Update on the Proteasome Inhibitor Bortezomibin Hematologic Malignancies

Comprehensive Review Update on the Proteasome Inhibitor Bortezomib in Hematologic Malignancies Andre Goy,1 Frederic Gilles2

Abstract The ubiquitin–proteasome system plays a crucial role in eukaryotic cells in maintaining protein homeostasis. Through the disruption of a variety of pathways and cell cycle checkpoints, proteasome inhibition leads to apoptosis and in experimental models can overcome chemoresistance. Bortezomib is the first of its class of proteasome inhibitors tested in humans that showed promising activity in several tumor types, and especially in hematologic malignancies, in phase I studies. The remarkable results obtained in phase II studies in multiple myeloma (MM) led to its fast-track approval by the US Food and Drug Administration in May 2003 for relapsed MM. More recent observation also revealed promising activity in non-Hodgkin’s lymphoma. This review will explore the rationale for the use of bortezomib in hematologic malignancies as well as provide an update on the results of ongoing studies and future directions for the use of this new agent in hematologic malignancies. The mechanism of action of bortezomib and its nonoverlapping toxicity profile make it a very appealing drug for combination with other chemotherapeutic or biologic agents. Bortezomib represents an excellent example of how progress in understanding the biology of cancer cells can impact clinical practice and lead toward a new era of rational therapeutics.

Clinical Lymphoma, Vol. 4, No. 4, 230-237, 2004 Key words: Leukemia, Multiple myeloma, Non-Hodgkin’s lymphoma, Nuclear factor–κB, Proteasome inhibition, Ubiquitin

Introduction Progress in understanding the biology of cancer cells translates into the identification of key molecular targets for the development of new drugs with novel antitumor mechanisms of action. One of the best examples of these breakthroughs in cancer therapy is bortezomib, previously known as PS-341, the first compound of a new class of agents called proteasome inhibitors. Bortezomib was recently approved by the US Food and Drug Administration (FDA) for relapsed multiple myeloma (MM). This review will summarize the rationale behind the development and success of bortezomib and provide an update of its clinical development in hematologic malignancies.

Rationale for the Clinical Use of Proteasome Inhibitors Structure and Function of Proteasome Intracellular proteolysis is an essential process1 that allows the constant renewal and maintenance of the proteasome. In 1 Department 2Department

of Lymphoma/Myeloma of Molecular Pathology The University of Texas M. D. Anderson Cancer Center, Houston Submitted: Sep 12, 2003; Revised: Jan 27, 2004; Accepted: Feb 9, 2004 Address for correspondence: Andre Goy, MD, Department of Lymphoma/ Myeloma, Box 429, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, Texas 77030 Fax: 713-745-5656; e-mail: [email protected]

eukaryotes, proteins are degraded by the ubiquitin–proteasome pathway.2 A major component of this system is the 26S proteasome, a 2.5-Md molecular machine containing 31 different subunits. Proteasomes, initially described in the late 1980s,3-5 are large multiple-subunit proteases that are found in the cytosol, both free or attached to the endoplasmic reticulum, as well as in the nucleus of all eukaryotic cells. Their ubiquitous presence and high abundance in these compartments reflect their central role in cellular protein turnover. The structure and function of the 26S proteasome have been reviewed in detail previously.6,7 The proteasome is formed by a cylinder-shaped multimeric barrel-like complex referred as the 20S proteolytic core, which, upon activation, is capped at one or both ends by another multimeric component known as the 19S regulatory unit.6,7 The proteolytic core is composed of 28 subunits that are arranged as 4 stacked 7-membered rings. The 2 outer α rings form a narrow channel that allows only denatured proteins to enter the inner core. The 2 central β rings, which each carry 3 active proteolytic sites, form the inner chamber, where proteolysis occurs.8,9 The target proteins are cleared through the proteasome in a 3-step process. Proteins are first ubiquitinated on lysine residues by the ubiquitin–enzyme complex. The tagged proteins are then captured by the 19S subunit of the proteasome and processed through the core of the proteasome. The ubiquitin residues are subsequently removed, and the proteins are unfolded, degraded, and recycled into small peptides within the catalytic unit of the

