(P 292) CHARACTERIZATION OF TELOMERE MAINTENANCE MECHANISMS IN OSTEOSARCOMA PATIENT-DERIVED LABORATORY MODELS

Objective: Osteosarcoma (OS) is the most common primary malignant tumor of the bone. We have previously demonstrated that ~70 % of OS patient tumors employ alternative lengthening of telomeres (ALT) mechanism to maintain their telomere length. Currently, there is a paucity of well-characterized, low-passage OS patient-derived cell lines (PDCLs) and patient-derived xenografts (PDXs) with defined telomere maintenance mechanisms (TMM). Additionally, the mechanisms underlying TMM activation in OS remain poorly understood. The objective of this study is to comprehensively characterize TMM in low passage OS PDCLs and PDXs and to elucidate the underlying mechanisms of TMM activation.

Methods: All PDCLs were cultured under hypoxic conditions (5% O₂ tension). PDXs were established subcutaneously in NSG mice and subsequently serially passaged in nu/nu mice. Short tandem repeat verification was used to confirm the authenticity of PDCLs and PDXs. Quantitative PCR (qPCR) was used to measure MYC mRNA, TERT mRNA, MYC copy number, and c-circle content. Greater than 10 copies of MYC when normalized to a single copy gene in a PDCL or PDX were considered as MYC amplified. MYC and ATRX protein expression in PDCLs was analyzed via immunoblotting, and immunohistochemistry was performed on PDX samples to assess ATRX expression. Lastly, transient MYC knockdown was performed using siRNA.

Results: We assessed DNA from 13 OS PDCLs and 52 OS PDXs for ALT positivity using the C-circle assay. Among these, 7 of 13 (54%) PDCLs and 30 of 52 (57%) PDXs were C-circle positive. Using a subset of the PDCLs and PDXs we carried out in depth analysis of telomere maintenance mechanisms (TMM). C-circle negative PDCLs (n=6) and PDXs (n=5) exhibited higher TERT mRNA expression compared to C-circle positive PDCLs (n=7) and PDXs (n=13) (P < 0.001). None of the PDCLs or PDXs showed both C-circle positivity and high TERT mRNA expression concurrently, indicating mutually exclusive activation of ALT and telomerase (TA) in OS laboratory models. We observed that 3 out of 7 ALT PDCLs and 7 out of 11 ALT PDXs had no ATRX protein expression, while TA+ PDCLs and PDXs expressed ATRX protein (Fisher's exact test: P < 0.01). It has been previously reported that ALT tumors are indolent compared to TA-activated tumors in other cancers. Consistent with these reports, we observed that the median time from engraftment to endpoint (1500 mm³) in mice was 3.5 months for ALT PDXs (n=11) compared to 1.6 months for TA+ PDXs (n=4) (Log-Rank P-value = < 0.001).
MYC amplification was previously reported in OS. MYC is a pleiotropic oncogene and has been shown to transcriptionally activate TERT in other cancers. We assessed MYC copy number in 13 PDCLs (7 ALT and 6 TA+) and 18 PDXs (13 ALT and 5 TA+). We observed that none of ALT PDCLs or PDXs had MYC genomic amplification. Conversely, 5 of 6 TA PDCLs and 4 of 5 TA PDXs had MYC genomic amplification (Fisher-exact test P < 0.001). Higher levels of MYC mRNA were detected in MYC amplified PDCLs (P = 0.02) and PDXs (P = 0.007) and MYC protein expression was higher in MYC amplified PDCLs. Transient knockdown of MYC in a MYC amplified OS cell line (OS-833) reduced TERT mRNA expression (>12-fold decrease in TERT mRNA expression; P < 0.001), indicating that MYC can transcriptionally drive TERT mRNA expression in OS cells.

Conclusion: This study indicates that ALT and TERT activation are mutually exclusive in OS. Specifically, loss of ATRX expression is observed exclusively in ALT OS models, whereas MYC genomic amplification occurs exclusively in models with TERT activation. Our findings support the hypothesis that MYC genomic amplification or loss of ATRX provides TMM activation for most OS. Some OS models activate TMM in the absence of ATRX loss or MYC amplification, indicating that there are other, yet to be identified, mechanisms responsible for TMM activation in OS.