Leukemia (2010) 24, 51–57 & 2010 Macmillan Publishers Limited All rights reserved 0887-6924/10 $32.00 www.nature.com/leu ORIGINAL ARTICLE

TNF-a-converting enzyme (TACE/ADAM17)-dependent loss of CD30 induced by proteasome inhibition through reactive oxygen species

AM Vahdat1, KS Reiners1, VL Simhadri1, DA Eichenauer1,BBo¨ll1, A Chalaris2, VR Simhadri1, K Wiegmann3, H-W Krell4, S Rose-John2, A Engert1, EP von Strandmann1 and HP Hansen1

1Laboratory of Immunotherapy, Department I of Internal Medicine, University Clinic Cologne, Cologne, Germany; 2Department of Biochemistry, University of Kiel, Kiel, Germany; 3Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Cologne, Germany and 4Roche Diagnostics Pharma Research, Penzberg, Germany

Combinations with proteasome inhibitors are currently being inhibitors of nuclear factor-kB (I-kB species).7 We previously investigated to improve the therapy of hematological malig- tested the combination of bortezomib and the fully human anti- nancies. We previously found that proteasome inhibition by CD30 antibody (Ab), MDX-060, in the anti-CD30 Ab-responsive bortezomib failed to sensitize anti-CD30 antibody (Ab)-based 8 lymphoma cell killing. In this study, we demonstrate in L540 HL cell line L540, both in vitro and in a SCID mouse model. Hodgkin’s lymphoma cells that proteasome inhibition not only Although preincubation with MDX-060 enhanced the cytotoxic communicates apoptosis but also more rapidly causes a loss of potency of bortezomib, simultaneous or preincubation with CD30 antigen from cell membrane and a simultaneous release bortezomib failed to improve Ab therapy. Currently, there is no of soluble CD30, a targeting competitor. This shedding was explanation for this defect. The reports on endothelial and catalyzed by the tumor necrosis factor (TNF)-a-converting enzyme (TACE, ADAM17) and blocked by the ADAM17-selective epithelial cells have shown that proteasome inhibition can lead inhibitor, Ro32-7315. In parallel with CD30 shedding, bortezo- to a metalloproteinase-dependent loss of cell surface tumor 9,10 mib caused the generation of reactive oxygen species (ROS). necrosis factor (TNF) receptor I/II (CD120a/b). Both, CD120a As apoptosis and shedding were inhibited by the radical and CD30 are released by TNF-a-converting enzyme (TACE, scavenger, N-acetyl-L-cysteine, ROS might have a pivotal ADAM17).4,11 We therefore tested whether bortezomib impairs function in both effects. In contrast, the pan-caspase inhibitor, Ab-based CD30 targeting by reducing the antigen density. In this zVAD-fmk, blocked bortezomib-induced apoptosis but not CD30 shedding, and Ro32-7315 blocked shedding but allowed study, we show that bortezomib stimulates ADAM17-dependent apoptosis. This suggests independent terminal signaling path- shedding of CD30 in L540 HL cells involving reactive oxygen ways that are conflicting in Ab-based immunotherapy. Conse- species (ROS). ADAM17 inhibition blocked this CD30 loss and quently, shedding inhibition substantially improved the improved the synergism of MDX-060 and bortezomib. synergistic antitumor efficacy of the human anti-CD30 Ab, MDX-060, and bortezomib. As proteasome inhibition also stimulated loss of TNF receptors, interleukin-6 receptor and syndecan-1 in different leukemia and lymphoma cell lines, we concluded that proteasome inhibition might impede targeted Materials and methods therapy against antigens susceptible to shedding. Leukemia (2010) 24, 51–57; doi:10.1038/leu.2009.230; Cells and reagents published online 5 November 2009 Keywords: human; ADAM17; interleukin-6 receptor; We used the CD30-positive HL cell lines L540 and L428, the immunotherapy; proteasome inhibition; bortezomib large cell anaplastic lymphoma cell line Karpas299, as well as the CD30-negative acute lymphoblastic leukemia cell line Reh, Jurkat (T-acute lymphoblastic leukemia) and RPMI 8226 (multiple myeloma). Cells were cultivated at 37 1C and 5% CO2 in RPMI-1640 containing 10% heat-inactivated fetal calf serum, Introduction penicillin (50 mg/ml) and streptomycin (50 mg/ml). The ADAM17 (À/À) mouse embryonal fibroblast cells were received from CD30 is selectively overexpressed in the malignant cell Dr R Black (Amgen Incorporated, Seattle, WA, USA) and