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proteasome. The resulting polypeptides, which are 3-22 residues in length, can then be recycled within the cell.8,10 The proteasome–ubiquitin system maintains intracellular protein homeostasis through 2 main functions. First, this system allows for the degradation of unassembled, damaged, misfolded, or mutated proteins, which are more abundant in tumor cells than in normal cells.11 Second, the ubiquitin–proteasome pathway coordinates the degradation of short-lived proteins, many of which are involved in critical functions such as cell cycle regulation, transcription regulation, apoptosis, antigen processing, chemotaxis, angiogenesis, and cell adhesion. The proteasome is thus an essential component of cellular metabolism, and, as such, a novel and appealing target for cancer treatment.10,11 Molecular Targets of Proteasome Inhibitors Proteasome inhibition leads to the accumulation of a variety of substrates, many of which are relevant to the biology of cancer cells (Figure 1). Proteasome inhibition leads first to the suppression of the natural oscillation of multiple cyclins (A, B, D, and E)6,12-16 as well as the accumulation of cell cycle inhibitors such as p2117 or p27.18 Proteasome inhibition also leads to the accumulation of other key cell cycle regulators and apoptotic factors, including the tumor suppressor p5319 gene and the cmyc oncogene.15,20 Several models have shown that proteasome inhibition can also induce apoptosis through changes in pro- and antiapoptotic molecules, among which are Bcl-2 cleavage,21,22 Bax, Bak, and Bad accumulation,23,24 abrogation of Mcl-1 and Bcl-xL, and XIAP upregulation.23 Proteasome inhibition also leads to the accumulation and activation of caspases 3, 8, and 9.23,25,26 Regulation of nuclear factor–κB (NF-κB) by the proteasome is of particular interest. The NF-κB pathway is constitutively active in several cancer subtypes and is associated with proliferation, cell survival, and protection from chemotherapyinduced apoptosis.7,25 Nuclear factor–κB is physiologically trapped in the form of an inactive complex with IκB in the cytoplasm. Under stress conditions (eg, exposure to chemotherapy, hypoxia, and radiation), IκB becomes phosphorylated and releases NF-κB, which then translocates into the nucleus where it can induce a variety of transcriptional reactions that will prevent apoptosis and induce cell proliferation and survival. Several mechanisms that may lead to the activation of NF-κB have been described in a variety of lymphoma subtypes as well as in chronic lymphocytic leukemia (CLL) and MM.27,28 These mechanisms include gene rearrangements (amplification, translocation)29 of NF-κB as well as mutations or polymorphisms of IκB rendering it unable to repress NF-κB activation.30,31 Bortezomib prevents IκB degradation, maintaining NF-κB in an inactive complex with IκB and preventing the downstream effects of NF-κB activation. Proteasome inhibition has also been shown in preclinical models to suppress protective signals from stromal cells32,33 and to inhibit angiogenesis.34-36 The accumulation of unassembled, misfolded, or mutated proteins, which are more abundant in cancer cells than in their normal counterparts,34,37,38 is also a consequence of proteasome inhibition that can be damaging for the cells.

Figure 1 Molecular Targets of Proteasome Inhibition Cyclin-dependent kinases Inhibitors p21, p27 Cyclins: A, B, D, E

Tumor suppressor (p53)

Accumulation of unassembled misfolded or mutated proteins

Oncogene (c-myc) Inhibitors of apoptosis and caspases cytochrome-c release Bcl-2 cleavage and phosphorylation

Inhibition of angiogenesis

Inhibition of NF-kB

Suppression of supportive signals from microenvironment Disruption of many pathways and checkpoints

Apoptosis Proteasome inhibition results in the accumulation of a variety of substrates many of which are essential for cancer cells survival: suppression of natural oscillations of cyclins A, B, D, E; cyclindependent kinases inhibitors accumulation; p53 and c-myc stabilization; Bcl-2 cleavage and phosphorylation; inhibition of NF-kB. Other mechanisms of action include inhibition of angiogenesis and suppression of supportive signals from the microenvironment. The disruption of multiple pathways and checkpoints leads to cell apoptosis. Abbreviation: NF-kB = nuclear factor–kB

