Probing the Specificity and Activity Profiles of the Proteasome

Mar 20, 2012 - *The Netherlands Cancer Institute, Division of Cell Biology II, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. Phone: +3120512197...
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Probing the Specificity and Activity Profiles of the Proteasome Inhibitors Bortezomib and Delanzomib Celia R. Berkers,† Yves Leestemaker,† Karianne G. Schuurman,† Bruce Ruggeri,‡ Susan Jones-Bolin,‡ Michael Williams,‡ and Huib Ovaa*,† †

Division of Cell Biology II, The Netherlands Cancer Institute, Amsterdam, The Netherlands Cephalon, Inc., Discovery Research, West Chester, Pennsylvania 19380, United States

‡

ABSTRACT: The ubiquitin proteasome system is an attractive pharmacological target for the treatment of cancer. The proteasome inhibitor bortezomib has been approved for the treatment of multiple myeloma and mantle cell lymphoma but is associated with substantial adverse effects and the occurrence of resistance, underscoring the continued need for novel proteasome inhibitors. In this study, bortezomib and the novel proteasome inhibitor delanzomib were compared for their ability to inhibit proteasome activity using both fluorogenic substrates and a recently developed fluorescent proteasome activity probe. Bortezomib and delanzomib were equipotent in inhibiting distinct subunits of the proteasome in a panel of cell lines in vitro. In a preclinical multiple myeloma model, both inhibitors inhibited the proteasome in normal tissues to a similar extent. Tumor proteasome activity was inhibited to a significantly higher extent by delanzomib (60%) compared to bortezomib (32%). In addition, delanzomib was able to overcome bortezomib resistance in vitro. The present findings demonstrate that proteasome activity probes can accurately monitor the effects of proteasome inhibitors on both normal and tumor tissues in preclinical models and can be used as a diagnostic approach to predict resistance against treatment with proteasome inhibitors. Furthermore, the data presented here provide rationale for further clinical development of delanzomib. KEYWORDS: activity profiling, bortezomib, delanzomib, proteasome, proteasome inhibitor

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INTRODUCTION The ubiquitin proteasome system is responsible for the degradation of proteins involved in a wide variety of cellular processes, including cell proliferation and survival, cell-cycle control, transcriptional regulation, and cellular stress responses, as well as for the destruction of abundant and misfolded proteins.1,2 The catalytic activity of the 26S proteasome resides within its 20S core and is mediated by three catalytically active constitutive β-subunits termed β1, β2, and β5. These have three different catalytic activities, caspaselike, tryptic, and chymotryptic activity, respectively. In lymphoid tissues or after induction with interferon-γ, these subunits can be replaced by their immunoproteasomal counterparts, termed β1i, β2i, and β5i, to form the immunoproteasome, while mixed-type hybrid proteasomes have also been reported.3−6 The increased sensitivity of malignant cells to proteasome inhibitors has resulted in proteasome inhibition emerging as a novel strategy for cancer treatment.2 In addition to eliciting direct apoptotic effects, proteasome inhibition may also enhance sensitivity and overcome drug resistance to standard chemo- and radiation therapy.7 Bortezomib8 (Figure 1), the active ingredient of velcade, is the first proteasome inhibitor to be approved as a single agent for the treatment of relapsed or refractory multiple myeloma (MM)9−11 and mantle cell lymphoma12 and, in combination with PEGylated doxorubicin, for the treatment of relapsed MM.13 Bortezomib treatment is associated with manageable but substantial © 2012 American Chemical Society

Figure 1. Structures of bortezomib, delanzomib, and proteasome activity probe 1.

side effects, for example, thrombocytopenia and peripheral neuropathy,10 and the occurrence of both primary and acquired drug Received: Revised: Accepted: Published: 1126

