Unscrambling the genomic chaos of osteosarcomas reveals a recurrent gene fusion

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. 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. RNA Sequencing Susanne Lorenz, PhD 517 candidate fusion transcripts identified in 11 osteosarcoma cell lines

5. Candidate fusion transcript validation

6. Whole genome sequencing • WG Sequencing • IOR/OS15 • MG-63 • ZK-58 • IOR/OS18 • Large heterogeneity

7. Comparison RNA-Seq and WGS

8. PMP22-ELOVL5 fusion exon6 exon1 exon2 exon4 exon3 exon5 5’ PMP22 3’ exon8 7 exon1 exon2 exon4 exon3 5 6 5’ ELOVL5 3’ ATG (Start) 1 2 3 4 5 2 3 4 5 6 7 8 Exons 106 aa 1aa 326aa out of 160 aa wt full protein sequence Phe Fusion transcript In frame fusion 5` 3`

9. PMP22-ELOVL5 fusion PMP22 3` adapter 5` adapter Fusion transcript 1 2 3 4 5 2 3 4 5 6 7 8 ELOVL5 RACE confirmed structure of fusion gene PMP22 ELOVL5 FISH Validation extended osteosarcoma panel Osteosarcoma cell lines (4/21) IOR/OS15, IOR/MOS, MHM, U-2OS Ostesarcomas xenografts (1/20) OKTx Clinicalsamples (2/9) primary tumours Recurrence of 14% K. Szuhai, Leiden IOR/OS15

10. PMP22 and 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 Mutations in Charcot-Marie-Tooth Disease Amino acids lost by the fusion with ELOVL5

11. Summary • 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

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

13. Transcriptome sequencing Gene B Gene A Fused mRNA Gene junction Paired-end 250-300 bp 2x100bp • Fusion genes • Biology • Biomarkers • RNA-Seq • 11 osteosarcoma cell lines Susanne Lorenz, PhD