Tumor suppressor gene inactivation in myeloid malignancies

Best Practice & Research Clinical Haematology Vol. 21, No. 4, pp. 601–614, 2008 doi:10.1016/j.beha.2008.09.001 available online at http://www.sciencedirect.com

1 Tumor suppressor gene inactivation in myeloid malignancies Jasmine C. Wong

PhD

Postdoctoral Fellow Department of Pediatrics, University of California, San Francisco, 513 Parnassus Ave, HSE 302, San Francisco, CA 94143 USA

Michelle M. Le Beau

PhD

Professor Section of Hematology/Oncology and Director, the Cancer Research Center, University of Chicago, IL, USA

Kevin Shannon *

MD

Professor of Pediatrics Department of Pediatrics, University of California, San Francisco, 513 Parnassus Ave, HSE 302, San Francisco, CA 94143 USA Comprehensive Cancer Center, University of California, San Francisco, CA 94143 USA

Our molecular understanding of the how tumor suppressor gene (TSG) abnormalities contribute to myeloid malignancies is relatively limited. While the NF1 and TP53 TSGs follow the Knudson two-hit paradigm and undergo biallelic inactivation, there is increasing evidence that inactivation of a single allele of TSG such as RUNX1, PU.1 and RPS14 (haploinsufficiency) can also contribute to leukemogenesis. New technologies including high density single nucleotide polymorphism (SNP) arrays, RNA interference (RNAi) and chromosome engineering to develop mouse models with defined genetic rearrangements are emerging as potent tools for cloning and studying the function of TSGs. Notwithstanding these advances, the role of many chromosomal deletions that are commonly observed in myeloid malignancies remains uncertain, particularly the deletion of chromosomes 5, 7, 9 and 20. Since these deletions are often associated with resistance to current therapies, discovering the relevant TSGs and determining how they function in cell growth are high priorities. Key words: tumor suppressor genes; myeloid malignancies.

* Corresponding author. Tel.: þ1 415 476 7932; Fax: þ1 415 502 5127. E-mail address: [email protected] (K. Shannon). 1521-6926/$ - see front matter ª 2008 Elsevier Ltd. All rights reserved.

602 J. C. Wong et al

INTRODUCTION For over 40 years, recurring cytogenetic abnormalities have provided a starting point for identifying genes that are altered by translocations, inversions, or deletions in myeloid malignancies and other human cancers. The recent development of dense single nucleotide polymorphism (SNP) array platforms has markedly increased the resolution of genome-wide analysis and now provide a powerful new tool for uncovering subtle structural aberrations that correspond to pathogenic mutations. The application of this approach to pediatric acute lymphoblastic leukemia (ALL) unexpectedly revealed alterations in an interrelated group of genes that regulate B lineage development in w40% of cases.1 In myeloid leukemia, major advances have been made by cloning genes located at translocation breakpoints and by identifying point mutations in genes that regulate signaling pathways.2-7 Genetic and biochemical studies performed in primary bone marrow cells, in leukemia-derived cell lines, and in animal models have shown that mutations that perturb gene expression or alter protein function play a central role in leukemogenesis by activating cellular proto-oncogenes. Point mutations (eg, in NRAS, KRAS, and PTPN11), internal tandem duplications (eg, in FLT3), and chromosomal translocations (eg, MLL fusions and AML1-ETO) act as dominant genetic traits, such that altering a single allele is sufficient to perturb cell growth and differentiation. Genes that are mutated in myeloid malignancies encode proteins that play integral roles in normal cellular processes including growth factor binding and receptor activation, signal transduction, and transcription pathways.2-7 Whereas tumor suppressor gene (TSG) inactivation is a common mechanism that contributes to the development of solid tumors such as carcinomas of the colon, pancreas, and lung, the role of TSG alterations in hematologic cancers is less certain. The proteins encoded by TSGs normally restrain cell growth, and these genes are frequently inactivated during tumorigenesis. As a result, cancers with TSG alterations often demonstrate either loss of a whole chromosome or smaller cytogenetic deletions of the DNA segment that encodes the relevant gene. Recent studies have also implicated acquired uniparental disomy as a mechanism of TSG inactivation in some myeloid malignancies.8,9 Molecular investigation has shown that inactivation of TSGs is commonly associated with cytogenetically invisible (submicroscopic) deletions that are detected by demonstrating loss of constitutional heterozygosity (LOH) with polymorphic markers located within or near tumor suppressor loci. Germline mutations of TSGs account for most known heritable cancer predispositions because inactivation of one allele is usually compatible with normal development. Most of the TSGs discovered to date follow the Knudson paradigm. That is, these genes are recessive at the cellular level and both alleles are deleted or mutated in cancer (Figure 1A). However, there is increasing evidence from studies of human leukemia specimens and in mutant strains of mice that inactivation of a single TSG allele (haploinsufficiency) can also contribute to tumorigenesis (Figure 1B).10-13 Here we concisely review known TSG alterations in myeloid malignancies, the functions of the encoded proteins, and discuss new approaches for uncovering putative TSGs from deleted intervals that have been identified in myeloid malignancies. TSG INACTIVATION IN MYELOID MALIGNANCIES Neurofibromatosis type 1 (NF1). NF1 is a common dominantly-inherited muIti-system disease characterized by pigmented lesions of the skin and eye and a high incidence

Tumor suppressor gene inactivation in myeloid malignancies 603

Figure 1. Mechanisms of TSG inactivation. (A) Knudson paradigm. Mutation or epigenetic silencing on one allele followed by a ‘‘second hit’’ such as loss of chromosomal segment (shown here) will lead to biallelic inactivation of gene function. (B) Haploinsufficiency. In this example, both alleles are initially wild-type. Loss of a chromosomal segment harboring a haploinsufficient TSG leads to reduced gene expression and is sufficient to contribute to tumorigenesis.

