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.

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Figure 1: Gene expression profile and ERG deletions in DUX4/ERG ALL.
Figure 2: Rearrangement of DUX4.
Figure 3: Structural and sequence alterations in DUX4/ERG ALL.
Figure 4: Expression of ERGalt in DUX4/ERG ALL.
Figure 5: DUX4 induces deregulation of ERG.
Figure 6: Expression of ERGalt induces ALL.

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Acknowledgements

We thank L. Yang (University of Washington, Seattle) for the gpIX reporter construct and the Genome Sequencing Facility, the Hartwell Center for Bioinformatics and Biotechnology, the Flow Cytometry and Cell Sorting core facility and the Biorepository of St. Jude Children's Research Hospital.

This work was supported in part by the American Lebanese Syrian Associated Charities of St. Jude Children's Research Hospital, by a Stand Up to Cancer Innovative Research Grant and a St. Baldrick's Foundation Scholar Award (to C.G.M.), by a St. Baldrick's Consortium Award (to S.P.H.), by a Leukemia and Lymphoma Society Specialized Center of Research grant (to S.P.H. and C.G.M.), by a Lady Tata Memorial Trust Award (to I.I.), by a Leukemia and Lymphoma Society Special Fellow Award and Alex's Lemonade Stand Foundation Young Investigator Awards (to K.G.R.), by American Society of Hematology Scholar Awards (to C.G.M., P.N. and K.G.R.), by Dutch Cancer Society Fellowship KUN2012-5366 (to E. Waanders), by a St. Luke's Life Science Institute grant (to H.Y.), by National Cancer Institute grants P30 CA021765 (St. Jude Cancer Center Support Grant), U10 CA180820 (ECOG-ACRIN Operations), and CA180827 and CA196172 (to E.P.); U10 CA180861 (to C.D.B. and G.M.); U24 CA196171 (The Alliance NCTN Biorepository and Biospecimen Resource); CA145707 (to C.L.W. and C.G.M.); U01 CA157937 (to C.L.W. and S.P.H.), R00 CA188293 (to P.N.); and grants to the Children's Oncology Group: U10 CA98543 (Chair's grant and supplement to support the COG ALL TARGET project), U10 CA98413 (Statistical Center) and U24 CA114766 (Specimen Banking); and by National Institute of General Medical Sciences grant P50 GM115279 (to J.Z., J.Y., W.E.E., M.V.R., M.L.L. and C.G.M.). This project has been funded in whole or in part by federal funds from the National Cancer Institute, US National Institutes of Health, under contract HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the US Department of Health and Human Services, nor does mention of trade names, commercial products or organizations imply endorsement by the US government.

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J.Z., B.X., G.W., Yu Liu, L.W., Y. Li, C.Q., J. Wen, M.E., J.M., G.S., X.C., S.N., X.M., M.R., P.G., L.D., C.L., K.G.R., Y.T., R.C.H. and C.G.M. analyzed genomic data. K. McCastlain, I.I., H.Y., Y.C., D.P.-T., M.L.C., K.B.K., S.T., E. Waanders, E. Wienholds, P.N., S.B., J. Wang, I.A., K.G.R., J.E., H.L.M., K.B., B.V., J.D., Yanling Liu, M.L.V., R.C.H. and I.-M.L.C. performed experiments. R.S.F., L.F., K.O., E.R.M., R.K.W. and J.R.D. performed genome sequencing. M.D., D.P. and C.C. performed biostatistical analysis. J.Y., W.E.E., M.V.R., C.-H.P., S.J., C.L.W., G.M., C.D.B., J.K., K. Mrózek, E.P., M.S.T., W.S., M.C.F., J.R., J.M.R., S.L., S.M.K., S.A.S., S.C.R., S.P.H., M.L.L. and J.R.D. provided patient samples and data. J.E.D. provided reagents. J.Z., J.E.D. and C.G.M. designed experiments. J.Z. and C.G.M. wrote the manuscript.

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Correspondence to Jinghui Zhang or Charles G Mullighan.

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A list of members appears in the Supplementary Note.

Integrated supplementary information

Supplementary Figure 1 ERG is temporally regulated during B cell development.

(a) Schema of Hardy stages of mouse B cell maturation. (b) Representative schema for FACS of B cell progenitor populations. (c) Heat map of genes upregulated at the Hardy A to B transition, showing upregulation of Erg as well as key genes in B cell development, including Ebf1, Pax5 and Rag1. (d) Heat map of the genes significantly upregulated at each developmental transition. Data were first reported in Holmfeldt et al.35.

Supplementary Figure 2 Unsupervised clustering of Affymetrix U133A microarray data identifies a distinct subtype of B-ALL.

Unsupervised clustering of 16,395 U133A expression probe sets passing the Affymetrix MAS5 absent call filter for 199 ALL cases, showing clustering of the novel (DUX4/ERG) subtype of ALL. Clustering of arrays and probe sets was performed in Genemaths XT 2.1 using a Pearson similarity coefficient and the UPGMA clustering algorithm.