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with one volume of glass beads (30 nM (Figure 2c), although in cell lysates bortezomib and delanzomib do not inhibit the β2 subunits but rather enhance the activity of this site at elevated concentrations (Figure 2a). The exact reason for the reduced labeling of the β2 subunits in intact cells is not clear at this point, but it is likely resulting from a cellular response to prolonged incubation with high concentrations of these toxic compounds and not from direct inhibition of the tryptic activity. Taken together, these results suggest that, in live cells, bortezomib and delanzomib are equipotent in inhibiting β1 and β5 subunits, confirming results in cell lysates and demonstrating that probe 1 can be used to reliably assay proteasome (subunit) inhibition in live cells. Validating ARP-1 Cells as a Model System To Compare Bortezomib and Delanzomib Treatment in Vivo. In a previous report, the in vivo efficacies of delanzomib and bortezomib were compared in a RPMI8226 subcutaneous

xenograft model of human MM in SCID mice and in an ARP-1 systemic model of human MM.21 In these models, differences were found between bortezomib and delanzomib in the extent and duration of tumor proteasome inhibition and antitumor efficacy. To assess whether the use of specific MM cell lines that respond differentially to delanzomib or bortezomib treatment were responsible for these observations, the effects of bortezomib and delanzomib were further studied in ARP-1 cells using probe 1. In accord with results from the other cell lines tested, bortezomib and delanzomib showed near-identical inhibition profiles in ARP-1 cells (Figure 3a). After a 2 h incubation period, both compounds dose-dependently inhibited β1 and β5 subunits with IC50 values between 3 and 10 nM, while β2 subunits remained relatively unaffected. After 24 h, also the labeling of β2 subunits was largely abolished at higher concentrations of both compounds. To investigate whether ARP-1 MM cells show differences in proteasome activity recovery after either bortezomib or delanzomib treatment, ARP-1 cells were incubated with 10 or 100 nM bortezomib or delanzomib, washed, and allowed to recover for the indicated time periods before being analyzed with probe 1 (Figure 3b). Proteasome activity recovered slowly but comparably after withdrawal of either bortezomib or delanzomib from ARP-1 cells. After treatment with 10 nM of either inhibitor, recovery of activity was hardly observed up to four hours after withdrawal of the inhibitor, but activity returned to baseline values within 20 h. After treatment with 100 nM inhibitor, immediate recovery of β5(i)/1 subunit activity was found, but activity of none of the subunits completely returned to baseline levels within the 20 h recovery period (Figure 3b). Together, these data demonstrate that bortezomib and delanzomib induce similar responses in ARP-1 cells. Delanzomib Induces an Improved Response in Tumors Compared to Bortezomib. Next, proteasome probe 1 was used to profile proteasome inhibition in a preclinical model of human MM using an ex vivo approach24 to establish whether bortezomib and delanzomib show differences in proteasome inhibition in vivo. NOD/scid mice bearing subcutaneous ARP-1 tumor xenografts (n = 5/group) were administered with the maximum tolerated dose (MTD) of bortezomib (1.2 mg/kg intravenous), delanzomib (4 mg/kg intravenous), or Solutol/ PBS vehicle. At different time points, proteasome activity in various healthy tissues and tumor tissue was assayed (Figure 4a). In all tissues, vehicle treatment had no effect on proteasomal subunit activity over a 24 h period (data not shown). Residual proteasome subunit activities in delanzomib- and bortezomib-treated samples were calculated as a percentage of the activities in vehicle-treated samples and averaged (Figure 4b). Total proteasome activity was defined as the sum of all individual activities. In normal murine tissues, delanzomib and bortezomib showed comparable inhibition patterns, although subtle differences could be observed between the two inhibitors as well as between different tissue types. Bortezomib and delanzomib inhibited β1 and β5 subunits to a similar degree in all tissues at all time points, with residual activities ranging between 20 and 50% for the β5(i)/1 subunits and between 0 and 10% for the β1i subunit (Figure 4b). The β2 subunit was least affected by compound treatment. A small decrease in labeling was only observed in lung tissue, whereas labeling of the β2i subunit was decreased in lung, liver, and spleen. Little recovery of the activity of individual subunits from either inhibitor was seen in this model over a period of 24 h, although in individual mice, proteasome activity appeared to recover in spleen, liver, and heart after treatment with both compounds (data not 1129