Specificities of Cell Death Induced by Proteasome Inhibition Proteasome inhibition results in changes of a large number of substrates, which translates into the disruption of a variety of pathways and checkpoints and leads to cell apoptosis.39 The disrupted targeted pathways can vary among cell types and cell cycle phases. Contrary to conventional chemotherapy agents, cell death by proteasome inhibition does not appear to depend on cell proliferation.6 In normal cells, proteasome inhibition leads preferentially to cell cycle arrest at the G1/S boundary,40,41 whereas in transformed or malignant cells it induces apoptosis.33 For example, fibroblasts transformed with ras and/or c-myc genes or Epstein-Barr virus lymphoblastoid cells transformed with c-myc were as much as 40 times more susceptible to apoptosis induced by the proteasome inhibitor ZLLF-CHO than their untransformed parental cell lines.20 Bortezomib has also been shown to overcome cancer cell resistance in a variety of models. In cell cultures and xenograft tumor models, bortezomib was found to enhance the sensitivity of cancer cells to traditional chemotherapeutic agents and to overcome drug resistance.11,41 Similarly, studies have shown that stabilization of NF-κB inhibition with use of an IκB superrepressor (ie, one not subject to ubiquitination and proteasomal degradation) leads to a greater extent of apoptosis after stress exposure by chemotherapy agents or radiation.42 Bortezomib was able to induce apoptosis even in p53-negative cell lines or cell lines overexpressing Bcl-2.6 Moreover, acquired or intrinsic multidrug-resistant (MDR) protein expression is very common

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Bortezomib in Hematologic Malignancies among all cancer cell types. Bortezomib appears as a poor substrate of MDR protein, contrary to most conventional chemotherapeutic agents.43 Bortezomib was found to be equally effective in myeloma cell lines with low or high levels of expression of homologues of the MDR protein MDR1/ MRP1.44,45 In addition, apoptosis induced by bortezomib occurred even in cells that failed to undergo apoptosis when irradiated.46 Finally, bortezomib has been shown to markedly sensitize resistant prostate, colon, and bladder cancer cells to tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) even in Bax and caspase 9–negative or Bcl-xL–overexpressing cells.47,48 Further insight into the complex molecular targets of bortezomib will be provided by ongoing pharmacogenomic studies in patients with MM,49 as well as by experimental models in leukemia50 or lymphoma cell lines.51

Clinical Development of Bortezomib Bortezomib as First Proteasome Inhibitor A number of natural and synthetic compounds have been shown to have proteasome-inhibition activity. For example, the bacterial metabolite lactacystin acts as a natural proteasome inhibitor.52 The first synthetic proteasome inhibitors were derived from small peptides carrying an aldehyde moiety related to calpain inhibitor–I.33 However, compounds derived from calpain inhibitor–I were not found to be metabolically active. Substitution of boronic acid for the aldehyde moiety produced proteasome inhibitors that were ≥ 1000 times more potent than their aldehyde analogues.33 Moreover, the peptide boronic acids are very specific to a number of common serine proteases. They block specifically the chymotrypsin-like activity of the proteasome in a reversible manner and dissociate from the proteasome at a slower rate than other proteasome inhibitors.10 Among the dipeptidyl-boronic acids, the compact water-soluble and cell-permeable agent initially described as PS-341 and now known as bortezomib is of particular interest. It is a potent (K = 0.6 nmol/L) but selective proteasome inhibitor10 that binds to the proteasome with high affinity and dissociates slowly, thus conferring stable but reversible proteasome inhibition.11 Pharmacokinetics and Preclinical Toxicity After bolus injection, bortezomib was shown to rapidly distribute from the plasma in < 10 minutes into all tissues except in the brain, spinal cord, eyes, and testes.11 The estimated terminal elimination half-life of bortezomib ranged from 9 to 15 hours at doses of 1.45-2.0 mg/m2. Preclinical data showed that, 1 hour after intravenous dosing, bortezomib produced a doserelated decrease in 20S proteasome activity in rat mononuclear cells.6 At 24 hours after a single intravenous dose, there was also inhibition of 20S proteasome activity, but the magnitude of the effect and the range of active doses were less than those seen at 1 hour. After 48 hours, the 20S proteasome activity had returned to basal levels.11 Repeated administration of bortezomib showed that 20S activity was also decreased in a doseand time-dependent manner. In a prolonged schedule, there was cumulated inhibition but no evidence of desensitization of the proteasome to the proteasome inhibitor.6 Plasma clearance

