The Proteasome As a Target for Cancer Therapy

6316 Vol. 9, 6316–6325, December 15, 2003 Clinical Cancer Research

Review The Proteasome as a Target for Cancer Therapy

Peter M. Voorhees,1,2 E. Claire Dees,1,2 and clinical data on proteasome inhibitors alone, and in Bert O’Neil,1,2 and Robert Z. Orlowski1,2 combination with conventional chemotherapeutics. 1The Lineberger Comprehensive Cancer Center and 2Department of Medicine, Division of Hematology/Oncology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina Introduction The Ubiquitin-Proteasome Pathway (UPP). Precise control of protein turnover is essential to cellular survival. In Abstract eukaryotes, the majority of protein degradation occurs through The proteasome is a multicatalytic proteinase complex the UPP, which consists of the ubiquitin-conjugating system and responsible for the degradation of most intracellular pro- the proteasome. The proteolytic activities of the proteasome are teins, including proteins crucial to cell cycle regulation and located within a 20S multisubunit structure consisting of four programmed cell death, or apoptosis. In preclinical cancer stacked rings arranged around an inner channel. This macro- models, proteasome inhibitors induce apoptosis, have in vivo molecule by itself is involved in turnover of some proteins, and antitumor efficacy, and sensitize malignant cells and tumors also forms the active proteolytic core of up to three other to the proapoptotic effects of conventional chemotherapeu- complexes (Fig. 1). When capped by the 19S regulatory com- tics and radiation therapy. Interestingly, transformed cells plex at each end, the 20S complex forms the core of the 26S display greater susceptibility to proteasome inhibition than proteasome, the major extralysosomal mediator of protein deg- nonmalignant cells. Therefore, proteasome inhibition holds radation. The ubiquitin-conjugating system targets proteins for promise as a novel approach to the treatment of cancer. degradation by the covalent attachment of multiple ubiquitin Inhibitors of the proteasome impact on cells in part through (Ub) chains (Fig. 2). Ubiquitination is mediated by the sequen- ␬ down-regulation of nuclear factor B, but also through tial action of an E1 Ub-activating enzyme, an E2 Ub-conjugat- modulation of cell cycle proteins and other pro- and anti- ing enzyme, and an E3 Ub-ligase. Once Ub-tagged, proteins apoptotic pathways. Bortezomib (VELCADE; formerly PS- bind to subunits in the 19S regulatory cap of the proteasome, 341), the first such inhibitor to undergo clinical testing, has where they are deubiquitinated and unfolded in an energy- demonstrated impressive antitumor activity and managea- dependent manner. These are then fed into the catalytic inner ble toxicities in Phase I and II trials both as a single agent, chamber of the 20S complex, which generates peptides of 3–22 and in combination with other drugs. It has been approved amino acids in size (1–3). recently by the Food and Drug Administration for therapy The Proteasome and Apoptosis. Programmed cell of patients with multiple myeloma who have received at least death, or apoptosis, is necessary to the survival of all multicel- two prior regimens and progressed on the last of these. lular organisms through roles in normal growth and develop- Ongoing preclinical evaluations of the mechanisms that un- ment. The discovery that a family of proteases, the caspases, derlie the antitumor effects of proteasome inhibitors, and mediated the execution of apoptosis generated interest in a clinical trials in a variety of tumor types, will allow addi- possible role of the proteasome in this process. Studies were tional refinement of the role these agents will play in cancer facilitated by the advent of pharmacological inhibitors of the therapy. Below we discuss the rationale behind targeting the proteasome, including synthetic peptidyl aldehydes and the bac- proteasome for cancer therapy, and review the preclinical terial compound lactacystin. Perhaps surprisingly, incubation of a human monoblast cell line with lactacystin led to apoptosis, demonstrating that proteasome inhibitors possess antitumor activity (4). Fujita et al. (5) provided the first evidence that Received 4/1/03; revised 7/29/03; accepted 8/26/03. proteasome inhibitors sensitize tumors to conventional therapies Grant support: General Clinical Research Centers program of the by showing that treatment of U937 cells with lactacystin alone division of Research Resources, NIH (RR00046), the National Center did not induce apoptosis, but potentiated tumor necrosis factor- for Research Resources, NIH (1K23 RR16536–01; E. C. D.), the mediated cell death. Shortly thereafter, the first in vivo demon- Lineberger Comprehensive Cancer Center SPORE for Breast Cancer (E. C. D. and R. Z. O.), the Department of Defense Breast Cancer Re- stration of the antitumor activity of proteasome inhibitors was search Program (BC991049; R. Z. O.), and the Leukemia and Lym- reported, in which a peptidyl aldehyde induced tumor growth phoma Society (R6206–02; R. Z. O.). The Phase I trial of the combi- delay in a murine xenograft model of Burkitt’s lymphoma (6). nation of pegylated liposomal doxorubicin and bortezomib is being An additional advance was made in the synthesis of the protea- supported in part by Millennium Pharmaceuticals, Inc. some inhibitor PS-341, since renamed bortezomib, a dipeptide The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked boronic acid analogue that possesses pharmacological charac- advertisement in accordance with 18 U.S.C. Section 1734 solely to teristics better suited for clinical testing in patients (7). Bort- indicate this fact. ezomib demonstrated broad and unique antitumor activity in a Requests for reprints: Robert Z. Orlowski, The University of North National Cancer Institute tumor cell line screen, as well as in Carolina at Chapel Hill, 22–003 Lineberger Comprehensive Cancer Center, CB # 7295/Mason Farm Road, Chapel Hill, NC 27599-7295. several murine xenograft models. These findings have paved the Phone: (919) 966-9762; Fax: (919) 966-8212; E-mail: R_Orlowski@ way for many exciting preclinical and clinical studies of the med.unc.edu. proteasome as a target for cancer therapy. Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2003 American Association for Cancer Research. Clinical Cancer Research 6317