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Figure 3. Bortezomib and delanzomib induce near-identical proteasome inhibition profiles and recovery patterns in ARP-1 cells. (a) In-gel fluorescence measurements showing representative proteasome activity profiles in ARP-1 cells after a 2 or 24 h incubation period with increasing concentrations of bortezomib or delanzomib (top panel). Quantification of gel images, with results plotted as percentage subunit activity compared to nontreated cells (bottom panel). (b) In-gel fluorescence measurements showing representative proteasome activity profiles in ARP-1 cells after a 1 h incubation with 10 or 100 nM of bortezomib or delanzomib, followed by the indicated recovery periods (top panel). Quantification of gel images, with results plotted as percentage subunit activity compared to nontreated cells (bottom panel). Results were obtained by incubating cells with inhibitor, followed by active proteasome labeling by probe 1 and SDS-PAGE analysis. bor: bortezomib; del: delanzomib.

to 60−100% in healthy tissues). These data underscore the strength of chemical profiling reagents and show that they can be used to accurately monitor both the activity of individual proteasome subunits and total proteasome activity in response to proteasome inhibitor treatment in preclinical models in vivo. In addition, these data indicate that delanzomib induces an improved response in the ARP-1 MM tumor model as compared to bortezomib at their respective MTDs. We cannot fully explain this remarkable difference. It could, at least in part, be due to the higher concentration of delanzomib that was used. Delanzomib exhibits a favorable cytotoxicity profile toward normal human endothelial cells, bone marrow progenitors, and bone marrowderived stromal cells and is tolerated at a higher dose in mouse models.21 Also differences in the pharmacodynamic properties (shape, polarity, and charge) of these compounds may contribute to the difference in response observed in tumor tissue. Taken together, our observations clearly suggest that significant improvements can be made to the current available proteasome inhibitors and provide rationale for further clinical development of delanzomib. Delanzomib Displays Activity against Cells That Are Resistant to Bortezomib. Finally, the ability of delanzomib to overcome resistance in the bortezomib resistant cell line THP1/ BTZ100 was studied.28 In addition, we set out to investigate whether probe 1 can be used to predict resistance of MM cells to bortezomib and/or delanzomib treatment. THP1/BTZ100 cells acquired bortezomib resistance by exposure to stepwise increasing

shown). Total proteasome activity was only partially inhibited in all normal tissues (Figure 4b). The largest effects on total activity were observed in the lungs, where 40% residual proteasome activity was measured. In the heart and liver 60−70% of total proteasome activity remained unaffected by delanzomib and bortezomib treatment, while in the spleen, total proteasome activity was hardly decreased compared to vehicle. No significant differences in total proteasome inhibition were observed between delanzomib and bortezomib in healthy murine tissue at all time points. In ARP-1 tumor xenografts, differences between the inhibition produced by delanzomib and bortezomib were more pronounced. In primary tumor tissues β5 and β1 subunits were inhibited to a significantly higher extent by delanzomib treatment compared to bortezomib treatment at all time points, with residual activities ranging between 10 and 20% after delanzomib treatment and between 60 and 80% after bortezomib treatment (Figure 4b). Delanzomib induced maximal inhibition of these subunits within 2 h, while maximal inhibition by bortezomib occurred only after 8−24 h. Additionally, treatment with either compound resulted in small decreases in β2 and β2i labeling. As a result, the residual total proteasome activity was significantly lower in delanzomib-treated tumors as compared to bortezomibtreated tumors at 2 and 24 h (Figure 4b). Delanzomib reduced total proteasome activity to 40−50% (as compared to 60−100% in healthy tissues). In contrast, the residual activity in bortezomib-treated tumor samples was 70−100% (as compared 1130

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Figure 4. Delanzomib induces an improved response in tumors compared to bortezomib in an ARP-1 MM tumor model. NOD/scid mice (n = 5/ group) bearing ARP-1 tumor xenografts received a single injection of the maximum tolerated dose of bortezomib or delanzomib or vehicle, and tissues were removed at t = 2, 8, or 24 h post dosing. Tissues were lysed, followed by active proteasome labeling by probe 1 and SDS-PAGE analysis. (a) In-gel fluorescence measurements showing representative proteasome activity profiles in mouse heart, lung, liver, spleen, and tumor tissues after bortezomib or delanzomib treatment for the indicated time periods. (b) Quantification of proteasome activities profiles in mouse heart, lung, liver, spleen, and tumor tissues after bortezomib or delanzomib treatment for the indicated time periods. The proteasome subunit activities in treated samples are plotted as a percentage of the subunit activity in vehicle-treated mice, and total proteasome activity is defined as the sum of all individual activities. Error bars represent the SEM. Statistical analysis was performed using GraphPad Prism software. *p < 0.05; **p < 0.01; ***p < 0.001.

