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Location and function of critical genes in leukemogenesis inferred from cytogenetic abnormalities in hematologic malignancies
Location and Function of Critical Genes in Leukemogenesis Inferred From Cytogenetic Abnormalities in Hematologic Malignancies Olivia A. Bernard and Rohnd Berger Dramatic advances in the cytogenetic analysis of chromosomal rearrangements of hematopoietic malignancies have occurred over the past years. These are due to considerable improvement in the techniques of molecular cytogenetics. Various applications of fluorescence in situ hybridization (FISH), used in conjunction with conventional cytogenetics, make the recognition of some abnormalities easier, and the localization of chromosomal breakpoints in structural rearrangements more precise. Under many circumstances, accurate breakpoint localization is the first step toward the identification of genes involved in translocations and inversions. Some of the genes recently discovered may be rearranged with several partner genes. These promiscuous genes are natural experiments that generate mutants which help to identify the function of genes rearranged in hematopoietic malignancies as well as that of their normal counterparts. The diversity of the genes implicated in leukemogenesis makes their functional study a challenge, but, as recently shown by their role in chromatin remodeling, increasing recognition of cross-talk between many of these genes justifies the development of analyses of leukemia-associated chromosome abnormalities and of their functional consequences. Semin Hemato/37:412419. Copyright 0 2000 by W.B. Saunders Company.
YTOGENETICS HAS LONG BEEN considered by many biologists involved in cancer researchfar too imprecise an approach to deserve scientific consideration. Changes in this view occurred, first from the finding of nonrandomnessof chromosomalabnormalities in malignant cells, and later from correlations between chromosome and generearrangements.The relationshipsbetween gene and chromosomeabnormalitieswere not obvious in the early 1980sbecausethe resolutionprovided by the banding techniquesused in cytogenetics was much less precise than could be achieved by molecular biology. An averagechromosomeband (-330 bands per neutral genome, or 22 autosomes +X, Paris Conference 1971) containsapproximatively lo7 base pairs-a potentially very large number of genes. Recurrence of some chromosome abnormalities as
C
From INSERiVl U434 and CNRS SD 401 No. 434, Institut de Ghahique A4okc&ire, Paris, France. Supported in part by Ligue Nation&e Contre le Canceu,Ligue Nationale Contre le Cancer (Corn& de Paris), and Associution de Rechrche Contre le Cancer (ARC). Address reprint requeststo Roland Berger, MD, Dr SC, U434, Institut de GPnbtique Molt&hire, 27 rue Juliette Dodu, 7’010 Paris, France. CoBtight 0 2000 by WB. Saw&s Company 0037-19G3/00/3704-0009$10.00/0 hi:1 0.1053/shm.2000.16449
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well asimprovement in molecular techniquesled to the identification of specific genesinvolved in the chromosome rearrangementsof malignancies.The study of Burkitt’s lymphoma-leukemiatranslocations had a stimulating effect on the evolution of attitudes toward this problem. The breakpointsof three recurrent translocationsfound in thesetumors wereshown to be localizedwithin the MyClocus on chromosome 8 and the immunoglobulin gene loci on chromosomes14, 2, and 22. These results acceleratedthe identification of other genes involved in chromosomalrearrangements. More recently, the use of fluorescencein situ hybridization (FISH) asa complementto cytogenetic methods has provided a new tool to facilitate gene identification and allowed the discovery of new chromosomal rearrangements.A large number of genesinvolved in normal hematopoieisishave now been identified from molecularcytogenetic studiesof structural chromosomal rearrangementsin various hematopoieticmalignancies.
Location of Genes Involved in Chromosome Rearrangements of Hematopoietic Malignancies The usual first step toward the characterization of genesinvolved in a hematopoietic malignancy is the
Seminars in Hematology Vo137, No 4 (October), 2000:~~ 412-419
Genes and Chromosomes in Leukemogenesis
identification by banding techniquesof a structural chromosomalabnormality, most often a translocation but on occasionan inversion, either recurrent or nor. The study of interstitial deletions, although it has been performed by means of FISH and DNA walking strategies,now is carried out mainly by polymerasechain reaction @‘CR)-basedand lossof heterozygosiry(LOH) approaches.The localization of a translocationor inversion chromosomalbreakpoint to a band or sometimesa subbandis indeedpossible, but with uncertainty createdby the uneven quality of banded chromosome preparations from malignant cells. Although classicalcytogeneticsremainsnecessary the useof moleculartechniquesenhancesthe possibilities of localization of the chromosomal breakpoints and rearrangementsand consequently the identification of the implicated genes.Among the first FISH techniques, whole-chromosome painting” remains useful for confirming a rearrangementdetected by banding techniquesor for uncovering an abnormality not seen in the banding analysis.42Other FISH techniquesapplicableto the whole genomecan now be used.Comparative genomic hybridization (CGH) is a quantitative method for determining chromosomal DNA distribution. Because CGH detects quantitative imbalances,it is not suitableto the study of balanced reciprocal translocations or inversions, which are among the most common rearrangements in hematopoieticmalignancies.CGH ishelpful in the study of partial deletions, either related or not to tumor-suppressorgenes,or amplified chromosomal segments,especiallyin the initial identification of amplicon genecontent. More recently, techniquesfor differential staining of the 24 human chromosomes have been developed. In the present state of the art, thesetechniquesmainly usewhole-chromosomepainting probes.The method’sprecision is no better than that of painting but allowsrecognition of rearrangements that are overlooked with conventional banding.54 Many efforts are under way to increasethe diversity of the probes used. An interesting new approach is based on the dissection of individual bands of a given chromosome for preparation of molecular probesspecific for each band followed. by differential staining.7 The result is that the chromosomeanalyzedlooks asif it consistedof a sequenceof horizontal bands (like classicalbands) of different colors; the successionof colors makes the analysis