Fig. 1 Forms of the proteasome found in eukaryotes. The multicatalytic proteinase complex, or 20S proteasome, is composed of four stacked rings oriented around a central channel, or pore. Each of the two outer rings contains seven nonidentical ␣-subunits, which are thought to be structural, whereas the two inner rings contain seven nonidentical ␤-subunits. Within the latter two rings are proteins that carry out the five characterized proteolytic activities of the proteasome (60). By itself, the free 20S proteasome can degrade some short-lived regulatory proteins, unfolded proteins, and damaged or mis- folded proteins (61). When combined with either 11S or 19S regulatory complexes, also called the PA28 and PA700 activators, respec- tively, the proteolytic role of the proteasome is modified. In its 26S incarnation the proteasome plays a critical role in the ubiquitin-proteasome pathway, as well as in ubiquitin-independent proteolysis.

Differential Susceptibility of Transformed Cells viding cells appear to be more susceptible to proteasome inhi- The proteasome is responsible for Ͼ80% of intracellular bition than quiescent cells, other elements must be at play. protein degradation (1–3), and its inhibition would therefore One factor that may contribute is the cyclin-dependent Kip1 seem incompatible with life. Remarkably, several studies have kinase inhibitor p27 .Anet al. (15) found that the induction demonstrated a selective susceptibility of transformed cells to of apoptosis in SV40-transformed fibroblasts correlated with the proteasome inhibition. For example, fibroblasts transformed accumulation of p27. In contrast, the parental normal fibroblast with ras and c-myc, and lymphoblasts transformed with c-myc, cell line was resistant to proteasome inhibition and did not were up to 40-fold more susceptible to proteasome inhibitor- accumulate significant levels of p27. induced apoptosis than primary fibroblasts or immortalized, Strong evidence exists supporting a role for the deregula- nontransformed human lymphoblasts (6). Patient-derived tion of various UPP functions in malignant transformation. For chronic lymphocytic leukemia (CLL) cells were ϳ10-times example, CLL cells have 3-fold higher levels of chymotrypsin- more sensitive to lactacystin than normal lymphocytes (8). like proteasome activity than normal lymphocytes (16). In- CD34ϩ hematopoietic progenitor cells from chronic myeloid creased proteasomal degradation of p27 leading to low levels of leukemia patients were roughly three times more susceptible to this cyclin-dependent kinase inhibitor confers a worse prognosis proteasome inhibitor-mediated apoptosis than cells from normal on patients with colon cancer and mantle cell lymphoma (17, volunteers (9). Malignant plasma cells from multiple myeloma 18). Similarly, decreased levels of the proapoptotic protein Bax patients were 20–40-times more sensitive to bortezomib-medi- are associated with increased proteasome activity and higher ated apoptosis than blood mononuclear cells (10). Finally, leu- Gleason scores in prostate cancer (19). Recent global gene kemic stem cells from acute myeloid leukemia patients were expression profiling of patient-derived myeloma cells has pro- more susceptible to the peptidyl aldehyde MG132 than normal vided some additional interesting insights (20). Hierarchical hematopoietic stem cells (11). clustering identified four distinct patient subgroups, and one of Whereas the molecular basis of this differential suscepti- several genes differentially expressed in the poor prognostic bility remains elusive, and may differ from one model system to subgroup was POH1, a 26S proteasome-associated protein. another, several interesting possibilities exist. Drexler (12) POH1 overexpression confers resistance to several chemother- showed that HL60 leukemia cells underwent proteasome inhib- apeutics, including doxorubicin, and to UV radiation (21). An- itor-mediated apoptosis when they were proliferating, but not other interesting finding was that, compared with normal plasma when they were induced to differentiate into macrophages. cells, the CDC34 gene transcript was strongly up-regulated in Similarly, Lopes et al. (13) found that actively proliferating myeloma samples. CDC34 is part of an E2/E3 complex respon- Rat-1 fibroblasts underwent apoptosis when exposed to protea- sible for ubiquitination of a number of important substrates, some inhibitors, but not when they were quiescent. However, including p27. More recently, pharmacogenomic analysis of the same investigators noted that pheochromocytoma cells un- patient samples from a Phase II trial of bortezomib in multiple derwent apoptosis regardless of whether they were proliferating myeloma suggested that transcription of cullin4A, which func- or had undergone differentiation. Similarly, patient-derived tions as part of an E3 Ub ligase complex, may be a predictor of