of learning disabilities. Persons with NF1 are at increased risk of specific benign and malignant neoplasms that primarily arise in cells derived from the embryonic neural crest.14,15 In addition, children (but not adults) with NF1 are strongly predisposed to myeloid malignancies, particularly juvenile myelomonocytic leukemia (JMML).16,17 JMML is a relentless cancer of young children characterized by over-production of myeloid lineage cells that infiltrate hematopoietic and non-hematopoietic tissues.18,19 JMML cells selectively form abnormal numbers of colony forming unit granulocytemacrophage (CFU-GM) colonies in methylcellulose cultures containing low concentrations of the growth factor granulocyte-macrophage colony-stimulating factor (GMCSF).20 Mutations in the NF1 gene, which encodes a GTPase activating protein called neurofibromin that negatively regulates Ras signaling, cause NF1. Genetic analysis of JMML bone marrows revealed loss of the normal parental allele in familial cases, and homozygous inactivation in many patients.21,22 Interestingly, acquired uniparental disomy for the mutant NF1 allele and for an extensive region of flanking DNA appears to be the most common mechanism underlying NF1 inactivation in myeloid malignancies.23 Biochemical investigation of primary leukemia cells from children with NF1 showed a reduction of neurofibromin-specific GAP activity, elevated levels of RasGTP, and activation of the Raf1 effector ERK.24 Together these data identified NF1 as a myeloid TSG that functions by negatively regulating Ras signaling. Consistent with these human data, inactivating Nf1 in murine hematopoietic cells results in a myeloproliferative disorder that shares clinical and biologic features with JMML and chronic myelomonocytic leukemia (CMML). Strains of Nf1 mutant mice have been harnessed to investigate the role of deregulated growth factor signaling in the pathogenesis of MPD, to test targeted therapeutics, and to investigate the interaction of germline Nf1 mutations with mutagenic chemotherapeutic agents and radiation in leukemogenesis.25-28 Whereas somatic NF1 mutations are reported infrequently in hematologic cancers, the gene is very large and the true incidence is uncertain.29 Furthermore, although NF1 clearly functions as a ‘‘classic’’ TSG that undergoes biallelic inactivation in myeloid malignancies, studies of primary human leukemia cells and

604 J. C. Wong et al

mouse models also suggest a haploinsufficient mechanism of action in some contexts.15,21,22,26-28,30 TP53. TP53 is the most common target of somatic mutation in human cancer. The p53 protein has transcriptional activity and has been called the ‘‘guardian of the genome’’ because it coordinates responses to genotoxic stress through a complex network of effectors.31 Loss of p53 function is thought to play a central role in the widespread genomic instability found in many advanced epithelial cancers. Interestingly, with the exception of therapy-related myelodysplastic syndrome and acute myeloid leukemia (t-MDS and t-AML), myeloid malignancies rarely display complex cytogenetic aberrations and it is therefore perhaps not surprising that TP53 mutations are relatively uncommon in these disorders. TP53 mutations are associated with cytogenetic abnormalities of the short arm of chromosome 17, and with a poor prognosis in a subset of patients with de novo AML.32-35 TP53 mutations also frequently coexist with monosomy 5 and del(5q) in patients with alkylator-induced t-MDS/t-AML.36-39 In myeloid diseases, abnormalities of chromosome 5 are associated with the highest level of karyotypic instability (patients with a 5q abnormality have an average of 8.0 abnormalities, whereas patients with an abnormality of 7q have an average of 3.0 abnormalities), which may be linked to the presence of TP53 abnormalities in the former group (Le Beau and Tennant, unpublished data). Finally, somatic TP53 mutations have been reported as a secondary genetic change in the blast crisis phase of chronic myelogenous leukemia (CML).40 An interesting study in mice also showed that Tp53 mutant bone marrow cells that are engineered to express BCR-ABL are relatively resistant to imatinib in vitro and in vivo, thereby implicating p53 function as important for the therapeutic response to this molecularly targeted agent.41 In summary, although studies to date indicate that TP53 mutations are relatively uncommon in myeloid malignancies, they appear to be associated with a high risk of treatment failure. The role of p53-regulated pathways in modulating the responses to conventional and targeted therapeutics in myeloid malignancies and in the development of resistance are interesting areas for further study. HAPLOINSUFFICIENT MYELOID TSGS RUNX1/AML1/CBFA2. The AML1 gene (also known as RUNX1 or CBFA2) plays a number of distinct roles in leukemogenesis. AML1 encodes a component of the core binding factor (CBP) complex that is essential for normal hematopoietic development. The t(8; 21) results in the production of an AML1-ETO fusion protein that enhances selfrenewal and can initiate myeloid leukemogenesis.42 AML1 is also targeted by translocations in some cases of t-AML that follow treatment with epipodophyllotoxins, and the TEL-AML1 fusion is the most common molecular abnormality in pediatric ALL.43-45 Interestingly, germline and somatic AML1 mutations that inactivate the protein are also associated with myeloid malignancies. Germline AML1 mutations cause familial platelet disorder (FPD), which is also associated with a predisposition to acute myeloid leukemia (AML).46 Affected individuals have thrombocytopenia, functional platelet abnormalities, and a prolonged bleeding time. Importantly, the leukemias associated with FPD/AML consistently retain one normal AML1 allele. Biallelic and heterozygous somatic AML1 mutations also occur in both de novo AML and in t-MDS/t-AML.47-50 In the latter group of patients, these mutations are associated with previous treatment with alkylating agents, with chromosome 7 loss or deletion, and with a poor prognosis.50 Overall RUNX1/AML1/CBFA2 is a particularly intriguing leukemia gene in that

