Oncogenomics

Oncogene (2008) 27, 2249–2256; doi:10.1038/sj.onc.1210857; published online 29 October 2007

Identification of the novel AML1 fusion partner gene, LAF4, a fusion partner of MLL, in childhood T-cell acute lymphoblastic leukemia with t(2;21)(q11;q22) by bubble PCR method for cDNA

Y Chinen1,2, T Taki1, K Nishida2, D Shimizu2, T Okuda2, N Yoshida2, C Kobayashi3, K Koike3, M Tsuchida3, Y Hayashi4 and M Taniwaki1,2

  1. 1Department of Molecular Laboratory Medicine, Kyoto Prefectural University of Medicine Graduate School of Medical Science, Kamigyo-ku, Kyoto, Japan
  2. 2Department of Molecular Hematology and Oncology, Kyoto Prefectural University of Medicine Graduate School of Medical Science, Kamigyo-ku, Kyoto, Japan
  3. 3Department of Pediatrics, Ibaraki Children’s Hospital, Futabadai, Mito, Japan
  4. 4Gunma Children's Medical Center, Shimohakoda, Hokkitsu, Shibukawa, Gunma, Japan

Correspondence: Dr T Taki, Department of Molecular Laboratory Medicine, Kyoto Prefectural University of Medicine Graduate School of Medical Science, 465 Kajii-cho Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan. E-mail: taki-t@umin.net

Received 4 May 2007; Revised 13 September 2007; Accepted 17 September 2007; Published online 29 October 2007.

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Abstract

The AML1 gene is frequently rearranged by chromosomal translocations in acute leukemia. We identified that the LAF4 gene on 2q11.2–12 was fused to the AML1 gene on 21q22 in a pediatric patient having T-cell acute lymphoblastic leukemia (T-ALL) with t(2;21)(q11;q22) using the bubble PCR method for cDNA. The genomic break points were within intron 7 of AML1 and of LAF4, resulting in the in-frame fusion of exon 7 of AML1 and exon 8 of LAF4. The LAF4 gene is a member of the AF4/FMR2 family and was previously identified as a fusion partner of MLL in B-precursor ALL with t(2;11)(q11;q23), although AML1-LAF4 was in T-ALL. LAF4 is the first gene fused with both AML1 and MLL in acute leukemia. Almost all AML1 translocations except for TEL-AML1 are associated with myeloid leukemia; however, AML1-LAF4 was associated with T-ALL as well as AML1-FGA7 in t(4;21)(q28;q22). These findings provide new insight into the common mechanism of AML1 and MLL fusion proteins in the pathogenesis of ALL. Furthermore, we successfully applied bubble PCR to clone the novel AML1-LAF4 fusion transcript. Bubble PCR is a powerful tool for detecting unknown fusion transcripts as well as genomic fusion points.

Keywords:

AML1/RUNX1, LAF4, T-cell acute lymphoblastic leukemia, MLL

Abbreviations:

AML, acute myeloid leukemia; ALL, acute lymphoblastic leukemia

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Introduction

A large number of leukemias have been found to be associated with specific chromosomal aberrations. Recent studies have demonstrated that several chromosomal rearrangements and molecular abnormalities are strongly associated with distinct clinical subgroups and can predict clinical features and therapeutic responses (Rowley, 1999; Taki and Taniwaki, 2006). Some genes have been associated with recurrent rearrangements and have many fusion partner genes, such as MLL at 11q23, TEL (ETV6) at 12p13 and NUP98 at 11p15; AML1 (RUNX1, CBFA2) at 21q22 is one of the most frequent targets of these chromosomal rearrangements in both acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) (Miyoshi et al., 1991; Hayashi, 2000; Kurokawa and Hirai, 2003). To date, a number of in-frame fusion partners of AML1 have been cloned: YTHDF2 at 1p35 (Nguyen et al., 2006), ZNF687 at 1q21.2 (Nguyen et al., 2006), MDS1/EVI1 at 3q26 (Mitani et al., 1994), FGA7 at 4q28 (Mikhail et al., 2004), SH3D19 at 4q31.3 (Nguyen et al., 2006), USP42 at 7p22 (Paulsson et al., 2006), MTG8 (ETO, CBFA2T1) at 8q22 (Erickson et al., 1992; Miyoshi et al., 1993), FOG2 at 8q23 (Chan et al., 2005), TRPS1 at 8q24 (Asou et al., 2007), TEL (ETV6) at 12p13 (Golub et al., 1995), MTG16 at 16q24 (Gamou et al., 1998) and PRDX4 at Xp22 (Zhang et al., 2004). Most AML1 translocations, except for TEL-AML1, are associated with AML, involving the N-terminus Runt domain and lacking the C-terminus transactivation domain (Kurokawa and Hirai, 2003). AML1 fusion proteins are associated with leukemogenesis by dominantly interfering with normal AML1-mediated transcription and acting as a transcriptional repressor (Okuda et al., 1998; Wang et al., 1998). Clinically, patients with AML harboring t(8;21) in both children and adults show a high rate of complete remission, and its prognosis is considered better than that of patients with a normal karyotype or other chromosomal aberrations (Grimwade et al., 1998).

In the present study, we analysed pediatric T-ALL with t(2;21)(q11;q22) and identified the LAF4 gene, which is one of the fusion partners of MLL, as a novel fusion partner of the AML1 gene.

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Results

Case report

A 6-year-old boy with a high leukocyte count (64700μl−1), containing 84% blasts in peripheral blood and with a mediastinal mass, was diagnosed as having T-ALL. A bone marrow smear was hypercellular with 69% blasts and negative for myeloperoxidase. The leukemic cells, after gating of CD45-positive cells, were positive for CD5 (90.7%), CD7 (90.7%), CD58 (69.9%) and cytoplasmic CD3 (92.8%), and negative for HLA-DR, IgG, IgM, Igκ, Igλ, CD8, CD13, CD14, CD19, CD20 and CD33. He was treated on the Tokyo Children's Cancer Study Group (TCCSG) L04-16 extremely high-risk (HEX) protocol, including stem cell transplantation, because the response to initial 7-day prednisolone (60mgm−2) monotherapy was poor. He achieved complete remission after the induction phase. After the early consolidation phase and two courses of the consolidation phase, he received allogeneic bone marrow transplantation from an unrelated HLA-matched donor 4 months after diagnosis. He has been in complete remission for 17 months.

The patient's leukemic cells at diagnosis were analysed after written informed consent was obtained from his parents, and the ethics committee of Kyoto Prefectural University of Medicine approved this study.

Identification of the AML1-LAF4 fusion transcript

Cytogenetic analysis of the leukemic cells of the patient using routine G-banding revealed 47, XY, add(1)(p36), +der(2)t(2;21)(q13;q22), t(2;21)(q13;q22), −9, −9, +mar1, +mar2, and spectral karyotyping (SKY) analysis revealed 47, XY, der(1)t(1;17)(p36.1;q23), der(2)t(2;21)(q11.2;q22), +der(2)t(2;21)(q11.2;q22), del(5)(p15.1), del(9)(q22), del(9)(p13), der(21)t(2;21)(q11.2;q22) (Supplementary Figure S1). Since AML1 is located at 21q22, we inferred that AML1 was rearranged in this case. Fluorescence in situ hybridization analysis using AML1-specific BAC (bacterial artificial chromosome) clones showed split signals of AML1 on two der(2)t(2;21)(q11.2;q22) and der(21)t(2;21)(q11.2;q22) chromosomes (Figure 1a).

Figure 1.
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Fluorescence in situ hybridization analysis of the leukemic metaphase. (a) Both RP11-272A3 (green, 3′ side of AML1) and RP11-994N6 (red, 5′ side of AML1) were hybridized to normal chromosome 21 (arrowhead), RP11-272A3 to der(21)t(2;21)(q11.2;q22) (arrow, green signal) and RP11-994N6 to two der(2)t(2;21)(q11.2;q22) chromosomes (arrows, red signal). (b) Two fusion signals of RP11-994N6 (5′ of AML1, red signals) and RP11-527J8 (3′ of LAF4, green signals) were detected on two der(2)t(2;21)(q11.2;q22) chromosomes (arrows).

