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In the last few years, the cancer field hasgained greater insights into andincreased appreciation for the genes andpathways involved in the maintenance ofgenome integrity. Indeed, it has longbeen recognized that most, if not all,human cancers display aberrations inchromosome count and structure whoserecurrent nature belies a pathogeneticlink to cancer development (Lengauer etal., 1998). For certain human cancers,such as those of the hematopoeitic sys-tem, there exists ample genetic evidence

that certain recurrent chromosomaltranslocations are diagnostic andcausative (Rowley, 2001). While debatecontinues over the origins of suchchanges and their precise contributionsto carcinogenesis (Marx, 2002), mount-ing experimental evidence has strength-ened the view that periods of geneticinstability can propel aspiring cancercells toward a lethal malignant endpoint.

The path to our current understand-ing has been illuminated largely throughthe systematic analysis of mouse models

defective for telomere maintenance, DNArepair, and tumor suppressor pathwayfunction (Hakem and Mak, 2001; Maserand DePinho, 2002). These model sys-tems have demonstrated that mice, defi-cient in the various “caretakers” of thegenome, sustain a spectrum of tumorsthat recapitulate more closely certaincytogenetic aspects of human cancers.Particularly informative have been mousemodels harboring deficiencies of compo-nents of the nonhomologous end-joiningpathway (NHEJ) of DNA double-strandbreak (DSB) repair (Ferguson and Alt,2001). Unable to complete the pro-grammed rearrangements initiated byDSBs induced by the RAG-1/2 nuclease,these mutant mouse strains have offereda novel view of the potential conse-quences of a free DSB—namely, thecheckpoints elicited by unrepaired DSBsand the detrimental impact of inappropri-ate DSB repair. Mice deficient for p53checkpoint function alone sustain highrates of T cell lymphomas and sarcomas,yet remarkably these tumors lack charac-teristic chromosome structural anomaliesand exhibit only modest levels of aneu-ploidy. In contrast, mice with dual impair-ments in NHEJ and p53 functionsuccumb to highly aggressive pro-B celllymphomas possessing signaturechromosomal rearrangements whoseanatomy points to a defective V(D)Jrecombination process (Difilippantonio etal., 2002; Gladdy et al., 2003 [this issue

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Take care of your chromosomes lest cancer takes care of you

The analysis of compound mouse mutants for nonhomologous end-joining DNA double-strand break repair and those defi-cient for the p53 checkpoint pathway has provided a fascinating look at the carcinogenic consequences of the failure toproperly repair DNA damage and to elicit appropriate checkpoints.

Figure 1. Creating a cancerous genome inthe absence of NHEJ and p53, and path-ways to pro-B cell lymphoma in miceA: In cells lacking p53 checkpoint function,the failure of NHEJ to properly repair the DNADSB created by RAG-induced cleavage atIgH leads to the formation of aberrant chro-mosomes (translocations and dicentrics). Inturn, these can result in c-myc amplification,BFB cycles, and further genomic instability,creating the genetic recipe for pro-B celllymphoma in mice.B: Once initiated, improper completion ofV(D)J recombination in mice lacking NHEJand p53 function results in a path that leadsto pro-B cell lymphoma. Alternatively, in theproper genetic context, generalizedgenomic instability in NHEJ-p53 doublemutants may also lead to a path to pro-B celllymphoma.

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of Cancer Cell];Vanasse et al., 1999; Zhuet al., 2002).