CLL cells, which are predominantly in the G0 phase of the cell response to therapy (22). While speculative, the above evidence cycle, can undergo apoptosis (14). Thus, whereas actively di- suggests that deregulated UPP function may play a direct role in

arrest, and promotion of apoptosis via the inhibition of Bcl-2 by Bax. Proteasome inhibitors also down-regulate signaling through the p44/42 mitogen-activated protein kinase (MAPK), a pathway crucial to the promotion of tumorigenesis in a number of model systems. A detailed description of all of the potential targets of proteasome inhibitors is beyond the scope of this review; however, below we concentrate on some of the better- studied mechanisms through which proteasome inhibitors achieve their antitumor activity. In addition to these, other pathways that may also be of importance, but for which data are still somewhat preliminary, are included in Table 1. NF␬B. The NF␬B transcription factor family is an important modulator of immune and inflammatory responses. More recent evidence has accumulated supporting their role in tumorigenesis through induction of cell proliferation, suppression of apoptosis, enhancement of tumor cell invasiveness and metastasis, and induction of angiogenesis (reviewed in Ref. 23). The UPP plays a critical role in regulation of the NF␬B family of transcription factors in that NF␬B1 and -␬B2 are processed into their mature forms through an UPP-dependent pathway. In addition, I␬B binds NF␬B in the cytoplasm, thereby blocking its nuclear translocation. A variety of stimuli lead to the phosphorylation of critical serine residues on I␬B, targeting it for ubiquitination and degradation by the proteasome, which then allows NF␬B to enter the nucleus and mediate transcription (23). Ex- pression of a “super-repressor” I␬B in which these serines are mutated to alanines, and cannot therefore be phosphorylated, Fig. 2 The ubiquitin-proteasome protein degradatory pathway. Pro- ␬ teins destined for degradation through the ubiquitin-proteasome path- prevents proteasome-mediated degradation of I B and seques- way are first labeled by the ubiquitin conjugation pathway. An E1 ters NF␬B in the cytoplasm, thereby mimicking the impact of ubiquitin-activating enzyme activates ubiquitin via the generation of a proteasome inhibitors on this pathway. thiol ester bond between the COOH-terminal glycine on ubiquitin and Given the role of the proteasome in I␬B degradation, an internal E1 cysteine residue. One of several known E2 ubiquitin- proteasome inhibitors might induce apoptosis by stabilizing conjugating enzymes transfers the activated ubiquitin moiety to an E3 ␬ ␬ family member, of which there are many. The E3 ubiquitin ligase cytoplasmic I B and blocking NF B nuclear translocation. In subsequently mediates the covalent attachment of multiple ubiquitin support of this, infection of a myeloma cell line with the moieties to the protein destined for degradation by the 26S proteasome. super-repressor I␬B led to increased apoptosis (24). Similarly, This target protein is recognized through its ubiquitin tag by the 19S cap treatment of myeloma cell lines and patient samples with bort- structure of the 26S proteasome, isopeptidases cleave off the ubiquitin moieties, which are recycled, and the target protein enters the protea- ezomib resulted in growth inhibition, even in drug-resistant cell some. Inside the proteasome the target is cleaved into oligopeptides, lines. Growth inhibition and apoptosis correlated with I␬B sta- which exit the proteasome, and may be subject to additional degradation bilization and decreased NF␬B activity. However, other poten- into amino acids. Both the 26S and 20S proteasomes can also function tial targets of proteasome inhibition were impacted upon as well, in ubiquitin-independent proteolysis (data not shown). including p21Cip1 and p27Kip1 (10). In a murine xenograft model of head and neck squamous cell carcinoma, expression of an I␬B super-repressor, or treatment with bortezomib, led to inhibition of tumor growth and angiogenesis, correlated with inhi- tumorigenesis, which may in part explain the differential sus- bition of NF␬B activity, and down-regulation of growth- regu- ceptibility of transformed cells. lated oncogene ␣ and vascular endothelial growth factor, NF␬B- dependent proangiogenic cytokines (25). It is also interesting to Molecular Targets of Proteasome Inhibitors note that, given the role of NF␬B in inflammatory responses, Given the myriad of proteasome substrates, it is not sur- proteasome inhibitors hold promise as anti-inflammatory agents prising that inhibition of its function affects many pathways. (reviewed in Ref. 26). Several important proteins that are regulated by the proteasome Not all studies, however, have confirmed a role for NF␬B include the inhibitor of nuclear factor ␬B (NF␬B; I␬B), the attenuation as a mechanism of proteasome inhibitor-induced cell tumor suppressor p53, the cyclin-dependent kinase inhibitors death. Treatment with lactacystin sensitized B-cell lymphoma p21 and p27, and the proapoptotic protein Bax (Table 1). Ac- cells to radiation-mediated apoptosis, but inhibition of NF␬B cumulation of these substrates on proteasome inhibition leads to activity was not demonstrated (27). In another study, treatment decreased NF␬B-dependent transcription of genes crucial to the of CLL cells with lactacystin did not lead to cytoplasmic accu- promotion of tumorigenesis, increased p53-mediated transcrip- mulation of I␬B or nuclear loss of NF␬B (16). Given the broad tion of genes important to apoptosis and negative regulation of number of substrates of the proteasome, it seems unlikely that the cell cycle, p21- and p27-mediated induction of cell cycle inhibition of NF␬B is the sole mechanism of antitumor activity.

