Original Manuscript

Leukemia (2005) 19, 1948–1957. doi:10.1038/sj.leu.2403891; published online 18 August 2005

Oncogenes

CALM-AF10+ T-ALL expression profiles are characterized by overexpression of HOXA and BMI1 oncogenes

W A Dik1,5, W Brahim2,3,5, C Braun2,3, V Asnafi2,3, N Dastugue4, O A Bernard3, J J M van Dongen1, A W Langerak1, E A Macintyre2,3 and E Delabesse2,3

  1. 1Department of Immunology, Erasmus MC, Rotterdam, The Netherlands
  2. 2Department of Hematology, Université Paris-Descartes, Faculté de Médecine, Hôpital Necker-Enfants-Malades, Paris, France
  3. 3INSERM EMI0210, Hôpital Necker-Enfants-Malades, Paris, France
  4. 4Department of Hematology, Université Toulouse III Paul-Sabatier, Hôpital Purpan, Toulouse, France

Correspondence: Dr E Delabesse, Laboratoire d'Hématologie and INSERM EMI 0210, Hôpital Necker 149-161, rue de Sèvres, 75743 Paris cedex 15, France. Fax : +33 1443 81745; E-mail: eric.delabesse@nck.aphp.fr

5These authors contributed equally to this work

Received 3 June 2005; Accepted 31 June 2005; Published online 18 August 2005.

Top

Abstract

The t(10;11)(p13;q14–21) is found in T-ALL and acute myeloid leukemia and fuses CALM (Clathrin-Assembly protein-like Lymphoid-Myeloid leukaemia gene) to AF10. In order to gain insight into the transcriptional consequences of this fusion, microarray-based comparison of CALM-AF10+ vs CALM-AF10- T-ALL was performed. This analysis showed upregulation of HOXA5, HOXA9, HOXA10 and BMI1 in the CALM-AF10+ cases. Microarray results were validated by quantitative RT-PCR on an independent group of T-ALL and compared to mixed lineage leukemia-translocated acute leukemias (MLL-t AL). The overexpression of HOXA genes was associated with overexpression of its cofactor MEIS1 in CALM-AF10+ T-ALL, reaching levels of expression similar to those observed in MLL-t AL. Consequently, CALM-AF10+ T-ALL and MLL-t AL share a specific HOXA overexpression, indicating they activate common oncogenic pathways. In addition, BMI1, located close to AF10 breakpoint, was overexpressed only in CALM-AF10+ T-ALL and not in MLL-t AL. BMI1 controls cellular proliferation through suppression of the tumor suppressors encoded by the CDKN2A locus. This locus, often deleted in T-ALL, was conserved in CALM-AF10+ T-ALL. This suggests that decreased CDKN2A activity, as a result of BMI1 overexpression, contributes to leukemogenesis in CALM-AF10+ T-ALL. We propose to define a HOXA+ leukemia group composed of at least MLL-t, CALM-AF10 and HOXA-t AL, which may benefit from adapted management.

Keywords:

BMI1, HOX, MLL, CALM-AF10, PICALM, MLLT10

Top

Introduction

The t(10;11)(p13;q14–21) is a recurrent translocation found in acute myeloid leukemias (AML) and T-ALL. This translocation fuses CALM (Clathrin-Assembly protein-like Lymphoid-Myeloid leukemia gene, also called PICALM or CLTH) to AF10 (also called MLLT10).1, 2, 3 The presence of the CALM-AF10 fusion transcript is associated to immature features along lymphoid and myeloid differentiation and to poor prognosis,3 similar to that found in acute leukemias (AL) with MLL (mixed lineage leukemia gene, also called HRX, HTRX, TRX1 or ALL-1) translocations.2

CALM was first identified as the partner of AF10 in t(10;11)(p13;q14–21) leukemias.1 CALM is an ubiquitously expressed 652 amino-acid (aa) protein containing an Epsin N-terminal homologous (ENTH) motif, which mediates the interaction with clathrin during endocytosis.4 Defective CALM function blocks hematopoiesis and iron metabolism due to defective transferrin endocytosis.5 The ENTH domain is maintained in all CALM-AF10 fusion proteins. CALM has also been shown to be fused to MLL, in a unique case of t(10;11)(p13;q23) observed in an infant AML.6

AF10 was first identified as a fusion partner of MLL in AMLs.7, 8 AF10 is expressed ubiquitously and codes for a protein of 1027 aa. AF10 functions as transcription factor, binding DNA through an AT-hook motif.9 AF10 contains two zinc-fingers and one bipartite nuclear location signal, both located in the N-terminal region, and a leucine zipper domain and a glutamine-rich domain in the C-terminal region.9, 10 The C-terminal leucine zipper domain is conserved following AF10 fusion to MLL or CALM and has been demonstrated to be essential to the transformation properties of the MLL-AF10 fusion protein.11, 12

The analyses of rare cases of t(10;11) with the expression of CALM-AF10 but not of the AF10-CALM reciprocal transcript indicated that CALM-AF10 transcripts encode the crucial fusion transcript.3 Indeed, retroviral expression of CALM-AF10 was reported to induce biphenotypic AL in mice (Deshpande A et al. Blood 2003;102: 216a; abstract #758). We recently reported CALM-AF10 fusion in 9% of T-ALL, restricted to TCRitalic gammadelta-expressing and immature cases (IM, defined by the expression of cytoplasmic CD3 and CD7 and the absence of cytoplasmic TCRbeta or a TCR).13, 14 To gain insight into the consequences of the expression of the CALM-AF10 fusion transcript, we performed microarray analysis to compare TCRitalic gammadelta-expressing and immature T-ALL samples, with and without CALM-AF10 fusion transcripts. We show here that CALM-AF10+ T-ALL are associated with elevated expression of a subset of HOXA genes and some of their transcriptional and functional regulators, as well as a specific overexpression of the oncogene BMI1.

Top

Materials and methods

T-ALL samples used for DNA microarray analysis

In all, 23 T-ALL samples with greater than 90% blasts (except for sample T135) were analyzed using Affymetrix U133A microarrays (Table 1). They include 14 adults and nine children aged 15 years or less. Four cases were purified using either positive (CD34+ for UPN4925) or negative (CD3- for UPN1439, UPN792 and UPN3954) selection. Six CALM-AF10+ T-ALL cases (four IM, two TCRitalic gammadelta+) with four different breakpoints, as detailed in Table 1, were analyzed. These samples were compared to 17 CALM-AF10- T-ALL cases with similar phenotypic features (Table 1). They included nine IM T-ALL, three TCRitalic gammadelta+ T-ALL and five cases likely to be IM T-ALL based on a sCD3-, CD4-, CD8-, CD1a- phenotype. Nine out of 13 tested were LYL1+ by quantitative RT-PCR, one out of 23 was HOX11L2+ (UPN3580) and none of 18 analyzed were HOX11+ or SIL-TAL1. One out of 18 patients tested by Southern for MLL was rearranged (UPN3776, MLL-AF6+).