These compound NHEJ p53 mutantmodel systems have offered a penetrat-ing view of the cytogenetic and onco-genic consequences of an introducedDNA DSB (created by RAG nuclease)and its aberrant repair in a setting ofattenuated DNA damage checkpoints.Among these studies, several commonthemes have emerged. In each case, formice lacking p53 and either Ku70 orKu80 (Difilippantonio et al., 2002),XRCC4 or LIG4 (Zhu et al., 2002), orcarrying a hypomorphic DNA-PKcs

“SCID” mutation (DNA-PKSCID) (Gladdyet al., 2003; Vanasse et al., 1999), thereemerges lymphomas bearing clonal andrecurrent chromosomal rearrangements.The advent of high-resolution cytogenet-ic technologies, such as spectral kary-otyping and array-comparative genomichybridization, has allowed the field tomolecularly define these rearrange-ments and thereby infer the mechanismof repair that produced them, gaininginsight into the molecular forces guidingcancer cell evolution. In nearly everylymphoma, rearrangements appear toinitiate from the site of RAG-inducedcleavage in the immunoglobulin heavychain locus (IgH) on chromosome 12,and subsequently involved the c-myclocus on chromosome 15 (Figure 1A).Repair of the RAG-induced DSB andgeneration of the (12;15) translocationappear to have resulted from a type ofend repair utilizing short homologies(Difilippantonio et al., 2002; Gladdy etal., 2003; Zhu et al., 2002). In addition,evidence suggests that dicentric forma-tion and ensuing bridge-fusion-breakage(BFB) cycles are involved in creating anamplified c-myc locus and more complexchromosomal rearrangements (Figure1A) (Difilippantonio et al., 2002; Gladdyet al., 2003; Zhu et al., 2002).The reasonthat c-myc is so frequently involved isunclear, but may reflect the presence offragile sites, cryptic sequences recog-nized by RAG-1/2, and/or the chanceoccurrence of a DSB generated by thebreakage of dicentrics.With regard to thelatter, it is interesting to note that certainNHEJ deficiencies are associated withdecreased telomere capping functionand the production of telomeric end-to-end fusions (Bailey et al., 1999; d’Addadi Fagagna et al., 2001) that would inturn set the stage for BFB-induced DSBsthroughout the genome. In this scenario,the critical role of c-Myc dysfunction in

lymphomagenesis would presumablylead to the selection of rare cells in thepopulation that harbor an amplified c-myc locus. Combined, these results pointto the central importance of DSBs ingenomic instability, particularly aberra-tions in chromosome structure, whichcan fuel pro-B cell lymphomagenesis.

More recent work has taken a stepfurther by assessing the role of RAG ininitiating the DSBs and specific chromo-some abnormalities that drive pro-B celltumors (Difilippantonio et al., 2002;Gladdy et al., 2003;Vanasse et al., 1999;Zhu et al., 2002), and unexpectedly,some different outcomes were observed.The absence of RAG-1/2 prevents theinitiation of V(D)J recombination due tothe failure to create the initial cleavage atimmune loci; hence, the lack of RAG-1/2might be expected to prevent pro-B celllymphomas in mice deficient for bothNHEJ and p53 function. This was testedpreviously (Difilippantonio et al., 2002;Vanasse et al., 1999; Zhu et al., 2002)and in the study in this issue of CancerCell (Gladdy et al., 2003) by breedingRAG-2 deficiency into the variousNHEJ/p53 mutant strains. Not surpris-ingly, pro-B cell lymphomas with therecurrent (t12;15) translocations weresuppressed; however, beyond this com-mon finding, interesting differences wereobtained among the various studies.Mice mutant for Xrcc4-p53 combinedwith RAG-2 deficiency died young,before they would be expected to devel-op the p53-associated T cell lymphomaobserved in NHEJ-proficient mice (Zhuet al., 2002), whereas Ku80-p53 mutantmice with RAG-2 deficiency did developT cell lymphomas at a later age, as dotheir Ku80 wild-type p53-deficient coun-terparts (Difilippantonio et al., 2002).Vanasse et al. (1999) observed thatDNA-PKSCID-p53-RAG-2 mutant micebehaved similarly, developing late T cellrather than early B cell lymphomas.Whereas this apparent difference in can-cer phenotype may reflect the shorterlifespan of the triple mutant Xrcc4-p53-RAG-2 mice compared to the DNA-PKSCID-p53-RAG-2 mutant mice, we aretempted to speculate that this distinctioncould reflect in part the differentialimpact of the various NHEJ deficiencieson telomere maintenance (d’Adda diFagagna et al., 2001). In striking con-trast, Gladdy et al. (2003) observed noreduction in either incidence or latency ofpro-B cell lymphomas in their DNA-PKSCID-p53-RAG-2 mutant mice com-

