Genetic Events & Their Implications
Many of these primary and secondary genetic events define biological subgroups of disease with distinct clinical behavior. In addition, the identification of dysregulated genes and pathways defines potential therapeutic targets.
Myeloma With t(4;14). The t(4;14) has been shown to lead to the simultaneous overexpression of two genes, FGFR3 and MMSET, and therefore provides two potential therapeutic targets.[7,8] The regulation of a number of other genes in the translocation area may be affected, although their precise role is yet to be determined. The t(4;14) group constitutes 10–15% of presenting cases and has been associated with adverse prognosis in a variety of clinical settings and is widely accepted to constitute an adverse prognostic factor in the context of high-dose therapy with autologous stem cell transplant (ASCT).[9–15] There is emerging evidence that bortezomib may ameliorate this poor prognosis, implying that the short survival associated with some adverse biomarkers may be context specific, and may be improved by alternative therapies.[16,17]
Upregulation of FGFR3 results in ectopic expression of the FGFR3 tyrosine kinase receptor. Several tyrosine kinase inhibitors exist that are active against the FGFR3 receptor and have shown effects in preclinical models, including PRO-001, CHIR 258 and PKC412.[18–20] However, the role of FGFR3 in MM pathogenesis has been questioned as 30% of tumors with the t(4;14) lack expression of FGFR3 due to the loss of der(14). Interestingly, the poor prognosis associated with t(4;14) in such patients lacking FGFR3 expression remains unchanged, lending support for a role for MMSET.[9,21] MMSET has recently been shown to have histone methyltransferase activity, with high levels of MMSET corresponding with an open chromatin structure. By this mechanism of epigenetic regulation, increased MMSET expression has been suggested to contribute to cellular adhesion, clonogenic growth, tumorigenicity and DNA repair.[22–24] The enzymatic activity of the histone methyltransferase containing SET domain of MMSET constitutes a possible drug target for this biological subgroup. In addition, recent work has identified a snoRNA ACA11 encoded within intron 18–19 of the MMSET gene that is highly expressed in t(4;14) myeloma. Its function is not yet clear, but the only study conducted so far suggested that it binds to proteins involved in postsplicing intron complexes.
Myeloma With Increased MAF Expression. Gene expression classification identifies 5–10% of presenting myeloma patients as having increased expression of the MAF oncogene, which is usually a result of the t(14;16) that upregulates c-MAF or the t(14;20) that upregulates MAFB.[13,26–28] t(14;16) is a rare translocation that has been linked with impaired clinical outcome in three patient series, although this prognostic significance has recently been questioned.[29,30] t(14;20) is the rarest IGH@ translocation and it also appears to be associated with short survival.
In a recent study, myeloma cell lines were treated with a panel of inhibitors of several pathogenically relevant cellular pathways and decreased expression of MAF, and its target genes were only seen with inhibition of MAP kinase signaling using MEK inhibitors. In this study, MEK inhibition caused downregulation of MAF in patients with the t(14;16) and t(4;14), suggesting a common pathway of MAF regulation in these two groups. MEK inhibition stopped proliferation and induced apoptosis in cell lines with these translocations and may represent a possible specific therapy for the high MAF expression subgroup.
Myeloma With t(11;14) & t(6;14). t(11;14) and t(6;14) result in direct dysregulation of cyclin D genes, which are key cell-cycle regulators. CCND1 is upregulated by t(11;14) and occurs in 17% of patients, whereas t(6;14) occurs in 2% of cases and upregulates CCND3. The prognostic impact of these events is neutral. While these translocations directly increase the expression of cyclin D genes, most myeloma cases without these translocations also have upregulation of cyclin D genes, and it has been proposed that cyclin D dysregulation is a unifying event in myeloma pathogenesis. A number of cyclin D inhibitors with various specificities have been shown to block cell cycle and induce specific myeloma cell apoptosis in laboratory studies and are now undergoing early-phase trials.
Myeloma With Increased MYC Expression. Upregulation of c-MYC is found in 15% of myeloma tumors and is linked to aggressive features such as plasmablastic disease. The increased c-MYC expression is often mediated through secondary translocations involving 8q24, which are common in human myeloma cell lines (HMCLs), and as such are thought to represent a progression event or a late genetic event. As secondary translocation events, c-MYC translocations do not always involve the IGH@ gene, with 40% of translocations involving different partner genes. Moreover, when the IGH@ locus is involved, the breakpoint does not usually occur within switch regions or V(D)J sequences, and the translocation is often complex.
