 36 ± 0 41 ± 5 1.1 52 ± 3 1.4  K562 35 ± 4 32 ± 3 0.9 38 ± 2 1.1 
Untreated
Depsipeptide
Bortezomib
UntreatedDepsipeptideBortezomib
Lysis ± SD (%)Lysis ± SD (%)Fold change*Lysis ± SD (%)Fold change*Lysis ± SD (%)Lysis ± SD (%)Fold change*Lysis ± SD (%)Fold change*
Renal cell carcinoma JOHW 7 ± 0 22 ± 3 3.1 41 ± 3 5.7 Melanoma STEW 8 ± 4 23 ± 6 2.9 33 ± 9 4.1 
 SAUJ 13 ± 1 27 ± 4 2.1 24 ± 1 1.8  SK23 2 ± 2 36 ± 0 7.2 34 ± 1 6.8 
 PORJC 41 ± 2 46 ± 2 1.1 48 ± 2 1.1  888 38 ± 1 39 ± 3 1.0 44 ± 2 1.2 
 STAR 9 ± 1 27 ± 2 3.0 21 ± 1 2.3  MIRF 8 ± 4 31 ± 2 3.9 15 ± 2 1.9 
 ORT 15 ± 3 34 ± 4 2.3 39 ± 2 2.6 Lung carcinoma 2228 12 ± 2 44 ± 5 3.7 36 ± 3 3.0 
 SNY 27 ± 1 47 ± 1 1.7 50 ± 1 1.9  1355 14 ± 2 16 ± 1 1.1 13 ± 2 0.9 
 COL 22 ± 1 36 ± 6 1.6 55 ± 6 2.5  H1299 14 ± 3 23 ± 0 1.9 23 ± 4 1.6 
 URB 42 ± 3 45 ± 3 1.1 59 ± 3 1.4  2087 23 ± 0 42 ± 2 1.8 44 ± 0 1.7 
 STR 23 ± 5 31 ± 5 1.3 46 ± 2 2.0  CALU-6 59 ± 7 64 ± 9 1.1 66 ± 9 1.1 
 WIT 32 ± 3 47 ± 1 1.5 48 ± 0 1.5  H466 37 ± 5 44 ± 4 1.2 30 ± 2 0.8 
 MAR 31 ± 3 23 ± 1 0.7 33 ± 2 1.1 Leukemias KG1a 11 ± 0 13 ± 2 1.2 39 ± 3 3.5 
 BEN 33 ± 4 50 ± 8 1.5 46 ± 5 1.4  CCL119 50 ± 7 54 ± 5 1.1 71 ± 2 1.4 
 SEA 36 ± 0 41 ± 5 1.1 52 ± 3 1.4  K562 35 ± 4 32 ± 3 0.9 38 ± 2 1.1 

NOTE: Allogeneic NK cells were evaluated for their lytic activity against different tumor cell lines after 18 hours of treatment with depsipeptide (25 ng/mL) or bortezomib (10 nmol/L) in a 51Cr release assay at an E/T ration between 1:1 and 10:1. This assay was done at least twice for all cell lines tested. Data are presented as the percentage of lysis of untreated, depsipeptide-, or bortezomib-treated tumor cells. Numbers in boldface represent significant difference.

*

Fold change in cytotoxicity compared with untreated tumor cells.

Potentiation of autologous NK cell–mediated cytotoxicity against patient tumor cells treated with depsipeptide or bortezomib. We next evaluated if the augmentation in tumor killing observed with allogeneic NK cells also occurred with autologous, “KIR-compatible” NK cells. NK cells were first isolated and expanded from patients with metastatic RCC and then tested in vitro for cytotoxicity against RCC tumor cell lines established from the same patient following exposure to depsipeptide or bortezomib. As observed with allogeneic NK cells, autologous NK cells were significantly more cytotoxic against the patient's own tumor in vitro following pretreatment with either agent (Fig. 2B). In 50% (two of four) of experiments, the fold increase in tumor cell lysis after exposure to bortezomib and depsipeptide was comparable to the fold increase in NK cell killing observed when KIR ligands on the tumor were blocked with a pan-MHC class I monoclonal antibody (data not shown).