Full figure and legend (81K)

To isolate fusion transcripts of AML1, we performed the bubble PCR method for cDNA (Figure 2) and obtained various-sized products (Figure 3a). Four different-sized products were sequenced and two products contained AML1 sequences fused to unknown sequences. Basic local alignment search tool (BLAST) search revealed that the unknown sequences were part of the LAF4 gene and both products had the same in-frame junctions (Figure 3b). LAF4 was located on chromosome 2q11.2–12, which was compatible with the result of spectral karyotyping analysis. We next performed reverse transcription-PCR to confirm AML1-LAF4 fusion transcripts, and obtained three different-sized AML1-LAF4 fusion products, including only one in-frame product (Figures 3c and d); however, reciprocal LAF4-AML1 fusion transcripts were not generated (Figure 3c). Type 2 transcript is an out-of-frame fusion and generated premature termination in exon 9 of LAF4 (Figure 3d). On the other hand, type 3 transcript is an in-frame fusion of exon 7 of AML1 and exon 8 of LAF4, the same as the type 1 transcript; however, the type 3 transcript contained an 85-bp intronic sequence between exons 9 and 10 of LAF4, which might be due to splicing error, and appeared as a premature termination codon within the intronic sequences (Figure 3d). AML1-LAF4 fusions were also confirmed by fluorescence in situ hybridization analysis (Figure 1b).

Figure 2.
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Outline of bubble PCR for cDNA. Bubble PCR primers (NVAMP-1 and NVAMP-2) can only anneal with one complementary sequence for bubble oligo synthesized with AML1 primer, but not bubble oligo itself; therefore, this single-stranded bubble provides the specificity of the reaction.

Full figure and legend (34K)

Figure 3.
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Identification of AML1-LAF4 fusion transcript. (a) Bubble PCR products by nested PCR using AML1-5S and NVAMP1 for first PCR, and AML1-E6S and NVAMP2 for second PCR (lane 1). M, size marker. (b) Sequence analysis of AML1-LAF4 fusion transcript. The single letter amino-acid sequences surrounding the fusion point are shown at the bottom of the figure. (c) Detection of AML1-LAF4 fusion transcripts by reverse transcription-PCR. Primers were AML1-PR7 and LAF4-11AS (lanes 1 and 3), AML1-PR8 and LAF4-PR5 (lanes 2 and 4), and β-actin, respectively. Lanes 1, 3 and 5, patient's leukemic cells; lanes 2, 4 and 6, normal peripheral lymphocytes. (d) Three fusion transcripts of AML1-LAF4 are schematically depicted. Gray/dotted boxes denote predicted AML1 exons and white boxes represent predicted LAF4 exons. Type 3 contains the LAF4 intron 9 splicing donor site. AML1-PR7 and LAF4-11AS indicate the primers used for reverse transcription-PCR. Asterisk shows the termination codon.

Full figure and legend (115K)

Detection of AML1-LAF4 genomic junctions

Southern blot analysis using a cDNA probe within exon 7 of AML1 detected a rearranged band derived from an approximately 11kb BglII germline fragment on chromosome 21 (data not shown). To isolate the fusion point of chromosomes 2 and 21, we next performed bubble PCR on genomic DNA and detected nested PCR products using primers AML1-GNM8-2S and NVAMP2 (Figure 4a). Sequence analysis of the subcloned PCR product revealed the genomic junction of 5′-AML1-LAF4-3′ (Figures 4c and d), and the result was confirmed by PCR analysis using primers AML1-GNM8-4S and LAF4-GNM11-2AS (Figure 4b); however, no 5′-LAF4-AML1-3′ product was generated, suggesting interstitial deletion near genomic break points (Figure 4b). These sequences near the break points did not contain any lymphoid heptamer/nonamer sequences, Alu sequences or consensus topoisomerase II cleavage sites.