Tumor suppressor gene inactivation in myeloid malignancies 605

dominantly acting chromosomal translocations, haploinsufficiency and biallelic inactivation are found in distinct subsets of myeloid malignancies. The respective biochemical consequences of each class of mutation are under investigation. Sfpi1/PU.1. Studies in genetically-engineered mice have uncovered putative TSGs that either undergo homozygous inactivation or contribute to leukemogenesis due to reduced levels of protein expression. The Sfpi1 gene, which encodes the PU.1 transcription factor, is an elegant example of the latter mechanism. PU.1 (Spi-1) is a hematopoietic-specific ETS family member whose expression is regulated by a highly conserved upstream regulatory element (URE) located 14 kb upstream of the transcriptional start site.51 PU.1 function has been extensively characterized in vivo using mouse models that express variable levels of Sfpi1 in hematopoietic cells, and dysregulation of PU.1 leads to loss of lineage development and/or leukemia.52,53 Homozygous Sfpi1 mutant mice demonstrate a block in lymphoid and myeloid differentiation and die late in gestation or shortly after birth, but do not develop leukemia.54,55 By contrast, Sfpi1þ/ mice show normal peripheral blood counts and do not spontaneously develop hematologic cancers. However, the penetrance of acute promyelocytic leukemia induced by expressing a PML-RARa transgene in mice is increased by heterozygous Sfpi1 inactivation.56 Although heterozygous Sfpi1 mutant mice do not spontaneously develop leukemia, a strain of mice harboring a hypomorphic Sfpi1 allele that reduces PU.1 expression in the bone marrow by w80% mice causes AML at high frequency after a short preleukemic phase.57 Studies in a mouse model of myeloid leukemia induced by g-irradiation provided additional evidence implicating Sfp1 as a myeloid TSG. In this system, murine leukemias exhibit loss of the region of chromosome 2 harboring the Sfpi1 gene, as well as a point mutation in the ETS domain of the retained allele that impairs DNA binding.58 It is unknown if the PU.1 peptides encoded by these mutant Sfp1 alleles are partially functional, as might be expected from the knock-out studies described above. The issue of whether Sfp1 can function as a ‘‘classical’’ TSG that undergoes biallelic inactivation under some circumstances is also raised by a study showing that AML develops when the DNA binding domain of PU.1 is conditionally deleted in adult mice.59 While this study and data from the g-irradiation model raise the possibility that somatic loss of Sfp1 function might contribute to leukemogenesis in adult hematopoietic cells, the studies conducted to date do not exclude the possibility that these mutant alleles retain some biologic activity. While it is clear that reduced Sfp1/PU.1 activity predisposes to leukemia in mice, the role of PU.1 mutations in human leukemia is less certain. Heterozygous, but not homozygous, PU.1 mutations were reported in human AML, but subsequent studies did not confirm this finding.60-64 The reasons for these discordant results are unknown. PU.1 is expressed at low levels in most cases of human AML, and PU.1 expression and/or function is down-regulated by several important oncogenic fusion proteins including AML1-ETO, PML-RARa and FLT3/ITD.65-68 These data raise the possibility that PU.1 might contribute to human myeloid leukemogenesis by mechanisms that do not require somatic mutation. The compelling evidence from mouse models that Sfp1/PU.1 functions as a myeloid TSG provides a strong rationale for continuing to evaluate the role of this gene in human leukemogenesis. RPS14 Haploinsufficiency in 5q– Syndrome. The 5q syndrome is a specific subtype of MDS that is associated with somatic deletions affecting chromosome bands 5q31– 33, anemia due to ineffective erythropoiesis, a protracted course, and clinical responsiveness to lenalidomide. Based on cytogenetic and molecular analyses of multiple independent patient specimens, Boultwood and colleagues identified a commonly

606 J. C. Wong et al

deleted segment (CDS) of 1.5 kb within 5q32 as harboring a putative myeloid TSG.69 However, systematic analysis of candidate TSGs from this interval did not reveal ‘‘second hit’’ mutations in 5q syndrome patient specimens, implicating haploinsufficiency of one or more genes within the CDS. Ebert et al. recently used RNA interference to implicate RPS14 as a haploinsufficient TSG in the 5q syndrome.70 This approach involved generating short hairpin RNA sequences that were complementary to each candidate gene, expressing each molecule in normal CD34þ bone marrow cells, and inducing these cells to differentiate into red blood cell precursors in culture. Reducing RPS14 levels resulted in impaired differentiation that was reminiscent of the 5q syndrome. RPS14 is crucial for efficient formation of the 40S ribosomal subunit complex, and subsequent studies of cells from 5q syndrome patients showed that ribosome production is impaired. However, loss of RPS14 does not account for all phenotypic features associated with the 5q syndrome, which include thrombocytosis, megakaryocytic dysplasia, neutropenia, and clonal dominance. This suggests that one or more additional genes on 5q plays a role in leukemic transformation. It is of interest that mutations in the ribosomal genes RPS19 and RPS25 cause Diamond Blackfan anemia, which is also associated with erythropoietic defects.71 These provocative new data raise questions about the general role of impaired ribosome biogenesis and protein translation in the pathogenesis of other forms of MDS that will be addressed over the next few years. They also establish RNA interference as a powerful new strategy for evaluating candidate TSGs that might function by haploinsufficiency. DNA SEGMENTS IMPLICATED AS HARBORING MYELOID TSGs Cytogenetic and molecular studies have defined intervals on chromosome bands 7q22, 5q31, 20q12, and 9q that are deleted in many myeloid malignancies. It has been hypothesized that each CDS contains a putative myeloid TSG that contributes to leukemogenesis by biallelic inactivation, haploinsufficiency, or both mechanisms. Despite intensive investigation, the relevant myeloid TSG (or TSGs) within these intervals remain uncertain. Here we summarize the current status of each CDS. Chromosome Band 7q22. Monosomy 7/del(7q) are among the most common cytogenetic alterations found in MDS and AML. Monosomy 7/del(7q) occurs in three general contexts: (1) de novo MDS and AML; (2) leukemias associated with known acquired risk factors; and (3) leukemias arising in patients with a constitutional predisposition.72 Adults who develop de novo AML with monosomy 7 demonstrate adverse clinical features such as advanced age and antecedent MDS. Importantly, the outcome for these patients is dismal and has not improved over the past two decades. To facilitate the identification of candidate myeloid TSGs from the long arm of chromosome 7, Le Beau et al performed a comprehensive analysis of the breakpoints and mapped the extent of deletions in 81 patients with MDS or AML who had a del(7q) detected by cytogenetic analysis.73 These studies demonstrated that 7q deletions are interstitial, and implicated 2 distinct segments as critical regions. In 65 patients, the proximal breakpoints were in q11.2-q22, the distal breakpoints were in q22-q36, and the smallest overlapping deleted segment was within q22. In 16 other cases, interstitial deletions of a more distal segment were detected with a common overlapping region of q32-q33.73 FISH analysis of metaphase cells from 15 of these patients whose leukemia cells had a del(7q) with proximal or distal breakpoints in band q22 refined the commonly deleted region of 7q22 to an interval between YACs yWSS1668 (proximal) and yWSS3710 (distal).73 Subsequent completion of the human genome sequence

