Molecular targeted therapies for multiple myeloma.

Multiple myeloma, a malignant plasma cell disorder, remains largely incurable despite conventional chemotherapy and hematopoietic stem cell transplantation. Recent studies have characterized the molecular mechanisms by which multiple myeloma plasma cells interact with the bone marrow microenvironment to regulate tumor cell growth, survival, and migration. This new understanding of the disease pathogenesis has provided a framework for developing novel therapies that target these molecular pathways. The first drug of its class, bortezomib, a proteasome inhibitor, ushered in this new era of drug development, and a variety of other novel agents are now in clinical and preclinical testing.

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Multiple myeloma is a malignant hematologic disorder that until now has remained almost universally fatal. It represents approximately 1% of all malignancies and 13% of hematologic malignancies in adults. In the United States, about 13,200 cases were diagnosed in 2000, resulting in 11,200 deaths.

The diagnosis of multiple myeloma is based on the detection of an abnormal monoclonal paraprotein in the serum or urine, bone lytic lesions, and >10% plasma cells in the bone marrow differential, in the absence of causes of secondary marrow plasmacytosis such as infection or an autoimmune process. The clinical manifestations result primarily from marrow failure, immunosuppression, lytic lesions of the axial skeleton and long bones, and renal failure.

The immunosuppression is multifactorial in etiology, involving neutropenia, decreased antigen-presenting cell and T-cell function, reduced production of normal immunoglobulin, and compromised immunoglobulin function due to the presence of paraprotein. The overgrowth of myeloma cells in bone marrow leads to marrow failure. Thus, defects of both innate and acquired immunity occur.

Without treatment, the average survival of patients with myeloma is approximately 6 months. Since the introduction of melphalan and steroids

in the 1960s and autologous transplantation in the 1980s, the average survival with treatment has been extended to 3 to 5 years.

In recent years, advances in understanding the pathogenesis of myeloma have led to the development of novel therapies targeting key molecules in its pathogenesis, both in the myeloma cell and in the bone marrow microenvironment. Since 2000, new therapies based on molecular targeting, including proteasome inhibitors and immunomodulatory agents, have significantly improved the outcome of patients with end-stage disease. While these agents are currently under evaluation as first-line therapy, their full impact on survival is yet to be realized.

Ig Gene Translocations Lead to Oncogene Overexpression

Although much has been learned about the biology of myeloma in recent years, the causes remain unknown. The malignant cells are derived from plasma cells whose Ig VDJ genes have undergone the processes of somatic hypermutation and isotype switch recombination in the germinal centers of lymph nodes. These cells subsequently migrate and expand in the bone marrow.

The malignant plasma cells characteristically produce a single abnormal serum monoclonal paraprotein, or M-protein, which is a type of immunoglobulin with a constant isotype and light-chain restriction. The paraprotein is uniform in migration pattern when subjected to electrophoresis.

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The isotype is IgG in 53% of patients, IgA in 25%, and IgD in 1%. Approximately 15% of cases show Bence-Jones protein, in which immunoglobulin light-chain only is expressed by the malignant clone. In 1% of cases there is no paraprotein production.

Karyotypic abnormalities are being increasingly characterized. Normally involved in class switch recombination, the immunoglobulin heavy chain region on chromosome 14q32 is involved in illegitimate translocations in 50% of patients with monoclonal gammopathy of uncertain significance (MGUS, a predecessor of multiple myeloma), 65% of myeloma patients, and >90% of myeloma cell lines.

Chromosome 14q32 translocations involving the immunoglobulin heavy chain region therefore are often early events in the pathogenesis of myeloma and result in aberrant overexpression of oncogenes such as cyclin D1, cyclin D3, and fibroblast growth factor receptor 3 (FGFR3). Karyotypic instability is seen at the earliest stage of the disease and increases with disease progression. In addition, as the disease progresses, there are activating mutations of proliferation factors such as N- and K-RAS and c-myc, as well as inactivation of antiproliferation factors p18 and p53, Rb, and PTEN.