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fasterand more preciseif the chromosomedissection was sufhciently accurate. Chromosome dissection oriented by banding analysisand followed by preparation of molecular probesfrom the rearrangedregions (or chromosomemarkers)is another possiblestrategy to identify genesimplicated in a rearrangement.The STAT5B-RAE4 fusion describedrecentlyZ is an example of such an approach:first microdissectionof a rearrangedchromosome17 with a breakpoint closeto the RARA locus,followed by verification by Southern blotting of an &4&l rearrangement,PCR cloning for recognition of the rearrangementof STATSB, and finally confirmation of the gene rearrangementby FISH with a P1 artificial chromosome(PAC) encompassingSTAT5B asa probe. Once the breakpoint localizations have been refined, FISH helps to locate the rearrangementmore precisely.The generalmethod is to find a DNA probe that gives a split signal between the two partner chromosomes.A good exampleisthe identification of the yeastartificial chromosome(YAC) containing the MU gene on baud 1lq23, which is rearrangedin a variety of acute leukemias.45Another recent proposal for determining a chromosomalbreakpoint with the FISH technique is basedon various methodsmaking use of extended chromatin. Multicolor DNA fiber FISH hasbeenused,for example,to visualizechromosomeband 1lq13 breakpointsthat are dispersedover the BCLl-PR4LIl locus in mantle cell lymphoma.52 Experimentson comparativehybridization to combed DNA fibers have shown that the method can be applied to fluorescenceratio measurementson single DNA molecules.19,24 The identification of a locus or loci involved in chromosomalrearrangementsis obviously difficult in the absenceof a candidategene.For suchcases,newly availablereliablehuman genomemapscan be usedin combination with existing YAC, PAC, and bacterial artificial chromosome (BAC) clones to localize a breakpoint precisely.The internet, data banks, and genome sequencingcenter serversare of great assistance for this purpose.FISH data generatedby useof theseprobesresult in more preciselocalization, until splitting of a probe is observed. Narrowing the breakpoint interval allowsthe identification of transcribedsequences and/or DNA rearrangements.Once one partner is recognized,either as a known or new gene, the identification of the secondpartner gene is straightforward.
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Based on studies of the past decade, several chromosomal bands can be regarded as the location of critical genes for leukemogenesis. Among the earliest recognized were the T-cell-receptor (TCR) and immunoglobulin (1G) genes, which are normally rearranged during T-cell differentiation and B-cell maturation, respectively 8~39The breakpoint location within the corresponding chromosomal band predicts the involvement of these loci with confidence, if they are observed in a corresponding lymphoid malignancy, Although several translocations involving TCR genes have been described in B-cell malignancies, as well as immunoglobulin genes in T-cell proliferations, none of the genes of the immunoglobulin superfamily has been involved in nonlymphoid tumor proliferationassociated chromosomal abnormalities. Translocations of TCR and IG usually result in the expression of a normal protein from the translocated partner gene, ectopically expressed or overexpressed. Some point mutations, whose functional significance is not always understood, have been found to be associated with these rearrangements. Many of the genes isolated during the study of translocations involving TCNIG genes were ultimately shown to be transcription factors. The list of involved genes is still growing with further advances made in molecular analysis of chromosome rearrangements. Functional investigation of the proteins involved is relatively direct because its cellular role is approximately the same under normal conditions as in the malignant process. The problem of gene identification became more complicated with attempts to analyze the second type of translocations, those resulting in the creation of chimeric genes and expression of fusion proteins. The first example of a translocation resulting in a hybrid gene was t(9;22), leading to the expression of the BCR-ABL fusion protein.@J6 Because of BCRmediated oligomerization of the ABL moiety the fusion protein exhibits constitutive tyrosine kinase activity. In many instances, cytogenetic location of the breakpoint combined with hematologic typing of the malignancy is helpful in the selection for a candidate gene on one of the partner chromosomes. Many genes have been identified that are rearranged with several partner genes, and the partners may correlate with a specific clinical phenotype. “Promiscuous” genes that rearrange with multiple partners are listed in Table 1 and three examples are described in detail below.
Table
1. Promiscuous
Genes
Involved
in Hematopietic
Malignancy
Gene
Chromosomal Location
NUP 98 TEL AMLl
llp15 12~13 21q22
MLL TCRAID TCRB IGH IGK IGL FGFRI E2A BCL6 RARA PDGFRB ALK
llq23
14qll 7q35 14q32 2p12 22qll Sqll
19p13 6q27 17q21 5q33 5q35
Hematologic Subtype
V V AML-M2 AML-M4 B-ALL V T-ALL + T-ALL + B-ALL + B-ALL + B-ALL + V B-ALL B-ML AML-MB CMML ML
ML ML ML ML ML
Abbreviations: V, variable; AML, acute myeloid leukemia; ALL, acute lymphoblastic leukemia; ML, malignant lymphoma; CMML, chronic myelomonocytic leukemia.
