DNA repair pathways in human multiple myeloma

Review

Paper type

Cell Cycle 12:17, 1–14; September 1, 2013; © 2013 Landes Bioscience

DNA repair pathways in human multiple myeloma

Role in oncogenesis and potential targets for treatment Claire Gourzones-Dmitriev1,2, Alboukadel Kassambara 1,2, Surinder Sahota3, Thierry Rème1,2, Jérôme Moreaux1,2, Pascal Bourquard4, Dirk Hose5, Philippe Pasero6, Angelos Constantinou6, and Bernard Klein1,2,7,* CHU Montpellier; Institute of Research in Biotherapy; Montpellier, France; 2INSERM; U1040; Montpellier, France;3Cancer Sciences Unit; Faculty of Medicine; University of Southampton; Southampton, UK;4CHU Nimes; Clinical Hematology; Nimes, France;5Medizinische Klinik V; Universitaetsklinikum Heidelberg; Heidelberg, Germany;6Institute of Human Genetics; CNRS-UPR1142; Montpellier, France;7Université Montpellier 1; UFR Médecine; Montpellier, France

Keywords: multiple myeloma, DNA repair, genetic instability, drug resistance, non-homologous end joining, homologous recombination, melphalan, bortezomib Abbreviations: 3′-P, 3′-phosphate; 3′-PUA, 3′-phospho-α,β-unsaturated aldéhyde; alt NHEJ, alternative non-homologous end joining; AP, apurinic/apyrimidinic; APE1, AP endonuclease 1; BER, base excision repair; CAK, CDK-activating kinase; CSR, class switch recombination; CT, chromosomal translocation; FA, Fanconi anemia; GG-NER, global genome nucleotide excision repair; HR, homologous recombination; ICL, interstrand DNA crosslink; Ig, immunoglobulin; IGH, immunoglobulin heavy chain; MGUS, monoclonal gammopathy of undetermined significance; MM, multiple myeloma; MMCs, multiple myeloma cells; MMR, mismatch repair; MRN, Mre11, Rad50, Nbs1; NER, nucleotide excision repair; NGS, next generation sequencing; NHEJ, non-homologous end joining; PI, proteasome inhibitor; PIDD, p53-induced protein with a death domain; PNKP, polynucleotide kinase 3′-phosphate; RIP1, receptor-interacting protein 1; ROS, reactive oxygen species; RPA, replication protein A; SHM, somatic hypermutation; SSA, single strand annealing; SSB, single-strand break; TC-NER, transcription-coupled nucleotide excision repair

Every day, cells are faced with thousands of DNA lesions, which have to be repaired to preserve cell survival and function. DNA repair is more or less accurate and could result in genomic instability and cancer. We review here the current knowledge of the links between molecular features, treatment, and DNA repair in multiple myeloma (MM), a disease characterized by the accumulation of malignant plasma cells producing a monoclonal immunoglobulin. Genetic instability and abnormalities are two hallmarks of MM cells and aberrant DNA repair pathways are involved in disease onset, primary translocations in MM cells, and MM progression. Two major drugs currently used to treat MM, the alkylating agent Melphalan and the proteasome inhibitor Bortezomib act directly on DNA repair pathways, which are involved in response to treatment and resistance. A better knowledge of DNA repair pathways in MM could help to target them, thus improving disease treatment.

In multiple myeloma (MM), malignant plasma cells display marked genomic abnormalities that arise during pathogenesis of disease and accrue during progression, and implicate aberrant DNA repair pathways in both phases. Extrinsic and intrinsic

*Correspondence to: Bernard Klein; Email: [email protected] Submitted: 06/30/2013; Revised: 07/28/2013; Accepted: 07/29/2013 http://dx.doi.org/10.4161/cc.25951

www.landesbioscience.com

factors initiate DNA repair. Extrinsic stimuli during germinal center transit in tumor origins result in recurrent chromosomal translocations, where double-strand DNA breaks are erroneously resolved. Distinctive features of ploidy arise early and associate with cell cycling and dysregulated DNA repair. Intrinsic cellular mechanisms mediate pervasive ongoing DNA damage at disease onset and during progression that recruits multiple DNA repair pathways. Further extrinsic challenges arise when DNA-modifying drugs, such as melphalan or Bortezomib, are delivered as therapy and contribute to disease progression and relapse. In this review, we examine the major cellular and molecular pathways that mediate DNA repair, and evaluate existing evidence for imbalanced mechanisms in MM cells. The evidence suggests that activity of DNA repair pathways in malignant plasma cells is highly likely to be pivotal in determining survival and therapeutic outcome. It specifically identifies key dysregulated pathways, the nature of cross-talk between central signaling pathways and DNA repair, polymorphisms in DNA repair genes, or deregulated expression of these genes, and shows that they frequently correlate with treatment response and patients’ survival. Furthermore, it reveals that therapeutic drugs used to treat MM can either activate or inhibit DNA repair pathways. The findings finally suggest that novel strategies to regulate DNA repair pathways have the potential to augment current therapies in resolving disease. Thus, understanding the DNA damage response in MM is important to improving patients’ survival and to progress the next phase of effective patient-specific therapies.

Cell Cycle

1

©2013 Landes Bioscience. Do not distribute.

1

Etiology MM remains in the main an incurable malignant plasma cell disease, with 22 000 new patients per year in the EU or in the USA and a median survival of 5 years.1 Seminal observations have established that it is preceded by monoclonal gammopathy of undetermined significance (MGUS), a premalignant phase2-6 that occurs in 3% of individuals over 50 years of age.7 MGUS can persist before developing to MM at a 1% rate/year, independently of the duration of the MGUS phase.2,4,6 MGUS or MM plasma cells are clonal cells, with molecular evidence that the same clone evolves during transformation between disease states.8 In individuals with MGUS, proliferation of clonal plasma cells remains below detection levels.9-11 In patients, MM cells (MMCs) cycle poorly (median percentage of MMCs in the S phase = 0.4%),12 and MMC proliferation is one of the strongest prognostic factors in MM, predicting for short overall survival.10,11,13 Molecular abnormalities and genomic instability in MM MMCs originate from post-follicular B cells, which have undergone multiple immunoglobulin (Ig) gene rearrangements during maturation: early recombination of V(D)J genes, and subsequent somatic hypermutation (SHM) of IGV genes that code for variable Ig domains, and deletional isotype class switch recombination (CSR) of Ig heavy chain (IGH) genes at the 14q32 locus.14 SHM in IGV genes is also a feature of MGUS,8 many of which undergo CSR. MGUS or MM can be grossly divided into non-hyperdiploid (75 chromosomes, half of patients) or hyperdiploid tumors (48–75 chromosomes, trisomies of odd chromosomes 3, 5, 7, 9, 11, 19, 21).3,5,6,15-17 Primary 14q32 chromosomal translocations (CTs) are found in the vast majority (>70%) of non-hyperdiploid MM and less frequently in hyperdiploid tumors (15% of cases). These primary translocations are recurrent in subsets of disease, juxtaposing CCND1 gene – t(11q13;14q32), 15–18% of tumors; MMSET/FGFR3 genes – t(4p16;14q32), 10–15% of tumors; MAF, MAFB, or MAFA genes – t(14q32;16q23), t(14q32;20q11), t(8q23;14q32), 8% of tumors; or CCND3 gene – t(6p23;14q32), 2% of tumors, under the control of strong Eμ intronic or 3′ IGHα enhancers that dysregulate gene expression.15-17 Most 14q32 CTs map into the switch IgH region, implicating deletional CSR as a pathogenic mechanism. Resolution of double-strand DNA breaks during CSR requires DNA repair pathways, including non-homologous end joining (NHEJ) (discussed below).3,5,14,18 Double-strand DNA breaks can also occur during SHM, to generate deletions and insertions in IGV genes, which surprisingly occur in general as multiples of nucleotide triplets.19 The aberrant resolution of SHM strand breaks by DNA repair specifically has been implicated in generating some CTs in MM.20 Some14q32 CTs have as-yet-undefined partner chromosomes, suggesting that multiple transcriptionally active loci are captured and targeted for translocated lesions by dysregulated DNA repair. Importantly, these CTs are present in the MGUS phase,8,21 indicating that abnormalities in the DNA repair spectrum is manifest early in pathogenesis and may be a pre-requisite for malignant transformation.

2

A resulting hallmark of the CT abnormalities is a deregulated expression of at least 1 of the 3 cyclin D genes in virtually all patients with MGUS or MM, either by constitutive activation of CCND1 or CCND3 genes or by upregulation of pathways (FGFR3/MMSET or MAF) resulting in high CCND2 expression.6,22-25 High cyclin D expression is found also in hyperdiploid MM, but the mechanisms are less clear and may involve gene amplification or downregulation of miRNA targeting genes (CCND1, FGFR3, MAF, TCC3), which are partners for IGH translocations in non-hyperdiploid tumors.9,26 This high expression of cyclin D may lead to deregulation of cell cycle checkpoints, as well as DNA repair pathways,12,27 to accentuate genetic instability, a hallmark of MM disease.10,11,13,28 Transposon-like elements can also integrate at abnormal sites to dysregulate cyclin D expression.29 Little is known as yet of the role of DNA repair pathways in mediating these transposon-like events in MM. Other translocations and genetic alterations involve TP53, NFκB, MYC, and KRAS genes, whose gene products are critical players in DNA replication and repair, are detected in MM tumors, with an increased incidence with disease evolution.14,17,20,25,30,31 Monoallelic deletion of chromosome 17p, including TP53 gene, is found in 7–10% of newly diagnosed patients in association with reduced survival, in 50% of patients with extramedullary disease, and increases with disease activity.6,15-17,32 In 30% of patients with monoallelic del17p, TP53 mutations are found in the remaining allele.33 P53 protein may also be degraded in MMCs by upregulation of MDM2 through methylation of the promoter of miRNAs targeting MDM2.34 Constitutive NFκB activity is observed in primary MMCs from about 20% of newly diagnosed patients,35-38 in part due to alterations of various genes coding for proteins of the NFκB pathway.36,37,39 Both canonical and non-canonical NFκB pathways are activated in MM,40 in association with drug resistance and/or with relapse.38 Finally, whereas translocations of MYC gene are found in MMCs of 15% of newly diagnosed patients,41 amplifications of MYC gene are found in 70% of patients with hyperdiploid MM, and 20% of non-hyperdiploid ones.42,43 MYC translocations are found in the majority of MM cell lines (90%).44 CTs can be late events in the 14q32 locus, notably involving MYC, and often appear as subclonal events.21 These observations reveal an ongoing relevance of DNA repair pathway abnormalities for disease progression in MM. A role for these pathways appears to persist under therapy. Recent evidence from aCGH studies serially tracking MM progression has shown that subclonal competition marked by specific genomic abnormalities is a common feature of disease, and that therapeutic pressure may be an important driver.45,46 Some of these subclones may have existed at onset of therapy at below detection levels, and others reflect a further tumor diversification that recruits aberrant DNA repair. Comparable features are also emerging from next generation sequencing (NGS) of the tumor genome, where sets of gene mutations are identifiable only in relapse samples, also implicating dysregulated DNA repair pathways, and persist in progeny.47,48 High-resolution SNP arrays have delineated wide-ranging structural alterations in the MM genome, including loss of heterozygosity,49 further highlighting a lack of corrective measures in the MM DNA repair machinery,

