Abstract 1656: MK-1775 (WEE1 inhibition) lacks efficacy against DNA repair deficient pancreatic cancer cells
Authors: Lal Shruti , Chand Saswati N. , Dylgjeri Emanuela , Yeo Charles J. , Winter Jordan M. , Brody Jonathan R. 
Published: August 2015

Introduction: In this study, we explored the possibility of developing a personalized approach of using MK-1775, a potent WEE1 inhibitor, as mono- or combination therapy to treat pancreatic ductal adenocarcinoma (PDA) cells with DNA repair deficiency.

Experimental Methods: We treated PDA cells harboring diverse genetic backgrounds with IC50 doses of clinically-relevant DNA damaging agents: mitomycin C (MMC) and the WEE1 inhibitor MK-1775. Drug sensitivity assays were performed in isogenic PDA cells that are either proficient or deficient in Fanconi Anemia (FA)/BRCA2 pathway. In addition, proficient cell lines were treated with siRNA oligos targeted against FANCD2 and BRCA2 genes. Mechanistic studies such as Annexin V staining were performed to measure apoptotic cells upon MMC and MK-1775 treatments. The degree of DNA damage was measured by immunofluorescence (IF) assay using γH2AX antibody. Mitotic entry was analyzed by both IF and flow cytometry analyses using pH3 antibody.

Results: Drug sensitivity assays demonstrated that FA/BRCA2-pathway proficient PDA cell lines (MiaPaCa2 and Panc1) are sensitive to the cytotoxic effect of MK-1775 compared to DNA repair-deficient cell lines (Capan1:BRCA2 deficient and Hs766T:FANCG deficient) which showed acute resistance to MK-1775. Immunoblotting showed that MK-1775 efficiently reduces the expression of WEE1 and accordingly phosphorylation of CDK1 in all cell lines. Annexin V staining showed a higher percentage of cell death in the FA/BRCA2-pathway proficient cell lines compared to deficient cell lines when exposed to MK-1775. Furthermore, IF experiments demonstrated that MMC treatment induces γH2AX foci in all cell lines, however, the FA/BRCA2 proficient cell lines showed a higher degree of nuclear abnormality and multi-nucleation after MK-1775 treatment compared to deficient cell lines. In addition, the FA/BRCA2 proficient cell lines showed significantly higher phospho histone 3 (pH3) staining, a marker of mitotic cells. FACS analyses validated that proficient cell lines showed a higher percentage of cells in the mitotic phase when exposed to MK-1775. In addition, we evaluated the cytotoxic effect of MK-1775 in combination with MMC and showed that FA/BRCA2 proficient cells are more sensitive to dual treatment than deficient cells. In addition, immnobloting detected cleaved caspase 3, a marker of apoptosis, after MK-1775 treatment alone or in combination with MMC, demonstrating cells undergoing mitotic catastrophe. We validated the results in the FA/BRCA2-pathway proficient cell lines pre-treated with siFANCD2 or siBRCA2 oligos as compared to the proficient control lines.

Conclusions: These results support a paradigm in which identified high risk FA/BRCA2-mutated patients would not benefit from WEE1 inhibitor monotherapy; and thus, would most likely respond better to conventional DNA damaging agent-based therapies (e.g., oxaliplatin or MMC).

Citation Format: Shruti Lal, Saswati N. Chand, Emanuela Dylgjeri, Charles J. Yeo, Jordan M. Winter, Jonathan R. Brody. MK-1775 (WEE1 inhibition) lacks efficacy against DNA repair deficient pancreatic cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1656. doi:10.1158/1538-7445.AM2015-1656