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of the drug occurs predominantly through metabolic inactivation by oxidative deboronation. Deboronated bortezomib metabolites are inactive as 26S proteasome inhibitors.11 Elimination occurs through bile and urine. In vitro studies indicate that bortezomib is a poor inhibitor of cytochrome P450 (CYP) isoenzymes 1A2, 2C9, 2D6, and 3A4, and that it does not induce CYP3A4 or CYP1A2.33 There is no clear evidence at this time of any requirements for dose adjustment in cases of liver or renal failure, but this is still being evaluated in ongoing clinical trials.53 Toxicity studies in cynomolgus monkeys were performed on animals treated by bolus intravenous injection twice weekly for 4 weeks at increasing doses followed by a 2-week recovery period.11 The major toxicity was gastrointestinal and included anorexia, vomiting, and diarrhea. Toxicity was dose dependent and cumulative: the highest dose level that did not cause severe irreversible toxicity was 0.067 mg/kg. Cardiovascular studies showed that a single dose of ≥ 0.25 mg/kg was lethal because of profound hypotension observed within 4-12 hours after treatment.7,54 Phase I Studies In 1998, bortezomib became the first proteasome inhibitor to be tested in clinical trials. Orlowski et al conducted a phase I trial to determine the maximum tolerated dose and the dose-limiting toxicity as well as the pharmacodynamics of bortezomib in 27 patients with refractory hematologic malignancies.55 In this study, bortezomib was administered intravenously (I.V.) twice weekly for 4 weeks followed by a 2-week rest. These 6-week cycles were repeated a maximum of 3 times. Bortezomib was given at escalating doses from 0.4 mg/m2 to 1.38 mg/m2.55 Twenty-seven patients received 293 doses of the drug, including 24 complete cycles. Dose-limiting toxicities at the dose greater than the 1.04mg/m2 maximum tolerated dose included thrombocytopenia (74%), fatigue (59%), nausea (52%), diarrhea (37%), anemia (48%), neutropenia (37%), electrolyte imbalance (hyponatremia or hypokalemia, 22%), and neuropathy (19%).55 The toxicity profile observed required therapy discontinuation mostly during the third week of the first cycle of therapy. This observation led to a new schedule for the phase II studies, with bortezomib injected twice weekly for 2 of 3 weeks. Consistent with preclinical data, pharmacologic studies revealed that bortezomib induced 20S proteasome inhibition in a time-dependent manner and confirmed that the inhibition was related to the dose in mg/m2 as well as to the absolute dose of the drug. Among 9 of 11 evaluable patients with heavily pretreated plasma cell dyscrasias, one experienced a complete response (CR) and 8 others showed some reduction in paraprotein levels and/or marrow plasmacytosis. In addition, one patient with relapsed mantle cell lymphoma (MCL) and another with refractory follicular lymphoma had a partial response (PR) with treatment at the dose of 1.38 mg/m2.56 One patient with Waldenstrom macroglobulinemia showed a 79% decrease of the lymphoplasmacytic infiltration in the bone marrow. Activity in Multiple Myeloma Several patients with MM showed very promising responses, including one CR in the phase I study mentioned earlier,55 leading to further exploration of the activity of this agent in phase