Cell Cycle Volume 12 Issue 17


Multiple Myeloma


To understand these genomic issues, we review the links between DNA repair pathways and genetic abnormalities in MM, which are likely to have a significant impact on disease occurrence, drug resistance, and/or relapse. Normal DNA Repair Pathways As described above, each cell must resolve up to 105 DNA lesions each day arising by spontaneous decays, replication errors, cellular metabolism, and exposure to irradiation or chemicals. To ensure cell survival and functionality, these lesions have to be repaired by specific pathways, which involve sensor proteins that nucleate the assembly of proteins and trigger a cascade of posttranslational modifications to initiate DNA repair.60,61 We begin with a brief overview of the essential features of the major DNA repair pathways that operate in normal cells. Excision repair Base excision repair (BER) eliminates the 104 base damages that occur spontaneously and daily in each healthy cell due to hydrolysis, oxidation, or methylation reactions (Fig. 1).55 Conventional BER is initiated by specific mono- or bi-functional DNA glycosylases, which recognize and hydrolyze the N-glycosylic bond between the damaged base and the sugar phosphate backbone, creating an apurinic/apyrimidinic (AP) intermediate site. The monofunctional glycosylases create a hydrolytic AP site, which is cleaved 5′ to the AP site by AP endonuclease 1 (APE1), and 3′ to the AP site by an AP lyase, to yield a single nucleotide gap that can be filled in by polymerase β and ligated by XRCC1/ligase 3 (Fig. 1).62 Following base removal by a bifunctional DNA glycosylase, the protein can incise the DNA backbone immediately 3′ to the AP site product via a β- or β,δ-elimination reaction, producing a single-strand break (SSB) with a 3′-phospho- α,βunsaturated aldehyde (3′-PUA) or 3′-phosphate (3′-P) group. APE1 removes the 3′-PUA residue and polynucleotide kinase 3′-phosphatase (PNKP) excises the 3′-P moiety. Nucleotide excision repair (NER) NER corrects a wide spectrum of bulky helix-disturbing DNA lesions (Fig. 1).63 Examples of lesions are cyclobutane pyrimidine dimers and pyrimidine-(6,4)-pyrimidone photoproducts generated by UV radiation, base adducts created by exogenous chemical agents such as melphalan, cisplatin, or benzopyrene, base lesions produced by reactive oxygen species (ROS) or endogenous lipid peroxidation products. NER can be coupled to transcription, a process known as transcription-coupled nucleotide excision repair (TC-NER), as opposed to global genome nucleotide excision repair (GG-NER), whereby the transcribed strand of active genes is repaired faster than in the genome in general and is triggered when the RNA polymerase is blocked at the lesion. In GG-NER, DNA lesions are sensed by XPC/RAD23/CENT2 and UV-DDB complexes, which recognize distortions of the DNA double helix. In TC-NER, the nucleotide excision repair process is coupled to transcription by CSA and CSB, 2 proteins implicated in Cockayne syndrome.64 In both GG-NER and TC-NER, the damaged duplex DNA is opened around the lesion by TFIIH, a multi-subunit helicase complex that includes XPB, p62, p52, p44, p34, p8, XPD, and the CDK-activating-kinase (CAK). Next, the

Cell Cycle

3


or masking to generate these lesions. The propensity for the t(11;14) CT in MM in particular is also markedly increased by a CCND1 polymorphism from GWAS studies,50 raising the question whether DNA repair mechanisms may differ in this constitutive genetic background and route to MM, and by extension in MM subsets segregated by specific CT abnormalities. The molecular mechanisms that yield non-hyperdiploid or hyperdiploid states are as yet unknown, but are faithfully recapitulated in tumor progeny in MGUS and MM. Aneuploidy results from chromosomal instability, which can affect segregation of whole chromosomes or cause specific chromosomal abnormalities, including deletions, insertions, and translocations.51 In relation to cancer, a question that has persisted is whether aneuploidy is a causal driver of transformation or a by-product, and is as yet not fully resolved. Whole chromosome segregation occurs during mitosis and begins during S phase, when DNA strands must be replicated precisely, involving specific components of the DNA repair pathway. Subsequently, in early mitosis, a spindle-assembly checkpoint ensures correct attachment of sister chromatids to the mitotic spindle prior to the initiation of chromosome segregation.52 Several mechanisms can aberrantly impact on mitotic segregation of chromosomes, including mutations in spindleassembly checkpoint genes which bypass p53-mediated cell arrest and lead to ploidy defects. These genes include the BUB and MAD spindle checkpoint gene families and have been found to be mutated in some cancers, but are extremely rare in MM, suggesting that comparable mutations do not play a role in chromosome instability in MM.53 In contrast, genes regulating centrosome assembly, a microtubule-organizing structure that is essential for generating dual spindle poles in mitosis, have been implicated in MM-associated chromosome instability. By establishing a gene expression-based index (centrosome index) that correlated with centrosome amplification, it has been shown that a high centrosome index is associated with a markedly poor prognosis in MM.54 Of note, genes regulating centrosome duplication and function and relevant to centrosome index include genes in DNA repair, such as ATM, ATR, RAD51, XRCC2, BRCA2, and others.54 A further backdrop to the genomic landscape in MM is the essentially perpetual cascade of “intrinsic” molecular lesions that target DNA as part of normal physiological processes. About 105 DNA hits can occur on a daily basis in a cell, caused by spontaneous decay (depurination, deamination, and methylation reactions), replication errors, cellular metabolism, reactive oxygen species, and by environmental DNA damaging agents such as UV light, ionizing radiation, or chemicals.55,56 It is not clear whether the rate of these intrinsically generated defects are increased or decreased in MMCs. However, if unrepaired, these chemical modifications in the DNA may ultimately undermine genetic integrity and deregulate cell death inducing pathways, and may be selectively resolved for survival by cancer cells, for which they may be particularly well adapted. Gene deregulation in MMCs may also due to epigenetic controls. Aberrant DNA methylation is observed in MMCs from patients and can affect tumor suppressor genes.57,58 In agreement, DNA methyltransferase (DNMT)3A and DNMT3B genes are overexpressed in MMCs compared with normal plasma cells.59

between 53BP1 and BRCA1. 53BP1 favors NHEJ and is antagonized by the product of the breast cancer susceptibility gene BRCA1, which promotes HR68,69 (Figs. 2 and 3). Methylation of histone H4 by MMSET leads to recruitment of 53BP1,70 which blocks DNA end resection by the Mre11/Rad50/Nbs1 (MRN) complex, CtIP, and BRCA1.69 In contrast, histone H4 acetylation by Tip60 blocks 53BP1 recruitment and promotes BRCA1 occupancy and, subsequently, HR.71 HR is also promoted by PARP1 and by proteins implicated in the inherited disorder, Fanconi anemia, from the toxic interference of NHEJ proteins.72-74 Cell cycle-regulated proteins such as cyclin-dependent kinases also play a key role in choice of pathway to resolve DSBs.60 Non-homologous end joining (NHEJ) NHEJ is a process that is active in all phases of the cell cycle, whereby broken ends are simply spliced back together (Fig. 2). As NHEJ functions independently of a DNA template for repair, it is intrinsically prone to altering the genetic information of the repaired DNA molecules. NHEJ starts with the recognition of DNA ends by Ku70/80 and is followed by the recruitment and activation of the DNA-dependent protein kinase DNA-PKcs, and of DNA end-processing enzymes such as ARTEMIS and template-independent polymerases (polymerases λ and μ) that might be necessary for end ligation by the XLF-XRCC4-DNA ligase 4 complex.18,60 DSBs are also generated during normal SHM and CSR modifications of B-lymphocyte Ig genes and repaired mainly by NHEJ.75 Homologous recombination (HR) HR is active in the S or G2 phases of the cell cycle, when sister chromatids templates are available for repair (Fig. 2). HR begins with nucleolytic resection of DNA ends, mediated by the MRN complex, CtIP, and BRCA1, which yields 3′ singlestranded DNA tails that are stabilized by replication protein A (RPA). Next, the breast cancer susceptibility gene product BRCA2 catalyzes the displacement of RPA and the formation of a RAD51 nucleoprotein filament. The RAD51 filament promotes homology search and catalyzes an exchange strand between the broken duplex and the intact sister chromatid. The 3′ end of the invading strand is extended by DNA synthesis using the sister chromatid as a template, and intact duplexes are eventually restored using one of multiple resolution mechanisms.76 Single strand annealing (SSA) Figure 1. DNA repair pathways of single strand DNA damages. Every day, cells are faced with thousand Extensive DNA resection within of damages involving base damages or bulky adducts, which have to be repaired to preserve cell funcrepetitive DNA sequences can result tion. Base damages induced by depurination or deamination are repaired by base excision repair, bulky in RAD52-mediated annealing of adducts (in particular pyrimidine dimers induced by UV radiation) by nucleotide excision repair and base mismatch occurring during DNA replication by mismatch repair. Repair pathways involve first sensing resected ends, followed by removal of of damage, followed by cleavage of damaged DNA, and synthesis of the correct DNA sequence using DNA flaps by XPF-ERCC1 nucleases complementary strand as template.

4



structure-specific endonucleases XPG and XPF/ERCC1 excise DNA 3′ and 5′ to the lesion, respectively, to eliminate a 20–30 nt-long oligonucleotide that comprises the DNA lesion. Finally, new DNA is synthetized by polymerases δ, κ, and e, using the intact strand as a template, and terminated by XRCC1/ligase3. Mismatch repair (MMR) MMR removes nucleotides that have been misincorporated during DNA replication and prevents promiscuous recombination between divergent sequences (Fig. 1).65 Mispaired bases are sensed by the MutSα heterodimer (MSH2-MSH6), whereas MutSβ (MSH2-MSH3) recognizes insertions/deletions of more than 2 extra bases. Lesion recognition is followed by the recruitment of MutLα (MLH1/PMS2) or MutLβ (MLH1/MLH3), which have endonuclease activity that can incise DNA near the mismatch. The nick is used by the 5′ exonuclease Exo1 as an entry point to degrade DNA past the mismatch, and the resulting single-stranded DNA gap is filled in by polymerase δ and sealed with DNA ligase I.65,66 Double-strand break (DSB) repair DSBs can be generated by DNA replication when cells enter S phase before completing SSB repair. They can also be generated by reactive oxygen species, ionizing radiation, or chemicals such as inhibitor of topoisomerase I or during the repair of interstrand DNA crosslinks (ICLs).60,67 The choice between NHEJ and homologous recombination (HR) repair pathways is regulated by complex regulatory mechanisms and involves competition

MGUS or healthy plasma cells (Fig. 4). Association between polymorphisms or deregulated expression of the XRCC5 (encoding Ku80), XRCC6 (encoding Ku70), ARTEMIS, LIG4 (encoding DNA ligase 4), or XRCC4 genes and the potential risk of developing MM has been described (Fig. 4). The polymorphic XRCC5 (Ku80) sequence is located within an exon splicing enhancer sequence and may result in exon skipping, yielding splicing errors and affecting mRNA stability and protein translation.84 The alanine-3-valine or threonine-9-isoleucine DNA ligase 4 allele is associated with a decreased risk of developing MM.85 A 4–5-fold increase in XRCC4 gene expression was found in MMCs compared with other lymphoma cells (mantle cell lymphoma, follicular cell lymphoma, diffuse large B-cells lymphoma, marginal zone lymphomas) or normal B cells from excised benign reactive

Deregulation of DNA Repair in Multiple Myeloma Cells

DNA repair pathways are critical to repair both the developmental impact of DSBs generated by SHM and CSR in Ig genes in MM origins and resolve the plethora of DNA lesions that occur each day in cells due to endogenous and exogenous stress. We review here the current knowledge on changes in DNA repair pathways in MM that could trigger disease development, perpetuate genomic instability, and affect response to diverse treatment regimen. Unresolved data on NHEJ deregulation in multiple myeloma cells Although several reports have documented polymorphisms or deregulation in genes/proteins involved in NHEJ pathway in MMCs, no definitive conclusion can be drawn as to whether the NHEJ pathway is deregulated in MMCs as compared with


Figure 2. DNA repair pathways of double-strand DNA breaks. DNA doublestrand breaks (DSB) occur in transcriptionally active or replicating cells in the presence of reactive oxygen species, ionizing radiation, or when the replication machinery encounters unrepaired single-strand damage. DSBs also enable DNA rearrangements in order to select for antigen-specific T or B lymphocytes. In addition, the main drugs used in cancer target cell replication machinery resulting in DSB and cell death. Two major pathways compete to repair DSBs. The accurate homologous recombination (HR) requires a duplicated sister chromatid and is active only at the end of S phase or in the G2 phase. Non-homologous end joining (NHEJ) is active in all phases of cell cycle and involved partial resection and ligation of DNA ends. The choice between HR and NHEJ in replicating cells is dictated by 53BP1 and BRCA1, which compete to bind DNA ends and block mutually. alt-NHEJ, alternative less accurate pathways, and SSA, single-strand annealing, can repair DSBs and may yield to DNA deletion, amplification, or translocation.