In one experiment, NK cells were isolated and expanded from PBMC collected from a patient with metastatic RCC before and after the patient had undergone allogeneic hematopoietic stem cell transplantation from his HLA-matched sibling. Both NK cell populations (i.e., 100% autologous and 100% allogeneic HLA-matched donor) showed significantly higher cytotoxicity against the patient's RCC cells after treatment with depsipeptide or bortezomib (Fig. 2C).

Enhanced NK cell activity against depsipeptide- or bortezomib-treated tumor cells is mediated by TRAIL. We next investigated the mechanism through which NK cell cytotoxicity was potentiated following tumor exposure to these drugs. No significant increase in either MHC class I, MIC-A/B, DR4 (TRAIL-R1), or Fas (CD95) expression was observed in tumor cells exposed to depsipeptide or bortezomib (Fig. 3A). In contrast, surface expression of DR5 (TRAIL-R2, ligated by TRAIL expressed on NK cells) was significantly up-regulated on RCC cells after treatment with either depsipeptide or bortezomib (P < 0.05). Moreover, the expression of DR5 on tumor cells correlated with their susceptibility to NK killing. RCC cells with low to absent DR5 expression at baseline (JOHW) showed a marked increase in DR5 expression and a significant enhancement in susceptibility to NK cell cytotoxicity following pretreatment with either drug (Fig. 3B). In contrast, in RCC cells with a high baseline expression of DR5 (PORJC), both depsipeptide and bortezomib failed to further increase DR5 expression and sensitize to NK cell cytotoxicity. A positive correlation between the expression of DR5 on tumor cells and their baseline susceptibility to NK cell–mediated cytotoxicity was observed (Fig. 3C); among 15 tumor cell lines analyzed, DR5 expression was significantly higher (P < 0.001) in those sensitive at baseline to NK cell–mediated lysis (i.e., >25% lysis at a 10:1 E/T ratio) compared with tumor lines where low baseline NK cell lysis was observed (<20% lysis). Tumor sensitization to NK cell killing also correlated with changes in the surface expression of DR5 following treatment with bortezomib and depsipeptide; a significantly higher increase from baseline in DR5 expression was observed in cell lines sensitized to NK cell killing (i.e., lysis was significantly higher after drug treatment compared with baseline) following drug exposure compared with cell lines were no enhancement occurred (P = 0.048 for depsipeptide and P = 0.002 for bortezomib; Table 3). Skin-derived fibroblasts had a low baseline expression of DR5 which did not increase following exposure to either drug.

Figure 3.

Cell surface phenotype and cytotoxicity after treatment with depsipeptide and bortezomib. JOHW-RCC was treated with depsipeptide (25 ng/mL) or bortezomib (10 nmol/L) for 18 hours and analyzed for cell surface expression of HLA-ABC, MIC-A/B, DR4 (TRAIL-R1), and Fas by flow cytometry (A). Human RCC cell lines JOHW (top), PORJC (middle), and skin-derived fibroblasts (bottom) were treated with depsipeptide (25 ng/mL) or bortezomib (10 nmol/L) for 18 hours, and then analyzed for cell surface expression of DR5 (B; left). Black lines, isotype control; blue lines, untreated cells; green lines, bortezomib-treated cells; and red lines, depsipeptide-treated cells. Allogeneic NK cells were tested against untreated (•), bortezomib- (▴) or depsipeptide-treated (▪) cells, respectively, in a 4-hour 51Cr release assay (B; right). Expression of DR5 in relation to NK cell cytotoxicity at baseline (C). Columns, mean of seven cell lines with low (<20%), and eight cell lines with high (>25%) tumor cytotoxicity and the relationship with DR5 expression; bars, ±SD. One of two (for A) and one of three (for B) representative experiments is shown.

Figure 3.