Modifying MYC expression therapeutically is difficult; however, a recent study has demonstrated that it is possible to modulate the function of the c-MYC oncoprotein by altering MYC transcription. This is achieved by interfering with chromatin-dependent signal transduction to RNA polymerase, specifically by inhibiting the acetyl-lysine recognition domains (bromodomains) of putative coactivator proteins implicated in transcriptional initiation and elongation. In laboratory studies, JQ1 has been demonstrated to be a selective small-molecule BET bromodomain inhibitor, which downregulates MYC transcription. This is followed by genome-wide downregulation of MYC-dependent target genes resulting in potent anti-myeloma proliferative effects associated with cell-cycle arrest and cellular senescence. Studies are underway to modify this molecule to a clinical formulation applicable for clinical trial use.
Chromosomal Deletions or Gains
Myeloma With +1q. Gain of the long arm of chromosome 1 is a frequent event in myeloma, which is described in 39% of newly presenting patients. In patients with specific IGH@ translocations such as t(4;14) and t(14;16), this rate increases to approximately 70%, and +1q may be particularly important in the progression process of these etiological subgroups. The prognostic importance of +1q was initially highlighted by the University of Arkansas for Medical Sciences (UAMS) high-risk gene expression signature, which was enriched with overexpressed genes on 1q and underexpressed genes on 1p. Gain of 1q21 detected by FISH has been linked to adverse prognosis in patients treated intensively and nonintensively, and in patients treated with or without thalidomide.[37–39]
1p21 has been identified as a minimally amplified region that contains the potential candidate oncogenes CKS1B, PDZK1 and BCL9.[40,41] In addition, ANP32E has been identified as an upregulated gene linked to adverse overall survival (OS) in this region.CKS1B overexpression has been shown to activate STAT3 and MEK/ERK signaling, so targeting these signaling pathways with specific inhibitors may benefit this patient group. The strong association of +1q21 with adverse prognosis using both FISH and array-based gene expression technologies means that it is central to a risk-adapted therapeutic approach, as outlined later.
Deletion of 1p. Approximately 32% of presenting myeloma patients have a deletion of 1p, and several different regions and genes have been identified as being of potential pathogenic importance. 1p32.3 is hemizygously deleted in 11% of presenting cases and is also frequently homozygously deleted, with homozygous deletions affecting two genes, FAF1 and CDKN2C. Deletion of 1p32.3 is associated with impaired survival in the treatment context of ASCT; but in nonintensively treated patients, it has a neutral prognostic impact. 1p22.1–21 is the most frequently deleted region of 1p, and del(1)(p21) has been linked to short survival in newly diagnosed patients treated with high-dose chemotherapy, and in relapsed patients treated with lenalidomide and dexamethasone.[43,45,46] 1p12 has also been identified as a region with recurrent homozygous deletions. FAM46C appears to be the target of these deletions, and it has been shown in two studies to be frequently mutated in myeloma, which suggests a pathogenic role.[44,47] As this region is deleted, it is unlikely to represent a region amenable to therapeutic targeting; however, information regarding deletion of 1p could be incorporated into risk-adapted therapeutic strategies.
Deletion of 13q. Deletion of chromosome 13 is the most common chromosomal abnormality in presenting patients, being found in 45% of cases tested by interphase FISH (iFISH), although this is lower at 19% when tested by conventional cytogenetics. The relationship between del(13q) and adverse prognosis is difficult to assess due to its association with other high-risk genetic lesions such as t(4;14). Early studies showed an association between del(13q) and impaired survival.[49–51] Recently, in studies using iFISH, the association with short survival in multivariate analyses is lost, as the short survival is mainly due to the presence of t(4;14). However, when tested by conventional cytogenetics, the association of del(13q) and poor prognosis remains.
In the majority of cases, deletion constitutes monosomy 13, making the identification of a critically deregulated region or gene difficult. One of the minimally deleted regions that has been defined is at 13q14, which contains a number of candidate genes including RB1. As mentioned earlier, targeting a minimally deleted region is difficult, although therapeutic approaches targeting mutant RB1 are being developed that may play a role in myeloma.
