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REVIEW Novel targeted drugs for the treatment of multiple myeloma: from bench to bedside B Bruno1, L Giaccone1, M Rotta1, K Anderson2 and M Boccadoro1, on behalf of the Multiple Myeloma Research Foundation 1

Divisione di Ematologia dell’Universita` di Torino, Ospedale San Giovanni Battista, Torino, Italy; and 2Jerome Lipper Multiple Myeloma Center, Dana-Farber Cancer Institute, Boston, MA, USA

Multiple myeloma remains an incurable plasma cell neoplasm. New insights into its pathogenesis have identified signaling pathways that have become potential therapeutic targets. It has clearly been established that intracellular regulatory proteins and interactions between malignant plasma cells and the bone marrow microenvironment play an important role in their survival and drug resistance. Several new agents associated with molecular targets are currently being investigated to design new treatment strategies aimed at prolonging survival and improving quality of life. This review illustrates their mechanisms of action and the possible future clinical applications. Leukemia (2005) 19, 1729–1738. doi:10.1038/sj.leu.2403905; published online 11 August 2005 Keywords: mutliple myeloma; new drugs; signaling pathways

Introduction Multiple myeloma (MM) is the second most frequent hematological malignancy in the United States and accounts for some 13% of all such diseases.1 It is currently incurable. Recent studies employing high-dose melphalan followed by autologous stem cell transplantation have indicated a survival benefit when compared to conventional chemotherapy.2 The curative potential of allogeneic stem cell transplantation coupled with reduced intensity or nonmyeloablative conditioning regimens remains to be determined.3 MM is a plasma cell disorder characterized by specific genetic aberrations and abnormal gene expression of several proto-oncogenes and tumor suppressor genes. Consequently, several abnormal events in the MM cell and its crosstalk with the bone marrow (BM) microenvironment promote cell survival, tumor progression, and eventually drug resistance. Recent advances in the understanding of the pathophysiology of the disease have identified specific signaling pathways originating from both mutated genes in MM cells and from the BM microenvironment. These have become targets for biologically based drugs that strike the many pathways through which MM cells escape immunosurveillance and chemotherapy.4 Whether as single agents or in combination, they overcome resistance to conventional chemotherapy in large numbers of heavily pretreated patients.4 Close collaboration between basic researchers and clinicians is required to translate advances from the bench to the bedside with the ultimate goal of converting MM into a chronic condition with much longer survival and a better quality of life. In the last couple of years, specialists in basic and clinical research convened in Torino, Italy (April 2004), for a Multiple Myeloma Research Foundation Correspondence: Dr B Bruno, Divisione Universitaria di Ematologia, Azienda Ospedaliera S Giovanni Battista di Torino, Via Genova 3, 10126 Torino, Italy; Fax: þ 39 11 6963737; E-mail: [email protected] Received 6 April 2005; accepted 7 July 2005; published online 11 August 2005

(MMRF) Roundtable on novel targeted agents for the treatment of MM and in Sydney, Australia (April 2005), for the 10th International Myeloma Workshop. Proceedings of the 2004 meeting of the American Society of Hematology also reported interesting and novel information on the use of new agents in MM. This report highlights the mechanisms of action of the ‘rational’ drugs illustrated during these meetings and the recently reported clinical outcomes of a new treatment paradigm.

The microenvironment Clonal MM cells, extracellular matrix proteins, and accessory cells (ie, BM stromal cells) form a complex and dynamic network of events that determine MM cell proliferation, migration, survival, and eventually drug resistance (Figure 1). MM cells bind to matrix proteins such as fibronectin, through integrins VLA-4 and VLA-5, which induces drug resistance and inhibits Fas-mediated apoptosis.5,6 MM cell to stromal cell binding, through VLA-4/VCAM-1 and LFA-1/ICAM-1 molecules, induces the secretion of several cytokines. IL-6 increases cell proliferation via the JAK/STAT and the Ras/MAPK signaling pathways; prevents dexamethasone-induced apoptosis via the PI3K/AKT signaling pathway; induces vascular endothelial growth factor (VEGF) secretion; and inhibits the differentiation of monocytes to dendritic cells, thereby impairing host anti-MM immunity.7–9 VEGF promotes angiogenesis, augments IL-6 production, and hampers the antigen-presenting-cell function of dendritic cells, while tumor necrosis factor a (TNF-a) promotes MM cell to BM stroma cell crosstalk.10–12 Furthermore, stromal cells secrete insulin-like growth factor 1 (IGF-1) and stroma-derived factor 1 (SDF-1), which activate the PI3K/ AKT and the Ras/MAPK signaling pathways.13,14 All these pathways and many others underlie MM pathogenesis and hence are potential targets of agents that may overcome resistance to conventional chemotherapy (Figure 2, Table 1).

Targeting growth factors and their receptors

Insulin growth factors The interactions between IGFs and their receptor IGF-1R stimulate MM cell growth and promote cell survival and migration. Since IGF-1R is expressed on several normal tissues and highly homologous with the insulin receptor, its inhibition was regarded as unsuitable for therapeutic purposes. However, the functional role of the IGF/IGF-1R pathway has recently been characterized in detail as a potential therapeutic target. It has been demonstrated that IGF-1R inhibition, whether by neutralizing anti-IGF-1R-specific monoclonal antibodies, antagonistic peptides, or selective IGF-1R kinase inhibitors: (i) prevents MM

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1730

Figure 1 The myeloma microenvironment: the complexity of the interplay between myeloma cells and microenvironmental cells and the network of cytokine-induced intracellular signaling pathways governs growth, survival, and drug resistance of the malignancy. A better understanding of the pathophysiology of the disease has allowed the development of ‘targeted therapies’ (see text).