Cell Cycle

5


(Fig. 2). SSA is inaccurate because it results in the deletion of one of the repeats and the intervening sequence. SSA may also lead to translocations when 2 DSBs occur within or near repeats located on different chromosomes.60,77 Alternative NHEJ (alt NHEJ) Defects in NHEJ promote DSB repair by a robust but less accurate alternative NHEJ pathway, whereby DNA ends are resected up to regions of microhomology, annealed, and ligated by XRCC1/ligase 3 (Fig. 2).60,66 The 53BP1 protein protects DNA ends from nucleolytic degradation and thereby prevents microhomology mediated repair.78 Interstrand-crosslink (ICL) repair ICLs are covalent bounds between the 2 antiparallel strands in duplex DNA. These lesions are induced by treatment with bifunctional alkylating agents, such as melphalan, which is frequently used to treat patients with MM.67 In non-replicating cells, the repair of mono- and di-adducts is mediated by the nucleotide excision repair machinery79 and by the DNA translocase FANCM, which facilitates the access of nucleases to the lesion (Fig. 3).80 In S-phase cells, crosslink repair is coupled to DNA replication, features DSBs as repair intermediates, and depends on the homologous recombination machinery Fig. 3).67 The products of genes that are defective in the chromosomal instability disorder Fanconi anemia (FA) orchestrate the coordinated action of the multiple enzymatic activities that are necessary to couple the recombination-dependent ICL repair with DNA replication.81 In response to genotoxic insults, a complex of 8 FA proteins in the nucleus (FANCA, -B, -C, -E, -F, -G, -L, and -M) catalyzes the conjugation of an ubiquitin moiety to FANCD2 and FANCI protein to activate their DNA repair functions. Ubiquitinated FANCD2 and FANCI are necessary for nucleolytic incisions to unhook the ICLs and for the insertion of nucleotides opposite the unhooked crosslinks.82,83 However, it is not yet entirely clear which of the structure-specific endonuclease FAN1, MUS81/ EME1, SLX1-SLX4 (FANCP), and ERCC1/XPF are responsible for ICL unhooking; translesion DNA synthesis across the unhooked ICL depends on REV1 and polymerases ζ.67

Figure 3. DNA repair of interstrand crosslinks (ICLs). Toxic compounds can create DNA Interstrand Crosslinks (ICLs), which impair transcription and replication machinery, and yield to cell death if unrepaired. ICL formation is the active principle of many cancer drugs, in particular alkylating agents. The removal of ICL involves the Fanconi anemia pathway, with sensing of ICL by FANCM, and then recruitment of protein complex resulting in ICL removal, creation of DSB, which is repaired by homologous recombination. In non-cycling cells, the initial sensing by FANCM is followed by ICL removal and DSB repair by poorly known mechanisms.

6

repair assay, NHEJ activity was found to be corrupted in one (RPMI8226) and functional in two (U266, OPM2) MM cell lines.93 A link between NHEJ defect and radiation sensitivity was unclear, since both RPMI8266 and U266 cells were radiationsensitive, and addition of a DNA-PKcs inhibitor was toxic in untreated cells but remained innocuous to irradiated cells. On the contrary, OPM2 cells were radiation-resistant, and the DNAPKcs inhibitor abrogated radiation resistance while exhibiting no toxicity to untreated OPM2 cells.93 Presumably, the inactivation of a specific DNA repair pathway may be compensated by the activity of alternative pathways, as discussed above. A recent report shows an increased affinity of Ku80 for DNA ends, and constitutive activation of NHEJ in MM cell lines compared with healthy peripheral blood mononuclear cells.87 Surprisingly, however, MM cell lines showed little or no ability to repair radiation-induced damages, suggesting that constitutive activation of NHEJ pathway in these MM cell lines constitutes a response to endogenous stress.87 As reviewed above (Fig. 2), NHEJ is favored by 53BP1, which blocks DNA end resection.69 Recruitment of 53BP1 to DSBs is dependent on histone H4 methylation by MMSET,70 the product of WHSC1 gene that is translocated and highly expressed in 15% of patients with MM and t(4p16;14q32) translocation in association with poor prognosis15(Fig. 4). Of note, the 5′ region of WHSC1 (MMSET ) gene is deleted in 30–40% of these patients with t(4p16;14q32) translocation,25,94 resulting in production of a MMSET protein lacking the serine in position 102. This residue is phosphorylated by ATM, which is a necessary event for the recruitment of MMSET to DSB.70 How WHSC1 (MMSET ) translocation and spiked expression impact on HR and NHEJ pathways in MM cells has yet to be investigated. Given the association of t(4;14) with poor outcome in MM, this potential perturbation of DNA repair may prove highly relevant to disease progression. Increased homologous recombination activity in multiple myeloma cells HR activity is higher in primary MMCs from patients and MM cell lines compared with healthy plasma cells, as evidenced by higher recombination activity in plasmid assays and increased expression of genes/proteins involved in homologous recombination such as RAD50 and RAD51 (Fig. 4).86,95 Increased HR activity in MM cell lines resulted in resistance to dexamethasone and progressive accumulation of loss of heterozygosity.95 Unlike in healthy plasma cells, nuclear foci identified by labeling of phosphorylated histone H2AX (γH2AX), which are indicators of DSBs,96 are detected in primary MMCs of untreated patients or in MM cell lines, and ATR is constitutively phosphorylated in MM cell lines.97 Since HR is active in the S and G2 phases of the cell cycle, HR-mediated repair in MM cell lines or primary MMCs may occur in only a minor fraction of cycling MMCs. In the vast majority of patients, only a small percentage of myeloma cells can be found in S phase, so other DNA repair pathways may predominate and influence MMC development, and survival after drug treatment. However, as discussed above, even if just a small fraction of MMCs are engaged in the cell cycle, their abundance is highly correlated with adverse prognosis. Therefore, DNA repair pathways involved in this minority of “feeder” cells



lymph nodes.86 In that study, XRCC6 (Ku70) gene expression was found to be decreased in MMCs and in other lymphoma cells compared with normal B cells. Finally, a recent study points out an increased expression of XRCC5 and ARTEMIS genes in MMCs cells compared with MGUS plasma cells, in association with shorter patients’ overall survival.87 How these polymorphisms or gene expression variations affect NHEJ activity in MMC, however, remains elusive. One study reported the existence of a 69 kDa Ku86 variant (Ku86v) in a majority of patients’ primary MMCs. This short variant of Ku80 has reduced affinity for DNA ends and is unable to associate with and activate DNA-PKcs, which renders cells hypersensitive to chemotherapeutic drugs or to irradiation.88 These data, however, were challenged by Kato et al., who failed to detect the variant Ku86v in primary MMCs or MM cell lines.89 Subsequent studies pointed out that this variant could be generated by proteolytic cleavage when manipulating cells in vitro, both in human lymphocytes or MM cell lines.90-92 Using an in vitro DNA

or pathway activity. The existence of a truncated variant is indicated by digit 5.


Cell Cycle

7


in S and G2 might be of particular importance for MM evolution, cells with a constitutively high transcriptional activity to produce Ig. prognosis, and response to treatment. Crosstalk between NFκB pathway and DNA repair pathway Mismatch repair The NFκB pathway is frequently deregulated in MM and Relatively few observations on the mismatch repair pathway is selectively targeted for somatic mutations in MM as defined in MM have been published, but are discordant. In this pathway, by NGS.39 This may affect the interplay between NFκB signals the DNA lesion is sensed and recognized by the MutSα heterodiand activation of DNA repair pathways. Conversely, it may be mer (MSH2-MSH6) or by the MutSβ (MSH2-MSH3) complex, feasible for DNA repair mechanisms to promote NFκB activa- followed by the recruitment of MutLα (MLH1/PMS2) or MutLβ tion and MMC survival. These significant possibilities arise, as (MLH1/MLH3) to the repair foci. In a study of 30 patients with NFκB activation has been shown to stimulate HR, increase ATM MM and 13 with MGUS, MLH1 promoter was found methyland BRCA2 expression, and directly associate with and stimu- ated in plasma cells of 10% of patients with MM but not from late CtIP and BRCA1, thus enhancing DSB end resection and MGUS patients.108 MLH1 promoter methylation was also associRPA coating, which are mandatory steps for BRCA2-mediated ated with a decrease or loss of MLH1 gene expression in MMCs. RPA replacement by RAD51 and homologous strand invasion98 These data suggest that a decrease in MLH1 expression follows (Fig. 4). Consequently, it is conceptually feasible that DNA dam- promoter hypermethylation, and may play a role in the transiage and especially DSBs may induce NFκB activation in MMCs tion of MGUS to MM. MLH1 promoter hypermethylation, howto promote tumor survival. Additional mechanisms may facilitate ever, was not confirmed in another study of 53 MM patients. this cross-talk in MMCs. Irradiation or topoisomerase inhibitors Decreased expression of MSH2, MLH1, PMS1 genes, coding for induce NFκB through ATM recruitment and activation at DSBs. ATM binds and phosphorylates NEMO, inducing its ubiquitination and further translocation to the cytoplasm, where NEMO binds to and activates IKKα and IKKβ kinases, which, in turn, activates the NFκB pathway.99,100 Another mechanism involves association of PIDD (p53-induced protein with a death domain) with RIP1 (receptor-interacting protein 1) and NEMO, which leads to NEMO sumoylation. NEMO then associates with ATM resulting in NFκB activation.101 Finally, PARP1 may induce association between ATM and the SUMO-1 ligase PIASy, which, in turn, sumoylates and activates IKKγ.98,102 These data are illuminating. They reveal cross-talk that links central conduits for signal transduction to mechanisms that regulate genomic structural integrity, suggesting that somatic mutations in key genes that transduce external stimuli (e.g., RAS) may now need to be considered for their impact on genomic instability in MM. Base excision repair Very little data exists to implicate BER in MM. A polymorphism in OGG1 gene, linked to low BER activity, is associated with an increased risk for MM.103 Polymorphisms in APE1 or MUTYH (Fig. 5) genes are associated with shorter survival of patients with MM.104 In addition, a higher expression of APE1, using immunofluorescence staining, was detected in MMCs as compared with controls.105 Nucleotide excision repair Polymorphisms within 3 genes encoding the NER proteins XPA, XPG (ERCC5 gene), and ERCC1 were recently reported in MMCs in a large cohort of patients, Figure 4. Double-strand DNA repair pathways in multiple myeloma. Genes codwith a shorter survival associating with ERCC5 rs1047768 ing for homologous recombination or non-homologous end joining pathways, 106 and XPA rs1800975 (Fig. 5), and emphasizing the whose promoter methylation (digit 1), polymorphism (digit 2), expression (digit importance of NER to remove ICLs that are likely to be 3), pathway activity (digit 4) are modified in multiple myeloma cells, are indicated. induced by alkylating drugs, in particular melphalan, in They are highlighted in red if the change is associated with a bad prognosis, in green with a good one, and blue in case of no prognostic value. The upwards or non-replicating but transcriptionally active cells.67,107 This downwards arrows indicate an increased or decreased gene or protein expression is of importance for MMCs, which are malignant plasma