Cell surface phenotype and cytotoxicity after treatment with depsipeptide and bortezomib. JOHW-RCC was treated with depsipeptide (25 ng/mL) or bortezomib (10 nmol/L) for 18 hours and analyzed for cell surface expression of HLA-ABC, MIC-A/B, DR4 (TRAIL-R1), and Fas by flow cytometry (A). Human RCC cell lines JOHW (top), PORJC (middle), and skin-derived fibroblasts (bottom) were treated with depsipeptide (25 ng/mL) or bortezomib (10 nmol/L) for 18 hours, and then analyzed for cell surface expression of DR5 (B; left). Black lines, isotype control; blue lines, untreated cells; green lines, bortezomib-treated cells; and red lines, depsipeptide-treated cells. Allogeneic NK cells were tested against untreated (•), bortezomib- (▴) or depsipeptide-treated (▪) cells, respectively, in a 4-hour 51Cr release assay (B; right). Expression of DR5 in relation to NK cell cytotoxicity at baseline (C). Columns, mean of seven cell lines with low (<20%), and eight cell lines with high (>25%) tumor cytotoxicity and the relationship with DR5 expression; bars, ±SD. One of two (for A) and one of three (for B) representative experiments is shown.

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Table 3.

Fold change in DR5 expression in drug-sensitized and nonsensitized tumor cell lines

Depsipeptide
Bortezomib
Fold change in DR5 expression*Fold change in DR5 expression*
Sensitized JOHW 3.0 JOHW 4.6 
 SAUJ 2.3 SAUJ 3.2 
 STAR 1.2 STAR 1.2 
 MIRF 2.1 MIRF 3.1 
 PANC1 0.9 PANC1 2.6 
 2228 2.9 2228 2.6 
 PC3 1.0 SEA 1.5 
 Mean fold change 1.90  2.69 
Nonsensitized PORJC 1.0 PORJC 1.0 
 MAR 1.2 MAR 1.2 
 SEA 1.5 888 1.0 
 888 0.7 PC3 0.9 
 1355 1.1 1355 1.1 
 CALU6 1.0 CALU6 1.0 
 SW620 1.7 SW620 1.6 
 K562 1.2 K562 1.1 
 Mean fold change 1.17  1.12 
 P value 0.048  0.002 
Depsipeptide
Bortezomib
Fold change in DR5 expression*Fold change in DR5 expression*
Sensitized JOHW 3.0 JOHW 4.6 
 SAUJ 2.3 SAUJ 3.2 
 STAR 1.2 STAR 1.2 
 MIRF 2.1 MIRF 3.1 
 PANC1 0.9 PANC1 2.6 
 2228 2.9 2228 2.6 
 PC3 1.0 SEA 1.5 
 Mean fold change 1.90  2.69 
Nonsensitized PORJC 1.0 PORJC 1.0 
 MAR 1.2 MAR 1.2 
 SEA 1.5 888 1.0 
 888 0.7 PC3 0.9 
 1355 1.1 1355 1.1 
 CALU6 1.0 CALU6 1.0 
 SW620 1.7 SW620 1.6 
 K562 1.2 K562 1.1 
 Mean fold change 1.17  1.12 
 P value 0.048  0.002 

NOTE: DR5 expression was evaluated by flow cytometry before and after bortezomib or depsipeptide treatment in 15 tumor cell lines. The mean fold change in DR5 expression (shown in boldface) in tumor cell lines pre- and postdrug treatment were calculated in cell lines sensitized to NK cell–mediated lysis (defined as significantly higher lysis occurring after drug treatment compared with baseline) by either depsipeptide or bortezomib compared with nonsensitized cell lines.

*

The fold change from baseline in the percentage of cells expressing DR5 by FACS after treatment with either depsipeptide or bortezomib.

Unpaired Student's t test.

Pan-caspase inhibition in tumors treated with Z-VAD completely abolished the enhanced cytotoxic effect, demonstrating that the contribution of caspases is essential to the augmented cytotoxic effect by NK cells against drug-treated tumor cells. Inhibition of NK cell perforin with concanamycin A or blocking DR4 or Fas on tumor cells with monoclonal antibodies did not reduce NK cell tumor cytotoxicity against tumors treated with either drug compared with unblocked targets. In contrast, blocking TRAIL on NK cells completely abolished the enhanced cytotoxic effect of NK cells against RCC cells exposed to depsipeptide or bortezomib (Fig. 4). Tumor cells sensitized to NK cell killing also had enhanced susceptibility to killing by recombinant TRAIL. Furthermore, blocking tumor DR5 with monoclonal antibodies after tumors were exposed to either agent significantly reduced NK cell cytotoxicity compared with DR5 unblocked targets, confirming the up-regulation of DR5 as a possible mechanism potentiating NK cell cytotoxicity.