DNA microarray procedure and gene expression analysis

DNA microarray analysis was performed as described previously.15 Briefly, total RNA was isolated using RNeasy columns according to the manufacturer's instructions (Qiagen, Hilden, Germany). Double strand (ds) cDNA was then generated from 2 to 5 mug of RNA using SuperScript reverse transcriptase and a T7-oligo(dT) primer. An ENZO kit (Farmingdale, NY, USA) was used to convert the ds cDNA template into biotinylated cRNA utilizing T7 RNA polymerase and biotinylated ribonucleotides. The biotinylated cRNA was then cleaned from enzymes and unincorporated nucleotides using RNeasy 'clean-up' spin columns (Qiagen). An adjusted cRNA yield was calculated to correct for carryover of unlabeled total RNA. cRNA (11 mug) was fragmented and 10 mug of fragmented cRNA was subsequently hybridized to a U133A microarray (Affymetrix, Santa Clara, CA, USA) for 16 h at 45°C. After washing and staining with PE-conjugated streptavidin, the array fluorescence was determined using a HP/Affymetrix scanner.

The fluorescences of the U133A chips were analyzed using dChip 1.3 software (http://www.dchip.org/ and Li and Wong16). A supervised analysis based on CALM-AF10 fusion was performed after normalization using standard settings of dChip (experiment/baseline ratio higher or lower than 1.2 using lower 90% confidence bound of fold change, experiment (or baseline for lower expression) minus baseline (or experiment) higher than 100 and P-value for testing experiment=baseline lower than 0.05). Microarray data are available from Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/;17 GSE2117 CALM-AF10 T-ALL series; individual accession numbers from GSM38216 to GSM38238).

Quantitative RT-PCR analyses

In all, 37 T-ALL samples (10 CALM-AF10+ IM/TCRitalic gammadelta+ T-ALL and 27 CALM-AF10- T-ALL (five IM, nine TCRitalic gammadelta, 11 TCRalphabeta lineage and two not classified)) different from those used for the microarray experiments and 19 MLL-translocated acute leukemias (MLL-t AL) including five B-cell lineage ALL with MLL-AF4 and 14 AML (two MLL-AF6, six MLL-AF9, two MLL-AF10, three MLL-ELL and one MLL-ENL) were quantified using Taqman quantitative RT-PCR for HOXA5, HOXA9, HOXA10, ANXA2, BMI1, DNAJC1, COMMD3, SPAG6, CD69, NBS1 and MEIS1 (Supplementary Table 1). HOXA Taqman probe sets were selected for optimal specificity, with the forward primer and the probe being located in the first exon and the reverse primer in the second exon.

RNA was reverse transcribed as described.18 cDNA quality was checked using ABL and GUSB as control genes.18 cDNA with ABL Ct values higher than 30 were discarded. The expression of each gene was defined by calculating the ratio of expression of the gene of interest to the average of ABL and GUSB values. Quantitative RT-PCR was performed as described18 using an AppliedBiosystems 7700 PCR machine (Foster City, CA, USA).

Southern blot analysis of CDKN2A (p16INK4A/p19ARF) deletions

Southern blot analysis was performed as described previously.19 In all, 15 mug of isolated DNA were digested with EcoRI (Invitrogen). Restriction fragments were size separated in a 0.7% agarose gel and transferred by vacuum blotting to a Nytran SuPerCharge membrane (Schleicher & Schuell Bioscience GmbH, Dassel, Germany), which was subsequently hybridized with a 32P-labeled CDKN2A exon 2 probe.20

Top

Results

Genes underexpressed in CALM-AF10+ T-ALL

In the aim to understand the oncogenic process of CALM-AF10 in T-ALL, we performed a microarray analysis comparing six CALM-AF10+ T-ALL to 17 CALM-AF10- T-ALL with comparable phenotypic characteristics. Supervised analysis of the microarrays identified 102 probe sets, corresponding to 89 genes, which were significantly (P<0.05) underexpressed in CALM-AF10+ T-ALL compared to the other T-ALL samples (Figure 1a). Nine genes were detected by more than one probe set: AKR7A2, KIAA0368, NCF4, PLXND1, PRSS2, QKI, TNFSF10 and TRA1 (twice each) and NCR3 (six times). For all these nine genes, the different probe sets representing the same gene were always adjacent in the tree view (Figure 1a). The five genes most significantly associated with CALM-AF10- T-ALL (Pless than or equal to0.001) were GGH, ARL6IP4, NBS1, OGFR and TUBB. The five genes with the highest ratio between CALM-AF10+ and CALM-AF10- T-ALL were PRSS3 (18.4-fold lower in CALM-AF10+ T-ALL), UBE2E3 (nine-fold lower), PRSS2 (7.8-fold lower), IGJ (6.1-fold lower) and S100A8 (3.6-fold lower).

Figure 1.
Figure 1 - 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

(a) Underexpressed genes in CALM-AF10+ T-ALL compared to CALM-AF10- IM/TCRitalic gammadelta+ T-ALL. (b) Overexpressed genes in CALM-AF10+ T-ALL compared to CALM-AF10- IM/TCRitalic gammadelta+ T-ALL. Fluorescence of each sample is indicated by the variance around the mean. Red squares indicate overexpression and green squares underexpression. The scale is indicated at the bottom. Names of samples are indicated at the top. HUGO names or accession numbers are indicated on the right. A clustering tree view of the genes generated by dChip is shown on the left. Tables indicate the Affymetrix U133A probe set accession numbers, the genes, the GenBank accession numbers, the chromosomal locations, the mean and standard error of the normalized fluorescences of CALM-AF10+ and CALM-AF10- T-ALL samples, the fold changes between these two categories and the P-value generated by a t-test, as performed by dChip software.

Full figure and legend (691K)

Noteworthy, genes with lower expression in CALM-AF10+ T-ALL include CDK4, coding for a cycle-dependent kinase, CSF3R, coding for the G-CSF receptor, FANCL, coding for an ubiquitin E3 ligase mutated in Fanconi anemia, ICSBP1, coding for a protein involved in the interferon response, IFI16, coding for an interferon-inducible protein specifically expressed in lymphoid cells, LMO4, coding for a LIM only protein that is mainly expressed in T cells and neural cells, MCM2, MCM5 and MCM7, coding for members of a complex that licences the cell to do a replication cycle, NBS1, coding for a protein that is involved in DNA ds breakage repair and which is deficient in the Nijmegen breakage syndrome, NKG7, coding for a protein highly expressed in T and NK cells, POLD1, coding for the DNA polymerase delta whose mutation is associated with a mutator and cancer phenotype, PRSS2, coding for a trypsin and located inside the TCRB locus, TNFSF10, mainly known as TRAIL, coding for a ligand of TNF, which induces apoptosis when bound to TNF and TYROBP, coding for a protein containing an ITAM (Immunoreceptor Tyrosine-based Activation Motif) region.

