Abstract B57: Bosutinib and bosutinib-isomer are novel Chk1 and Wee1 kinase inhibitors that sensitize cells to DNA damaging agent by overriding cell cycle checkpoint arrest.

Abstract

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 took advantage of the promiscuous nature of kinase inhibitors and screened 9 clinically relevant kinase inhibitors for their ability to sensitize pancreatic cancer cells (Panc1) 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 for the treatment of chronic myelogenous leukemia (CML). Other Src-family kinase inhibitors did not exhibit chemosensitization activity. Unbeknownst to us, the bosutinib we purchased was an isomer (Bos-I) that was unknowingly synthesized and sold to the research community as "authentic" bosutinib. The two compounds differed only in the arrangement of the same R groups around the aniline ring. Authentic bosutinib is designated 2, 4 dichloro, 5-methoxy, while Bosutinib isomer is 3, 5 dichloro, 4-methyoxy. Although our screen identified the isomer, we found that authentic bosutinib also had chemosensitization activity In vitro. Both bosutinib and Bos-I inhibited DNA damage checkpoint kinases Chk1 and Wee1 in vitro, with Bos-I showing greater potency. These inhibitors enhanced sensitivity to genotoxic agents that either arrest cells in S phase (gemcitabine, cisplatin) or G2 (doxorubicin). Live-cell imaging and immunofluorescence staining showed that Bos-I forced drug-treated cells to override the checkpoint arrest and entered an aberrant mitosis. The in vivo efficacy of bosutinib and Bos-I were validated using cells derived directly from a pancreatic cancer patient's tumor. Notably, the xenograft studies showed that the combination of gemcitabine and bosutinib or Bos-I were significantly more effective in suppressing tumor growth than any of the agents used alone. Consistent with the in vitro data that showed Bos-I was more potent checkpoint inhibitor than bosutinib, higher concentrations of bosutinib were needed to suppress tumor growth by gemcitabine. Molecular modeling of bosutinib and Bos-I bound to Chk1 and Wee1 indicate that the gatekeeper residues and the position of the methoxy group in the aniline ring may be critical determinants of drug binding. The methoxy group of bosutinib (position 5) clashes within the binding pocket particularly near the gatekeeper residues Leu and Asn, in Chk1 and Wee1 respectively. The methoxy group in the Bos-I (position 3) is shifted away from the gatekeeper and allows the inhibitor to bind deeper in the binding site for both kinases. The structural predictions are supported by relative binding energy calculations of bosutininb and Bos-I bound to these kinases. In addition, in vitro kinase assays that show that changing the gatekeeper residue in Wee1 reduced its sensitivity to Bos-I but enhanced its sensitivity to the authentic bosutinib. 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. Checkpoint override by bosutinib and its isomer maybe due to ability to dually inhibit Chk1 and Wee1 kinases. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B57. Citation Format: Neil Beeharry, Eugenia Banina, Vladmir Khazak, Sean Deacon, Jeff Peterson, Mark Andrake, Igor Astsaturov, Tim J. Yen. Bosutinib and bosutinib-isomer are novel Chk1 and Wee1 kinase inhibitors that sensitize cells to DNA damaging agent by overriding cell cycle checkpoint arrest. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B57.