Figure 4.
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Cloning of the genomic junction of AML1 and LAF4. (a) Bubble PCR for genomic DNA. N, normal human lymphocytes; P, patient’s leukemic cells. (b) Detection of the genomic fusion point of AML1-LAF4 by PCR. Primers were AML1-GNM8-4S and LAF4-GNM11-2AS (lanes 1 and 3), and LAF4-GNM11-2S and AML1-GNM8-2AS (lanes 2 and 4). Lanes 1 and 2, patient's leukemic cells; lanes 3 and 4, normal peripheral lymphocytes. M, size marker. (c) Sequences of breakpoints in the patient's leukemic cells. (d) Physical map of the breakpoint regions. Open vertical boxes represent defined exons in each gene. Horizontal arrows show the primers used. Restriction sites are indicated by capital letters: G, BglII; H, HindIII. AML1c1 indicates the position of the cDNA probes for Southern blot analysis. A vertical arrow shows AML1-USP42 breakpoint.

Full figure and legend (133K)

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Discussion

In this study, we identified that LAF4 was fused to AML1 in pediatric T-ALL with t(2;21)(q11;q22). Other regions with chromosomal aberrations in this patient were not considered to be associated with recurrent cytogenetic changes involving T-ALL, except for the deletion of the short arm of chromosome 9. Spectral karyotyping analysis detected del(9)(p13), and additional analysis of genome array (Human Mapping 50K Hind Array, Affymetrix, Tokyo, Japan) revealed homozygous deletion of 4.5Mb within the 9p21 region, including the CDKN2A/p16/p14 locus (data not shown), which is frequently deleted in T-ALL (Ohnishi et al., 1995).

Although the patient showed a complex chromosomal abnormality, t(2;21)(q11;q22) can form regular head-to-tail fusion transcripts of both AML1 and LAF4, because the transcription direction of AML1 and LAF4 is telomere to centromere. Furthermore, fluorescence in situ hybridization analysis revealed two der(2)t(2;21)(q11.2;q22) creating 5′-AML1-LAF4-3′, suggesting that 5′-AML1-LAF4-3′ is critical for leukemogenesis.

LAF4 was previously reported to be a fusion partner of MLL in pediatric B-precursor ALL with t(2;21)(q11;q23) (von Bergh et al., 2002; Bruch et al., 2003; Hiwatari et al., 2003). LAF4 is the first gene fused to both AML1 and MLL, and both AML1-LAF4 and MLL-LAF4 contained the same domains of LAF4 (Figure 5). During the preparation of this manuscript, we found another pediatric T-ALL patient with AML1-LAF4 reported in the Meeting Abstract (Abe et al., Blood (ASH Annual Meeting Abstracts) 2006; 108: 4276), suggesting that t(2;21)(q11;q23) is a recurrent cytogenetic abnormality and that the AML1-LAF4 fusion gene is associated with the T-ALL phenotype. Both putative fusion proteins of AML1-LAF4 observed in two patients contained the Runt domain of AML1, and the transactivation domain, nuclear localization sequence and C-terminal homology domain of LAF4, although the fused exon of LAF4 differed in the two cases. Several studies have reported that the fusion partners of MLL fused with different genes such as MLL-AF10 and CALM-AF10, MLL-CBP and MOZ-CBP or MLL-p300 and MOZ-p300 (Ida et al., 1997; Taki et al., 1997; Chaffanet et al., 2000). Comparison of the structure and function between AML1-LAF4 and MLL-LAF4 will facilitate our understanding of the molecular mechanisms underlying AML1- and MLL-related leukemia.

Figure 5.
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Schematic representation of putative AML1, LAF4 and AML1-LAF4 fusion proteins. Putative MLL-LAF4 fusion protein is also indicated for comparison. Arrows, break points or fusion points; AD, transactivation domain; AT, AT hooks; CHD, C-terminal homology domain; DNA, methyltransferase homology region; RD, RUNT domain; MT, DNA methyltransferase homology region; NLS, nuclear localization sequence.