FGFR3 is a tyrosine kinase receptor for the fibroblast growth factor family of ligands, which are expressed ubiquitously by cells of mesodermal origin. These receptors regulate many cellular processes and have been strongly implicated in tumorigenesis and angiogenesis, with signaling via signal transducer and activator of transcription 3 (STAT3).

Deregulated FGFR3 mutants in myeloma cell lines appear to activate mainly the mitogen-activated protein kinase (MAPK) pathway, although other pathways also may be activated.

Cyclins 1, 2, and 3 are commonly overexpressed in myeloma. In normal cells, Rb protein, in association with histone deacetylase (HDAC), inhibits transcription factor E2F in the quiescent cell. On mitogenic stimulation, cyclin-dependent kinases such as cdk4/6 become activated and induce phosphorylation of Rb, which then dissociates from E2F, relieving the HDAC inhibition.

This allows E2F to induce transcription of key enzymes involved in cell cycle regulation and progression past the G1 cell cycle restriction point, facilitating the replicative cycle without the requirement of a mitogenic stimulus.

In myeloma, constitutive over-expression of cyclin D, as well as abnormalities in Rb and over-expression of growth factors and growth factor receptors, contribute to uncontrolled cell cycle progression of the malignant clone.

Interaction with Bone Marrow Induces Growth and Survival Pathways

The pathogenic processes of multiple myeloma are localized predominantly in the bone marrow. Significant interactions between the myeloma cells and bone-marrow microenvironment amplify constitutive signaling pathways in the myeloma cells. These interactions induce upregulation of cytokines such as IL-1[alpha], IL-6, IGF-1, and VEGF. These cytokines in turn induce multiple growth signaling cascades, such as Ras-Raf-MAPK, JAK-STAT, and PI3K/Akt pathways.

Survival mechanisms involved in malignant transformation and resistance to apoptosis include up-regulation of bcl-2, mcl-1, NF-[kappa]B, and Akt and signaling through PI3K and MAPK kinase pathways. Moreover, chemotherapy itself induces drug resistance by activating NF-[kappa]B and upregulating bcl-2 in multiple myeloma cells.

On osteoclasts, there is upregulation of RANKL (receptor activator of NF-[kappa]B ligand) and down-regulation of osteoprotegrin (OPG, the inhibitor of RANKL). RANKL also has been found on myeloma cells.

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This stromal cell interaction activates osteoclast activity, resulting in increased osteolysis, while increasing drug resistance, proliferation, and migration in plasma cells. High serum levels of IL-6 and IGF-1 seen in patients with myeloma contribute to chemoresistance and treatment failure. For example, dexamethasone-induced apoptosis in myeloma cells is abrogated in vitro by IL-6 or IGF-1.

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The malignant plasma cells adhere to bone marrow stromal cells. Adhesion triggers stromal cells to secrete various cytokines that also promote myeloma cell growth and block chemotherapy cytotoxicity by inducing antiapoptotic signaling. Adhesion of multiple myeloma cells to fibronectin via the VLA-4 integrin results in upregulation of p27 and cell cycle arrest, thereby increasing resistance to cytotoxic agents such as doxorubicin and melphalan.

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CD40 is present on myeloma plasma cells and bone marrow stromal cells. Binding by CD40 ligand on T cells increases endogenous IL-6 secretion by myeloma and stromal cells through activation of NF-[kappa]B and also increases expression of bcl-2, which increases drug resistance.

Cytokine stimulation at the cell surface activates transmembrane receptors that signal transduction pathways to the cytoplasm via an elaborate network of cytokine-associated proteins and adaptors.

In myeloma, the Ras-Raf-MEKERK pathways are stimulated by cytokines such as IL-6, VEGF, and IGF-1. In addition, IL-6 receptors also activate JAK-STAT pathways. IL-6-induced activation of SHP-2 is an important mediator of resistance to dexamethasone via inhibition of dexamethasone-induced activation of RAFTK (related adhesion focal tyrosine kinase).

