review

Cancer Biology & Therapy 10:9, 830-838; Novermber 1, 2010; © 2010 Landes Bioscience

830 Cancer Biology & Therapy volume 10 issue 9

Introduction

Multiple myeloma is a molecularly heterogeneous disease with a high degree of genomic instability in which specific genetic changes can be linked to clinical presentation and prognosis. Despite recent improvements in event-free survival and overall survival with the use of high-dose chemotherapy and stem cell support as well as the development of novel agents such as tha-lidomide, lenalidomide and Bortezomib, MM remains an incur-able disease. The development of effective targeted therapies requires a detailed knowledge of various genetic and signaling pathways governing MM genesis. This review will focus on the current understanding of the molecular pathogenesis of MM and the intracellular signaling pathways and their regulations, with emphasis on the rationale for identifying therapeutic targets that can be applied in the clinic.

Plasma Cell Development

Plasma cells (PC) are terminally differentiated B-cells that are responsible for the production of immunoglobulins with a high degree of specificity in response to microbial pathogens. Stem cell commitment to the B-cell lineage starts in the bone marrow, where the rearrangement of the immunoglobulin heavy (IgH) and light chain genes leads to the expression of surface immunoglobulins. These immature B cells leave the bone marrow and migrate in the peripheral blood to the lymph nodes, resting in the G

0 phase of the cell cycle until exposure

to antigenic stimuli. These stimuli lead to the generation of short-lived pre-germinal center plasma cells “lymphoblasts”, which differentiate into a short-lived, non-switched (IBM).1,2 Later on, the lymphoblasts enter the Germinal Center of the lymph nodes (GC) where somatic hypermutation and heavy chain class switching occur leading to high affinity antibody production, specifically switched (IgG, IgA, IgE) plasma cells. The transformed cells of the GC either undergo apoptosis or

*Correspondence to: Ashraf Badros; Email: abadros@umm.eduSubmitted: 08/31/10; Accepted: 09/14/10Previously published online:www.landesbioscience.com/journals/cbt/article/13622DOI: 10.4161/cbt.10.9.13622

Multiple myelomaImplementing signaling pathways and molecular biology

in clinical trials Mouhamad Bazzi and Ashraf Badros*

University of Maryland; Greenebaum Cancer Center; Baltimore, MD USA

Key words: multiple myeloma, nuclear factor pathway, heast shock protein/HSP90, HDAC inhibitor, AKT inhibitor, mTOR, refractory myeloma

mature to centrocytes, which in turn transform into long-lived plasma cells that leave the lymph nodes and home back to the bone marrow as terminally differentiated plasma cells. These cells secrete most of the serum immunoglobulins and have a life span approximately of 1 month (these steps are illustrated in Fig. 1).3,4

The Bone Marrow Microenvironment

The bone marrow (BM) “microenvironment” plays a crucial role in MM pathogenesis.5 The BM microenvironment is com-posed of a variety of extracellular matrix (ECM) proteins, such as fibronectin, collagen, laminin, osteopontin, as well as cellular components including hematopoetic stem cells, immune cells, erythrocytes, BM endothelial cells, osteoblasts and osteoclasts. These cells constitutively secrete high levels of the chemokine, CXCL-12 (SDF-1, stromal derived factor) and various cytokines including IL6, IGF-1, VEGF, FGF, BAF, SDF-1, which attract circulating MM cells to the bone marrow. Several adhesion mol-ecules such as CD40, VLA 4 (very late antigen), VLA5, LFA 1 (leukocyte function-associated antigen), NCAM (neuronal adhesion molecule), ICAM (intracellular adhesion molecule) are expressed on both myeloma and bone marrow stromal cells. Their interactions activate multiple intracellular signaling pathways (STAT3, PI3K, MAPK, NFκB) leading to further upregulation of adhesion molecules and secretion of cytokines, which increase the proliferation drive and survival of MM cells, stimulate osteo-clasts and suppress osteoblasts. These events result in the forma-tion of lytic bone lesions, stimulation of blood vessels formation and promotion of drug resistance.6-8 The relationship is sustained via cytokines (indirect) or cell-cell interaction (direct)9,10 (Fig. 2).

