PGBD5 promotes site-specific oncogenic mutations in human tumors

  • An Erratum to this article was published on 01 October 2017

Abstract

Genomic rearrangements are a hallmark of human cancers. Here, we identify the piggyBac transposable element derived 5 (PGBD5) gene as encoding an active DNA transposase expressed in the majority of childhood solid tumors, including lethal rhabdoid tumors. Using assembly-based whole-genome DNA sequencing, we found previously undefined genomic rearrangements in human rhabdoid tumors. These rearrangements involved PGBD5-specific signal (PSS) sequences at their breakpoints and recurrently inactivated tumor-suppressor genes. PGBD5 was physically associated with genomic PSS sequences that were also sufficient to mediate PGBD5-induced DNA rearrangements in rhabdoid tumor cells. Ectopic expression of PGBD5 in primary immortalized human cells was sufficient to promote cell transformation in vivo. This activity required specific catalytic residues in the PGBD5 transposase domain as well as end-joining DNA repair and induced structural rearrangements with PSS breakpoints. These results define PGBD5 as an oncogenic mutator and provide a plausible mechanism for site-specific DNA rearrangements in childhood and adult solid tumors.

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Figure 1: Human rhabdoid tumors exhibit genomic rearrangements associated with PGBD5-specific signal-sequence breakpoints.
Figure 2: PGBD5 is physically associated with human genomic PSS sequences that are sufficient to mediate DNA rearrangements in rhabdoid tumor cells.
Figure 3: Ectopic expression of PGBD5 in human cells leads to oncogenic transformation both in vitro and in vivo.
Figure 4: PGBD5 transposase activity is necessary to transform human cells.
Figure 5: Transient PGBD5 transposase expression is sufficient to transform human cells.
Figure 6: DNA end-joining repair is required for survival of cells expressing active PGBD5.
Figure 7: PGBD5-induced cell transformation involves site-specific genomic rearrangements associated with PGBD5-specific signal-sequence breakpoints.

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  • 24 May 2017

    In the version of this article initially published online, the affiliations for Jiali Zhuang listed an incorrect present address instead of an equal contribution. The error has been corrected in the print, PDF and HTML versions of this article.

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Acknowledgements

We are grateful to A. Gutierrez, M. Mansour, D. Bauer, T. Look, H. Zhu, C. Feschotte, M. Kharas, J. Petrini, and M. Gil Mir for critical discussions, and J. Gilbert for editorial advice. We thank T. Westbrook (Baylor College of Medicine) and M. Aldaz (MD Anderson Cancer Center) for materials. This work was supported by funding from NIH K08 CA160660, P30 CA008748, U54 OD020355, UL1 TR000457, P50 CA140146, Spanish Ministerio de Economía y Competitividad SAF2014-60293-R, Cancer Research UK, the Wellcome Trust, the Starr Cancer Consortium, the Burroughs Wellcome Fund, the Sarcoma Foundation of America, the Matthew Larson Foundation, the Josie Robertson Investigator Program, and the Rita Allen Foundation. A.G.H. is supported by the Berliner Krebsgesellschaft e.V. and the Berlin Institute of Health. A.K. is supported as a Damon Runyon–Richard Lumsden Foundation Clinical Investigator.

Author information

Affiliations

Authors

Contributions

A.G.H. contributed to the study design and collection and interpretation of the data. R.K. performed ChIP–seq, whole-genome sequencing, and FLEA PCR data analysis. J.Z. analyzed tumor genome-sequencing data with laSV. E.J., C. Reed, A.E., and E.S. performed in vitro transformation assays and vector design and cloning. I.C.M. performed experiments and analyzed data. E.R.-F., S.G., M.P., C.E.M., A.-K.E., M.S., K.A., C. Reeves, N.D.S., D.T., and Z.W. analyzed genome-sequencing data. A.N.B. and S.P.J. contributed to creation of PAXX-deficient cells and study design. E.d.S. contributed to mouse-xenograft study design. M.G. performed statistical analysis of data sets. C.R.A. performed histological analysis of tumor samples. E.P., C.W.M.R., H.S., E.M., and S.A.A. contributed to study design. A.K. contributed to study design, data analysis, and interpretation. A.K. and A.G.H. wrote the manuscript, to which all authors contributed.

Corresponding author

Correspondence to Alex Kentsis.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 PGBD5 is highly expressed in rhabdoid and other pediatric and childhood solid tumors.

(a) Bar graph showing relative expression of PGBD5 in tumors (red), as compared to normal tissues (blue). Median expression is indicated by horizontal line, boxes indicate 25% and 75% quartiles; whiskers indicate minimum and maximum values. (b) Dot plot showing the relative PGBD5 mRNA expression in atypical teratoid/rhabdoid tumor (ATRT) molecular subgroups (SHH, Sonic hedgehog pathway activation; TYR, tyrosinase overexpression; MYC, MYC and HOX overexpression). Bars denote mean. (c) Dot plot showing the relative PGBD5 mRNA expression in medulloblastoma tumor molecular subgroups. (d) Dot plot showing the relative PGBD5 mRNA expression in ependymoma tumor molecular subgroups. (e) Dot plot showing the relative PGBD5 mRNA expression in ATRT tumors relative to the age of patients at diagnosis. Bars denote mean.