Table 1 Molecular targets of proteasome inhibitors Target Sequelae of proteasome inhibition Contribution to antitumor effect NF␬Ba Stabilization of I␬B, which inhibits nuclear Decreases NF␬B-dependent transcription of genes translocation of NF␬B important in tumor cell survival, proliferation, invasion and metastasis, and angiogenesis p53 Accumulation of p53 protein by inhibition of Increases p53-dependent transcription of cell cycle proteasome-mediated p53 degradation inhibitors (p21) and proapoptotic factors (Bax)

p21 and p27 Accumulation of both p21 and p27, and increased Induces G1/S cell cycle arrest and apoptosis transcription of p21 through accumulation of p53

Bax Accumulation of Bax by inhibition of proteasome- Increases Bax interaction with Bcl-2 and Bcl-xL, mediated Bax degradation and through increased promoting release of mitochondrial cytochrome c p53-mediated transcription and apoptosis p44/42 MAPK Transcriptional activation of the MKP-1 phosphatase, Down regulates p44/42-dependent cell proliferation leading to p44/42 dephosphorylation and and survival signals, and possibly angiogenesis inactivation tBid (62) Accumulation through decreased proteasomal tBid induces conformational changes in Bak, degradation promoting mitochondrial release of cytochrome c Smac/Diablo (63) Accumulation through decreased proteasomal Smac and Diablo bind to and inhibit members of the degradation XIAP protein family a NF␬B, nuclear factor ␬B; I␬B, inhibitor of nuclear factor ␬B; MAPK, mitogen-activated protein kinase; Diablo, direct IAP binding protein with low pI; Smac, Second mitochondria-derived activator of caspase; XIAP, X-chromosome-linked inhibitor of apoptosis.

p44/42 MAPK. Like NF␬B, the p44/42 MAPK pathway negative p53 attenuated proteasome inhibitor-induced apopto- plays important roles in cell proliferation, suppression of apo- sis. Comparable studies in other model systems have been ptosis, and angiogenesis through activation of a signaling cas- confirmatory of a role for p53 accumulation. It is important to cade that ultimately phosphorylates p44 and p42 (reviewed in note, however, that whereas p53 is an attractive and logical Refs. 28–30). Through additional phosphorylation events, these candidate contributing to proteasome inhibitor-mediated apo- kinases modulate the activity of several important signaling ptosis, many studies have shown that proteasome inhibition molecules and transcription factors, including c-Myc and NF␬B activates apoptosis regardless of the p53 status of a cell (e.g., itself. The p44/42 pathway is important in a variety of tumors, Refs. 12, 35). This is likely a testament to the multiple molecular including both hematological malignancies such as leukemias targets of these agents. In addition, because many p53 targets and solid tumors such as breast cancer (reviewed in Refs. 31, such as p21 and Bax are themselves degraded through the UPP, 32). In the latter, where p44/42 has been particularly well they would accumulate in response to proteasome inhibition studied, signaling through the receptor tyrosine kinase, Her2, is even in the absence of p53. mediated in part through p44/42 MAPK. Overexpression of Cyclin-Dependent Kinase Inhibitors p21 and p27. p21 Her2 portends a worse prognosis in breast cancer patients, and and p27, members of the Cip/Kip family of cyclin-dependent increased p44/42 activity may be associated with poorer patient kinase inhibitors, stop cell cycle progression at the G1-S phase outcomes independent of Her2 expression. Proteasome inhibi- by binding to, and inactivating, a number of cyclin/cyclin- tors act in part through down-regulation of the p44/42 MAPK dependent kinase complexes. The UPP plays a crucial role in the signaling cascade. Treatment of myeloma cells with bortezomib turnover of both proteins. Therefore, proteasome inhibitors in- decreased activation of p44/42, which correlated with inhibition crease levels of p21 and p27 by decreasing proteasomal degra- of myeloma growth (10). More extensive studies of the impact dation of both proteins, and by increasing p53-mediated tran- of proteasome inhibitors on p44/42 have been performed in scription of p21. Evidence that p21 plays an important role in breast cancer model systems. Treatment of breast cancer cell the antitumor effect of proteasome inhibitors is largely circum- lines with proteasome inhibitors led to a loss of dually phos- stantial. Many investigators have shown that p21 accumulates phorylated, active p44/42 MAPK (33). Dephosphorylation of on proteasome inhibition, but others have found that p21 is p44/42 correlated with transcriptional induction of the MAPK dispensable to inhibitor-induced cell death (36). Perhaps more phosphatases 1 and 2. Studies with pharmacological agents and interesting is the role that p27 may play. As described above, dominant-negative mutant constructs inhibiting p44/42 signal- down-regulation of p27 activity plays an important role in the ing enhanced apoptosis, whereas activation of p44/42 signifi- oncogenesis of a number of tumor types, and low levels of p27 cantly opposed apoptosis. in some tumors are the result of increased proteasome-mediated The p53 Tumor Suppressor. Degradation of the p53 degradation. The fact that p27 up-regulation is directly involved protein occurs predominantly through the UPP (reviewed in Ref. is supported by the finding that introduction of antisense p27 34), and proteasome inhibitors may exert some of their effects oligonucleotides into an oral squamous cancer cell line partially through p53 accumulation. Increased p53 levels would increase blocked proteasome inhibitor-mediated apoptosis (37). Thus, at transcription of cell cycle targets such as p21 and the proapo- least in some systems, accumulation of p21, p27, or both may ptotic protein Bax. Many studies have demonstrated increased contribute to cell-cycle arrest of tumor cells and eventual apo- levels of p53 or its targets upon proteasome inhibition. Direct ptosis. evidence that p53 plays a role came from an early study on Bax. Whereas the proteasome modulates apoptosis indi- Rat-1 fibroblasts (13), in which expression of a dominant- rectly through NF␬B-dependent transcription of a number of anti-

