Original Article

Leukemia (2006) 20, 224–229. doi:10.1038/sj.leu.2404076; published online 15 December 2005

A novel and cytogenetically cryptic t(7;21)(p22;q22) in acute myeloid leukemia results in fusion of RUNX1 with the ubiquitin-specific protease gene USP42

K Paulsson1, A N Békássy2, T Olofsson3, F Mitelman1, B Johansson1 and I Panagopoulos1

  1. 1Department of Clinical Genetics, Lund University Hospital, Lund, Sweden
  2. 2Department of Pediatrics, Lund University Hospital, Lund, Sweden
  3. 3Department of Hematology, Lund University Hospital, Lund, Sweden

Correspondence: Dr K Paulsson, Department of Clinical Genetics, University Hospital, SE-221 85 Lund, Sweden. E-mail: kajsa.paulsson@med.lu.se

Received 4 July 2005; Revised 10 October 2005; Accepted 18 October 2005; Published online 15 December 2005.

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Abstract

Although many of the chromosomal abnormalities in hematologic malignancies are identifiable cytogenetically, some are only detectable using molecular methods. We describe a novel cryptic t(7;21)(p22;q22) in acute myeloid leukemia (AML). FISH, 3'RACE, and RT-PCR revealed a fusion involving RUNX1 and the ubiquitin-specific protease (USP) gene USP42. The genomic breakpoint was in intron 7 of RUNX1 and intron 1 of USP42. The reciprocal chimera was not detected – neither on the transcriptional nor on the genomic level – and FISH showed that the 5' part of USP42 was deleted. USP42 maps to a 7p22 region characterized by segmental duplications. Notably, 17 kb duplicons are present 1 Mb proximal to USP42 and 3 Mb proximal to RUNX1; these may be important in the genesis of t(7;21). This is the second cryptic RUNX1 translocation in hematologic malignancies and the first in AML. The USPs have not previously been reported to be rearranged in leukemias. The cellular context in which USP42 is active is unknown, but we here show that it is expressed in normal bone marrow, in primary AMLs, and in cancer cell lines. Its involvement in the t(7;21) suggests that deregulation of ubiquitin-associated pathways may be pathogenetically important in AML.

Keywords:

acute myeloid leukemia, RUNX1, USP42, cryptic translocation

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Introduction

The acquired, clonal chromosomal aberrations frequently found in hematologic malignancies are closely associated with leukemogenesis. For example, the strong correlations that exist between certain balanced rearrangements and specific clinical, immunophenotypic, and morphologic patterns strongly indicate that these fusion gene-generating abnormalities play a major role in the transformation process.1 However, cytogenetically identifiable chromosomal aberrations are seen only in approximately half of all acute myeloid leukemias (AMLs). This notwithstanding, it has been suggested that primary, balanced rearrangements – often resulting in chimeric genes – are necessary for malignant hematologic disorders to arise.2 It should be stressed that the absence of rearrangements does not exclude the presence of chromosomal changes because the resolution of standard cytogenetics is quite poor. In fact, some cryptic aberrations, detectable only with molecular methods, have been shown to be quite common, for example, the t(12;21)(p13;q22), occurring in 20% of B-lineage acute lymphoblastic leukemias (ALLs),3 the t(5;14)(q35;q32), found in 10–20% of T-ALLs,3 and the del(4)(q12q12), which is characteristic for hypereosinophilic syndrome.4 Considering the diagnostic, prognostic, and biologic impact of leukemia-associated translocations and fusion genes, it is important to search for other cryptic rearrangements that may be present in hematologic malignancies.