Insulin-like growth factors and their receptor are expressed in solid tumors and various hematologic malignancies, including myeloma. IGF-1 receptor stimulation is important to the pathophysiology of myeloma by inducing MAPK-mediated proliferation as well as survival factors Akt and NF-?B. Telomerase activity, important for maintaining DNA integrity in immortalized cells, is also upregulated by IGF receptor stimulation.

VEGF is secreted by myeloma plasma cells as well as bone marrow stromal cells. VEGF upregulation occurs when myeloma cells bind to stroma. VEGF receptors on myeloma cells, when activated by VEGF, induce PKC-dependent cell migration as well as MAPK-dependent proliferation in the myeloma cells.

Conventional Therapies Are Not Uniformly Effective

Although oral therapy with melphalan and prednisone has extended the median survival of patients with multiple myeloma from 6 months to 3 years (using low doses of oral melphalan), the response rates have been only 50 to 60%. Complete remissions are rare, and myeloma remains incurable with conventional chemotherapy.

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Infusional VAD (vincristine, doxorubicin [Adriamycin], dexamethasone) was introduced in 1984 in an attempt to overcome drug resistance by using prolonged infusions of combination chemotherapy. VAD resulted in significant increases in response rate and complete remission rate over those seen with melphalan and prednisone. However, overall survival with VAD or other forms of infusional chemotherapy has not significantly improved. The 5-year survival rate remains about 28%, and the bone marrow toxicity of conventional chemotherapy is dose-limiting.

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Hematopoietic cell transplantation with either autologous or allogeneic (from a histocompatible family member or non-blood relative) marrow has been explored extensively as a means to increase doses of conventional chemotherapy. Allogeneic transplantation has been associated with treatment-related mortality in up to 40% of patients and therefore is performed infrequently.

In contrast, autologous transplantation has been much safer, with a treatment-related mortality of <3%. Autologous transplantation is superior to conventional chemotherapy for the treatment of multiple myeloma, achieving a median survival of 55 to 72 months. Some studies suggest the benefit of a second auto-transplantation in those patients having less than a very good partial response to a single autograft.

However, only 50% of patients aged 60 or under, and less than 20% of patients aged 60 to 70, receive transplantation because of early death, comorbidity, poor response to therapy, or failed peripheral blood stem cell harvests. For these patients, new therapies are urgently needed.

Novel Biologically Based Therapies Are Entering Clinical Use

Recently, new understanding of the molecular biology of myeloma has lead to major developments in novel biologically based therapies for the disease. In particular, proteasome inhibitors, thalidomide and immunomodulatory analogues of thalidomide, 2-methoxyestradiol, arsenic trioxide, monoclonal antibodies, and histone deacetylase inhibitors have entered into clinical trials.

These novel therapies were initially evaluated in patients who had relapses after transplantation. Due to their efficacy and tolerability, some of these agents are now being evaluated to treat patients with earlier stage disease, as initial therapy or treatment of first relapse, and in combination with other therapies.

Proteasome Inhibition

The proteasome is a large intracellular molecule with multicatalytic protease activities found in both the cytoplasm and nucleus. It is essential for the nonlysosomal, ATP-dependent proteolytic pathway, catalyzing the rapid degradation of intracellular proteins that regulate cell adhesion, transcription, angiogenesis, and antigen presentation by MHC class I molecules. The proteasome also is essential for the rapid elimination of abnormally folded or oxidized proteins.

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Among the proteins mediated by this pathway, the growth and survival factor NF-[kappa]B is regulated by I[kappa]B[alpha], which serves to prevent its nuclear localization and transcriptional activity. In response to growth signals or stress, I[kappa]B[alpha]becomes ubiquitinated, which is the signal for its proteasomal degradation. NF-[kappa]B is released, resulting in transcriptional upregulation of growth and survival factors.