Vascular Endothelial Growth Factor (VEGF), secreted by plasma cells in response to IL 6 stimulation, is found in high levels in the plasma of MM patients Its receptor (VEGF-R) is highly expressed on MM cells and may be part of an auto-crine signaling pathway. Specifically, VEGF activates the PI3K pathway mediating MM cell migration, MEK/ERK pathway mediating MM cell proliferation, and upregulates the expres-sion of survivin and Mcl-1 thereby mediating survival.11,12 It drives angiogenesis, enhances osteoclastic bone resorbing activity, increases microvessel marrow density (MVD) and

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review review

Nuclear Factor-κB Pathway

The nuclear factor-κB pathway is a major regulator of the immune and inflammatory responses of the cell. Its activation is essential for the expression of a wide variety of genes (TNFalpha, IL-6, adhesion molecules and others), regulation of the cell cycle, as well as the activation and differentiation of lymphocytes.20 The adhesion of MM cells to bone MSCs triggers NF-κB-mediated transcription. NF-κB target genes promote survival, proliferation, confer chemo resistance of MM cells and increase bone resorption and angiogenesis. Higher levels of NF-κB have been reported in MM cells derived from patients relapsing after chemotherapy as well as in drug-resistant MM cells compared to drug sensitive myeloma cells.5,21,22 The NF-κB family, a com-plex of proteins that control the transcription of DNA, includes RelA (p65), RelB, c-Rel, p50 (NF-κB1) and p52 (NFB2). They are normally kept inactive in the cytoplasm by interaction with their inhibitors, called IκB or kept in the unprocessed forms. The transcriptional activity of nuclear factor dimers is regulated by at least two signaling pathways: the “canonical” pathway and the “non-canonical” pathway.

The canonical pathway, activated by several agonists such as TNFalpha, LPS, IL-1, CD40L, leads to a conformational change of the IKK (α,β,Ý) protein complex. The activated IKK complex phosphorylates IκBα (an inhibitor of p50 and p65) and this leads to its ubiquitination and degradation. Nuclear factor dimers are thereby released to translocate into the nucleus where they activate target genes. The inactivation of IκB is the rate-limiting step in the activation of canonical pathway. In contrast, the non-canonical pathway requires NF-κB-induced Kinase (NIK) and IKKα to mediate the processing of precur-sor nuclear factor into active isoforms. In the resting phase, p100 binds to and keeps RelB in the cytosol and NIK is main-tained at a low level owing to proteasomal degradation medi-ated by the TRAF2-TRAF3/cIAP1/2 (inhibitory of apoptosis) complex. In the active phase, activation of the receptor leads to recruitment of the cIAP1/2-TRAF2/3 complex to the recep-tor followed by proteasomal degradation of the complex. NIK accumulates in the cytoplasm and activates IKKα, which leads to proteasome-mediated p100 processing into p52, which then migrates to the nucleus and regulates transcription of its target genes.20 It is worthwhile to mention that the non-canonical pathway can activate the canonical pathway in several ways: for example, the high level of NIK results in direct activation of IKK, which in turn phosphorylates IκBα thereby activating the classical pathway. Additionally, prolonged activation of the non-canonical pathway may result in overproduction of cyto-kines that activate the canonical pathway.20,22

NFκB regulates several pathways relevant to cancer including (a) anti-apoptotic genes: BCLx