Tumor suppressor gene inactivation in myeloid malignancies 607

delineated the size of this interval as 2.52 Mb. Molecular analysis has not uncovered either biallelic inactivation or epigenetic silencing of candidate genes located within this CDS in human leukemia specimens.74-76 Based on these data, it appears likely that any myeloid TSG located within this 2.5 Mb CDS functions by haploinsufficiency. However, cytogenetic, fluorescence in situ hybridization (FISH), and LOH studies of monosomy 7 and del(7q) cases performed by many different research groups paint a complex picture of 7q deletions in myeloid malignancies with some data implicating DNA segments that are either more centromeric or more telomeric as potentially harboring myeloid TSGs.77-81 Uncovering the gene (or genes) on 7q that contribute to myeloid leukemogenesis and understanding how these TSGs regulate hematopoietic growth are important research questions due to the high prevalence of monosomy 7 and del(7q) deletions in myeloid malignancies and the association of these cytogenetic abnormalities with adverse clinical features. Chromosome Band 5q31. Loss of a whole chromosome 5, or a deletion of the long arm of this chromosome, del(5q), are observed in w10% of patients with de novo MDS or AML, and occur in w40% of t-MDS/t-AML. Abnormalities of chromosomes 5 and/or 7 are a hallmark of t-MDS/t-AML arising subsequent to alkylating agent therapy. Le Beau and colleagues used a combination of cytogenetic analysis and FISH to analyze myeloid malignancies with a del(5q) and defined a CDS spanning 970 kb of genomic DNA that is flanked by the genetic markers D5S479 and D5S500.82,83 This interval contains 20 candidate genes all of which were analyzed for ‘‘second hit’’ mutations in leukemia samples. No pathologic mutations were detected, a situation that is reminiscent of the 7q22 CDS. EGR1, which encodes a transcriptional regulator that is essential for the differentiation of monocytic cells, is a particularly interesting candidate 5q31 gene. To address if Egr1 inactivation might contribute to leukemogenesis by haploinsufficiency, Joslin et al exposed wild-type, Egr1þ/, and Egr1/ mice to the alkylating agent ethylnitrosourea (ENU).13 Interestingly, heterozygous and homozygous Egr1 mutant mice were predisposed to the development of lymphoid leukemia and to a myeloproliferative disorder (MPD). Importantly, the incidence of hematologic malignancy was similar in Egr1þ/ and Egr1/ mice, and mutational analysis of leukemic cells from Egr1þ/ mice did not reveal mutations in the wild-type allele. Together, these data provide strong evidence that Egr1 is a haploinsufficient TSG in mice. The relevance of these data to human myeloid malignancies and the role of other genes within the 5q31 CDS in leukemogenesis are areas of active investigation. The CTNNA1 gene, which encodes a catenin, is also located on 5q31, though distal to the 1 Mb CDS that was defined by cytogenetic and FISH analysis. Whereas CTNNA1 is not mutated in leukemias with a del(5q), Liu and colleagues found that this gene is expressed at lower levels in AML or MDS with a del(5q), than in other AMLs or in normal hematopoietic stem cells.84 These studies raise the possibility that loss of expression of CTNNA1 in HSCs may contribute to the pathogenesis of some cases of AML with a del(5q). Chromosome Band 20q12. A deletion of the long arm of chromosome 20, or del(20q), is a recurring cytogenetic abnormality that is found in w10% of patients with polycythemia vera as well as in other MPDs, w4% of patients with MDS, and in w12% of patients with AML.85-89 Although del(20q) is often observed as the sole cytogenetic abnormality, it is also associated with a complex karyotype, particularly 5del(5q) or 7/del(7q) in AML and MDS.86,90 Cytogenetic analysis of del(20q) revealed that the deletions are variable in size, with many resulting in the loss of most of 20q.91 Studies performed by various groups provide supportive evidence for a CDS

608 J. C. Wong et al

that extends from 20q11.2 to 20q12.91-94 In a later study that included 113 patients with myeloid malignancies associated with a del(20q), Bench and colleagues refined the region to two different but overlapping CDS of 2.7 Mb for MPD, and 2.6 Mb for MDS/AML.95 Wang and colleagues used FISH to examine cells from 34 patients with myeloid disorders with structural rearrangements of 20q, and found that breakpoints can occur either proximal, distal or within the CDS, suggesting that there may be multiple myeloid tumor suppressor genes on 20q.96 However, in two patients, they identified molecular deletions of DNA sequences within the established CDS and refined the region to w250-kb within band 20q12. These studies allowed the identification of a set of candidate myeloid TSGs. However, homozygous inactivation of any of these genes has not been reported to date. Chromosome Band 9q21. Deletion of a portion of the long arm of chromosome 9, or del(9q), is a recurring abnormality found in w2% of AML.97 Although it can occur as a sole abnormality, it is frequently associated with other cytogenetic abnormalities, particularly t(8;21).98-101 Therefore it is likely that the critical gene products affected by del(9q) cooperates with the AML1/ETO fusion oncoprotein. Sweetser and colleagues narrowed the 9q CDS to a w2.4 Mb interval of 9q21.32-q21.33 by studying 43 del(9q) AML samples with high-density microsatellite markers. However, no homozygous loss was detected, and sequencing analysis of the 10 known genes and 3 putative genes within or immediately adjacent to the interval failed to identify any pathogenic mutations in the retained allele.102 Expression of 7 of these genes were lower in del(9q) AML samples than in CD34-purified progenitors from normal individuals, suggesting that haploinsufficiency or reduced expression of one or more of these critical genes may contribute to leukemogenesis.102 Dayyani and colleagues developed a set of shRNAs directed against the genes in the 9q CDS, and shRNAs to TLE1 (transducin like enhancer of split)-1 and TLE4 were capable of rescuing AML1ETO expressing U937T-A/E cells from AML1-ETO induced cell-cycle arrest and apoptosis, and increased the rate of cell division in the Kasumi-1 cell line, which expresses AML1-ETO.103 Forced expression of either TLE1 or TLE4 caused apoptosis and cell death. Furthermore, knockdown of Gro3, a TLE homolog in zebrafish, cooperated with AML1-ETO to cause an accumulation of noncirculating hematopoietic blast cells, implicating TLE1 and TLE4 as potential haploinsufficient TSGs in myeloid malignancies. CONCLUSIONS AND FUTURE DIRECTIONS While some progress has been made in uncovering TSGs that contribute to myeloid leukemogenesis and in elucidating the mechanisms of action of some of the mutated proteins, there are substantial gaps in our current knowledge. Of particular importance is the need to characterize the relevant genes within commonly deleted intervals on chromosomes 5, 7, 9, and 20, particularly as some of these genetic lesions are very common and are associated with resistance to current therapies. Intensive investigation of these intervals by a number of labs supports haploinsufficiency as a likely mechanism of action in at least some cases. The relatively large size of many of these deleted intervals and the molecular and clinical heterogeneity that is observed in patients with the same chromosomal deletion represent formidable obstacles for TSG discovery. However, recent technical advances provide powerful new tools. High density SNP arrays may prove useful for uncovering novel submicroscopic deletions and for refining the boundaries of known CDSs. The rapid progress of high throughput sequencing