The RUNX1 gene (previously AML1, CBFA2), located at 21q22, was first identified as one of the fusion partners in the t(8;21)(q22;q22) in AML-M2.5 Since then, RUNX1 has been shown to be involved in numerous translocations as well as harboring mutations – germ line or acquired – in a substantial proportion of AML cases.6, 7 RUNX1 codes for a transcription factor belonging to the 'RUNT domain' gene family, and is a key regulator of hematopoiesis.8 The wild-type RUNX1 forms a heterodimer with CBFB – encoded by a gene targeted by the inv(16)(p13q22) in AML-M49 – and the active complex functions both as a transcriptional activator and as a repressor.8 Apart from the t(12;21), which results in an ETV6/RUNX1 fusion in B-lineage ALL,10 and the recently described t(4;21)(q28;q22), leading to a RUNX1/FGA7 chimera in T-ALL,11 RUNX1 is typically rearranged through translocations in AML and myelodysplastic syndromes (MDSs). To date, five in-frame fusion gene partners have been reported: RUNX1T1 (previously ETO, MTG8, CBFA2T1) at 8q22,12 MDS1/EVI1 at 3q26,13 CBFA2T3 (previously MTG16) at 16q24,14 PRDX4 at Xp22,15 and ZFPM2 (previously FOG2) at 8q23.16 The translocations resulting in these fusions have all been detectable with G-banding. In the present study, we describe a novel and cytogenetically cryptic t(7;21)(p22;q22) in AML, fusing RUNX1 with the ubiquitin-specific protease (USP) gene USP42, which is involved in the ubiquitin pathway.17 This is the second cryptic RUNX1 translocation described in hematologic malignancies, and the first in AML. It is also the first report of a fusion involving a USP gene in hematologic malignancies.

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Patients, materials, and methods

Case history

A previously healthy 7-year-old boy was admitted to the hospital in March 1995 because of pyrexic tonsillitis and cervical adenitis. The clinical examination was unremarkable. The peripheral blood values were hemoglobin 102 g/l, white blood cell count 35.6 times 109, with 1.4 times 109/l granulocytes and 24.9 times 109 atypical monocytic cells, and thrombocytes 69 times 109/l. The bone marrow (BM) aspirate was hypercellular, containing blasts difficult to classify morphologically. Flow cytometric two-color analysis showed that 75–80% of the BM cells were positive for HLA-DR, CD34, CD33, CD13, CD11c, CD7, CD5, and CD4, and partly positive for CD56 but negative for B-lymphocytic markers. The diagnosis was AML-M0 with aberrant expression of T-lymphocyte-associated markers. The chromosome analysis revealed a seemingly normal male karyotype. The patient received treatment according to the NOPHO-AML-93 protocol.18 After induction therapy, the BM still contained 25% blasts; three additional chemotherapy blocks were needed to achieve complete remission (CR) (May 1995). Allogeneic stem cell transplantation, with an HLA identical sister as a donor, was performed in June 1995. BM relapse occurred in March 2000, at which time the karyotype was 46,XY,t(4;6)(q24;p11),del(5)(q15),t(11;18)(q23;q21)[24]. The immunophenotype was essentially the same as at diagnosis. A second CR was achieved with a single cytoreductive chemotherapy course in April 2000. Then, donor lymphocyte infusions were given on three occasions. The patient is a complete donor chimera, with no signs of the disease on the latest follow-up in April 2005.

FISH analyses

The t(7;21) was originally detected with a whole chromosome paint (WCP) probe for chromosome 7. To characterize the translocation, FISH was performed according to standard protocols on BM cells, obtained at diagnosis and relapse. For multicolor-FISH (M-FISH), WCP probes for combined binary ratio labeling-FISH were used.19 RUNX1 rearrangements were investigated with the LSI AML1/ETO and the LSI TEL/AML1 probes, covering the whole RUNX1 gene (Vysis, Downers Grove, IL, USA). BACs and PACs used for mapping of the chromosome 7 breakpoint were generously supplied by Dr M Rocchi, Bari, Italy (Supplementary Table 1). The fosmids G248P88689C2 and G248P8048B11, specific for the 5' and 3' parts of USP42, respectively, were also applied (BACPAC Resources, St Oakland, CA, USA). In addition, WCP probes for chromosomes 7 and 21 were utilized (Vysis).