Andre Goy, Frederic Gilles II trials. Additional rationale for the use of bortezomib was supported by experimental data. Activation of NF-κB signaling appears to be particularly important for the survival of MM cells. High NF-κB activity was found in MM cells isolated from patients as well as from bone marrow stromal cells. Furthermore, the cell lines of chemoresistant MM cells showed increased NF-κB activity compared with chemosensitive lines. Moreover, NF-κB is known to regulate the transcription of interleukin (IL)–6 in bone marrow stromal cells. It also modulates the expression of adhesion molecules (CD54 and CD106)16 as well as the expression of cyclin D1 and antiapoptotic proteins (IAPs, Bcl-xL) on MM cells.34 In addition to its capacity to antagonize the activation of protective NF-κB in MM cells, bortezomib can also prevent protective interactions from bone marrow stromal cells by interfering with IL-6–triggered signaling,57 by inhibiting angiogenesis,35,36,58 and by inducing modulation of vascular endothelial growth factor secretion and activity in MM.28,32 Phase II Studies The safety and efficacy of bortezomib were assessed in 2 phase II trials in patients with relapsed and/or refractory MM: the SUMMIT trial (for relapsed MM and/or MM refractory to most recent therapy)59 and the CREST trial (for relapsed MM and/or MM refractory to first-line therapy).60 Most dose-limiting toxicity in the initial phase I study appeared during the third week of the first cycle at a dose > 1.04 mg/m2 on a 4-week schedule and at a dose of 1.56 mg/m2 on a 3-week schedule. Consequently, in the phase II SUMMIT study, bortezomib was given at a dose of 1.3 mg/m2 on days 1, 4, 8, and 11 followed by a 10-day rest period. The 21-day cycles were repeated for a maximum of 8 cycles. Patients with suboptimal response were allowed to receive dexamethasone in addition to bortezomib after 2 cycles (for patients with progressive disease) or after 4 cycles (for patients with stable disease). The trial was evaluated and reviewed by an independent committee with strict response criteria according to Blade et al.61 The criteria for a complete response were (1) no detectable M protein in blood and urine by immuno-electro-fixation (IEF) for a minimum of 2 determinations at 6-week intervals, (2) < 5% plasma cells in the bone marrow, (3) stable bone disease, and (4) normal calcium. The population enrolled in this study was heavily pretreated; of 193 patients who could be evaluated, 92% had previously received ≥ 3 of the major classes of agents for MM, and disease in 91% of patients was refractory to the most recent therapy received. The response rate to bortezomib was 35%, with 7 patients (3.6%) exhibiting a CR and 12 additional patients (6.2%) in whom myeloma protein was detectable only by IEF. The median overall survival was 16 months, with a median response duration of 12 months. Importantly, the response rate was independent of performance status, myeloma type, α2microglobulin, and chromosome 13 deletion status.62 The CREST trial, the other phase II trial performed in MM, was an open-label, multicenter, randomized phase II doseresponse study. Fifty-four patients were enrolled in this study, which compared 2 doses of bortezomib (1.0 mg/m2 vs. 1.3

mg/m2) on the same schedule (days 1, 4, 8, and 11) every 21 days for a total of 8 cycles. The patients had received a median of 3 previous therapies, and 48% had experienced relapse after autologous stem cell transplantation (ASCT). The results of the CREST trial showed that patients could experience a CR at doses of 1.3 mg/m2 and 1.0 mg/m2, although the overall response rate was higher with a dose of 1.3 mg/m2 (38%) than a dose of 1.0 mg/m2 (30%).60 The remarkable results seen in these phase II trials led to approval of bortezomib by the FDA for relapsed MM in May 2003.63 Toxicity Profile Toxicity analysis in the SUMMIT trial showed that grade 3 adverse events included thrombocytopenia (28%), fatigue (12%), peripheral neuropathy (12%), and neutropenia (11%). Grade 4 events occurred in 14% of patients. The incidence of tumor lysis syndrome (approximately 1%) did not differ from what has been seen with standard or high-dose chemotherapy in this population.64 The toxicity profile in the CREST trial was similar.60,62 The SUMMIT and CREST MM trials were pooled (N = 256) in a recent update on toxicity analysis; the occurrence of grade 3/4 thrombocytopenia is dependent on baseline platelet count, which will decrease by approximately 60% overall.65 When serum thrombopoietin levels were measured in a subset of patients receiving bortezomib, no change was observed. The mechanism of thrombocytopenia appears different from that of conventional chemotherapy and dose not appear to be a result of direct toxicity on BM progenitors. However, bortezomib might interfere with platelet release, as the budding of platelets from megakaryocyte progenitors appear to be an NF-κB–dependent process resulting in transient thrombocytopenia. The other main toxicity seen in patients with MM was peripheral neuropathy. Based on the pooled data, peripheral neuropathy occurred in one third of patients. However, approximately 80% of patients had baseline evidence of peripheral neuropathy before entering the trial. Among the 60 patients who had no evidence of baseline neuropathy, only 3% developed a grade 3 neuropathy while receiving bortezomib. Nerve conduction studies showed that this was a length-dependent axonal sensory polyneuropathy with predominant small-fiber involvement.66 Future Directions As recommended by the FDA, a confirmatory phase III trial has been started to confirm the phase II data. This ongoing study, known as the Assessment of Proteasome Inhibition for Extending Remissions (APEX) trial, is a randomized study between dexamethasone versus bortezomib as first-line therapy in newly diagnosed MM with the possibility for patients in the dexamethasone arm to cross over to the bortezomib arm in case of progression. The APEX trial was closed in December 2003 after the interim analysis showed the superiority of the bortezomib arm. Further details on response rates and toxicity profiles will be presented at the 40th Annual Meeting of the American Society of Clinical Oncology in June 2004. Additional ongoing studies will address the role of bortezomib in a variety of combined regimens. The combination of thalido-

