Drug Resistance in Multiple Myeloma: How to Cross the Border

Abstract

Multiple myeloma is a hematological malignancy of plasma cells, characterized by a high level of genetic instability, with various gene mutations and chromosomal translocations. Despite the introduction of effective novel agents in the treatment of myeloma, i.e., proteasome inhibitors and Immunomodulatory Drugs (IMiDs), disease relapse and progression occur almost universally, due to innate and acquired drug resistance. We here describe molecular mechanisms and pathways of resistance so far elucidated. Due to the genetic complexity of myeloma, characterized by intra-clonal heterogeneity and progressing with branching evolutionary patterns, we believe that a single general strategy to overcome drug resistance in all patients cannot be developed. However, it is possible that in the future drug resistance will be prevented or treated with the individualized application of genomic and proteomic analyses, targeting the vulnerable pathways of a specific patient in a specific phase of the disease.

Keywords: Drug resistance; Multiple myeloma; Thalidomide; Lenalidomide; Pomalidomide; Bortezomib; Carfilzomib

Introduction

Multiple myeloma (MM) is a hematological malignancy of plasma cells (PCs), characterized by lytic bone lesions, anemia, renal insufficiency, and the presence of monoclonal proteins in the blood and urine [1]. It is the second most common hematopoietic malignancy, with approximately 22,350 new cases and 10,710 deaths reported in 2013 [2]. The median age at diagnosis is approximately 70 years; 37% of patients are younger than 65 years, 26% are between the ages of 65 and 74 years, and 37% are 75 years of age or older [1]. Therapeutic advancement in recent years has increased the overall survival of myeloma patients. This is due not only to autologous Stem Cell Transplantation (SCT), but also to the availability of novel agents, such as the Immunomodulatory Drugs (IMiDs) thalidomide, lenalidomide, and pomalidomide, and the proteasome inhibitors bortezomib and carfilzomib [3]. Despite the use of these new agents, MM relapses in virtually all patients [4].

The unique feature of myeloma cells is their intimate relationship with the bone marrow (BM) microenvironment. This consists of various Extracellular Matrix (ECM) proteins, cell components, osteoclasts, and osteoblasts [5]. The BM microenvironment provides a specialized niche that supports growth of myeloma and maintain their long-term survival by secreting growth factors such as Interleukin-6 (IL-6), Insulin Like Growth Factor-1 (IGF-1), Vascular Endothelial Growth Factor (VEGF), B-cell Activating Factor (BAFF), Fibroblast Growth Factor (FGF), Stromal cell-Derived Factor 1a (SDF1a), and Tumor Necrosis Factor-a (TNFa) [6-10]. The direct interaction of the BM microenvironment with myeloma is responsible for myeloma cells growth and survival, angiogenesis, osteolytic lesions, and drug resistance [11-13]. Not surprisingly, multiple efforts have been made to target the interaction of myeloma cells to BM microenvironment for its treatment [14].

As in other malignant neoplasms, myeloma patients initially respond to chemotherapy; however, during the course of the disease, either intrinsic or acquired resistance to chemotherapy will emerge. MM is characterized by a complex genomic instability and cytogenetic constitution, which predisposes the malignant cells to intrinsic resistance [15-17]. The acquired resistance is mediated by several mechanisms, including overexpressed drug transporter proteins, such as P-glycoprotein (P-gp) -also known as Multidrug Resistance Protein 1 (MDR1)-[18], overexpression of anti-apoptotic proteins, such as B-cell leukemia protein 2 (Bcl-2), B-cell lymphoma-extralarge (Bcl-XL), Bcl2-related protein A1 (A1/Bfl1), and constitutive expression of transcription factor nuclear factor kappa B (NF-κB). Thus, therapeutic interventions that can induce apoptosis of tumor cells could become useful approaches to treat MM.

