Abstract 1843: Combination therapy targeting Rb/Wee1 kinase pathways for rhabdomyosarcoma treatment

Abstract

Abstract Introduction Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. New therapeutic strategies are needed particularly for high-risk patients where the survival rate is 20-40%. The FDA approved CDK4/6 inhibitor palbociclib (PD), and the WEE1 kinase inhibitor MK-1775 have anticancer effects when used as single agents. PD causes cell arrest at G1 phase (requiring intact Rb), while MK-1775 promotes cell death by abrogating DNA damage during the G2/M checkpoint phase by forcing premature mitosis entry. As the two drugs target tumor cells at different phases of the cell cycle, cells that are not in the targeted phase may escape single therapy. We hypothesized that sequential combination therapy will increase therapeutic efficacy. The use of PD first, will induce the cells to go under G1 arrest. Upon recovery, more cells will synchronize in the S/G2 phase, increasing their susceptibility to MK1775, resulting in synergistic cell killing. Methods Rb+ RMS (Rh-30) and Rb- osteosarcoma (OS) cell lines (CCH-D, LM7, and SASO2) were treated with increasing doses of PD for 6 days, then stained with propidium iodide (PI) and analyzed for cell cycle status to determine whether PD induced G1 arrest. To determine the optimal recovery time for Rb+ cells to be synchronized in S/G2 phase, cells were treated with PD (IC50-IC75) for 6 days and then were harvested either immediately or after 3-24 h recovery in drug free medium. Cells were stained with PI for cell cycle analysis. A 12-day long combination therapy assay was performed with or without recovery time between the two treatments. Combination index (CI) was calculated by using the statistical program calcusyn to determine whether the combinatorial drug treatment had acted in an antagonistic, additive or synergistic manner. Results Six days of treatment with PD resulted in reversible G1 arrest only in Rb+ cell lines, confirming that Rb, the downstream target of CDK4, must be intact to induce G1 arrest by palbociclib. Six days of treatment with PD followed by 9-12 h recovery in fresh medium (the optimal recovery time prior to treatment with MK-1775), led to re-entry of the Rb+ Rh-30 cells into the cell cycle and successful synchronization at S/G2 phase. By contrast, Rb- cells failed to arrest in G1 phase following treatment with PD and were not synchronized at S/G2 phase. Since cells synchronized in S/G2 phase are more vulnerable to MK1775, combination therapy resulted in a synergistic effect in the Rb+ cells but an antagonistic effect in the Rb- cells. Conclusion: Our results suggest that the combination therapy strategy of PD preceding MK1775 may be a novel therapeutic approach for patients with Rb+ RMS. This strategy will be investigated in xenograft and PDX RMS model. Citation Format: Simin Kiany, Gangxiong Huang, Melanie Justice, Khandan Keyomarsi, Eugenie Kleinerman. Combination therapy targeting Rb/Wee1 kinase pathways for rhabdomyosarcoma treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1843.