DNA Repair Pathways: Targets For Drugs Used to Treat MM and Role in Resistance Melphalan and DNA repair pathways Melphalan is used in patients receiving high-dose therapy (i.e., high dose melphalan 2 × 100 mg/m2) and autologous stem cell transplantation, as well as in non-transplantable patients in combination with steroids and either a proteasome inhibitor Bortezomib (VMP-regimen) or an immunomodulatory drug Thalidomide (MPT-regimen).4 Melphalan is a nitrogen mustard (4-[bis{2-chloroethyl}amino]-1-phenylalanine), which induces alkylation of the guanine in N-7 and adenine in N-3 and interstrand DNA crosslinks between guanine and guanine or guanine and adenine.111 Ninety-five percent of the lesions are monoadducts and 5% interstrand DNA crosslinks (ICLs),112 and these lesions impair DNA transcription and replication, yielding cell death if unrepaired. ICL number in MMCs increases with the melphalan concentration used to treat MMCs in vitro,113 and

this is likely to also occur in vivo (Fig. 6A). Although ICLs are infrequent, they are highly toxic for cells. The mechanisms of resistance to melphalan therapy have been extensively examined in MMC. One mechanism of resistance to alkylating drugs is the ability of MMCs to export the drug through upregulation of membrane efflux proteins. This has been well documented for many cancers114 and in particular for MM.115 A second resistance mechanism is the ability of MMCs to excise melphalan monoadducts and ICLs through DNA repair pathways, supported by 3 lines of evidence. (1) MMCs from patients relapsing from high-dose or continuous melphalan therapy are able to efficiently repair ICLs in vitro, whereas MMCs from untreated patients are not.113 (2) Upon in vivo melphalan treatment, the efficiency of ICL repair within the TP53 gene in patients’ nontumor leukocytes can efficiently predict for patient sensitivity to melphalan.116 (3) Associations between melphalan resistance and polymorphisms in genes encoding DNA repair proteins have been documented: in a study of 169 patients treated with vincristineadriamycine-dexamethasone then high-dose melphalan and stem cell transplantation, patients’ outcome was significantly associated with SNPs in OGG1, PARP, PCNA, RAD51, and XPC genes and patients’ survival with BARD1, BRCA1, ERCC1, PCNA, and TP53BP1 genes (Fig. 6A). Moreover SNPs in BRCA1, CDKN1A, and XRCC1 genes were associated with therapy-associated mucositis.103 Many of these genes encode for proteins implicated in the HR or NER pathways that are necessary for melphalan-induced ICL repair. In another study, polymorphisms in the ERCC2 and

Figure 5. Single-strand DNA repair pathways in multiple myeloma. The changes in genes implicated in single-strand damage repair (BER, NER, or MMR) and modified in MM cells compare with healthy cells are highlighted. As indicated in Figure 2A, digit 1 indicates changes in promoter methylation, digit 2 the existence of polymorphisms, digit 3 changes in protein expression and digit 4 changes in activity. The upwards or downwards arrows indicate an increased or decreased gene expression or activity.

8



proteins involved in MMR, was reported in another study on MMCs from a small cohort of patients and requires further confirmatory studies.109 Somatic mutations in DNA repair genes Interestingly, acquired somatic mutations in MMCs, excluding p53, appear to be markedly limited in the spectrum of genes that mediate DNA repair, as exemplified in the genome NGS data of a large cohort of 38 MM cases.39,110

methasone or IMiDs (lenalidomide, thalidomide, and pomalidomide).


Cell Cycle

9


XRCC3 genes were implicated in the repair of bulky adducts and also observed in a melphalan-resistant cell line126 (Figs. 2 and DSBs and associated with treatment failure.117 6A). Defining the precise array of DNA repair proteins with a More detailed studies of mechanisms of resistance to melpha- role in melphalan resistance may allow additional targeted drugs lan have utilized MM cell lines, which proliferate highly unlike to be developed that can block culprit proteins. primary MMCs in the majority of patients. Increased expressions Proteasome inhibitor (PI) and DNA repair pathways of genes coding for Fanconi anemia (FA) or HR pathways such Bortezomib is a proteasome inhibitor (PI) that reversibly taras BRCA1, BRCA2, FANCA, FANCC, FANCF, FANCL, and gets the catalytic β5 and β1 proteasome subunits and inhibits RAD51C and upregulation of FA and HR pathways were found in melphalan-resistant myeloma cell lines.118 FANCF is a key player in melphalan resistance, since knockdown of FANCF can reverse resistance and overexpression of FANCF reduces melphalan sensitivity118 (Fig. 6A). Constitutive NFκB activation is in part responsible for transcriptional activation of genes encoding FA proteins and for melphalan resistance (further highlighting cross-talk with DNA repair).119 Thus, the FA pathway confers resistance of MMCs to melphalan and presents a potential therapeutic target. Curcumin, for instance, inhibits both the NFκB and the FA pathways120,121 and increases drug concentration inside MMCs.121 Hence, combinatorial therapies using curcumin may overcome resistance of MM cell lines to melphalan. Blocking autophagy may also improve the efficiency of melphalan in MM cell lines and patients’ primary MMCs.122 Autophagy is activated after DNA damage by melphalan, and several studies show that autophagy could have some protective effect in part by increasing ATP production.123 As BER is involved in the repair of psoralen-induced ICLs,124 it may contribute to melphalan resistance (Figs. 1 and 6A). A siRNA to APE1 sensitized MMCs to melphalan.105 Overexpression of a dominant-negative form of APE1, however, impaired the ability of CHO cells to repair DNA lesions induced by alkylating agents such as decarbazine, thiotepa, busulfan carmustine, but, surprisFigure 6. DNA repair pathways and drugs used to treat patients with multiple myeloma. ingly, not by melphalan.125 On the contrary, a (A) Velcade and Melphalan, 2 of the main drugs used to treat MM patients, modify DNA repair decrease in the expression of 3 DNA glycosylpathways in 2 opposite ways. Melphalan induces adducts and ICLs that need to be repaired ases (NEIL1, UNG2, MPG) associated with mainly by the Fanconi anemia pathway and homologous recombination. Base excision repair a decrease repair of 8-oxodG and acquisition (BER), non-homologous end joining and nucleotide excision repair (NER) pathways are also involved in ICLs repair. Changes in activity or expression of genes involved in these pathways of melphalan resistance in a cell line (Fig. or the existence of polymorphisms may activate and thus ease the repair of the lesions leading 6A). The authors hypothesized that decreased to cell survival and resistance to Melphalan. Some polymorphisms are associated with a better activity of these glycosylases in resistant cells response after treatment. Changes in BER pathway can affect ICLs repair: an increased activinduced less AP sites caused by endogenous ity could help to repair the lesions but a decreased activity may also facilitate ICLs repair by the Fanconi anemia pathway by improving DNA accessibility. Velcade inhibits Fanconi anemia reactive oxygen species. Therefore, fewer propathway, homologous recombination and NER. In red are summarized the features and dereguteins involved in AP site repair were mobilized lations associated with disease progression and resistance to melphalan, in green the features closed to the sites of ICLs, facilitating the and deregulations associated with response to treatment and a better outcome after treatment accessibility of ICL repair proteins to ICLs.126 with melphalan. (B) Very few data show a possible association between treatment with IMiDs and dexamethasone and DNA repair pathways. In green are summarized polymorphisms and Finally, an increase expression of 2 proteins changes in protein expression associated with a better outcome after treatment with dexainvolved in NHEJ (XRCC4 and ligase 4) was

10

DNA repair pathway features or deregulations and sensitivity or resistance to dexamethasone and IMiDs. Polymorphisms in ERCC1, ERCC5, or XRCC5 (Ku70) genes (coding for NER or NHEJ proteins) (Figs. 1, 2, and 6B) are associated with a better response to Thalidomide as a single agent in MM patients, and polymorphisms in ERCC1 and XRCC5 with a longer overall survival.143 Gene expression profiling of MMCs from patients harvested before and immediately (48 h) after treatment with dexamethasone (40 mg/day) or with thalidomide (400 mg/day) has revealed an overall increase in ERCC1 expression (NER pathway) after dexamethasone treatment and a downregulation by thalidomide. An increased ERCC1 expression after treatment with dexamethasone or Thalidomide was associated with better patients’ outcome.144 Of note, IMIDs can increase ROS production, DNA oxidation that will require DNA repair. In MMC, polymorphic variants of NER and NHEJ proteins, and modulation of their levels of expression by specific drugs will affect functional activities that appear to associate with antitumor effects: again, the precise mechanisms remain to be elucidated. The expression of heme oxydase 1 HO-1, an enzyme implicated in cellular response to oxidative stress, was enhanced after 3 months of treatment with Lenalidomide and Bortezomib,145 and treatment with Thalidomide or Lenalidomide induces upregulation of the oxidative-stress responsive 1 (OXSR1) gene. This upregulation is associated with a poor outcome and could therefore be a mechanism of resistance after prolonged exposure to drugs.144 New therapeutic strategies: Synthetic lethality As reviewed above, DNA repair pathways can be deregulated in MMCs and modulate the activity of the major drugs currently used in disease. Thus, one can speculate that targeting DNA repair pathways may potentiate the efficacy of current drugs and reverse drug resistance. This strategy, called “synthetic lethality” is currently being developed in breast cancers with mutated BRCA1 or BRCA2 genes. In these cancer cells, DSBs repair by HR fails due to a lack of functional BRCA1 or BRCA2 proteins. Treatment of these cancer cells with PARP1 inhibitors such as Olaparib inhibits SSB repair, yielding DSBs, which cannot be repaired by a defective HR and are consequently highly toxic for cancer cells.66,146 Such a strategy has been investigated for MMCs, in which HR was inhibited by Bortezomib treatment, as co-treatment of MMCs with Bortezomib and PARP1 inhibitor resulted in DSB accumulation and MMC killing.132 Importantly, many inhibitors targeting DNA repair pathways (PARP1, DNA-PK, ATM, ATR, MGMT, APE) or cell cycle checkpoints (CHK1, CHK2) have now been developed66,146-148 and could be useful to induce the death of MMCs in combination with DNA damage inducing drugs to develop therapeutic synthetic lethality.148-150 These inhibitors could be of particular interest for improving response and survival of patients treated with high-dose melphalan and autologous stem cell transplantation. In these patients, only few MMCs (5.5 MMCs/μL after 7 days) may survive in an almost empty bone marrow 9 days after melphalan treatment.151 These few resistant MMCs likely harbor melphalan monoadducts or crosslinks, which have to be repaired to get MMC survival and tumor regrowth. Targeting DNA repair pathways with specific drugs could help killing MMCs, preventing or delaying relapse.