Supplementary Figure 2 PGBD5-specific signal sequences.

Sequence logos detected near the breakpoints of genomic rearrangements in the HPRT1 forward genetic screen (top)32, as compared to those observed in primary rhabdoid (middle) and engineered RPE cell tumors (bottom).

Supplementary Figure 3 Distribution and structure of somatic genomic rearrangements in primary rhabdoid tumors.

(a) Distribution of somatic deletions, duplications, insertions, inversions and translocations observed in 31 primary rhabdoid tumors. (b) Distribution of predicted mechanisms at the rearrangement breakpoints as homologous recombination (HR), microhomology-mediated end joining (MMEJ), mobile element rearrangements (ME), and non-template insertions (NI). (c) Tile plot showing recurrence of somatic translocations (blue), deletions (red), duplications (light blue), and inversions (green) affecting specific genes, excluding SMARCB1, in individual rhabdoid tumor specimens. (d) Validation of specific somatic rearrangements of TENM3 and CNTNAP2 genes, as assessed using variant and wild-type allele-specific PCR in matched tumor and normal primary patient specimens. (e-h) Schematics of gene structure of CNTNAP2 and TENM3 before and after rearrangements, and Sanger DNA sequencing chromatograms of the individual rearrangement breakpoints detected by variant allele-specific PCR in individual primary rhabdoid tumor specimens (arrowheads mark the breakpoints). (i) Validation of t(5;22) translocation using variant and allele-specific PCR. (j) Schematic of the chromosomes 5 and 22 before and after rearrangement, leading to the translocation breakpoint detected by variant allele-specific PCR (arrowhead marks the breakpoint).

Supplementary Figure 4 Schematic of flanking-sequence exponential anchored polymerase chain reaction (FLEA PCR).

Biotinylated primer specific for the NeoR cassette is used for linear extension, followed by streptavidin purification, and nested PCR to amplify integration breakpoints, followed by DNA sequencing.

Supplementary Figure 5 Ectopic expression of PGBD5 transforms immortalized BJ and RPE cells in vivo.

(a) Tumor volume as a function of time of RPE (right) and BJ cells (left) stably expressing GFP-PGBD5 and GFP only, compared to non-transduced cells and cells expressing SV40 large T antigen (LTA) and HRAS (n = 10 per group). (b) Kaplan-Meier analysis of tumor-free survival of mice with subcutaneous xenografts of RPE and BJ cells expressing GFP-PGBD5 or GFP only, as compared to non-transduced cells or cells expressing SV40 LTA and HRAS (n = 10 per group, P < 0.0001 by log-rank test).

Supplementary Figure 6 GFP-PGBD5 expression does not induce global chromosomal instability.

Representative karyotype of BJ (lower panel) and RPE cells (upper panel) stably expressing GFP-PGBD5 (right) and GFP (left).

Supplementary Figure 7 Doxycycline-inducible PGBD5 expression in RPE cells leads to penetrant subcutaneous tumor formation in xenograft models.

(a) GFP-T. ni piggyBac is expressed at similar relative mRNA levels as GFP-PGBD5 in RPE cells as measured by quantitative RT-PCR (n = 3, P = 0.79 for GFP-PGBD5 vs. GFP-T. ni piggyBac). (b) Western blot against PGBD5 showing inducible expression of PGBD5 protein in RPE cells stably transduced with pINDUCER21-PGBD5 after 48 h of treatment with doxycycline (0-600 ng/mL) compared to RPE cells stably expressing GFP-PGBD5. (c) Tumor size of RPE xenografts as a function of time, with PGBD5 expression induced using doxycycline (+/- Dox) in RPE cells stably transduced with pINDUCER21-PGBD5 compared to GFP-PGBD5 expressing RPE cells and non-transduced cells. Cells were treated with doxycycline for 10 days prior to subcutaneous injection (n = 10 per group).

Supplementary Figure 8 PGBD5-mediated genome remodeling requires NHEJ repair.

(a) Flow cytometric analysis of cleaved caspase-3 expression in PAXX+/+ and PAXX−/− RPE cells before and after 48 h of doxycycline-induced PGBD5 expression (500 ng/ml doxycycline). (b) Representative images of PAXX+/+ and PAXX−/− RPE cells stained for DAPI (blue) and γ-H2AX (red) 3 h, 6 h, 24 h and 30 h after doxycycline-induced PGBD5 expression (500 ng/ml doxycycline, scale bar = 50 μm). (c) Number of viable PAXX+/+ and PAXX−/− RPE cells per cm2 in monolayer culture as measured by trypan blue staining after 72 h of doxycycline-induced expression of PGBD5, as compared to untreated control cells (n = 3). *P = 1.52 x 10-4 for PAXX−/−; +Dox vs. PAXX−/−; -Dox. Error bars represent standard deviations of three independent experiments. (d) Fraction of γ-H2AX-positive cells over time in PAXX+/+ and PAXX−/− RPE cells before and after doxycycline-induced PGBD5 expression (500 ng/ml doxycycline, n = 3 per group).