The CALM gene itself is detectably underexpressed in CALM-AF10+ T-ALL (1.6-fold lower). The CALM probe set, 212506_at, is located in the final CALM exon 20, which is missing in all CALM-AF10 fusion transcripts identified so far.

Genes overexpressed in CALM-AF10+ T-ALL

Supervised analysis of the microarrays revealed that 48 out of 22 215 probe sets, corresponding to 44 genes, were significantly (P<0.05) overexpressed in CALM-AF10+ T-ALL when compared with CALM-AF10- T-ALL (Figure 1b). Three genes were identified by more than one probe set: HOXA9, HOXA10 (twice each) and ANXA2 (three times). The different probe sets representing each of these genes were always adjacent in the tree view (Figure 1b). The five genes with the most significant association with CALM-AF10+ T-ALL were HOXA9 (twice), BMI1, SOX4, SFRS6 and COMMD3 (Pless than or equal to0.001). The five genes with the highest ratio between CALM-AF10+ and CALM-AF10- T-ALL were TUBB (7.2-fold higher in CALM-AF10+T-ALL), HOXA9 (twice, 4.1- and 3.4-fold higher), HOXA5 (3.4-fold higher), PNMA2 (2.8-fold higher) and TCF8 (2.7-fold higher). Three HOXA genes, HOXA5, HOXA9 and HOXA10, were identified as overexpressed. HOXA5 and HOXA9 are clustered together in the tree view, whereas HOXA10 is located at the other end of the tree (Figure 1b). Two overexpressed genes, BMI1 and COMMD3 (also known as BMI1 upstream protein), are located at the 10p12.2 chromosomal band, the region of AF10 breakpoint and are clustered close to each other within the same branch (Figure 1b). Finally, TUBB was found twice in the array analysis, once underexpressed (probe set 212320_at) and once overexpressed (probe set 204141_at) in CALM-AF10+ T-ALL. The beta-tubulin genes constitute a large family of genes with up to 20 members.21 The probe set 212320_at matches completely with the TUBB gene located in 6p21.33 in the final exon and also with two pseudogenes in 13q14.11 and 1p32.3. The probe set 204141_at matches completely only with the TUBB gene located in 6p25, in the final exon. These two probe sets did not crosshybridize with each other according to Blast analysis. So, one TUBB gene located at 6p25 is significantly overexpressed in CALM-AF10+ T-ALL, and one, probably located at 6p21.33, is significantly underexpressed (although signal coming from pseudogenes cannot be formally excluded).

Noteworthy, genes overexpressed in CALM-AF10+ T-ALL include BMI1, coding for a member of the Polycomb (PcG) family, CD69, coding for an early T-cell activation antigen, CDKN1B (also known as KIP1), coding for a CDK inhibitor, FTH1, coding for the ferritin heavy chain, GADD45A, coding for a gene induced by p53, HOX genes (HOXA5, HOXA9 and HOXA10), JUNB, coding for a transcription factor, PIK3R1 (also known as p85), coding for a regulatory chain of the phosphatidylinositol 3 kinase, PSCD1, coding for a gene specifically expressed in T and NK cells, SKIL, coding for an oncogene also known as SNO, involved in the TGFbeta pathway regulation, SOX4, a frequent retroviral integration site involved in mouse retroviral insertional mutagenesis and TCF8, coding for a negative regulator of interleukin-2 expression.

HOX expression in CALM-AF10+ T-ALL

As CALM-AF10 expression appeared to be associated with overexpression of HOXA5, HOXA9 and HOXA10 genes, we analyzed more precisely the expression of HOX genes using data coming from the microarrays. We extracted the normalized fluorescence of all HOX genes present on the U133A chip, calculated the average and the standard error for CALM-AF10+ and CALM-AF10- cases and plotted it according to their genomic location. Only six HOX genes out of 39 are absent from the U133A microarray (Figure 2, Supplementary Table 2). HOXA5, HOXA9 and HOXA10 were clearly overexpressed when compared to the other members of the HOXA cluster. Surprisingly, HOXA6 and HOXA7, which are located between HOXA5 and HOXA9, were not overexpressed. None of the paralogous genes for HOXA5 (HOXB5, HOXC5), HOXA9 (HOXB9, HOXD9) and HOXA10 (HOXC10, HOXD10) were overexpressed in CALM-AF10+ T-ALL. A slight underexpression of HOXB9, HOXC11 and HOXC13 was seen (Figure 2, Supplementary Table 2). Therefore, the overexpression of HOX genes in CALM-AF10+ T-ALL seems to be restricted to HOXA5, HOXA9 and HOXA10, as judged from microarray data analyses.

Figure 2.
Figure 2 - 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

Plotting of the Affymetrix U133A normalized fluorescence of HOX genes according to their location (average and standard error). Representation of the four clusters of HOX genes found in the human genome, HOXA at 7p15, HOXB at 17q21, HOXC at 12q13 and HOXD at 2q31. The names and orientation of HOX genes are indicated at the top of each cluster. Open arrows indicate the data available and closed arrows indicate data missing from microarrays. CALM-AF10+ T-ALL data are drawn with a continuous line and CALM-AF10- T-ALL data are drawn with a dashed line.

Full figure and legend (134K)

Overexpression of HOXA5, HOXA9 and HOXA10 by independent analysis in CALM-AF10+ T-ALL

Owing to the important role of genes such as HOXA and BMI1 in oncogenesis, we decided to validate the association between CALM-AF10 and genes overexpressed according to the microarray analysis by quantitative RT-PCR in an independent group of T-ALL samples composed of 10 CALM-AF10+ and 27 CALM-AF10- T-ALL samples (14 IM/TCRitalic gammadelta and 11 TCRalphabeta lineage cases). As HOXA overexpression was previously shown to be associated with MLL-t AL,22, 23, 24, 25, 26 an additional set of 19 MLL-t AL samples was also analyzed.