3'RACE and gene-specific RT-PCR

Total RNA was extracted according to standard methods from BM cells, obtained at relapse. First-strand cDNA synthesis was performed with the SMART RACE cDNA Amplification kit (BD Biosciences, San Jose, CA, USA). The GC-RICH PCR System (Roche Diagnostics, Penzberg, Germany) was used for the 3'RACE PCR reaction, with the forward primer RUNX1-765F (Supplementary Table 2), specific for exon 4 of RUNX1. The PCR products were separated by gel electrophoresis, excised from the gel, and used as template for a nested PCR reaction (Supplementary Table 2). The resulting fragments were sequenced (Supplementary Table 2) using the BigDye terminator v1.1 cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) on an ABI PRISM 3100-Avant Genetic Analyzer (Applied Biosystems). All sequences were tested against reported sequences (http://www.ncbi.nlm.nih.gov/BLAST/). To confirm the RACE findings, nested PCR was performed using forward RUNX1-specific primers and reverse USP42-specific primers (Supplementary Table 2). In addition, nested PCR was performed for the reciprocal fusion transcript (Supplementary Table 2).

Genomic fusion

DNA was extracted according to standard methods from BM cells, obtained at relapse. To identify the genomic fusion breakpoints in RUNX1 and USP42, nested PCR was performed with the GeneAmp XL PCR kit (Applied Biosystems), using 400 ng of genomic DNA as template, and the amplified fragments were sequenced (Supplementary Table 2). Attempts were also made to amplify the reciprocal genomic fusion with primer pairs listed in Supplementary Table 2.

RT-PCR and Northern blot expression analyses

To investigate the expression of USP42, RT-PCR was performed (Supplementary Table 2) using cDNA from two normal BM, two childhood AMLs (one with a normal karyotype and one with a t(8;21)), and two adult AMLs (one with a complex karyotype and one with an add(7)(p22)) as template. Northern blot analysis was performed, utilizing the Human Cancer Cell Line MTN Blot (BD Biosciences Clontech, Palo Alto, CA, USA), containing poly A+ RNA, on the cancer cell lines HL-60 (AML), HeLa S3, K-562 (CML), MOLT-4 (T-ALL), Raji (Burkitt's lymphoma), SW480 (colorectal adenocarcinoma), A549 (lung carcinoma), and G-361 (melanoma). A probe for USP42 was obtained by RT-PCR using the primers USP42-127F and USP42-562R (Supplementary Table 2) with K-562 cDNA as template. The probe was labeled with 32P using the RediprimeII labelling system (Amersham, Little Chalfont, UK).

Additional cases

As the t(7;21)(p22;q22) is not visible with G-banding, we screened additional cases for the RUNX1/USP42 fusion with RT-PCR (Supplementary Table 2). These included 25 childhood AMLs with available RNA, regardless of karyotypic features, four adult AML-M0 with various chromosome abnormalities, and four adult AMLs with 7p aberrations.

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Results

FISH reveals a translocation of RUNX1 to 7p and narrows down the chromosome 7 breakpoint region to 1.2 Mb

The karyotype was normal at the time of diagnosis (Figure 1a); at relapse, several abnormalities were identified, none of which involved chromosomes 7 or 21. The t(7;21) was first identified with WCP probes (Figure 1b). M-FISH analysis did not reveal any additional chromosomal aberrations. FISH with a probe for RUNX1 and WCP7 disclosed that a part of this gene was translocated to 7p (Figure 1c). To map the breakpoint on 7p, BACs and PACs were used (Supplementary Table 1). All BACs/PACs proximal to RP11-577O18 hybridized to the der(7), and all distal to RP5-1163J12 to the der(21) (Supplementary Table 1), suggesting a 7p22 breakpoint. Analyses of the clones in between were difficult to interpret due to the presence of a 100 kb inverted duplication in this region,20 involving RP11-577O18 and RP11-611L7 at 6.6–6.5 Mb and RP5-1163J12 and RP1-42M2 at 5.7–5.8 Mb from the 7p telomere. Three of these four clones yielded signals on both the der(7) and the der(21), indicating that the breakpoint was localized between or in near proximity to the duplications (Supplementary Table 1). RP5-1163J12 hybridized only to the der(7), and may thus have been, at least partly, deleted. This was confirmed by use of two fosmids. G248P8048B11, corresponding to the 3' part of USP42, hybridized to the der(7); no signals for the more telomeric G248P88689C2, specific for the 5' part of this gene, were detected. The FISH investigations narrowed down the breakpoint region to 1.2 Mb. As the involved sequence contained more than 20 genes (www.ensembl.org), 3'RACE was used to detect the fusion partner of RUNX1.