Figure 2 Signaling cascades involved in myeloma pathogenesis: IL-6 effects are mediated by the mitogen-activated protein kinase (MAPK), phosphatidylinositol-3 kinase (PI3K)/Akt kinase, and Janus kinase/signaling transducer and activator of transcription 3 (JAK/STAT3) signaling pathways; vascular endothelial growth factor (VEGF) triggers the PI3K-dependent and the MAPK pathways. Insulin like growth factor-1 (IGF-1) triggers the MAPK and PI3K/Akt kinase pathways.

cell proliferation by blocking the Ras/Raf/MAPK and phosphatidylinositol 3-kinase dependent (PI3K)/Akt-1 pathways; (ii) induces phosphorylation of proapoptotic Forkhead transcripLeukemia

tion factor (FKHR); (iii) downregulates intracellular antiapoptotic proteins such as FLIP, survivin, cIAP-2, and XIAP; (iv) as well as increases telomerase activity.13,15–19 In addition to being present

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1731 Table 1 myeloma

Novel targeted drugs for the treatment of multiple

Targeting signaling events in myeloma cell development Targeting insulin growth factor NVP-AEW541 Targeting fibroblast growth factor SU5402 CHIR258 PRO-001 Targeting vascular endothelial growth factor PTK787/ZK222584 GW654652 Targeting histone-deacetylase: SAHA NVP-LAQ824 NVP-LBH589 Targeting myeloma cells and myeloma cell to stromal cell crosstalk Thalidomide ImiDs (thalidomide analogs) Bortezomib Arsenic trioxide Aplidin Targeting bone disease Humanized anti-RANK ligand antibodies RANK.Fc Fc.OPG

in the circulation, IGFs mediate autocrine and paracrine growth, since they are produced by both malignant plasma cells and stromal cells. These in vitro findings have since been substantiated in an in vivo 5TMM murine model.20 Overall, MM is the most sensitive malignancy to IGF-1R inhibitors and thus an ideal testing ground for their clinical application. These inhibitors can be administered orally without adverse side effects on glucose metabolism. NVP-AEW541 is a new orally bioavailable representative IGF-1R inhibitor that distinguishes between cell IGF-1R (IC50 ¼ 0.086 mM) and the closely related cell insulin receptors (IC50 ¼ 2.3 mM). This compound inhibits the IGF/IGF-1R pathway in a tumor xenograft and reduces the growth of IGF-1R-driven fibrosarcomas.21 It is a member of the first class of selective IGF-1R kinase inhibitors with a potentially useful therapeutic window.

Fibroblast growth factor The t(4;14)(p16;q32) translocation occurs in 15% of MM and dysregulates fibroblast growth factor receptor 3 (FGFR3). In the mouse, it confers a growth advantage on B cells, enhanced either by IL-6 or activating FGFR3 mutations. These mutations are uncommon in newly diagnosed MM patients and present in less than 5% of those carrying t(4;14). The clinical impact of t(4;14) as a parameter associated with reduced overall survival (OS) has been shown in large studies.22 Stewart and coworkers22 reported the frequency and prognostic relevance of the t(11;14) and t(4;14) on 120 MM patients treated with highdose melphalan (200 mg/m2) followed by autologous stem cell transplantion. Both translocations were evaluated by immunofluorescence detection of cytoplasmic light chain combined with fluorescence in situ hybridization (cIg-FISH), and were detected in 13.5 and 13% of patients, respectively. Patients with t(4;14) showed a significantly higher relapse rate (79%) and

shorter event-free survival (EFS) (median 9.5 months) than those without (49% and 25.8 months, P ¼ 0.0001), and significantly shorter overall survival (median 18 months vs 46.3 months; P ¼ 0.0053). These findings confirmed that t(4;14) is associated with a poor prognosis, even for patients receiving intensive chemotherapy. To target FGFR3, two FGFR-specific tyrosine kinase inhibitors, SU5402 and CHIR258, have been assessed in MM.23 Both compounds inhibited FGFR3 phosphorylation both in vitro and in in vivo murine models. FGFR3-dependent murine B cells were sensitive to both drugs. After 72 h treatment with 10 M SU5402, human MM cell lines which express both t(4;14) translocation and constitutively active mutant FGFR3, such as the KMS11 line, displayed an 85% decrease in S-phase cells, a 95% increase in G0/G1 cells, and a 4.5-fold increase in apoptotic cells. CHIR258 induced dose-dependent apoptosis. Furthermore, in cell lines expressing wild-type FGFR3, the stimulating effect of FGF ligand was abrogated by treatment with FGFR3 inhibitors. In contrast, MM cells without t(4;14) or with t(4;14) plus a secondary ras mutation did not respond. In vivo activity of CHIR258 was also demonstrated in MM xenograft mice. Primary patient tumor cells with known t(4;14) also responded. Overall, these data demonstrate SU5402 and CHIR258 activity against human MM cells and support the design of clinical trials of early intervention with FGFR3 inhibitors in patients with t(4;14). A unique class of recently characterized high-affinity anti-FGFR3 human monoclonal antibodies (ProChon Biotech, Rehovot, Israel) neutralizes both wild-type and constitutively activated mutant FGFR3 of several cell lines. The monoclonal antibody PRO-001 in particular completely blocks FGFR3 activity in vitro and inhibits the growth of ectopic, FGFR3-dependent mouse tumors. These findings point to PRO-001 as a promising agent for the treatment of patients carrying the t(4;14) translocation.