L, BCL2, IAP, XIAP, Survivin; (b)

cell cycle regulators: Cyclin D1, ICAM1, VCAM1, E Selectin; and (c) genes involved in drug resistance: c Myc, MDR1. In addi-tion, NF-κB counteracts p53 and directly inhibits its function by protein-protein interaction. Furthermore, NF-κB activation is implicated in de novo acquired resistance to chemotherapy. NF-κB inhibitors serve as sensitizers to anticancer drugs.23,24

promotes monoclonal gammopathy of undetermined signifi-cance (MGUS) progression into MM stage, which appears to be a prognostic factor.13,14 Bone damage is caused by both activating osteoclasts and blocking the differentiation of mes-enchymal stem cells to mature osteoblasts, which results in increased bone destruction and decreased bone formation.15,16 Mesenchymal stem cells (marrow stromal cells, MSC) express the receptor activator of NF-κB ligand (RANKL)+, alkaline phosphatase (ALP)- and Wnt receptors FZD and LRP5. They differentiate into osteoblasts, characterized by RANKL- and ALP-. During MM progression, plasma cells secrete increasing levels of DKK1, a potent inhibitor of Wnt signaling, which binds to the LRP co-receptor and blocks the differentiation of MSCs into mature osteoblasts.17,18 The continuous exposure of bone marrow to elevated DKK1 results in overproduction of RANKL by MSC that leads to osteoclast differentiation. In normal individuals, the actions of RANKL is regulated by osteoprotegerin; however, in MM patients osteoprotegerin is trapped and inactivated by syndecan, which is secreted by the plasma cells.19 Knowledge of the events occurring in the bone marrow microenvironment of MM patients has resulted in the development of several drugs that target the BM microenvi-ronment. Table 1 summarizes several novel agents and their targets, currently in clinical trials.

Figure 1. Normal plasma cell development. Functional v(D)J rear-rangements of igH and igL genes in pre-B cells in the bone marrow generate an immature B cell that expresses a functional ig on the cell surface, which then exits the BM as a virgin (mature) B cell and homes to the secondary lymphoid tissues. interaction with antigen stimulates the formation of a lymphoblast which differentiates into a short-lived, nonswitched (igM), or switched (igG, igA, ige or igD) PC. Later on, the lymphoblast generated by a productive interaction with antigen enters a germinal center where it undergoes somatic hypermuation of its igH and igL genes and antigen selection of cells with high affinity ig recep-tor. A germinal center plasmablast that undergoes a productive igH switch recombination typically homes to the BM where it differentiates into a long-lived plasma cell.

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of resistance. One mode of primary resistance to bortezomib is conveyed by constitutively high levels of heat shock protein.88

Irreversible proteasome inhibitors, such as carfilzomib, have shown efficacy in MM. Carfilzomib (PR-171) is a tetrapeptide epoxyketone that irreversibly inhibits the chymotrypsin-like activity of the 20S proteasome subunit and decreases prolif-eration in interleukin-6-dependent and independent MM cell lines.30 It is more potent than bortezomib in proteasome inhibi-tor naïve patients and can overcome bortezomib resistance. Carfilzomib monotherapy has yielded durable responses in heav-ily pretreated patient populations in three phase II trials; most notably, including those with bortezomib-refractory disease with ORR of 60–70% in bortezomib naïve and 30–40% in bortezo-mib pretreated patients with PFS 7–8 months.31,32

Carlfizomib, in combination with lenalidomide and low-dose dexamethasone, has been tested in phase Ib in relapsed/refractory MM patients with 1–3 prior treatments and showed CR/nCR of 21%, ≥PR: 55%, ≥MR: 76%, with total disease control rate of 93%. In that study, DVT and neuropathy were not observed with prolonged administration of treatment (>16 months) along with manageable expected hematological events.33 Furthermore, carlfizomib has been shown to be safe in renal insufficiency, with-out the need of dose adjustment, and in contrast to bortezomib, CFZ-related neuropathy is uncommon and not dose-limiting.34,35