chymotrypsin-like and peptidylglutamyl peptide hydrolyzing activities.127 Bortezomib can thus induce proteotoxic stress by preventing misfolded protein degradation, and its efficiency can be improved by co-treatment with inducers of misfolded protein such as puromycine.128 Bortezomib preferentially targets proliferating cells but is also active in quiescent cells in the G0 phase of the cell cycle.129 As protein ubiquitination is critical for building DNA repair foci through protein recruitment and degradation, it is not altogether surprising that PI affects DNA repair, as has been shown in the main for FA and HR pathways. PIs block NFκB mediated activation of FA pathway,119 decrease expression of FANC proteins, and inhibit FA pathway by preventing FANCD2 monoubiquitination and FANCD2 focus formation, and also inhibit HR by blocking recruitment of NBS1, phospho-ATM, BRCA1, and Rad51 (Fig. 6A).130,131 The decrease in FANCD2 and H2Ax ubiquination by PI is explained by the accumulation of poly-ubiquitinated proteins and, consequently, the decrease of the pool of available nuclear ubiquitin.132 Bortezomib treatment may prevent DNA resection through inhibiting proteasomal degradation of proteins involved in chromatin relaxation, thus preventing recruitment of RPA onto ssDNA.130,131 This effect of Bortezomib on blocking DNA repair could explain why a phase II study has shown that the combination of Bortezomib together with highdose melphalan before autologous stem cell transplantation could increase by 3-fold the complete remission rate (35% vs. 11%).133 The underlying mechanisms inferred here from improved remission rates may include a reduced potential of DNA repair pathways to clear lesions, thereby increasing susceptibility to apoptotic clearance and/or reducing the efficacy of DNA repair in genomic adaptations to resist therapy. The clinical benefit of combination of Bortezomib and high-dose melphalan is currently being tested in large clinical trials according to clinicaltrial.gov. New proteasome inhibitors entering clinical development are Carfilzomib or MLN2238. Carfilzomib bind irreversibly to proteasome subunits and can overcome resistance to Bortezomib.134 These new PIs targeting the proteasome are expected to also target DNA repair pathways. The effect of PIs on other DNA repair pathways is less studied. It is noteworthy that in human fibroblasts, PIs block the repair of UV-induced lesions by NER.135 Immunodulatory drugs, dexamethasone, and DNA repair pathways IMiDs (Thalidomide, Lenalidomide, or Pomalidomide) are immunomodulatory drugs that bind to an F-box protein Cereblon, a member of an ubiquitin ligase complex.136,137 Dexamethasone, a major drug in MM, is a corticosteroid that binds to intracellular glucocorticoid receptors, which migrate to the nucleus and act as a transcription factor. Resistance to lMiDs involves mostly mutations or downregulation of cereblon expression.47,137 Various mechanisms have been described for dexamethasone resistance: expression of a truncated glucocorticoid receptor,138 mutations in receptor gene,47 epigenetic silencing of RASD1, cell growth inhibitor induced by dexamethasone treatment,139 overexpression of Hsp27,140 or change in the secretion of cytokines and chemokines in the microenvironment.141 Overexpression of Bruton tyrosine kinase could also play a role in resistance according to a recent study.142 But very few data report a possible association between

Concluding Remarks

be exploited to selectively kill MMCs by targeting the remaining repair pathways that are critical for their survival.

References 1. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2012. CA Cancer J Clin 2012; 62:10-29; PMID:22237781; http://dx.doi.org/10.3322/ caac.20138 2. Kyle RA, Therneau TM, Rajkumar SV, Offord JR, Larson DR, Plevak MF, Melton LJ 3rd. A longterm study of prognosis in monoclonal gammopathy of undetermined significance. N Engl J Med 2002; 346:564-9; PMID:11856795; http://dx.doi. org/10.1056/NEJMoa01133202 3. Landgren O, Kyle RA, Pfeiffer RM, Katzmann JA, Caporaso NE, Hayes RB, Dispenzieri A, Kumar S, Clark RJ, Baris D, et al. Monoclonal gammopathy of undetermined significance (MGUS) consistently precedes multiple myeloma: a prospective study. Blood 2009; 113:5412-7; PMID:19179464; http://dx.doi. org/10.1182/blood-2008-12-194241 4. Rajkumar SV. Treatment of multiple myeloma. Nat Rev Clin Oncol 2011; 8:479-91; PMID:21522124; http://dx.doi.org/10.1038/nrclinonc.2011.63 5. Weiss BM, Abadie J, Verma P, Howard RS, Kuehl WM. A monoclonal gammopathy precedes multiple myeloma in most patients. Blood 2009; 113:541822; PMID:19234139; http://dx.doi.org/10.1182/ blood-2008-12-195008 6. Rajkumar SV, Gupta V, Fonseca R, Dispenzieri A, Gonsalves WI, Larson D, Ketterling RP, Lust JA, Kyle RA, Kumar SK. Impact of primary molecular cytogenetic abnormalities and risk of progression in smoldering multiple myeloma. Leukemia 2013; 27:1738-44; PMID:23515097; http://dx.doi. org/10.1038/leu.2013.86 7. Kyle RA, Therneau TM, Rajkumar SV, Larson DR, Plevak MF, Offord JR, Dispenzieri A, Katzmann JA, Melton LJ 3rd. Prevalence of monoclonal gammopathy of undetermined significance. N Engl J Med 2006; 354:1362-9; PMID:16571879; http://dx.doi. org/10.1056/NEJMoa054494 8. Zojer N, Ludwig H, Fiegl M, Stevenson FK, Sahota SS. Patterns of somatic mutations in VH genes reveal pathways of clonal transformation from MGUS to multiple myeloma. Blood 2003; 101:41379; PMID:12531815; http://dx.doi.org/10.1182/ blood-2002-09-2825 9. Witzig TE, Timm M, Larson D, Therneau T, Greipp PR. Measurement of apoptosis and proliferation of bone marrow plasma cells in patients with plasma cell proliferative disorders. Br J Haematol 1999; 104:131-7; PMID:10027725; http://dx.doi. org/10.1046/j.1365-2141.1999.01136.x 10. Hose D, Rème T, Hielscher T, Moreaux J, Messner T, Seckinger A, Benner A, Shaughnessy JD Jr., Barlogie B, Zhou Y, et al. Proliferation is a central independent prognostic factor and target for personalized and risk-adapted treatment in multiple myeloma. Haematologica 2011; 96:87-95; PMID:20884712; http://dx.doi.org/10.3324/haematol.2010.030296


Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed. Acknowledgments

This work was supported by grants from the European community (FP7-OVERMYR project), the INCA (Institut National du Cancer) institute (2012-109/087437, Paris, France) and from the Association pour la Recherche sur le Cancer (ARC) (SL220110603450, Paris France). CG is supported by a French Labex EpiGenMed fellowship.

11. Greipp PR, Katzmann JA, O’Fallon WM, Kyle RA. Value of beta 2-microglobulin level and plasma cell labeling indices as prognostic factors in patients with newly diagnosed myeloma. Blood 1988; 72:219-23; PMID:3291982 12. Greipp PR, Leong T, Bennett JM, Gaillard JP, Klein B, Stewart JA, Oken MM, Kay NE, Van Ness B, Kyle RA. Plasmablastic morphology–an independent prognostic factor with clinical and laboratory correlates: Eastern Cooperative Oncology Group (ECOG) myeloma trial E9486 report by the ECOG Myeloma Laboratory Group. Blood 1998; 91:2501-7; PMID:9516151 13. Paiva B, Vídriales M-B, Montalbán M-Á, Pérez JJ, Gutiérrez NC, Rosiñol L, Martínez-López J, Mateos MV, Cordón L, Oriol A, et al. Multiparameter flow cytometry evaluation of plasma cell DNA content and proliferation in 595 transplant-eligible patients with myeloma included in the Spanish GEM2000 and GEM2005A polymorphism is a risk factor for t(11;14)(q13;q32) multiple myeloma. Nat Genet 2013; 45:522-5; PMID:23502783; http:// dx.doi.org/10.1038/ng.2583 51. Ricke RM, van Deursen JM. Aneuploidy in health, disease, and aging. J Cell Biol 2013; 201:11-21; PMID:23547028; http://dx.doi.org/10.1083/ jcb.201301061 52. Siegel JJ, Amon A. New insights into the troubles of aneuploidy. Annu Rev Cell Dev Biol 2012; 28:189214; PMID:22804579; http://dx.doi.org/10.1146/ annurev-cellbio-101011-155807 53. Chng WJ, Fonseca R. Centrosomes and myeloma; aneuploidy and proliferation. Environ Mol Mutagen 2009; 50:697-707; PMID:19739237; http://dx.doi. org/10.1002/em.20528 54. Chng WJ, Braggio E, Mulligan G, Bryant B, Remstein E, Valdez R, Dogan A, Fonseca R. The centrosome index is a powerful prognostic marker in myeloma and identifies a cohort of patients that might benefit from aurora kinase inhibition. Blood 2008; 111:1603-9; PMID:18006703; http://dx.doi. org/10.1182/blood-2007-06-097774