The first proteasome inhibitor to enter clinical trials was a boronic acid dipeptide called bortezomib (PS-341, Velcade). Bortezomib acts on both myeloma cells and bone marrow.

Specifically, bortezomib inhibits MAP kinase signaling and myeloma cell proliferation, triggers caspase-mediated apoptosis, and overcomes drug resistance by inhibiting NF-[kappa]B. This inhibition results in decreased adhesion molecule expression and myeloma cell binding to bone marrow stromal cells, as well as abrogation of related IL-6 secretion from stromal cells.

Bortezomib ushered in a new era of drug development, being the first drug in a new class of compounds to reach clinical trials and the first new drug in over 3 decades to be FDA approved for the treatment of myeloma.

In a phase II clinical trial, 202 patients with relapsed and refractory myeloma received 1.3 mg/[m.sup.2] of bortezomib twice weekly for 2 weeks, followed by 1 week without treatment, for up to eight cycles (24 weeks). The response rate was 35%, and 68% of patients had either decreased or stable paraprotein levels. The median duration of response was 12 months, and the median overall survival was 16 months. This was a significant improvement over an historical median survival of 9 months in patients with relapsed and refractory myeloma.

In May 2003, bortezomib was the first proteasome inhibitor to be approved by the FDA for use in patients with relapsed refractory myeloma.

As a single agent, bortezomib represents a significant advance in the treatment of myeloma. A phase III clinical trial comparing bortezomib with dexamethasone for treatment of relapsed myeloma (APEX trial) has shown a statistically significant prolongation in time to progression in the bortezomib-treated cohort. Based on this study, FDA approval has been extended to patients with relapsed myeloma.

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Clinical trials are also underway to explore the activity of bortezomib combined with conventional therapy such as doxorubicin or melphalan.

Thalidomide and Immunomodulatory Analogues

The antiangiogenic properties of thalidomide, together with the increased angiogenesis observed in marrow of patients with myeloma, led to the use of thalidomide in myeloma in the late 1990s. While thalidomide was initially used for its antiangiogenic properties, subsequent investigations have found that it also has direct cytotoxic activity against myeloma cells in vitro, via activation of caspase-8.

Single-agent thalidomide in myeloma achieved a response rate of over 30%, predominantly in patients with refractory disease following autologous transplantation. The addition of dexamethasone to thalidomide also enhances its anti-myeloma activity. In clinical trials, the response rate was 36% for patients treated with thalidomide alone and 72% for those treated with the combination of thalidomide-dexamethasone, including 16% complete responses in the combined treatment group.

Significant anti-myeloma activity has been observed even after prior resistance to thalidomide and dexamethasone given separately, as well as after intensive therapy supported by autologous marrow transplantation.

However, at least one third of patients treated with thalidomide have experienced mild or moderate constipation, fatigue, or somnolence. More potent thalidomide analogues have been developed that have a much more favorable side effect profile and may have an even greater impact on response rates to induction chemotherapy.

Thalidomide analogues with potent immunomodulatory properties have been identified that markedly stimulate T-cell proliferation, as well as IL-2 and interferon[gamma] (IFN-[gamma]) production. One such agent, lenalidomide (CC-5013, Revlimid), is 50 to 2000 times more potent than thalidomide in stimulating T-cell proliferation triggered via the T-cell receptor and 50 to 100 times more potent than thalidomide in augmenting IL-2 and IFN-[gamma] production.

Lenalidomide inhibited tumor growth, decreased angiogenesis, and prolonged host survival in a human plasmacytoma model in mice. In patients with myeloma, lenalidomide stimulates host antimyeloma natural killer cell immunity and augments antibodydependent cellular cytotoxicity in vitro. Importantly, it causes no significant somnolence, constipation, or neuropathy.

In clinical trials, at least a 25% reduction in paraprotein was achieved in 17 of 24 patients (71%), and 4% achieved complete remission. This anti-myeloma activity was particularly impressive, because 15 patients (60%) had prior high-dose therapy with autologous marrow transplantation and 16 patients (64%) had progressive disease despite treatment with thalidomide.