the population of Hodgkin’s lymphoma (HL). Despite this selec- À/À ADAM10 ( ) cells were from Dr D Hartmann (Center for tivity, the clinical benefit of anti-CD30 immunotherapy in HL Human Genetics, Leuven, Belgium). They were cultivated in patients is poor.1,2 This is at least in part due to CD30 shedding Dulbecco’s modified Eagle’s medium/5% fetal calf serum. that leads to the generation of a soluble serum competitor.3,4 The anti-CD30 monoclonal Abs, Ki-1, Ki-2, Ki-3 and Ki-4, were In addition, aberrant transcription factor nuclear factor-kB 12 used for the detection of CD30 and soluble CD30 (sCD30). survival activity, governed by the overexpression of nuclear MDX-060 was supplied by Medarex (Bloomsbury, NJ, USA). factor-kB-inducing kinase, is a hallmark of HL and participates Other reagents were goat anti-human Fc Ab (GaH-IgG; in apoptosis resistance of tumor cell lines.5,6 Dianova, Hamburg, Germany) and the proteasome inhibitor, The proteasome inhibitor, bortezomib, inhibits the nuclear bortezomib (Velcade or PS-341, Millennium Pharmaceuticals, factor-kB signaling pathway by blocking the degradation of Cambridge, MA, USA). Marimastat (BB-2516) was from British Biotech Pharmaceuticals (Oxford, UK). GI254023X (Glaxo- Correspondence: Dr HP Hansen, Department of Internal Medicine I, SmithKline, Stevenage, UK) was kindly provided by Dr A Ludwig, University Hospital Cologne, LFI, Ebene 4, room 703, Kerpener Aachen, Germany. The ADAM17-selective inhibitor, Ro32- Str. 62, Cologne 50924, Germany. 13 E-mail: [email protected] 7315, and the gelatinase-selective inhibitor, Ro28-2653, were Received 13 May 2009; revised 1 September 2009; accepted 30 from Roche Diagnostics GmbH, Pharma Research (Penzberg, September 2009; published online 5 November 2009 Germany). Loss of membrane proteins by proteasome inhibition AM Vahdat et al 52 Transfection Results and discussion In 24-well plates, aliquots of 1 Â 105 cells were cultivated over- night. Medium was removed from adherent cells and 400 ml Bortezomib stimulates ADAM17-catalyzed shedding Opti-MEM I (Invitrogen, Karlsruhe, Germany) was added. of CD30 DNA (0.8 mg DNA/50 ml Opti-MEM I) and LipofectAmin Bortezomib caused a loss of CD30 from L540 cells. The effect (Invitrogen; 2/48 ml Opti-MEM I) were prepared separately and was minute after 7 h but increased after 14 and 20 h of incubated for 5 min. Then, DNA and LipofectAmin solutions incubation (Figure 1a). The portion of dead cells increased were mixed and incubated for 30 min before being added to under treatment, that is, 1, 8 and 18% after 7, 14 and 20 h, the cells. Cells were incubated for 36 h before use. respectively (data not shown). However, the loss was not restricted to dead cells, as 7-amino-actinomycin D-negative cells showed an effect similar to the whole cell population. In Western blot all cases, receptor reduction was clearly inhibited by the broad- Whole cell lysates generated after 2 min boiling with SDS spectrum metalloproteinase inhibitor, marimastat, indicating a sample buffer and cell-free supernatants were developed in function of metalloproteinases. Soluble CD30 was determined SDS-polyacrylamide gel electrophoresis and transferred to in the cell-free supernatants from the same experiment. As nitrocellulose. Filters were blocked with phosphate-buffered shown in Figure 1b, 1.4-fold elevated sCD30 levels (Po0.0001) saline containing 1% fat-free dry milk and then incubated at were already found after 7 h of stimulation. After 14 h, the gain 4 1C with anti-CD30 and anti-b-actin Abs as indicated. Finally, was 2.3-fold (Po0.0001) and after 20 h the gain was 2.9-fold they were stained with peroxidase-coupled goat anti-mouse (P ¼ 0.0129). Western blot analysis confirmed the stimulated immunoglobulin-G and ECL reagent (GE Healthcare, Freiburg, conversion from 120 kDa CD30 to 90 kDa sCD30 after 14 h Germany). of bortezomib treatment (Figure 1c). Here, the effects were also inhibited by metalloproteinase inhibition. Despite the Reactive oxygen species detection enhanced sCD30 release, the CD30 surface expression Cells were incubated with or without bortezomib ±100 mM remained stable after 7 h of stimulation. This might be explained N-acetyl-L-cysteine (NAC; Sigma-Aldrich, Taufkirchen, Germany) by an activated receptor transport after bortezomib treatment, 9 as indicated. For the final 15 min of incubation, 20,70- which compensates the loss in the first hours of stimulation. dichlorodihydro-fluorescein diacetate (Sigma-Aldrich; 5 mM) Because of the invariant receptor density after 7 h and an was added and incubated at 37 1C. ROS were analyzed using appreciable cell death after 20 h of bortezomib stimulation, flow cytometry (BD FACSCalibur, Becton Dickinson GmbH, we stimulated for 14 h in subsequent experiments, unless Heidelberg, Germany). otherwise indicated. Shedding stimulation was dose-dependent and reached plateau values at 10 ng/ml in L540 cells resulting in a 70% reduction of cell surface CD30 and a threefold gain Determination of sCD30 of sCD30 compared with non-treated controls (Figure 1d). 14 The test was performed as previously described. Stimulation of CD30 shedding was also found in L428 (HL) and Karpas299 (large cell anaplastic lymphoma) cells, as indica- ted by a reduction of cell surface CD30 and an increased release XTT viability assay of sCD30 (P ¼ 0.0372 and 0.0041, respectively) (Figures 1e and f). 4 m In 96-well plates, cells (2 Â 10 /well) were cultivated in 200 l This treatment caused a 10 and 8% loss of viability in L428 of RPMI-1640 medium containing 10% fetal calf serum. The test and Karpas299 cells, respectively. Owing to the lack of was started by preincubating cells with anti-CD30 Ab MDX-060 malignant Hodgkin’s cells in the blood stream of patients, it is (50 ng/ml) þ GaH-IgG (100 ng/ml). One hour later, increasing very difficult to test primary cells. However, bortezomib was concentrations of bortezomib were added. The same was also tested in clinical trials with HL patients. No clinical benefit was carried out in reverse order. Ro32-7315 (1.5 mM) was applied to observed as single agent or in combination with gemcitabine experiments when indicated. Then, cells were incubated for or dexamethasone.15–17 The trial combining bortezomib and 48 h and pulsed for additional 4 h with 50 ml of fresh serum-free dexamethasone was prematurely closed after 12 patients due medium containing 1.5 mM XTT (Sigma-Aldrich) and 0.025 mM to lack of response. Serum was available from three patients N-methyl-phenazonium-methyl-sulfate (Sigma-Aldrich). Absor- who received treatment according to the protocol until the end bance of the reaction was determined using m-Quant-ELISA of study. In all cases sCD30 levels clearly increased under reader (Bio-Tek Instruments, Winooski, VT, USA) at 450 versus therapy (3-, 5.3- and 6-fold; data not shown). Despite the low 650 nm. The concentration required to achieve a 50% reduction number of cases and although it could not be finally of XTT turnover (IC50) compared with untreated controls was calculated. demonstrated that this effect was caused by bortezomib alone, this finding provides some evidence for bortezomib- stimulated shedding in vivo. Analysis of apoptosis The metalloproteinases, ADAM10 and ADAM17, have been Cells (2 Â 105/ml) were cultivated in RPMI-1640/10% fetal calf shown to catalyze CD30 shedding, their individual participa- 4,18 serum with or without agents as indicated. Then, cells were tion depends on the way of triggering. We tested the role of washed once with phosphate-buffered saline and subsequently these candidate enzymes in bortezomib-stimulated CD30 À À suspended in annexin binding buffer (10 mM HEPES/NaOH, shedding using either ADAM10-deficient (ADAM10 / )or À À 140 mM NaCl, 2.5 mM CaCl2, pH 7.4). They were incubated for ADAM17-defective (ADAM17 / ) murine embryonic fibro- 10 min on ice with annexin V-phycoerythrin and 7-amino- blasts. Both showed some constitutive release of sCD30, actinomycin D. Cells without any previous cytotoxic treatment indicating a certain releasing enzyme redundancy (Figure 2a). were used to determine background apoptosis. Assays were Although 100 ng/ml of bortezomib doubled the sCD30 release performed in triplicate and analyzed using flow cytometry and (707.5–1569 U/ml, Po0.0001) in ADAM10-defective cells with Cellquest software (BD FACSCalibur, Becton Dickinson GmbH). functional ADAM17, it failed to stimulate CD30 shedding in