Deletion of 17p. Hemizygous deletion of 17p is found in approximately 10% of newly presenting myeloma cases. Gene mapping shows that in most of these cases, the whole of 17p is deleted. However, the tumor suppressor gene TP53, which maps to 17p13, is thought to be the relevant deregulated gene in these cases. In presenting myeloma cases without del(17p), the rate of TP53 mutations is <1%, which rises to 25–37% in cases with deletion, and this provides some evidence of the importance of TP53 deregulation in these cases.[54–56]TP53 is activated in times of cellular stress, inducing cell-cycle arrest and apoptosis. Consequently, functional TP53 is important in mediating the apoptotic response to chemotherapy.
Del(17p) has been reported to be associated with short survival in newly presenting patients treated with conventional induction chemotherapy and ASCT.[14,15,54,58] Del(17p) remains associated with impaired survival in newly diagnosed patients treated with thalidomide and in relapsed patients treated with lenalidomide, suggesting that Immunomodulatory drugs may not improve the poor outcome of this patient group.[53,59–61] The impact on bortezomib in this patient group is uncertain. A recent analysis of patients with del(17p) treated with bortezomib or conventional induction followed by ASCT showed that bortezomib failed to improve the outcome of this group. Conversely, two independent studies found that the administration of bortezomib before and after ASCT improved the survival of patients with del(17p).[62,63]
There are several possible therapeutic strategies for specifically targeting the TP53 response. The main inhibitory molecule of TP53, MDM2, can be targeted by selective small-molecule antagonists such as nutlin. Nutlin has been shown to increase TP53 levels in myeloma cells that retain wild-type TP53 protein, which is the majority of presenting myeloma patients, driving the cell to apoptosis. Nutlin has also demonstrated synergy with bortezomib, which may contribute by blocking proteasomal degradation of TP53.[64,65] An alternative approach for targeting the TP53 axis is to use the concept of synthetic lethality. Loss of TP53 function specifically inactivates the G1S checkpoint, making cells sensitive to agents targeting the G2M checkpoint in the presence of DNA damage. Treating cells with a defect in the G1S checkpoint with a CHK1 inhibitor, which inhibits the G2 checkpoint, may sensitize them to the induction of cell death.
Proliferative Myeloma. In comparison with many other tumors, myeloma cells are characterized by a relatively low rate of cell division. However, a subset of myeloma patients has a higher proliferative rate, and these patients have more aggressive disease.[67–69] Proliferation can be quantified by the plasma cell labeling index, which identifies cells in S-phase using a monoclonal antibody (BU-1) reactive with 5-bromo-2-deoxyuridine. However, this technique has failed to find widespread application and alternative techniques such as gene expression have been used to define proliferative disease.[13,33,71,72] As well as identifying patients with poor prognosis, the high proliferation rate means that this group may benefit from agents that target cells in mitosis. This includes some traditional agents such as vinca alkaloids, which affect spindle formation, and newer agents such as aurora kinase inhibitors. Aurora kinases regulate the G2 cell-cycle checkpoint, and are intimately involved in centrosome and spindle formation. Preclinical data are available on several active compounds, demonstrating that these aurora kinase inhibitors induce apoptosis of myeloma cell lines and patient samples.
Until recently, mutational studies have been limited to studies of selected candidate genes such as TP53, N-RAS, K-RAS and NF-κB pathway genes. The advent of next-generation sequencing technologies, which sequence either the whole genome or exome of a tumor, will illuminate recurrent mutational events and enhance our understanding of gene mutations in myeloma. The initial results of sequencing 38 myeloma genomes have recently been reported, showing how these data may be used to inform myeloma biology in the future. Mutations were found in NF-κB pathway members, highlighting the recognized importance of dysregulation of this pathway in myeloma, but also in genes involved in protein modification, histone methylation and blood coagulation, suggesting novel oncogenic mechanisms in myeloma. A number of groups have confirmed these findings[73,74] and in the future it is likely that sequencing results will be used to define deregulated genes and pathways for the purposes of therapeutic targeting.
