Integrated genome-wide genotyping and gene expression profiling reveals BCL11B as a putative oncogene in acute myeloid leukemia with 14q32 aberrations

Abstract

Acute myeloid leukemia is a neoplasm characterized by recurrent molecular aberrations traditionally demonstrated by cytogenetic analyses. We used high density genome-wide genotyping and gene expression profiling to reveal acquired cryptic abnormalities in acute myeloid leukemia. By genome-wide genotyping of 137 cases of primary acute myeloid leukemia, we disclosed a recurrent focal amplification on chromosome 14q32, which included the genes BCL11B, CCNK, C14orf177 and SETD3, in two cases. In the affected cases, the BCL11B gene showed consistently high mRNA expression, whereas the expression of the other genes was unperturbed. Fluorescence in situ hybridization on 40 cases of acute myeloid leukemia with high BCL11B mRNA expression [2.5-fold above median; 40 out of 530 cases (7.5%)] revealed 14q32 abnormalities in two additional cases. In the four BCL11B-rearranged cases the 14q32 locus was fused to different partner chromosomes. In fact, in two cases, we demonstrated that the focal 14q32 amplifications were integrated into transcriptionally active loci. The translocations involving BCL11B result in increased expression of full-length BCL11B protein. The BCL11B-rearranged acute myeloid leukemias expressed both myeloid and T-cell markers. These biphenotypic acute leukemias all carried FLT3 internal tandem duplications, a characteristic marker of acute myeloid leukemia. BCL11B mRNA expression in acute myeloid leukemia appeared to be strongly associated with expression of other T-cell-specific genes. Myeloid 32D(GCSF-R) cells ectopically expressing Bcl11b showed decreased proliferation rate and less maturation. In conclusion, by an integrated approach involving high-throughput genome-wide genotyping and gene expression profiling we identified BCL11B as a candidate oncogene in acute myeloid leukemia.

Figure 1.

Research design. GEP: gene expression profiling.

Figure 2.

Identification of interstitial amplifications on 14q32.2 using SNPExpress. (A) Sequential alignment of the genotypes with copy numbers from the Affymetrix DNA mapping array of chromosome 14q32.2 of four AML samples. The copy numbers are shown for each individual patient by horizontal lines (n=0, 1, 2, 3, 4). The single nucleotide polymorphism genotypes are sequentially aligned along the chromosome (AA: red; BB: yellow; AB: blue, no call: white). Gains (default n>2.5) are depicted as the pink background. Gene expression levels are visualized as vertical white bars at the chromosomal position of the gene-specific probe set. In the event that multiple probe sets span the same region in the chromosome-wide view the vertical gene expression bars are green and proportional to the highest expression value. The green boxes represent exons of the encoded genes, and the arrows indicate the orientation. In AML #2301 and AML #7073 clear amplifications are visible, whereas these aberrations are absent in the two control case of AML. (B) Snapshot of SNPExpress illustrating the amplified region in AML cases #2301 and #7073 from Figure 2A, showing the genes located within the amplified regions. C14orf177 and BCL11B are amplified in both AML cases, whereas SETD3 and CCNK are amplified only in AML #7073. BCL11B expression is increased in AML #2301 and #7073 as indicated by the green bar (multiple probe sets), whereas BCL11B expression is absent in control AML cases (Figure 2A).

Figure 3.

FISH analysis of AML cases #2301 and #7073 using probes specific for BCL11B and flanking BCL11B. (A) Schematic representation of the fluorescein isothiocyanate-labeled BAC probe (RP11 431B1) covering the BCL11B locus and Texas red-labeled BAC probe (RP11 242A7) covering the region adjacent to BCL11B. (B) Microscope images of FISH analysis performed on metaphase chromosomes of AML cases #2301 and #7073 showing additional green signal (RP11431B1) indicative of an extra copy of the BCL11B locus.

Figure 4.

Western blot analysis of BCL11B in AML case #2301. Western blot analysis with a BCL11B-specific antibody demonstrates high expression of full-length BCL11B in AML case #2301, in the nuclear compartment (upper panel). Whole cell lysates from Jurkat, an acute T-cell leukemia cell line, and AML #2238 show high BCL11B expression. AML cases #2195 and #2240 with low BCL11B mRNA expression were used as negative controls (#2301_w:whole cell lysate; #2301_c: cytoplasmic lysate; #2301_n: nuclear lysate). β-actin was used as a loading control (lower panel).

Figure 5.

Correlation view based on gene expression profiling of 530 AML cases. (A) Pearson correlation view of 530 AML cases showing gene expression correlation based on 2847 probe sets. The black bars indicate expression of BCL11B 1: BCL11B expression: 219528_s_at and 2: BCL11B expression: 222895_s_at, where the size of the bars is proportional to the levels of BCL11B expression; 3: selected AML with BCL11B overexpression (>2.5-fold mean). The BCL11B-rearranged cases #2301, #6451, #6366 and #7073 are indicated by arrows. (B) FISH analysis performed on metaphase spreads of AML case #6451 showing disassociation of the probe RP11242A7 (red) and RP11431B1 (green) indicating translocation of BCL11B.

Figure 6.

Western blot analyses for BCL11B primary AML. Immuno-detection of the BCL11B protein in AML cases with elevated levels of BCL11B mRNA (+) and cases with undetectable levels of BCL11B mRNA (−) (upper panel; Jurkat cell lysate as positive control). GAPDH was used as the loading control (lower panel).

Figure 7.

Effects of BCL11B overexpression in murine 32D(GCSF-R) cells. (A) Western blot analyses for BCL11B in 32D(GCSFR) cells. 32D(GCSFR) clones overexpressing BCL11B are indicated by #2, #4, and #12 (interleukin 3 for 1 and 10 days). Lysates obtained from these clones were immunostained for BCL11B at day 1 and day 10 [Jurkat cells: positive control; 32D: 32D(GCSF-R) cells]. GAPDH was used as the loading control (lower panel). (B) Growth curve of 32D(GCSFR) cells with (squares, dashed line) and without (round, solid line) BCL11B expression and parental 32D(GCSFR) cells (triangles, dotted line) incubated with interleukin 3. 32D cells were counted every 24 h for 10 days. (C) May-Grünwald-Giemsa-stained cytospins of 32D(GCSF-R) cells with (upper panel) and without (lower panel) BCL11B expression incubated with granulocyte colony-stimulating factor for 7 days. Granulocytic differentiation is monitored by the presence of cells with segmented nuclei.