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Bortezomib in Hematologic Malignancies mide, dexamethasone, and bortezomib is being tested as firstline therapy as well as in relapsed disease after ASCT.67 Conventional chemotherapy plus bortezomib is also being evaluated in the context of salvage therapy, including high-dose melphalan followed by ASCT.68 Preliminary results of a phase I study of the combination of liposomal doxorubicin plus bortezomib in refractory hematologic malignancies suggest very promising activity in MM: 3 of 8 patients with MM experienced a CR, including 1 patient who had experienced progression after a previous CR with bortezomib alone.56 Additional questions remain to be answered regarding the bortezomib schedule, especially in the combination regimens, the role of bortezomib as maintenance therapy, and the mechanisms of resistance to proteasome inhibition. In this setting, important studies are under way with gene profiling of purified myeloma cells in patients treated with bortezomib.69 Preliminary results suggest that it will be possible from molecular profiling data to predict response to bortezomib in patients with MM.49 These molecular studies will also shed some light on the mechanisms of action of bortezomib in vivo,69 in addition to the mechanisms of resistance to proteasome inhibitors.28,70-72

Activity in Non-Hodgkin’s Lymphoma Bortezomib has also demonstrated activity against other Bcell malignancies in laboratory studies and in the phase I study55 in which 2 patients with relapsed non-Hodgkins’s lymphoma (NHL)—one with MCL, the other with follicular lymphoma— experienced a PR. Moreover, treatment of MCL cells with bortezomib in vitro led to cell growth inhibition and rapid induction of apoptosis at the molecular level, stabilization of IκB, and reduced binding of activated NF-κB to its promoter.73 Bortezomib also exhibited activity against MCL tumors in a xenograft model in severe combined immunodeficient mice.74 In addition, bortezomib had shown activity against a variety of cell lines in the initial screening, which included several diffuse large-cell lymphoma (DLCL) cell lines.10,75 Gene profiling studies have shown that diffuse large B-cell lymphoma (DLBCL) is composed of 2 subgroups, germinal center Bcell–like (GCB) and activated B-cell–like (ABC), each of which presents distinct pathogenetic mechanisms and clinical outcomes.76 Patients with ABC-type DLBCL have a poorer prognosis, and ABC-type DLBCL is frequently refractory to chemotherapy. Interestingly, in vitro, ABC-DLBCL cell lines show higher NF-κB activation, constitutive IκB kinase (IKK) activity, and IκB degradation compared with GCB lines.77 Furthermore, ABC-DLBCL cell lines treated with a superrepressor IκB (unphosphorylatable by IKK and therefore nondegradable) showed cell death and G1-phase growth arrest.78 These results suggest that proteasome inhibition via downregulation of the NF-κB pathway could be a potential treatment of at least a subset of lymphomas.79,80 Preliminary Results in Non-Hodgkin’s Lymphoma Two ongoing phase II studies have confirmed promising activity in NHL, one at Memorial Sloan-Kettering Cancer Center81 and another at M. D. Anderson Cancer Center.82 In

