02.01 Cell Cycle Dependent Inhibition of G9a Induces Replication Catastrophe in Pancreatic Cancer

G. Urrutia1, J. Toro-Zapata1, A. Salmonson1, G. Lomberk1  1Medical College Of Wisconsin,Research/Surgery,Milwaukee, WI, USA

Introduction:  Pancreatic ductal adenocarcinoma (PDAC) presents a significant health burden as the third leading cause of cancer-related deaths in the United States. Despite significant efforts to develop better therapeutics to treat PDAC, the 5-year survival rate for patients has improved only marginally. Thus, there remains an urgent need to further understand the molecular mechanisms underlying PDAC development to identify innovative therapeutic targets. Our laboratory is focused on utilizing epigenetic inhibitors for this purpose.

Methods:  IncuCyte® Live-Cell Analysis and clonogenic assays were utilized to monitor the in vitro effect of drug treatments (LY2606368 and BRD4770) on L3.6 and primary PDAC cell lines from human patients. Cellular effects were observed by immunofluorescence of phosphorylated H2A.X and FACS analyses, while molecular effects were assessed by western blot for various cell cycle and apoptosis markers. Subcutaneous xenografts were implanted and subsequently treated with vehicle, individual drugs, or in combination to evaluate in vivo tumor growth, and immunohistochemistry was performed on the harvested tumor tissue.

Results: Here, we sought to combine targeting of Checkpoint kinase 1 (Chk1), a key regulator of cell cycle transition in the DNA damage response pathway, and G9a, an epigenetic regulator of histone H3 lysine 9 methylation (H3K9me), which is necessary for reforming chromatin during DNA replication. Using live cell imaging, we found that the growth of PDAC cells, both L3.6 and primary cell lines from PDAC patients, is reduced by the combined inhibition of Chk1 (LY2606368) and G9a (BRD4770), achieving a synergistic effect. This result was recapitulated by clonogenic assays. To determine the underlying mechanism, we evaluated the extent of DNA damage as measured by H2AX phosphorylation and found the combination of LY2606368 and BRD4770 induces activation of the ATR-Chk1 axis, but fails at the Chk1 checkpoint, leading to replication stress and DNA damage. FACS analysis of cells treated with the combination further supported this observation, which demonstrated a significant increase of cells in S-phase, as well as a substantial sub-G1 fraction, indicating cell death. Moreover, cell death coincided with increased levels of cleaved caspase 3, as confirmed by fluorescent detection and western blot. Interestingly, pan-caspase inhibition, using Z-VAD-FMK, did not rescue the effect, indicating that the main mechanism involved in this process is not caspase-dependent. In vivo treatment of subcutaneous pancreatic cancer xenografts demonstrated that combined targeting of these pathways reduces tumor growth, which involves a reduction in proliferation observed by Ki67 staining along with an increase in overall DNA damage.

Conclusion: In summary, our results demonstrate that targeting the epigenetic regulator G9a in combination with inhibition of the DNA damage response checkpoint offers a novel therapeutic approach for pancreatic cancer through triggering DNA replication catastrophe.