Leukemia Loss of membrane proteins by proteasome inhibition AM Vahdat et al 53

Figure 2 ADAM17-catalyzed shedding of CD30 after bortezomib treatment. (a) ADAM10- (open bars) or ADAM17-defective murine embryonic fibroblasts (filled bars) were transfected with human CD30 cDNA. 36 h after transfection, cells were stimulated with bortezomib as indicated. After another 14 h, sCD30 in the cell-free supernatants was determined by CD30 ELISA. The results show U/ml as means± s.d. for two independent experiments. The dashed and drawn lines represent the constitutive release of the cells. (b and c) L540 cells were stimulated with bortezomib (10 ng/ml, dotted lines, bo þ ) or not stimulated (dotted lines, 0). Bortezomib-treated cells were incubated in the presence or absence of a dilution of Ro32-7315, marimastat, Ro28-2653 (b) or GI254023X (c). Soluble CD30 (sCD30) was deter- mined. The curve shows means of three independent experiments. The dashed line indicates half- maximal stimulation of the sCD30 release and the arrow indicates the IC50 of the inhibitors Ro32-7315 and marimastat.

Figure 1 Bortezomib stimulates shedding of CD30. (a and b) L540 cells were cultivated with bortezomib (Bo, 10 ng/ml) for 7, 14 or 20 h ±marimastat (I, 1.5 mM). They were stained with 7-amino-actinomycin D (7-AAD) and phycoerythrin-coupled anti-CD30 antibodies (Ab). ADAM17-defective cells. We also applied the ADAM17- CD30 expression was determined by flow cytometry and is shown selective inhibitor, Ro32-731513 (Figure 2b). It blocked borte- from whole cell population or living (7-AAD-negative) cells. zomib-stimulated sCD30 release from L540 cells with an IC50 The histograms show one typical experiment out of three. Soluble of approximately 230 nM. This concentration is compara- CD30 (sCD30) was determined in the cell-free supernatants of the experiment using CD30 ELISA. Untreated cells are depicted as filled ble with the IC50 of the potent broad-spectrum inhibitor, gray curves or bars, bortezomib treatment is symbolized as fine marimastat. As the gelatinase inhibitor (Ro28-2653) and the borderline/open bar and metalloproteinase inhibition of bortezomib ADAM10-selective inhibitor (GI254023X) showed only weak treatment is in bold/black. (c) CD30 and sCD30 were also detected inhibition at higher concentrations, we concluded that borte- by western blot. Here, L540 cells were treated for 14 h with zomib triggers CD30 shedding in a ADAM17-dependent m bortezomib (10 ng/ml) and marimastat (1.5 M) as indicated. Cell manner (Figures 2b and c). lysates were used for the detection of CD30 and b-actin and cell-free supernatants for sCD30. A mixture of peroxidase-coupled Ki-2 and We tested the effect of bortezomib on other surface proteins Ki-4 Abs was used for the detection of CD30 or sCD30. (d) L540 cells that are both, interesting targets for immunotherapy and subjects were incubated for 14 h with a dilution series of bortezomib. Again, of proteolytic cleavage. Among them, syndecan-1 (CD138) and CD30 expression was determined by flow cytometry on viable cells, the interleukin-6 (IL-6) receptor (CD126) are expressed on L540 by forward/side scatter gating. The fluorescence of cells is shown as cells (Figure 3). CD138 is expressed on B cells and used as target ± the mean of three independent experiments s.d. In parallel, sCD30 in plasma cell malignancies.19 CD126 is expressed on was determined by ELISA showing the mean of three experiments. (e and f) Karpas299 and L428 cells were incubated for 14 h hepatocytes, leukocytes and its signaling participates in the with bortezomib (10 ng/ml) in the presence or absence of marimastat pathogenesis of rheumatoid arthritis and some tumors. Targeting (1.5 mM). CD30 expression was determined by flow cytometry CD126 was found to improve the outcome of rheumatoid from viable cells after forward/side scatter gating and sCD30 arthritis.20 Both antigens were downregulated on bortezomib was determined by ELISA from three or four independent treatment on L540 HL cells and on Jurkat T-acute lympho- experiments. blastic leukemia cells. Here, the loss was also inhibited by ADAM17-selective inhibition. However, downregulation of cell