Figure 4.

Blocking TRAIL inhibits depsipeptide- and bortezomib-mediated augmentation in NK cell tumor cytotoxicity. Allogeneic NK cells were tested for cytotoxicity in a 16-hour 51Cr release assay against RCC cells (JOHW) at baseline or following exposure to either depsipeptide or bortezomib. Significantly higher lysis against depsipeptide-treated (▪) and bortezomib-treated (▴) tumor cells compared with untreated tumor cells (•) was observed. NK cell–mediated cytotoxicity was abolished in drug-exposed tumor cells when incubated with 10 μg/mL of anti-TRAIL antibody (⧫), whereas incubation with an isotype-matched control antibody in depsipeptide- treated (□) or bortezomib- treated (△) tumor cells did not decrease cytotoxicity (A). Effects on NK cell–mediated tumor cytotoxicity after blocking perforin with concanamycin A (), or Fas (), DR4 (), and DR5 (×) with monoclonal antibodies in bortezomib- and depsipeptide-treated tumors (B). *The inhibition of NK cell–mediated cytotoxicity in bortezomib- and depsipeptide-treated tumor cells after blocking with DR5 antibody compared with unblocked target cells (E/T ratio, 10:1) was 54% and 50%, respectively. Recombinant TRAIL-mediated lysis of RCC cells (JOHW) exposed to bortezomib and depsipeptide compared with untreated tumor cells (C). The pan-caspase inhibitor Z-VAD was added to the 51Cr assay at a concentration of 20 μmol/L (D). White columns, untreated cells; black columns, Z-VAD-treated cells. NO, untreated cells; DP, depsipeptide-treated cells; B, bortezomib-treated cells. E/T ratio, 4:1. One of three representative experiments is shown.

Figure 4.

Blocking TRAIL inhibits depsipeptide- and bortezomib-mediated augmentation in NK cell tumor cytotoxicity. Allogeneic NK cells were tested for cytotoxicity in a 16-hour 51Cr release assay against RCC cells (JOHW) at baseline or following exposure to either depsipeptide or bortezomib. Significantly higher lysis against depsipeptide-treated (▪) and bortezomib-treated (▴) tumor cells compared with untreated tumor cells (•) was observed. NK cell–mediated cytotoxicity was abolished in drug-exposed tumor cells when incubated with 10 μg/mL of anti-TRAIL antibody (⧫), whereas incubation with an isotype-matched control antibody in depsipeptide- treated (□) or bortezomib- treated (△) tumor cells did not decrease cytotoxicity (A). Effects on NK cell–mediated tumor cytotoxicity after blocking perforin with concanamycin A (), or Fas (), DR4 (), and DR5 (×) with monoclonal antibodies in bortezomib- and depsipeptide-treated tumors (B). *The inhibition of NK cell–mediated cytotoxicity in bortezomib- and depsipeptide-treated tumor cells after blocking with DR5 antibody compared with unblocked target cells (E/T ratio, 10:1) was 54% and 50%, respectively. Recombinant TRAIL-mediated lysis of RCC cells (JOHW) exposed to bortezomib and depsipeptide compared with untreated tumor cells (C). The pan-caspase inhibitor Z-VAD was added to the 51Cr assay at a concentration of 20 μmol/L (D). White columns, untreated cells; black columns, Z-VAD-treated cells. NO, untreated cells; DP, depsipeptide-treated cells; B, bortezomib-treated cells. E/T ratio, 4:1. One of three representative experiments is shown.