Table 2 Chemo/radiosensitization by proteasome inhibitors Target Sequelae of proteasome inhibition Contribution to antitumor effect NF␬Ba Inhibits nuclear translocation of NF␬B that would Blocks NF␬B-mediated transcription of antiapoptotic

ordinarily be stimulated by many chemotherapeutics genes such as Bcl-2, Bcl-xL, XIAPs and radiation p44/42 MAPK Inhibits activation of p44/42 that is mediated by Down regulates p44/42-dependent survival signals several chemotherapeutics, including taxanes P-gp Inhibits processing of P-glycoprotein precursors, May prevent efflux of cytotoxic agents through the resulting in accumulation of immature forms P-gp pump, increasing their intracellular concentrations TopI Stabilizes TopI, of which the degradation is stimulated May stabilize the TopI-DNA cleavable complex, by agents derived from camptothecins, such as prevent its repair and DNA religation, promoting irinotecan apoptosis TopII␣ Accumulation of TopII, of which the degradation in Increased abundance of the target for TopII inhibitors, some tumors is increased as a mechanism of such as anthracyclines and epipodophylotoxins resistance Genotoxic stress response Decreased transcription of the DNA repair machinery Sensitization of tumors to DNA-damaging agents (alkylating agents, anthracyclines, epipodophylotoxins, platinum-based compounds) a NF␬B, nuclear factor ␬B; MAPK, mitogen-activated protein kinase; P-gp, P-glycoprotein; TopI, topoisomerase I; TopII␣, topoisomerase II␣.

apoptotic genes, the UPP likely plays a direct role as well by therapy led to complete NF␬B inhibition. Proteasome inhibitors degrading important proapoptotic proteins. Upon dephosphoryl- may also play a role as radiation sensitizers. In a mouse colon ation, Bax interacts with and inhibits the antiapoptotic proteins cancer xenograft model, tumors were treated with a single dose

Bcl-2 and Bcl-xL in mitochondria, leading to cytochrome c release of radiation or bortezomib alone or in combination. The post- and activation of the caspase cascade. Li and Dou (19) demon- treatment to pretreatment tumor volume ratios, and the levels of strated recently that Bax levels are regulated by proteasome-medi- apoptosis, suggested a synergistic interaction between the ther- ated degradation. Apoptosis in Bcl-2 overexpressing Jurkat cells apies, and treatment with the I␬B super-repressor led to similar treated with lactacystin correlated with the accumulation of mito- results (41). Ma et al. (42) showed recently that treatment of chondrial-associated Bax. Other studies have shown that bort- myeloma cell lines resistant to melphalan, mitoxantrone, and ezomib can induce Bcl-2 phosphorylation and cleavage in associ- doxorubicin with a subtoxic dose of bortezomib sensitized them

ation with G2-M phase arrest (38), supporting the possibility that to treatment with these agents, which became cytotoxic at several mechanisms may be responsible for the ability of these 10,000–100,000-fold lower concentrations in the presence of agents to abrogate the antiapoptotic effects of Bcl-2. bortezomib. NF␬B activity in these resistant cell lines was higher than that of nonresistant myeloma cell lines. ␤1 integrin- Proteasome Inhibitors Overcome Resistance to mediated adhesion of myeloma cells to fibronectin also plays an Standard Therapies important role in de novo drug resistance. Landowski et al. (43) Both de novo and acquired resistance to chemotherapy and showed that treatment with proteasome inhibitors attenuated ␬ radiation therapy limit the effectiveness of many treatments adhesion-mediated drug resistance and nuclear NF B activity. currently available to patients, and proteasome inhibitors may Proteasome inhibitors may down-regulate other resistance impact on both mechanisms (Table 2). Strong evidence exists pathways as well. Several groups of chemotherapeutics, most supporting a role for NF␬B as part of inducible chemoresistance notably the taxanes and anthracyclines, activate signaling (23), whereby proapoptotic stimuli also activate antiapoptotic through the p44/42 MAPK pathway, and in some model systems survival pathways. For example, treatment of fibrosarcoma cells this has been shown to be antiapoptotic. The ability of protea- with tumor necrosis factor ␣, ionizing radiation, or the anthra- some inhibitors to induce MKPs might indicate that these agents cycline daunorubicin led to nuclear NF␬B accumulation. Inhi- would be useful in enhancing taxane- or anthracycline-mediated bition of NF␬B nuclear translocation with an I␬B super-repres- apoptosis by down-regulating p44/42 MAPK activation. In sup- sor dramatically potentiated apoptosis to these agents. The port of this possibility, early evidence from our laboratory camptothecin CPT-11 also activated NF␬B in fibrosarcoma shows that the proteasome inhibitor bortezomib blocks doxoru- cells, and whereas xenografts were unresponsive to CPT-11 or bicin-mediated activation of p44/42 MAPK in a murine xe- the super-repressor I␬B alone, the combination of the two led to nograft model of breast cancer. Furthermore, this correlates with the ability of the combination therapy to enhance apoptosis and significant tumor growth inhibition (39). 3 Down-regulation of NF␬B likely plays an important role in antitumor efficacy. the ability of proteasome inhibitors to abrogate drug resistance. Anthracyclines and the epipodophyllotoxins work in part Using a colon cancer xenograft model, Cusack et al. (40) by inhibiting topoisomerase II (TopII), stabilizing the TopII- demonstrated that the combination of bortezomib and CPT-11 was more effective than either agent alone. Tumor regression correlated with synergistic induction of apoptosis. Treatment with CPT-11 alone activated NF␬B, whereas the combination 3 R. Orlowski, unpublished observations.