HOXA5, HOXA9 and HOXA10 expression levels (medians of 134, 197 and 126%, respectively) were significantly higher in CALM-AF10+ than in CALM-AF10- T-ALL samples (medians of 0% (P<0.001, using a Mann–Whitney U-test for an identical hypothesis), 2% (P<0.001) and 0% (P<0.001), respectively, Figure 3a and Supplementary Table 3), thus confirming our microarray data. The expression remained significantly different when CALM-AF10+ T-ALL were compared separately to CALM-AF10- IM/TCRitalic gammadelta or TCRalphabeta lineage T-ALL samples.

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

Independent quantitative RT-PCR. (a) Quantitative RT-PCR of HOXA5, HOXA9, HOXA10, MEIS1 and BMI1. (b) Quantitative RT-PCR of DNAJC1, COMMD3, BMI1 and SPAG6. The ratio of genes expression relative to two ubiquitously expressed genes (GUSB and ABL) was calculated for each sample. Three categories of samples are compared: CALM-AF10+ T-ALL (CA+, n=10), CALM-AF10- T-ALL (CA-, n=27) and MLL-t AL (MLL, n=19). Results are presented as a box plot graph using a logarithmic scale. The boundary of the box closest to zero indicates the 25th percentile, a line within the box marks the median, and the boundary of the box farthest from zero indicates the 75th percentile. Narrow horizontal bars above and below the box indicate the 90th and 10th percentiles. Outliers are indicated as dots, but are included in the calculation. P-values of the results are indicated only if significant (P<0.05), otherwise labeled as NS (nonsignificant), at the top using a Mann–Whitney U-test for an identical hypothesis. At the bottom of (b), the genomic organization around the AF10 breakpoint is presented either as germline (upper) or translocated (lower).

Full figure and legend (150K)

Importantly, expression levels of HOXA5, HOXA9 and HOXA10 in CALM-AF10+ T-ALL were similar to those detected in MLL-t AL samples (medians of 61% (P=0.164), 223% (P=0.668) and 119% (P=0.875), respectively, Figure 3a and Supplementary Table 3). Together, these data confirm that CALM-AF10+ T-ALL are significantly associated with HOXA5, HOXA9 and HOXA10 overexpression, and in that respect resemble to MLL-t AL.

Overexpression of MEIS1 in CALM-AF10+ T-ALL

MEIS1 expression is tightly associated to HOXA9 expression in MLL-t AL27, 28, 29 and encodes an essential cofactor of HOXA9 function in normal and malignant processes. Owing to the close relationship between HOXA9 and MEIS1, we decided to quantify MEIS1 expression. Although MEIS1 was not identified using stringent statistical assays, its expression level accessed by microarray showed a 1.9-fold increase in CALM-AF10+ T-ALL with respect to CALM-AF10- samples T-ALL (data not shown). In the independent cohort of AL samples, MEIS1 expression was significantly higher in CALM-AF10+ T-ALL compared to CALM-AF10- T-ALL and MLL-t AL (medians of expression of 386%, 2%, (P<0.001) and 144% (P=0.035), respectively, Figure 3a and Supplementary Table 3). The expression was also significantly different when CALM-AF10+ T-ALL were compared separately to either CALM-AF10- IM/TCRitalic gammadelta or TCRalphabeta lineage T-ALL samples. We therefore demonstrate that the association between HOXA9 and MEIS1 expression described in MLL-t AL is also found in CALM-AF10+ T-ALL.

Overexpression of BMI1 by independent analysis in CALM-AF10+ T-ALL

Beside HOXA genes, we were interested to confirm the overexpression of BMI1 in CALM-AF10+ T-ALL as BMI1 was shown recently to be essential to the proliferation of leukemic stem cells.30, 31 We quantified BMI1 expression levels on the same independent cohort used for the quantification of HOXA genes. BMI1 expression in CALM-AF10+ T-ALL was significantly higher than in CALM-AF10- T-ALL (medians of expression of 1099% and 196% (P<0.001), Figure 3a and Supplementary Table 3). The expression was also significantly different when CALM-AF10+ T-ALL were compared separately to either IM/TCRitalic gammadelta or TCRalphabeta lineage CALM-AF10- T-ALL samples. BMI1 expression in CALM-AF10+ samples was also higher than in MLL-t AL samples (median of 61% (P<0.001)). Noteworthy, the two MLL-AF10 samples analyzed in the MLL-t AL group had a BMI1 expression of 296 and 42%, lower than the median of expression in CALM-AF10+ samples and within the range of values from the MLL-t AL group. BMI1 is therefore specifically overexpressed in CALM-AF10+ T-ALL.

Overexpression of genes close to the der(10) breakpoint by quantitative RT-PCR

The BMI1 gene is located close to the AF10 gene on chromosome 10, as is COMMD3, which was also overexpressed in CALM-AF10+ T-ALL. For that reason, we decided to investigate the expression of the genes located within a 500 kb genomic area of chromosome 10, between AF10 and SPAG6 (a gene immediately centromeric to BMI1; see Figure 3b). This region contains four genes, DNAJC1, COMMD3, BMI1 and SPAG6 (Figure 3b).

The expression of all four genes (DNAJC1, COMMD3, BMI1 and SPAG6) was significantly increased in CALM-AF10+ T-ALL compared to CALM-AF10- T-ALL (medians of expression of 168% vs 35% (P=0.005), 167% vs 75% (P=0.003), 1099% vs 196% (P<0.001), 144% vs 1% (P<0.001), respectively). This result is in keeping with an upregulation resulting from a cis-activation effect due to the vicinity of the translocation breakpoint (Supplementary Table 3 and Figure 3b).

The CDKN2A (p16INK4A/p19ARF) region is not deleted in CALM-AF10+ T-ALL

The CDKN2A locus encodes the cell cycle regulators and tumor suppressors p16INK4A and p19ARF and BMI1 has been demonstrated to be a potent negative regulator of this locus.32 As CDKN2A is usually deleted in more than 70% of T-ALL,33 we decided to verify if this main target of BMI1 was conserved in CALM-AF10+ T-ALL. Owing to limited amounts of DNA, the CDKN2A locus could only be examined in 13 T-ALL cases of which four contained a CALM-AF10 fusion. Southern blot analysis revealed that the locus was maintained in all tested CALM-AF10+ T-ALL samples, while it was deleted in six out of nine CALM-AF10- T-ALL. As described previously, CDKN2A deletions in the CALM-AF10- T-ALL cases were accompanied by deletions of the CDKN2B locus (encoding p15INK4B), which is also recognized by the probe used.20

Top

Discussion

We demonstrated in this study that CALM-AF10 T-ALL are characterized by overexpression of HOXA5, HOXA9, HOXA10, MEIS1 and BMI1.