CBF The CBF &ore Binding Factor), alsonamed PEBP2 (rolyoma Enhancer Binding rrotein 2) is a heterodimerit transcription factor composedof two subunits, CBFa and CBFP, which regulatestranscription of various genes,particularly in hematopoiesis.CBFencoding genes have been characterized simultaneously in studiesof viral transcriptional regulation and chromosomaltranslocations.50 The CBFP coding gene is fused to SMMHC (Smooth &scle &Iyosin Heavy chain) in inv( 16) of acute myelomonocytic leukemia with bone marrow eosinophilia (AMLM~Eo).~‘,~~ Of the three genescoding for the CBFa subunit, only Ah4Ll has been involved in human leukemogenesis,both myeloid and lymphoid, by chromosomaltranslocationsand point mutations.38 AMLI is involved in several myeloid malignancyassociatedtranslocations.The amino-terminal part of the protein, containing its runt DNA binding domain, is fused to the ET0 protein as a result of t(8;21)(q22;q22)34,36and to an ETO-related gene, MTGl6; asa resultof t(16;21)(q24;q22).12/lJYLI is alsorecombinedwith the EV71 locus in t(3;21) all of which should result in the expressionof a short form of AMLl that lacks its transactivation domain.35
Genesand Chromosomesin Leukmogenesis
t(12;21), w h’rc h IS ’ fr eq uent in and specific for childhood B-cell acute lymphoblastic lymphoma (B-ALL), leads to the expression of a TEL-AMLl fusion product that contains almost all of the AMLl protein. 15,43In all cases, fusion proteins contain the runt domain of AMLl and are predicted to exhibit dominant-negative activity for normal CW’transcription,ll while point-mutations in AMU alleles demonstrate loss of mnction. These mutations are found in sporadic and in some inherited hematopoietic abnormalities, and they indicate that haploinsufhciency of the AMLl gene is leukemogenic.49 Other translocations involving AMLI, mainly in therapy-related AML, await molecular identification.44
MLL/HRX The MLL/HRXgene, localized to band 1 lq23, can be considered as a human homolog to the Drosophih trithordx gene, a transcriptional regulator involved in control of homeotic genes such as Bitbomx and Antenmpediu in fly developmental biology. MLL is fused with more than 30 identified partner genes, including itself, in a large variety of malignancies.4,23,58Curiously, the translocation breakpoints are clustered in a restricted area (8.3 kb) ofMLL4,23,51; the 5 ’ region of MLL, which remains on chromosome 11 after the balanced reciprocal translocations, is crucial for the malignant process. In each translocation, the rearrangement leads to an in-frame fusion, and presumably to a functional fusion protein, suggesting that disruption of MLL is not itself sufficient for leukemogenesis. MLL usually contributes about the same amino-terminal amino acids to the fusion protein, but the partner genes cannot be characterized as belonging to a single structural or functional category. The normal functions of some of the partners are known, and they include a wide range of activities: a cytoplasmic protein involved in signal transduction (AFG); an elongation factor (ELL); and a transcription factor (AFGq2 UFKHRL) . The nudeotide sequences of the partner protein suggested that they might all provide an oligomerization domain, and this hypothesis has some support from recent experimental data. to It is also possible that the partner gene is partly responsible for the association observed between some translocations and the clinical features of the associated leukemic subtypes.
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TEL/El-W The TEL/ETVGgene located on chromosome band 12~13 exemplifies a more complex situation.‘4a46 It codes for a member of the ETS family of transcription factors, which possess oligomerization and transcriptional repression properties.2130 TEL is rearranged in a variety of hematologic malignancies, and chromosome breakpoints are scattered all along the gene. Similar to MLL, the genes fused to TEL after chromosomal translocation are not homogeneous in terms of function. In contrast to MLL, the partner proteins usually do provide crucial functional domains. TEL has been hypothetized to be involved in leukemogenesis by at least four different mechanisms. The most frequently observed TEL-involving translocation is t(12;21), which fuses TEL to AiML1.15z43 The TEL moiety contributes a transcriptional repression domain and the fusion protein is thought to exert a dominant-negative effect on normal CBF activity.2o Several tyrosine kinase proteins, such as PDGFR& ABL, JAK2, ARG, and TRIWNTRK3, are also fused to TEL. In these cases, the TEL portion causes the fusion protein to oligomerize and thus exhibit constitutive tyrosine kinase activity. lL21 Whether all of these tyrosine kinases activate the same signal transduction pathway is not yet established. However, activation of the STAT5 factor could be an important common step in leukemogenic processes.26 Third, there are several examples predicting a TEL fusion protein that contains the ETS DNA binding domain of TEL, exemplified by the MNl-TEL fusion protein, which is thought to deregulate TEL target genes. Finally, molecular analysis of several TEL abnormalities does not predict the formation of the fusion protein. In these instances, the main role of these abnormalities would be to inactivate by some mechanism TEti normal functions. Such loss of activity may also follow the deletion of TEL associated with t(12;21).