Leukemia Loss of membrane proteins by proteasome inhibition AM Vahdat et al 54

Figure 3 Cell surface expression of CD126 and CD138. L540 and Jurkat cells were stimulated with bortezomib (bo, 10 ng/ml) in the presence or absence of Ro32-7315 (I, 1.5 mM). CD126 and CD138 expression was determined by flow cytometry after 14 h of incuba- tion. Expression on untreated cells is depicted as a filled gray curve, bortezomib treatment is symbolized as fine borderline curve and metalloproteinase-inhibited bortezomib treatment is in bold/ black. The FACS histograms show one representative experiment out of three.

surface proteins by bortezomib might not represent a general phenomenon. RPMI8226, a multiple myeloma cell line that was successfully killed upon CD138 targeting, showed no detectable loss of CD138 after treatment (data not shown). Thus, it is tempting to speculate that proteasome inhibition not only Figure 4 Kinetic study. L540 cells were incubated in the presence of governs tumor survival by inhibiting nuclear factor-kB signaling bortezomib (10 ng/ml) or without (control) for periods of time as but also modifies the expression of surface proteins, a process indicated. Cells were stained with annexin V-phycoerythrin and which might negatively influence immunotherapeutic ap- 7-amino-actinomycin D to determine apoptosis by flow cytometry. proaches in certain malignancies. Percentage of apoptotic cells (lower and upper right) was evaluated for bortezomib-treated and untreated cells after eight different periods of time as exemplarily shown for 12 h of incubation. In a kinetic, the differences in the percentage of apoptotic cells of untreated and bortezomib-treated L540 is shown (filled circle). In parallel, sCD30 Mechanism of bortezomib-stimulated shedding was determined in cell-free supernatants of four independent induction experiments. The differences of the means of treated and untreated To better understand the downstream effects, we compared the ones±s.d. are shown (open circle). kinetics of bortezomib-induced shedding and apoptosis. Significantly elevated sCD30 levels were first detectable after 3 h of incubation (P ¼ 0.0474) (5–20 h; Po0.0001), with a maximum gain between 3 and 7 h (Figure 4). However, an elevated annexin V staining, indicating increased apoptosis We tested the function of elevated ROS levels in CD30 was not observed before 7 h of incubation. This shows that the shedding from L540 cells and found that bortezomib (10 ng/ml) contribution of bortezomib to receptor release sets in more and H2O2 (1 mM) stimulated sCD30 release (Figure 5b). In both rapidly than its contribution to cell killing. We therefore cases it was inhibited by NAC (1 mM, P ¼ 0.0012) and Ro32- speculate that CD30 shedding is not a consequence of apoptosis 7315 (1.5 mM, Po0.0001), indicating a role of ROS and but occurs earlier or in parallel. ADAM17. On the other hand, bortezomib and H2O2 also Apoptosis induction by bortezomib in multiple myeloma or caused apoptosis as demonstrated by annexin V staining. mantle cell lymphoma is at least in part communicated by Bortezomib- and H2O2-induced apoptosis could be inhibited elevated and sustained levels of ROS participating in down- by NAC, indicating a pivotal function of ROS in shedding as stream generation of DNA fragments.21,22 Moreover, ROS well as in induction of apoptosis. production may cause ectodomain shedding involving In neutrophils, apoptotic cell death is the natural stimulus of ADAM17 or ADAM10.23,24 We tested ROS production in ADAM17-dependent IL-6 receptor (CD126) shedding. For this L540 cells in response to bortezomib (Figure 5a). Comparing shedding event prior activation of caspases 3, 8 and 9 is stimulated and untreated cells in a kinetic, elevated ROS levels required.25 We tested the role of caspases in bortezomib- were detectable after 3 h of incubation (relative gain ¼ stimulated CD30 shedding using the pan-caspase inhibitor, 1.188±0.048). The gain failed when cells were preincubated zVAD-fmk. In contrast to IL-6 receptor release in neutrophils, with the radical scavenger NAC (relative gain ¼ 1.03±0.01) sCD30 release in L540 cells was not inhibited by pan-caspase indicating in a significant generation of NAC-sensitive ROS at inhibition (Figure 5c). As zVAD-fmk was able to inhibit this time point (P ¼ 0.0098). ROS levels increased after bortezomib-induced apoptosis in L540 cells as detected by prolonged incubation periods, resulting in a 1.6-fold versus annexin V staining, we concluded that sCD30 release is not 1.1-fold (P ¼ 0.0308) elevation after 4 h and a 1.9-fold versus caspase-dependent. In addition, apoptosis induction by 1.1-fold (Po0.0001) gain after 6 h of incubation. Thus, in kinetic CD95(Fas) ligation also caused caspase-independent CD30 evaluations, the generation of ROS from bortezomib-stimulated shedding in L540 cells (data not shown). Thus, CD30 shedding L540 cells correlated with elevated sCD30 levels in the cell-free in L540 cells correlates with different kinds of apoptosis supernatants (Figure 4). induction but is not dependent on apoptosis itself because