Bona fide transcription factors of the homeobox family (HOX genes) share 61 aa helix–turn–helix DNA-binding domain, the homeodomain34 and are clustered in four genomic regions (HOXA, HOXB, HOXC and HOXD), containing a total of 39 different HOX genes grouped in 13 paralogous groups. Their expression is tightly controlled at the transcription level. The involvement of HOX genes during normal and malignant cellular proliferation has been demonstrated in experimental models and in humans. In mice, the homozygous deletion of HOXA9 exclusively disrupted hematopoiesis, reducing the number of leukocytes, lymphocytes and thymocytes, without affecting the number of earlier progenitors.35, 36

Overexpression of HOXA9 induces myeloid leukemias, as demonstrated by retroviral insertional mutagenesis in BXH2 mice28 and retroviral transfection of HOXA9 in mouse bone marrow.37 Overexpression of HOXA9 was shown to enhance myelopoiesis and to block the differentiation of B cells but not T cells.38 Specific overexpression of HOXA9 directed to B or T cells did not induce leukemia, suggesting that HOXA9 per se is preferentially a myeloid oncogene.38 In humans, HOX genes, especially HOXA9, have clearly been shown to be upregulated in MLL-t AL samples.22, 23, 24, 25, 26 Transformation by MLL-ENL was shown to be dependent on HOXA9.39 In keeping with this observation, wild-type MLL binds directly to the HOXA9 promoter and methylates histone H3 at lysine by its C-terminal SET domain.40, 41, 42 More directly, HOX genes are overexpressed after fusion with T-cell receptor genes in T-ALL43, 44 or when fused to the NUP98 gene45, 46 as a result of chromosomal translocations. Our results demonstrate a clear association of HOXA5, HOXA9 and HOXA10 overexpression in T-ALL and the CALM-AF10 fusion gene, indicating a common target between MLL-t and CALM-AF10 oncoproteins. Recently, leukemic transformation by MLL-AF10 was shown to be mediated by the recruitment of a methyltransferase, hDOT1L, to its AF10 region. The upregulation of HOXA9 induced by MLL-AF10 was dependent of the MLL binding to the HOXA9 locus and the association of AF10 with hDOT1L.12 As no obvious DNA-binding domain exists in CALM, the mechanism of activation of HOXA9 by CALM-AF10 should be analyzed further.

HOXA9 function is modulated by interaction with several cofactors like MEIS1. MEIS1 is a member of the TALE (3 aa loop extensions) homeodomain proteins composed of MEIS1, MEIS2, MEIS3, PBX1, PBX2, PBX3 and PREP1/KNOX1.28, 37, 47, 48 Coinfection of HOXA9 and MEIS1 enhances the capacity of myeloid transformation by HOXA937 and synergistically blocks myeloid differentiation.49 Probably due to the relatively small size of our population, upregulation of MEIS1 was not identified upon stringent statistical criteria used for the analysis of our microarray data (despite a 1.9-fold increase in CALM-AF10+ T-ALL). However, in our study overexpression of MEIS1 in CALM-AF10+ T-ALL was clearly demonstrated by quantitative PCR on a larger number of samples, demonstrating a very strong association between HOXA9 and MEIS1. In addition to HOXA9, CALM-AF10+ T-ALL also overexpressed HOXA5 and HOXA10. Overexpression of HOXA10 has previously been shown to interfere with myeloid and lymphoid differentiation and can induce AML.50, 51 Recently, HOXA10 was also found to be one of the most prominent transcriptional activation targets in T-ALL that harbor fusions between TCRB and the HOXA cluster.43, 44 Together, these observations strongly support a role for HOXA genes in T-ALL oncogenesis.

Overexpression of HOXA9 and MEIS1 is a recurrent feature of MLL fusion leukemia, but upregulation of BMI1 has not been reported in MLL-translocated samples. We demonstrated here that the overexpression of BMI1 is a unique feature of CALM-AF10+ T-ALL. BMI1 was first identified as a target in retroviral mutagenesis, inducing T-cell lymphoma in association with c-MYC.52 Recently, BMI1 was shown to be essential to the self-renewal of normal and leukemic stem cells.30, 31, 53 The BMI1 protein is part of a Polycomb complex including also MEL18, Mph1/Rae28, M33, SCMH1 and Ring1A/B.53 BMI1 is the only member of this complex that is specifically upregulated in CALM-AF10+ T-ALLs (data not shown). Keeping with this result, BMI1 was shown to be the only Polycomb gene whose forced expression was associated with a marked increase of self-renewal of hematopoietic stem cells.53

BMI1 controls cellular proliferation by inhibiting the expression of the CDKN2A locus that encodes the tumor suppressors p16ink4a and p19arf.32 CDKN2A is more frequently deleted in T-ALL than in other subtypes of AL, namely in more than 70% of cases.20 Such a deletion would obviously preclude an antiproliferative effect of BMI1 in the CALM-AF10+ samples. However, using Southern blot analysis, we found no CDKN2A deletion in CALM-AF10+ T-ALL. This suggests that overexpression of BMI1 has functional effects similar to that of CDKN2A deletion.

In CALM-AF10+ T-ALL, aberrant expression of BMI1 could be due to a cis effect, as the BMI1 gene is located in the vicinity of the chromosome 10 breakpoint. In keeping with this hypothesis, transcriptional cis activation is a classical form of activation in T-ALL.54 CALM-AF10+ T-ALL showed overexpression of several AF10 downstream neighboring genes (DNAJC1, COMMD3, BMI1 and SPAG6) located close to the AF10 gene breakpoint. To our knowledge, this is the first study to report on upregulation of genes downstream from a fusion gene in leukemia. From these four AF10 downstream genes, BMI1 is the only one known to be involved in oncogenesis. Two MLL-AF10 AML samples were analyzed in our series, but they did not demonstrate high expression of these genes. In fact, the MLL and AF10 genes lie in an opposite orientation on their respective chromosome and their in-frame fusion requires at least two chromosomal breaks. Therefore, MLL-AF10 translocations are frequently associated with complex karyotypes, rearrangements and deletions at the breakpoints.55, 56, 57, 58, 59 It is conceivable that t(10;11)(p12;q23) translocation frequently results in the deletion of BMI1 from the translocated chromosome 10 or an opposite orientation regarding MLL-AF10 fusion. Possible deletion of BMI1 by t(10;11)(p12;q23) could be assessed by FISH analysis, but unfortunately we were unable to do so because of lack of material for the two MLL-AF10 cases.

Finally, activation of HOXA genes by CALM-AF10, MLL fusions or HOXA translocations seems to be a common leukemic pathway. The difference of phenotype (myeloid, B- or T-cell markers expression) could merely reflect the stage of maturation arrest, without reflecting the oncogenic mechanism. We propose to define a HOXA+ leukemia group composed of at least MLL-translocated, CALM-AF10 and HOXA-translocated ALs, which may benefit from adapted management (Figure 4).