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Cellular inactivation through KIR inhibits NK cell cytotoxicity against tumor cells. This inhibitory mechanism represents a major barrier limiting the therapeutic potential of adoptively infused autologous NK cells in patients with cancer. As a consequence, we sought to develop a method that would potentiate both autologous and allogeneic NK cell–mediated tumor killing. We found that NK cell cytotoxicity was potentiated in vitro by pre-exposing tumor cells to the proteasome inhibitor bortezomib or the histone deacetylase inhibitor depsipeptide. We also found that NK cells from patients with RCC were significantly more cytotoxic against autologous tumor cells following treatment with these agents. In contrast, normal fibroblasts were relatively resistant to NK cell–mediated lysis, even when exposed to these drugs. The observation that fibroblasts exposed to bortezomib or depsipeptide did not have enhanced susceptibility to NK cells suggests that this sensitizing effect may be relatively specific to malignant cells. If these observations are reproducible in animal models, they would provide the rationale for protocols that pretreat cancer patients with bortezomib or depsipeptide to potentiate the anticancer effects of subsequent adoptive NK cell infusions.

Depsipeptide and bortezomib have been shown to activate executioner caspases (16, 17) and up-regulate the cyclin-dependent kinase inhibitor p21/Waf-1 (18, 19). Accordingly, we observed apoptosis in vitro in multiple myeloma cell lines treated with either depsipeptide or bortezomib. Each agent, when given alone, induced minimal apoptosis in RCC cell lines. In contrast, the combination of drugs induced apoptosis in most RCC lines tested. Synergistic apoptotic effects induced by combining depsipeptide and bortezomib have been reported to occur in gastrointestinal adenocarcinoma cells (20). Although both depsipeptide and bortezomib suppressed the proliferation of RCC cells in vitro, they have thus far been of limited benefit in the treatment of patients with metastatic RCC (21, 22).

Bortezomib and depsipeptide are known to up-regulate death receptors on tumor cells. Furthermore, depsipeptide and bortezomib increase susceptibility to recombinant TRAIL-mediated apoptosis in a number of different tumor cells (12, 2325). Sayers et al. showed that bortezomib-treated murine RCC (RENCA) was sensitized to TRAIL-mediated apoptosis via down-regulation of c-FLIP (13). Others have recently reported that bortezomib and depsipeptide can directly up-regulate the expression of TRAIL-R2 (DR5), sensitizing tumor cells to TRAIL-induced apoptosis (25, 26).

The current study extends these observations: we found that the augmented antitumor effects of NK cells occurred as a consequence of bortezomib and depsipeptide sensitizing tumor cells to NK cell TRAIL-mediated cytotoxicity. Killing via perforin and Fas pathways seemed to be uninvolved, as tumor sensitization to bortezomib and depsipeptide persisted in cytotoxicity assays using concanamycin A–treated NK cells or tumor cells in which Fas was blocked. Furthermore, surface expression of MHC class I and MIC-A/B did not change in these cell lines, making it unlikely that disruption of NK cell inhibition via KIR ligands or NK cell activation via NKG2D play a role in this sensitizing effect. In contrast, tumors sensitized to NK cell cytotoxicity by bortezomib or depsipeptide up-regulated surface expression of the TRAIL receptor DR5 and had increased susceptibility to killing by recombinant TRAIL. Furthermore, tumor sensitization to NK cell killing following treatment with bortezomib and depsipeptide correlated with changes in the surface expression of DR5. Significantly higher increases from baseline in DR5 expression were observed in cell lines sensitized to NK cell killing following drug exposure compared with cell lines where no enhancement occurred. The enhanced susceptibility to NK cell killing was completely abolished by blocking TRAIL on NK cells and partially abolished by blocking DR5 on tumor cells. In some cell lines, only a minor up-regulation in surface expression of DR5 occurred despite a significant increase in tumor killing by NK cells after drug exposure. These findings suggest other pathways influenced by TRAIL besides DR5 up-regulation are involved in augmenting NK cell cytotoxicity against tumor cells exposed to these agents. It has been reported that certain chemotherapeutic agents could redistribute death receptors, such as CD95, DR4, and DR5 into lipid rafts, facilitating the caspase cascade activation response to death receptor stimulation (27). Whether depsipeptide or bortezomib induce the accumulation of death receptors in lipid rafts was not evaluated.