Full figure and legend (43K)

The only AML1 fusion partners in T-ALL are LAF4 and FGA7. It is not known how FGA7 is associated with T-ALL leukemogenesis, because FGA7 does not show any significant sequence homology to any known protein motifs and/or domains (Mikhail et al., 2004). Both patients with AML1-LAF4 and MLL-LAF4 fusions were diagnosed as having ALL, but they have different lymphoid lineages. MLL-LAF4 is associated with B-lineage ALL; however, AML1-LAF4 generates T-ALL. Our previous study showed that LAF4 was expressed not only in B-lineage ALL but also in T-lineage ALL cell lines (Hiwatari et al., 2003). LAF4 showed strong sequence similarity to AF4 (Ma and Staudt, 1996), which has a role in the differentiation of both B and T cells in mice (Isnard et al., 2000). Furthermore, it was reported that AML1 also plays an important role in T- and B-cell differentiation, because AML1-deficient bone marrow increased defective T- and B-lymphocyte development (Ichikawa et al., 2004). These findings support that both AML1 and LAF4 are associated with T-ALL, respectively. Further functional analysis of the AML1-LAF4 fusion gene will provide new insights into the leukemogenesis of AML1-related T-ALL. Recently, it has been reported that C-terminal truncated AML1-related fusion proteins play critical roles in leukemogenesis (Yan et al., 2004, Agerstam et al., 2007), suggesting that the two additional types of fusion transcripts observed in our patient (types 2 and 3 in Figures 3d and 5) have an additional function in leukemogenesis other than that of the entire AML1-LAF4 fusion protein.

In this study, we first applied the panhandle PCR method, which is usually used for cloning the fusion partners of MLL or NUP98 (Megonigal et al., 2000; Taketani et al., 2002); however, no fusion transcripts could be obtained. Therefore, we searched for another method to clone the fusion transcripts and adapted the bubble PCR method for cDNA cloning. To date, bubble PCR has been performed for cloning unknown genomic fusion points but not fusion cDNAs (Zhang et al., 1995). Using double-stranded cDNA, we could apply the bubble PCR method for cloning fusion cDNA with fewer nonspecific products. The bubble PCR primer can only prime DNA synthesis after a first-strand cDNA has been generated by an AML1-specific primer because of the bubble-tag with an internal non-complementary region (Zhang et al., 1995). Although bubble PCR for genomic DNA generated one or two amplification products (Smith, 1992), bubble PCR for cDNA generated a complex set of amplification products that appeared as a smear by SYBR green staining, suggesting that a random hexamer generated various double-stranded cDNA containing the AML1 sequence. This means that various fusion points can be estimated, even if after bubble oligo ligation was generated. Furthermore, bubble PCR for cDNA could amplify in both 5′–3′ and 3′–5′ directions of the gene or transcript, and easily handle any exons fused to unknown partners for amplification. Once-ligated cDNAs are also available for cloning any genes, other than AML1, as the target. We demonstrated the efficiency and specificity of bubble PCR for cDNA (Table 1 and Supplementary Figure S2).


To date, a great number of fusion genes associated with chromosomal translocations have been cloned, although these fusion genes are found as a minor part of various malignancies. Recently, high frequencies of mutations in NOTCH1 in T-ALL (James et al., 2005), NPM in AML with normal karyotype (Weng et al., 2004) and JAK2 in myeloproliferative disorders (polycythemia vera, essential thrombocythemia and idiopathic myelofibrosis) (James et al., 2005) have been reported, and these mutations are considered to be a good target for therapy. These genes were first identified as associated with chromosomal translocations in a small subset of specific phenotypes of hematologic malignancies (Ellisen et al., 1991; Morris et al., 1994; Lacronique et al., 1997). These findings suggest that continuing attempts to identify genes associated with chromosomal translocations can be expected to provide further insights into the significance of various gene alterations in cancer and the development of novel-targeted therapies (Taki and Taniwaki, 2006). The bubble PCR method for cDNA will contribute to identifying numerous novel translocation partners more easily and further functional analysis of chimeric transcripts.

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Materials and methods

Spectral karyotyping analysis

Spectral karyotyping analysis was performed with a SkyPainting kit (Applied Spectral Imaging, Migdal Ha’Emek, Israel). Signal detection was performed according to the manufacturer's instructions.