Figure 4.
Figure 4 - 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

The pattern of overexpression in CALM-AF10+ T-ALL is similar to that of MLL-t AL, essentially overexpression of HOXA genes, as also in AML with NUP98-HOXA9. These fusion genes could be seen as members of a common transformation pathway dysregulating HOXA9 and its downstream targets.

Full figure and legend (12K)

Top

References

  1. Dreyling MH, Martinez-Climent JA, Zheng M, Mao J, Rowley JD & Bohlander SK. The t(10;11)(p13;q14) in the U937 cell line results in the fusion of the AF10 gene and CALM, encoding a new member of the AP-3 clathrin assembly protein family. Proc Natl Acad Sci USA 1996; 93: 4804−4809. | Article | PubMed | ChemPort |
  2. Dreyling MH, Schrader K, Fonatsch C, Schlegelberger B, Haase D & Schoch C et al.. MLL and CALM are fused to AF10 in morphologically distinct subsets of acute leukemia with translocation t(10;11): both rearrangements are associated with a poor prognosis. Blood 1998; 91: 4662−4667. | PubMed | ISI | ChemPort |
  3. Narita M, Shimizu K, Hayashi Y, Taki T, Taniwaki M & Hosoda F et al.. Consistent detection of CALM-AF10 chimaeric transcripts in haematological malignancies with t(10;11)(p13;q14) and identification of novel transcripts. Br J Haematol 1999; 105: 928−937. | Article | PubMed | ISI | ChemPort |
  4. Tebar F, Bohlander SK & Sorkin A. Clathrin assembly lymphoid myeloid leukemia (CALM) protein: localization in endocytic-coated pits, interactions with clathrin, and the impact of overexpression on clathrin-mediated traffic. Mol Biol Cell 1999; 10: 2687−2702. | PubMed | ISI | ChemPort |
  5. Klebig ML, Wall MD, Potter MD, Rowe EL, Carpenter DA & Rinchik EM. Mutations in the clathrin-assembly gene Picalm are responsible for the hematopoietic and iron metabolism abnormalities in fit1 mice. Proc Natl Acad Sci USA 2003; 100: 8360−8365. | Article | PubMed | ChemPort |
  6. Wechsler DS, Engstrom LD, Alexander BM, Motto DG & Roulston D. A novel chromosomal inversion at 11q23 in infant acute myeloid leukemia fuses MLL to CALM, a gene that encodes a clathrin assembly protein. Genes Chromosomes Cancer 2003; 36: 26−36. | Article | PubMed | ISI | ChemPort |
  7. Chaplin T, Ayton P, Bernard OA, Saha V, Della Valle V & Hillion J et al.. A novel class of zinc finger/leucine zipper genes identified from the molecular cloning of the t(10;11) translocation in acute leukemia. Blood 1995; 85: 1435−1441. | PubMed | ISI | ChemPort |
  8. Chaplin T, Bernard O, Beverloo HB, Saha V, Hagemeijer A & Berger R et al.. The t(10;11) translocation in acute myeloid leukemia (M5) consistently fuses the leucine zipper motif of AF10 onto the HRX gene. Blood 1995; 86: 2073−2076. | PubMed | ISI | ChemPort |
  9. Linder B, Newman R, Jones LK, Debernardi S, Young BD & Freemont P et al.. Biochemical analyses of the AF10 protein: the extended LAP/PHD-finger mediates oligomerisation. J Mol Biol 2000; 299: 369−378. | Article | PubMed | ISI | ChemPort |
  10. Saha V, Chaplin T, Gregorini A, Ayton P & Young BD. The leukemia-associated-protein (LAP) domain, a cysteine-rich motif, is present in a wide range of proteins, including MLL, AF10, and MLLT6 proteins. Proc Natl Acad Sci USA 1995; 92: 9737−9741. | PubMed | ChemPort |
  11. DiMartino JF, Ayton PM, Chen EH, Naftzger CC, Young BD & Cleary ML. The AF10 leucine zipper is required for leukemic transformation of myeloid progenitors by MLL-AF10. Blood 2002; 99: 3780−3785. | Article | PubMed | ISI | ChemPort |
  12. Okada Y, Feng Q, Lin Y, Jiang Q, Li Y & Coffield VM et al.. hDOT1L links histone methylation to leukemogenesis. Cell 2005; 121: 167−178. | Article | PubMed | ISI | ChemPort |
  13. Asnafi V, Radford-Weiss I, Dastugue N, Bayle C, Leboeuf D & Charrin C et al.. CALM-AF10 is a common fusion transcript in T-ALL and is specific to the TCRgammadelta lineage. Blood 2003; 102: 1000−1006. | Article | PubMed | ISI | ChemPort |
  14. Asnafi V, Beldjord K, Libura M, Villarese P, Millien C & Ballerini P et al.. Age-related phenotypic and oncogenic differences in T-cell acute lymphoblastic leukemias may reflect thymic atrophy. Blood 2004; 104: 4173−4180. | Article | PubMed | ISI | ChemPort |
  15. Staal FJ, van der Burg M, Wessels LF, Barendregt BH, Baert MR & van den Burg CM et al.. DNA microarrays for comparison of gene expression profiles between diagnosis and relapse in precursor-B acute lymphoblastic leukemia: choice of technique and purification influence the identification of potential diagnostic markers. Leukemia 2003; 17: 1324−1332. | Article | PubMed | ISI | ChemPort |
  16. Li C & Wong WH. Model-based analysis of oligonucleotide arrays: expression index computation and outlier detection. Proc Natl Acad Sci USA 2001; 98: 31−36. | Article | PubMed | ChemPort |
  17. Barrett T, Suzek TO, Troup DB, Wilhite SE, Ngau WC & Ledoux P et al.. NCBI GEO: mining millions of expression profiles − database and tools. Nucleic Acids Res 2005; 33: D562−D566. | Article | PubMed | ISI | ChemPort |
  18. Beillard E, Pallisgaard N, van der Velden VH, Bi W, Dee R & van der Schoot E et al.. Evaluation of candidate control genes for diagnosis and residual disease detection in leukemic patients using 'real-time' quantitative reverse-transcriptase polymerase chain reaction (RQ-PCR) − a Europe against cancer program. Leukemia 2003; 17: 2474−2486. | Article | PubMed | ISI | ChemPort |