TRAIL and DR5 antibody therapy have been shown to eradicate established tumor metastases in mice (28, 29). Our findings, as well as findings by others, would suggest the combination of either bortezomib or depsipeptide with recombinant TRAIL or agonistic TRAIL receptor antibodies would likely enhance the antitumor effect. However, because immune cells in the tumor microenvironment produce cytokines that may contribute to tumor eradication, the combination of either of these drugs with cell-based therapies using cells having high surface expression of TRAIL (i.e., NK or T cells) could have theoretical advantages over the use of recombinant TRAIL-targeting proteins.

Inducing tumor cytotoxicity via TRAIL may have advantages in terms of safety compared with immunotherapeutic strategies using recombinant tumor necrosis factor or Fas ligand; although administration of the latter agents can induce apoptosis in transformed cells, both have been associated with extreme toxicity in humans precluding their further development. Recombinant TRAIL and agonistic antibodies to TRAIL receptors induces apoptosis in a variety of tumor cells. However, in contrast to malignant cells, expression of TRAIL decoy receptors (DcR1 and DcR2) seems to predominate in normal tissues protecting them from TRAIL-induced apoptosis (30, 31). In a murine model of human mammary adenocarcinoma, TRAIL was reported to suppress tumor growth in vivo without affecting normal tissues (32). In contrast to tumor cells, we observed that bortezomib and depsipeptide did not increase the susceptibility of fibroblasts to killing by NK cells.

Altogether, these findings suggest that bortezomib and depsipeptide might selectively sensitize tumors to NK cell TRAIL while avoiding NK cell–mediated toxicity of normal tissues. Our study focused on in vitro findings using human tumors and NK cells. The development of animal models to confirm (in vivo) our in vitro findings could help optimize the efficacy and safety of trials testing in humans whether or not these agents sensitize tumors to NK cell cytotoxicity.

Recently, Skov and colleagues reported that treatment of multiple myeloma and mantle cell lymphoma tumor cell lines with histone deacetylase inhibitors led to a functional increase in the expression of MIC-A/B molecules rendering them more sensitive to NK cell–mediated lysis (33). However, in their experiments, blocking the MIC-A/B pathway using monoclonal antibodies to MIC-A/B or NKG2D-Fc did not completely inhibit NK cell–mediated cytotoxicity, suggesting that other pathways are likely involved in this sensitizing effect. Although we did not evaluate the expression of MIC-A/B in multiple myeloma and mantle cell lymphoma lines, the histone deacetylase inhibitor depsipeptide did not alter surface expression of MIC-A/B on RCC cell lines. Furthermore, the majority of our RCC cell lines had no surface expression of MIC-A/B at baseline or after treatment with either depsipeptide or bortezomib, suggesting that the MIC-A/B pathway did not account for the enhanced susceptibility of RCC cells to NK cell cytotoxicity following exposure to these drugs.

Bortezomib and depsipeptide represent two different classes of antineoplastic drugs that directly induce tumor apoptosis via distinct pathways. Although we found that both agents enhanced tumor susceptibility to NK cell cytotoxicity, some tumor cells were selectively sensitized to only one of the two drugs. Therefore, it is possible that these agents have both shared and unique effects on specific pathways that intensify TRAIL-mediated apoptosis.

In summary, this is the first study to show that tumor cells could be sensitized to NK cell TRAIL–mediated cytotoxicity by pretreatment with bortezomib or depsipeptide. Based on these findings, cancer immunotherapy strategies that evaluate the efficacy of adoptively infused NK cells in conjunction with bortezomib or depsipeptide as tumor sensitization in animal models, and subsequently in humans, should be considered. Although NK cell proliferation was inhibited, relatively little apoptosis occurred following NK cell exposure to either agent. Because depsipeptide and bortezomib have relatively short half-lives (34, 35), delaying adoptive cellular therapy until the plasma levels of these drugs have decreased could offer a strategy to minimize any deleterious effects of these agents on NK cells.

Grant support: Intramural research program of NIH, National Heart, Lung and Blood Institute, Hematology Branch.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The authors acknowledge Drs. Ram Srinivasan, Yoshiyuki Takahashi, Jason Wynberg, Takehito Igarashi, and Anthony Suffredini for their valuable contributions and insight related to this work.

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