NF-κB Pathway Inhibitors

Bortezomib is a reversible inhibitor of the NF-κB pathway that targets primarily the β5-subunit (PSMB5) subunit/chymotryp-sin-like activity of the 26S proteasome and, to a somewhat lesser extent, also the caspase-like activity harbored by the β1 (PSMB6) proteasome subunit. Bortezomib also can lead to canonical NF-κB pathway activation rather than inhibition in both MM and endothelial cancer cell lines. A possible mechanism includes direct or indirect activation of IKKβ, leading to the phosphory-lation of IκBα and ultimately, its non-proteasomal degradation. The p50/p65 dimer translocates to the nucleus, thereby activat-ing the canonical pathway.25 This provides the rational frame-work for combining a direct IKKβ inhibitor with bortezomib. Table 2 provides a summary of the potential anti-myeloma mech-anisms of bortezomib.

Bortezomib has changed the management and natural history of MM, has proven to be safe and effective in elderly patients, in patients with renal failure (can contribute to its reversal) and can overcome certain high-risk features such as 13 q-, 4:14, high b2 microalbumin levels. Most recently, bortezomib seems to negate the adverse consequences of the deletion of p53.26-28 Re-treatment of patients who have been exposed to bortezomib showed a lower response rate from 60 to 22%, indicating the emergence

Figure 2. interaction of MM cells with BM microenvironment. The homing of MM cells to their BM milieu is mediated by stromal-derived factor (SDF-1α) and its receptor CXCr4 on MM cells. The adhesion of MM cells and BM stromal cells activates many intracellular signaling pathways (STAT3, Pi3K, MAPK, NF-κB) leading to upregulation of adhesion molecules as well as increased cytokine secretion from both MM cells and BMSC. Osteoclasts are activated by rANKl and OPGL from BMSC and MiP-1α from MM cells. Osteoblasts are inhibited by DKK1 and iL-7 from MM cells, thereby forming bone lesions. SDF-1α, stromal-derived factor; rANKl, receptor activator of NF-κB ligand; OPGL, osteprotegerin lignad; BMSC, bone marrow stromal cells; MiP-1α, macrophage inflamatory protein; DKK 1, dockkopf 1; veGF, vascular endothelial growth factor

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resistant to other drugs, such as immunomodulatory agents and bortezomib. Hsp90 inhibition not only targets the MM cells directly but also targets the bone marrow microenvironment by suppressing the expression and function of growth factor recep-tors ( IGFs/IGF-1R, IL-6/IL-6R) and decreases the produc-tion of proangiogenic cytokines. This may explain why Hsp90 inhibitors can overcome the protective effect conferred to MM cells by the bone marrow milieu.41 Richardson and colleagues conducted a phase I/II clinical trial to evaluate the efficacy and safety of combining tanespimycin and bortezomib in relapsed/refractory MM and showed ORR of 48% in bortezomib-naïve and 22% in bortezomib-pretreated patients along with PFS of 7.2 months and 3.2 months respectively and with the median dura-tion of response being 12 months even in bortezomib pretreated patients. The therapy was well tolerated with a low incidence of severe treatment-related adverse events.42 A phase III trial assess-ing the combination in first-line therapy of MM is being planned.