DNA cleavable complex and inhibiting religation of single- and than bortezomib in that they inhibit serine and cysteine pro- double-stranded DNA breaks. Evidence suggests that tumors teases in addition to the proteasome, and also have poor stability may develop resistance to these agents through the down-regu- and bioavailability. The bacterial product lactacystin, whereas lation of their preferred target, TopII␣, a proteasome substrate. more proteasome specific, irreversibly blocks several of the Therefore, proteasome inhibitors might overcome resistance to proteolytic activities of the proteasome, whereas bortezomib TopII inhibitors by up-regulating levels of TopII␣. Consistent reversibly inhibits the chymotrypsin-like activity. These unique with this possibility, Ogiso et al. (44) demonstrated that TopII␣ features of bortezomib may make it easier to administer in the in depletion in colon and ovarian cancer cells occurred in response vivo setting and also may decrease its toxicity. Over 90% of to glucose deprivation and hypoxia, two conditions encountered bortezomib is cleared from the plasma compartment within 15 frequently in tumors. Lactacystin prevented down-regulation of min, and therefore to better guide dosing in Phase I clinical trials TopII␣ in colon cancer cells deprived of glucose and oxygen, a bioassay was developed that allows measurement of protea- potentiating the effects of etoposide and doxorubicin, but not of some function in whole blood or tumor samples. Using this other agents. Using a colon cancer xenograft model, these assay, investigators showed that normal proteasome activity is investigators demonstrated increased tumor inhibition with the restored 2–3 days after the administration of bortezomib i.v. combination treatment of etoposide and lactacystin. Interest- They were also able to demonstrate drug delivery to all of the ingly, the camptothecins, which target topoisomerase I, induce tissues except the central nervous system, eyes, and testes, UPP-dependent topoisomerase I degradation, suggesting that suggesting that therapeutic benefits from treatment of tumors in proteasome inhibitors may potentiate the activity of these agents these locations may be limited in the setting of, for example, an not only by down-regulating NF␬B activation, but also by intact blood-brain barrier. stabilizing topoisomerase I (45). Single Agent Phase I Studies with Bortezomib. Several Perhaps the best-known mechanism of chemotherapy re- Phase I clinical trials have evaluated bortezomib for the treat- sistance is P-glycoprotein (P-gp), a cell surface transporter that ment of refractory and relapsed solid tumors and hematological functions as an efflux pump for intracellular toxins. Several malignancies. In the first published data, 43 patients with solid classes of drugs, including the anthracyclines, Vinca alkaloids, tumors were treated with bortezomib given as an i.v. bolus and epipodophyllotoxins, are P-gp substrates. Interestingly, pro- injection on days 1, 4, 8, and 11 of a 3-week cycle (49). In teasome function is required for the normal maturation of P-gp. preclinical studies it was found that more frequent therapy Proteasome inhibition leads to accumulation of immature P-gp increased toxicity, indicating the need for some recovery of that is incapable of transporting drugs out of cells efficiently. proteasome function between dosing for normal cellular home- Thus, whereas it has not been tested directly, proteasome inhib- ostasis. Dose-limiting toxicities included diarrhea and sensory itor-mediated blockade of P-gp maturation may function to neurotoxicity, the latter of which occurred in patients with abrogate resistance to P-gp substrates (46). antecedent neuropathy. The maximum tolerated dose was de- 2 Recent studies have suggested that proteasome inhibitors fined as 1.56 mg/m , and pharmacodynamic studies showed a synergize with DNA damaging agents by inhibiting the tran- dose-dependent proteasome inhibition in vivo. One patient with scription of genes involved in DNA damage repair. Mitsiades et non-small cell lung carcinoma had a partial response (PR) to al. (47) showed that subtoxic concentrations of bortezomib therapy, whereas 3 other patients had stable disease as their best sensitized chemotherapy-resistant myeloma cell lines and pa- responses. tient samples to doxorubicin and melphalan. Gene expression Results of another Phase I trial have been reported in profiling demonstrated down-regulation of a number of tran- patients with advanced hematological malignancies (50). Using scripts involved in DNA repair. The authors proposed that the a more intensive schedule in which bortezomib was dosed twice abrogation of resistance to melphalan and doxorubicin was weekly for 4 consecutive weeks over a 6-week cycle, the max- 2 mediated in part by transcriptional down-regulation of the pro- imum tolerated dose was 1.04 mg/m . Dose-limiting toxicities tective response to genotoxic stress. included thrombocytopenia, hyponatremia, hypokalemia, fa- It should be noted, however, that proteasome inhibition has tigue, and malaise, whereas peripheral neuropathy was seen in 5 not overcome chemoresistance in all cases. For example, bort- patients, all of whom had received neurotoxic agents previously. ezomib was unable to reverse breast cancer cell resistance to Bortezomib again induced a dose- and time-dependent inhibi- cyclophosphamide or cisplatin in vitro (48). These findings tion of proteasome function. Evidence of antitumor efficacy was point to the need for additional elucidation of the mechanisms seen in 9 out of 9 evaluable patients with plasma cell dyscrasias, involved in the action of these agents, so that their optimal use including 1 patient with multiple myeloma who had a durable can be identified. complete response (CR). Partial responses were also seen in 2 patients with non-Hodgkin’s lymphoma, including 1 with mantle cell lymphoma, and another with low-grade follicular B-cell Clinical Data lymphoma. Given the broad spectrum of activity of proteasome inhib- Phase II Studies with Single-Agent Bortezomib. En- itors in in vitro and in vivo models, interest in the proteasome as couraging preclinical and Phase I studies in plasma cell dyscra- a target for cancer therapy has increased dramatically. To date, sias led to a Phase II trial evaluating bortezomib in patients with the first and only such inhibitor to have entered clinical trials is multiple myeloma (51). Heavily pretreated patients (202) re- bortezomib, a peptide boronic acid derivative, which offers ceived 1.30 mg/m2 of bortezomib on days 1, 4, 8, and 11 of a several advantages over some of the other available proteasome 3-week cycle for up to eight cycles. Complete responses, meas- inhibitors. Peptidyl aldehydes such as MG132 are less specific ured by the stringent Blade´ criteria (52), were seen in 4% of