Vascular endothelial growth factor Angiogenesis is an important mechanism for tumor growth. VEGF plays a pivotal role in MM biology,10,11 since VEGF serum levels, degree of angiogenesis, and microvessel density are all correlated with disease severity and survival. However, VEGF may act by means of several other mechanisms. It stimulates the growth and migration of MM cells, increases IL-6 production, and prevents dendritic cells from presenting antigens to host T cells. Binding of VEGF to MM cells through tyrosine-kinase receptors, in particular VEGF receptor 1 (also defined as FMSlike tyrosine kinase receptor or FLT1), triggers important downstream signaling pathways: a PI3-kinase/protein kinase Ca (PKCa)-dependent cascade mediating MM cell migration on fibronectin; MEK-extracellular-signal-regulated protein kinase (ERK) pathway mediating cell proliferation, as well as survival signaling through upregulation of Mcl-1 and survivin. PTK787/ ZK 222584 (PTK787) is designed to bind specifically to the tyrosine kinase domain of VEGF receptor and inhibit angiogenesis and other VEGF cell effects. In in vitro studies, it enhanced the inhibitory effect of dexamethasone on MM cell growth and overcame IL-6 protection against dexametasone-induced apoptosis. These findings demonstrate that PTK787 acts directly on both MM cells and their microenvironment by inhibiting the IL-6 paracrine loop that mediates tumor cell growth. These preclinical findings provide the rationale for clinical trials seeking to overcome drug resistance and improve outcome in patients with relapsed MM. VEGF inhibitors have been designed to bind to VEGF receptors and block VEGF signaling cascades. PTK787/ZK 222584 (PTK/ZK) (Novartis Pharma AG, Basel, Leukemia

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1732 Switzerland) is an oral inhibitor of angiogenesis. In preclinical models, it has inhibited the growth of MM cells in the BM microenvironment. Objective responses have been reported in glioblastoma, colorectal and renal cell cancer patients in phase I/II clinical trials using PTK/ZK as single agent. PTK/ZK significantly reduced the tumor blood supply, measured by dynamic-contrast-enhanced magnetic resonance imaging. This finding was correlated with improved patient outcome. Chronic, once-daily administration of oral PTK/ZK, alone or in combination with standard chemotherapy, has generally been well tolerated in over one thousand patients so far. The most commonly reported complaints of mild to moderate severity have been nausea, fatigue, vomiting, and dizziness. PTK/ZK (1.25 mg/day) is currently being evaluated in clinical phase I trials for MM. Moreover, GW654652, another recently developed pan-inhibitor of VEGF receptors, blocks the growth and migration of MM cells in the BM microenvironment and the stage is thus set for its clinical application.

tumor cell line growth at submicromolar concentrations. Moreover, they induced tumor cell apoptosis at low concentrations in cell proliferation assays. Even at higher concentrations, they only caused reversible cell cycle arrest in normal human dermal fibroblasts. In in vivo nude mice studies, NVP-LAQ824 inhibited or stabilized the growth of established human lung, colon, or breast tumor xenografts, regardless of their p53 tumor suppressor gene status. NVP-LAQ824-treated cells did not undergo histone acetylation, suggesting that its antitumor activity is based on HDAC inhibition. However, it was also observed that NVPLAQ824 affected protein stability through hsp90 protein acetylation, and that this led to decreased levels of oncogenic proteins (eg, Her2/neu and BCR-abl) as well as depletion of antiangiogenic factors such as VEGF. These findings suggest that NVP-LAQ824 may operate through several anti-MM mechanisms.

Targeting myeloma cells and myeloma cell to stromal cell crosstalk Targeting signaling pathways in tumor cell development

Thalidomide (Table 2) Histone deacetylase inhibitors Histone acetylation plays a key role in gene expression, cell differentiation, and survival. It is regulated by the activity of histone acetyltransferases and histone deacetylases (HDAC). Abnormal acetylation has been described in the development of several solid tumors and leukemias. HDAC regulate the acetylation status of nucleosomal histones and the function of transcription factor complexes at the transcriptional level. Their inhibition causes differentiation and/or apoptosis in transformed cells. Mitsiades et al24 showed that HDAC inhibitors, such as the suberoylanilide hydroxamic acid (SAHA), induce potent apoptosis on both MM cell lines and tumor cells from patients, both sensitive and resistant to conventional or new treatments. HDAC function is critical for MM cells, since it maintains a transcriptional program indispensable for their uncontrolled proliferation and/or inappropriate resistance to proapoptotic stimuli. By primarily targeting the regulation of gene expression, HDAC inhibitors affect both MM cells and their interactions with the microenvironment. Gene expression profiling studies showed that HDAC inhibition triggers a distinct transcriptional signature hallmarked by the suppression of signaling pathways crucial for MM cell proliferation, survival, and drug resistance. These pathways downregulate the IGF/IGF-1 receptor axis and the IL-6 receptor signaling cascades; as well as suppress DNA and antiapoptotic molecule (eg, caspase inhibitors) synthesis, oncogenes, as well as DNA repair enzyme synthesis, transcription factors, nucleocytoplasmic transport regulators, and adhesion molecules. Importantly, HDAC inhibitors enhance MM cell sensitivity to other agents, including dexamethasone and cytotoxic chemotherapy, as well as new drugs such as thalidomide analogs, proteasome inhibitors, and heat shock protein (hsp) 90 inhibitors. Oral SAHA is bioavailable and well tolerated, objective responses have been observed in phase I clinical trials, providing the rationale for its wider application. A phase I trial of oral SAHA in relapsed or refractory MM patients is currently in progress at the Jerome Lipper Multiple Myeloma Center in Boston. The anticancer effects of two novel HDAC inhibitors, NVPLAQ824 and NVP-LBH589 (Novartis Institute for BioMedical Research, Basel, Switzerland), have been shown to partially inhibit HDAC activity in purified H1299 lung carcinoma cells, transcriptionally activated the p21 promoter, and inhibited Leukemia

