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Patients with monosomy 7 (−7) or del(7q) comprise a heterogeneous subgroup in acute myeloid leukemia (AML) but no specific target genes have been identified [1–4]. Recently, we detected a commonly deleted region on the long arm of chromosome 7 in a large cohort of Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) at the time of leukemic transformation using single-nucleotide polymorphism array (SNP-A) [5, 6]. This region was located on 7q22.1 encompassing only two genes, CUX1 and SH2B2. Currently, we screened for acquired mutations in these two candidate genes using 15 secondary AML cases with preceding MPN and loss-of-heterozygosity (LOH) on chromosome 7q. We detected a novel hemizygous missense mutation (V1288L) in the HOX domain of the transcriptional regulator CUX1 in one case with primary myelofibrosis (PMF) at time of leukemic evolution. Although only detected in 1/15 (6.5%) of the cases with secondary AML, an acquired mutation of CUX1 may play a critical role in myeloid malignancies with 7q aberrations.

Abnormalities involving chromosome 7q are frequently detectable in myeloid malignancies [1–4]. The association of losses in 7q with AML suggests that this region contains a tumor suppressor gene or genes whose loss of function contributes to leukemic transformation or tumor progression. Based on several preceding chromosome banding studies, two minimal deleted regions have been identified; one locus at centromeric band 7q22 and the other at telomeric breakpoint varying from q32 to q36 [7–9]. The complexity of 7q rearrangements suggests that a synergy of different genetic factors, rather than the alteration of a single tumor suppressor gene, could be involved in the pathogenesis of 7q− in myeloid disorders. Two recent studies identified monoallelic or biallelic loss-of-function mutations in the histone H3 methyltransferase EZH2 on 7q36.1 in patients with myelodysplastic/s (12%) and in those with myelofibrosis (13%); the authors suggested EZH2 functions as a tumor-suppressor gene in these malignancies rather than an oncogene as in some other malignancies [10, 11]. Notably, no EZH2 mutations were found so far in de novo or secondary patients with AML having complete or partial monosomy for chromosome 7.

Using high-density SNP-A provides a robust and detailed approach to detect large and small copy-number changes, as well as copy-number neutral (CNN-) LOH. We recently applied this interrogational method and performed a systemic analysis of 159 samples obtained from patients with either MPN or secondary AML with preceding MPN to obtain a comprehensive profile of genomic alterations associated with leukemic transformation in MPN disease [5]. Complete or partial deletion (−7/7q−) and CNN-LOH of the long arm of chromosome 7 were one of the most common abnormalities detected by SNP-A analysis in 25% of samples with secondary AML, and associated with inferior outcome. The minimal deleted region spanned a small region at 7q22.1.

In this study, we used 15 secondary AML cases with chromosome seven aberrations and prior MPN (Table I) to screen for novel tumor suppressor genes. Using both cytogenetics and SNP-A, 11 samples (73%) showed heterozygous deletions on 7q; three samples (20%) had CNN-LOH on 7q; and the analysis of one case (7%; #138) revealed both CNN-LOH on 7q21.13-qter and a small homozygous deletion on 7q22.1 (0.88 Mbp) encompassing only two candidate genes, CUX1 and SH2B2 (Fig. 1). One of the major protein function of SH2B2 (alias APS) is the recruitment of c-Cbl into the receptor/JAK complex, and thereby inhibiting JAK/STAT signaling activity [12, 13]. CUX1 (alias Cut homeobox 1 or CUTL1) belongs to a family of transcription factors with homeodomain (HOX) involved in the control of cell proliferation and differentiation [14].

Table I. Genetic Aberrations on Chromosome 7 in 15 Patient Samples from Time of Leukemic Transformation after Prior MPN
Case #DiagnoseJAK2V617FPrior MPNCytogeneticsSNP-A (breakpoints)Aberration size [Mbp]Genetic Variation in CUX1
  1. Indicated are detected aberrations on chromosome 7 and in CUX1 on 7q22.1. ET, essential thrombocythemia; PMF, primary myelofibrosis; PV, polycythemia vera; sAML, secondary acute myelogenous leukemia; Mbp, megabase pairs.