  19. van Dongen JJ & Wolvers-Tettero IL. Analysis of immunoglobulin and T cell receptor genes. Part I: basic and technical aspects. Clin Chim Acta 1991; 198: 1−91. | Article | PubMed | ChemPort |
  20. Hebert J, Cayuela JM, Berkeley J & Sigaux F. Candidate tumor-suppressor genes MTS1 (p16INK4A) and MTS2 (p15INK4B) display frequent homozygous deletions in primary cells from T- but not from B-cell lineage acute lymphoblastic leukemias. Blood 1994; 84: 4038−4044. | PubMed | ISI | ChemPort |
  21. Lee MG, Lewis SA, Wilde CD & Cowan NJ. Evolutionary history of a multigene family: an expressed human beta-tubulin gene and three processed pseudogenes. Cell 1983; 33: 477−487. | Article | PubMed | ISI | ChemPort |
  22. Rozovskaia T, Feinstein E, Mor O, Foa R, Blechman J & Nakamura T et al.. Upregulation of Meis1 and HoxA9 in acute lymphocytic leukemias with the t(4:11) abnormality. Oncogene 2001; 20: 874−878. | Article | PubMed | ISI | ChemPort |
  23. Armstrong SA, Staunton JE, Silverman LB, Pieters R, den Boer ML & Minden MD et al.. MLL translocations specify a distinct gene expression profile that distinguishes a unique leukemia. Nat Genet 2002; 30: 41−47. | Article | PubMed | ISI | ChemPort |
  24. Yeoh EJ, Ross ME, Shurtleff SA, Williams WK, Patel D & Mahfouz R et al.. Classification, subtype discovery, and prediction of outcome in pediatric acute lymphoblastic leukemia by gene expression profiling. Cancer Cell 2002; 1: 133−143. | Article | PubMed | ISI | ChemPort |
  25. Ferrando AA, Armstrong SA, Neuberg DS, Sallan SE, Silverman LB & Korsmeyer SJ et al.. Gene expression signatures in MLL-rearranged T-lineage and B-precursor acute leukemias: dominance of HOX dysregulation. Blood 2003; 102: 262−268. | Article | PubMed | ISI | ChemPort |
  26. Rozovskaia T, Ravid-Amir O, Tillib S, Getz G, Feinstein E & Agrawal H et al.. Expression profiles of acute lymphoblastic and myeloblastic leukemias with ALL-1 rearrangements. Proc Natl Acad Sci USA 2003; 100: 7853−7858. | Article | PubMed | ChemPort |
  27. Zeisig BB, Milne T, Garcia-Cuellar MP, Schreiner S, Martin ME & Fuchs U et al.. Hoxa9 and Meis1 are key targets for MLL-ENL-mediated cellular immortalization. Mol Cell Biol 2004; 24: 617−628. | Article | PubMed | ISI | ChemPort |
  28. Nakamura T, Largaespada DA, Shaughnessy JD, Jr, Jenkins NA & Copeland NG. Cooperative activation of Hoxa and Pbx1-related genes in murine myeloid leukaemias. Nat Genet 1996; 12: 149−153. | Article | PubMed | ISI | ChemPort |
  29. Lawrence HJ, Rozenfeld S, Cruz C, Matsukuma K, Kwong A & Komuves L et al.. Frequent co-expression of the HOXA9 and MEIS1 homeobox genes in human myeloid leukemias. Leukemia 1999; 13: 1993−1999. | PubMed | ISI | ChemPort |
  30. Park IK, Qian D, Kiel M, Becker MW, Pihalja M & Weissman IL et al.. Bmi-1 is required for maintenance of adult self-renewing haematopoietic stem cells. Nature 2003; 423: 302−305. | Article | PubMed | ISI | ChemPort |
  31. Lessard J & Sauvageau G. Bmi-1 determines the proliferative capacity of normal and leukaemic stem cells. Nature 2003; 423: 255−260. | Article | PubMed | ISI | ChemPort |
  32. Jacobs JJ, Kieboom K, Marino S, DePinho RA & van Lohuizen M. The oncogene and Polycomb-group gene bmi-1 regulates cell proliferation and senescence through the ink4a locus. Nature 1999; 397: 164−168. | Article | PubMed | ISI | ChemPort |
  33. Cayuela JM, Madani A, Sanhes L, Stern MH & Sigaux F. Multiple tumor-suppressor gene 1 inactivation is the most frequent genetic alteration in T-cell acute lymphoblastic leukemia. Blood 1996; 87: 2180−2186. | PubMed | ISI | ChemPort |
  34. Abate-Shen C. Deregulated homeobox gene expression in cancer: cause or consequence? Nat Rev Cancer 2002; 2: 777−785. | Article | PubMed | ISI | ChemPort |
  35. Lawrence HJ, Helgason CD, Sauvageau G, Fong S, Izon DJ & Humphries RK et al.. Mice bearing a targeted interruption of the homeobox gene HOXA9 have defects in myeloid, erythroid, and lymphoid hematopoiesis. Blood 1997; 89: 1922−1930. | PubMed | ISI | ChemPort |
  36. Izon DJ, Rozenfeld S, Fong ST, Komuves L, Largman C & Lawrence HJ. Loss of function of the homeobox gene Hoxa-9 perturbs early T-cell development and induces apoptosis in primitive thymocytes. Blood 1998; 92: 383−393. | PubMed | ISI | ChemPort |
  37. Kroon E, Krosl J, Thorsteinsdottir U, Baban S, Buchberg AM & Sauvageau G. Hoxa9 transforms primary bone marrow cells through specific collaboration with Meis1a but not Pbx1b. EMBO J 1998; 17: 3714−3725. | Article | PubMed | ISI | ChemPort |
  38. Thorsteinsdottir U, Mamo A, Kroon E, Jerome L, Bijl J & Lawrence HJ et al.. Overexpression of the myeloid leukemia-associated Hoxa9 gene in bone marrow cells induces stem cell expansion. Blood 2002; 99: 121−129. | Article | PubMed | ISI | ChemPort |
  39. Ayton PM & Cleary ML. Transformation of myeloid progenitors by MLL oncoproteins is dependent on Hoxa7 and Hoxa9. Genes Dev 2003; 17: 2298−2307. | Article | PubMed | ISI | ChemPort |
  40. Nakamura T, Mori T, Tada S, Krajewski W, Rozovskaia T & Wassell R et al.. ALL-1 is a histone methyltransferase that assembles a supercomplex of proteins involved in transcriptional regulation. Mol Cell 2002; 10: 1119−1128. | Article | PubMed | ISI | ChemPort |
  41. Milne TA, Briggs SD, Brock HW, Martin ME, Gibbs D & Allis CD et al.. MLL targets SET domain methyltransferase activity to Hox gene promoters. Mol Cell 2002; 10: 1107−1117. | Article | PubMed | ISI | ChemPort |