concentrations of bortezomib were required to inhibit the chymotryptic activity in THP1/BTZ100 cells as compared to THP1/ WT cells (IC50 values of 83 and 10 nM, respectively), whereas delanzomib inhibited the chymotryptic activity in THP1/BTZ100 cells with equal potency compared to the WT cell line (IC50 values of 18 and 14 nM, respectively) (Figure 5a,b). To investigate whether probe 1 can predict the sensitivity of proteasomal subunits to specific proteasome inhibitors, THP1/ WT and THP1/BTZ100 cells were pulsed with increasing concentrations of bortezomib and delanzomib, followed by probe labeling and analysis. In line with results from other cell lines tested, bortezomib and delanzomib showed near identical inhibition profiles in THP1/WT cells (Figure 5c, left panels). After a 3 h incubation period, both compounds dose-dependently inhibited β1 and β5 subunits with IC50 values between 3 and 10 nM. Labeling of β2 subunits by probe 1 remained relatively unaffected at lower concentrations but was hampered at higher concentrations of bortezomib and delanzomib, especially after 24 h. In contrast, delanzomib and bortezomib

concentrations of bortezomib (2.5−100 nM). Study of the molecular mechanism of bortezomib resistance in these cells revealed an Ala49Thr mutation in the β5 subunit protein, resulting from a G322A point mutation in the PSMB5 gene.28 This point mutation has been described in multiple bortezomib-resistant cell lines of hematopoietic lineage, including KMS-11, OPM-2 (both MM), and Jurkat cells (human lymphoblastic T cells).29,30 THP1/BTZ100 cells were shown to be cross-resistant to other proteasome inhibitors that target the β5 subunit.28 First, we compared the proteasome inhibitory potential of delanzomib and bortezomib in cell lysates of THP1/BTZ100 and THP1 wild type (THP1/WT) cells using fluorogenic substrates for the different proteasomal activities as described above (Figure 5a). Differences between bortezomib (black lines) and delanzomib (gray lines) were observed in the degree of inhibition of the chymotryptic activity, but not of the tryptic and caspase-like activities of the proteasome (Figure 5a). Bortezomib and delanzomib showed near-identical inhibition profiles in THP1/WT cells (dotted lines), in accordance with data obtained from other cell types (Figure 2a). Higher 1131

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Figure 5. Delanzomib can overcome bortezomib resistance in vitro. (a) Proteasome inhibitory profiles in THP1/WT and THP1/BTZ100 cell lysates after the addition of increasing concentrations of bortezomib (black lines) or delanzomib (gray lines), obtained using fluorogenic substrates for the different proteasomal activities. Results are plotted as percentage activity compared to nontreated lysates. Solid lines: THP1/BTZ100 cells; dotted lines: THP1/WT cells. (b) IC50 values of delanzomib (gray bars) and bortezomib (black bars) in THP1/WT and THP1/BTZ100 cell lysates for inhibition of the chymotryptic activity. (c) In-gel fluorescence measurements showing representative proteasome activity profiles in THP1/WT and THP1/BTZ100 cells after a 3 or 24 h incubation with increasing concentrations of bortezomib and delanzomib. Results were obtained by incubating cells with inhibitor, followed by active proteasome labeling by probe 1 and SDS-PAGE analysis. (d) Viability of THP1/WT and THP1/BTZ100 cells after incubation with increasing concentrations of bortezomib (black lines) or delanzomib (gray lines) for 48 h. Results are plotted as percentage viability compared to DMSO treated control cells in each cohort. Solid lines: THP1/BTZ100 cells; dotted lines: THP1/WT cells. (e) EC50 values of delanzomib (gray bars) and bortezomib (black bars) in THP1/WT and THP1/BTZ100 cells.

