Abstract
In contrast to many other sarcoma subtypes, the chaotic karyotypes of osteosarcoma have precluded the identification of pathognomonic translocations. We here report hundreds of genomic rearrangements in osteosarcoma cell lines, showing clear characteristics of microhomology-mediated break-induced replication (MMBIR) and end-joining repair (MMEJ) mechanisms. However, at RNA level, the majority of the fused transcripts did not correspond to genomic rearrangements, suggesting the involvement of trans-splicing, which was further supported by typical trans-splicing characteristics. By combining genomic and transcriptomic analysis, certain recurrent rearrangements were identified and further validated in patient biopsies, including a PMP22-ELOVL5 gene fusion, genomic structural variations affecting RB1, MTAP/CDKN2A and MDM2, and, most frequently, rearrangements involving TP53. Most cell lines (7/11) and a large fraction of tumor samples (10/25) showed TP53 rearrangements, in addition to somatic point mutations (6 patient samples, 1 cell line) and MDM2 amplifications (2 patient samples, 2 cell lines). The resulting inactivation of p53 was demonstrated by a deficiency of the radiation-induced DNA damage response. Thus, TP53 rearrangements are the major mechanism of p53 inactivation in osteosarcoma. Together with active MMBIR and MMEJ, this inactivation probably contributes to the exceptional chromosomal instability in these tumors. Although rampant rearrangements appear to be a phenotype of osteosarcomas, we demonstrate that among the huge number of probable passenger rearrangements, specific recurrent, possibly oncogenic, events are present. For the first time the genomic chaos of osteosarcoma is characterized so thoroughly and delivered new insights in mechanisms involved in osteosarcoma development and may contribute to new diagnostic and therapeutic strategies.
Highlights
The chaotic chromosomes and heterogeneity of osteosarcomas has made the identification of pathognomonic mutations difficult
Fusion transcripts were identified by sequencing the transcriptomes of 11 osteosarcoma cell lines
The identified microhomology pattern for all rearrangement types differed significantly (p-values < 1.00e-05) from patterns identified by analyzing a random set of 500 sequences derived from the human genome, demonstrating a specific enrichment of 1-5 bp and 6-25 bp microhomologies at the breakpoints. These findings suggest that somewhat different microhomology mediated DNA repair mechanisms, as i.e. non-homologous end-joining (NHEJ) and microhomology-mediated end-joining (MMEJ) repair, are involved in the generation of the genomic rearrangements and deletions shorter than 5 kb
Summary
The chaotic chromosomes and heterogeneity of osteosarcomas has made the identification of pathognomonic mutations difficult. PMP22-ELOVL5, was recurrent, detected in four cell lines (IOR/OS15, IOR/ MOS, MHM, U-2 OS), one xenograft (OKTx), and two patient samples (OS29 and OS83). The cell line IOR/OS15, harboring the PMP22-ELOVL5 gene fusion and a genomic amplification of the whole PMP22 locus, was higher expressed only for the part of the transcript involved in the fusion (Fig. 3c).
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