The recurrent SET-NUP214 fusion as a new HOXA activation mechanism in pediatric T-cell acute lymphoblastic leukemia - PubMed (original) (raw)

. 2008 May 1;111(9):4668-80.

doi: 10.1182/blood-2007-09-111872. Epub 2008 Feb 25.

Martine van Grotel, Joëlle Tchinda, Charles Lee, H Berna Beverloo, Peter J van der Spek, Andrew Stubbs, Jan Cools, Kyosuke Nagata, Maarten Fornerod, Jessica Buijs-Gladdines, Martin Horstmann, Elisabeth R van Wering, Jean Soulier, Rob Pieters, Jules P P Meijerink

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The recurrent SET-NUP214 fusion as a new HOXA activation mechanism in pediatric T-cell acute lymphoblastic leukemia

Pieter Van Vlierberghe et al. Blood. 2008.

Abstract

T-cell acute lymphoblastic leukemia (T-ALL) is mostly characterized by specific chromosomal abnormalities, some occurring in a mutually exclusive manner that possibly delineate specific T-ALL subgroups. One subgroup, including MLL-rearranged, CALM-AF10 or inv (7)(p15q34) patients, is characterized by elevated expression of HOXA genes. Using a gene expression-based clustering analysis of 67 T-ALL cases with recurrent molecular genetic abnormalities and 25 samples lacking apparent aberrations, we identified 5 new patients with elevated HOXA levels. Using microarray-based comparative genomic hybridization (array-CGH), a cryptic and recurrent deletion, del (9)(q34.11q34.13), was exclusively identified in 3 of these 5 patients. This deletion results in a conserved SET-NUP214 fusion product, which was also identified in the T-ALL cell line LOUCY. SET-NUP214 binds in the promoter regions of specific HOXA genes, where it interacts with CRM1 and DOT1L, which may transcriptionally activate specific members of the HOXA cluster. Targeted inhibition of SET-NUP214 by siRNA abolished expression of HOXA genes, inhibited proliferation, and induced differentiation in LOUCY but not in other T-ALL lines. We conclude that SET-NUP214 may contribute to the pathogenesis of T-ALL by enforcing T-cell differentiation arrest.

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Figures

Figure 1

Figure 1

Gene expression profiles of 92 T-ALL patients. (A) Differentially expressed genes among the major molecular cytogenetic T-ALL subgroups (TAL1, LMO2, HOXA, HOX11/TLX1, and HOX11L2/TLX3; n = 67). The significance level (Wilcoxon P value) and FDR corrected P value for the top 100 genes in each T-ALL subgroup is indicated. TAL1, HOX11, and HOX11L2 subgroups show significant differentially expressedprobesets. (B) Cluster analysis of 92 patients with T-ALL (67 with known, 25 with unknown) based upon the top 25 most significant probesets for the TAL1, TAL1/LMO2, HOX11, and HOX11L2 subgroups combined with 15 HOXA probesets as previously described. (C) Principal component analyses shows clustering of the patients with unknown T-ALL along the molecular cytogenetic known patients: 1 _HOX11L2_-like, 19 _TAL1/LMO2_-like, and 5 _HOXA_-like patients.

Figure 2

Figure 2

Submicroscopic del(9)(q34.11q34.13) in T-ALL. (A) Chromosome 9 ideogram and corresponding oligo array-CGH plots of test DNA–control DNA ratios (blue tracing) versus the dye-swap experiment (red tracing) for patient no. 126. Detailed analyses of the centromeric and telomeric breakpoints show involvement of SET and NUP214. (B) Similar array-CGH plot for patient no. 120. Centromeric and telomeric breakpoints show involvement of SET and ABL1. (C) Overview of oligoarray-CGH results in the potential breakpoint regions for 3 patients with T-ALL with del(9)(q34.11q34.13). The 60-mer oligonucleotide probes present on the array-CGH slide and located in the telomeric and centromeric breakpoint regions, as well as the specific genes located in this region with their transcription direction, are shown. Abbreviations: N; normal, L; loss. Dual-color FISH analysis of patient no. 125 (D) and no. 120 (E) using the LSI BCR-ABL ES translocation probe.

