Abstract 5340: Simultaneous targeting of CDK4/6 and BETs is independent of RB status in osteosarcoma

Osteosarcoma (OS) is the most prevalent primary bone cancer in children and adolescents, characterized by genetic instability and aggressive progression leading to poor survival. Despite the use of multimodal therapies, survival rates have stagnated around 60% highlighting a critical need for the identification of effective drug targets to improve outcomes. Sequencing efforts reveal cell-cycle dysregulation which may be targeted via CDK4/6 inhibitors (CDK4/6i) such as palbociclib and abemaciclib. While Retinoblastoma proficiency (RB+) has been used as a biomarker of response to CDK4/6i clinically, over 70% of OS patients exhibit RB deficiency (RB-). Therefore, it is imperative to understand to what degree RB- affects response to CDK4/6i in OS. Based on in vitro screening data, we hypothesize a combination of CDK4/6i with bromodomain and extraterminal domain inhibitors (BETi) will potentiate the CDK4/6i anti-tumorigenic effects in both RB+ and RB- OS. A hallmark of OS is high levels of genomic instability and BETi may further induce DNA damage to kill DNA repair-deficient cells. In-vitro combination studies identified dose ratios of CDK4/6i and bivalent BETi (AZD5153) that induce synergistic growth inhibition in a panel of monoallelic and biallelic RB OS cell lines. CRISPR-mediated RB-knockout (KO) clones were developed in human (143B, MG63.3) and murine (K7M2) metastatic OS cell lines. In comparison to wildtype (WT) clones, KO clones were 2-10-fold more resistant to palbociclib and at most 2-fold more resistant to abemaciclib; with differences attributed to broader selectivity profile of abemaciclib. However, dual CDK4/6i and BETi therapy resulted in synergistic growth inhibition in both WT and KO clones. To track emergence of therapeutic resistance, a longitudinal growth assay indicated that dual therapy induced cytotoxicity over time with greater effects observed in KO clones, presumably due to increased genome instability. CDK4/6i therapy with abemaciclib, but not palbociclib, increased C-PARP expression by 2-fold in WT cells and 50-fold in KO cells. Annexin V flow cytometry corroborated these findings, showing apoptosis in 25% of WT cells and 75% of KO cells. Limiting dilution assays revealed that dual CDK4/6i and BETi therapy reduced clonogenic cell frequency, with a more pronounced effect in KO clones. Additionally, RB monoallelic OS PDX models TT2 (pretreated) and PDX96 (naïve), were treated for 6 weeks with CDK4/6i (palbociclib, 40mg/kg, BETi (AZD5153,1 mg/kg), or their combination and significantly reduced tumor growth compared to single agents in both models (p<0.05) and was well tolerated. A trend for increased probability of survival was observed in PDX96. Future studies will extend dosing duration and focus on metastatic and RB- variant models. These findings provide rationale for further study of novel therapeutic options which may expand the clinical utility of CDK4/6i regardless of RB status. Citation Format: Rada Malko, Pankita H. Pandya, Harlan E. Shannon, Erika A. Dobrota, Barbara J. Bailey, Niknam Riyahi, Keiko E. Kreklau, Ryli E. Justice, Melissa A. Trowbridge, Kathryn L. Coy, Anthony L. Sinn, Khadijeh Bijangi-Vishehsaraei, M R. Saadatzadeh, Micheal J. Ferguson, Kyle W. Jackson, Brian D. Weiss, Karen E. Pollok. Simultaneous targeting of CDK4/6 and BETs is independent of RB status in 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 5340.