55. Lindahl T. Instability and decay of the primary structure of DNA. Nature 1993; 362:709-15; PMID:8469282; http://dx.doi. org/10.1038/362709a0 56. Hoeijmakers JHJ. DNA damage, aging, and cancer. N Engl J Med 2009; 361:1475-85; PMID:19812404; http://dx.doi.org/10.1056/NEJMra0804615 57. Galm O, Wilop S, Reichelt J, Jost E, Gehbauer G, Herman JG, Osieka R. DNA methylation changes in multiple myeloma. Leukemia 2004; 18:168792; PMID:15318245; http://dx.doi.org/10.1038/ sj.leu.2403434 58. Walker BA, Wardell CP, Chiecchio L, Smith EM, Boyd KD, Neri A, Davies FE, Ross FM, Morgan GJ. Aberrant global methylation patterns affect the molecular pathogenesis and prognosis of multiple myeloma. Blood 2011; 117:553-62; PMID:20944071; http:// dx.doi.org/10.1182/blood-2010-04-279539 59. Amodio N, Leotta M, Bellizzi D, Di Martino MT, D’Aquila P, Lionetti M, Fabiani F, Leone E, Gullà AM, Passarino G, et al. DNA-demethylating and anti-tumor activity of synthetic miR-29b mimics in multiple myeloma. Oncotarget 2012; 3:1246-58; PMID:23100393 60. Ciccia A, Elledge SJ. The DNA damage response: making it safe to play with knives. Mol Cell 2010; 40:179-204; PMID:20965415; http://dx.doi. org/10.1016/j.molcel.2010.09.019 61. Iyama T, Wilson DM 3rd. DNA repair mechanisms in dividing and non-dividing cells. DNA Repair (Amst) 2013; 12:620-36; PMID:23684800; http:// dx.doi.org/10.1016/j.dnarep.2013.04.015 62. Caldecott KW. Single-strand break repair and genetic disease. Nat Rev Genet 2008; 9:619-31; PMID:18626472 63. Friedberg EC. How nucleotide excision repair protects against cancer. Nat Rev Cancer 2001; 1:22-33; PMID:11900249; http://dx.doi. org/10.1038/35094000 64. Hanawalt PC, Spivak G. Transcription-coupled DNA repair: two decades of progress and surprises. Nat Rev Mol Cell Biol 2008; 9:958-70; PMID:19023283; http://dx.doi.org/10.1038/nrm2549 65. Jiricny J. The multifaceted mismatch-repair system. Nat Rev Mol Cell Biol 2006; 7:335-46; PMID:16612326; http://dx.doi.org/10.1038/ nrm1907 66. Shaheen M, Allen C, Nickoloff JA, Hromas R. Synthetic lethality: exploiting the addiction of cancer to DNA repair. Blood 2011; 117:607482; PMID:21441464; http://dx.doi.org/10.1182/ blood-2011-01-313734 67. Deans AJ, West SC. DNA interstrand crosslink repair and cancer. Nat Rev Cancer 2011; 11:467-80; PMID:21701511; http://dx.doi.org/10.1038/nrc3088 68. Bunting SF, Callén E, Wong N, Chen H-T, Polato F, Gunn A, Bothmer A, Feldhahn N, FernandezCapetillo O, Cao L, et al. 53BP1 inhibits homologous recombination in Brca1-deficient cells by blocking resection of DNA breaks. Cell 2010; 141:243-54; PMID:20362325; http://dx.doi.org/10.1016/j. cell.2010.03.012 69. Chapman JR, Taylor MRG, Boulton SJ. Playing the end game: DNA double-strand break repair pathway choice. Mol Cell 2012; 47:497-510; PMID:22920291; http://dx.doi.org/10.1016/j.molcel.2012.07.029 70. Pei H, Zhang L, Luo K, Qin Y, Chesi M, Fei F, Bergsagel PL, Wang L, You Z, Lou Z. MMSET regulates histone H4K20 methylation and 53BP1 accumulation at DNA damage sites. Nature 2011; 470:124-8; PMID:21293379; http://dx.doi. org/10.1038/nature09658 71. Tang J, Cho NW, Cui G, Manion EM, Shanbhag NM, Botuyan MV, Mer G, Greenberg RA. Acetylation limits 53BP1 association with damaged chromatin to promote homologous recombination. Nat Struct Mol Biol 2013; 20:317-25; PMID:23377543; http:// dx.doi.org/10.1038/nsmb.2499



31. Chng WJ, Gonzalez-Paz N, Price-Troska T, Jacobus S, Rajkumar SV, Oken MM, Kyle RA, Henderson KJ, Van Wier S, Greipp P, et al. Clinical and biological significance of RAS mutations in multiple myeloma. Leukemia 2008; 22:2280-4; PMID:18528420; http://dx.doi.org/10.1038/leu.2008.142 32. Avet-Loiseau H, Attal M, Campion L, Caillot D, Hulin C, Marit G, Stoppa AM, Voillat L, Wetterwald M, Pegourie B, et al. Long-term analysis of the IFM 99 trials for myeloma: cytogenetic abnormalities [t(4;14), del(17p), 1q gains] play a major role in defining long-term survival. J Clin Oncol 2012; 30:194952; PMID:22547600; http://dx.doi.org/10.1200/ JCO.2011.36.5726 33. Lodé L, Eveillard M, Trichet V, Soussi T, Wuillème S, Richebourg S, Magrangeas F, Ifrah N, Campion L, Traullé C, et al. Mutations in TP53 are exclusively associated with del(17p) in multiple myeloma. Haematologica 2010; 95:1973-6; PMID:20634494; http://dx.doi.org/10.3324/haematol.2010.023697 34. Pichiorri F, Suh S-S, Rocci A, De Luca L, Taccioli C, Santhanam R, Zhou W, Benson DM Jr., Hofmainster C, Alder H, et al. Downregulation of p53-inducible microRNAs 192, 194, and 215 impairs the p53/MDM2 autoregulatory loop in multiple myeloma development. Cancer Cell 2010; 18:36781; PMID:20951946; http://dx.doi.org/10.1016/j. ccr.2010.09.005 35. Bharti AC, Shishodia S, Reuben JM, Weber D, Alexanian R, Raj-Vadhan S, Estrov Z, Talpaz M, Aggarwal BB. Nuclear factor-kappaB and STAT3 are constitutively active in CD138+ cells derived from multiple myeloma patients, and suppression of these transcription factors leads to apoptosis. Blood 2004; 103:3175-84; PMID:15070700; http://dx.doi. org/10.1182/blood-2003-06-2151 36. Annunziata CM, Davis RE, Demchenko Y, Bellamy W, Gabrea A, Zhan F, Lenz G, Hanamura I, Wright G, Xiao W, et al. Frequent engagement of the classical and alternative NFkappaB pathways by diverse genetic abnormalities in multiple myeloma. Cancer Cell 2007; 12:115-30; PMID:17692804; http:// dx.doi.org/10.1016/j.ccr.2007.07.004 37. Keats JJ, Fonseca R, Chesi M, Schop R, Baker A, Chng W-J, Van Wier S, Tiedemann R, Shi CX, Sebag M, et al. Promiscuous mutations activate the noncanonical NFkappaB pathway in multiple myeloma. Cancer Cell 2007; 12:131-44; PMID:17692805; http://dx.doi.org/10.1016/j.ccr.2007.07.003 38. Markovina S, Callander NS, O’Connor SL, Kim J, Werndli JE, Raschko M, Leith CP, Kahl BS, Kim K, Miyamoto S. Bortezomib-resistant nuclear factor-kappaB activity in multiple myeloma cells. Mol Cancer Res 2008; 6:1356-64; PMID:18708367; http://dx.doi.org/10.1158/1541-7786.MCR-08-0108 39. Chapman MA, Lawrence MS, Keats JJ, Cibulskis K, Sougnez C, Schinzel AC, Harview CL, Brunet JP, Ahmann GJ, Adli M, et al. Initial genome sequencing and analysis of multiple myeloma. Nature 2011; 471:467-72; PMID:21430775; http://dx.doi. org/10.1038/nature09837 40. Demchenko YN, Glebov OK, Zingone A, Keats JJ, Bergsagel PL, Kuehl WM. Classical and/or alternative NFkappaB pathway activation in multiple myeloma. Blood 2010; 115:3541-52; PMID:20053756; http:// dx.doi.org/10.1182/blood-2009-09-243535 41. Avet-Loiseau H, Gerson F, Magrangeas F, Minvielle S, Harousseau JL, Bataille R; Intergroupe Francophone du Myélome. Rearrangements of the c-myc oncogene are present in 15% of primary human multiple myeloma tumors. Blood 2001; 98:3082-6; PMID:11698294; http://dx.doi.org/10.1182/blood. V98.10.3082

13

87. Calimeri T, Fulcineti M, Lin J, Samur MK, Calkins AS, Vahia AV, et al. Aberrant non-homologous end joining in multiple myeloma: a role in genomic instability and as a potential prognostic marker. Blood ASH meeting abstracts 2012. 120:2932 88. Tai YT, Teoh G, Lin B, Davies FE, Chauhan D, Treon SP, Raje N, Hideshima T, Shima Y, Podar K, et al. Ku86 variant expression and function in multiple myeloma cells is associated with increased sensitivity to DNA damage. J Immunol 2000; 165:6347-55; PMID:11086072 89. Kato M, Iida S, Komatsu H, Ueda R. Lack of Ku80 alteration in multiple myeloma. Jpn J Cancer Res 2002; 93:359-62; PMID:11985783; http://dx.doi. org/10.1111/j.1349-7006.2002.tb01264.x 90. Han Z, Johnston C, Reeves WH, Carter T, Wyche JH, Hendrickson EA. Characterization of a Ku86 variant protein that results in altered DNA binding and diminished DNA-dependent protein kinase activity. J Biol Chem 1996; 271:14098-104; PMID:8662896; http://dx.doi.org/10.1074/jbc.271.24.14098 91. Łanuszewska J, Widłak P. The truncation of Ku86 in human lymphocytes. Cancer Lett 2004; 205:197205; PMID:15036652; http://dx.doi.org/10.1016/j. canlet.2003.10.016 92. Gullo CA, Ge F, Cow G, Teoh G. Ku86 exists as both a full-length and a protease-sensitive natural variant in multiple myeloma cells. Cancer Cell Int 2008; 8:4; PMID:18442416; http://dx.doi. org/10.1186/1475-2867-8-4 93. Yang C, Betti C, Singh S, Toor A, Vaughan A. Impaired NHEJ function in multiple myeloma. Mutat Res 2009; 660:66-73; PMID:19028508; http://dx.doi.org/10.1016/j.mrfmmm.2008.10.019 94. Keats JJ, Reiman T, Belch AR, Pilarski LM. Ten years and counting: so what do we know about t(4;14) (p16;q32) multiple myeloma. Leuk Lymphoma 2006; 47:2289-300; PMID:17107900; http://dx.doi. org/10.1080/10428190600822128 95. Shammas MA, Shmookler Reis RJ, Koley H, Batchu RB, Li C, Munshi NC. Dysfunctional homologous recombination mediates genomic instability and progression in myeloma. Blood 2009; 113:22907; PMID:19050310; http://dx.doi.org/10.1182/ blood-2007-05-089193 96. Mah L-J, El-Osta A, Karagiannis TC. gammaH2AX: a sensitive molecular marker of DNA damage and repair. Leukemia 2010; 24:679-86; PMID:20130602; http://dx.doi.org/10.1038/ leu.2010.6 97. Walters DK, Wu X, Tschumper RC, Arendt BK, Huddleston PM, Henderson KJ, Dispenzieri A, Jelinek DF. Evidence for ongoing DNA damage in multiple myeloma cells as revealed by constitutive phosphorylation of H2AX. Leukemia 2011; 25:134453; PMID:21566653; http://dx.doi.org/10.1038/ leu.2011.94 98. Volcic M, Karl S, Baumann B, Salles D, Daniel P, Fulda S, Wiesmüller L. NFκB regulates DNA doublestrand break repair in conjunction with BRCA1CtIP complexes. Nucleic Acids Res 2012; 40:181-95; PMID:21908405; http://dx.doi.org/10.1093/nar/ gkr687 99. Janssens S, Tschopp J. Signals from within: the DNAdamage-induced NFkappaB response. Cell Death Differ 2006; 13:773-84; PMID:16410802; http:// dx.doi.org/10.1038/sj.cdd.4401843 100. Miyamoto S. Nuclear initiated NFκB signaling: NEMO and ATM take center stage. Cell Res 2011; 21:116-30; PMID:21187855; http://dx.doi. org/10.1038/cr.2010.179 101. Habraken Y, Piette J. NFkappaB activation by doublestrand breaks. Biochem Pharmacol 2006; 72:113241; PMID:16965765; http://dx.doi.org/10.1016/j. bcp.2006.07.015