Genes and Function More than the half of the genes found to be involved in chromosomal translocations were unknown at the time of their discovery, at least in man (Table 2). Subsequently, as their functions were uncovered, they fell into several categories, including: control of cellular differentiation (TALlKCL, MLL/HRX), proliferation and cell cycle regulation (MC CCNDl), pro-
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Table 2. Primary Gene ABL E2A FGFRl MLL MYC NUP98 PDGFRB RARA TAN CBF ALK BCL2 BCL6 BCR TALlILM02 TCLl/MTCPl TEL P16, PWRF
Genes
Located
at Chromosome
Localization
9q34 19p13 8~11-12 llq23 8q24 llp15 5q33 17q21 9q34 21q22 and 16q22 5q35 18q21 3q27 22qll lp32/11p13 14q32lXq28 12~13 9p21
grammed cell death (&X2), and signal transduction pathway members (ABL, ALK) . There is great functional diversiT of the genes involved in hematopoietic37 and leukemogenic processes. Nevertheless some putative unifying mechanisms have now been uncovered. The first example is the recently demonstrated TALI-IA402 connection.3,40,47 Both gene products are found in a large protein complex that also contains GATAl , E47, and LDB l/NLI, all of which are normally present during erythroid dif5erentiation.55 Another common mechanism involves control of chromatin structure. Balance between acetylase and deacetylase activities is important for transcriptional regulation during cell growth and differentiation. Many of the oncogenic fusion proteins have been shown to interact with components of acetylase/ deacetylase complexes. For example, the two fusion proteins, PML-RARA and PLZF-RARA, resulting from t(15;17) and t(11;17) of acute promyelocytic leukemia (APL) and of acute myeloid leukemia (AML) with resemblance to APL,5”,28 are in a protein complex that includes histone deacetylase 1 (HDACl) and the corepressors mSin3A and NcoRISMRT. At pharmacologic doses, retinoic acid (RA) may dissociate the complex, including PML-RARA, and thus mediate transactivation of RARA target genes, while PLZF-RARA is insensitive to W17~27 The difference in the response of the two fusion proteins to RA explains the efficacy of RA in the treatment of APL
Abnormality Function
Signal transducer Transcription factor Growth factor receptor Transcription factor Transcription factor Nucleopore protein Growth factor receptor Nuclear receptor Signal transducer Transcription factor Signal transducer Antiapoptotic factor Transcription factor Signal transducer Transcription factor Unknown Transcription factor Cell cycle control
Breakpoints Status
at Discovery
Known Known Known Known (Drosophila) Known Known Known Known Known (Drosophila) Same time Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown
and its lack of therapeutic activity in PLZF-RARAassociated leukemia. ‘* One practical consequence of these findings is the proposal to use inhibitors of deacetylases (butyrate, trischostatin A, trapoxin) in the treatment of APL.59 The fusion protein AMLI-ETO, resulting from t(8;21) of AML, also is able, like ETO, to interact with corepressor complexes (HDACl, N-coR/ SMRT).13 Moreover, whereas the fusion proteins repress transcription, a histone deacetylase inhibitor impairs this property. 31,56,57Similarly, the CBFBMYH 11 (SMMHC) fusion, resulting from inv( 16) of M4Eo, acts as a repressor in recruiting Sin3A.32 The ET0 protein also has recently been shown to act as a corepressor for the PLZF protein.33 Additionally, BCL6 andTEL can function as transcriptional repressors. Other proteins, such as NUP98, interact with the CBP/P300 proteins, which exhibit acetylase activity22 The genes encoding these CBP/P300 are themselves both involved in chromosomal translocations with MLL, and CBP also partners with MOzl Consistent with the involvement of these activities in leukemogenesis, mice heterozygous for an inactivated (knock-out) allele of CBP develop hematopoietic malignancies, and the cancer cells have lost the wild-type CBP allele 25 Furthermore, MLL itself has been demonstrated to interact with components of the SWI/SNF complex, underscoring the role of chromatin remodeling complex as a whole in the leukemic process.’
Genes und Chromosomes in Leukemogenesis
Conclusion The importance of traditional chromosomeanalysis and now molecular cytogeneticsin the location and identification of genes involved in hematopoietic malignanciescannot be underestimated.That role has beendecisivein the identification of new normal and fused genesand in the accurate definition of new hematologicsubtypesof malignancies.The contribution of the field in identifying new translocationsand gene rearrangementsprompt in turn the analysisof their functional consequencesand the identification of new targetsfor specifictherapy. Many novel geneshave been isolated, some of which have been shown to play a role in normal hematopoiesis.In rearrangementsoccurring in hematopoietic malignancies,the existenceof promiscuous genesemphasizes gene-geneand protein-protein interrelationsand helps to reveal the complex network of their functions. If the presentchallengeis the definition of the functional consequences of chromosomal abnormalitiesin preparation for future targetedtherapies,it remainsthe role of cytogeneticsto define new chromosomalabnormalities,both structural and numerical, and to understand the mechanismsof their genesis.Systematicapplication of 24-color FISH and of telomericprobesprovidesa promisingdirection for researchin this field. New techniquesusedin association with more classicalcytogenetic methods will contribute to fulfilling theseexpectations.
