A. M. Francis1, A. Alexander2, J. P. Carey2, V. Ravi3, K. Keyomarsi2, K. K. Hunt1 1University Of Texas MD Anderson Cancer Center,Surgical Oncology,Houston, TX, USA 2University Of Texas MD Anderson Cancer Center,Experimental Radiation Oncology,Houston, TX, USA 3University Of Texas MD Anderson Cancer Center,Sarcoma Medical Oncology,Houston, TX, USA
Introduction:
The retinoblastoma (RB) gene pathway is one of the most frequently altered pathways in human cancer. This pathway is regarded as one of the central regulators of cell proliferation through the coordination of the RB protein, cyclin-dependent protein kinases (CDK4, CDK6), D-type cyclins, the INK4 family of cyclin-dependent kinase inhibitors and the E2F-family of transcription factors. CDK4/6 inhibitors such as PD-03329991 (PD-991) have been shown to cause a G1 arrest in RB-positive cells while WEE1 inhibitors such as MK-1775 can cause premature mitotic entry of S-phase cells. We hypothesized that sarcoma cells could be synchronized in S-phase using PD-991 followed by a period of recovery to facilitate synergistic demise with MK-1775.
Methods:
Sarcoma cell lines (HT-1080, SK-LMS-1, SaOS-2) were evaluated for RB-pathway alterations using western blot analysis. Cells were treated with PD-991 for 6 days and MK-1775 for 2 days to determine IC50 values. FACS analysis was used to determine the optimal recovery timepoint for synchronization in S-phase after treatment with PD-991. Combination experiments involved application of PD-991 with or without a period of recovery followed by application of MK-1775. CalcuSyn 2.0 was used to yield a combination index (CI) to determine if the combination was synergistic (CI<1), additive (CI=1) or antagonistic (CI>1).
Results:
HT-1080 and SK-LMS-1 were RB-positive while SaOS-2 was RB-negative. PD-991 was more effective as single agent therapy in RB-positive cells (1.5-2.4uM) than RB-negative cells (7.0uM). MK-1775 had similar efficacy across all cell lines tested (1.4uM [HT-1080], 0.7uM [SK-LMS-1], 1.0uM [SaOS-2]). The greatest proportion of S-phase cells was observed after 6-9 hours of recovery from PD-991 (HT-1080: 21-25% vs 16% [vehicle], SK-LMS-1: 30-43% vs 21% [vehicle], SaOS-2: not obtainable due to its RB-negativity). PD-991 followed by MK-1775 was strongly synergistic in RB-positive cells (Figure). A period of recovery after treatment with PD-991 enhances this synergism. In RB-negative SaOS-2, the combination of PD-991 and MK-1775 was additive to antagonistic (CI 1.0-1.5).
Conclusion:
Synchronization of cells in S-phase using PD-991 followed by treatment with MK-1775 causes synergistic demise of RB-positive sarcoma cells. In vivo studies are warranted to test this novel treatment strategy and are currently underway.