23sAMLPos.PV7q−del(7)(q11.21 qter)96
24sAMLPos.PVNormal7qCNN-LOH(q11.23 qter)86
27sAMLPos.PVNormaldel(7)(q22 1)3.8
28sAMLPos.PVNormal7qCNN-LOH(q22.1-qter)59
34sAMLPos.PV7q−del(7)(q11.21 qter)96rs76202142 G/T (SNP; Intron 16-17)
36sAMLNeg.PV7q−del(7)(q22 1qter)59
72sAMLNeg.ET7q−del(7)(q11 .22 qter)87
79sAMLNeg.ET7q−7qCNN-LOH(q21.3qter)63rs73712454 G/T (SNP; Intron 22-23)
80sAMLPos.ET7q−del(7)(p12 3qter)109rs73712454 G/T (SNP; Intron 22-23)
87sAMLNeg.ETNormaldel(7)(q22 1)3.5
90sAMLNeg.ET7q−del(7)(q11 21 qter)96V1288L (rnissense mutation; Exon 24)
130sAMLPos.PMF−7−7158
134sAMLPos.PMF7q−del(7)(q11 21qter)96
138sAMLPos.PMFNormal7qCNN-LOH(q21.13qter), del7q22.169, 0.88Homozygously deleted
139sAMLNeg.PMF7q−del(7)(q21 3qter)64
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Figure 1. Aberrations on chromosome 7 in patients with transformed MPN. (A) Indicated are SNP-Chip findings on Chromosome 7 in 15 patients (see Table I) with transformed MPN. Each horizontal line represents abnormality detected in each patient; green: CNN-LOH; blue: deletion. (B) One patient, #138, with transformed MPN showed CNN-LOH on 7q, as well as a homozygous deletion on 7q22.1 encompassing only CUX1 and SH2B2. AsCN, allele-specific copy number; CN, copy number; CNN-LOH, copy-number neutral loss-of-heterozygosity; SNP, single nucleotide polymorphism. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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First, we investigated all coding exons of SH2B2 in the 15 patients with secondary AML by direct sequencing, but no genetic variation, either SNP or mutation, could be detected. Instead, after screening the coding regions of the three Cut domains and the HOX domain of the CUX1 gene, we detected not only a SNP in case #34, #79, and #80 (Table I) but also a genetic variation in a highly conserved nucleotide position in case #90 (V1288L; Table I), which has not been previously described in the “Database of Genomic Variants” (http://projects.tcag.ca/variation/), the “UCSC Genome Browser” (http://genome.ucsc.edu/), or the “Cosmic Catalogue for Somatic Mutations in Cancer” (http://www.sanger.ac.uk/genetics/CGP/cosmic/). We were able to characterize the novel nucleotide variant (GTC [RIGHTWARDS ARROW] CTC) as a somatic mutation by showing a normal genetic code in a serial sample of case #90 originating from the chronic phase of MPN before leukemic evolution (Fig. 2A). Interestingly, not only the V1288L missense mutation in CUX1 was acquired at the time of transformation to secondary AML but also the loss of the normal allele with del(7)(q11.21qter) as indicated by SNP-A [5]. The V1288L mutation is located in the coding region of the DNA-binding HOX domain of CUX1 (Fig. 2B) and has statistically a detrimental effect on the CUX1 protein (non-neutral, reliability index 2, accuracy 70%) according to the SNAP software, a neural-network–based method to make prediction regarding the functionality of a mutated protein [15].

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Figure 2. Novel somatic mutation of CUX1 in a paient with transformed MPN and 7q−. (A) Nucleotide sequencing of MPN case #90 revealed one missense mutation (V1288L) in the CUX1 gene after leukemic transformation. (B) Indicated is the schematic overview of the CUX1 protein including its three CUT domains, the HOX domain, as well as the three known isoforms p75, p110, and p200 (according to Sansregret et al, 2008) [16]. AA, amino acid. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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The human CUX1 spans at least 340 kb, contains 33 exons, and at least three protein isoforms can be expressed as the result of proteolytic processing or transcription initiation at an alternative start site (Fig. 2B) [14]. The full-length protein, p200, is a complex protein with four evolutionarily conserved DNA-binding domains: three Cut repeats and a Cut homeodomain (HOX; Fig. 2B). CUX1 was originally shown to function in precursor cells of various lineages as a transcriptional repressor that down-modulates lineage specific genes that later become expressed in terminally differentiated cells [14, 16]. Noteworthy, homozygous mutant mice expressing a hypomorphic and non-functional HOX domain of the CUX1 protein demonstrated myeloid hyperplasia, suggesting that CUX1 functions as a tumor suppressor and that its loss is a significant event in the generation or progression of myeloid disorders [17]. However, one study performing a mutational analysis of childhood samples with AML and monosomy 7 revealed no somatic mutations in CUX1 [18]. In contrast, transgenic mice expressing the short isoform of CUX1, p75, displayed heightened susceptibility to mammary tumors and a myeloproliferative disease-like myeloid leukemia, pointing to an oncogenic role of CUX1 [19, 20]. In consequence, regarding to present literature, CUX1 has a critical regulatory influence on the myeloid development and may act as either transcriptional repressor or activator depending on its isoform.

In summary, this is the first report of an acquired missense mutation of CUX1 in a patient with secondary AML and 7q−. Although detected in only one case (6.5%), it may contribute to the pathogenesis of AML in a subset of patients including the transformation or progression of chronic myeloid diseases in association with 7q−. Larger cohorts of myeloid diseases and further functional analysis are required to explore the impact of the novel mutation in the CUX1 gene.

Acknowledgements

H.P.K. is the holder of the Mark Goodson endowed Chair in Oncology Research at Cedars Sinai Medical Center and is a member of the Jonsson Cancer Center and the Molecular Biology Institute, UCLA.