Agents capable of extending disease control with a minimum impact on quality of life are urgently needed for patients ineligible for high-dose treatments because of age or comorbidities. Thalidomide, withdrawn from the market in the 1960s due to its teratogenicity, has recently been used to treat malignant diseases. Its remarkable effects on the MM microenvironment include: (i) inhibition of angiogenesis, induced by VEGF and basic FGF; (ii) reduction of MM cell adhesion to extracellular matrix proteins and stromal cells; (iii) downregulation of several cytokines involved in cell survival and proliferation; enhancement of host anti-MM immunity. It was first employed as a single agent in refractory MM.25 Barlogie et al26 reported X50% reduction of the monoclonal paraprotein in 30%, and near-CR in 14% of 169 heavily pretreated and refractory MM patients. After 2 years, EFS and OS were 20 and 48%, respectively, and 34 and 69%, in responding patients. As a single agent, its efficacy was confirmed in smaller series of patients.27–31 The observation of synergism formed the rationale for its use with dexamethasone, where potent antitumour effects were seen in patients refractory to both drugs given previously as single agents, and with chemotherapy.32–35 The efficacy of thalidomide as salvage treatment prompted to explore its use in untreated MM.36–41 Rajkumar et al36 reported significant activity in indolent and smouldering MM. Weber et al38 employed it in asymptomatic MM and in combination with dexamethasone in symptomatic untreated MM. Palumbo et al,40 on behalf of the Italian Multiple Myeloma Study Group, reported 18% complete remission and 6% near-complete remission in newly diagnosed elderly patients treated with monthly courses of oral melphalan and prednisone for 7 days, and low-dose thalidomide until progression or relapse. A retrospective matched case-control analysis of 200 patients who entered two consecutive studies and received thalidomide plus dexamethasone or vincristine– doxorubicin–dexamethasone as primary therapy in preparation for autografting showed that the thalidomide arm had a significantly higher response rate (76 vs 52.5%, P ¼ 0.0004).41 Overall, severe hematological toxicity has rarely been reported. Venous thromboembolism has emerged as the most prominent adverse effect when thalidomide was used in combination with other agents in untreated MM, whereas its incidence was low when employed as single agent in advanced MM.26 Rajkumar et al36 reported a 10% incidence with

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1733 Table 2

Thalidomide containing regimens in relapsed, refractory and untreated multiple myeloma Patients Age (in years) Disease status

Singhal et al (1999) Rajkumar et al (2000) Barlogie et al (2001) Hus et al (2001) Neben et al (2002) Tosi et al (2002) Mileshkin et al (2003) Palumbo et al (2001) Dimopoulos et al (2001) Kropff et al (2003) Lee et al (2003) Rajkumar et al (2001) Rajkumar et al (2002) Weber et al (2003) Rajkumar et al (2003)

84 16 169 53 83 60 75 77 44 60 236 16 50 40 28 31

38%460 64 (48–85) 40%460 63 (32–79) 59 (34–86) 60 (35–78) 64 (36–83) 65 67 18–75 60 (31–84) 60 (38–75) 61 (33–78) NR NR 61 (40–74)

Cavo et al (2005)

200

F

Regimen

Relapse/refractory Relapse/refractory Relapse/refractory Relapse/refractory Relapse/refractory Relapse/refractory Relapse/refractory Relapse/refractory Relapse/refractory Relapse/refractory Relapse/refractory Diagnosis Diagnosis Diagnosis Diagnosis Diagnosis

Thal Thal Thal Thal Thal Thal Thal Thal+Dex Thal+Dex Hyper CTD DT-PACE Thal Thal+Dex Thal+Dex Thal Thal

Diagnosis

Thal+Dex vs VAD

Overall response a

32% 25%c 30%c 36%c 42% 28%c 28%c 41%c 55% 68%c 32%c 38%c 64%c 72%e 36%c 34%c 75 vs 52%

PFS (follow-up)

OS (follow-up)

b

50% (3 mo) 51/84 (12 mo) F F 20% (24 mo) 48% (24 mo) F F 45% (17 mo) 86% (17 mo) d d E50% (9 mo) 22/28 (9 mo) 50% (6 mo) 50% (15 mo) 50% (12 mo) F 450% (10 mo) 50% (12 mo) b 50% (11 mo) 50% (19 mo) F F 2/16 (12 mo) F F F 1/40 (9 mo) 3/40 (9 mo) 3/28 (25 mo) 2/28 (25 mo) 80% (12 mo) 93% (12 mo) 63% (24 mo) F F

a

X25% paraprotein reduction. Event-free survival. X50% paraprotein reduction. d In patients with response 425%. e X75% paraprotein reduction. PFS: progression-free survival; OS: overall survival; mo: months. b c

thalidomide and dexamethasone as in early stage myeloma. The incidence of venous thromboembolism was evaluated in untreated patients randomly assigned to receive induction chemotherapy with or without thalidomide. In the arm with additional thalidomide, the incidence was 28% compared to 4% in the nonthalidomide arm.42 Another study showed that patients treated with thalidomide and doxorubicin were at a high risk of venous thromboembolism.43 These findings show that prophylaxis with low-molecular-weight heparin or other anticoagulants is mandatory in newly diagnosed MM.

