Unscrambling the genomic chaos of osteosarcoma reveals extensive transcript fusion, recurrent rearrangements and frequent novel TP53 aberrations

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.

Increased PD-L1 and T-cell infiltration in the presence of HLA class I expression in metastatic high-grade osteosarcoma: a rationale for T-cell-based immunotherapy

“…Osteosarcoma’s genomic complexity is one of the major explanations for the lack of specific targetable mutations or molecular pathways. On the other hand, this genomic characteristic and the associated high mutational burden may generate specific tumour neoantigens, therefore providing potential targets for T-cell-based immunotherapies [5–7]. …”

Substitutions of short heterologous DNA segments of intragenomic or extragenomic origins produce clustered genomic polymorphisms

“…During repair, MMEJ often generates short, direct, or inverted repeats (19) and occasionally incorporates ectopic DNA at the recombinant joints (20). MMEJ results in highly variable clustered polymorphisms at the recombinant joint and is now recognized as a driving force in rapidly evolving oncogenic cells (21). DNA polymerase theta (POLQ) has recently been identified as the key enzyme in MMEJ-directed error-prone repair, but many mechanistic details of its function remain elusive (22).…”

Osteosarcoma: Molecular Pathogenesis and iPSC Modeling

“…Chromothripsis -- the presence of thousands of clustered chromosomal rearrangements -- is observed in 25% of clinical osteosarcoma human samples compared to 2–3% of cancers overall, such as chronic lymphocytic leukemia (CLL) [4]. Chromosomal translocations and mutations may juxtapose proto-oncogenes with constitutively active promoters, cause deletion of tumor suppressor genes, or produce chimeric oncogenes (e.g., PMP22-ELOVL5 ) [2–5]. Germline and somatic genome sequencing efforts have revealed potential pathological mechanisms involved in both osteosarcoma and syndromes with a genetic predisposition to osteosarcoma.…”

“…Hundreds of genomic rearrangements have been identified in osteosarcomas using genomic and transcriptomic analysis, including recurrent rearrangements of TP53, RB1, MDM2 and CDKN2A as well as PMP22-ELOVL5 gene fusions [2–5]. The most frequent TP53 rearrangements (e.g., TP53-VAV1, TP53-EMR1, TP53-PPRAD and TP53-KPNA3 ) resulted in the inactivation of p53 in osteosarcoma, explaining how TP53 gene function can be consistently disrupted in osteosarcoma despite the low observed prevalence of TP53 mutations in sporadic osteosarcomas, as shown in traditional mutation analyses [5, 7].…”

“…The most frequent TP53 rearrangements (e.g., TP53-VAV1, TP53-EMR1, TP53-PPRAD and TP53-KPNA3 ) resulted in the inactivation of p53 in osteosarcoma, explaining how TP53 gene function can be consistently disrupted in osteosarcoma despite the low observed prevalence of TP53 mutations in sporadic osteosarcomas, as shown in traditional mutation analyses [5, 7]. A study of transcriptome analysis on untreated clinical osteosarcoma samples revealed that two other osteosarcoma-specific fusion gene products, LRP1-SNRNP25 and KCNMB4-CCND3 , were associated with osteosarcoma cell motility [8] and that a TP53-KPNA3 translocation was associated with chemotherapy resistance and metastasis [7].…”