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Unscrambling the genomic chaos of osteosarcomas reveals a recurrent gene fusion Leonardo A. Meza-Zepeda, PhD Department of Tumor Biology Institute for.

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Presentation on theme: "Unscrambling the genomic chaos of osteosarcomas reveals a recurrent gene fusion Leonardo A. Meza-Zepeda, PhD Department of Tumor Biology Institute for."— Presentation transcript:

1 Unscrambling the genomic chaos of osteosarcomas reveals a recurrent gene fusion Leonardo A. Meza-Zepeda, PhD Department of Tumor Biology Institute for Cancer Research The Norwegian Radium Hospital

2 Osteosarcomas, a genomic chaos? Harald Rieder, Duesseldorf

3 The Norwegian Radium Hospital Tumour panel 19 Osteosarcoma cell lines (EuroBoNeT panel) Well characterised preclinical model Genomics and phenotypic (Poster 107, Myklebost) Aim Identify fusion genes in osteosarcomas, to improve the knowledge about pathways involved in osteosarcoma biology Strategy Sequence the transcriptome (RNA-Seq) to identify recurrent fusion transcripts Whole genome sequencing (DNA-Seq) to confirm the fusion transcripts at the genomic level

4 The Norwegian Radium Hospital Sample Candidate fusion transcripts after filtering IOR/MOS5 IOR/OS15110 IOR/OS18160 MHM11 OSA 56 ZK-58 113 KPDN 56 MG-63 102 SaOS-S2 36 IOR/OS10 15 IOR/SARG 36 517 candidate fusion transcripts identified in 11 osteosarcoma cell lines RNA Sequencing Susanne Lorenz, PhD

5 The Norwegian Radium Hospital Candidate fusion transcript validation Candidate fusion transcript Found in RNA-Seq data Samples tested by breakpoint PCR Transcript validation by Sanger sequencing IOR/OS15IOR/MOSMHMNorm boneOsteoblast COL1A2-MMP14IOR/OS15, IOR/OS18+++--+ FUS-LGMNIOR/OS15, MG-63++---+ DHFR-PABPC1IOR/OS15++---+ PTMA-NPM1IOR/OS15, IOR/OS18+++--+ ELOVL5-PMP22IOR/OS15++---+ CDC5L-BTBD9IOR/OS15+----+ RAI1-CLIC5IOR/OS15+----+ TP53-PPRADIOR/OS15+----+ MRPL39-MAML3IOR/OS15+----+ PLXNA2-RUNX1IOR/OS15+----+ EIF5A-HMGN2IOR/OS15, MG-63, Saos2++++++ EEF1A1-VIMIOR/OS15, IOR/OS18, SARG++++++ EIF2S1-RBM25IOR/OS15+----+ COL1A1-HSP90AB1IOR/OS15, MG-63+-+--- SCO1-CLIC5IOR/OS15+++++- CREB3L1-HNRPAIOR/OS15-----NA PDZRN4-PKM2 IOR/OS15, IOR/OS18, MHM -----NA

6 The Norwegian Radium Hospital Whole genome sequencing WG Sequencing IOR/OS15 MG-63 ZK-58 IOR/OS18 Large heterogeneity

7 The Norwegian Radium Hospital Comparison RNA-Seq and WGS FusionIdentified inType of translocation ELOVL5-PMP22IOR-OS15inter-chromosomal CDC5L-BTBD9IOR-OS15inter-chromosomal RAI1-CLIC5IOR-OS15inter-chromosomal SCO1-CLIC5IOR-OS15inter-chromosomal MRPL39-MAML3IOR-OS15inter-chromosomal PLXNA2-RUNX1IOR-OS15inter-chromosomal EIF2S1-RBM25IOR-OS15inter-chromosomal TP53-PPRADIOR-OS15inter-chromosomal BAZ1A-NUMBIOR-OS15intra-chromosomal MRPL39-NRIP1IOR-OS15intra-chromosomal CLIP4-EPHB4MG63intra-chromosomal DNER-ELL2MG63inter-chromosomal TP53-VAV1*MG63inter-chromosomal TP53-EMR1*MG63inter-chromosomal BNC2-MTAPMG63intra-chromosomal HEATR7A-PARP10MG63intra-chromosomal CREBBP-TFAP4ZK58intra-chromosomal GDPD5-MAP6ZK58intra-chromosomal

8 The Norwegian Radium Hospital PMP22-ELOVL5 fusion Fusion transcript 5`3` 5’ 3’ exon1 exon2 exon3 exon4 exon5 5’ 3’ exon1 exon2 exon3 exon4 65 PMP22 ELOVL5 exon6 7 exon8 124325435768 full protein sequence 106 aa out of 160 aa wt Phe 1 aa326 aa ATG (Start) Exons In frame fusion

9 The Norwegian Radium Hospital PMP22-ELOVL5 fusion PMP22 ELOVL5 3` adapter 5` adapter Fusion transcript 124354357628 RACE confirmed structure of fusion gene IOR/OS15 K. Szuhai, Leiden PMP22 ELOVL5 FISH Validation extended osteosarcoma panel Osteosarcoma cell lines (4/21) IOR/OS15, IOR/MOS, MHM, U-2OS Ostesarcomas xenografts (1/20) OKTx Clinical samples (2/9) primary tumours Recurrence of 14%