Supplementary Figure 3 NALM-6 cells exhibit the gene expression profile of DUX4/ERG ALL.

Clustering of the top 100 differentially expressed Affymetrix U133A/B gene expression probe sets for DUX4/ERG ALL in 32 acute leukemia cell lines examined in duplicate.

Supplementary Figure 4 Mapping and verification of IGHDUX4 rearrangement breakpoints.

Mapping of IGH breakpoints from whole-genome sequencing data. (a) The number of cases with discordant paired-end reads between chromosome 14 and one of the chromosomes with a DUX array (e.g., chromosomes 4 and 10). (b) CNVs identified by CONSERTING77 at the IGH locus. (c) Histone 3 lysine 27 acetylation data from ENCODE indicating the locations for super-enhancers. (d) IGH gene annotation. Representative sequencing electropherograms from genomic PCR in a and RT–PCR and capillary sequencing in b from two cases showing the C-terminal breakpoint of DUX4 rearranged to IGH, with intervening non-template nucleotides arising from RAG-mediated recombination and read-through into the IGH locus.

Supplementary Figure 5 Clinical outcome of DUX4/ERG ALL.

Kaplan–Meier plots of event-free survival (EFS) for children with ALL treated on St. Jude Children’s Research Hospital and Children’s Oncology Group protocols, showing the excellent outcome of DUX4/ERG cases (red lines). (a,b) Stratification by ALL subtype. (c,d) EFS for DUX4/ERG cases stratified by the presence or absence of IKZF1 alterations.

Supplementary Figure 6 Mutational spectrum of DUX4/ERG ALL.

Representative protein-domain plots showing the location of mutations for selected genes.

Supplementary Figure 7 RT–PCR and immunoblotting for ERG isoforms.

(a) RT–PCR for ERG transcripts using primers specific for exons 1 and 10, showing amplification of internally deleted transcripts. (b) Immunoblotting using an antibody specific to the C terminus showing the aberrant ERG peptide fragment (approximately 28 kDa) in novel cases in the test cohort, but not in non-novel B- and T-lineage ALL cases. (c) Immunoblotting of lysates from HEK293T cells transfected with MSCV-IRES-GFP vector expressing ERG alt (lane 2), several DUX4/ERG samples with (lanes 3 and 4) or without (lanes 5–7) deletion, and non-DUX4/ERG ALL samples (lanes 8 and 9). The size of the protein expressed from the cloned ERGalt transcript is identical to that of the protein expressed in patients, confirming that this transcript encodes the aberrant C-terminal ERG protein fragment observed in leukemic cells.

Supplementary Figure 8 Detailed analysis of ERG exon 6 alt, the alternative first exon in the ERGalt transcript.

RNA–seq read coverage, the region mapped by RACE and RT–PCR, and read pair mapping at the ERGalt first exon and canonical exon 7 in SJERG026. Coverage for RNA–seq is shown at the top, with the black line marking the exon boundary determined by RACE and RT–PCR. RNA–seq read pairs were sorted by ERGalt exon start site. The forward and reverse reads in a read pair are shown in red and blue, respectively. Cyan color indicates a ‘skipped’ region, i.e., a spliced intron, while gray color indicates a region where a forward read overlaps with a reverse read in a read pair. The black arrow points to the transcription initiation site determined on the basis of RACE that shows a strong bias for reverse reads, as expected from a first exon. The initiation site also matches the highest RNA–seq coverage peak in this region. A small proportion of reads aligned in forward orientation were from unspliced read pairs for which the reverse reads clustered to two additional potential alternative transcription start sites for ERGalt (marked by gray arrows).

Supplementary Figure 9 ERG transcriptional levels and intron retention in DUX4/ERG cases.

RNA–seq coverage (y axis) in the genomic region encoding ERG exons 5–10 (RefSeq accession NM_182918) for three tumors with ERG alt expression (top) and three tumors without ERG alt expression (bottom). The location of the ERGalt exon is marked in red and indicated by an arrow. Samples appended with an asterisk (e.g., SJERG016 above and SJERG031 below) are those that harbor ERG focal deletion. Aberrant expression of intron 6 is higher in ERGalt-negative samples (bottom) than in ERGalt-positive samples (top).

Supplementary Figure 10 Identification, expression and localization of ALE, a long noncoding RNA in DUX4/ERG ALL.