Supplementary Figure 9 Conventional alignment-based variant analysis of structural variants in PGBD5-transformed RPE cells.

(a) Venn diagrams showing the number of identified SNVs and indels detected by Strelka, LoFreq and Pindel in genomes of RPE cells expressing GFP-PGBD5 compared to GFP. (b) Venn diagrams showing the number of identified exonic SNVs and indels detected by Strelka, loFreq and Pindel in GFP-PGBD5 expressing RPE cells. (c) Venn diagrams showing the number of identified large structural variants detected by DELLY, BreakDancer (BD) and CREST (filtered high-confidence set) in GFP-PGBD5 expressing RPE cells.

Supplementary Figure 10 GFP-PGBD5-expressing cells exhibit a low frequency of copy-number variants across the genome.

(a) Copy number profile in RPE cells expressing GFP-PGBD5 compared to GFP expressing cells, computed by BIC-Seq2. (b) Relative chromosomal sequence coverage in GFP-PGBD5 expressing cells (left) compared to GFP expressing cells (right) as a function of chromosome number.

Supplementary Figure 11 Single-nucleotide-variant mutational signatures of GFP-PGBD5-expressing cells.

(a) Fraction of SNVs involving each nucleotide in GFP-PGBD5 expressing RPE cells compared to GFP expressing cells as detected by Mutect, LoFreq and Strelka (left to right). (b) Mutational signature in GFP-PGBD5 expressing RPE cells measured as the relative fraction of SNVs (union of Mutect, LoFreq and Strelka) in each substitution class and sequence context immediately 3′ and 5′ to the mutated base. (c) Genomic distribution of SNVs in GFP-PGBD5 expressing RPE cells according to their mutational class (upper panel) and variant allele frequency (lower panel).

Supplementary Figure 12 PGBD5-induced genomic rearrangements in RPE cells and primary malignant rhabdoid tumors.

(a) Histogram showing the genomic size distribution of deletions (excluding small indels) detected by SMuFin in PGBD5-transformed RPE cells. (b) Distribution of somatic deletions, duplications, insertions, inversions and translocations observed in PGBD5-expressing RPE cell tumors. (c) Distribution of predicted mechanisms at the rearrangement breakpoints as homologous recombination (HR), microhomology-mediated end joining (MMEJ), mobile element rearrangements (ME), and non-template insertions (NI). (d) Variant allele-specific PCR of genomic rearrangements detected in PGBD5-expressing RPE cell tumors of RMST (#1), WWOX (#2), FHOD3 (#3), XRN2 (#4), and SERINC5 (#5). (e-h) Schematics of gene structure of RMST, WWOX, FHOD3, and SERINC5 genes before and after rearrangements, and Sanger DNA sequencing chromatograms of the individual rearrangement breakpoints detected by variant allele-specific PCR in individual primary RPE cell tumor specimens (arrowheads mark the breakpoints).

Supplementary Figure 13 Inactivation of WWOX is necessary but not sufficient for clonogenic maintenance of PGBD5-transformed RPE tumor cells.

(a) Western blot of WWOX showing shRNA-mediated depletion of WWOX in RPE-GFP cells stably transduced with pGIPZ-shWWOX, as compared to pGIPZ-shScramble control. Actin serves as loading control. (b,c) Representative photographs of Crystal violet-stained colonies (b) and clonogenic efficiency (c) of RPE-GFP cells expressing pGIPZ-shWWOX, as compared to pGIPZ-shScramble control. (P = 0.44). (d) Western blot of WWOX showing doxyclycline-induced expression of wild-type WWOX in RPE-GFP cells stably transduced with tetOn-advanced-WWOX vector, as compared to RPE-GFP-PGBD5 xenograft tumor-derived cells with PGBD5-induced WWOX mutation. (e,f) Representative photographs of Crystal violet-stained colonies (e) and clonogenic efficiency (f) of RPE-GFP cells and RPE-GFP-PGBD5 xenograft tumor-derived cells stably transduced with tetOn-advanced-WWOX and treated with doxycycline (500 ng/ml) or vehicle control. PGBD5-transformed cells with WWOX mutations, but not control GFP cells, exhibit significantly reduced clonogenic efficiency upon ectopic expression of wild-type WWOX (*P = 0.0098). Error bars represent standard deviations of three independent experiments.

Supplementary Figure 14 Schematic of PGBD5-induced genomic rearrangement mechanisms.

(a) Schematic of intragenic deletion with the PSS sequences colored in red. (b) Schematic of possible mechanisms of PGBD5-induced rearrangements.

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Henssen, A., Koche, R., Zhuang, J. et al. PGBD5 promotes site-specific oncogenic mutations in human tumors. Nat Genet 49, 1005–1014 (2017). https://doi.org/10.1038/ng.3866

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