Background

Multiple Myeloma (MM) is an incurable malignant plasma cell disease with an incidence of 5 per 100,000 inhabitants, affecting approximately 25,000 new patients per year in the EU, with a debilitating impact on personal lives and health management. MM locates primarily to the bone marrow (BM) - in multiple “niches” that provide a microenvironment which promotes tumor survival. In turn, multiple myeloma cells (MMCs) profoundly alter the BM, resulting in osteolytic lesions, anemia, and immunosuppression. On a molecular level, MM is characterized by a marked disease heterogeneity as defined by chromosomal aberrations and gene expression profiles, with at least 7 groups definable at presentation (Zhan et al. 2006). Concordantly, intraclonal variation between tumor cells in a given patient, as defined for example by interphase fluorescence in situ hybridization (iFISH) (Hose et al. 2009b), are indicative of on-going disease evolution.


The current treatment paradigm differentiates between symptomatic patients (Durie et al. 2006) eligible for intensive treatment, and those not, which include elderly patients above the age of about 65-70 years. Intensive treatment typically comprises an induction regimen to decrease tumor mass and improve patient’s status including high dose corticosteroids (dexamethasone, D), and at least one so-called “novel agent”. The latter comprise the proteasome inhibitor (bortezomib, B, Velcade) and the immunomudulatory drugs thalidomide (T) or lenalidomide (L, also termed Revlimid) (Cavo et al. 2010; Goldschmidt et al. 2003; Harousseau et al. 2010). These compounds are likely to be the mainstay of treatment in MM for the foreseeable future. They act on MMCs, “niche cells”, and their interaction (Davies et al. 2001; Gorgun et al. 2010; Perez et al. 2010). After induction treatment, MMCs are further eliminated with high dose of an alkylating agent (high dose melphalan, HDM) with hematopoietic stem cells rescue by autologous stem cell transplantation (ASCT). The overall survival (OS) of intensively treated patients below 65-70 is 8-9 years and the event free survival (EFS) is 3-4 years (Barlogie et al. 2010; Harousseau and Moreau 2009).


Patients invariably relapse after each subsequent treatment regimen, become resistant to treatment, and succumb to their disease.


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I.    Clinical consideration of drug resistance


Therapy

Knowledge of the mode of action of current drugs in use in MM is necessary to assess emergence of resistant MMCs in patients. D is cytotoxic to MMCs and blocks the production of inflammatory factors supporting MMC growth. B blocks the degradation of proteins through the proteasome resulting in blockade of cell cycle and apoptosis of MMCs. T and L show pleiotropic mechanisms of action (anti-angiogenesis, T and NK cell stimulation, apoptosis induction and cell cycle arrest (Escoubet-Lozach et al. 2009; Huang et al. 2010; Li et al. 2010b; Verhelle et al. 2007), that are not fully elucidated (Li et al. 2010a). HDM is a DNA intercalating molecule that blocks DNA replication and induces death of replicating MMCs. HDM also destroys the proliferating hematopoietic stem cells that need to be rescued by ASCT. All three “novel agents” (B, T, L) are under investigation as consolidation or maintenance therapy after HDM and ASCT for variable periods of time to control or eliminate remaining MMCs (Palumbo et al. 2010b; Sonneveld et al. 2010). For patients ineligible for intensive treatment, combinations of MP (Melphalan and Prednisone) are used in combination with a novel agent, i.e. T, or B (Facon et al. 2007; San Miguel et al. 2008) and eventually L (Palumbo et al. 2010a). Both groups of patients are treated with bisphosphonates that decrease bone turnover by inducing osteoclast apoptosis (Berenson et al. 2002; Weinstein et al. 2002). Relapse treatment is typically based on the use of combinations of novel agents with D, mainly TD, LD, BD, and eventually HDM and ASCT. BD and LD are used in most patients in first and second relapse (Engelhardt et al. 2010; Raab et al. 2009). Thereafter, therapy becomes more and more heuristic including the use of less effective (e.g. bendamustine) or experimental agents (Michael et al. 2010; Raab et al. 2009). Typically, the time to relapse in responsive patients decreases in each sequential relapse. Ultimately MMCs become refractory to all treatment options, or patients do not tolerate further treatment due to side effects, most often hematological. Systematic studies on the percentage in MM in which a drug is active in relapse if used in induction treatment are lacking.


Conventional and molecular prognostic factors

MMCs harbor a high median number of chromosomal aberrations (Fonseca et al. 2004) and multiple changes in gene expression compared to normal BM plasma cells (Hose et al. 2009a; Hose et al. 2009b; Seckinger et al. 2009; Zhan et al. 2002; Zhan et al. 2006). This molecular heterogeneity is thought to transmit into the very different survival times ranging from a few months to 15 or more years (Barlogie et al. 2006). Relapse appears at any time from early, intermediately to lately. Two main approaches are currently used to predict when relapse appears, that is for risk stratification in myeloma: i) readily obtainable clinical prognostic factors (Greipp et al. 2005) and ii) molecular diagnostics assessing chromosomal aberrations and changes in gene expression.


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II.    Drug resistance in cancer and in MM


Tumor drug resistance involves both intrinsic mechanisms in tumor cells and extrinsic microenvironment-mediated protection.