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trithorax by 1 lq23 translocations: Leukemogenic and transcriptional implications, in Rauscher FJ III, Vogt PK (eds): ChromosomaITi-anslocations and Oncogenic Factors. Berlin, Germany, Springer, 1997, pp 1-23 59. WarreII RPJr, He L-Z, Richon V, et al: Targeted transcription therapy of acute promyelocytic leukemia using an inhibitor of histone deacetylase. J NatI Cancer Inst 90:1621-1625, 1998
YTOGENETICS HAS LONG BEEN considered by many biologists involved in cancer researchfar too imprecise an approach to deserve scientific consideration. Changes in this view occurred, first from the finding of nonrandomnessof chromosomalabnormalities in malignant cells, and later from correlations between chromosome and generearrangements.The relationshipsbetween gene and chromosomeabnormalitieswere not obvious in the early 1980sbecausethe resolutionprovided by the banding techniquesused in cytogenetics was much less precise than could be achieved by molecular biology. An averagechromosomeband (-330 bands per neutral genome, or 22 autosomes +X, Paris Conference 1971) containsapproximatively lo7 base pairs-a potentially very large number of genes. Recurrence of some chromosome abnormalities as
C
From INSERiVl U434 and CNRS SD 401 No. 434, Institut de Ghahique A4okc&ire, Paris, France. Supported in part by Ligue Nation&e Contre le Canceu,Ligue Nationale Contre le Cancer (Corn& de Paris), and Associution de Rechrche Contre le Cancer (ARC). Address reprint requeststo Roland Berger, MD, Dr SC, U434, Institut de GPnbtique Molt&hire, 27 rue Juliette Dodu, 7’010 Paris, France. CoBtight 0 2000 by WB. Saw&s Company 0037-19G3/00/3704-0009$10.00/0 hi:1 0.1053/shm.2000.16449
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well asimprovement in molecular techniquesled to the identification of specific genesinvolved in the chromosome rearrangementsof malignancies.The study of Burkitt’s lymphoma-leukemiatranslocations had a stimulating effect on the evolution of attitudes toward this problem. The breakpointsof three recurrent translocationsfound in thesetumors wereshown to be localizedwithin the MyClocus on chromosome 8 and the immunoglobulin gene loci on chromosomes14, 2, and 22. These results acceleratedthe identification of other genes involved in chromosomalrearrangements. More recently, the use of fluorescencein situ hybridization (FISH) asa complementto cytogenetic methods has provided a new tool to facilitate gene identification and allowed the discovery of new chromosomal rearrangements.A large number of genesinvolved in normal hematopoieisishave now been identified from molecularcytogenetic studiesof structural chromosomal rearrangementsin various hematopoieticmalignancies.
Location of Genes Involved in Chromosome Rearrangements of Hematopoietic Malignancies The usual first step toward the characterization of genesinvolved in a hematopoietic malignancy is the
Seminars in Hematology Vo137, No 4 (October), 2000:~~ 412-419
Genes and Chromosomes in Leukemogenesis
identification by banding techniquesof a structural chromosomalabnormality, most often a translocation but on occasionan inversion, either recurrent or nor. The study of interstitial deletions, although it has been performed by means of FISH and DNA walking strategies,now is carried out mainly by polymerasechain reaction @‘CR)-basedand lossof heterozygosiry(LOH) approaches.The localization of a translocationor inversion chromosomalbreakpoint to a band or sometimesa subbandis indeedpossible, but with uncertainty createdby the uneven quality of banded chromosome preparations from malignant cells. Although classicalcytogeneticsremainsnecessary the useof moleculartechniquesenhancesthe possibilities of localization of the chromosomal breakpoints and rearrangementsand consequently the identification of the implicated genes.Among the first FISH techniques, whole-chromosome painting” remains useful for confirming a rearrangementdetected by banding techniquesor for uncovering an abnormality not seen in the banding analysis.42Other FISH techniquesapplicableto the whole genomecan now be used.Comparative genomic hybridization (CGH) is a quantitative method for determining chromosomal DNA distribution. Because CGH detects quantitative imbalances,it is not suitableto the study of balanced reciprocal translocations or inversions, which are among the most common rearrangements in hematopoieticmalignancies.CGH ishelpful in the study of partial deletions, either related or not to tumor-suppressorgenes,or amplified chromosomal segments,especiallyin the initial identification of amplicon genecontent. More recently, techniquesfor differential staining of the 24 human chromosomes have been developed. In the present state of the art, thesetechniquesmainly usewhole-chromosomepainting probes.The method’sprecision is no better than that of painting but allowsrecognition of rearrangements that are overlooked with conventional banding.54 Many efforts are under way to increasethe diversity of the probes used. An interesting new approach is based on the dissection of individual bands of a given chromosome for preparation of molecular probesspecific for each band followed. by differential staining.7 The result is that the chromosomeanalyzedlooks asif it consistedof a sequenceof horizontal bands (like classicalbands) of different colors; the successionof colors makes the analysis