Histone Deacetylase Inhibitors in MM

Histones, proteins found in all eukaryotic cells, package and order the DNA into structural units called nucleosomes. Histones are highly conserved and can be grouped into five major classes: H1/H5, H2A, H2B, H3 and H4. Two of each of the core histones (H2A, H2B, H3 and H4) assemble to form one octameric nucleosome core particle by wrapping 200 base pairs of DNA. The linker histone, H1, binds the nucleosome at the entry and exit sites of the DNA, thus locking the DNA into place. Histones act as spools around which DNA winds; this enables the compaction necessary to fit the large genomes of eukaryotes inside cell nuclei. The compacted molecule is 40,000 times shorter than an unpacked molecule. By compact-ing DNA, histones make it difficult for transcription factors to access DNA binding sites, hence limiting and regulating transcription. Histones undergo post-translational modifi-cations, which alter their interaction with DNA and nuclear proteins. Modifications of the tail include methylation, acety-lation, phosphorylation and sumoylation. Histone modifica-tions affect diverse biological processes such as gene regulation and DNA repair. The acetylation of histones, crucial to regu-lating their ability to interact with DNA, is regulated by the balance of the activities of two key enzyme classes: histone acetyltransferases (HATs) and histone deacetylases (HDACs). Hypoacetylation of histones is associated with condensed chromatin, resulting in the repression of gene transcription. Therefore, HATs allow transcription to occur, whereas his-tone deacetylases (HDACs) prevent transcription.43,44 HDACs, depending on sequence identity, are divided into four classes: class I consists of HDAC1, 2, 3, 8; class II consists of HDAC 4, 5, 6, 7, 9, 10; Class IV consists of HDAC11, Class III is a distinct class and consist of NAD+-dependent enzymes known as silent information regulator 2 (SIR2). HDAC I and IV are constitutively localized in the nucleus, whereas HDAC II can shuttle between the nucleus and the cytoplasm. In general class I HDACs are widely expressed, whereas class II and IV HDACs show various degree of tissue specificity.

These promising data, have led to the initiation of a phase III trial evaluating lenalidomide-dexamethasone, with or without carfil-zomib, in relapsed MM.

Heat Shock Protein 90 as a Therapeutic Target for MM

Cells respond to environmental stress by increasing synthesis of a number of molecular chaperones (also known as heat shock proteins). Heat shock proteins (HSPs) are required for the sta-bility and function of a number of conditionally activated and/or expressed signaling proteins needed for the growth and sur-vival of normal and malignant cells. They facilitate proper pro-tein folding, prevent misfolding or aggregation, and preserve the 3-dimensional conformation of proteins in a functionally competent state.36 One of the key components of the chaperone complex, HSP90, is critical for the proliferation and survival of cancer cells. Clients of Hsp90 participate, frequently in overlap-ping pathways, in mediating cancer cell survival. These clients include Akt, Her2 and HIF-1, mutant p53, c-Raf-1, CdK4, EGFR and Src family kinases. Thus, targeting HSP90 can inhibit mul-tiple survival pathways used by myeloma cells.37 Hsp90 inhibitors bind to the ATP binding pocket, inhibit chaperone function and could potentially result in cytostasis or cell death. Consequently, many client proteins are targeted for degradation via the ubiq-uitin-proteasome pathway. Furthermore, proteasome inhibition does not protect Hsp90 clients in the face of chaperone inhibi-tion; instead, client proteins become insoluble and toxic to the cell.38,39 Figure 3 highlights Hsp90 functions.

Interest has arisen in combining proteasome inhibition with inhibition of Hsp90; the idea being that dual treatment will lead to enhanced accumulation of insoluble proteins and trigger apop-tosis.40 Initial experimental support for such a hypothesis was provided by Mitsiades et al. who reported that Tanespimycin, a potent Hsp90 inhibitor, has shown antitumor activity in in vitro and in vivo preclinical models of MM. Tanespimycin disrupts the function of Hsp90 and decreases the viability of MM cells

Table 1. Therapeutic agents currently in clinical trials for MM, which target cell surface molecules and cytokines

Target Agent

CXCr4 antagonsit AMD3100.69

CD40 SGN 40/phase i.70

FGF, PDGFr SU5402 and PD173074/Preclinical.71,72

iL6, iL6-r Phase i/Altizumab73

iGF-r Ave 164274,75/Preclinical.

rANKL Denosumab (AMG 162).76

recombinant OPG construct Phase i/AMGN-0007.77

veGF, veGFrvalatinib (PTK 787) NCT00240162

Study has been completed Zactima (ZD 6474)/phase ii.78

Kappa Myeloma AntigenMDX-1097 an anti-kappa light chain

chimeric antibody Phase i.79

NK cell inhibitory Kir (killer immunoglobulin-like receptor)

iPH2101 a novel inhibitory Kir monoclonal antibody phase i.80

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certain pathological conditions, they can escape proteasomal degradation and form toxic aggregates. These misfolded/aggre-gated proteins are recognized and bound by HDAC6, which loads them onto the dynein motor complex, leading to degra-dation and clearance of cytotoxic protein aggregates. Preclinical studies have shown that dual inhibition of proteasomal and aggregosomal protein degradation pathways by bortezomib and HDAC II inhibitors (Tubacin or LBH589) results in synergistic cytotoxicity against MM cell lines.50-52