Intrinsic mechanisms of drug resistance in tumor cells.

These mechanisms can be classified into 4 categories.


1) Efflux transporters in tumor cells and drug resistance.


Multidrug resistant transporters extrude large hydrophobic cytotoxic agents from cancer cells, and are ATP binding cassette (ABC) proteins, of which 3 major types found in humans are members of the ABCB (ABCB1/MDR1/P-glycoprotein), ABCC (ABCC1, ABCC2, probably ABCC306, 10-11) and ABCG (ABCG2) subfamilies, with multiple members in each (Sarkadi et al. 2006). Upregulated transporter expression mediates resistance in cancer cells by ATP-dependent extrusion of chemotherapeutic drugs.


2) Gene mutations, gene and miRNA expression in tumor cells and drug resistance.


The most striking example of mutation conferring drug resistance is in the BCR-ABL fusion protein, which is oncogenic and drives chronic myeloid leukemia (CML) growth. The BCRABL fusion protein can be potently blocked by Imatinib kinase inhibitor, but mutations in it allow its escape (Quintas-Cardama et al. 2009). The hypoxia-inducible factor HIF1α has been associated with chemosensitivity and regulates p53 activity, raising the potential for p53 mutations to restrict efficacy of use of HIF1α inhibitors (Rohwer et al. 2010).


Genomic mutations and their role in cancer are now being examined at a hitherto unparalleled level, made possible by the availability of the draft human genome in 2000 (McDermott et al. 2011) and decreasing costs by several orders of magnitude. Many cancer genomes are under evaluation by the International Cancer Genome Consortium [http://icgc.org]. Two models have emerged of cancer origins. In the first, mutations are characterized as either ‘drivers’ or ‘passengers’ in transformation, and cancer progression occurs by sequential stepwise acquisition of drivers mutations in subclones which dominate growth (Stratton et al. 2009). In the second, seen less frequently, i.e. in 2%-3% of all cancers and in ~25% of bone cancers, malignant transformation is driven by a single catastrophic event, termed chromothripsis (‘chromosome’ – ‘shattering’), where genomic aberrations occur at a scale incompatible with stepwise acquisition (Stephens et al. 2011).


The role of miRNA in drug resistance in cancer is also emerging as significant (Zheng et al. 2010). Imbalanced miRNA expression can mediate a multitude of cellular effects, such as regulating apoptosis. In Multiple Myeloma, miRNA expression in primary MMCs is linked with high risk (Zhou et al. 2010), but the mechanisms of action of the prognostic miRNA have yet to be characterized.


3) Signaling pathways, apoptosis and drug resistance.


Apoptosis and its abrogation in cancer cells have also been delineated as mechanisms that promote resistance to drugs. MCL-1 leukemic cells are more sensitive to chemotherapy than their BCL-2 counterparts (Brunelle et al. 2009). Autophagy is a complex response in cancer cells involving pro-death and pro-survival signals in response to cell death inducing agents and is currently under evaluation as a mechanism of drug resistance (Dalby et al. 2010).


4) Tumor stem cell and drug resistance


The stem cell question in cancer has attracted much attention recently, and has been proposed as relevant to understanding drug resistance, as a quiescent cell that escapes therapy (Blanpain et al. 2011; Dick 2008). In Multiple Myeloma, there is no formal identification of tumor stem cells able to propagate the disease. Most MM research teams, including OVER-MyR partners, failed to identify any such precursor (Perez-Andres et al. 2010; Pfeifer et al 2011).


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Extrinsic mechanisms - Tumor environment and drug resistance


The role of niche cells in supporting tumorigenesis and survival in cancer is now widely accepted, and key studies are prominent. For example, cancer-associated fibroblasts (CAFs) co-injected with tumor cells alter the niche cell state and stimulate angiogenesis and tumor invasion (Erez et al. 2010). Many niche cell communication signals promoting tumor survival and repair can likewise induce drug resistance in cancer cells. These include:


1) Cell communication molecules (e.g. growth factor receptors) that activate signaling pathways (MAP kinase, JAK/STAT, PI3-kinase/akt, NF-κB) can be directly altered to resist drug therapy (Sierra et al. 2010).


2) Chemokines (e.g. SDF-1, CCL2) can play a role in binding to G-receptors and are critical in recruiting tumor cells in close vicinity to the tumor environment.


3) Specific adhesion molecules are best exemplified by integrin α4β1 that binds VCAM1/ICAM1 and fibronectin and confers drug resistance in various tumors (Damiano et al. 1999).


In Multiple Myeloma little is known of the role or mechanisms by which specific niche cells protect MMCs from drug assault to potentiate resistance. Evidence to date centers on 3 main reports:


1. Tumor-associated macrophages (TAMs) mediate protection of MMCs from M (Zheng et al. 2009).


2. Components of BM stromal cells enhance NF-κB activity in MMCs and can enhance B resistance (Markovina et al. 2010).


3. Recent observations suggest that APRIL drives a subset of MMCs bearing a specific IgH-translocation into cell cycle (Quinn et al. 2011) and that this may underlie drug resistance.

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