Table 3 Phase I combination studies with bortezomib A more comprehensive listing of bortezomib clinical trials is provided by Wright et al. (64). Standard agent Population DLTsa MTD (mg/m2) Lead PI/Institution Last Update Irinotecan Solid tumors Report pending Report pending Ryan ASCO 2002 Massachusetts General Gemcitabine Solid tumors Report pending Report pending Ryan ASCO 2002 Massachusetts General 5-Fluorouracil/leucovorin Solid tumors Diarrhea Bortezomib 1.00 Lenz ASCO 2002 5-fluorouracil 500 University of Southern Leucovorin 20 California Paclitaxel Solid tumors Study ongoing Shapiro Ohio State University Docetaxel Solid tumors Study ongoing Armstrong Johns Hopkins Paclitaxel/carboplatin Solid tumors Study ongoing Adjei Mayo Clinic Carboplatin/etoposide Solid tumors Study ongoing Eckhardt University of Colorado Doxorubicin Solid tumors Study ongoing Wilding ASCO 2002 University of Wisconsin Liposomal doxorubicin Solid tumors Study ongoing Dees/Orlowski ASCO 2003 University of North Carolina Liposomal doxorubicin Hematologic malignancies Study ongoing Orlowski/Dees ASCO 2003 University of North Carolina a DLT, dose-limiting toxicity; MTD, maximum tolerated dose; PI, principal investigator; ASCO, American Society of Clinical Oncology.

patients, and near-CRs with residual immunofixation-positive some inhibitor-mediated apoptosis, and studies in Hodgkin’s paraprotein were seen in another 6%. The combined CR, near- disease, mantle cell lymphoma, and chronic myeloid leukemia CR, and PR rate was 27%, whereas 59% of patients had stable are also underway. Single-agent bortezomib is being evaluated disease or better. Median overall survival was 16 months, and in a variety of solid tumors, including hepatocellular carcinoma, the median duration of response among patients with a CR, PR, melanoma, metastatic breast carcinoma, metastatic colorectal or minor response was 12 months. Also of note, the median time carcinoma, metastatic renal cell carcinoma, neuroendocrine tu- to progression for all of the patients was 6.6 months, more than mors, ovarian and primary peritoneal carcinoma, non-small cell twice the 3-month progression time on the previous therapy. lung carcinoma, and sarcoma. Common toxicities included gastrointestinal symptoms, fatigue, Bortezomib-Based Combination Therapy. The ability thrombocytopenia, and peripheral neuropathy, with 18% of pa- of proteasome inhibition to enhance chemo- and radiosensitiv- tients having to discontinue therapy due to drug-related adverse ity, and overcome drug resistance in preclinical models, has events. These encouraging findings led to the approval of bort- engendered great excitement about the potential of combination ezomib by the Food and Drug Administration as therapy for regimens with standard chemotherapeutics. To explore this pos- patients with multiple myeloma who had received at least two sibility further, a number of Phase I studies evaluating bort- prior therapies and who had progressed on the last of these. A ezomib in combination with standard chemotherapeutic agents Phase III randomized international multicenter trial comparing have now been conducted or are currently underway. For ex- bortezomib with dexamethasone in this patient population is ample, a clinical trial of the combination of irinotecan and currently underway. bortezomib has been completed recently that defined the dose Because proteasome inhibitors seem to be able to over- limiting toxicities and the dose for additional testing of this come Bcl-2-mediated suppression of apoptosis and showed ev- combination (55). Likewise, a Phase I trial of the combination of idence of activity against non-Hodgkin’s lymphoma in Phase I pegylated liposomal doxorubicin (Doxil) and bortezomib in studies, O’Connor et al. (53) initiated a Phase II trial of bort- patients with refractory solid tumors is currently ongoing at our ezomib in patients with indolent lymphomas. A preliminary institution. Twenty patients with refractory metastatic solid tu- report of the results indicated several responses in patients with mors have been treated thus far with bortezomib, which is follicular lymphomas, including one CR, and major responses in administered on days 1, 4, 8, and 11 of a 3-week cycle, and patients with mantle cell lymphoma as well. The encouraging pegylated liposomal doxorubicin at 30 mg/m2, which is admin- early efficacy data in mantle cell lymphoma is not surprising istered on day 4. Doses up to 1.50 mg/m2 of bortezomib in this given the important roles of constitutive NF␬B activity and combination have been administered, with low-grade fatigue, proteasome-mediated down-regulation of p27 in mantle cell nausea, and thrombocytopenia having been the most frequent lymphomagenesis. Early data from one other clinical trial of toxicities. Accrual is ongoing to define a dose for additional bortezomib shows excellent activity in mantle cell lymphoma as testing. These and other trials focusing on a general population well, with patients achieving responses and one CR noted (54). of solid tumor patients, or those with hematological malignan- Another Phase II study of bortezomib is specifically focusing on cies, are summarized in Table 3. On the basis of these study CLL given the differential sensitivity of these cells to protea- results, it appears that the toxicities of bortezomib in combina-