The evaluation of lenalidomide has forged ahead to phase II multi-center trials. In two trials now completed, the results confirm both responses in relapsed/refractory myeloma and the favorable side effect profile. Two phase III trials comparing lenalidomide/dexamethasone versus dexamethasone alone for relapsed myeloma were recently unblinded due to a significant prolongation in time to progression in the combination cohort.

2-Methoxyestradiol

2-Methoxyestradiol (2ME2) is an estrogen derivative that induces cell death independently of the estrogen receptor. 2ME2 mediates anti-myeloma activity directly on myeloma cells and in the bone marrow microenvironment. While 2ME2 is a corticosteroid-derived compound, death signaling induced by 2ME2 differs from that of the corticosteroid dexamethasone.

Furthermore, 2ME2 induces apoptosis in myeloma cells resistant to dexamethasone. 2ME2induced apoptosis is mediated by the release of mitochondrial cytochrome c and Smac (second mitochondrial activator of caspases), followed by the activation of caspases-9 and -3.

In contrast to its effects on myeloma cells, 2ME2 does not reduce the survival of normal peripheral blood lymphocytes. Moreover, 2ME2 enhances dexamethasone-induced apoptosis, and its effect is not blocked by IL-6.

In a murine model, 2ME2 inhibited myeloma cell growth, prolonged survival, and decreased angiogenesis. A multicenter phase II trial of 2ME2 in patients with chronic-phase myeloma is currently underway in the United States.

Arsenic Trioxide

In the early 1990s, impressive results were observed with arsenic trioxide in patients with acute promyelocytic leukemia, leading to its approval for use in the United States for relapsed or refractory APL. Subsequent preclinical studies showed significant activity of arsenic trioxide in myeloma.

Arsenic induces superoxide production while reducing glutathione levels, resulting in mitochondrial-mediated apoptosis. The activity of arsenic may be enhanced by ascorbic acid-mediated cellular glutathione depletion.

In a phase I/II trial of 6 patients with stage IIIA relapsed/refractory myeloma, 0.25 mg/kg/day of arsenic together with 1,000 mg/day of ascorbic acid was given for 25 days over a 35-day period. No dose-limiting toxicity was seen, and only one episode of grade 3 hematologic toxicity (leukopenia) and no grade 3 non-hematologic toxicities were observed.

Two patients (both with thalidomide-refractory disease) had partial responses; four patients had stable disease. Therefore, the combination of arsenic with ascorbic acid has acceptable toxicity and is active in refractory/relapsed myeloma. Arsenic with ascorbic acid is now being evaluated in combination with either melphalan or dexamethasone in separate phase II studies.

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Cell Growth and Survival Signaling Pathways Are Drug Targets

Histone Deacetylase Inhibitors

Histone deacetylase inhibitors are a promising new treatment strategy in hematologic malignancies. Gene expression, cellular differentiation, and survival are regulated by the opposing activities of histone acetyltransferases and histone deacetylases (HDACs). HDAC inhibition results in accumulation of acetylated nuclear histones and induces differentiation and/or apoptosis in transformed cells.

HDAC inhibitors mediate antimyeloma activity and overcome drug resistance in the bone marrow milieu. They sensitize myeloma cells to death receptor-mediated apoptosis and inhibit the secretion of IL-6 induced in marrow stromal cells by the binding of myeloma cells, thus overcoming cell adhesion-mediated drug resistance.

HDAC inhibitors are synergistic with dexamethasone, bortezomib, and IGF-1 inhibitors against myeloma and may one day be incorporated into combinations of novel and conventional therapies in the treatment of myeloma.

Anti-CD40 Monoclonal Antibodies

The humanized anti-CD20 monoclonal antibody rituximab has been useful in the treatment of non-Hodgkin's lymphoma. Most myeloma cells do not express significant levels of CD20, but CD40, which is expressed on B-cell malignancies, is expressed on human myeloma cells. CD40 stimulation upregulates VEGF secretion and myeloma cell migration.