Thalidomide analogs (Table 3) Lenalidomide (Revlimids) is a novel agent that targets both MM cells and their microenvironment. Preclinical studies have shown that it is approximately 50–2000 times more potent than its parent compound. It stimulates T-cell proliferation, increases IL-2 and IFN-a secretion up to 100 times, strongly inhibits TNF-a activity, induces apoptosis of drug-resistant MM cells, and inhibits their adhesion to stromal cells and angiogenesis. Animal studies have demonstrated the absence of teratogenic effects. In a phase I dose-finding study (5–50 mg/day) on relapsed/ refractory MM patients, the best balance between efficacy and toxicity was obtained at the dose of 25 mg/day. The doselimiting toxicity was myelosuppression and no instances of the well-known dose-limiting toxicities of thalidomide, such as sedation, constipation or neuropathy, were reported. In all, 71% of patients had a 425% reduction in monoclonal paraprotein.44 A randomized comparison of a 3-week course of 15 mg twice a day vs 30 mg a day, followed by a 1-week rest, was conducted in a series of 101 relapsed/refractory patients. High-dose dexamethasone was combined with lenalidomide for patients with stable disease or progression after 8 weeks of single-agent treatment. Response rates were similar in both cohorts. The overall response rate was 36% with 10% complete remission. In all, 43% of patients achieved stable disease (Richardson PG, Blood 2002; 100: 386, abstract). In a multicenter trial on 222

patients with advaced disease, single-agent lenalidomide at 30 mg/day led to 28% of responses, defined as a monoclonal paraprotein reduction of at least 25% (Richardson PG, Haematology 2005; 90: 737, abstract). Interestingly, lenalidomide in combination with dexamethasone in newly diagnosed MM has been evaluated in 13 patients with a response rate of 85% (Rajkumar SV, Blood 2004; 104: 331, abstract). Two phase III studies comparing lenalidomide plus dexamethasone vs dexametasone plus placebo in previously treated MM have recently been concluded (Dimopoulous M, Haematology 2005; 90: 0402, abstract). In both studies, the MM-009 study by Weber et al38 and the MM-010 study by Dimopoulous et al, the median time to progression for the lenalidomide arm was not reached (460 weeks and 447 weeks, respectively). In contrast, the median time to progression in the dexamethasone/placebo arm was 19.9 and 20.4 weeks, respectively (Po0.00001). The overall response rate was significantly higher in the lenalidomide/dexamethasone group compared to the dexamethasone group (51.3 vs 22.9%, Po0.0001, and 47.6 vs 18.4%, Po0.001, in MM-009 and MM-010, respectively).

Bortezomib (Table 3) The proteasome is a key regulator of many cellular processes. Preclinical studies have strongly indicated it as a highly effective therapeutic target in MM. Bortezomib (VELCADEs, formerly known as PS-341) is the only proteasome inhibitor so far approved for clinical trials. In 2003, it was approved for the treatment of MM patients who had received at least two lines of treatment and were progressing after the last. Two major trials (SUMMIT and CREST) contributed to this approval.45 SUMMIT was a phase II study for patients with relapsed and refractory MM. Bortezomib was administered i.v. at 1.3 mg/m2 twice weekly for 2 weeks every 3 weeks for a maximun of six cycles. The cumulative response rate, including complete, partial, and minimal response, was 35% in patients who had undergone a median of six previous lines of chemotherapy. CREST was a Leukemia

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Leukemia

Len Len Len+Dex Len+Dex vs Dex+placebo Len+Dex vs Dex+placebo Bor vs Dex Bor+Dex Bor+Len Bor+Adr+Thal+Dex Bor Bor+MP Bor+Adr+Dex Bor+Dex Bor+Dex+Thal+PACE Relapse/refractory Relapse/refractory Diagnosis Relapse/refractory Relapse/refractory Relapse/refractory Relapse/refractory Relapse/refractory Relapse/refractory Diagnosis Diagnosis Diagnosis Diagnosis Diagnosis F F 61 (32–78) F F F 67%460 F 14465 60 100%X65 55 (37–66) 63 F 34 (19) 222 31 (13) 351 354 333 15 9 (6) 20 (14) 28 (22) 24 (11) 21 38 (23) 57

Valued as X50% reduction in paraprotein levels. Valued as X25% reduction in paraprotein levels. Phase III trial. d Time to progression. e Valued as X75% reduction in paraprotein levels. f Peripheral blood stem cell harvest not affected. g Near CR or CR. Bor: bortezomib; Dex: dexamethasone; Len: lenalidomide; Adr: adriamycin; Thal: thalidomide; MP: oral melphalan and prednisone; PACE: continuous infusion of cisplatin–adriamycin– cyclophosphamide–etoposide; PFS: progression-free survival; OS: overall survival; mo: months. c

b

a

F F F F F 80 vs 66% (12 mo) F F F F F F F F F F F d 447 vs 20.4 weeks d 460 vs 19.9 weeks 6.22 vs 3.49 mo 5/15 (5 mo) F F F F F F F 21% 28%b 85%a 47.6 vs 18.4% 51.3 vs 22.9% 38 vs 18%a 73%a 4 stable; 2 minor response 50%e 41% 91% 95%a 83%a 26%g