Cellular Signaling Pathways Deregulated in Myeloma
A summary of cell signaling pathways discussed in this section is shown (Figure 2). It should be noted that much of the work carried out to elucidate these pathways has been performed in cell lines. Although groups have validated these results in primary patient cells, parallels between in vivo and cell lines may be limited. Much work is ongoing to improve these in vitro/in vivo model systems to ensure the effective translation of in vitro work to the development of effective new therapies.
NF-κB. NF-κB is a transcription factor that is upregulated in both myeloma tumor cells and the bone marrow stromal cells that surround the tumor. In myeloma cells, upregulation of NF-κB causes disruption of cell-cycle and apoptotic pathways; while in stromal cells, it triggers production of important cytokines such as IL-6 and BAFF that cause paracrine stimulation of the myeloma cells.[75,76] Homozygous deletion events affecting NF-κB regulatory genes including TRAF3 at 14q32, CYLD at 16q12.1 and BIRC2/BIRC3 at 11q22.2 have been reported, and mutations of NFKB2, NFKB1, CYLD, TACI, NIK, TRAF2, TRAF3, BIRC2, BIRC3 and CD40 are also described.[77–80] The preliminary results of whole-genome sequencing of 38 myeloma tumors found 14 mutations in NF-κB genes, highlighting the potential importance of deregulation of this pathway in some myeloma tumors.
Gene expression signatures related to increased activation of NF-κB signaling have been described, and it may be possible to adapt these as diagnostic tools for measuring NF-κB activity. Alternatively, patients with the deletional or mutational events described above could be targeted with agents that act through downregulation of NF-κB signaling. Within the tumor cells, there are two NF-κB pathways, the canonical and noncanonical, both of which involve the proteasome suggesting that the effectiveness of proteosome inhibitors in myeloma may be due to altered NF-κB signaling. Agents that target that NF-κB pathway more specifically have been developed, such as MLN120B that is an inhibitor of IKKβ. MLN120B has been shown to selectively inhibit the canonical NF-κB pathway, resulting in an inhibition of myeloma cell growth in vitro and an augmentation of the effects of dexamethasone, doxorubicin and melphalan.[81,82] Upregulation of the NF-κB pathway in bone marrow stromal cells has also been shown to be important in myeloma pathogenesis. MLN120B decreased IL-6 secretion from bone marrow stromal cells, suggesting that agents that target the NF-κB pathway may modulate the protective environment of the bone marrow milieu as well as targeting the myeloma cell.
The MAPK Pathway. The MAPK cascade is involved in regulation of cell differentiation, proliferation and survival. The pathway can be stimulated by various cytokines including IL-6, IGF-1 and TNFα, which activate the RAS/RAF kinases. RAF activates MEK, which in turn activates ERK.
RAS mutation prevalence is variable across studies, but in the majority, it is between 20 and 35%, making it one of the most commonly mutated genes in myeloma [MORGAN G, PERS. COMM.,] Initial studies suggested that NRAS was more commonly mutated than KRAS, but more recent data suggest that they are of equal prevalence [MORGAN G, PERS. COMM. Mutated RAS has been associated with poor prognosis and more aggressive disease features. As RAS mutations are found rarely in MGUS they are likely to be a secondary transforming event in myeloma. The first signaling stage of the pathway involves transferring farnesyl groups from farnesyl diphosphate to RAS, allowing RAS to attach to the intracellular membrane. This step can be targeted by farnesyl transferase inhibitors, and several of these have demonstrated some efficacy in myeloma, including perillic acid, FTI-277 and Tipifarnib.[87–89] Further downstream, MEK has been targeted by agents such as AZD6244 and AS703026. Increased MEK signaling promotes steroid resistance, and MEK inhibition with AZD6244 has been shown to sensitize cells to dexamethasone.[91,92] AZD6244 treatment of HMCLs also inhibited growth and survival, as well as downregulating the secretion of osteoclast-activating factors, suggesting that targeting MEK may also have a beneficial effect on myelomatous bone disease. Given the prevalence of the RAS mutation in myeloma, MEK inhibition has promising therapeutic potential.
Next-generation sequencing identified BRAF mutations in 4% of tumors.[47,73] This finding has potential clinical relevance, as BRAF kinase inhibitors are active in melanoma patients who harbor the V600E BRAF mutation.