Intrinsic resistance in multiple myeloma

Genomic instability and complex cytogenetic constitution are hallmarks of MM. The genetic lesions leading to myeloma have been recognized as inherited variations, translocations, copy number abnormalities, mutations, methylation, and microRNA (miRNA) abnormalities [15,16,19]. These lesions are associated with the myeloma proliferation and drug resistance [15,17]. Based on karyotype abnormalities, MM can be divided in two main groups: hyperdiploid and non-hyperdiploid. Studies suggested that only 10% of hyperdiploid group show a primary IgH translocation at 14q32 locus, whereas 70% of cases in the non-hyperdiploid group carry that [20]. Of note, patients in the hyperdiploid group have a better survival and prognosis of those in the non-hyperdiploid group. The IgH translocation usually involves juxtaposition of the immunoglobulin gene to an oncogene on partner chromosomes creating several reciprocal translocations. The more frequent ones are the t(4;14) (p16;q32) and t(11;14)(q13;q32) translocations, present in 15% and 17% of myeloma patients, respectively [21]. There are several other translocations involving aberrant expression of oncogenes, such as c-MAF, c-Myc, and Cyclin D [21-24]. Whether these cytogenetic aberrations are directly associated with drug resistance and relapse is still debatable and warrants further investigations; nonetheless, recent studies have provided evidence that some of these cytogenetic abnormalities predispose to drug resistance and are associated to disease relapse. For example, studies of Chang-Yew et al. showed that blocking c-MAF, the oncogene overexpressed in patients with t(14;16) and t(14;20) translocations, inhibits cell proliferation and sensitizes myeloma cells to chemotherapy drugs [25,26]. Moreover, 1q21 gain, found in almost 70% of multiple myeloma patients harboring the t(14;16) or t(4:14) translocations, has been correlated with an adverse outcome [27,28].

Acquired resistance in multiple myeloma

Role of P-glycoprotein in acquired resistance

One of the most important players for acquiring drug resistance is the P-gp, produced by the MDR/ATP-Binding Cassette (ABC) B1 gene. This protein is the best known family member of pump transporters that mediate cellular efflux of peptides and drugs. Studies have shown that almost 75% of patients treated with doxorubicin, dexamethasone, and vincristine do increase the expression of P-gp [29]. Importantly, the expression of P-gp is found to be very low in untreated multiple myeloma patients, suggesting that the cumulative dose increases the expression of this protein in myeloma cells. A second drug-efflux protein, the Multidrug Resistance Protein-1 (MRP1), has not been found to be overexpressed in myeloma cells [30,31], making P-gp as the candidate protein in drug resistance. In untreated patients, P-gp does not seem to be expressed de novo. Interestingly, studies have shown that patients treated with melphalan do not increase P-gp expression [29], but increase in expression of P-gp was associated with treatment of other conventional chemotherapy agents, such as vincristine, doxorubicin, and dexamethasone [32], as well as novel agents, such as bortezomib and carfilzomib [33].

Because of the critical role of p-gp in drug resistance, inhibition of P-gp activity has become a major focus in clinical studies, and several MDR modulators have been tested to reverse drug resistance. For example, a phase I/II study was conducted using the MDR modulator verapamil, in combination with vincristine, doxorubicin, and dexamethasone, to treat patients with refractory MM [31]. This strategy induced a partial response rate of 50%, but this combination therapy resulted in excessive toxicity, and P-gp inhibitors do not constitute standard of care in the current management of MM.

Activation of nuclear factor-κB (NF-κB) and acquired resistance

Another major pathway that plays an important role in drug resistance is the NF-κB signaling pathway. The NF-κB term refers to a family of signal-responsive transcription factors that includes RelA/ p65, c-Rel, RelB, NF-κB1/p50 and NF-κB2/p52 [34]. In normal resting cells, NF-κB transcription factors are maintained in an inactive state within the cytoplasm through binding to inhibitory proteins called IκBa, which also include the unprocessed p105 and p100 forms of NF-κB1 and NF-κB2 [34]. The activation of NF-κB is divided into two pathways, canonical and non-canonical. The canonical pathway is induced by physiological NF-κB stimuli including TNF-a or IL- 1, and antigen receptors [35-37]. These cytokines bind and induce Tumor Necrosis Factor Receptor 1 (TNFR1) signaling. Stimulation of TNFR1 leads to the binding of tumor necrosis factor receptor Type1-Associated Death Domain protein (TRADD), which provides an assembly platform for the recruitment of Fas-Associated protein with Death Domain (FADD) and TNF Receptor-Associated Factor 2 (TRAF2) [38]. TRAF2 couples with receptor-interacting serine/ threonine-protein kinase 1 (RIP1) for IκB kinase (IKK) activation. IKK complex consists of two catalytically active kinases, IKKa and IKKβ, and the regulatory subunit IKKγ (NEMO) [39,40]. In the canonical pathway, IκBa is phosphorylated in an IKKβ- and NEMO-dependent manner followed by ubiquitination and proteasomal degradation, which thus releases the bound NF-κB dimers so they can translocate to the nucleus. In contrast, the non-canonical pathway, inducedby certain TNF family cytokines -such as CD40L, B-cell Activating Factor (BAFF) and lymphotoxin-β (LT-β)-, involves IKKa-mediated phosphorylation of p100 associated with RelB, which leads to partial processing of p100 and the generation of transcriptionally active p52-RelB complexes. IKKa activation and phosphorylation of p100 depends on Nuclear factor κB-Inducing Kinase (NIK), which is tightly regulated by TRAF3, TRAF2 and additional ubiquitin ligases [35-38]. Once in the nucleus, NF-κB dimers are further regulated by protein phosphorylation [41] and other post-translational modifications, such as protein acetylation [42], and activate genes whose products inhibit cell death [43-46], stimulate cell proliferation [47], and promote migratory and invasive phenotypes that are associated with tumor progression [48]. The transcription factor NF-κB could also contribute to drug resistance in myeloma through up-regulation of some Bcl-2 anti-apoptotic family members including Bcl-XL [49].

