Simultaneous Targeting of CDK4/6 and BETs is Independent of RB Status in Osteosarcoma

Abstract

Osteosarcoma (OS), the third most common malignancy in adolescents, is characterized by heterogeneity and genomic instability. Despite aggressive multimodal therapy, survival rates remain around 60% for localized and between 5-30% for metastatic disease. OS has a high propensity for early hematogenous and recurrence, indicating a critical need for novel treatment options. Genomic analysis from the Pediatric Cancer Precision Genomics Program at our institution and others have identified dysregulation in replication stress (RS) pathways (MYC, RAD21) and cell cycle regulators (CDKN2A, CDK4/6), pointing to CDK4/6 inhibitors (CDK4/6i), like palbociclib and abemaciclib, as promising therapeutic strategies. However, monotherapy often fails, highlighting the need for effective combination approaches. While functional RB has traditionally been viewed as necessary for CDK4/6i sensitivity, emerging evidence challenges this paradigm and suggests that RB-deficient (RB-) tumors may respond through alternative, RB-independent mechanisms. Bromodomain and extraterminal (BET) proteins are targeted by BET inhibitors (BETi), like AZD5153, are epigenetic readers that regulate the transcription of oncogenes like MYC, E2F and mTOR, and increase transcription-replication errors promoting genomic instability, complementing CDK4/6 blockade. Based on functional in vitro drug screens in the Pollok lab, my dissertation investigates whether combining CDK4/6i with a BETi enhances therapeutic efficacy regardless of RB status. My research demonstrates that dual BETi and CDK4/6i therapy produces additive-to-synergistic anti-tumor effects in both RB-proficient (RB+) and RB- OS cell lines, including CRISPR-engineered RB-knockout clones. Functional studies show enhanced apoptosis, DNA damage, altered cell cycle progression, and reduced clonogenicity. In vivo, combination therapy significantly suppressed tumor growth in both treatment-naïve and previously treated OS patient-derived xenograft models. In a lung-metastatic OS model, CDK4/6i alone markedly reduced metastatic burden; however, at the tested BETi exposure, the addition of BETi did not further decrease metastatic foci. These results underscore the therapeutic potential of CDK4/6 blockade and establish a foundation for continued mechanistic and translational investigation. Building upon these functional studies, ongoing evaluation of in vitro and in vivo mechanistic profiling, using transcriptomic, kinome, and histologic analysis aim to refine combination strategies, optimize BETi dosing and sequencing, and define the contexts where dual CDK4/6i and BETi therapy most effectively limit OS progression and dissemination.