SGN-40, a humanized anti-CD40 monoclonal antibody, has shown preclinical activity against dexamethasone-sensitive and -resistant myeloma cell lines and patient myeloma cells expressing CD138 and CD40. SGN-40-mediated cytotoxicity is associated with upregulation of cytotoxic ligands of the TNF family (Fas/FasL, TNF-related apoptosis-inducing ligand [TRAIL], and TNF[alpha]).

Pretreatment of myeloma cells with SGN-40 blocks CD40 ligand-mediated PI3K/AKT and NF-[kappa]B activation, and it also inhibits proliferation triggered by IL-6. In addition, SGN-40 pretreatment blocks the ability of IL-6 to protect against dexamethasone-induced inhibition of DNA synthesis. A phase I, multidose study of SGN-40 in patients with refractory or relapsed myeloma has begun accrual at the Dana-Farber Cancer Institute.

VEGF

Human myeloma cell lines and myeloma patient cells express high-affinity VEGF receptor-1 or Fms-like tyrosine kinase-1 (Flt-1). VEGF inhibitors PTK787 and GW654652 are novel agents currently under preclinical and clinical evaluation for the treatment of myeloma.

These small-molecule tyrosine-kinase inhibitors block VEGFinduced signaling and migration in myeloma cells that are both sensitive and resistant to conventional therapy. Importantly, GW654652 also inhibits IL-6 and VEGF secretion and proliferation of myeloma cells induced by tumor cell binding to bone marrow stromal cells.

The activity of a pan-VEGF receptor inhibitor against myeloma cells in the bone marrow milieu, coupled with its lack of major toxicity in preclinical mouse models, provides the framework for clinical trials of this drug class to improve patient outcome in myeloma.

IGF-1

Specific IGF-1 receptor inhibition with neutralizing antibody, antagonistic peptide, or the selective kinase inhibitor NVP-ADW742 has induced in vitro activity against myeloma, including those resistant to conventional therapies. NVP-ADW742, alone or combined with cytotoxic chemotherapy, has shown significant antitumor activity in a xenograft myeloma murine model, providing the rationale for the therapeutic use of selective IGF-1 receptor inhibitors in myeloma.

Telomerase

Telomeres are DNA protein complexes at the ends of eukaryotic chromosomes that protect linear DNA ends from erosion over multiple replication cycles. They also protect chromosome ends from recognition as double-strand DNA breaks and subsequent "repair" mechanisms, which may induce apoptosis by exonucleolytic trimming and end-to-end fusion.

Telomerase is a ribonucleoprotein with reverse transcriptase activity that compensates for the telomere loss associated with cell division. Telomerase is overexpressed in human malignancies but is undetectable in most other somatic tissues, and it is therefore an attractive therapeutic target.

In normal human diploid cells lacking telomerase, progressive telomere shortening leads to an early crisis in which short dysfunctional telomeres are recognized as DNA damage and may cause apoptosis.

Telomerase inhibitors, such as anti-sense RNA complementary to telomerase RNA, small interfering RNA (siRNA), and G quadruplex-interacting agents, result in loss of telomerase activity, telomere shortening, and cell death in myeloma cells, providing the rationale for therapeutic trials in myeloma.

Apoptotic Signaling-Based Strategies Warrant Further Research

Apoptosis is a mechanism of controlled cell death critically important in many biologic processes. Damaged or senescent cells initiate an intrinsic death signal that activates a cascade of caspase enzymes that target cellular proteins, resulting in programmed cell death.

Drugs or radiotherapy initiate apoptosis by targeting vital cellular processes, such as proteasomal degradation of abnormal or ubiquitinated proteins. Apoptosis then proceeds via common downstream effector pathways, which represent a balance between proapoptotic caspases and antiapoptotic proteins. These processes can be enhanced by combining different mediators upstream or by releasing inhibitors to apoptosis downstream.