Tumor suppressor gene inactivation in myeloid malignancies 609

now enables investigators to rapidly sequence many candidate genes from multiple specimens. As the costs of DNA sequencing decreases, it will soon be feasible to consider sequencing all of the genes located on 5q or 7q in a well-characterized group of myeloid malignancies. The experience obtained to date with these technologies underscore that it is imperative to sequence matched normal germline DNA in parallel with the leukemia specimen to distinguish novel mutations that are likely to be pathogenic. The elegant studies of Ebert and coworkers implicating RPS14 as a haploinsufficient TSG in the 5q syndrome established RNAi as a potent tool for TSG discovery.70 A critical aspect of RNAi screens is the availability of a robust functional readout that correlates with biologic activity. The screen that identified RPS14 assayed candidate knock-down constructs for the ability to increase myeloid proliferation and impair erythroid growth and differentiation, which are highly characteristic of the 5q syndrome. However, many of the other segmental deletions found in myeloid malignancies are not associated with a consistent cellular phenotype, which poses a challenge for devising relevant read-outs. On the other hand, RNAi-based screens have the great advantage of identifying a specific gene from a CDS that is likely to function as a haploinsufficient TSG. As described above, mouse models are another potent strategy for investigating candidate myeloid TSGs that either function through the ‘‘classic’’ Knudson mechanism or by haploinsufficiency. The chromosome engineering technique pioneered by Allan Bradley now enables investigators to delete large DNA segments in the mouse germline.104-106 This strategy involves three steps, and makes use of a novel selection system. The essence of this is to embed two complementary, but nonfunctional, fragments of a hypoxanthine phosphoribosyl transferase (HPRT) minigene cassette within the loxP-containing targeting vectors. Cre recombination between the chromosomal loxP sites then joins the two HPRT fragments together and creates a functional HPRT minigene. If the gene targeting is performed in HPRT -deficient embryonic stem (ES) cells, then hypoxanthine/aminopterin/thymidine (HAT) medium can be used to select for the desired deletion events. Sib-selection for the loss of other selectable markers, Southern analysis, FISH, and karyotype analysis allow for a definitive assessment of chromosomal structure before blastocyst injection of the mutant ES cells. This strategy effectively overcomes concerns about the efficiency of Cre recombination over large distances and permits undesirable recombination products (eg, inversions) to be excluded. This general approach has a number of advantages. First, it provides a viable, function-based alternative to traditional positional cloning strategies. Second, it represents an unbiased approach for the identification of TSGs within the commonly deleted segments that should be advantageous if any of the segments contain more than one TSG whose combined loss is required for the tumor phenotype. Third, it offers the potential to identify TSGs that deregulate cell growth through haploinsufficiency. Finally, chromosome engineering generates reagents and, ultimately, murine models of human disease that can be used immediately to test new therapies and to study the biology of the TSGs. Elegant studies by Bagchi and colleagues that identified CHD5 as the elusive 1p36.3 TSG underscore the power of this strategy for cloning human cancer genes.107 Given these complementary new experimental techniques, it seems likely that the next few years will witness substantial progress in our understanding of the role of TSGs in the initiation and progression of myeloid malignancies. Elucidating the relevant disease genes and interrogating how they function in hematopoietic growth control are essential steps toward achieving the long-term goal of developing more effective and less toxic therapeutic strategies for these cancers.

610 J. C. Wong et al

ACKNOWLEDGEMENTS The work in our laboratories on myeloid TSGs is supported by NIH grants P01CA40046, U01CA84221, R37CA72614, by project X81XWH-05-1-0265 from the U.S. Army NF Research Program, and by a Leukemia and Lymphoma Society Research Fellowship. REFERENCES *1. Mullighan CG, Goorha S, Radtke I et al. Genome-wide analysis of genetic alterations in acute lymphoblastic leukaemia. Nature 2007; 446: 758–764. 2. Rowley JD. The role of chromosome translocations in leukemogenesis. Seminars in Hematology 1999; 36: 59–72. 3. Look A. Oncogenic transcription factors in the human acute leukemias. Science 1997; 278: 1059–1064. 4. Shannon KM. The Ras signaling pathway and the molecular basis of myeloid leukemogenesis. Current Opinion in Hematology 1995; 3: 305–308. 5. Kelly L, Clark J & Gilliland DG. Comprehensive genotypic analysis of leukemia: clinical and therapeutic implications. Current Opinion in Oncology 2002; 14: 10–18. 6. Gilliland DG & Tallman MS. Focus on acute leukemias. Cancer Cell 2002; 1: 417–420. 7. Van Etten RA & Shannon KM. Focus on myeloproliferative diseases and myelodysplastic syndromes. Cancer Cell 2004; 6: 547–552. 8. Kralovics R, Guan Y & Prchal JT. Acquired uniparental disomy of chromosome 9p is a frequent stem cell defect in polycythemia vera. Experimental Hematology 2002; 30: 229–236. 9. Fitzgibbon J, Smith LL, Raghavan M et al. Association between acquired uniparental disomy and homozygous gene mutation in acute myeloid leukemias. Cancer Research 2005; 65: 9152–9154. 10. Cook WD & McCaw BJ. Accommodating haploinsufficient tumor suppressor genes in Knudson’s model. Oncogene 2000; 19: 3434–3438. 11. Quon KC & Berns A. Haplo-insufficiency? Let me count the ways. Genes & Development 2001; 15: 2917–2921. 12. Vives V, Su J, Zhong S et al. ASPP2 is a haploinsufficient tumor suppressor that cooperates with p53 to suppress tumor growth. Genes & Development 2006; 20: 1262–1267. *13. Joslin JM, Fernald AA, Tennant TR et al. Haploinsufficiency of EGR1, a candidate gene in the del(5q), leads to the development of myeloid disorders. Blood 2007; 110: 719–726. 14. Cichowski K & Jacks T. NF1 tumor suppressor gene function: narrowing the GAP. Cell 2001; 104: 593–604. 15. Side L, Emanuel P, Taylor B et al. Mutations of the NF1 gene in leukemias from children without evidence of neurofibromatosis, type 1. Blood 1998; 92: 267–273. 16. Bader JL & Miller RW. Neurofibromatosis and childhood leukemia. The Journal of Pediatrics 1978; 92: 925–929. 17. Stiller CA, Chessells JM & Fitchett M. Neurofibromatosis and childhood leukemia/lymphoma: A population-based UKCCSG study. British Journal of Cancer 1994; 70: 969–972. 18. Emanuel PD, Shannon KM & Castleberry RP. Juvenile myelomonocytic leukemia: molecular understanding and prospects for therapy. Molecular Medicine Today 1996; 2: 468475. 19. Arico M, Biondi A & Pui C-H. Juvenile myelomonocytic leukemia. Blood 1997; 90: 479–488. 20. Emanuel PD, Bates LJ, Castleberry RP et al. Seletive hypersensitivity to granulocyte-macrophage colony stimulating factor by juvenile chronic myeloid leukemia hematopoietic progenitors. Blood 1991; 77: 925–929. 21. Shannon KM, O’Connell P, Martin GA et al. Loss of the normal NF1 allele from the bone marrow of children with type 1 neurofibromatosis and malignant myeloid disorders. The New England Journal of Medicine 1994; 330: 597–601. 22. Side L, Taylor B, Cayouette M et al. Homozygous inactivation of the NF1 gene in bone marrow cells from children with neurofibromatosis type 1 and malignant myeloid disorders. The New England Journal of Medicine 1997; 336: 1713–1720.