Fluorescence in situ hybridization analysis

Fluorescence in situ hybridization analysis of the patient’s leukemic cells using AML1-specific BAC clones (RP11-272A3, 3′ of AML1 and RP11-994N6, 5′ of AML1) was carried out as described previously (Taniwaki et al., 1994). Fusion of AML1 and LAF4 was analysed with the patient’s leukemic cells using RP11-994N6 (5′ of AML1) and RP11-527J8 (3′ of LAF4).

Bubble PCR for cDNA

We modified the original bubble PCR method to apply for cDNA cloning (Figure 2; Supplementary Figure S2) (Smith, 1992; Zhang et al., 1995).

Poly(A)+ RNA was extracted from the patient’s leukemic cells using a QuickPrep Micro mRNA Purification Kit (GE Healthcare, Buckinghamshire, UK). Two hundred nanograms of poly(A)+ RNA was reverse transcribed to cDNA in a total volume of 33μl with random hexanucleotide using the Ready-To-Go You-Prime First-Strand Beads (GE Healthcare). Double-stranded cDNAs were synthesized from 10μl of single-stranded cDNA with a phosphorylated random hexanucleotide, blunt ended with T4 DNA polymerase, digested with RsaI endonuclease and ligated with bubble oligo. RsaI, a 4-bp blunt-ended cutter, was chosen to shorten the bubble oligo-ligated fragments, so that almost all bubble oligo-ligated fragments would be easy to clone by standard PCR reaction. This suggests that poor-quality samples are also suited to this method, although it is unsuitable for cloning long products.

The sequences of the primers used are listed in Supplementary Table S1 and their positions in the AML1 gene are shown in Supplementary Figure S2. Nested PCR was performed using primers NVAMP-1 (bubble oligo) and AML1-5S (exon 5) for first round PCR, and NVAMP-2 (bubble oligo) and AML1-E6S (exon 6) for nested PCR. NVAMP1 and NVAMP2 can only anneal to the newly synthesized unique sequence of the bubble oligo by AML1-5S.

We used poly(A)+ RNA in bubble PCR for cDNA with the expectation that this approach could amplify fewer transcripts; however, total RNA is also suitable for this method.

Bubble PCR for genomic DNA

Bubble PCR for genomic DNA was performed as described previously (Smith, 1992; Zhang et al., 1995). Primers were as follows: NVAMP-1 and AML1-GNM8S for first round PCR, and NVAMP-2 and AML1-GNM8-2S for second round PCR (Supplementary Table S1).

Reverse transcription–PCR and genomic PCR analyses

Reverse transcription–PCR and genomic PCR analyses were performed as described previously. After 35 rounds of PCR (30s at 94°C, 30s at 55°C, 1min at 72°C), 5μl of PCR product were electrophoresed in a 3% agarose gel. Primers were as follows: AML1-PR7 and LAF4-11AS, and AML1-PR8 and LAF4-PR5 for reverse transcription-PCR; and AML1-GNM8-4S and LAF4-GNM11-2AS, and LAF4-GNM11-2S and AML1-GNM8-2AS for genomic PCR (Supplementary Table S1).

Nucleotide sequencing

Nucleotide sequences of PCR products and, if necessary, subcloned PCR products were analysed as described previously (Hiwatari et al., 2003).

Southern blot analysis

High-molecular-weight DNA was extracted from the patient’s leukemic cells by proteinase K digestion and phenol/chloroform extraction. DNA (10μg) was digested with BglII, subjected to electrophoresis on 0.7% agarose gel and transferred to a nylon membrane. Blots were hybridized to probes that were labeled by the Dig-labeled PCR method according to the manufacturer's instructions (Roche Applied Science, Tokyo, Japan). Probes were 112bp AML1 cDNA fragments (AML1c1, nucleotides 1233–1344; GenBank accession no. NM_001754).

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Acknowledgements

We express our appreciation for the outstanding technical assistance of Kozue Sugimoto, Minako Goto and Kayoko Kurita. This work was supported by a grant-in-aid for Scientific Research (B) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and the Takeda Science Foundation.

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