Figure 3

Figure 3

SET-NUP214 fusion transcript in T-ALL. (A) RT-PCR analysis using _SET_- and _NUP214_-specific primers and GAPDH primers as internal control, reveals a specific SET-NUP214 fusion gene in T-ALL patient nos. 125, 126, and 120; the patient with AUL; and the T-ALL cell line LOUCY. NUP214-ABL1 fusion was detected in patient nos. 120 and no.88 and in the T-ALL cell line PEER (B) Sequence analysis confirmed an identical fusion between exon 7 of SET and exon 18 of NUP214 in all SET-NUP214+ patients with T-ALL, the patient with AUL, and the LOUCY cell line. (C) Western blot analysis of T-ALL cell lines revealed a SET-NUP214 fusion in the cell line LOUCY. (D) At the protein level, the breakpoints are situated in the acidic tail of SET and the coiled-coil domain of NUP214.

Figure 4

Figure 4

HOXA activation in SET-NUP214+ T-ALL. (A) Relative HOXA9 expression levels by RQ-PCR for _MLL_-rearranged, CALM-AF10+, inv(7)(p15q34), _TAL1_-, _LMO2_-, _HOX11L2_-, or _HOX11_-rearranged patients and T-ALL cell lines including LOUCY, MOLT13, SKW3, HPB-ALL, HSB2, and PEER. (B) Comparison of HOXA9 expression levels between the HOXA T-ALL subgroup (MLL, CALM-AF10, inv(7)(p15q34), and SET-NUP214; n = 10) and other T-ALL subgroups (TAL1, LMO2, HOX11L2, or HOX11). The horizontal lines represent the medians. (C) Relative expression levels of HOXA genes by RQ-PCR for the 3 SET-NUP214+ patients with T-ALL, the LOUCY cell line, the patient with AUL, and SKW3. (D) Heatmap of the 20 significant and differentially expressed probesets with a false discovery rate (FDR) lower than 5% for the HOXA cluster compared with the other patients with T-ALL.

Figure 5

Figure 5

siRNA knockdown of SET and SET-NUP214 in the LOUCY T-ALL cell line. (A) SET expression using Western blot analysis 4 days after electroporation with the following conditions: no siRNA, control siRNA, siRNA SET exon 8, or siRNA SET exon 5. (B) Relative SET and SET-NUP214 expression by RQ-PCR after 2, 4, and 7 days for conditions as mentioned in panel A. (C) Relative expression of all members of the HOXA clusters by RQ-PCR (except for HOXA11 and HOXA13) after 4 days for conditions as mentioned in panel A. (D) Optical density (OD) values relative to control cells without pulse after 7 days for conditions as mentioned in panel A. (E) Total cell numbers after 7 days for conditions as mentioned in panel A. (F) Percentage of cell death relative to control cells without pulse after 7 days for conditions as mentioned in panel A. Error bars visualize the standard error of the mean (SEM). (G) FACS analysis 6 days after electroporation with either no siRNA, siRNA SET exon 8, or siRNA SET exon 5 for TCRγδ and membrane CD3. The percentage of cells positive for TCRγδ or mCD3 expression are visualized in quadrant 4.

Figure 6

Figure 6

ChIP and coIP analysis of T-ALL cell lines LOUCY and SKW3. (A) ChIP analysis of SKW3, LOUCY, and LOUCY 4 days after electroporation with siRNA SET exon 5, for promoter sequences of HOXA1, HOXA3, HOXA9, HOXA10, and HOXA11. The amount of bound DNA was calculated relative to the 5% input DNA in anti-NUP214 and anti-acetyl histone H3 immunoprecipitates, whereas no antibody and anti-FLAG immunoprecipitates were used as negative control. Error bars represent SEM. (B) Western blot analysis of NUP214 and PP32 immunoprecipitates of the cell line LOUCY using anti-SET. (C) Similar Western blot analysis as in panel B for CRM1 and DOT1L immunoprecipitates in LOUCY. (D) Western blot analysis of NUP214 immunoprecipitates of the cell line LOUCY using anti-AF10.

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