OS (follow-up) Age (in years) Patients

a

Richardson et al (2002) Richardson et al (2005) Rajkumar et al (2004) Dimopoulous et al (2005)c Weber et al (2005)c Richardson et al (2005)c Kropff et al (2005) Richardson et al (2005) Hollmig et al (2004) Richardson et al (2004) Mateos et al (2005) Oakervee et al (2005)f Jagannath et al (2005)f Barlogie et al (2004)f

Table 3

Lenalidomide and bortezomib in multiple myeloma

Disease status

Regimen

Overall response

PFS (follow-up)

1734 phase II trial for relapsed or refractory patients after first-line therapy. Bortezomib was given at 1.0 or 1.3 mg/m2 according to the SUMMIT schedule. CREST’s preliminary results suggest that efficacy and toxicity are potentially dose-related. In APEX, a large phase III trial, bortezomib was compared to high-dose dexamethasone in 669 relapsed or refractory MM patients. Patients treated with bortezomib had higher response, longer time to progression, and longer survival. The overall response rate, including CR and PR, was 38% in the bortezomid arm vs 18% in the dexamethasone arm (Po0.001). In all, 1 year survival was 80% in the bortezomid arm vs 66% in the dexamethasone arm (P ¼ 0.002). The rates of grade 4 toxicities and discontinuations were similar in both arms.46 These and other trials (Table 3) provide the rationale for the development of new protocols aimed at evaluating the safety and efficacy of bortezomib to treat newly diagnosed MM (Richardson PG, Haematology 2005; 90: 504, abstract; Hollmig K, Blood 2004; 104: 2399, abstract; Richardson PG, Blood 2004; 104: 336, abstract; Mateos MV, Haematology 2005; 90: 726, abstract; Barlogie B, Blood 2004; 104: 538, abstract).47–49 Jagannath et al49 illustrated the results obtained in 38 newly diagnosed patients. Bortezomib was given according to the SUMMIT schedule, whereas 40 mg dexamethasone was added on the day of and after each bortezomib dose when at least a partial remission was not reached after two courses, or after four courses for patients not in complete remission. The most common adverse events (grade 1–3) were peripheral neuropathy (56%), fatigue (56%), diarrhea (44%), constipation (38%), and neurophatic pain (12%). Grade 4 neutropenia was reported in one patient. Major responses according to the EBMT criteria were seen in 83% of the first 23 patients who completed six courses. The best responses were observed after cycles 2 (43%) and 4 (39%). Furthermore, bortezomib did not affect successive peripheral hematopoietic cell collection.50 Another study recently evaluated an induction regimen based on bortezomib in combination with doxorubicin and dexamethasone (PAD regimen) prior to stem cell mobilization and high-dose therapy with autologous transplantation.51 After induction alone, 20 out of 21 achieved at least PR. Importantly, PAD did not hamper effective peripheral hematopoietic cell mobilization. Mateos et al are currently investigating the most appropriate dose and efficacy of bortezomid in combination with melphalan and prednisone in untreated MM patients older than 65. Results on the first 24 patients have been reported. Bortezomib was used at a dose of 1.3 mg/m2. No dose-limiting toxicities have been observed. Significant responses have been seen in 10 out of 11 evaluable patients.52 Overall, the results of these clinical studies support the benefit of the use of bortezomib in relapsed/ refractory and untreated MM.

Arsenic trioxide Arsenic trioxide (AsO3) exerts its antitumor activity in hematological malignancies through several pathways. Its many postulated mechanisms include: (i) induction of apoptosis by Bcl-2 downregulation; (ii) suppression of NF-kB and VEGF functions; (iii) reduced adhesion of MM cells to stromal cells; and (iv) increased sensitivity to chemotherapy. However, in in vitro assays, a major role is played by the inhibition of STAT3 activation and JAK/STAT signaling pathways in MM cells. AsO3 anti-MM activity also appears to be inversely correlated with intracellular glutathione (GSH) levels, possibly because of the repair of the mitochondrial damage. Moreover, its effects are enhanced by agents such as ascorbic acid that reduce GSH