References

  1. Top of page
  • 1
    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: 13121320.
  • 2
    Larson RA. Is secondary leukemia an independent poor prognostic factor in acute myeloid leukemia? Best Pract Res Clin Haematol 2007; 20: 2937.
  • 3
    Brezinová J,Zemanová Z,Ransdorfová S, et al. Structural aberrations of chromosome 7 revealed by a combination of molecular cytogenetic techniques in myeloid malignancies. Cancer Genet Cytogenet 2007; 173: 1016.
  • 4
    Iwanski GB,Thoennissen NH,Koeffler HP. Therapy-related Acute myelogenous leukemia. In: Neoplastic Diseases of the Blood, 5th ed. Cambridge University Press. 2011, p 370394.
  • 5
    Thoennissen NH,Krug UO,Lee DHT, et al. Prevalence and prognostic impact of allelic imbalances associated with leukemic transformation of Philadelphia chromosome-negative myeloproliferative neoplasms. Blood 2010; 115: 28822890.
  • 6
    Thoennissen NH,Koeffler HP. Leukaemic transformation of philadelphia-chromosome-negative myeloproliferative neoplasms—A review of the molecular background. Euro Oncol Haematol 2011; 7: 5962.
  • 7
    Fischer K,Fröhling S,Scherer SW, et al. Molecular cytogenetic delineation of deletions and translocations involving chromosome band 7q22 in myeloid leukemias. Blood 1997; 89: 20362041.
  • 8
    Liang H,Fairman J,Claxton DF, et al. Molecular anatomy of chromosome 7q deletions in myeloid neoplasms: Evidence for multiple critical loci. Proc Natl Acad Sci USA 1998; 95: 37813785.
  • 9
    González MB,Gutiérrez NC,García JL, et al. Heterogeneity of structural abnormalities in the 7q31.3 approximately q34 region in myeloid malignancies. Cancer Genet Cytogenet 2004; 150: 136143.
  • 10
    Ernst T,Chase AJ,Score J, et al. Inactivating mutations of the histone methyltransferase gene EZH2 in myeloid disorders. Nat Genet 2010; 42: 722726.
  • 11
    Nikoloski G,Langemeijer SM,Kuiper RP, et al. Somatic mutations of the histone methyltransferase gene EZH2 in myelodysplastic syndromes. Nat Genet 2010; 42: 665667.
  • 12
    Wakioka T,Sasaki A,Mitsui K, et al. APS, an adaptor protein containing Pleckstrin homology (PH) and Src homology-2 (SH2) domains inhibits the JAK-STAT pathway in collaboration with c-Cbl. Leukemia 1999; 13: 760767.
  • 13
    Hu J,Hubbard SR. Structural characterization of a novel Cbl phosphotyrosine recognition motif in the APS family of adapter proteins. J Biol Chem 2005; 280: 1894318949.
  • 14
    Rong Zeng W,Soucie E,Sung Moon N, et al. Exon/intron structure and alternative transcripts of the CUTL1 gene. Gene 2000; 241: 7585.
  • 15
    Bromberg Y,Yachdav G,Rost B. SNAP predicts effect of mutations on protein function. Bioinformatics. 2008; 24: 23972398.
  • 16
    Sansregret L,Nepveu A. The multiple roles of CUX1: Insights from mouse models and cell-based assays. Gene 2008; 412: 8494.
  • 17
    Sinclair AM,Lee JA,Goldstein A, et al. Lymphoid apoptosis and myeloid hyperplasia in CCAAT displacement protein mutant mice. Blood 2001; 98: 36583667.
  • 18
    Hindersin S,Niemeyer CM,Germing U, et al. Mutation analysis of CUTL1 in childhood myeloid neoplasias with monosomy 7. Leuk Res 2007; 31: 13231324.
  • 19
    Cadieux C,Kedinger V,Yao L, et al. Mouse mammary tumor virus p75 and p110 CUX1 transgenic mice develop mammary tumors of various histologic types. Cancer Res 2009; 69: 71887197.
  • 20
    Cadieux C,Fournier S,Peterson AC, et al. Transgenic mice expressing the p75 CCAAT-displacement protein/Cut homeobox isoform develop a myeloproliferative disease-like myeloid leukemia. Cancer Res 2006; 66: 94929501.

Nils H. Thoennissen* †, Terra Lasho‡, Gabriela H. Thoennissen* †, Seishi Ogawa§, Ayalew Tefferi‡, H. Phillip Koeffler* ¶, * Division of Hematology and Oncology, Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles, California, † Department of Hematology and Oncology, University Hospital of Münster, Münster, Germany, ‡ Department of Hematology, Mayo Clinic, Rochester, Minnesota, § Department of Regeneration Medicine for Hematopoiesis, Graduate School of Medicine, University of Tokyo, University of Tokyo Hospital, Tokyo, Japan, ¶ National Cancer Institute of Singapore, National University of Singapore, Singapore.