Tumor suppressor gene inactivation in myeloid malignancies 611 *23. Stephens K, Weaver M, Leppig KA et al. Interstitial uniparental isodisomy at clustered breakpoint intervals is a frequent mechanism of NF1 inactivation in myeloid malignancies. Blood 2006; 108: 1684–1689. 24. Bollag G, Clapp DW, Shih S et al. Loss of NF1 results in activation of the Ras signaling pathway and leads to aberrant growth in murine and human hematopoietic cells. Nature Genetics 1996; 12: 144–148. 25. Birnbaum RA, O’Marcaigh A, Wardak Z et al. Nf1 and Gmcsf interact in myeloid leukemogenesis. Molecular Cell 2000; 5: 189–195. 26. Chao RC, Pyzel U, Fridlyand J et al. Therapy-induced malignant neoplasms in Nf1 mutant mice. Cancer Cell 2005; 8: 337–348. 27. Mahgoub N, Taylor B, Le Beau M et al. Myeloid malignancies induced by alkylating agents in Nf1 mice. Blood 1999; 93: 3617–3623. 28. Mahgoub N, Taylor BR, Gratiot M et al. In vitro and In vivo effects of a farnesyltransferase inhibitor on Nf1- deficient hematopoietic cells. Blood 1999; 94: 2469–2476. 29. Balgobind BV, Van Vlierberghe P, van den Ouweland AM et al. Leukemia-associated NF1 inactivation in patients with pediatric T-ALL and AML lacking evidence for neurofibromatosis. Blood 2008; 111: 4322–4328. 30. Ingram DA, Yang FC, Travers JB et al. Genetic and biochemical evidence that haploinsufficiency of the Nf1 tumor suppressor gene modulates melanocyte and mast cell fates in vivo. The Journal of Experimental Medicine 2000; 191: 181–188. 31. Riley T, Sontag E, Chen P et al. Transcriptional control of human p53-regulated genes. Nature Reviews. Molecular Cell Biology 2008; 9: 402–412. 32. Fenaux P, Jonveaux P, Quiquandon I et al. P53 gene mutations in acute myeloid leukemia with 17p monosomy. Blood 1991; 78: 1652–1657. 33. Kurosawa M, Okabe M, Kunieda Y et al. Analysis of the p53 gene mutations in acute myelogenous leukemia: the p53 gene mutations associated with a deletion of chromosome 17. Annals of Hematology 1995; 71: 83–87. 34. Nahi H, Lehmann S, Bengtzen S et al. Chromosomal aberrations in 17p predict in vitro drug resistance and short overall survival in acute myeloid leukemia. Leukemia & Lymphoma 2008; 49: 508–516. 35. Stirewalt DL, Kopecky KJ, Meshinchi S et al. FLT3, RAS, and TP53 mutations in elderly patients with acute myeloid leukemia. Blood 2001; 97: 3589–3595. 36. Horiike S, Misawa S, Kaneko H et al. Distinct genetic involvement of the TP53 gene in therapy-related leukemia and myelodysplasia with chromosomal losses of Nos 5 and/or 7 and its possible relationship to replication error phenotype. Leukemia 1999; 13: 1235–1242. 37. Side LE, Curtiss NP, Teel K et al. RAS, FLT3, and TP53 mutations in therapy-related myeloid malignancies with abnormalities of chromosomes 5 and 7. Genes, Chromosomes & Cancer 2004; 39: 217–223. 38. Lothe RA, Smith-Sorensen B, Hektoen M et al. Biallelic inactivation of TP53 rarely contributes to the development of malignant peripheral nerve sheath tumors. Genes, Chromosomes & Cancer 2001; 30: 202–206. 39. Christiansen DH, Andersen MK & Pedersen-Bjergaard J. Mutations with loss of heterozygosity of p53 are common in therapy- related myelodysplasia and acute myeloid leukemia after exposure to alkylating agents and significantly associated with deletion or loss of 5q, a complex karyotype, and a poor prognosis. Journal of Clinical Oncology 2001; 19: 1405–1413. 40. Skorski T, Nieborowska-Skorska M, Wlodarski P et al. Blastic transformation of p53-deficient bone marrow cells by p210bcr/abl tyrosine kinase. Proceedings of the National Academy of Sciences of the United States of America 1996; 93: 13137–13142. *41. Wendel HG, de Stanchina E, Cepero E et al. Loss of p53 impedes the antileukemic response to BCR-ABL inhibition. Proceedings of the National Academy of Sciences of the United States of America 2006; 103: 7444–7449. 42. Higuchi M, O’Brien D, Kumaravelu P et al. Expression of a conditional AML1-ETO oncogene bypasses embryonic lethality and establishes a murine model of human t(8;21) acute myeloid leukemia. Cancer Cell 2002; 1: 63–74. 43. Slovak ML, Bedell V, Popplewell L et al. 21q22 balanced chromosome aberrations in therapy-related hematopoietic disorders: report from an international workshop. Genes, Chromosomes & Cancer 2002; 33: 379–394. 44. Loh ML, Goldwasser MA, Silverman LB et al. Prospective analysis of TEL/AML1-positive patients treated on Dana-Farber Cancer Institute Consortium Protocol 95-01. Blood 2006; 107: 4508–4513.

