Therapeutic targeting of BET bromodomain proteins increases DNA damage and potentiates salvage therapy in osteosarcoma xenografts derived from patients with replication stress signatures

Abstract Osteosarcoma (OS) is an aggressive cancer of the bone with high metastatic potential in pediatric as well as adolescent and young adults. The survival rate for metastatic and relapsed OS patients is <30% and there is currently no effective standardized salvage therapy. Lack of efficacy is attributed to genetic complexity present in OS that is partly due to moderate levels of replication stress (RS). While high levels of RS can induce cell death, moderate RS levels may cause genomic instability that contributes to OS progression. Therefore, induction of RS to high levels that cause cell death could be a promising therapeutic strategy. Bromodomain and extra-terminal domain [BET proteins (BRD2,3, and 4)] are a family of epigenetic readers that not only regulate gene expression networks, but also regulate DNA replication and RS. Thus, we tested the hypothesis that BET inhibition will potentiate the efficacy of salvage therapy through exacerbation of RS in xenograft models of aggressive OS. The effect of the bivalent BET inhibitor (BETi), AZD5153, as a single agent and in combination with cytotoxic agents such as topotecan and ifosfamide was evaluated. Combination index and Bliss independence analyses demonstrated additive to synergistic cell growth inhibition in OS cell lines upon treatment with clinically relevant concentrations of AZD5153+ topotecan/ifosfamide. Treatment with PROTAC ARV825 that degrades BET proteins, resulted in similar growth inhibitory effects. Significant increase in PARP cleavage was observed following AZD5153+topotecan treatment compared to single agent, indicating enhancement of apoptosis. In addition, western blot and comet assays showed that BETi+topotecan induces its effect, at least partly, through increased DNA damage and RS in vitro. In vivo efficacy and safety studies focused on patient-derived xenografts (PDXs) of naive and pre-treated OS that harbor RS signatures. AZD5153 as a single agent significantly suppressed tumor growth in both naïve (PDX96) and pretreated (TT2) OS PDX models compared to vehicle (p<0.05, Two-way ANOVA; Holm-Sidak). Anti-tumor effect correlated with increased γ-H2AX following AZD5153 exposure in PDX, indicative of increased RS. Moreover, RNA-seq analysis integrated with kinome profiling data from BETi-treated PDX exhibited deregulation of factors involved in RS. Combination treatments of BETi+topotecan/ifosfamide indicated that AZD5153 potentiated the anti-cancer effect of salvage therapy in TT2 OS PDX, was well tolerated, and increased the probability of survival in mice. Efficacy in an OS RS+ metastatic lesion model is in progress. These data collectively suggest that BET inhibition as a single agent and in combination with low-dose salvage therapy holds promise as novel treatment strategies for inducing RS-mediated cell death in aggressive OS. Citation Format: Niknam Riyahi, Pankita H. Pandya, Barbara J. Bailey, Erika A. Dobrota, Courtney Young, Harlan E. Shannon, Farinaz Barghi, Rada Malko, Khadijeh Bijangi-Vishehsaraei, Melissa A. Trowbridge, Kathy Coy, Felicia M. Kennedy, Anthony L. Sinn, Steve Angus, Michael J. Ferguson, M. Reza Saadatzadeh, Karen E. Pollok. Therapeutic targeting of BET bromodomain proteins increases DNA damage and potentiates salvage therapy in osteosarcoma xenografts derived from patients with replication stress signatures. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6727.