413
fasterand more preciseif the chromosomedissection was sufhciently accurate. Chromosome dissection oriented by banding analysisand followed by preparation of molecular probesfrom the rearrangedregions (or chromosomemarkers)is another possiblestrategy to identify genesimplicated in a rearrangement.The STAT5B-RAE4 fusion describedrecentlyZ is an example of such an approach:first microdissectionof a rearrangedchromosome17 with a breakpoint closeto the RARA locus,followed by verification by Southern blotting of an &4&l rearrangement,PCR cloning for recognition of the rearrangementof STATSB, and finally confirmation of the gene rearrangementby FISH with a P1 artificial chromosome(PAC) encompassingSTAT5B asa probe. Once the breakpoint localizations have been refined, FISH helps to locate the rearrangementmore precisely.The generalmethod is to find a DNA probe that gives a split signal between the two partner chromosomes.A good exampleisthe identification of the yeastartificial chromosome(YAC) containing the MU gene on baud 1lq23, which is rearrangedin a variety of acute leukemias.45Another recent proposal for determining a chromosomalbreakpoint with the FISH technique is basedon various methodsmaking use of extended chromatin. Multicolor DNA fiber FISH hasbeenused,for example,to visualizechromosomeband 1lq13 breakpointsthat are dispersedover the BCLl-PR4LIl locus in mantle cell lymphoma.52 Experimentson comparativehybridization to combed DNA fibers have shown that the method can be applied to fluorescenceratio measurementson single DNA molecules.19,24 The identification of a locus or loci involved in chromosomalrearrangementsis obviously difficult in the absenceof a candidategene.For suchcases,newly availablereliablehuman genomemapscan be usedin combination with existing YAC, PAC, and bacterial artificial chromosome (BAC) clones to localize a breakpoint precisely.The internet, data banks, and genome sequencingcenter serversare of great assistance for this purpose.FISH data generatedby useof theseprobesresult in more preciselocalization, until splitting of a probe is observed. Narrowing the breakpoint interval allowsthe identification of transcribedsequences and/or DNA rearrangements.Once one partner is recognized,either as a known or new gene, the identification of the secondpartner gene is straightforward.
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Based on studies of the past decade, several chromosomal bands can be regarded as the location of critical genes for leukemogenesis. Among the earliest recognized were the T-cell-receptor (TCR) and immunoglobulin (1G) genes, which are normally rearranged during T-cell differentiation and B-cell maturation, respectively 8~39The breakpoint location within the corresponding chromosomal band predicts the involvement of these loci with confidence, if they are observed in a corresponding lymphoid malignancy, Although several translocations involving TCR genes have been described in B-cell malignancies, as well as immunoglobulin genes in T-cell proliferations, none of the genes of the immunoglobulin superfamily has been involved in nonlymphoid tumor proliferationassociated chromosomal abnormalities. Translocations of TCR and IG usually result in the expression of a normal protein from the translocated partner gene, ectopically expressed or overexpressed. Some point mutations, whose functional significance is not always understood, have been found to be associated with these rearrangements. Many of the genes isolated during the study of translocations involving TCNIG genes were ultimately shown to be transcription factors. The list of involved genes is still growing with further advances made in molecular analysis of chromosome rearrangements. Functional investigation of the proteins involved is relatively direct because its cellular role is approximately the same under normal conditions as in the malignant process. The problem of gene identification became more complicated with attempts to analyze the second type of translocations, those resulting in the creation of chimeric genes and expression of fusion proteins. The first example of a translocation resulting in a hybrid gene was t(9;22), leading to the expression of the BCR-ABL fusion protein.@J6 Because of BCRmediated oligomerization of the ABL moiety the fusion protein exhibits constitutive tyrosine kinase activity. In many instances, cytogenetic location of the breakpoint combined with hematologic typing of the malignancy is helpful in the selection for a candidate gene on one of the partner chromosomes. Many genes have been identified that are rearranged with several partner genes, and the partners may correlate with a specific clinical phenotype. “Promiscuous” genes that rearrange with multiple partners are listed in Table 1 and three examples are described in detail below.
Table
1. Promiscuous
Genes
Involved
in Hematopietic
Malignancy
Gene
Chromosomal Location
NUP 98 TEL AMLl
llp15 12~13 21q22
MLL TCRAID TCRB IGH IGK IGL FGFRI E2A BCL6 RARA PDGFRB ALK
llq23
14qll 7q35 14q32 2p12 22qll Sqll
19p13 6q27 17q21 5q33 5q35
Hematologic Subtype
V V AML-M2 AML-M4 B-ALL V T-ALL + T-ALL + B-ALL + B-ALL + B-ALL + V B-ALL B-ML AML-MB CMML ML
ML ML ML ML ML
Abbreviations: V, variable; AML, acute myeloid leukemia; ALL, acute lymphoblastic leukemia; ML, malignant lymphoma; CMML, chronic myelomonocytic leukemia.