Leukemia Loss of membrane proteins by proteasome inhibition AM Vahdat et al 55

Figure 5 Mechanism of CD30 shedding. (a) Determination of reactive oxygen species (ROS) subsequent to bortezomib stimulation. L540 cells were incubated with bortezomib (bo, 10 ng/ml) in the presence or absence of 100 mM N-acetyl-L-cysteine (NAC) or the solvent alone for different periods of time. For the final 15 min cells were stained with H2DCFDA, washed and ROS activity of cells was detected by flow cytometry. The relative production of ROS was determined by comparing the mean fluorescence of treated versus untreated cells at each time point of four independent experiments (NS, not significant). The relative (b þ c) L540 cells were stimulated for 14 h with bortezomib (10 ng/ml) or H2O2 (1 mM) in the presence or absence of NAC (1 mM), Ro32-7315 (Ro; 1.5 mM) or zVAD-fmk (100 mM). Soluble CD30 was determined in the cell-free supernatants by ELISA. Cell viability/apoptosis was evaluated by flow cytometry after 7-amino-actinomycin D and annexin V staining. Dot blot analysis shows the percentage of viable (lower lift) and apoptotic (upper and lower right) cells. caspase activation is no prerequisite for this process. Instead, at bortezomib (Figure 6b). Both L540 and Reh control cells were least upon bortezomib stimulation, ROS seem to represent a sensitive to bortezomib in XTT viability assays (IC50: 6 and common player in the apoptosis and shedding pathway. 10 ng/ml, respectively). In agreement with our previous data, the combination of bortezomib and MDX-060 showed synergistic cytotoxicity in L540 cells but not in the control cells lacking Inhibition of ADAM17 increases the cytotoxicity of CD30.8 Pretreatment with Ab is essential for synergy which is bortezomib and MDX-060 against L540 not seen after bortezomib coapplication or pretreatment. We also tested the role of shedding stimulation on the impact of However, inhibition of CD30 shedding by Ro32-7315 sup- anti-CD30 immunotherapy. The antitumor efficacy of the pressed this failure of synergy. human anti-CD30 Ab, MDX-060, in L540 cells is enhanced by To verify the role of ADAM17-catalyzed shedding in anti-human IgG Abs (GaH-IgG).26 Such crosslinking of CD30 bortezomib-supported immunotargeting of L540 cells, we forms visible cell aggregates as indicated in Figure 6a. Aggregate analyzed the impact of Ro32-7315 by simultaneous staining of formation depends on antigen density. We observed that late apoptosis with 7-amino-actinomycin D and early apoptosis bortezomib pretreatment abrogates clot formation that was with annexin V-phycoerythrin in flow cytometry. To minimize a reverted by concomitant ADAM17 inhibition. As inhibition of direct impact of bortezomib on apoptosis, we used a subtoxic ADAM17 suppressed bortezomib-induced CD30 shedding, we concentration (4 ng/ml) that was still able to induce CD30 speculated that downregulation of cell surface CD30 by bor- shedding. As demonstrated in Figure 6c, ADAM17 inhibition tezomib pretreatment might negatively influence CD30-targeted alone (Ro) had no effect on cell viability but was able to triple immunotherapy. the rate of early apoptotic cells (lower right gate) after To confirm this speculation, we evaluated the influence of bortezomib preincubation þ MDX-060 incubation (bo þ MDX subtoxic concentrations of Ro32-7315 (1.5 mM) and MDX-060 and bo þ Ro þ MDX) from 8.34 to 29.01% and more than on the cytotoxic potential of increasing concentrations of double the rate of late apoptotic/necrotic cells from 6.38 to