Figure 1.
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G-banding and FISH findings. (a) Partial karyotype, showing apparently normal chromosomes 7 and 21. In hindsight, the der(7) is probably to the right and the der(21) to the left. (b) The t(7;21) was discovered using WCP probes for chromosomes 7 (green) and 21 (red). Derivative chromosomes are indicated by arrows. (c) A RUNX1-specific probe (red) revealed that this gene was translocated to 7p (green). Derivative chromosomes are indicated by arrows. For clarity, the ETV6 probe included in the hybridization is not shown.

Full figure and legend (106K)

3'RACE and RT-PCR amplifies a RUNX1/USP42 fusion mRNA

3'RACE was performed with forward primers for RUNX1 (Supplementary Table 2) and reverse universal primers. The first PCR resulted in a band of 1.6 kb after gel electrophoresis (Supplementary Figure 1A), which was used as a template for a second PCR, resulting in four distinct bands, ranging in size from 0.4 to 1.5 kb (Supplementary Figure 1B). The three shorter fragments were shown to be splice variants of RUNX1. The 1.5 kb fragment contained an in-frame fusion between exon 7 of RUNX1 in the 5' position and exon 2 of the USP42 gene in the 3' position (Supplementary Figure 2A). USP42 is present in the PAC RP4-810E6, located at 7p22 between the inverted duplications (Supplementary Table 1). RUNX1 and USP42 are oriented in opposite directions (http://www.ncbi.nlm.nih.gov/mapview/). Thus, a fusion between these two genes can occur by a simple translocation between 7p and 21q, with the RUNX1/USP42 chimeric mRNA being transcribed from the der(7) locus. RT-PCR with gene-specific primers (Supplementary Table 2) confirmed the RUNX1/USP42 fusion, and revealed an additional transcript missing exon 6 of RUNX1 (Supplementary Figure 1C–D). RT-PCR with primers for the reciprocal fusion did not amplify a USP42/RUNX1 transcript.

Genomic PCR reveals that the breakpoints occurred in RUNX1 intron 7 and USP42 intron 1

The genomic RUNX1/USP42 fusion was amplified, using primers in the surrounding exons (Supplementary Table 2), and sequenced. The analysis disclosed that nucleotide (nt) 4968 of the 6690 bp RUNX1 intron 7 was fused to nt 3006 of the 3948 bp USP42 intron 1, with insertion of one thymine in the junction (Supplementary Figure 2B). The genomic breakpoint in USP42 intron 1 was located in a repetitive LINE/L1 sequence (http://woody.embl-heidelberg.de/repeatmask/). No homologous sequences were found in the vicinity of the breakpoints. PCR in order to amplify the reciprocal genomic fusion yielded no products.

USP42 is expressed in normal BM, in primary AMLs, and in eight cancer cell lines

RT-PCR with USP42-specific primers revealed expression in normal BM, as well as in four primary AML samples (Figure 2a). Northern blot analysis of eight cancer cell lines disclosed the presence of two USP42 transcripts in all cell lines; one 5 kb and one 2 kb (Figure 2b). The first of these may correspond to the aAug05 transcript (http://www.ncbi.nih.gov/IEB/Research/Acembly/), which is 5093 bp and contains the sequences that hybridize to the USP42 blot probe. The latter transcript may be a previously not described USP42 splice variant.

Figure 2.
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Expression analyses of USP42. Transcripts are indicated by arrows. (a) RT-PCR demonstrating expression of USP42 in normal bone marrow and in primary AMLs. M, molecular marker; lanes 1–2, normal bone marrow; lanes 3–6, AMLs; lane 7, K-562; and lane 8, negative control. (b) Northern blot analysis of eight cancer cell lines. Two USP42 transcripts are visible; one approximately 5 kb and one approximately 2 kb.