612 J. C. Wong et al 45. Rubnitz JE, Wichlan D, Devidas M et al. Prospective analysis of TEL gene rearrangements in childhood acute lymphoblastic leukemia: a Children’s Oncology Group study. Journal of Clinical Oncology 2008; 26: 2186–2191. 46. Song WJ, Sullivan MG, Legare RD et al. Haploinsufficiency of CBFA2 causes familial thrombocytopenia with propensity to develop acute myelogenous leukaemia [see comments]. Nature Genetics 1999; 23: 166–175. 47. Osato M, Asou N, Abdalla E et al. Biallelic and heterozygous point mutations in the runt domain of the AML1/PEBP2alphaB gene associated with myeloblastic leukemias. Blood 1999; 93: 1817–1824. 48. Preudhomme C, Warot-Loze D, Roumier C et al. High incidence of biallelic point mutations in the Runt domain of the AML1/PEBP2 alpha B gene in Mo acute myeloid leukemia and in myeloid malignancies with acquired trisomy 21. Blood 2000; 96: 2862–2869. 49. Harada H, Harada Y, Niimi H et al. High incidence of somatic mutations in the AML1/RUNX1 gene in myelodysplastic syndrome and low blast percentage myeloid leukemia with myelodysplasia. Blood 2004; 103: 2316–2324. 50. Christiansen DH, Andersen MK & Pedersen-Bjergaard J. Mutations of AML1 are common in therapy-related myelodysplasia following therapy with alkylating agents and are significantly associated with deletion or loss of chromosome arm 7q and with subsequent leukemic transformation. Blood 2004; 104: 1474–1481. 51. Okuno Y, Huang G, Rosenbauer F et al. Potential autoregulation of transcription factor PU.1 by an upstream regulatory element. Molecular and Cellular Biology 2005; 25: 2832–2845. 52. DeKoter RP, Kamath MB & Houston IB. Analysis of concentration-dependent functions of PU.1 in hematopoiesis using mouse models. Blood Cells, Molecules & Diseases 2007; 39: 316–320. 53. Kastner P & Chan S. PU.1: a crucial and versatile player in hematopoiesis and leukemia. The International Journal of Biochemistry & Cell Biology 2008; 40: 22–27. 54. Scott EW, Simon MC, Anastasi J et al. Requirement of transcription factor PU.1 in the development of multiple hematopoietic lineages. Science 1994; 265: 1573–1577. 55. McKercher SR, Torbett BE, Anderson KL et al. Targeted disruption of the PU.1 gene results in multiple hematopoietic abnormalities. The EMBO Journal 1996; 15: 5647–5658. 56. Walter MJ, Park JS, Ries RE et al. Reduced PU.1 expression causes myeloid progenitor expansion and increased leukemia penetrance in mice expressing PML-RARalpha. Proceedings of the National Academy of Sciences of the United States of America 2005; 102: 12513–12518. *57. Rosenbauer F, Wagner K, Kutok JL et al. Acute myeloid leukemia induced by graded reduction of a lineage-specific transcription factor, PU.1. Nature Genetics 2004; 36: 624–630. *58. Cook WD, McCaw BJ, Herring C et al. PU.1 is a suppressor of myeloid leukemia, inactivated in mice by gene deletion and mutation of its DNA binding domain. Blood 2004; 104: 3437–3444. 59. Metcalf D, Dakic A, Mifsud S et al. Inactivation of PU.1 in adult mice leads to the development of myeloid leukemia. Proceedings of the National Academy of Sciences of the United States of America 2006; 103: 1486–1491. 60. Mueller BU, Pabst T, Osato M et al. Heterozygous PU.1 mutations are associated with acute myeloid leukemia. Blood 2002; 100: 998–1007. 61. Mueller BU, Pabst T, Osato M et al. Heterozygous PU.1 mutations are associated with acute myeloid leukemia. Blood 2003; 101: 2074. 62. Dohner K, Tobis K, Bischof T et al. Mutation analysis of the transcription factor PU.1 in younger adults (16 to 60 years) with acute myeloid leukemia: a study of the AML Study Group Ulm (AMLSG ULM). Blood 2003; 102: 3850.. author reply 3850–3851. 63. Lamandin C, Sagot C, Roumier C et al. Are PU.1 mutations frequent genetic events in acute myeloid leukemia (AML)? Blood 2002; 100: 4680–4681. 64. Vegesna V, Takeuchi S, Hofmann WK et al. C/EBP-beta, C/EBP-delta, PU.1, AML1 genes: mutational analysis in 381 samples of hematopoietic and solid malignancies. Leukemia Research 2002; 26: 451–457. 65. Steidl U, Rosenbauer F, Verhaak RG et al. Essential role of Jun family transcription factors in PU.1 knockdown-induced leukemic stem cells. Nature Genetics 2006; 38: 1269–1277. 66. Vangala RK, Heiss-Neumann MS, Rangatia JS et al. The myeloid master regulator transcription factor PU.1 is inactivated by AML1-ETO in t(8;21) myeloid leukemia. Blood 2003; 101: 270–277. 67. Mueller BU, Pabst T, Fos J et al. ATRA resolves the differentiation block in t(15;17) acute myeloid leukemia by restoring PU.1 expression. Blood 2006; 107: 3330–3338.

