Osteosarcoma (OS) is an aggressive bone cancer in pediatric adolescent and young adult patients. Survival rate for metastatic and relapsed OS patients remains dismal at <30%. Additionally, no effective standardized salvage therapy currently exists for these patients, in part due to genomic complexities arising from moderate levels of replication stress (RS). Bromodomain and extra-terminal domain protein inhibitors (BETi) are an underexplored option to target RS, as BET inhibition creates an imbalance between transcription-replication kinetics thereby exacerbating oncogenic RS and cell death. BETs (BRD2,3,4) are epigenetic readers that play a role in regulating gene expression networks as well as DNA replication and repair. We hypothesized that BET inhibition with AZD5153 would suppress OS growth by inducing tumor suppressive mechanisms that exacerbate DNA damage and sensitize tumors to DNA-damaging chemotherapeutic agents. AZD5153 monotherapy significantly suppressed OS tumor growth compared to vehicle (p<0.05) in female and male patient-derived xenografts (PDXs) established from both treatment-naïve (PDX96, PDX115, PDX112) and metastatic OS (PDX77-TT2). Mechanistic evaluation showed increased gamma-H2AX and p-RPA2 S8 following AZD5153 exposure in PDX96, indicative of enhanced RS. RNA-seq and protein analyses revealed dysregulation of DNA damage response genes, including upregulation of TXNIP, a tumor suppressor that promotes DNA damage and apoptosis, and downregulation of PDGFRA. Similar TXNIP upregulation and PDGFRA suppression were observed in AZD5153-treated PDX115. Increased TXNIP expression was also evident in vitro when BET/BRD4 activity was inhibited in OS cell lines by siRNA, PROTAC-mediated degradation, or disruption of BRD4-histone interactions via AZD5153. These effects were accompanied by elevated c-PARP levels, a marker of cell death. Ongoing studies suggest BRD4 inhibition elevates TXNIP via AKT pathway suppression, as indicated by reduced phospho-AKT in AZD5153-treated tumors. In vitro combination studies showed additive-to-synergistic effects between AZD5153 and a variety of salvage agents (etoposide, SN38, and topotecan). In particular, AZD5153 in combination with topotecan resulted in enhanced apoptosis, increased TXNIP, decreased AKT, and elevated RS markers (gamma-H2AX, comet assays). In vivo, dose finding studies indicated that PDX77-TT2 was resistant to salvage agents ifosfamide and SN38 but moderately sensitive to topotecan. Furthermore, AZD5153 alone or in combination with topotecan improved survival compared to single agents (p<0.05) and was well tolerated. Collectively, these data support BET inhibition alone or with salvage therapy as a promising therapeutic approach for aggressive OS, potentially mediated through TXNIP upregulation and AKT pathway suppression.