pared to DNA-PKSCID-p53 mutant con-trols. Whereas lymphomas of the DNA-PKSCID-p53 mutant cohort harbored thecharacteristic RAG-induced rearrange-ments, this tumor was suppressed in thetriple mutant background. In its absence,however, a different type of pro-B celllymphoma was revealed which lackedthe (t12;15) translocation, possessed dif-ferent translocations, and had differentbiological properties. In the latter con-text, the Gladdy et al. DNA-PKSCID-p53-RAG-2 mutant pro-B cell lymphomaswere unique in their capacity to spread tothe meninges—the membranes envelop-ing the central nervous system (Gladdyet al., 2003). This interesting and novelexperimental outcome suggests thatgeneralized genomic instability associat-ed with NHEJ deficiency in the appropri-ate context can cooperate with p53checkpoint deficiency to create lesionscapable of endowing pro-B cell lym-phomas with the capacity to invade andreside in the meningial membranes—although, as discussed below, additionalfactors may be operative. Nonetheless,expression of c-Myc was elevated com-pared to normal lymphoid cells, pointingto its central importance in lymphomapathogenesis and the capacity of c-Mycto be dysregulated via multiplemechanisms.

What are we to make of the differ-ences in the pro-B cell tumor phenotypearising in the Gladdy et al. triple mutantDNA-PKSCID-p53-RAG-2 cohort com-pared to the other studies? As far as canbe discerned, the mutant allelesemployed were identical: RAG-2, DNA-PKcs (SCID), and p53 (regardless, twodifferent alleles of p53 have been shownto behave similarly [Difilippantonio et al.,2002]). Is it possible that the phenotypicdifferences relate to various modifierspresent in these heavily mixed geneticstrains? The fact that NHEJ and p53 defi-ciencies result in a near-universal pro-Bcell lymphoma condition suggests thatthese combined mutations drive thistumor type regardless of genetic back-ground in RAG-proficient animals. In theGladdy et al. study (Gladdy et al., 2003),one might assume that a modifier differ-ence may influence the phenotypic out-come of the triple mutant mice, althoughthis seems unlikely given the heavilymixed composition of the large experi-mental cohorts examined. Nonetheless,a specific genetic modifier in the Gladdyet al. triple mutant cohort cannot be dis-counted, nor can we exclude the modu-

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lating effects of environmental anddietary factors. Despite these differ-ences, it appears that DSBs, whetherinduced by the RAG nuclease or by therandom BFB process enabled byNHEJ/p53 deficiency, can lead to pro-Bcell lymphomas (Figure 1B). These unre-solved issues should motivate efforts toidentify the genetic and epigenetic fac-tors that endow lymphoma cells with thecapacity to hone to meningial mem-branes—an occurrence that is notuncommon in the lymphoma clinic.

Are there any unifying themes asso-ciated with these studies? For one, it isclear that NHEJ deficiency (as well asother DNA repair defects) results ingenomic instability that can provoke car-cinogenesis. This becomes abundantlyclear when programmed DNA DSBs areinitiated in the absence of NHEJ in thelymphoid compartment. Although the c-myc rearrangements found in theNHEJ/p53 double mutant murine tumorsare not identical to those typically foundin human cancers (Rowley, 2001), theyare informative nonetheless with respectto identifying mechanisms that con-tribute to the chromosomal aberrationsencountered in a wide variety of humanlymphoid and solid tumors. Gene amplifi-cation (and deletion) can contribute to awide spectrum of tumor types, and thegenetic experiments described here andprevious cell culture studies (Pipiras etal., 1998) indicate that DSBs are crucialinitiators of these events. Second, thep53 checkpoint mechanism in responseto aberrant DNA DSBs has once againshown its central importance and anadditional basis for why its loss of func-