102. Stilmann M, Hinz M, Arslan SC, Zimmer A, Schreiber V, Scheidereit C. A nuclear poly(ADPribose)-dependent signalosome confers DNA damage-induced IkappaB kinase activation. Mol Cell 2009; 36:365-78; PMID:19917246; http://dx.doi. org/10.1016/j.molcel.2009.09.032 103. Dumontet C, Landi S, Reiman T, Perry T, Plesa A, Bellini I, Barale R, Pilarski LM, Troncy J, Tavtigian S, et al. Genetic polymorphisms associated with outcome in multiple myeloma patients receiving high-dose melphalan. Bone Marrow Transplant 2010; 45:1316-24; PMID:19966851; http://dx.doi. org/10.1038/bmt.2009.335 104. Ushie C, Saitoh T, Iwasaki A, Moriyama N, Murakami H. The polymorphisms of base excision repair genes influence the prognosis of multiple myeloma. Blood ASH meeting abstracts 2012; 120 105. Yang Z-Z, Chen X-H, Wang D. Experimental study enhancing the chemosensitivity of multiple myeloma to melphalan by using a tissue-specific APE1-silencing RNA expression vector. Clin Lymphoma Myeloma 2007; 7:296-304; PMID:17324338; http://dx.doi. org/10.3816/CLM.2007.n.006 106. de Larrea CF, Navarro A, Tovar N, Pedrosa F, Díaz T, Cibeira MAT, et al. Impact of single nucleotide polymorphisms in genes involved in DNA repair and drug metabolism on survival after autologous stem cell transplantation in patients with multiple myeloma. Blood ASH meeting abstracts 2012; 120:2934 107. Episkopou H, Kyrtopoulos SA, Sfikakis PP, Fousteri M, Dimopoulos MA, Mullenders LHF, Souliotis VL. Association between transcriptional activity, local chromatin structure, and the efficiencies of both subpathways of nucleotide excision repair of melphalan adducts. Cancer Res 2009; 69:4424-33; PMID:19417135; http://dx.doi.org/10.1158/00085472.CAN-08-3489 108. Martin P, Santón A, García-Cosio M, Bellas C. hMLH1 and MGMT inactivation as a mechanism of tumorigenesis in monoclonal gammopathies. Mod Pathol 2006; 19:914-21; PMID:16607377; http:// dx.doi.org/10.1038/modpathol.3800590 109. Kotoula V, Hytiroglou P, Kaloutsi V, Barbanis S, Kouidou S, Papadimitriou CS. Mismatch repair gene expression in malignant lymphoproliferative disorders of B-cell origin. Leuk Lymphoma 2002; 43:393-9; PMID:11999575; http://dx.doi. org/10.1080/10428190290006215 110. Walker BA, Wardell CP, Melchor L, Hulkki S, Potter NE, Johnson DC, Fenwick K, Kozarewa I, Gonzalez D, Lord CJ, et al. Intraclonal heterogeneity and distinct molecular mechanisms characterize the development of t(4;14) and t(11;14) myeloma. Blood 2012; 120:1077-86; PMID:22573403; http://dx.doi. org/10.1182/blood-2012-03-412981 111. Osborne MR, Wilman DE, Lawley PD. Alkylation of DNA by the nitrogen mustard bis(2-chloroethyl) methylamine. Chem Res Toxicol 1995; 8:31620; PMID:7766817; http://dx.doi.org/10.1021/ tx00044a018 112. Muniandy PA, Liu J, Majumdar A, Liu S-T, Seidman MM. DNA interstrand crosslink repair in mammalian cells: step by step. Crit Rev Biochem Mol Biol 2010; 45:23-49; PMID:20039786; http://dx.doi. org/10.3109/10409230903501819 113. Spanswick VJ, Craddock C, Sekhar M, Mahendra P, Shankaranarayana P, Hughes RG, Hochhauser D, Hartley JA. Repair of DNA interstrand crosslinks as a mechanism of clinical resistance to melphalan in multiple myeloma. Blood 2002; 100:224-9; PMID:12070031; http://dx.doi.org/10.1182/blood. V100.1.224 114. Gottesman MM, Fojo T, Bates SE. Multidrug resistance in cancer: role of ATP-dependent transporters. Nat Rev Cancer 2002; 2:48-58; PMID:11902585; http://dx.doi.org/10.1038/nrc706



72. Hochegger H, Dejsuphong D, Fukushima T, Morrison C, Sonoda E, Schreiber V, Zhao GY, Saberi A, Masutani M, Adachi N, et al. Parp-1 protects homologous recombination from interference by Ku and Ligase IV in vertebrate cells. EMBO J 2006; 25:1305-14; PMID:16498404; http://dx.doi. org/10.1038/sj.emboj.7601015 73. Adamo A, Collis SJ, Adelman CA, Silva N, Horejsi Z, Ward JD, Martinez-Perez E, Boulton SJ, La Volpe A. Preventing nonhomologous end joining suppresses DNA repair defects of Fanconi anemia. Mol Cell 2010; 39:25-35; PMID:20598602; http://dx.doi. org/10.1016/j.molcel.2010.06.026 74. Pace P, Mosedale G, Hodskinson MR, Rosado IV, Sivasubramaniam M, Patel KJ. Ku70 corrupts DNA repair in the absence of the Fanconi anemia pathway. Science 2010; 329:219-23; PMID:20538911; http:// dx.doi.org/10.1126/science.1192277 75. Lieber MR. NHEJ and its backup pathways in chromosomal translocations. Nat Struct Mol Biol 2010; 17:393-5; PMID:20368722; http://dx.doi. org/10.1038/nsmb0410-393 76. West SC. Molecular views of recombination proteins and their control. Nat Rev Mol Cell Biol 2003; 4:435-45; PMID:12778123; http://dx.doi. org/10.1038/nrm1127 77. Hartlerode AJ, Scully R. Mechanisms of doublestrand break repair in somatic mammalian cells. Biochem J 2009; 423:157-68; PMID:19772495; http://dx.doi.org/10.1042/BJ20090942 78. Bothmer A, Robbiani DF, Feldhahn N, Gazumyan A, Nussenzweig A, Nussenzweig MC. 53BP1 regulates DNA resection and the choice between classical and alternative end joining during class switch recombination. J Exp Med 2010; 207:855-65; PMID:20368578; http://dx.doi.org/10.1084/jem.20100244 79. Williams HL, Gottesman ME, Gautier J. Replicationindependent repair of DNA interstrand crosslinks. Mol Cell 2012; 47:140-7; PMID:22658724 80. Wang Y, Leung JW, Jiang Y, Lowery MG, Do H, Vasquez KM, Chen J, Wang W, Li L. FANCM and FAAP24 maintain genome stability via cooperative as well as unique functions. Mol Cell 2013; 49:9971009; PMID:23333308; http://dx.doi.org/10.1016/j. molcel.2012.12.010 81. Kim N, Jinks-Robertson S. Transcription as a source of genome instability. Nat Rev Genet 2012; 13:20414; PMID:22330764 82. Knipscheer P, Räschle M, Smogorzewska A, Enoiu M, Ho TV, Schärer OD, Elledge SJ, Walter JC. The Fanconi anemia pathway promotes replicationdependent DNA interstrand cross-link repair. Science 2009; 326:1698-701; PMID:19965384; http:// dx.doi.org/10.1126/science.1182372 83. Constantinou A. Rescue of replication failure by Fanconi anaemia proteins. Chromosoma 2012; 121:21-36; http://dx.doi.org/10.1007/s00412-011-0349-2 84. Hayden PJ, Tewari P, Morris DW, Staines A, Crowley D, Nieters A, Becker N, de Sanjosé S, Foretova L, Maynadié M, et al. Variation in DNA repair genes XRCC3, XRCC4, XRCC5 and susceptibility to myeloma. Hum Mol Genet 2007; 16:3117-27; PMID:17901044; http://dx.doi.org/10.1093/hmg/ ddm273 85. Roddam PL, Rollinson S, O’Driscoll M, Jeggo PA, Jack A, Morgan GJ. Genetic variants of NHEJ DNA ligase IV can affect the risk of developing multiple myeloma, a tumour characterised by aberrant class switch recombination. J Med Genet 2002; 39:9005; PMID:12471202; http://dx.doi.org/10.1136/ jmg.39.12.900 86. Roddam PL, Allan JM, Dring AM, Worrillow LJ, Davies FE, Morgan GJ. Non-homologous end-joining gene profiling reveals distinct expression patterns associated with lymphoma and multiple myeloma. Br J Haematol 2010; 149:258-62; PMID:20148879; http://dx.doi.org/10.1111/j.1365-2141.2010.08088.x

14

126. Sousa MML, Zub KA, Aas PA, Hanssen-Bauer A, Demirovic A, Sarno A, Tian E, Liabakk NB, Slupphaug G. An inverse switch in DNA base excision and strand break repair contributes to melphalan resistance in multiple myeloma cells. PLoS One 2013; 8:e55493; PMID:23405159; http://dx.doi. org/10.1371/journal.pone.0055493 127. Crawford LJA, Walker B, Ovaa H, Chauhan D, Anderson KC, Morris TCM, Irvine AE. Comparative selectivity and specificity of the proteasome inhibitors BzLLLCOCHO, PS-341, and MG-132. Cancer Res 2006; 66:6379-86; PMID:16778216; http://dx.doi. org/10.1158/0008-5472.CAN-06-0605 128. Neznanov N, Komarov AP, Neznanova L, StanhopeBaker P, Gudkov AV. Proteotoxic stress targeted therapy (PSTT): induction of protein misfolding enhances the antitumor effect of the proteasome inhibitor bortezomib. Oncotarget 2011; 2:209-21; PMID:21444945 129. Voorhees PM, Orlowski RZ. The proteasome and proteasome inhibitors in cancer therapy. Annu Rev Pharmacol Toxicol 2006; 46:189-213; PMID:16402903; http://dx.doi.org/10.1146/ annurev.pharmtox.46.120604.141300 130. Jacquemont C, Taniguchi T. Proteasome function is required for DNA damage response and fanconi anemia pathway activation. Cancer Res 2007; 67:7395-405; PMID:17671210; http://dx.doi. org/10.1158/0008-5472.CAN-07-1015 131. Murakawa Y, Sonoda E, Barber LJ, Zeng W, Yokomori K, Kimura H, Niimi A, Lehmann A, Zhao GY, Hochegger H, et al. Inhibitors of the proteasome suppress homologous DNA recombination in mammalian cells. Cancer Res 2007; 67:8536-43; PMID:17875693; http://dx.doi.org/10.1158/00085472.CAN-07-1166 132. Neri P, Ren L, Gratton K, Stebner E, Johnson J, Klimowicz A, Duggan P, Tassone P, Mansoor A, Stewart DA, et al. Bortezomib-induced “BRCAness” sensitizes multiple myeloma cells to PARP inhibitors. Blood 2011; 118:6368-79; PMID:21917757; http:// dx.doi.org/10.1182/blood-2011-06-363911 133. Roussel M, Moreau P, Huynh A, Mary J-Y, Danho C, Caillot D, Hulin C, Fruchart C, Marit G, Pégourié B, et al.; Intergroupe Francophone du Myélome (IFM). Bortezomib and high-dose melphalan as conditioning regimen before autologous stem cell transplantation in patients with de novo multiple myeloma: a phase 2 study of the Intergroupe Francophone du Myelome (IFM). Blood 2010; 115:32-7; PMID:19884643; http://dx.doi.org/10.1182/blood-2009-06-229658 134. Steele JM. Carfilzomib: A new proteasome inhibitor for relapsed or refractory multiple myeloma. J Oncol Pharm Pract 2013; PMID:23292972; http://dx.doi. org/10.1177/1078155212470388 135. Wang Q-E, Wani MA, Chen J, Zhu Q, Wani G, El-Mahdy MA, Wani AA. Cellular ubiquitination and proteasomal functions positively modulate mammalian nucleotide excision repair. Mol Carcinog 2005; 42:53-64; PMID:15547920; http://dx.doi. org/10.1002/mc.20065 136. Ito T, Ando H, Suzuki T, Ogura T, Hotta K, Imamura Y, Yamaguchi Y, Handa H. Identification of a primary target of thalidomide teratogenicity. Science 2010; 327:1345-50; PMID:20223979; http://dx.doi. org/10.1126/science.1177319 137. Zhu Y-X, Braggio E, Shi C-X, Bruins LA, Schmidt JE, Van Wier S, Chang XB, Bjorklund CC, Fonseca R, Bergsagel PL, et al. Cereblon expression is required for the antimyeloma activity of lenalidomide and pomalidomide. Blood 2011; 118:47719; PMID:21860026; http://dx.doi.org/10.1182/ blood-2011-05-356063 138. Moalli PA, Pillay S, Weiner D, Leikin R, Rosen ST. A mechanism of resistance to glucocorticoids in multiple myeloma: transient expression of a truncated glucocorticoid receptor mRNA. Blood 1992; 79:21322; PMID:1728309