Table 4 Disease-targeted Phase I or II combination studies Standard agent Patient population PIa/institution Melphalan Multiple myeloma Berenson/Cedars Sinai Thalidomide Multiple myeloma Barlogie/University of Arkansas Docetaxel Metastatic breast cancer Piccart/Baselga/Europe Docetaxel Non-small cell lung cancer Gandara/Lara/University of California Docetaxel Prostate cancer Dreicer/Roth/Cleveland Clinic Mitoxantrone Prostate cancer Papandreou/M. D. Anderson Oxaliplatin Advanced colorectal cancer Hochster/New York University Carboplatin Ovarian or primary peritoneal carcinoma Aghajanian/Memorial Sloan Kettering Carboplatin Ovarian, primary peritoneal, and fallopian tube cancer Ramirez/M. D. Anderson Gemcitabine/carboplatin Non-small cell lung cancer Davies/Gandara/University of California Prednisone Hormone refractory prostate cancer Scher/Memorial Sloan Kettering Radiation Squamous cell carcinoma of the head and neck Sausville/NCI Bortezomib Ϯ gemcitabine Metastatic pancreatic adenocarcinoma Alberts/O’Connell/Mayo Clinic/NCCTG Bortezomib Ϯ irinotecan Advanced colorectal carcinoma Ryan/Massachusetts General Bortezomib Ϯ irinotecan Gastroesophageal junction or stomach Wadler/Cornell U. Medical College Bortezomib Ϯ EPOCH Non-Hodgkin’s lymphoma Wilson/NCI Bortezomib Ϯ Docetaxel Advanced non-small cell lung carcinoma Schiller/University of Wisconsin a PI, principal investigator; EPOCH, etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin chemotherapy; NCI, National Cancer Institute; NCCTG, North Central Cancer Treatment Group. tion with standard chemotherapy have not necessitated signifi- Conclusions cant reduction of the single-agent doses, implying that any The UPP impacts on a number of cellular processes crucial additive toxicities of bortezomib are very manageable. Addi- to oncogenesis. Proteasome inhibitors are able to induce apo- tionally, pharmacological interactions with other chemothera- ptosis and tumor regression as single agents in a broad spectrum peutics have not been reported. Phase I or II studies of a number of tumor cell lines, and in in vivo xenograft models. Perhaps of these combinations targeting specific solid tumor subtypes more importantly, they have shown the ability to overcome drug are either currently being planned or already underway based on resistance and to synergize with a number of conventional safety data and evidence of clinical activity from the Phase I therapies. Whereas down-regulation of NF␬B activity likely trials (Table 4). For example, encouraging evidence of activity plays an important role in their observed effects, a number of has been seen in a study of bortezomib and carboplatin in other factors have also been shown to be involved. The selec- patients with recurrent ovarian cancer. Major responses have tivity of these agents to cancer cells is intriguing, and may be been reported in this patient population, including one response partly explained by increased proteasome activity and/or in- in a patient with previously documented platinum-refractory creased activity of components of the Ub-conjugating system in disease (56). transformed cells. Additional investigation into the role that Combination regimens based on bortezomib are also being deregulated UPP activity plays in various malignancies, and studied in patients with hematological malignancies. The Phase whether it correlates with tumor responses to these agents, is II multiple myeloma study was designed to allow addition of warranted. These studies may also support the development of dexamethasone to patients with progressive disease after two drugs that target specific components of the UPP. Phase I and II cycles or stable disease after four. A recent analysis of these data trials with the proteasome inhibitor bortezomib have demon- indicated that dexamethasone appeared to have an additive strated tolerable toxicities and some clinical responses, with effect on response rate, albeit with some increased toxicity (57). significant activity in multiple myeloma. Data evaluating the Another interesting combination being evaluated in myeloma efficacy of bortezomib in solid tumors are less mature; however, patients is that of thalidomide and bortezomib. Early results it is likely that for these diseases proteasome inhibitors will from that Phase I study have shown tolerable toxicity with, work best as a component of combination therapy. Mature Phase interestingly enough, no significant increase in neurotoxicity. 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