HDAC inhibitors are emerging as promising new anticancer agents. An updated and combined analysis of two phase I trials designed to determine the maximum tolerated dose of vorinostat, an oral inhibitor of class I and II HDACs, when combined with bortezomib in relapsed/refractory MM showed ORR of 39% with a minimal response (MR) of 11% and stable disease (SD) of 43%.53 Vorinostat, in combination with Lenalidomide and dexamethasone, in a relapsed/refractory setting (including 50% of patients with prior lenalidomide treatment), in a phase I multi-center study showed: ≥minor response rate of 82%, ≥partial response rate of 64% with good tolerance; only 3/28 patients dis-continued treatment because of secondary effects.54 Furthermore,

HDAC inhibitors lead to histone hyperacetylation and neu-tralization of positive charges, which results in an open chromatin structure and transcriptional activation. This alters the expres-sion of genes responsible for tumor viability (VEGF, STAT5, p53, BCL6) and leads to growth arrest, differentiation and apop-tosis. In addition, HDAC inhibitors target non-histone proteins such as NF-κB, p53, Hsp90, growth factors and anti-apoptotic proteins, thereby contributing to their efficacy against cancer. HDAC inhibitors affect tumor cell growth and survival through multiple biological mechanisms: induction of the transcription of negative regulators of cell proliferation, induction of apoptosis via of both death receptor (extrinsic) and mitochondrial (intrinsic) apoptotic pathways, acetylation of mitotic spindle proteins, inhi-bition of angiogenesis, downregulation of proteasome activity, and disruption of Hsp90 chaperone activity (Fig. 4).45-47

Several HDAC inhibitory molecules such as SAHA, FR901228 and LAQ284 have shown inhibitory, pro-apoptotic and cyto-toxic effects on MM cells.48,49 HDAC6, a member of the class II HDACs, has been shown to play a crucial role in the aggresome/autophagy degradation system. Misfolded, polyubiquitinated proteins are normally degraded by proteasomes; however, in

Table 2. Overview of some of the molecular effects of proteasome inhibitors that contribut to their anti-myeloma activity in addition to NF-κB inhibition

Target Mechanism Consequence

Non-canonical NF-κB

inhibition of proteasome-dependent p100 conversion to p52

Survival, proliferation…

Cdkis Stabilize Cdkis such as p21 and p27 Cell cycle arrest81

alpha 4-integrin Down regulate expression of vLA-4 Overcome adhesion-mediated drug resistance82

Calcium Disrupt mitochondrial calcium uniporter Dysregulate intracellular calcium storage; induce caspase activation83

HSP-90 induce HSP-90 expression and cell-surface exposure Stabilize signaling protein needed for cell growth and survival40

JNK Activate JNK Upregulate Fas and activate caspase-8 and caspase-384

rOS induce reactive oxygen species production Promote mitochondrial injury with release of pro-apoptotic factors85

veGF, veGFr Suppress stromal cell production of veGF reduce myeloma cell migration and marrow angiogenesis86

Table 3. Agents showing efficacy in Bortezomib-pretreated myeloma patients

Class Agent Efficacy

irreversible proteasome inhibitor CarfilzomibMonotherapy: Orr 70% in bortezomib-naïve vs. 30% in bortezomib-ref/rel,

PFS of 7 months31,32 CFZ+ Lenalidomide+ Dex: Cr/nCr 21%, ≥Mr 76%33

HSP90 inhibitor TanespimycinMonotherapy: Orr 48% in bortezomib-naïve vs. 22% in bortezomib-ref/rel,