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the latter study, patients with relapsed or refractory indolent or aggressive B-cell NHL were eligible without limitation in the number of previous therapies; patients in whom ASCT had failed were also included. The schedule was as follows: bortezomib was given I.V. over 3-5 seconds at 1.5 mg/m2 on days 1, 4, 8, and 11, followed by a 10-day rest period. These 21-day cycles were repeated for a maximum of 6 cycles unless patients were removed from the study because of toxicity or failure to respond. Restaging was performed every 2 cycles during therapy and then every 3 months afterward. From July 2002 to September 2003, 31 patients were enrolled, of whom 29 were evaluable for toxicity or response. On the basis of the preclinical data, which suggested promising activity in MCL, the patient population was stratified into 2 groups: MCL and other B-cell NHLs. There were 18 MCLs and 12 other subtypes of lymphomas, including DLCL, follicular lymphoma, transformed follicular lymphoma, and small lymphocytic lymphoma. The toxicity profile was characterized by gastrointestinal toxicity (nausea, vomiting, and/or diarrhea) or fatigue and hypotension. Hematologic toxicity was mild and transient. Grade 3 neuropathy was seen in only 1 patient who had previously received vinca alkaloids and taxanes for relapsed DLCL. The incidence of neuropathy was found to be higher in patients with MM,66 among whom a significant fraction of patients had peripheral neuropathy related to underlying disease and exposure to thalidomide. Four patients developed grade 2 rash, which underwent biopsy in 2 cases and showed necrotizing vasculitis, as previously seen in the Memorial Sloan-Kettering study.81 In all 4 cases, patients were treated symptomatically, without oral steroids, and were able to continue treatment with bortezomib.82 Eight of the 15 patients with MCL responded, with 3 CRs and 5 PRs, for a response rate of 53%, and 1 patient with refractory DLCL had a PR. Two of the patients with MCL who experienced a CR were still without evidence of disease 4 and 7 months later; the third patient with MCL who had a CR underwent an ASCT consolidation 2 months after completing bortezomib and was still doing well at the time of this publication. The 5 additional MCL responders who had a PR showed a response duration of 1-11 months. One patient with DLCL in whom 3 prior regimens had previously failed, including up-front stem cell transplantation, had a PR with a response duration of 4 months. Similar promising results were observed in the trial developed at Memorial Sloan-Kettering Cancer Center, with activity seen in MCL and follicular lymphoma, including 1 CR in a patient with extensive and bulky relapsed follicular lymphoma.81 Future Directions in Non-Hodgkin’s Lymphoma Other directions are being explored with this new class of agents in NHL. The encouraging preliminary data observed in the 2 phase II studies, especially in MCL,82 led to the design of a multicenter national trial with bortezomib as a single agent for relapsed MCL. Moreover, recently reported in vitro and murine experimental models suggest a synergy between bortezomib and antisense Bcl-283 or rituximab.84 This synergy will be evaluated in separate clinical trials combining bortezomib with antisense anti–Bcl-2 or rituximab. Additional phase I/II studies have start-

Andre Goy, Frederic Gilles ed to explore toxicity and efficacy of bortezomib in combination with chemotherapeutic agents such as liposomal doxorubicin56 or other anthracycline-based regimens.85 As with myeloma, the same questions remain to be addressed regarding the type of schedule and the dosing in the combination studies, as well as the mechanisms of resistance to proteasome inhibition. We can also expect that molecular profiling will help us understand the mechanisms of resistance to bortezomib in lymphoma cells.51,86 Recent data suggest a role for heat-shock proteins Hsp27 and Hsp90 in the resistance to bortezomib in lymphoma cells.28,72,87

Activity in Other Hematologic Malignancies Hodgkin’s Disease The importance of NF-κB activation and its deregulation in Hodgkin’s disease (HD) is well documented.31,88,89 In vitro studies have also recently provided a rationale for the potential efficacy of bortezomib in HD. The malignant Reed-Sternberg cells of HD are known to constitutively express high levels of activated NF-κB, and in 4 well-characterized HD cell lines, bortezomib was found to reduce cell proliferation in a dose- and time-dependent manner.48 The antiproliferative effect was caused by induction of apoptosis, cell cycle arrest at the G2/M phase, or both. The drug inhibited nuclear localization of NF-κB in a time-dependent manner, induced poly(ADP-ribose) polymerase cleavage, and activated the caspase cascade. Furthermore, bortezomib cleaved Bid in a time-dependent manner and increased the intracellular level of the proapoptotic protein Bax, but had no significant effect on Bcl-2 or Bcl-X. The effect of bortezomib on chemotherapy and TRAIL-induced cell death was also studied. Bortezomib potentiated the effect of submaximal concentrations of doxorubicin and gemcitabine in these cell lines. Similarly, bortezomib potentiated the effects of TRAIL protein and the apoptotic activity of agonistic monoclonal antibodies to the TRAIL receptors R1 and R2.48 An ongoing phase II study of bortezomib in HD at M. D. Anderson Cancer Center will help to further evaluate the potential benefit of this drug in patients with relapsed or refractory HD. Chronic Lymphocytic Leukemia Previous laboratory studies have shown that proteasome inhibition can lead to apoptosis in lymphocytes isolated from patients with CLL but not in normal lymphocytes.90 In another study, proteasome inhibitors did not alter the levels of expression of the proapoptotic Bcl-2 family proteins, Bax and Bid, in CLL cells before the onset of apoptosis. Instead, proteasome inhibitors induced a caspase-independent conformational change of Bax, with its translocation to the mitochondria resulting in mitochondria perturbation, as evidenced by loss of the mitochondrial membrane potential and cytochrome-c release.91 However, as a single agent administered on the same schedule as in MM in a small multicenter study, bortezomib did not show any significant activity in patients with relapsed or refractory CLL, even though proteasome inhibition was detectable in patients’ leukemic cells.92 Additional studies, especially combination studies, are needed with regimens including nucleoside analogues such as fludarabine or antisense anti–Bcl-2 to explore potential synergy in

