Abstract 7045: Anti-tumor effects following BET inhibition in preclinical models of pediatric and AYA osteosarcoma

Abstract Osteosarcoma (OS) is the most common primary malignant bone sarcoma in pediatric, adolescent, and young adult patients with a five-year survival rate of <30%. This is due, in part, to genomic complexities arising from moderate levels of oncogenic replication stress (RS). While moderate levels of RS cause genome instability that contributes to OS progression, high levels of RS can lead to cell death. Notably, bromodomain and extra-terminal domain (BET) proteins (BRD2, 3, 4) are epigenetic readers that regulate not only transcriptional networks but also DNA replication and repair. Targeting RS through utilization of BET inhibitors (BETi) is an underexplored treatment option in OS and little is known about BETi-induced molecular changes and adaptive responses in OS. We hypothesize that BETi leads to decreased OS cell growth via not only remodeling of gene expression but also increased DNA damage and non-tolerated RS levels. Effect of BETi (AZD5153, BMS-986378, ZEN-3694) in clinical trials for pediatric and adult solid tumors were investigated. In vitro BETi screening of OS cell lines indicated that bivalent AZD5153 was the most potent BETi compared to monovalent BMS-986378 and ZEN-3694. However, in a longitudinal cell growth delay assay with repeat exposure of BETi at clinically relevant doses, all BETi decreased OS cell growth at similar rates. To interrogate mechanisms of action, RNA-seq analysis of BET/BRD4 inhibition via AZD5153, PROTAC (ARV-825), and BRD4 siRNA in OS cell lines revealed distinct and overlapping patterns of alterations in gene expression. A major challenge in OS therapy is mitigation of invasiveness and targeting OS metastatic foci. In all treatment groups, RNAseq analysis indicated upregulation of tumor suppressor DHRS2 an inhibitor of tumor invasion but also TMPRSS9, a promoter of tumor invasion. This observed duality in tumor invasion mechanisms is an example of the intra-heterogeneity of the tumor response and indicative that a second therapeutic hit is likely necessary for suppression of tumor invasiveness in remaining OS cells following BETi therapy. In vivo studies demonstrate that bivalent AZD5153 significantly suppressed tumor growth in OS patient-derived xenografts (PDXs) derived from both treatment-naïve (patient status: alive-PDX112, PDX124; deceased- PDX96, PDX115) and pre-treated metastatic (patient status: deceased-TT2/HT77) OS compared to vehicle (p<0.05). Therapy was well tolerated with similar drug sensitivities in female and male mice. Analysis of in-vivo PDX96 response following AZD5153 therapy indicated increased γ-H2AX, and expression of pro-apoptotic genes, indicative of increased RS and cell death. At the transcript level, increased DHRS2 was evident with no changes in TMPRSS9. These data sets reveal the promise of BETi for treatment of OS PDX derived from treatment naïve patients some of which go on to relapse, and in OS PDX derived from metastatic sites. Citation Format: Ryli E. Justice, Niknam Riyahi, Barbara J. Bailey, Erika A. Dobrota, Rada Malko, Harlan E. Shannon, Keiko E. Kreklau, Melissa A. Trowbridge, Kathryn L. Coy, Felicia M. Kennedy, Melinda R. Ervin, Anthony L. Sinn, Christopher Davis, Kyle W. Jackson, Brian D. Weiss, Steven D. Angus, M R. Saadatzadeh, Pankita H. Pandya, Karen E. Pollok. Anti-tumor effects following BET inhibition in preclinical models of pediatric and AYA osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7045.