CBF The CBF &ore Binding Factor), alsonamed PEBP2 (rolyoma Enhancer Binding rrotein 2) is a heterodimerit transcription factor composedof two subunits, CBFa and CBFP, which regulatestranscription of various genes,particularly in hematopoiesis.CBFencoding genes have been characterized simultaneously in studiesof viral transcriptional regulation and chromosomaltranslocations.50 The CBFP coding gene is fused to SMMHC (Smooth &scle &Iyosin Heavy chain) in inv( 16) of acute myelomonocytic leukemia with bone marrow eosinophilia (AMLM~Eo).~‘,~~ Of the three genescoding for the CBFa subunit, only Ah4Ll has been involved in human leukemogenesis,both myeloid and lymphoid, by chromosomaltranslocationsand point mutations.38 AMLI is involved in several myeloid malignancyassociatedtranslocations.The amino-terminal part of the protein, containing its runt DNA binding domain, is fused to the ET0 protein as a result of t(8;21)(q22;q22)34,36and to an ETO-related gene, MTGl6; asa resultof t(16;21)(q24;q22).12/lJYLI is alsorecombinedwith the EV71 locus in t(3;21) all of which should result in the expressionof a short form of AMLl that lacks its transactivation domain.35
Genesand Chromosomesin Leukmogenesis
t(12;21), w h’rc h IS ’ fr eq uent in and specific for childhood B-cell acute lymphoblastic lymphoma (B-ALL), leads to the expression of a TEL-AMLl fusion product that contains almost all of the AMLl protein. 15,43In all cases, fusion proteins contain the runt domain of AMLl and are predicted to exhibit dominant-negative activity for normal CW’transcription,ll while point-mutations in AMU alleles demonstrate loss of mnction. These mutations are found in sporadic and in some inherited hematopoietic abnormalities, and they indicate that haploinsufhciency of the AMLl gene is leukemogenic.49 Other translocations involving AMLI, mainly in therapy-related AML, await molecular identification.44
MLL/HRX The MLL/HRXgene, localized to band 1 lq23, can be considered as a human homolog to the Drosophih trithordx gene, a transcriptional regulator involved in control of homeotic genes such as Bitbomx and Antenmpediu in fly developmental biology. MLL is fused with more than 30 identified partner genes, including itself, in a large variety of malignancies.4,23,58Curiously, the translocation breakpoints are clustered in a restricted area (8.3 kb) ofMLL4,23,51; the 5 ’ region of MLL, which remains on chromosome 11 after the balanced reciprocal translocations, is crucial for the malignant process. In each translocation, the rearrangement leads to an in-frame fusion, and presumably to a functional fusion protein, suggesting that disruption of MLL is not itself sufficient for leukemogenesis. MLL usually contributes about the same amino-terminal amino acids to the fusion protein, but the partner genes cannot be characterized as belonging to a single structural or functional category. The normal functions of some of the partners are known, and they include a wide range of activities: a cytoplasmic protein involved in signal transduction (AFG); an elongation factor (ELL); and a transcription factor (AFGq2 UFKHRL) . The nudeotide sequences of the partner protein suggested that they might all provide an oligomerization domain, and this hypothesis has some support from recent experimental data. to It is also possible that the partner gene is partly responsible for the association observed between some translocations and the clinical features of the associated leukemic subtypes.
415
TEL/El-W The TEL/ETVGgene located on chromosome band 12~13 exemplifies a more complex situation.‘4a46 It codes for a member of the ETS family of transcription factors, which possess oligomerization and transcriptional repression properties.2130 TEL is rearranged in a variety of hematologic malignancies, and chromosome breakpoints are scattered all along the gene. Similar to MLL, the genes fused to TEL after chromosomal translocation are not homogeneous in terms of function. In contrast to MLL, the partner proteins usually do provide crucial functional domains. TEL has been hypothetized to be involved in leukemogenesis by at least four different mechanisms. The most frequently observed TEL-involving translocation is t(12;21), which fuses TEL to AiML1.15z43 The TEL moiety contributes a transcriptional repression domain and the fusion protein is thought to exert a dominant-negative effect on normal CBF activity.2o Several tyrosine kinase proteins, such as PDGFR& ABL, JAK2, ARG, and TRIWNTRK3, are also fused to TEL. In these cases, the TEL portion causes the fusion protein to oligomerize and thus exhibit constitutive tyrosine kinase activity. lL21 Whether all of these tyrosine kinases activate the same signal transduction pathway is not yet established. However, activation of the STAT5 factor could be an important common step in leukemogenic processes.26 Third, there are several examples predicting a TEL fusion protein that contains the ETS DNA binding domain of TEL, exemplified by the MNl-TEL fusion protein, which is thought to deregulate TEL target genes. Finally, molecular analysis of several TEL abnormalities does not predict the formation of the fusion protein. In these instances, the main role of these abnormalities would be to inactivate by some mechanism TEti normal functions. Such loss of activity may also follow the deletion of TEL associated with t(12;21).