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Most antitumor agents trigger apoptotic signaling, and defects in this process lead to the development of drug resistance. In multiple myeloma, loss of an apoptotic response to chemotherapy may result from overexpression of anti-apoptotic proteins, which may be secondary to genetic abnormalities, or may be acquired after exposure to cytotoxic chemotherapy due to upregulation of drug efflux pumps.

External signals also may contribute, such as the signaling initiated when myeloma cells bind to bone marrow extracellular matrix proteins and bone marrow stromal cells, and that due to cytokines in the bone marrow milieu, including IL-6 and IGF-1.

Two major pathways of apoptosis have been identified that are either mitochondrial-dependent or independent. The extrinsic pathway, mediated by death receptors at the cell surface membrane, can be activated by stimuli such as Fas ligand or TRAIL, inducing activation of caspase-8. Alternatively, the intrinsic pathway is induced via release of mitochondrial apoptogenic proteins to the cytosol.

Ionizing radiation, TRAIL, and thalidomide and its immunomodulatory analogues trigger caspase-8 activation, whereas arsenic trioxide and dexamethasone trigger caspase-9 activation. Bortezomib induces both the caspase-8 and -9 apoptotic pathways. IL-6 and IGF1 activate the PI3K/Akt cascade, which inhibits caspase-9/3 apoptotic signaling.

Mitochondria harbor two key modulators of apoptosis, cytochrome c and Smac. Translocation of JNK from cytosol to the mitochondria occurs in response genotoxic stress, thereby inhibiting the antiapoptotic function of proteins of the Bcl-2 family and allowing release of mitochondrial apoptogenic molecules.

JNK activation is required for irradiation, anti-Fas, ceramide, or TNF-induced apoptosis, and it is also an important component of bortezomib-induced apoptosis. In contrast, dexamethasone causes activation of RAFTK, thereby releasing Smac from the mitochondria and promoting caspase activation independently of cytochrome c. Importantly, IL-6 inhibits dexamethasone-induced, but not JNK-mediated, apoptosis.

Stress signaling pathways induced by anti-myeloma agents have been delineated, which allows for identification of novel molecular targets to amplify apoptosis. For example, anti-myeloma drugs induce apoptosis via release of mitochondrial cytochrome c or Smac, suggesting that peptides that activate these molecules may reduce the apoptotic threshold of drugs.

Smac mimetics have been synthesized that induce synergistic anti-myeloma activity when combined with other drugs. The finding that dexamethasone-induced apoptosis occurs without cytochrome c release or activation of JNK provides the rationale for amplifying apoptotic signaling by combining dexamethasone with agents that trigger cytochrome c release and activate JNK.

Inhibitor of apoptosis proteins are a family of apoptosis-suppressing proteins commonly overexpressed in human cancers. Some of these inhibitors, such as XIAP, directly bind and suppress caspases and therefore define a distal checkpoint for cell death control. Compounds have been generated that target the BIR2 domain of XIAP, inhibiting its interaction with caspase-3. These compounds are active as single agents, and inhibit tumor growth in mice with very little toxicity to normal tissue.

Normal cells appear less dependent on inhibitors of apoptosis proteins, thus providing a favorable therapeutic index. The proapoptotic Smac peptides target the BIR3 domain of XIAP, thereby inhibiting its caspase-9-suppressing activity. Smac peptides induce apoptosis as single agents and also sensitize tumor cells to apoptosis-inducing agents.

These novel molecular pathways provide the basis for rational drug development strategies to overcome drug resistance in myeloma. Signaling pathways also provide the framework for rationally combining novel therapeutics. Apoptotic mediators can be enhanced and inhibitors blocked by using small molecules, a strategy that seeks to maximize the inhibition of growth and survival pathways while overcoming the ability of myeloma cells to develop drug resistance. These therapies represent the beginning of a new age of translational research, when understanding of the molecular biology of malignant disease can be translated into a favorable therapeutic outcome for patients.