of delanzomib, to inhibit the β5 subunit in THP1/BTZ100 cells (Figure 5c). From the data obtained in THP1/BTZ100 and THP1/ WT cells we conclude that THP1/BTZ100 cells are resistant to bortezomib due to a mutation in the β5 protein, which prevents bortezomib binding. Remarkably, assays using probe 1 show that THP1/BTZ100 cells are still equally sensitive to proteasome inhibition by delanzomib as compared to THP1/WT cells and that this sensitivity correlates with a decrease in cell viability after delanzomib treatment. Taken together, our data suggest that delanzomib can overcome acquired bortezomib resistance, originating from a mutation in the β5 subunit protein, in vitro. In addition, profiling experiments using probe 1 demonstrate that resistance against treatment with proteasome inhibitors can be predicted with the use of activity reporter reagents.

showed different inhibition profiles in THP1/BTZ100 cells (Figure 5c, right panels). Delanzomib inhibited the β5 subunits in THP1/BTZ100 cells with IC50 values between 3 and 10 nM, while higher concentrations of bortezomib were required to reach a similar extent of β5 inhibition. These labeling patterns are in accordance with results found in cell lysates using fluorogenic substrates and suggest that probe 1 can be used to predict the sensitivity of the different proteasomal subunits to bortezomib and delanzomib. Finally, we investigated whether the proteasome inhibitory profiles determined using probe 1 in THP1/BTZ100 and THP1/ WT cells correlated to the viability of these cells in response to proteasome inhibitor treatment. Cell viability was measured using a cell titer blue assay (Figure 5d). EC50 values, defined as the concentration of inhibitor causing 50% cell death after 48 h, were determined using GraphPad Prism software (Figure 5e). THP1/WT cells responded similarly to bortezomib and delanzomib treatment, with EC50 values of 23 and 30 nM, respectively (Figure 5d). In THP1/BTZ100 cells on the other hand, EC50 values for bortezomib were higher (>200 nM) as compared to delanzomib (31 nM) (Figures 5d,e). These data are in accord with the proteasome inhibitory profiles of bortezomib and delanzomib, which demonstrate the inability of bortezomib, but not

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DISCUSSION Proteasome inhibition is a validated strategy for the treatment of cancer, with a precedence set by bortezomib, a boronic acidbased proteasome inhibitor. Bortezomib is currently the only proteasome inhibitor approved for human use. Bortezomib treatment is, however, associated with adverse effects,10 including thrombocytopenia31 and peripheral neuropathy,32 and with both primary and secondary resistance.14 This has led to the search for 1132

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reported improved antitumor efficacy may result in further benefit in a clinical setting. The data obtained in mice ex vivo show that probe 1 can be successfully used for detailed studies of the effects of proteasome inhibitors on both normal and tumor tissues in preclinical models. It has been shown that measuring only the chymotryptic activity, as is routinely done, may not accurately reflect total proteasome activity and that both the caspase-like and tryptic activities contribute considerably to overall proteasome activity.33 The use of chemical probes to assay proteasome activity has the advantage that the activity of all constitutive and immunoproteasome subunits can be profiled in a single measurement. In addition, probe 1 has equal affinity for all proteasome active sites and can therefore be used to reliably measure total proteasome activity. Finally, our data show that delanzomib displays activity against a bortezomib resistant cell line in vitro. The cell line THP1/BTZ100 is resistant to concentrations of bortezomib up to 100 nM. The study of the molecular mechanism of bortezomib resistance in these cells revealed an Ala49Thr mutation in the β5 subunit protein.28 Ala49 is a highly conserved residue among many prokaryotic and mammalian species and very likely involved in tight interactions with the pyrazine-2-carboxy-phenylalanyl backbone of bortezomib, as can be reasoned from the bortezomib liganded yeast 20S proteasome structure.34 Therefore, mutation of this residue to threonine is likely to have a profound effect on binding of bortezomib and of other proteasome inhibitors that target the β5 subunit. Our data show that delanzomib binding was not influenced by the Ala49Thr mutation. Bortezomib and delanzomib are structurally similar but differ in their P2 and P3 residues, suggesting that even small changes to the backbone structure of bortezomib can be sufficient to overcome bortezomib resistance. The data presented here suggest that the development of secondgeneration proteasome inhibitors will likely result in the generation of inhibitors that can overcome resistance and may ultimately contribute to better patient outcome, while the described activity profiling methods can be used to predict sensitivity to individual proteasome inhibitors. This suggests that chemical proteasome activity reporters may enable matching of tumor proteasome to a complementary inhibitor to contribute to the future selection of the most suitable inhibitors for clinical treatment.