PFS of 3.5–7 months42

histone deacetylase inhibitors (Selective inhibitor)

vorinostatvorinostat + bortezomib in relapsed/refractory: Orr 39%; Mr 11%, SD 43%53 vorinostat + Lenalidomie-Dexe in relapse/refractory: ≥Mr 82%, ≥Pr: 64%54

histone deacetylase inhibitors (Pan HDAC inhibitor)

PanobinostatPanobinostat + bortezomib in relapsed/refractory pts: ≥partial response:

50%, 15% iF-negative Cr55

AKT pathway inhibitor PerifosinePerifosine + bortezomib in relapsed Orr 40%, ≥minor response of and 55%,

TTP/OS: 8/25 months61 Perifosine + lenalidomide Orr: 33%/66% in ref/rel pts87

signal transduction inhibitor (including mTOri property)

Temsirolimus Temsirolimus + bortezomib in relapsed/refractory ≥Mr 79%, Cr 10%66

Mamalian target of rapamycin/mTOr i NvP-BeZ235 Preclinical,63 phase i64

Farnesyl transferase inhibitor FTi-277 Preclinical29

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degradation of IκBα and leads to the localization of NFκB to the nucleus where it can induce the transcription of anti-apoptotic genes, thus, Akt can inhibit apoptosis by activating NFκB.57,58 Although the direct effect of bortezomib on the AKT pathway is downregulation, the net effect is activation of this pathway via a positive feedback mechanism. Furthermore, AKT stimulates the NF-κB pathway, which can offset the effects of bortezomib, supporting the rational of combining bortezomib with an AKT inhibitor.59

The oral AKT inhibitor, perifosine, has shown to inhibit the growth of MM cells in vitro and in vivo.60 In a phase I/II Trial using perifosine with bortezomib in 84 refractory/relapsed MM pts, of whom 100% had prior bortezomib treatment and 75% of whom had prior lenalidomide treatment, the ORR (defined as ≥minor response) was 40% and OS was 16 months among bort-ezomib refractory, and ORR was 55% with TTP of 8.8 months and median OS of 25 months among bortezomib-relapsed patients.61 These encouraging results led to the initiation of a phase III study using bortezomib and dexamethasone with or without perifosine in bortezomib exposed patients with relapsed/refractory MM.

the use of panobinostat (LBH5989), a pan-HDAC inhibitor, in combination with Bortezomib in patients with relapsed/refrac-tory MM, showed ≥partial response rate of 50% including 15% IF negative CR.55 The promising data, combined with the man-ageable toxicity profile of HDAC inhibitor containing regimens, has led to instigation of a placebo-controlled phase III trial evalu-ating bortezomib plus vorinostat.

PI3K/AKT Pathway in MM

The PI3K/AKT pathway is important for MM cell oncogen-esis. By regulating p21Cip1 (a cyclin/cdk inhibitor), inactivat-ing GSK3 (a serine/threonine protein kinase implicated in the control of cellular response to DNA damage) and upregulating Mammalian Target of Rapamycin (mTOR), the PI3K/AKT pathway regulates cell cycle progression, induces DNA synthesis and promotes survival and migration of multiple myeloma cells. Furthermore, via inactivation of Bad (a pro-apoptotic protein) and Raf (a serine-threonine specific kinase), PI3K/AKT exert an anti-apoptotic effect.56 The anti-apoptotic effect is also medi-ated by AKT directly, which causes the phosphorylation and

Figure 3. HSP 90 (Heat Shock Protein) is a mediator of cellular homeostasis. Although a cytoplasmic protein, HSP 90 affects diverse nuclear processes including transcription, chromatin remodeling and cell growth. HSP 90 stabilizes, protects and activates many proteins and oncoproteins/clients critical for the proliferation of cancer cells. HSP 90 clients include protein kinases, anti-apoptotic proteins, transcription factors, steroid hormones and cytokine receptors. Clients also include many signaling pathway proteins involved in mediating MM cell survival and growth, including Akt, iKK, mutant p53, c-raf-1, HiF-1 and others, thereby making it a rational drug target.