Full figure and legend (79K)

Screening for additional AMLs possibly harboring the cryptic RUNX1/USP42 fusion

Screening for RUNX1/USP42 fusion transcripts with RT-PCR in 33 AMLs revealed no additional t(7;21)-positive cases.

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Discussion

We here describe a novel and cytogenetically cryptic t(7;21)(p22;q22) involving the RUNX1 and USP42 genes. This AML case had an apparently normal karyotype at diagnosis (Figure 1a); the t(7;21) was an unexpected finding while screening pediatric leukemias with FISH for the t(7;12)(q36;p13).21

FISH analyses to characterize the region surrounding the 7p breakpoint resulted in complex signal patterns (Supplementary Table 1). This was partly due to a 100 kb inverted duplicon present on both sides of the actual breakpoint site,20 leading to crosshybridization of some BACs/PACs (Supplementary Table 1). This crosshybridization was not visible on control slides or on the other chromosome 7 homologue in the present case, most likely because the duplicons were too close (<1 Mb) to be seen as separate signals when residing on the same chromosome. Furthermore, the FISH assays disclosed the presence of noncontinuous deletions in the breakpoint region (Supplementary Table 1), including deletion of the 5' part of USP42. In line with the latter, we did not detect a reciprocal USP42/RUNX1 chimera, neither on the transcriptional nor on the genomic level. Deletions surrounding translocation breakpoints have been reported in several other leukemia-associated rearrangements, such as the t(9;22)(q34;q11) in chronic myeloid leukemia (CML).22 The 7p region surrounding USP42 is characterized by a particularly large number of segmental duplications and has been described as highly rearrangement-prone.20 Interestingly, a 17 kb duplicated sequence is present approximately 0.8 Mb proximal to USP42 at 7p22 and 3 Mb proximal to RUNX1 at 21q22 (http://projects.tcag.ca/humandup/). It is in this context noteworthy that a duplicon of 76 kb has previously been shown to be situated close to BCR and ABL1, the genes involved in the t(9;22) in CML.23 It is possible that such homologous sequences are of importance in the formation of translocations.

USP42 contains 25 alternative exons and codes for five protein isoforms (http://www.ncbi.nih.gov/IEB/Research/Acembly/). The encoded protein belongs to the USP family, a large group of cysteine proteases with variable size and structure and with conserved motifs mainly in the catalytic region.17 They are involved in the ubiquitin pathway, which regulates important functions, such as the activity, degradation, and subcellular localization of proteins, and hydrolyze linear and branched ubiquitin modifications, including the removal of polyubiquitin from target proteins, thereby saving them from ubiquitin-mediated degradation.17 USP42 contains the conserved catalytic Cys, His, and Asp/Asn residues that form the catalytic triad of the UCH domain of USPs (Figure 3) and can remove ubiquitin from Ub-M-beta-galactosidase, strongly suggesting that it is a functional USP.17 The gene is highly expressed in skeletal muscle, liver, and pancreas, more weakly in placenta, brain, and heart, and not at all in ovary, testis, prostate, and thymus.17 In this study, we show that USP42 is expressed also in normal BM and primary AMLs (Figure 2a). Furthermore, Northern blot analyses revealed USP42 expression in all examined cancer cell lines (Figure 2b). Taken together, USP42 seems to be widely, albeit not ubiquitously, expressed in human tissues.

Figure 3.
Figure 3 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Schematic view of retained protein domains in the RUNX1/USP42 chimera. RUNT, RUNT domain; TA, transactivation domain; UCH, ubiquitin carboxyl-terminal hydrolase domain. The fusion protein retains the CBFB- and DNA-binding RUNT domain of RUNX1 and the catalytic UCH domain of USP42. The breakpoint is within the TA domain of RUNX1.