tion can participate in the developmentof so many tumor types. In the lymphoidcompartment, its function is to forceaberrant cells into apoptosis, rather thanto allow alternative pathways of DNArepair (Figure 1B). The link betweenDSBs, p53 status, dicentric formation,and increased cancer—particularlyepithelial cancers—was first forged inmice deficient for telomere function(Artandi et al., 2000; Chin et al., 1999), inwhich p53 loss provides a permissivecontext for the increased formation ofchromosomal fusions and recurrentrearrangements via BFB cycles that fuelcancer initiation. Analogously, in theNHEJ mutants, p53′s absence enablesexisting DNA damage to be replicatedand create the aberrant chromosomesfound in tumors. Lastly, this body of workcontinues to validate the use of geneti-cally engineered mice as models toelucidate mechanisms governing chro-mosome stability and to uncover novelgenetic pathways that promote or pre-vent human cancer.

Richard S. Maser* and Ronald A. DePinho

Department of Medical OncologyDana Farber Cancer InstituteDepartments of Medicine and GeneticsHarvard Medical SchoolBoston, Massachusetts 02115*E-mail: richard_maser@dfci.harvard.edu

Selected reading

Artandi, S.E., Chang, S., Lee, S.L., Alson, S.,Gottlieb, G.J., Chin, L., and DePinho, R.A.

(2000). Nature 406, 641–645.

Bailey, S.M., Meyne, J., Chen, D.J., Kurimasa, A.,Li, G.C., Lehnert, B.E., and Goodwin, E.H.(1999). Proc. Natl. Acad. Sci. USA 96,14899–14904.

Chin, L., Artandi, S.E., Shen, Q., Tam, A., Lee,S.L., Gottlieb, G.J., Greider, C.W., and DePinho,R.A. (1999). Cell 97, 527–538.

d’Adda di Fagagna, F., Hande, M.P., Tong, W.M.,Roth, D., Lansdorp, P.M., Wang, Z.Q., andJackson, S.P. (2001). Curr. Biol. 11, 1192–1196.

Difilippantonio, M.J., Petersen, S., Chen, H.T.,Johnson, R., Jasin, M., Kanaar, R., Ried, T., andNussenzweig, A. (2002). J. Exp. Med. 196,469–480.

Ferguson, D.O., and Alt, F.W. (2001). Oncogene20, 5572–5579.

Gladdy, R.A., Taylor, M.D., Williams, C.J.,Grandal, I., Karaskova, J., Squire, J.A., Rutka,J.T., Guidos, C.J., and Danska, J.S. (2003).Cancer Cell 3, this issue, 37–50.

Hakem, R., and Mak, T.W. (2001). Annu. Rev.Genet. 35, 209–241.

Lengauer, C., Kinzler, K.W., and Vogelstein, B.(1998). Nature 396, 643–649.

Marx, J. (2002). Science 297, 544–546.

Maser, R.S., and DePinho, R.A. (2002). Science297, 565–569.

Pipiras, E., Coquelle, A., Bieth, A., andDebatisse, M. (1998). EMBO J. 17, 325–333.

Rowley, J.D. (2001). Nat. Rev. Cancer 1,245–250.

Vanasse, G.J., Halbrook, J., Thomas, S.,Burgess, A., Hoekstra, M.F., Disteche, C.M., andWillerford, D.M. (1999). J. Clin. Invest. 103,1669–1675.

Zhu, C., Mills, K.D., Ferguson, D.O., Lee, C.,Manis, J., Fleming, J., Gao, Y., Morton, C.C., andAlt, F.W. (2002). Cell 109, 811–821.

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