(P 371) DISRUPTION OF THE TP53 LOCUS IN OSTEOSARCOMA LEADS TO TP53 PROMOTER GENE FUSIONS AND RESTORATION OF PARTS OF THE TP53 SIGNALLING PATHWAY

Objective: TP53 is the most frequently mutated gene in human cancer. This gene shows not only loss-of-function mutations, but also recurrent missense mutations with gain- or separation-of-function activity. We have studied the primary bone malignancy osteosarcoma, which harbours one of the most rearranged genomes of all cancers. This is odd since it primarily affects children and adolescents who have not lived the long life thought necessary to accumulate massive numbers of mutations. In osteosarcoma, TP53 is often disrupted by structural variants, and we hypothesize that such rearrangement of the TP53 locus leads to the induction of cancer-driving genes by gene fusion or promoter swapping events.

Methods: Whole-genome mate-pair sequencing technology (Nextera Mate Pair, Illumina) was used to detect structural alterations affecting TP53 in a discovery cohort of 36 chemotherapy-treated resection specimens and a validation cohort of 36 treatment-naïve diagnostic biopsies from paediatric and adult conventional osteosarcoma patients. Whole genome DNA copy number alterations were investigated in 120 osteosarcomas, including the aforementioned cases (Cytoscan HD arrays, Thermo Fischer Scientific). For 68 osteosarcomas, 13 control osteoblastomas and in vitro cell models, high-quality RNA was obtained, and global gene expression analysis was performed using RNA sequencing (TruSeq, Illumina). To monitor intratumor heterogeneity among individual cells, single cell low-pass whole genome sequencing (0.01x, 100 cells per tumour) was applied to osteosarcoma cells.

Results: We show that TP53 structural variants are early events that not only facilitate further chromosomal alterations, but also allow the TP53 promoter to upregulate genes erroneously placed under its control in a fashion accentuated by ongoing DNA damage. Paradoxically, many of the induced genes are part of the TP53-associated transcriptome, suggesting a need to counterbalance loss of TP53 function through separation-of-function mutations via promoter swapping. This mechanism was preferentially seen in patients with a younger age of osteosarcoma onset. Interestingly, introduction of a TP53 promoter gene fusion into a TP53-/- cell line shifted the global gene expression pattern back towards that of wild type cells, in line with the above findings.

Conclusion: Our findings demonstrate how massive genome errors can functionally turn the promoter region of a tumour suppressor gene into a constitutively active oncogenic driver. More specifically, we demonstrate that TP53 is not only lost in carcinogenesis, but its promoter can be selected for during tumour evolution and paradoxically rescue parts of the wildtype TP53-signalling pathway. This suggests that while the classical tumour suppressor activites of TP53 are lost, certain parts of the TP53 signalling pathway that are necessary for cancer cell survival and proliferation are retained. In conclusion, TP53 structural variants in osteosarcoma likely function similarly to separation-of-function hotspot TP53 missense variants seen in adult carcinomas.