Myeloma cells often show constitutive expression of NF-κB [50]. Most solid and lymphoid tumors show constitutive NF-κB activity [51]. Constitutive or overexpression of NF-κB has been associated with myeloma cell proliferation, survival, invasion and drug resistance [52,53]. Although the precise role of NF-κB activation in pathogenesis of myeloma has not been fully characterized, it has been shown that myeloma cell adhesion to bone marrow stromal cells induces NF- κB-dependent up-regulation of transcription of IL-6 [54,55], which is a major growth and survival factor for myeloma cells. Since IL-6 upregulates adherence of myeloma cells to fibronectin and it confers resistance to drug-induced apoptosis, blockade of NF-κB signaling represents a novel therapeutic strategy in MM. Recent studies have indicated that targeting NF-κB pathway in myeloma has a positive outcome [56,57]. Along these line, myeloma cells harboring TRAF3 gene mutation in NF-κB pathway are resistant to dexamethasone but sensitive to bortezomib [58], suggesting that apoptotic function of bortezomib is partly explained by blocking the canonical NF-κB pathway; however, at the same time it unexpectedly induces the alternative (non-canonical) pathway, which makes myeloma cells less responsive [59]. Moreover, recent studies have shown that though canonical NF-κB pathway can be successfully blocked by small-molecule inhibitors of IKKß, in myeloma cell lines in vitro, the anti-MM activity in vivo of these IKKß inhibitors is limited because of the compensatory activation of the non-canonical pathway [60]. Furthermore, it has been reported that myeloma cells tend to develop a bortezomib-resistant NF-κB phenotype through a Proteasome- Inhibitor Resistant (PIR) pathway [61]. The latter flaws of bortezomib may explain to a large extent why it should be applied in combined regimens for those myeloma patients who are refractory to it. Since bortezomib-induced cytotoxicity cannot be fully attributed to inhibition of canonical NF-êB activity in myeloma cells, inhibition of both canonical and noncanonical pathways would be a desirable strategy to combat resistance.

Cancer Stem Cells and Resistance

The association of cancer stem cells (CSCs) and drug resistance has been demonstrated in various types of cancer including MM [62]. The importance of targeting CSC population has been realized for many years, but this research has been limited by the fact that a specific CSC population in MM has not been properly defined yet [17]. Studies from the last decade have supported the notion that CSCs are resistant to chemotherapeutic agents. Various strategies have been proposed to target the molecular, metabolic, and epigenetic signatures, and the self-renewal signaling pathway characteristic of CSCs. These cells exhibit high levels of ABC activity, Aldehyde Dehydrogenases (ALDH1), and Retinoic Acid (RA) Receptor a (RARa) that have been associated with clonogenic potential and resistance to chemotherapy [63,64]. Further research is needed to understand the molecular mechanisms that keep myeloma cells clonogenic and drive the transition between stem-like and non-stemlike states in the local bone marrow microenvironment. These studies could provide us new approaches to target drug resistance in MM.

Drug resistance to specific antineoplastic agents

Although we are far from having a satisfactory knowledge of the different mechanisms of resistance to various chemotherapy agents, multiple studies have elucidated particular strategies and mutations developed by myeloma cells after exposure to specific drugs. We herein describe the most important published experience with the various class of agents used in the treatment of MM. A few examples are shown in Figure 1.

    

    

    Figure 1:  Examples of mechanisms of resistance of myeloma cells to
chemotherapeutics agents (details in text).
1: Increased repair of DNA interstrand cross links with alkylating agents;
2: Mutations of the hormone-binding region of the Glucocorticoid Receptor
(GR); 3: Decreased expression of the Cereblon (CRBN) protein with
the IMiDs drugs; 4: Mutations of the bortezomib-binding pocket of the
proteasome; 5: Multidrug Resistance (MDR), with cellular extrusion of a
variety of chemotherapy agents by the drug efflux transporter P-glycoprotein.