Deregulation of DUX4 and ERG in acute lymphoblastic leukemia

Abstract

Chromosomal rearrangements deregulating hematopoietic transcription factors are common in acute lymphoblastic leukemia (ALL). Here we show that deregulation of the homeobox transcription factor gene DUX4 and the ETS transcription factor gene ERG is a hallmark of a subtype of B-progenitor ALL that comprises up to 7% of B-ALL. DUX4 rearrangement and overexpression was present in all cases and was accompanied by transcriptional deregulation of ERG, expression of a novel ERG isoform, ERGalt, and frequent ERG deletion. ERGalt uses a non-canonical first exon whose transcription was initiated by DUX4 binding. ERGalt retains the DNA-binding and transactivation domains of ERG, but it inhibits wild-type ERG transcriptional activity and is transforming. These results illustrate a unique paradigm of transcription factor deregulation in leukemia in which DUX4 deregulation results in loss of function of ERG, either by deletion or induced expression of an isoform that is a dominant-negative inhibitor of wild-type ERG function.

Figure 1. Gene expression profile and ERG deletions in DUX4/ERG ALL

a, Hierarchical clustering of the top 100 Affymetrix U133A probe sets upregulated in each subtype of 199 B- and T-lineage ALL cases, including DUX4/ERG ALL. b, Principal component analysis of FPKM gene expression data derived from poly-A RNA sequencing identifying DUX4/ERG ALL c, SNP microarray data of cases with ERG deletions. Data for each case is shown as a column, and shown in a log₂ ratio scale where deletions are blue. The position of the ERG locus is shown as an arrow on the left of the panel, and genomic coordinates shown in megabases on the right. Cases are ordered from left to right according to the extent of the deletion, with focal exon 1 deletions on the left, the common exons 3–7 and 3–9 deletions in the middle of the panel, and whole gene deletion on the right. d, Representative DNA copy number data of four cases with ERG deletion also shown in log₂ ratio scale, with probe level data shown, deletion being below the x axis. The extent of deletion in each case is shown by horizontal bars.

Figure 2. Rearrangement of DUX4

a, deregulation of DUX4 is observed exclusively in the ALL cases with ERG deregulation. Gene expression data shown as fragments per kilobase of mapped reads (FPKM) from RNA-sequencing in box plots, with ALL cases grouped by subtype on the x axis. Horizontal lines show median, and the boxes, interquartile range. b, an example of the commonly complex rearrangements of IGH and DUX4. Each third of the panel shows RNA-seq gene expression data for three loci involved in the rearrangement. The top shows increased expression at DUX4. The dotted line shows the breakpoint of DUX4 that is juxtaposed to a small segment of CDH4 shown in the middle panel, which in turn is rearranged to IGH, shown in the lower panel. c, schematic showing the location of the breakpoints in DUX4, stratified by age group. d, Immunoblotting of cell line and primary leukemic sample lysates showing proteins of variable size corresponding to DUX4 C-terminal truncation and/or appending with amino acid residues encoded by read through into the IGH locus.

Figure 3. Structural and sequence alterations in DUX4/ERG ALL

Heatmap showing genomic data for DUX4/ERG ALL cases, each of which is represented in a column and genes are grouped by functional pathway. The colors for each type of genetic alterations are shown at the bottom of the figure. The genomic profiling performed, and presence/absence of ERGalt, ALE and DUX4 rearrangement are shown at the top of the figure, where yellow represents assay performed (or alteration present), white, not performed or absent, and gray, data not available.

Figure 4. Expression of ERGalt in DUX4/ERG ALL

a, read depth from mRNA-sequencing of an ERG ALL case with expression of exon 6 alt (red) and a case lacking ERG alt expression (gray). b, RT-PCR with PCR primers specific to exon 6 alt and exon 10, showing amplification of a larger amplicon in ALL cases lacking ERG alt, with amplification of the intervening intronic sequence between ERGalt and exon 7, and a smaller amplicon in ERGalt positive cases arising from splicing from exon 6 alt to exon 7, shown in a representative electropherogram. This transcript results in a novel N-terminus of ERG encoded by exon 6 alt comprising 7 residues spliced in frame to exons 7–10. c, structure of the canonical and ERG ALL-associated ERG transcripts and isoforms. d, relative abundance of ERG transcripts in ERG ALL and other B-ALL subtypes. Each column represents a case. The top panel represents expression of wild type (WT) ERG, and the middle panel expression of ERGalt, and the less abundant ERGalt isoforms. The lower panel shows the cumulative abundance of all isoforms as a proportion within each case. Transcripts arising from splicing across ERG deletion are low abundance in most cases with ERG deletion (with the exception of one case with biallelic deletion. Non-coding ERGalt isoforms are more common in ERG ALL cases lacking ERG deletion.

Figure 5. DUX4 induces deregulation of ERG

a, DUX4 ChIP-seq and ATAC-sequencing data of Reh, NALM6 and DUX4/ERG ALL xenograft showing DUX4 binding at ERG exon 6 alt (arrowed) in NALM6 but not in Reh. ATAC sequencing showing open chromatin at this peak of DUX4 binding at ERG exon 6 alt in the DUX4/ERG xenograft sample and NALM6 but not in Reh. ATAC-seq data is provided in Supplementary Figure 19. b–c, expression of two truncated DUX4 alleles, but not empty vector, results in expression of ERGalt by RT-PCR (b) and immunoblotting (c). b, the top panel shows RT-PCR for ERG using primers specific for exon 6 alt and exon 7. In untransduced or empty vector transduced Reh cells, a larger amplicon is observed that incorporates the intervening intronic transcript (lanes 3 and 4). In DUX4/ERG ALL cells (PARLRH, lane 2) and DUX4-transduced Reh cells (lanes 5 and 6), ERGalt is amplified, with in-frame splicing from exon 6 alt to exon 7 (lower part of panel). c, Immunoblotting showing expression of ERGalt in cells transduced with DUX4. The top part of the panel shows immunoblotting with an N-terminus DUX4-specific antibody; the middle part blotting with a C-terminus specific ERG antibody, and the lower part, actin control. DUX4 alleles induce expression of ERGalt in HEK293T cells (lanes 6 and 7) and Reh cells (lanes 11 and 12). Lane 5 shows 293T cells transfected with ERGalt virus as a positive control for ERGalt (and negative for DUX4); lane 8 shows DUX4/ERG patient sample PARLRH positive for DUX4 and ERGalt.

Figure 6. Expression of ERGalt induces ALL

a, Transplantation of lineage negative Arf^−/− progenitors induces erythromegakaryoblastic leukemia, and ERGalt expressing marrow a lymphoid progenitor, biphenotypic or B-lymphoid leukemia. *** P< 0.0001 (Mantel-Cox); n= 12 ERG WT, 19 ERGalt mice from 2 independent experiments. b, immunoblotting with a C-terminus specific ERG antibody for ERG ALL samples and splenocytes of mouse leukemias, showing expression of ERG alt in human ERG ALL and mouse tumors induced by expression of ERG alt. c, proportion of leukemias displaying erythromegakaryoblastic, lymphoid, or mixed immunophenotype (mixed = lymphoid and myeloid subpopulations). d, representative immunophenotyping of ERG-induced tumors showing expression of the erythroid marker Ter119 in an ERG WT induced tumor, and coexpression of B220 and CD19 in ERGalt induced leukemia. Cells were gated on GFP expression.