Leukemia Loss of membrane proteins by proteasome inhibition AM Vahdat et al 56

Figure 6 Cytotoxic synergy of MDX-060 in combination with bortezomib and Ro32-7315. (a) Aggregation of L540 cells after 14 h incubation with MDX-060/GaH-IgG (MDX-060), MDX-060/GaH-IgG after 1 h preincubation with bortezomib (MDX-060 þ bo), or MDX-060/GaH- IgG after 1 h preincubation with Ro32-7315 þ bortezomib (MDX-060 þ bo þ Ro). (b) XTT viability assays of L540 or Reh control cells after 48 h exposure to subtoxic concentrations of crosslinked MDX-060 (50 ng/ml MDX-060; 100 ng/ml GaH-IgG) and increasing concentrations of bortezomib in the presence or absence of Ro32-7315 (1.5 mM) as indicated. Cells were preincubated for 1 h, either with MDX-060±Ro32-7315 (L540/MDX first), (Reh/MDX first) or with bortezomib ±Ro32-7315 (L540/bo first), (Reh/bo first). MDX-060 and GaH-IgG (MDX-060/L540) were distributed in 100 ml aliquots in 96-well plates with 2 Â 104 L540 or Reh target cells and the plates were incubated for 1 h before addition of bortezomib (100 ml each) or vice versa as indicated. Means and s.d. of three independent experiments are given. (c) L540 cells were incubated for 14 h in the presence of Ro32-7315 (1.5 mM, Ro), MDX-060 (50 ng/ml MDX-060 and 100 ng/ml GaH-IgG; MDX), bortezomib (4 ng/ml; bo) or combinations as indicated and stained with annexin V and 7-amino-actinomycin D. The plots show data from a representative evaluation.

13.31% (upper right gate). These data confirm that induction of Conflict of interest CD30 shedding by bortezomib substantially perturbs anti- CD30-targeted immunotherapy. H-WK is a company employee of Roche Diagnostics Pharma Many membrane proteins regulate their density and func- Research Penzberg, Germany. tionality through metalloproteinase-dependent cleavage of their ectodomains. Some of them, CD30, CD44, CD138 and Acknowledgements HER2 for instance, are potential target antigens due to their 27 selective overexpression in certain tumor entities. Another We thank Gisela Scho¨n for excellent technical assistance. This example is CD126. After shedding, the soluble ectodomain study was supported by grants from Deutsche Krebshilfe to HPH is still able to bind IL-6. The resulting soluble receptor–ligand and AE (106473) and from the DFG to EPvS (Str 530/5-1). AMV, complexes contribute to chronic inflammation by initiating IL-6 28 KSR, VLS, KW, AC performed research and analyzed data EPvS, signaling in CD26-negative immune cells (transsignaling). DAE, BB, VRS, H-WK, SR-J, AE and HPH designed the research Others, such as released ErbB ligands, L1, CD44 and MICA, and analyzed data, HPH wrote the paper. promote tumor proliferation and migration or worsen immune surveillance after cleavage.27 ADAM17 participates in most cleavage events and is overexpressed in many tumors suggesting an adverse role of this enzyme in tumor progression and References treatment. We showed that the cleavable antigens, CD30, CD138 and CD126, were reduced in their cell membrane 1 Ansell SM, Horwitz SM, Engert A, Khan KD, Lin T, Strair R et al. density after bortezomib treatment. Using CD30 as an example, Phase I/II study of an anti-CD30 monoclonal antibody (MDX-060) we clearly demonstrated that bortezomib impedes immuno- in Hodgkin’s lymphoma and anaplastic large-cell lymphoma. therapy targeting CD30 in HL cells. This effect should be J Clin Oncol 2007; 25: 2764–2769. 2 Bartlett NL, Younes A, Carabasi MH, Forero A, Rosenblatt JD, carefully considered in therapeutic approaches, particularly Leonard JP et al. A phase 1 multidose study of SGN-30 immuno- when released proteins are involved in the pathomechanism of therapy in patients with refractory or recurrent CD30+ hemato- the malignancy. logic malignancies. Blood 2008; 111: 1848–1854.

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