  42. Martin ME, Milne TA, Bloyer S, Galoian K, Shen W & Gibbs D et al.. Dimerization of MLL fusion proteins immortalizes hematopoietic cells. Cancer Cell 2003; 4: 197−207. | Article | PubMed | ISI | ChemPort |
  43. Speleman F, Cauwelier B, Dastugue N, Cools J, Verhasselt B & Poppe B et al.. A new recurrent inversion, inv(7)(p15q34), leads to transcriptional activation of HOXA10 and HOXA11 in a subset of T-cell acute lymphoblastic leukemias. Leukemia 2005; 19: 358−366. | Article | PubMed | ISI | ChemPort |
  44. Soulier J, Clappier E, Cayuela JM, Regnault A, Garcia-Pedro M & Dombret H et al.. HOXA genes are included in genetic and biological networks defining human acute T-cell leukemia (T-ALL). Blood 2005; 106: 274−286. | Article | PubMed | ISI | ChemPort |
  45. Nakamura T, Largaespada DA, Lee MP, Johnson LA, Ohyashiki K & Toyama K et al.. Fusion of the nucleoporin gene NUP98 to HOXA9 by the chromosome translocation t(7;11)(p15;p15) in human myeloid leukaemia. Nat Genet 1996; 12: 154−158. | Article | PubMed | ISI | ChemPort |
  46. Borrow J, Shearman AM, Stanton VP, Jr, Becher R, Collins T & Williams AJ et al.. The t(7;11)(p15;p15) translocation in acute myeloid leukaemia fuses the genes for nucleoporin NUP98 and class I homeoprotein HOXA9. Nat Genet 1996; 12: 159−167. | Article | PubMed | ISI | ChemPort |
  47. Owens BM & Hawley RG. HOX and non-HOX homeobox genes in leukemic hematopoiesis. Stem Cells 2002; 20: 364−379. | Article | PubMed | ISI | ChemPort |
  48. Shen WF, Rozenfeld S, Kwong A, Kom ves LG, Lawrence HJ & Largman C. HOXA9 forms triple complexes with PBX2 and MEIS1 in myeloid cells. Mol Cell Biol 1999; 19: 3051−3061. | PubMed | ISI | ChemPort |
  49. Fujino T, Yamazaki Y, Largaespada DA, Jenkins NA, Copeland NG & Hirokawa K et al.. Inhibition of myeloid differentiation by Hoxa9, Hoxb8, and Meis homeobox genes. Exp Hematol 2001; 29: 856−863. | Article | PubMed | ISI | ChemPort |
  50. Thorsteinsdottir U, Sauvageau G, Hough MR, Dragowska W, Lansdorp PM & Lawrence HJ et al.. Overexpression of HOXA10 in murine hematopoietic cells perturbs both myeloid and lymphoid differentiation and leads to acute myeloid leukemia. Mol Cell Biol 1997; 17: 495−505. | PubMed | ISI | ChemPort |
  51. Bjornsson JM, Andersson E, Lundstrom P, Larsson N, Xu X & Repetowska E et al.. Proliferation of primitive myeloid progenitors can be reversibly induced by HOXA10. Blood 2001; 98: 3301−3308. | Article | PubMed | ISI | ChemPort |
  52. Haupt Y, Bath ML, Harris AW & Adams JM. bmi-1 transgene induces lymphomas and collaborates with myc in tumorigenesis. Oncogene 1993; 8: 3161−3164. | PubMed | ISI | ChemPort |
  53. Iwama A, Oguro H, Negishi M, Kato Y, Morita Y & Tsukui H et al.. Enhanced self-renewal of hematopoietic stem cells mediated by the polycomb gene product Bmi-1. Immunity 2004; 21: 843−851. | Article | PubMed | ISI | ChemPort |
  54. Rabbitts TH. Chromosomal translocation master genes, mouse models and experimental therapeutics. Oncogene 2001; 20: 5763−5777. | Article | PubMed | ISI | ChemPort |
  55. Beverloo HB, Le Coniat M, Wijsman J, Lillington DM, Bernard O & de Klein A et al.. Breakpoint heterogeneity in t(10;11) translocation in AML-M4/M5 resulting in fusion of AF10 and MLL is resolved by fluorescent in situ hybridization analysis. Cancer Res 1995; 55: 4220−4224. | PubMed | ISI | ChemPort |
  56. Angioni A, La Starza R, Mecucci C, Sprovieri T, Matteucci C & De Rossi G et al.. Interstitial insertion of AF10 into the ALL1 gene in a case of infant acute lymphoblastic leukemia. Cancer Genet Cytogenet 1998; 107: 107−110. | Article | PubMed | ISI | ChemPort |
  57. Chaplin T, Jones L, Debernardi S, Hill AS, Lillington DM & Young BD. Molecular analysis of the genomic inversion and insertion of AF10 into MLL suggests a single-step event. Genes Chromosomes Cancer 2001; 30: 175−180. | Article | PubMed | ISI | ChemPort |
  58. Van Limbergen H, Poppe B, Janssens A, De Bock R, De Paepe A & Noens L et al.. Molecular cytogenetic analysis of 10;11 rearrangements in acute myeloid leukemia. Leukemia 2002; 16: 344−351. | Article | PubMed | ISI | ChemPort |
  59. Klaus M, Schnittger S, Haferlach T, Dreyling M, Hiddemann W & Schoch C. Cytogenetics, fluorescence in situ hybridization, and reverse transcriptase polymerase chain reaction are necessary to clarify the various mechanisms leading to an MLL-AF10 fusion in acute myelocytic leukemia with 10;11 rearrangement. Cancer Genet Cytogenet 2003; 144: 36−43. | Article | PubMed | ISI | ChemPort |
Top

Acknowledgements

ED is supported by Fondation de France, Comité Leucémie, l'Association de Recherche contre le Cancer et la Ligue Nationale contre le Cancer, comité de Paris. WAD, JJMvD and AWL are supported by the Dutch Cancer Foundation (EMCR 2002-2707), AWL is supported in part by the Haak Bastiaanse Kuneman Foundation. We thank Dr Frank JT Staal from the Department of Immunology, Erasmus MC, Rotterdam, for technical advice on microarray analysis, Dr Jean-Philippe Jais from the Department of Biostatistics, Hôpital Necker-Enfants-Malades, for statistical support, Dr Jean-Michel Cayuela from the Laboratory of Hematology, Hôpital Saint-Louis, Paris, for the gift of CDKN2A probe and Pr Ali Saad from the Laboratory of Cytogenetics, Farhat Hached hospital, Sousse, Tunisia, for valuable help.

Supplementary Information

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