function. In addition, cells expressing the human papillomavirus E6 gene, which show increased degradation of p53 by ubiquitination and fail to accumulate p53 in response to DNA-damaging agents, do increase their p53 levels following heat and hypoxia. These results suggest that hypoxia is an example of a "nongenotoxic" stress which induces p53 activity by a different pathway than DNA-damaging agents.

It has been convincingly demonstrated that genotoxic stresses cause the accumulation of the tumor suppressor gene p53. One important consequence of increased p53 protein levels in response to DNA damage is the activation of a G1-phase cell cycle checkpoint. It has also been shown that G1-phase cell cycle checkpoints are activated in response to other stresses, such as lack of oxygen. Here we show that hypoxia and heat, agents that induce cellular stress primarily by inhibiting oxygen-dependent metabolism and denaturing proteins, respectively, also cause an increase in p53 protein levels. The p53 protein induced by heat is localized in the cytoplasm and forms a complex with the heat shock protein hsc70. The increase in nuclear p53 protein levels and DNA-binding activity and the induction of reporter gene constructs containing p53 binding sites following hypoxia occur in cells that are wild type for p53 but not in cells that possess mutant p53. However, unlike ionizing radiation, the accumulation of cells in G1 phase by hypoxia is not strictly dependent on wild-type p53 function. In addition, cells expressing the human papillomavirus E6 gene, which show increased degradation of p53 by ubiquitination and fail to accumulate p53 in response to DNA-damaging agents, do increase their p53 levels following heat and hypoxia. These results suggest that hypoxia is an example of a "nongenotoxic" stress which induces p53 activity by a different pathway than DNA-damaging agents.

Polymerization of metabolic enzymes into micron‐scale assemblies is an emerging phenomenon. However the role assembly plays in regulating metabolic flux differs between enzymes and in many cases is unknown. Furthermore, the signaling pathways that regulate assembly are often not well understood. Our work focuses on enzymes mediating pyrimidine and purine biosynthetic pathways that undergo regulated assembly. To address the biological function of these assemblies we have developed point mutants of CTP synthase and inosine monophosphate dehydrogenase (IMPDH), that prevent their assembly without eliminating their catalytic activity. We will present new data using these novel tools in mice and Drosophila to elucidate the role of polymerization of these enzymes in native contexts in which they assemble. Our findings demonstrate a critical role for assembly of CTPS and IMPDH in supporting the growth and proliferation of cells with elevated nucleotide demands. Thus, we propose that assembly of these nucleotide biosynthetic enzymes has been harnessed in metazoans as a homeostatic mechanism to ensure that metabolic biosynthetic flux meets demand. Support or Funding Information NIH GM083025

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.