Sequential targeting of retinoblastoma and DNA synthesis pathways is a therapeutic strategy for sarcomas that can be monitored in real-time.

Treatment strategies with a strong scientific rationale based on specific biomarkers are needed to improve outcomes in patients with advanced sarcomas. Suppression of cell cycle progression through reactivation of the tumor suppressor retinoblastoma (Rb) using CDK4/6 inhibitors is a potential avenue for novel targeted therapies in sarcomas that harbor intact Rb signaling. Here, we evaluated combination treatment strategies (sequential and concomitant) with the CDK4/6 inhibitor abemacicib to identify optimal combination strategies. Expression of Rb was examined in 1043 sarcoma tumor specimens, and 50% were found to be Rb-positive. Using in vitro and in vivo models, an effective 2-step sequential combination strategy was developed. Abemaciclib was used first to prime Rb-positive sarcoma cells to reversibly arrest in G1-phase. Upon drug removal, cells synchronously traversed to S-phase, where a second treatment with S-phase targeted agents (gemcitabine or WEE1-kinase inhibitor) mediated a synergistic response by inducing DNA damage. The response to treatment could be non-invasively monitored using real-time positron emission tomography (PET) imaging and serum thymidine kinase activity. Collectively, these results show that a novel, sequential treatment strategy with a CDK4/6 inhibitor followed by a DNA damaging agent was effective, resulting in synergistic tumor cell killing. This approach can be readily translated into a clinical trial with non-invasive functional imaging and serum biomarkers as indicators of response and cell cycling.