new proteasome inhibitors with an improved cytotoxicity profile and the potential to overcome bortezomib resistance. Delanzomib, a novel proteasome inhibitor that is structurally similar to bortezomib, is one of several second-generation inhibitors in clinical trials.20,21 In the present study the proteasome inhibitory profiles of bortezomib and delanzomib were compared in cell lysates, whole cells, and ex vivo in a mouse model of human MM, using both fluorogenic substrates and a chemical proteasome activity reporter that enables profiling of all proteasome activities in a single measurement in a wide range of tissue types.24 In addition, we investigated the proteasome inhibitory profiles of delanzomib and bortezomib in cells with acquired resistance to bortezomib and assessed whether our chemical proteasome probe could predict resistance against either inhibitor. The present study shows that chemical proteasome probe 1 can be used to profile the effects of proteasome inhibitors both in vitro and in preclinical in vivo models of human MM. Delanzomib and bortezomib showed comparable inhibition patterns in a panel of human tumor cell lines. Both compounds were equipotent in inhibiting the β1 (caspase-like activity) and β5 (chymotryptic activity) subunits and had little effect on the β2 subunit activity (tryptic activity), in agreement with previous reports.21,23 IC50 values in cell lysates were 40−75 nM and 10−25 nM for the inhibition of the caspase-like and chymotryptic activities, respectively. IC50 values for both the β1 and β5 subunits ranged between 3 and 10 nM in intact cells. These values correspond well to the antiproliferative activity of both compounds on representative human solid and hematological tumor cell lines with IC50 values ranging between 1 and 35 nM.21 Data obtained using either fluorogenic substrates in cell lysates or using probe 1 in intact cells were similar, validating the use of probe 1 as a reliable method to assay proteasome activity. Proteasome activity recovered similarly after withdrawal of either inhibitor from cells, with recovery depending on the dosage used. Treatment with a low dose resulted in little recovery up to four hours after withdrawal of the inhibitor, but complete recovery within 20 h. Treatment with a high dose resulted in immediate recovery of the β5(i)/1 subunit activities, but proteasome activity did not return to baseline levels within the 20 h recovery period. In contrast to the finding that delanzomib and bortezomib have near-identical effects on tumor cell proteasome activity in vitro, marked differences between delanzomib and bortezomib were found in vivo in an ARP-1 MM xenograft model. In tumor xenografts, delanzomib showed a significantly higher degree of inhibition of β1 and β5 subunits at the MTD as compared to bortezomib (80−90% for delanzomib as compared to 40−60% for bortezomib), resulting in a significantly higher degree of overall proteasome inhibition (60% for delanzomib as compared to 32% for bortezomib), while the response of normal, healthy murine tissues to treatment with the maximum tolerated dose of bortezomib or delanzomib was identical. These results are in agreement with Piva et al., who have shown that, in a RPMI8226 MM tumor xenograft model, delanzomib induced a similar degree of proteasome inhibition in normal mouse peripheral tissues compared to bortezomib, but a greater and moresustained inhibition of tumor proteasome activity.21 Consistent with these findings, delanzomib treatment was shown to have a greater in vivo antitumor efficacy, and delanzomib administration resulted in a dose-related induction of complete tumor regression, whereas bortezomib treatment only delayed tumor growth.21 While the antitumor efficacy of bortezomib results in effective antitumor therapy, the improved response of delanzomib in tumors that can be correlated with its previously

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AUTHOR INFORMATION

Corresponding Author

*The Netherlands Cancer Institute, Division of Cell Biology II, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. Phone: +31205121979. Fax: +31205122029. E-mail: h.ovaa@ nki.nl. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Dr. G. Jansen for providing the THP1/ BTZ100 cell line. This work was funded by the Dutch Cancer Society (Grant NKI 2005-3368).

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REFERENCES

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