10 The Norwegian Radium Hospital PMP22 and ELOVL5 Mutations in Charcot-Marie-Tooth Disease Amino acids lost by the fusion with ELOVL5 PMP22: peripheral myelin protein 22 Component of myelin in the peripheral nervous system Associated with Charcot–Marie–Tooth disease Osteosarcomas Overexpressed in a subset of tumour ELOVL5: fatty acid elongase 5 plasma membrane protein; elongation of long-chain polyunsaturated fatty acids

11 The Norwegian Radium Hospital Identified a large number of candidate fusion transcripts in osteosarcomas Identified a recurrent fusion in osteosarcomas PMP22-ELOVL5 Large number of genomic rearrangements and heterogeneity Discrepancies between genomic rearrangements and rearrangements at the RNA level Majority of fusion transcripts does not seem to be a direct consequence of the genomic chaos Summary

12 The Norwegian Radium Hospital Acknowledgements Dept. of Tumor Biology Susanne Lorenz Tale Barøy Ola Myklebost Leiden University Medical Center Karoly Szuhai Genomics Core Facility Jinchang Sun Jan-Christian Bryne Bioinformatics Core Facility Daniel Vodák Dept. Cancer Prevention Torfinn Nome Gard O. S. Thomassen Rolf Skotheim Ragnhild A. Lothe

13 The Norwegian Radium Hospital Most frequent translocation in regions of chromothripsis

14 The Norwegian Radium Hospital Top 10 frequent translocations cell linelocus Alocus Bfrequencyfeaturesgenes involvedcell linelocus Alocus Bfrequencyfeatures genes involved IOR/OS15chr1 (q32.2)chr21 (q21.1)82.5 LINC00478MG-63chr10 (q11.23)chr16 (q21)36.5 chr22 (q12.3)chr6 (p22.3)83.3 chr11 (q21)chr9 ( p21.1)44.1SESN3 chr3 (q26.1)chr9 ( p21.3)83.3 chr12 (q23.3)chr7(q31.1)35.5IMMP2L chr12 (q23.3)chr7(q31.1)83.5IMMP2L chr13 (q21.31)chr20 (p11.1)41.7 chr21 (q21.1)chr4 (q13.1)97.5chromothripsisLPHN3 chr3 (p11.1)chr7 (q11.21)38.9 chr11 (p12)chr18 (q12.2)100.0LRRC4C/FHOD3 chr4 (p13)chr9 ( p23)52.4chromothripsisKCTD8 chr21 (q21.2)chr4 (q28.1)100.0chromothripsis chr4 (p13)chr9 (p13.3)38.6chromothripsis ATP8A1/UN C13B chr21 (q21.2)chr4 (q28.3)100.0chromothripsis chr4 (q32.1)chr9 ( p21.3)37.9chromothripsis chr21 (q21.3)chr4 (q28.1)100.0chromothripsis miRNA155 chr4 (q35.2)chr9 (q13)38.2chromothripsis chr21 (q21.1)chr4 (q28.3)100.0chromothripsisCHODL chr6 (p22.1)chr9 ( p21.3)40.2chromothripsisMLLT3 IOR/OS18chr10 (q22.2)chr5 (q11.2)30.4BMS1P4ZK-58chr4 (q35.2)chr9 (q13)34.07079646 chr12 (q23.3)chr7(q31.1)47.7IMMP2L chr22 (q11.22)chr6 (q21)36.27906977HDAC2 chr13 (q21.31)chr20 (p11.1)32.0 chr12 (p12.3)chr19 (q13.43)37.56097561MZF1 chr14 (q32.31)chr7 (q21.3)41.1GNGT1 chr12 (q23.3)chr7 (q31.1)37.56613757IMMP2L chr14 (q32.31)chr17 (p13.1)49.8TP53 Chr17 (q25.2)chr3 (q13.11)39.28571429SEC14L1 chr22 (q12.3)chr6 (p22.3)37.7 chr11 (q12.3)chr2 (q31.3)39.62264151 FTH1/LINC0 0913 chr3 (p12.1)chr6 (q11.1)30.6KHDRBS2 chr14 (q32.33)chr2 (q13)40.5904059RPL23AP7 chr3 (p12.1)chr6 (q11.1)31.1 chr17 (p13.1)chr5 (q13.2)45.41484716 TP53/PTCD 2 chr3 (p12.1)chr6 (q12)32.7 chr14 (q32.33)chr22 (q13.33)46.09053498RPL23AP82 chr3 (p14.3)chr7 (q35)32.2 ARHGEF3/CNTN AP2 chr14 (q32.33)chr19 (q13.43)48.74551971 MGC2752

15 The Norwegian Radium Hospital Full length fusion transcript PMP22 ELOVL5 Gene SP1 Gene SP2 5` RACE3` RACE Fusion product Normal ELOVL5 Fusion product Normal PMP22 3` adapter 5` adapter Fusion transcript 124354357628 K. Szuhai, Leiden PMP22 ELOVL5 RACE confirmed structure of fusion gene IOR/OS15

16 The Norwegian Radium Hospital Transcriptome sequencing Paired-end 250-300 bp 2x100bp Fused mRNA Gene A Gene B Gene junction Fusion genes Biology Biomarkers RNA-Seq 11 osteosarcoma cell lines Susanne Lorenz, PhD

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