139. Nojima M, Maruyama R, Yasui H, Suzuki H, Maruyama Y, Tarasawa I, Sasaki Y, Asaoku H, Sakai H, Hayashi T, et al. Genomic screening for genes silenced by DNA methylation revealed an association between RASD1 inactivation and dexamethasone resistance in multiple myeloma. Clin Cancer Res 2009; 15:4356-64; PMID:19549772; http://dx.doi. org/10.1158/1078-0432.CCR-08-3336 140. Chauhan D, Li G, Hideshima T, Podar K, Mitsiades C, Mitsiades N, Catley L, Tai YT, Hayashi T, Shringarpure R, et al. Hsp27 inhibits release of mitochondrial protein Smac in multiple myeloma cells and confers dexamethasone resistance. Blood 2003; 102:3379-86; PMID:12855565; http://dx.doi. org/10.1182/blood-2003-05-1417 141. Moreaux J, Legouffe E, Jourdan E, Quittet P, Rème T, Lugagne C, Moine P, Rossi JF, Klein B, Tarte K. BAFF and APRIL protect myeloma cells from apoptosis induced by interleukin 6 deprivation and dexamethasone. Blood 2004; 103:314857; PMID:15070697; http://dx.doi.org/10.1182/ blood-2003-06-1984 142. Tai Y-T, Anderson KC. Bruton’s tyrosine kinase: oncotarget in myeloma. Oncotarget 2012; 3:913-4; PMID:22989914 143. Cibeira MT, de Larrea CF, Navarro A, Díaz T, Fuster D, Tovar N, Rosiñol L, Monzó M, Bladé J. Impact on response and survival of DNA repair single nucleotide polymorphisms in relapsed or refractory multiple myeloma patients treated with thalidomide. Leuk Res 2011; 35:1178-83; PMID:21435719; http://dx.doi. org/10.1016/j.leukres.2011.02.009 144. Burington B, Barlogie B, Zhan F, Crowley J, Shaughnessy JD Jr. Tumor cell gene expression changes following short-term in vivo exposure to single agent chemotherapeutics are related to survival in multiple myeloma. Clin Cancer Res 2008; 14:4821-9; PMID:18676754; http://dx.doi.org/10.1158/10780432.CCR-07-4568 145. Barrera LN, Rushworth SA, Bowles KM, MacEwan DJ. Bortezomib induces heme oxygenase-1 expression in multiple myeloma. Cell Cycle 2012; 11:224852; PMID:22617388; http://dx.doi.org/10.4161/ cc.20343 146. Lord CJ, Ashworth A. The DNA damage response and cancer therapy. Nature 2012; 481:287-94; PMID:22258607; http://dx.doi.org/10.1038/ nature10760 147. Smith J, Tho LM, Xu N, Gillespie DA. Chapter 3 The ATM-Chk2 and ATR-Chk1 Pathways in DNA Damage Signaling and Cancer. 1st ed. Elsevier Inc; 2010. 148. Pei X-Y, Dai Y, Youssefian LE, Chen S, Bodie WW, Takabatake Y, Felthousen J, Almenara JA, Kramer LB, Dent P, et al. Cytokinetically quiescent (G0/ G1) human multiple myeloma cells are susceptible to simultaneous inhibition of Chk1 and MEK1/2. Blood 2011; 118:5189-200; PMID:21911831; http:// dx.doi.org/10.1182/blood-2011-02-339432 149. Smith J, Tho LM, Xu N, Gillespie DA. Chapter 3 The ATM-Chk2 and ATR-Chk1 Pathways in DNA Damage Signaling and Cancer. 1st ed. Elsevier Inc; 2010. 150. Landau HJ, McNeely SC, Nair JS, Comenzo RL, Asai T, Friedman H, Jhanwar SC, Nimer SD, Schwartz GK. The checkpoint kinase inhibitor AZD7762 potentiates chemotherapy-induced apoptosis of p53mutated multiple myeloma cells. Mol Cancer Ther 2012; 11:1781-8; PMID:22653969; http://dx.doi. org/10.1158/1535-7163.MCT-11-0949 151. Caraux A, Vincent L, Bouhya S, Quittet P, Moreaux J, Requirand G, Veyrune JL, Olivier G, Cartron G, Rossi JF, et al. Residual malignant and normal plasma cells shortly after high dose melphalan and stem cell transplantation. Highlight of a putative therapeutic window in Multiple Myeloma? Oncotarget 2012; 3:1335-47; PMID:23154454



115. Yang HH, Ma MH, Vescio RA, Berenson JR. Overcoming drug resistance in multiple myeloma: the emergence of therapeutic approaches to induce apoptosis. J Clin Oncol 2003; 21:423947; PMID:14615454; http://dx.doi.org/10.1200/ JCO.2003.06.001 116. Dimopoulos MA, Souliotis VL, Anagnostopoulos A, Bamia C, Pouli A, Baltadakis I, Terpos E, Kyrtopoulos SA, Sfikakis PP. Melphalan-induced DNA damage in vitro as a predictor for clinical outcome in multiple myeloma. Haematologica 2007; 92:1505-12; PMID:18024399; http://dx.doi. org/10.3324/haematol.11435 117. Vangsted A, Gimsing P, Klausen TW, Nexø BA, Wallin H, Andersen P, Hokland P, Lillevang ST, Vogel U. Polymorphisms in the genes ERCC2, XRCC3 and CD3EAP influence treatment outcome in multiple myeloma patients undergoing autologous bone marrow transplantation. Int J Cancer 2007; 120:1036-45; PMID:17131345; http://dx.doi. org/10.1002/ijc.22411 118. Chen Q, Van der Sluis PC, Boulware D, Hazlehurst LA, Dalton WS. The FA/BRCA pathway is involved in melphalan-induced DNA interstrand cross-link repair and accounts for melphalan resistance in multiple myeloma cells. Blood 2005; 106:698705; PMID:15802532; http://dx.doi.org/10.1182/ blood-2004-11-4286 119. Yarde DN, Oliveira V, Mathews L, Wang X, Villagra A, Boulware D, Shain KH, Hazlehurst LA, Alsina M, Chen DT, et al. Targeting the Fanconi anemia/ BRCA pathway circumvents drug resistance in multiple myeloma. Cancer Res 2009; 69:9367-75; PMID:19934314; http://dx.doi.org/10.1158/00085472.CAN-09-2616 120. Bharti AC, Donato N, Singh S, Aggarwal BB. Curcumin (diferuloylmethane) down-regulates the constitutive activation of nuclear factor-kappa B and IkappaBalpha kinase in human multiple myeloma cells, leading to suppression of proliferation and induction of apoptosis. Blood 2003; 101:105362; PMID:12393461; http://dx.doi.org/10.1182/ blood-2002-05-1320 121. Xiao H, Xiao Q, Zhang K, Zuo X, Shrestha UK. Reversal of multidrug resistance by curcumin through FA/BRCA pathway in multiple myeloma cell line MOLP-2/R. Ann Hematol 2010; 89:399404; PMID:19756599; http://dx.doi.org/10.1007/ s00277-009-0831-6 122. Pan Y, Gao Y, Chen L, Gao G, Dong H, Yang Y, Dong B, Chen X. Targeting autophagy augments in vitro and in vivo antimyeloma activity of DNA-damaging chemotherapy. Clin Cancer Res 2011; 17:3248-58; PMID:21288924; http://dx.doi.org/10.1158/10780432.CCR-10-0890 123. Katayama M, Kawaguchi T, Berger MS, Pieper RO. DNA damaging agent-induced autophagy produces a cytoprotective adenosine triphosphate surge in malignant glioma cells. Cell Death Differ 2007; 14:54858; PMID:16946731; http://dx.doi.org/10.1038/ sj.cdd.4402030 124. Couvé S, Macé-Aimé G, Rosselli F, Saparbaev MK. The human oxidative DNA glycosylase NEIL1 excises psoralen-induced interstrand DNA cross-links in a three-stranded DNA structure. J Biol Chem 2009; 284:11963-70; PMID:19258314; http:// dx.doi.org/10.1074/jbc.M900746200 125. McNeill DR, Lam W, DeWeese TL, Cheng YC, Wilson DM 3rd. Impairment of APE1 function enhances cellular sensitivity to clinically relevant alkylators and antimetabolites. Mol Cancer Res 2009; 7:897-906; PMID:19470598; http://dx.doi. org/10.1158/1541-7786.MCR-08-0519

DNA repair pathways in human multiple myeloma

DNA repair pathways in human multiple myeloma

Suggest Documents

Deregulation of DNA Double-Strand Break Repair in Multiple Myeloma

Deregulation of DNA Double-Strand Break Repair in Multiple Myeloma

The NF-B Activating Pathways in Multiple Myeloma - MDPI

The NF-B Activating Pathways in Multiple Myeloma - MDPI

Multiple pathways are involved in DNA degradation

The NF-B Activating Pathways in Multiple Myeloma - MDPI

(DWI) in multiple myeloma

repair pathways keep mitochondrial DNA intact

Modelling DNA Repair Pathways: Recent Advances ...

Targeting DNA repair pathways for cancer treatment

Human Multiple Myeloma Cells Suppression of ...

DNA-Related Pathways Defective in Human

Synergistic DNA-damaging effect in multiple myeloma ...

Aging Results in Differential Regulation of DNA Repair Pathways in ...

Paraneoplastic Sarcoidosis in Multiple Myeloma

Thrombosis in Multiple Myeloma

Vaccination in Multiple Myeloma

Selective Induction of DNA Repair Pathways in ... - Semantic Scholar

The Impact of DNA Repair Pathways in Cancer ... - Semantic Scholar

Calmodulin Mediates DNA Repair Pathways Involving H2AX in ...

Myeloma Allogeneic transplantation for multiple myeloma - Nature

Genetic Interactions of DNA Repair Pathways in the Pathogen ...

Misregulation of DNA damage repair pathways in ...

Coordination of Altered DNA Repair and Damage Pathways in ...