inducing apoptosis between bortezomib and conventional chemotherapy or other biologic agents in CLL.46 Acute Myeloid Leukemia and Myelodysplastic Syndrome A phase I study of bortezomib in patients with relapsed or refractory acute myeloid leukemia, acute lymphoblastic leukemia, or myelodysplastic syndrome was conducted with doses of 0.75-1.5 mg/m2.93 Although there was detectable proteasome inhibition at 1 hour and increased apoptosis in patients’ blasts in vitro, there was only marginal activity in vivo, with only a transient decrease of blast counts in 2 patients treated at doses of 1.25 mg/m2 and 1.5 mg/m2, respectively. Laboratory data suggest that bortezomib might be more efficient in inducing apoptosis in leukemic cells in combination with other agents. TRAIL can selectively induce apoptosis in tumor cells. Bortezomib sensitizes myeloid leukemia cells to TRAIL-mediated apoptosis by reducing the level of FLICE-inhibitory protein (c-FLIP).94 The combination of bortezomib and TRAIL was much more effective than either agent alone in promoting apoptosis of the murine myeloid leukemia C149. Interestingly, apoptosis sensitization by bortezomib affected neither the activity of NF-κB nor the levels of most antiapoptotic proteins. Another model using human leukemia cells K562 and LAMA 84 showed that bortezomib interacts synergistically with histone deacetylase inhibitors to induce apoptosis in Bcr/Abl positive cells resistant to imatinib mesylate.95 Interactions between the bortezomib and histone deacetylase inhibitors have been examined in Bcr/Abl–positive cells with minimally toxic concentrations of bortezomib plus suberoylanilide hydroxamic acid or sodium butyrate. Such combinations resulted in an appreciable increase in mitochondrial injury, an increase in reactive oxygen species generation,96 diminished NF-κB activation, and caspase activation leading to apoptosis. Apoptosis was also observed in imatinib-resistant K562 cells and CD34+ mononuclear cells obtained from a patient with imatinib-resistant disease.95 In another leukemic model with U937 cells, proteasome inhibitors were shown to appreciably lower the apoptotic threshold of leukemic cells exposed to cyclin-dependent kinase inhibitors such as flavopiridol.97 Together, these findings suggest that combined proteasome inhibition with histone deacetylase inhibitors and/or other chemotherapeutic agents may represent a novel strategy in the treatment of resistant leukemia.

Conclusion Bortezomib is a promising new agent for the treatment of hematologic malignancies. Its remarkable activity in MM led to its fast-track approval by the FDA for relapsed MM. Promising activity was also found more recently in non-Hodgkin’s lymphoma, especially in MCL. As a result, a national multicenter trial with bortezomib as a single agent in relapsed or refractory MCL is ongoing. Encouraging activity of bortezomib was also seen in other subtypes of lymphomas, including follicular lymphoma and DLCL, as well as in Waldenstrom macroglobulinemia. Because of its distinct mechanisms of action and its nonoverlapping toxicity, bortezomib also appears to be a very promising agent in combination with conventional

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Bortezomib in Hematologic Malignancies chemotherapy or other biologic agents, and could help open new perspectives in the treatment of hematologic malignancies.

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