Full figure and legend (8K)

Several members of the USP family have previously been implicated in tumorigenesis, primarily by being deregulated in various neoplasms.24, 25, 26, 27 Most notably, Usp18 (previously Ubp43) levels have been shown to be specifically upregulated in mice expressing the RUNX1/CBFA2T1 chimera,25 and USP33 is overexpressed in B-ALL as compared with T-ALL,26 suggesting that deregulation of USPs may be important in leukemogenesis. Furthermore, a recurrent rearrangement involving USP6 has previously been described in aneurysmal bone cysts (ABC); the t(16;17)(q22;p13) which places the whole coding sequence of USP6 under the transcriptional control of the highly active CDH11 promotor.27 USP6 is also the 3' part of other fusion genes in ABC, suggesting that its increased expression may be pathogenetically important in this disorder.27

RUNX1 is involved in both in-frame and out-of-frame fusions in myeloid malignancies. All in-frame chimeras have RUNX1 in the 5' position.13, 14, 15, 16, 28 This results in proteins that retain the CBFB- and DNA-binding RUNT domain but lose the transactivation (TA) domain of RUNX1. Several of these fusions have been shown to act as dominant-negative inhibitors of normal RUNX1 function;8 the same is true for some mutations.7 Taken together, there is strong support for a leukemogenic effect resulting from a dominant-negative function of RUNX1 rearrangements/mutations. However, gain-of-function is most likely also involved in translocations. For example, the RUNX1/CBFA2T1 fusion protein upregulates Usp18 in mice.25

Like previously described chimeras in myeloid malignancies, the t(7;21) places RUNX1 in the 5'position, although its intron 7 breakpoint is 1–2 introns further downstream compared with these fusions.13, 14, 15, 28 Thus, the RUNT domain is retained, but the breakpoint is within, rather than 5' to, the TA domain (Figure 3). However, the latter is most likely nonfunctional because it is disrupted. Based on previous studies,13, 14, 15, 28 it seems likely that the RUNX1/USP42 fusion protein may have a dominant-negative effect on normal RUNX1 function. Since the present case was an AML-M0, it is also notable that biallelic RUNX1 mutations have been reported to be frequent in such cases, indicating that the absence of normal RUNX1 may be associated with this particular subtype.29 Furthermore, the RUNX1/USP42 chimera retains almost the entire USP42 open reading frame, including the catalytic UCH domain (Figure 3; http://www.sanger.ac.uk/Software/Pfam/). Thus, it cannot be excluded that the protease is still active. Although the pathogenetic consequences of the t(7;21) resulting from USP42 is difficult to interpret, since the specific cellular context in which USP42 is active has not been elucidated, the fact that deregulation of several members of the USP family have previously been described in neoplasias suggests that USP42 deregulation may have a leukemogenic effect.24, 25, 26, 27 RUNX1 has been shown to be subject to ubiquitin–proteasome-mediated degradation.30 This is interesting considering that some USPs can remove polyubiquitin from target proteins, thereby saving them from degradation by the proteasome. It is thus tempting to speculate that fusion with USP42 helps to stabilize the RUNX1 part of the chimera. This could possibly enhance the putative dominant-negative effects of RUNX1/USP42. Taken together, data from similar fusions and from other USPs in malignant disorders make it likely that the pathogenetically important outcome of the t(7;21) includes dominant-negative effects of normal RUNX1 function and possibly deregulation of USP42.

The present identification of the t(7;21) clearly demonstrates that lack of cytogenetically detectable chromosomal aberrations does not exclude chimeric genes. Previously, only three truly cryptic chromosomal abnormalities resulting in fusion genes have been described in AML: the del(11)(q23q23) leading to MLL/ARHGEF12 and MLL/CBL chimeras, respectively,31, 32 and the t(5;11)(q35;p15.5) resulting in a NUP98/NSD1 fusion.33 However, recent gene expression studies have shown that AMLs with normal karyotypes display heterogeneous expression patterns,34 indicating the presence of many different underlying, cryptic primary rearrangements. In the future, molecular methods such as M-FISH/SKY or array-based comparative genome hybridization (CGH) may identify more hidden abnormalities.

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Acknowledgements

This study was supported by grants from the Swedish Cancer Society, the Swedish Children's Cancer Foundation, and Gunnar Nilsson's Cancer Foundation.

Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)