The PI3K Pathway. IL-6, IGF-1 and HGF induce AKT phosphorylation, which in turn activates several downstream targets including mTOR, GSK-3B and forkhead transcription factor. AKT activation has been linked to resistance to dexamethasone-induced apoptosis, mediated through inactivation of capsase-9. mTOR, a serine/threonine protein kinase, upregulates the expression of several downstream targets such as the D-Cyclins, influencing G1/S phase transition and cellular proliferation. Inhibition of P13K signaling should result in G1 growth arrest and sensitization to steroid-induced apoptosis. The pathway can potentially be targeted at several points. Several P13K inhibitors have been characterized in HMCLs including SF1126, pichromene and CAL-101.[94,95] Phosphorylation and activation of AKT is inhibited by the synthetic alkylphospholipid perifosine, which has demonstrated in vitro antimyeloma activity and has moved into early-phase clinical trials. Downstream of AKT, the P13K pathway can be downregulated by mTOR inhibition. Rapamycin is an mTOR inhibitor produced by Streptomyces hygroscopius, which has demonstrated in vitro antimyeloma activity and synergism with bortezomib and lenalidomide. Rapamycin analogs have been developed, which mediate mTOR inhibition through binding to FKBP-12. Temsirolimus has moved into Phase II trials in combination with bortezomib following demonstration of a tolerable side-effect profile.[99,100] It has been suggested that inhibition of mTOR may upregulate upstream elements of the P13K pathway such as AKT through autoregulatory feedback loops, and that P13K or AKT may, therefore, be more suitable candidates for targeted inhibition. Dual inhibition of mTOR by rapamycin and AKT with perifosine may overcome this feedback mechanism, and this approach has been shown to be effective in vitro. New agents in development target the ATPase activity of PI3 kinase upstream of mTOR and AKT, for example, NVP-BEZ235 and PI-103 derivatives, and show dual inhibition of AKT and mTOR. These agents are in Phase I clinical trials and results are awaited.
JAK/STAT Pathway. The JAK/STAT3 pathway has been found to be constitutively activated in approximately 50% of primary myeloma samples; and in this setting, suppression of STAT3 with cucumin results in apoptosis. JAK inhibition has been shown to inhibit phosphorylation of STAT3, and to inhibit expression of STAT3 target genes including CCND2, and several JAK inhibitors have demonstrated antimyeloma efficacy in vitro including atimprimod, AZD1480, TG101209 and INCB16562.[104,105] Mouse models have confirmed the potential utility of atimprimod, which has moved into early-phase clinical trials.
The Unfolded Protein Response Pathway. The unfolded protein response is a critical process for the efficient production of immunoglobulin, making it essential for the normal functioning of mature plasma cells. A build-up of misfolded immunoglobulin triggers an increase in IRE1α endoribonuclease activity, which processes unspliced XBP1 (XBP1u) to its spliced, active form (XBP1s), enabling it to function as a transcription factor. The ratio of XBP1 s/u has been shown to be prognostically important, with a low ratio being associated with better OS. Moreover, patients with a low ratio who received thalidomide had superior survival compared with patients not receiving thalidomide, suggesting that XBP1 could be used as a predictor of thalidomide response. Molecules that target this pathway either nonspecifically (e.g., bortezomib) or specifically (e.g., IRE1α RNase inhibitors) are under development.[108,109]
Epigenetic Deregulation in Myeloma
DNA Methylation. DNA methylation is important in normal cell development and differentiation as well as being important in the progression of cancer. Analysis of cancer genomes has identified that global hypomethylation and gene-specific hypermethylation are common in many different cancer cell types. Methylation changes occur at CpG dinucleotides that are present at a higher frequency in promoter regions and within repeat sequences and transposable elements. Hypomethylation in cancer cells mainly occurs within the repeat sequences and transposable elements, whereas hypermethylation occurs in promoter regions, particularly of suspected tumor suppressor genes. Hypermethylation of DNA is associated with heterochromatin and correlates with regions of inactive transcription.