Genes and Function More than the half of the genes found to be involved in chromosomal translocations were unknown at the time of their discovery, at least in man (Table 2). Subsequently, as their functions were uncovered, they fell into several categories, including: control of cellular differentiation (TALlKCL, MLL/HRX), proliferation and cell cycle regulation (MC CCNDl), pro-
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Table 2. Primary Gene ABL E2A FGFRl MLL MYC NUP98 PDGFRB RARA TAN CBF ALK BCL2 BCL6 BCR TALlILM02 TCLl/MTCPl TEL P16, PWRF
Genes
Located
at Chromosome
Localization
9q34 19p13 8~11-12 llq23 8q24 llp15 5q33 17q21 9q34 21q22 and 16q22 5q35 18q21 3q27 22qll lp32/11p13 14q32lXq28 12~13 9p21
grammed cell death (&X2), and signal transduction pathway members (ABL, ALK) . There is great functional diversiT of the genes involved in hematopoietic37 and leukemogenic processes. Nevertheless some putative unifying mechanisms have now been uncovered. The first example is the recently demonstrated TALI-IA402 connection.3,40,47 Both gene products are found in a large protein complex that also contains GATAl , E47, and LDB l/NLI, all of which are normally present during erythroid dif5erentiation.55 Another common mechanism involves control of chromatin structure. Balance between acetylase and deacetylase activities is important for transcriptional regulation during cell growth and differentiation. Many of the oncogenic fusion proteins have been shown to interact with components of acetylase/ deacetylase complexes. For example, the two fusion proteins, PML-RARA and PLZF-RARA, resulting from t(15;17) and t(11;17) of acute promyelocytic leukemia (APL) and of acute myeloid leukemia (AML) with resemblance to APL,5”,28 are in a protein complex that includes histone deacetylase 1 (HDACl) and the corepressors mSin3A and NcoRISMRT. At pharmacologic doses, retinoic acid (RA) may dissociate the complex, including PML-RARA, and thus mediate transactivation of RARA target genes, while PLZF-RARA is insensitive to W17~27 The difference in the response of the two fusion proteins to RA explains the efficacy of RA in the treatment of APL
Abnormality Function
Signal transducer Transcription factor Growth factor receptor Transcription factor Transcription factor Nucleopore protein Growth factor receptor Nuclear receptor Signal transducer Transcription factor Signal transducer Antiapoptotic factor Transcription factor Signal transducer Transcription factor Unknown Transcription factor Cell cycle control
Breakpoints Status
at Discovery
Known Known Known Known (Drosophila) Known Known Known Known Known (Drosophila) Same time Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown
and its lack of therapeutic activity in PLZF-RARAassociated leukemia. ‘* One practical consequence of these findings is the proposal to use inhibitors of deacetylases (butyrate, trischostatin A, trapoxin) in the treatment of APL.59 The fusion protein AMLI-ETO, resulting from t(8;21) of AML, also is able, like ETO, to interact with corepressor complexes (HDACl, N-coR/ SMRT).13 Moreover, whereas the fusion proteins repress transcription, a histone deacetylase inhibitor impairs this property. 31,56,57Similarly, the CBFBMYH 11 (SMMHC) fusion, resulting from inv( 16) of M4Eo, acts as a repressor in recruiting Sin3A.32 The ET0 protein also has recently been shown to act as a corepressor for the PLZF protein.33 Additionally, BCL6 andTEL can function as transcriptional repressors. Other proteins, such as NUP98, interact with the CBP/P300 proteins, which exhibit acetylase activity22 The genes encoding these CBP/P300 are themselves both involved in chromosomal translocations with MLL, and CBP also partners with MOzl Consistent with the involvement of these activities in leukemogenesis, mice heterozygous for an inactivated (knock-out) allele of CBP develop hematopoietic malignancies, and the cancer cells have lost the wild-type CBP allele 25 Furthermore, MLL itself has been demonstrated to interact with components of the SWI/SNF complex, underscoring the role of chromatin remodeling complex as a whole in the leukemic process.’
Genes und Chromosomes in Leukemogenesis
Conclusion The importance of traditional chromosomeanalysis and now molecular cytogeneticsin the location and identification of genes involved in hematopoietic malignanciescannot be underestimated.That role has beendecisivein the identification of new normal and fused genesand in the accurate definition of new hematologicsubtypesof malignancies.The contribution of the field in identifying new translocationsand gene rearrangementsprompt in turn the analysisof their functional consequencesand the identification of new targetsfor specifictherapy. Many novel geneshave been isolated, some of which have been shown to play a role in normal hematopoiesis.In rearrangementsoccurring in hematopoietic malignancies,the existenceof promiscuous genesemphasizes gene-geneand protein-protein interrelationsand helps to reveal the complex network of their functions. If the presentchallengeis the definition of the functional consequences of chromosomal abnormalitiesin preparation for future targetedtherapies,it remainsthe role of cytogeneticsto define new chromosomalabnormalities,both structural and numerical, and to understand the mechanismsof their genesis.Systematicapplication of 24-color FISH and of telomericprobesprovidesa promisingdirection for researchin this field. New techniquesusedin association with more classicalcytogenetic methods will contribute to fulfilling theseexpectations.
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trithorax by 1 lq23 translocations: Leukemogenic and transcriptional implications, in Rauscher FJ III, Vogt PK (eds): ChromosomaITi-anslocations and Oncogenic Factors. Berlin, Germany, Springer, 1997, pp 1-23 59. WarreII RPJr, He L-Z, Richon V, et al: Targeted transcription therapy of acute promyelocytic leukemia using an inhibitor of histone deacetylase. J NatI Cancer Inst 90:1621-1625, 1998