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an mTOR inhibitor, NVP-BEZ235, in combination with an immunomodulatory agent (IMID).63,64

Temsirolimus, an ester of sirolimus, is a signal transduction inhibitor with antitumor properties, which specifically inhib-its mTOR. Recently, a phase II trial involving patients with relapsed or refractory multiple myeloma showed response rate of 40%.65 Temsirolimus, in combination with bortezomib, for

The PI3K/AKT/mTOR pathway mediates proliferation, sur-vival and drug resistance in MM cells, which makes it a rational target for an mTOR inhibitor. Significantly, PTEN, a nega-tive regulator of PI3K/AKT pathway, is mutated in 5–20% in myeloma patients. This mutation tends to occur in advanced disease and is associated with enhanced sensitivity to an mTOR inhibitor.62 Recent studies demonstrated promising results using

Figure 4. HDAC inhibitors lead to histone hyperacetylation, resulting in transcriptional activation and alteration of gene expression responsible for tumor viability. HDAC inhibition leads to growth arrest, differentiation and apoptosis. Among the non-histone substrates of HDACs are cell motility molecules (α Tubulin), chaperones (HSP90), gene transcription factors and co-regulators (p53, c-myc, Bcl2) and signaling mediators (NF-κB, STAT 3), which contribute further to the anti-myeloma activities of HDAC inhibitors. HDAC, histone deacetylase; HSP90, heat shock protein 90; NF-κB, nuclear factor-κB.

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mTOR inhibitor or FTI in combination with bortezomib/rev-limid represents a rational strategy to be pursued in clinical trials.

Summary

An important focus of cancer research is understanding the signal transduction pathways that regulate the proliferation of malignant cells, with the assumption that molecular signals required for tumor cell growth may be excellent therapeutic targets. MM management, in the era of novel agents, remains challenging as the majority of patients develop resistance over time by obscure mechanisms. Thus, finding and developing new agents with novel mechanisms of action or improving the second generation of novel agents (e.g., irreversible proteasome inhibitors) are pressing issues and require detailed knowledge of signaling pathways governing myelomagenesis. New drugs are showing promise are in clinical trials (e.g., carfilzomib, pomalidomide) while numerous other drugs that target dif-ferent pathways are tested in combination with novel agents in ongoing clinical trials (inhibitors of HDACs, AKT, HSP, mTOR) should expand the therapeutic arsenals and improve outcome.

relapsed/refractory MM is being evaluated in a phase II trial; so far, 27 patients have been treated with weekly I.V. Temsirolimus and bortezomib every 35 days. A minor response was observed in 79% of patients, with CR/nCR in 10% of patients and only 10% showed progressive disease. Overall, side effects proved manage-able with Grade 4 toxicity seen in 14% of patients.66

Ras can activate PI3K, which contributes to cell survival and suppression of apoptosis. Furthermore, gain-of-function muta-tions in RAS are relatively common in MM patients and their frequency increases from 27% at diagnosis to 46% as the disease progresses.67,68 Membrane localization of Ras has been inhibited with farnesyl transferase inhibitors (FTIs) (Ras becomes active only after the post-translational addition of farnesyl moieties to its carboxy terminus. Without such modification, Ras remains in the cytosol and cannot activate the PI3K or MAPK pathways). The use of an FTI inhibits the growth and induces apoptosis in drug resistant myeloma tumor cells.29 The sequence of bortezomib fol-lowed by an FTI (lonafarnib) induces synergistic cell death in both MM cell lines and patient samples. These preclinical studies suggest that FTIs may represent new therapeutic agents for the treatment of refractory MM.

In patients refractory to novel agents (IMID, bortezomib), targeting the PI3K/AKT pathway using an AKT inhibitor,

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