A recent study used a genome-wide approach to interrogate methylation status in myeloma and showed a clear distinction in methylation pattern between nonmalignant cells (B cells, normal plasma cells and MGUS cells) compared with malignant plasma cells (presentation myeloma, PCL and HMCLs). At the transition from MGUS to myeloma, the key feature was a marked loss of methylation. Genome hypomethylation is frequently linked to altered chromatin structure and increased frequencies of copy number abnormalities. Gene-specific hypo- and hyper-methylation was also identified at the transition of MGUS to myeloma.[110,111] Pathway analysis of the genes affected demonstrated involvement of developmental, cell-cycle and transcriptional regulatory pathways. At the transition from myeloma to PCL, rather than finding further hypomethylation as may have been anticipated, gene-specific hypermethylation of genes involved in cell signaling and cell adhesion pathways was found. Unsupervised clustering of myeloma samples defined several independent methylation profiles: one t(4;14) group, two separate t(11;14) groups and two separate hyperdiploid groups. The most distinct of these methylation profiles belonged to the t(4;14) cytogenetic subgroup that showed more frequent hypermethylation of genes compared with the other subgroups and is likely to be related to the histone methyltransferase activity of MMSET.
There is a potential to target DNA methylation changes with DNA methyltransferase inhibitors such as 5-azacytidine. 5-azacytidine is a pyrimidine analog that can lead to the removal of methyl groups from DNA. It has the potential to reverse some of the gene-specific hypermethylation that has been characterized at the transition of MGUS to myeloma, increasing the expression of silenced tumor suppressor genes. Conversely, such agents may also increase the global hypomethylation process, so whether the net biological effect would be beneficial is unknown. However, 5-azacytidine has been shown to induce apoptosis in myeloma cells, to overcome the survival advantage conferred by cytokines such as IL-6, and to enhance the activity of other drugs including doxorubicin and bortezomib, so the overall effects may be positive. Although there is extensive experience of DNA demethylating agents in other hematological conditions such as myelodysplasia, they have not been widely used in myeloma to date. However, these recent insights into the role of methylation in myeloma may provide a biological basis for the use of these agents in specific patient groups.
In addition to targeting DNA methylation, a number of groups have investigated the role of histone deacetylase inhibitors as a way of altering gene transcription and inducing myeloma cell death. Although these inhibitors 'theoretically' target histone, it is now known they have a number of other effects that may be important in mediating their antimyeloma activity.[113–115]
miRNA genes encode a class of small (17–25 base pairs) RNAs that act as epigenetic regulators as they do not code for proteins themselves, but regulate the translation of other proteins by complementary base pairing to specific mRNA protein-coding transcripts. They have been demonstrated to act as oncogenes or tumor suppressors in numerous tumor types, and their transcriptional control is regulated by promoter hypermethylation or global hypomethylation. Several groups have attempted to identify which miRNAs are differentially expressed in myeloma. When compared with normal plasma cells, upregulated miRNAs in cell lines included miR-32, miR-21, miR-17~92, miR-106~25 and miR-181a/b. miR-17~92 is MYC related and was only upregulated in myeloma, not MGUS, suggesting that expression could be related to MYC upregulation during myeloma progression. A global increase in miRNA expression in high-risk myeloma has also been demonstrated. Specific miRNA patterns have also been noted with different genetic supgroups. Deregulation of 16 miRNAs mapped to chromosomal regions frequently involved in ploidy changes were identified including miR-22 at 17p13.4 and miR-25 at 7q22.1. In addition, deregulation of miRNAs has been associated with chromosomal translocation including miR-1 and miR-133a upregulation with the t(14;16) translocation. This suggests an impact from chromosomal abnormalities on miRNA expression. In addition, miRNAs may target several pathways involved in cell-cycle progression and cell survival including the STAT3 pathway and the MDM2/p53 autoregulatory loop.[117,122] There is some discrepancy between studies as to which miRNAs are differentially expressed and at what stage of disease; however, the overall conclusions are consistant that deregulation of miRNAs are likely to be involved in myeloma pathogenesis and warrants further investigation both to further the understanding of the biology of myeloma and as potential targets for treatment.
Other genes involved in RNA processing have also been found to be mutated in the whole genome sequencing of myeloma. These include DIS3, encoding an RNA exonuclease, and FAM46C, thought to be functionally related to the regulation of translation. Evidence of alternative splicing dysregulation has also been reported in myeloma and was shown to have an effect on OS. It is thought that dysregulation of protein translation by these routes may be an oncogenic mechanism and could yield potential therapeutic targets in future.
Expert Rev Hematol. 2012;5(6):603-617. © 2012 Expert Reviews Ltd.