(a) UCSC WGL plot of transcriptome sequencing data at ERG for a representative case, SJERG003, showing peaks of coverage representing ERG exons, and a region of coverage proximal to the ERG isoform 1 (NM_182918) locus. (b,c) Stranded total RNA–seq data for the same case. (b) The sense strand shows coverage corresponding to a four-exon noncoding RNA, with exon junctions shown below the WGL plot as red bars and the numbers to the left of each bar corresponding to the number of exon–exon junctions. (c,d) Antisense sequencing data corresponding to coding ERG transcripts (c) and the number of annotated (green) and non-annotated (red) junctions shown as bars (d). Note the high number of reads corresponding to ERG alt (exon 6 alt to exon 7) junctions. (e) RT–PCR for expression of ALE (antisense long noncoding RNA associated with ERG) showing two spliced isoforms. (f) Sequential RNA and DNA FISH showing that expressed ALE transcripts are retained at the ERG locus, in contrast to coding ERG transcripts, which are dispersed throughout the nucleus.

Supplementary Figure 11 Expression of ERGalt and ALE in ALL and pediatric tumors.

(ad) Expression levels of ERGalt (a,b) and ALE (c,d) across ALL subtypes (a,c) and pediatric tumors (c,d). Expression of these transcripts was uncommon outside of DUX4/ERG ALL and at a lower level.

Supplementary Figure 12 DUX4 induces ERGalt expression.

(a) Analysis of previously published myoblast DUX4 ChIP–seq and transcriptome sequencing data23, depicting the region of ERG flanking the first exon of ERGalt and showing expression of canonical ERG transcripts in cells not expressing DUX4 and expression of ERGalt transcripts (red bracket) exclusively in cells expressing DUX4. (b) ChIP–PCR data confirming binding of DUX4 at ERG exon 6 alt in NALM-6 but not Reh cells. Primers for PCR at the DUX4 binding site amplify chr. 21: 39,764,747–39,764,881 (hg19), and negative-control primers amplify chr. 21: 39,769,872–39,769,942 (primers are listed in Supplementary Table 16). (c) DUX4 ChIP–seq and ATAC–seq data at CD200R1 in NALM-6 and Reh cells, showing peaks of DUX4 binding corresponding to regions of open chromatin (identified by ATAC–seq) in the DUX4/ERG cell line NALM-6 but not the ETV6-RUNX1 cell line Reh. This corresponds to overexpression of this gene in DUX4/ERG ALL. (d) Lentiviral expression of the rearranged DUX4 allele encoding Glu415*, but not empty vector, results in expression of ERGalt in three individual cord blood pools, as demonstrated by RT–PCR. Representative Sanger sequencing electropherograms confirming expression of ERGalt are shown below the RT–PCR gel image.

Supplementary Figure 13 Binding of DUX4 to the first exon of ERGalt.

(a) The ERG locus and ATAC–seq data for a representative DUX4/ERG case with expression of ERGalt, SJERG000016, showing an ATAC–seq peak (red arrow) not observed in the Reh cell line (ETV6-RUNX1 positive). (b) The genomic region highlighted in the red box in a, showing the overlapping ATAC–seq peak, DUX4 ChIP–seq peak, and two DUX4 binding motifs (TAAATCAATCA and TAATCTCATCA) at the first exon of ERGalt

Supplementary Figure 14 Nuclear localization and transactivation activity of ERGalt.

(a) Immunofluorescence of Arf–/– pre-B cells transduced with retroviral vectors with no ERG construct (MIG), wild-type ERG (MIG-ERG WT) or ERGalt (MIG-ERG e6alt) showing nuclear localization of ERG. Scale bars, 10 μm. (b) Competition assay to determine the transcriptional activity of wild-type (WT) ERG and ERGalt (MT) supplemented with empty vector (MIG) determined using the pGL3-gpIX luciferase reporter in HEK293T cells. Bars show means ± s.e.m. luciferase activity was derived from two individual experiments with triplicate measurements. (c) Immunoblotting of HEK293T cells transfected with empty vector or ERG plasmids confirming the expression shown in b.

Supplementary Figure 15 ERG and ERGalt colony-forming assays.

(a) Schema for colony assays. Mouse Arf–/– lineage-negative bone marrow was cotransduced with GFP-expressing bi- or tricistronic vectors also expressing NRAS Gly12Asp and/or wild-type ERG or ERGalt together with RFP-expressing empty vector or IK6-expressing vector. Flow-sorted GFP+RFP+ cells were plated in methylcellulose and lymphoid cytokines (IL-7, FLT3L and SCF), and colonies were counted and scored after 7–10 d, collected and replated. (b) Cells expressing NRAS Gly12Asp or WT ERG result in non-sustained replating in the absence of the dominant-negative form of IKZF1, IK6, but ERGalt results in sustained replating particularly in the presence of NRAS Gly12Asp with or without IK6. Representative colony morphology (showing GFP and RFP positivity) and immunophenotyping data are depicted showing pre-B immunophenotype.

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Zhang, J., McCastlain, K., Yoshihara, H. et al. Deregulation of DUX4 and ERG in acute lymphoblastic leukemia. Nat Genet 48, 1481–1489 (2016). https://doi.org/10.1038/ng.3691

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