In cancer, the mouse double minute 2 (MDM2) is an oncoprotein that contributes to the promotion of cell growth, survival, invasion, and therapeutic resistance. The impact of MDM2 on cell survival versus cell death is complex and dependent on levels of MDM2 isoforms, p53 status, and cellular context. Extensive investigations have demonstrated that MDM2 protein–protein interactions with p53 and other p53 family members (p63 and p73) block their ability to function as transcription factors that regulate cell growth and survival. Upon genotoxic insults, a dynamic and intricately regulated DNA damage response circuitry is activated leading to release of p53 from MDM2 and activation of cell cycle arrest. What ensues following DNA damage, depends on the extent of DNA damage and if the cell has sufficient DNA repair capacity. The well-known auto-regulatory loop between p53-MDM2 provides an additional layer of control as the cell either repairs DNA damage and survives (i.e., MDM2 re-engages with p53), or undergoes cell death (i.e., MDM2 does not re-engage p53). Furthermore, the decision to live or die is also influenced by chromatin-localized MDM2 which directly interacts with the Mre11-Rad50-Nbs1 complex and inhibits DNA damage-sensing giving rise to the potential for increased genome instability and cellular transformation.

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Osteosarcoma (OS) patients exhibit poor overall survival, partly due to copy number variations (CNVs) resulting in dysregulated gene expression and therapeutic resistance. To identify actionable prognostic signatures of poor overall survival, we employed a systems biology approach using public databases to integrate CNVs, gene expression, and survival outcomes in pediatric, adolescent, and young adult OS patients. Chromosome 8 was a hotspot for poor prognostic signatures. The MYC-RAD21 copy number gain (8q24) correlated with increased gene expression and poor overall survival in 90% of the patients (n = 85). MYC and RAD21 play a role in replication-stress, which is a therapeutically actionable network. We prioritized replication-stress regulators, bromodomain and extra-terminal proteins (BETs), and CHK1, in order to test the hypothesis that the inhibition of BET + CHK1 in MYC-RAD21+ pediatric OS models would be efficacious and safe. We demonstrate that MYC-RAD21+ pediatric OS cell lines were sensitive to the inhibition of BET (BETi) and CHK1 (CHK1i) at clinically achievable concentrations. While the potentiation of CHK1i-mediated effects by BETi was BET-BRD4-dependent, MYC expression was BET-BRD4-independent. In MYC-RAD21+ pediatric OS xenografts, BETi + CHK1i significantly decreased tumor growth, increased survival, and was well tolerated. Therefore, targeting replication stress is a promising strategy to pursue as a therapeutic option for this devastating disease.

Patients with aggressive osteosarcoma (OS) have poor prognosis due in part to copy number variations (CNVs) that contribute to dysregulation of gene expression (GE) and therapeutic resistance. The objective of the present study was to utilize the TARGET database to integrate CNV and corresponding GE with poor prognosis in pediatric OS (n=85) followed by functional validation of prioritized targets. Cox regression analysis indicated that CNVs in 2642 genes correlated with relapse risk in pediatric OS. Furthermore, the top 10 genes with CNVs significantly associated with increased risk for relapse were present on chromosome 8. The MYC and RAD21 copy number gain (MYC-RAD21 CNV+) located on chromosome 8q correlated with increased GE and poor survival in >90% of the relapsed patients. Based on network analysis, the MYC-RAD21 CNV+ was prioritized for development of targeted therapy. MYC, an oncogenic driver of OS growth, can be indirectly inhibited by bromodomain and extra-terminal domain inhibitors (BETi). RAD21 expression has been associated with increased sensitivity to cell cycle checkpoint kinase 1 inhibitors (CHK1i) in melanoma. Additionally, mechanistic links exist between MYC and CHK1, especially during replication stress. Our hypothesis was that the MYC-RAD21 CNV+ serves as a biomarker of poor prognosis and therapeutic response to BETi+CHK1i therapy. Cell growth response to BETi and CHK1i was evaluated in MYC-RAD21 CNV+ pediatric OS cell lines and a patient-derived xenograft (PDX)-derived xenoline (TT2-77). OS lines (G292, MG63, U2OS, and TT2-77) were highly sensitive to single agent BETi/OTX-015, CHK1i/ SRA737 or CHK1i/LY2606368 at clinically relevant concentrations. Combination index and Bliss independence analysis indicated that BETi+CHK1i did not result in synergistic or additive inhibition of growth at clinically relevant concentrations. However, in OS lines Saos2 and Saos2-LM7 BETi+CHK1i resulted in additive to synergistic inhibition of growth at multiple dose-ratios and at clinically relevant concentrations. In the TT2-77 PDX, whole genome sequencing indicated that the original OS biopsy and the TT2-77 PDX generated from a resection sample harbor the MYC-RAD21 CNV+ (4 copies/amplicon). PDX tumor fragments were implanted into the flank of immunodeficient NOD/SCID/IL2Rγ mice. Once tumor volumes reached 100-150 mm3, mice were randomized and treated with four 5-day cycles of BETi/OTX-015 and/or CHK1i/SRA737. BETi+CHK1i significantly decreased TT2-77 growth, increased probability of survival, and was well tolerated. BETi+CHK1i is a promising therapeutic approach for treatment of relapsed pediatric MYC-RAD21 CNV+ OS. It is possible that MYC, BETs, RAD21 and CHK1 protein levels could dictate sensitivity to combination BETi+CHK1i independent of MYC-RAD2 CNV+ status. Studies are in progress to identify responder versus non-responder signatures in OS. Citation Format: Khadijeh Bijangi-Vishehsaraei, Pankita Pandya, Cheng Lijun, Tang Shan, Anthony Sinn, Melissa Trowbridge, Kathy Coy, Courtney Hemenway, Barbara Bailey, Harlan Shannon, Jixin Ding, Erika Dobrota, M. Reza Saadatzadeh, Adily Elmi, Jeremiah Shultz, Mary Murray, Mark Marshall, Michael Ferguson, Todd Bertrand, L. Daniel Wurtz, Sandeep Batra, Lang Li, Jamie Renbarger, Karen Pollok. Systems biology approach provides rationale for dual-targeted inhibition of BET and CHK1 in aggressive pediatric osteosarcoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 450.