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1735 levels. Glutathione-S-transferases (GSTs) are a large family of phase II detoxification enzymes expressed in several isoforms. They catalyze the conjugation of GSH to many compounds, including chemotherapeutic agents. The isoform GSTP1 has been described as a direct inhibitor of the c-Jun N-terminal kinase (JNK). GSTP1 knockout mice show increased activity of the JNK and JAK/STAT signaling pathways. Specific GSTP1 inhibitors have been synthesized. TLK199 mimics the phenotype of GSTP-deficient mice in vivo. Several reports have now reported that overexpression of GSTP, or abnormal GSH levels, are frequent in cancer cell lines and confer increased drug resistance. GSTP1 polymorphism in MM patients correlates with the response to chemotherapy.53 Interestingly, MM patients carrying the 105Val allele, instead of 105Ile, show a better primary response and progression-free survival. In contrast, the 105Val GSTP1 variant is less active in conjugating GSH to cyclophosphamide metabolites and it has been suggested that improved responses may be due to reduced detoxification in malignant cells. These findings indicate that GSTP1 is involved in drug detoxification and drug-induced modulation of JNK activity, which could ultimately affect the activity of many drugs, including AsO3. AsO3 (Trisenoxs) was initially used as a single agent in patients with advanced MM, and subsquently combined with dexamethasone, ascorbic acid, and low-dose melphalan in various combinations.54 Most current protocols employ AsO3 at 0.25 mg/kg on a twice-weekly schedule for up to 12 weekly courses after a 4–5-day loading dose. The drug has a favorable safety profile and nonhematopoietic toxicities are rare. Single-agent therapy in 38 predominantly refractory MM patients produced a response rate of 29%. Addition of ascorbic acid in a similar group substantially increased the response. Partial remissions, defined as 450% reduction in monoclonal paraprotein, were only observed when AsO3 was used in combination with ascorbic acid, ascorbic acid and dexamethasone, or ascorbic acid and melphalan. In all studies, durable complete remissions and near-complete remissions were observed. Most importantly, additional phase II trials are currently evaluating the efficacy of AsO3 and ascorbic acid in combination with bortezomib and thalidomide.

Aplidins (Aplidium albicans) Aplidin (dehydrodidemnin B) is a cyclic depsipeptide isolated from the Mediterranean tunicate Aplidium albicans that has shown promising in vitro and in vivo antitumor activity in a variety of malignancies. Its apoptotic effects are determined by triggering Fas/CD95-, JNK-, and mitochondrial-mediated apoptotic signaling pathways and have been observed at nanomolar concentrations (10–100 nM) in human leukemic cell lines and primary leukemic cell cultures. Strong apoptotic activity on a panel of human MM cell lines, as well as on MM cell cultures from patients, including those resistant to conventional and novel therapies (Gajate C, Multiple Myeloma Research Foundation (MMRF) Roundtable. Torino, Italy, 2004; abstract). Importantly, normal cells were more resistant to Aplidin than tumor cells. Primary cultures of normal, resting blood lymphocytes, in fact, were spared or only weakly affected by Aplidin, although mitogen-stimulated T lymphocytes were sensitive to the drug-induced apoptosis. The preliminary results of 327 patients treated with Aplidin, 215 enrolled in phase I and 112 in phase II clinical trials, were recently reported (Lopez-Martin J, Multiple Myeloma Research Foundation (MMRF) Roundtable. Torino, Italy, 2004; abstract). The drug was administered intravenously over 3 h infusions in most patients. The phase I

program set out to deliver a similar dose intensity of approximately 2.5 mg/m2/week. When a central venous catheter was not used, injection-site reactions included erythema, pain, and phlebitis in about 20% of the patients. A median of two courses was given. The most important side effects concerned the muscles and bones and were sometimes associated with fatal acute renal failure. In all, 9% of the patients experienced reversible grade 3–4 increases in CPK; 3% complained of grade 3–4 myalgia, and 4% of muscular weakness. These symptoms usually resolved within 1–3 weeks. Liver toxicity was doselimiting. Grade 3–4 increases in AST/ALT in 13% of the patients were followed by elevations in alkaline phosphatase (8%). Hematological laboratory abnormalities were not dose-limiting. Grade 3–4 mucositis was not reported and severe vomiting and grade 3 diarrhea and constipation were described in less than 5%. Pulmonary embolism and deep venous thrombosis were reported in patients with progressive disease, but it was not possible to rule out a correlation with Aplidin treatment in two patients. Based on its in vitro activity and safety profile, several phase II clinical studies are in progress in solid tumors and hematological malignancies. A phase II trial in MM is being developed in the United States and Spain.

Targeting multiple pathways

New strategies Understanding of the pathogenesis of MM has led to the identification of multiple signaling pathways that together endow clonal plasma cells with survival advantages and eventually drug resistance. Strategies aimed at simultaneously hitting these pathways to prevent cells from escaping drug effects are being investigated. IL-6 is produced by both MM and stromal cells, is the major growth and survival factor for MM, and affects at least three major signaling pathways. It mediates growth and survival via the JAK/STAT3 pathway and activation of the MEK/MAPK signaling pathways. Moreover, IL-6 prevents dexamethasone-mediated apoptosis by activating the PI3K/AKT-1 signaling pathway. Human MM cells were shown to become independent of the IL-6/JAK/STAT3 pathway when cocultured with BM stromal cells. This stresses the fact that the crosstalk between MM cells and the BM microenvironment stimulates additional IL-6-independent pathways that induce a survival advantage. Further studies also showed that selective targeting of the MEK/MAPK pathway by exposure to either a small compound inhibitor of MEK or MAPK-directed siRNA constructs was not sufficient to induce apoptosis, regardless of the presence or absence of stromal cells. These findings showed that targeting IL-6/STAT3 or MAPK independently did not induce apoptosis in MM cells cocultured with stromal cells.55 Combined targeting of both pathways, however, efficiently induced apoptosis. Importantly, this effect was observed with both MM cell lines as well as freshly isolated MM cells from the patients’ BM aspirates. Biochemical analyses revealed that stromal cells stimulate the JAK/STAT3 pathway partly through IL-6-independent mechanisms. These data provide the experimental evidence that simultaneous targeting of different independently activated pathways plays a key role in efficiently inducing apoptosis of MM cells in the BM microenvironment. This underscores the fact that combinations of new drugs that hit multiple pathways will have direct implications in better MM management. Mcl-1, a member of the prosurvival Bcl-2 subfamily, is another key protein in MM cell biology, which is tightly regulated by IL-6. The use of Mcl-1 antisense triggers an Leukemia

