Re-purposing clinical kinase inhibitors to enhance chemosensitivity by overriding checkpoints

Abstract

Inhibitors of the DNA damage checkpoint kinase, Chk1, are highly effective as chemo- and radio-sensitizers in preclinical studies but are not well-tolerated by patients. We exploited the promiscuous nature of kinase inhibitors to screen 9 clinically relevant kinase inhibitors for their ability to sensitize pancreatic cancer cells to a sub-lethal concentration of gemcitabine. Bosutinib, dovitinib, and BEZ-235 were identified as sensitizers that abrogated the DNA damage checkpoint. We further characterized bosutinib, an FDA-approved Src/Abl inhibitor approved for chronic myelogenous leukemia. Unbeknownst to us, we used an isomer (Bos-I) that was unknowingly synthesized and sold to the research community as “authentic” bosutinib. In vitro and cell-based assays showed that both the authentic bosutinib and Bos-I inhibited DNA damage checkpoint kinases Chk1 and Wee1, with Bos-I showing greater potency. Imaging data showed that Bos-I forced cells to override gemcitabine-induced DNA damage checkpoint arrest and destabilized stalled replication forks. These inhibitors enhanced sensitivity to the DNA damaging agents’ gemcitabine, cisplatin, and doxorubicin in pancreatic cancer cell lines. The in vivo efficacy of Bos-I was validated using cells derived directly from a pancreatic cancer patient’s tumor. Notably, the xenograft studies showed that the combination of gemcitabine and Bos-I was significantly more effective in suppressing tumor growth than either agent alone. Finally, we show that the gatekeeper residue in Wee1 dictates its sensitivity to the 2 compounds. Our strategy to screen clinically relevant kinase inhibitors for off-target effects on cell cycle checkpoints is a promising approach to re-purpose drugs as chemosensitizers.

Keywords: :

• Checkpoint override
• DNA damage
• Kinase inhibitors
• Mitosis
• drug repurposing

Disclosure of Potential Conflicts of Interest

There are no competing interests for any of the authors. A patent application (US application # PCT/US2013/031344) was filed by Brian Cocca (Stradley Ronon Stevens and Young, LLP) and Inna Khartchenko (FCCC) on behalf of the applicants Neil Beeharry and Timothy Yen.

Acknowledgments

We appreciate the comments and critical reading from Drs Enders and Dunbrack. Dr Enders also provided the mouseWee1 cDNA and human Wee 1 siRNA. We wish to acknowledge the technical assistance of Drs Fink, Duong-Ly, and Yang. We also acknowledge the Core supported Imaging, Molecular Modeling, High-throughput Screening, Cell Sorting, Histopathology, Animal and Tissue Culture facilities at Fox Chase Cancer Center for equipment and technical support. This work is dedicated to the memory of Elena Gitelson MD PhD.

Financial Disclosure

This work was supported in part by NIH CA169706, core grant CA06927, DoD OC100172, an Appropriation from Common Wealth of PA, the PA CURE (T.J.Y.); the Plain and Fancy Board of Associates Fellowship, FCCC (N.B.); the Pew Charitable Fund, by a generous gift from Mrs Concetta Greenberg to Fox Chase Cancer Center, by Tobacco Settlement funding from the State of Pennsylvania, the Bucks County Board of Associates, NIH K22 CA160725, R21 CA164205, and a career development award from Genentech (I.A.) and R01 GM083025 (J.R.P.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.