Tumor suppressor gene inactivation in myeloid malignancies 613 68. Zheng R, Friedman AD, Levis M et al. Internal tandem duplication mutation of FLT3 blocks myeloid differentiation through suppression of C/EBPalpha expression. Blood 2004; 103: 1883–1890. 69. Boultwood J, Fidler C, Strickson AJ et al. Narrowing and genomic annotation of the commonly deleted region of the 5q- syndrome. Blood 2002; 99: 4638–4641. *70. Ebert BL, Pretz J, Bosco J et al. Identification of RPS14 as a 5q- syndrome gene by RNA interference screen. Nature 2008; 451: 335–339. 71. Ebert BL, Lee MM, Pretz JL et al. An RNA interference model of RPS19 deficiency in Diamond-Blackfan anemia recapitulates defective hematopoiesis and rescue by dexamethasone: identification of dexamethasone-responsive genes by microarray. Blood 2005; 105: 4620–4626. 72. Luna-Fineman S, Shannon K & Lange BJ. Childhood monosomy 7: epidemiology, biology and mechanistic implications. Blood 1995; 85: 1985–1999. 73. Le Beau MM, Espinosa 3rd R, Davis EM et al. Cytogenetic and molecular delineation of a region of chromosome 7 commonly deleted in malignant myeloid diseases. Blood 1996; 88: 1930–1935. 74. Kratz CP, Emerling BM, Donovan S et al. Candidate gene isolation and comparative analysis of a commonly deleted segment of 7q22 implicated in myeloid malignancies. Genomics 2001; 77: 171–180. 75. Emerling BM, Bonifas J, Kratz CP et al. MLL5, a homolog of Drosophila trithorax located within a segment of chromosome band 7q22 implicated in myeloid leukemia. Oncogene 2002; 21: 4849–4854. *76. Curtiss NP, Bonifas JM, Lauchle JO et al. Isolation and analysis of candidate myeloid tumor suppressor genes from a commonly deleted segment of 7q22. Genomics 2005; 85: 600–607. 77. Kere J, Ruutu T, Lahtinen R et al. Molecular characterization of chromosome 7 long arm deletions in myeloid disorders. Blood 1987; 70: 1349–1353. 78. Kere J, Donis-Keller H, Ruutu T et al. Chromosome 7 long-arm deletions in myeloid disorders: terminal DNA sequences are commonly conserved and breakpoints vary. Cytogenetics and Cell Genetics 1989; 50: 226–229. 79. Fischer K, Frohling S, Scherer S et al. Molecular cytogenetic delineation of deletions and translocations involving chromosome band 7q22 in myeloid leukemias. Blood 1997; 89: 2035–2041. 80. Liang H, Fairman J, Claxton DF et al. Molecular anatomy of chromosome 7q deletions in myeloid leukemia: Evidence for multiple critical loci. Proceedings of the National Academy of Sciences of the United States of America 1998; 95: 3781–3785. 81. Tosi S, Scherer SW, Giudici G et al. Delineation of multiple deleted regions in 7q in myeloid disorders. Genes, Chromosomes & Cancer 1999; 25: 384–392. 82. Zhao N, Stoffel A, Wang PW et al. Molecular delineation of the smallest commonly deleted region of chromosome 5 in malignant myeloid diseases to 1-1.5 Mb and preparation of a PAC-based physical map. Proceedings of the National Academy of Sciences of the United States of America 1997; 94: 6948–6953. 83. Lai F, Godley LA, Joslin J et al. Transcript map and comparative analysis of the 1.5-Mb commonly deleted segment of human 5q31 in malignant myeloid diseases with a del(5q). Genomics 2001; 71: 235–245. 84. Liu TX, Becker MW, Jelinek J et al. Chromosome 5q deletion and epigenetic suppression of the gene encoding alpha-catenin (CTNNA1) in myeloid cell transformation. Nature Medicine 2007; 13: 78–83. 85. Wattel E, Lai JL, Hebbar M et al. De novo myelodysplastic syndrome (MDS) with deletion of the long arm of chromosome 20: a subtype of MDS with distinct hematological and prognostic features? Leukemia Research 1993; 17: 921–926. 86. Davis MP, Dewald GW, Pierre RV et al. Hematologic manifestations associated with deletions of the long arm of chromosome 20. Cancer Genetics and Cytogenetics 1984; 12: 63–71. 87. Aatola M, Armstrong E, Teerenhovi L et al. Clinical significance of the del(20q) chromosome in hematologic disorders. Cancer Genetics and Cytogenetics 1992; 62: 75–80. 88. Fenaux P, Morel P & Lai JL. Cytogenetics of myelodysplastic syndromes. Seminars in Hematology 1996; 33: 127–138. 89. Heim S & Mitelman F. Cytogenetic analysis in the diagnosis of acute leukemia. Cancer 1992; 70: 1701–1709. 90. Kurtin PJ, Dewald GW, Shields DJ et al. Hematologic disorders associated with deletions of chromosome 20q: a clinicopathologic study of 107 patients. American Journal of Clinical Pathology 1996; 106: 680–688. 91. Nacheva E, Holloway T, Carter N et al. Characterization of 20q deletions in patients with myeloproliferative disorders or myelodysplastic syndromes. Cancer Genetics and Cytogenetics 1995; 80: 87–94.

614 J. C. Wong et al 92. Roulston D, Espinosa 3rd R, Stoffel M et al. Molecular genetics of myeloid leukemia: identification of the commonly deleted segment of chromosome 20. Blood 1993; 82: 3424–3429. 93. Asimakopoulos FA, White NJ, Nacheva E et al. Molecular analysis of chromosome 20q deletions associated with myeloproliferative disorders and myelodysplastic syndromes. Blood 1994; 84: 3086–3094. 94. Bench AJ, Aldred MA, Humphray SJ et al. A detailed physical and transcriptional map of the region of chromosome 20 that is deleted in myeloproliferative disorders and refinement of the common deleted region. Genomics 1998; 49: 351–362. 95. Bench AJ, Nacheva EP, Hood TL et al. Chromosome 20 deletions in myeloid malignancies: reduction of the common deleted region, generation of a PAC/BAC contig and identification of candidate genes. UK Cancer Cytogenetics Group (UKCCG). Oncogene 2000; 19: 3902–3913. 96. Wang PW, Eisenbart JD, Espinosa 3rd R et al. Refinement of the smallest commonly deleted segment of chromosome 20 in malignant myeloid diseases and development of a PAC-based physical and transcription map. Genomics 2000; 67: 28–39. 97. Grimwade D, Walker H, Oliver F et al. The importance of diagnostic cytogenetics on outcome in AML: analysis of 1,612 patients entered into the MRC AML 10 trial. The Medical Research Council Adult and Children’s Leukaemia Working Parties. Blood 1998; 92: 2322–2333. 98. Peniket A, Wainscoat J, Side L et al. Del (9q) AML: clinical and cytological characteristics and prognostic implications. British Journal of Haematology 2005; 129: 210–220. 99. Schoch C, Haase D, Haferlach T et al. Fifty-one patients with acute myeloid leukemia and translocation t(8;21)(q22;q22): an additional deletion in 9q is an adverse prognostic factor. Leukemia 1996; 10: 1288–1295. 100. Grimwade D, Walker H, Harrison G et al. The predictive value of hierarchical cytogenetic classification in older adults with acute myeloid leukemia (AML): analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial. Blood 2001; 98: 1312–1320. 101. Raimondi SC, Chang MN, Ravindranath Y et al. Chromosomal abnormalities in 478 children with acute myeloid leukemia: clinical characteristics and treatment outcome in a cooperative pediatric oncology group study-POG 8821. Blood 1999; 94: 3707–3716. 102. Sweetser DA, Peniket AJ, Haaland C et al. Delineation of the minimal commonly deleted segment and identification of candidate tumor-suppressor genes in del(9q) acute myeloid leukemia. Genes, Chromosomes & Cancer 2005; 44: 279–291. *103. Dayyani F, Wang J, Yeh JR et al. Loss of TLE1 and TLE4 from the del(9q) commonly deleted region in AML cooperates with AML1-ETO to affect myeloid cell proliferation and survival. Blood 2008; 111: 4338–4347. 104. Ramirez-Solis R, Liu P & Bradley A. Chromosome engineering in mice. Nature 1995; 378: 720–724. 105. van der Weyden L & Bradley A. Mouse chromosome engineering for modeling human disease. Annual Review of Genomics and Human Genetics 2006; 7: 247–276. 106. Shannon KM, Le Beau MM, Largaespada DA et al. Modeling myeloid leukemia tumor suppressor gene inactivation in the mouse. Seminars in Cancer Biology 2001; 11: 191–200. *107. Bagchi A, Papazoglu C, Wu Y et al. CHD5 is a tumor suppressor at human 1p36. Cell 2007; 128: 459–475.