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1736 important decrease in viability by inducing apoptosis in MM cell lines. The ability of different signaling pathway inhibitors to inhibit Mcl-1 protein expression was recently investigated.56 When AG490, an inhibitor of JAK2, was used, one-third of MM cell lines and plasma cells from MM patients displayed a remarkable downregulation of Mcl-1 expression that correlated with increased apoptosis. Similar results were also obtained by exposing MM cell lines to an inhibitor of IGF-1R. It has recently been demonstrated that the proapoptotic proteins with single Bcl-2 homology domain, BH-3-only proteins, are essential for apoptosis and regulation of antiapoptotic Bcl-2 family proteins. The three major isoforms of Bim, a BH-3-only protein, are expressed in MM cells and downregulated by IL-6. Upregulation of Bim with concomitant downregulation of Mcl-1 can be obtained by blocking IL-6 signaling pathways. Bim is strongly associated with Mcl-1, and, upon apoptosis, the endogenous Mcl-1/Bim complex falls to barely detectable levels. These observations strongly indicate that the decrease of Mcl-1/Bim interactions may be prompted by a decline of Mcl-1 level. In apoptotic MM cells, Bim level was increased while the Mcl-1/Bim complex was almost undetectable, suggesting that Bim is released and becomes available to exert its proapoptotic function. It can be concluded that a future clinical approach may be focused on compounds such as BH-3-peptides or BH-3peptidomimetics that disrupt the Mcl-1/Bim system.

Targeting pathways mediating bone disease Most MM patients present with osteoporosis, osteolytic lesions, and/or pathological fractures. Bisphosphonates are currently the main therapy for treatment of bone disease, with possible additional effects on gd T cells that enhance anti-MM activity.50,52,51,57 Bone is destroyed via osteoclast activation regulated by many cytokines, once defined as osteoclast activating factors (OAFs). However, a central role in abnormal bone resorption is played by the osteoprotegerin (OPG)/receptor activator of NKkB (RANK)/RANK-ligand (RANK-L) axis (Figure 3). In healthy individuals, stromal cells secrete OPG, a soluble decoy receptor, that prevents osteoclast activation by competing with RANK, expressed on osteoclasts, in binding to RANK-L. When RANK-L binds to its functional receptor RANK, osteoclasts are activated. The RANKL–OPG ratio determines the level of osteoclast activity. Binding of MM cells with stromal cells determines a remarkable imbalance between OPG and RANK-L that suppresses OPG levels and favors osteoclastogenesis and bone destruction. In vitro and in vivo studies to clarify the role of the OPG/RANKL/RANK axis are under way to identify new therapeutic strategies. The efficacy of recombinant OPG and RANK constructs in preventing bone disease was first illustrated in mice.58 MM was induced in C57BL/KaLwRij mice by i.v. injection of 5T2MM murine MM cells. After 8 weeks, serum monoclonal paraprotein concentrations showed that all these mice had MM. They were treated with Fc.OPG or vehicle from week 8 until killing at week 12, or with RANK.Fc from week 0 until killing. All animals developed MM bone disease characterized by typical osteolytic bone lesions. Treatment with Fc.OPG or RANK.Fc prevented the development and increase of osteolytic lesions by completely inhibiting osteoclast formation and also reduced the serum paraprotein concentration by 25%. Furthermore, treatment with Fc.OPG from the time of MM cell injection resulted in a significant decrease in this concentration and a significantly prolonged time to morbidity. These findings show that targeting the OPG/RANKL/RANK system with recombinant OPG or soluble RANK constructs may prevent Leukemia

Figure 3 (a) Osteoprotegerin (OPG), a decoy receptor synthesized by normal bone marrow stromal cells, prevents osteoclast activation by competing with receptor activator of NK-kB (RANK) on osteoclasts, in binding to RANK-ligand (RANK-L). (b) In myeloma bone disease, the binding of myeloma cells to stromal cells determines a remarkable imbalance between OPG and RANK-L with highly suppressed OPG levels that favor osteoclastogenesis and osteolysis. TNF-a, IL-6, and IL-11 are also potent stimulators of osteoclast formation.

bone disease. If translated into the clinical setting, this approach could result in a dramatic improvement of quality of life and enhanced disease-free survival of MM patients.

Conclusions The MMRF is funding several basic research projects to better understand the pathogenesis of MM. The biology of this disease

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1737 is dramatically complex as it cannot be associated with a single causative molecular mechanism. Indeed, our understanding of the signaling pathways that determine MM cell development, growth, survival, and drug resistance within the BM microenvironment is the key to the rapid identification of molecular targets and elaboration of new molecules against them so as to design phase I and II clinical trials. A vast array of advanced technologies (eg, gene expression profiling) is now greatly broadening our knowledge of the molecular mechanisms of MM biology and potential therapeutical targets. Their further characterization will allow the development of new generations of selective drugs. One of the challenges that lie ahead is identification of the best molecular synergism between new drugs, possibly in association with the other so-called ‘rational’ drugs, such as bortezomid, with already established roles in the management of MM. The new prospects for the treatment of MM will stem from fruitful collaboration between basic researchers and clinical investigators resulting in the transfer of new insights into MM biology from the bench to the bedside with the ultimate goal of prolonging survival and improving quality of life.

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