Karyotypic Abnormalities of Myeloma Plasma Cells

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The immunoglobulin heavy chain region (IgH) on chromosome 14q32 is involved in illegitimate translocations in 50% of patients with monoclonal gammopathy of uncertain significance (MGUS, a predecessor of multiple myeloma), 65% of myeloma patients, and >90% of myeloma cell lines. These translocations involve several partner chromosomes and result in aberrant overexpression of oncogenes such as cyclin D1, cyclin D3, or fibroblast growth factor receptor 3 (FGFR3).

Molecular Characteristics of Myeloma Cells

Recurrent Cytogenic Ig Heavy-Chain
Translocations and Their Oncogene Products
  t(11;14) -- Cyclin D1
  t(6;14)  -- Cyclin D3
  t(4;14)  -- FGFR3/MMSET
  t(16;14) -- c-MAF
  t(8;14)  -- c-MYC
  t(6;14)  -- MUM1/IRF4
  t(20;14) -- MAFB

Surface Antigens
  CD38
  CD138
  CD40

Surface Cytokine Receptors
  IL6R
  VEGFR
  IGFR

SUPPORTIVE CARE IMPROVEMENTS

The 1990s have seen significant advances in supportive care for myeloma patients. For example, the widespread use of bisphosphonates, in particular pamidronate and zoledronate, has been shown to significantly reduce the rate of skeletal events in patients with multiple myeloma, as well as effectively treat hypercalcemia, reduce bone pain, and improve quality of life.

Erythropoietin is being used increasingly to reduce transfusion requirements in patients with anemia. Kyphoplasty is a recently developed surgical technique that has achieved significant improvements in quality of life for patients with compression fractures.

Molecular Targetting by Novel Anti-myeloma Agents

Targeting myeloma cells and their interaction with
bone marrow microenvironment

  Proteasome inhibitors (bortezomib)
  Thalidomide and immunomodulatory analogues
    (lenalidomide)
  2-Methoxyestradiol
  Arsenic trioxide
  Lysophosphatidic acid acyltransferase-b inhibitor
  Triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic
    acid (CDDO)
  N-N-diethyl-8,8-dipropyl-2-azaspiro [4.5] decane-2-
    propanamine (Atiprimod)

Targeting bone marrow microenvironment

  I[kappa]B kinase inhibitor
  p38 MAPK inhibitor
  TGF-[beta] receptor inhibitor

Targeting cell growth and survival pathways
in myeloma cells

  Histone deacetylase inhibitor
  VEGF receptor tyrosine kinase inhibitors
  Telomerase inhibitor
  Farnesyl transferase inhibitor
  Heat shock protein-90 (hsp-90) inhibitor
  Bcl-2 antisense oligonucleotide
  Inosine monophosphate dehydrogenase
  Rapamycin
  DNA methyltransferase (DNAMT)

Targeting cell surface receptors

  TNF-related apoptosis-inducing ligand (TRAIL)/Apo2 ligand
  IGF-1 receptor inhibitor
  HMG-CoA reductase inhibitor
  Anti-CD40 antibody

ADAPTED FROM HIDESHIMA T, ET AL: BLOOD 104(3):607-618, 1 AUG 2004.

Publication date: 5 July 2005

LAURENCE CATLEY and KENNETH C. ANDERSON are in the Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston.

[e-mail]

Supported by the Doris Duke Distinguished Clinical Research Scientist Award (K.C.A.).

M-Protein Isotypes in MM

Isotype                                   Percent/Total

IgG                                          53%
IgA                                          25%   75%
IgD                                           1%
IgE                                       <0.01%
Bence-Jones                                  15%   15%
  (light chain only)
Heavy chains only                            <1%
  (G or A)
[greater than or equal to] 2 M-proteins      <1%   10%
No M-protein                                  1%
IgM                                          12%