Inhibition of heat shock protein 90 (HSP90) leads to inappropriate processing of proteins involved in cell survival pathways. We found that HSP90 inhibitor, 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (DMAG), is synergistic with radiation for non-small cell lung cancer cell lines, NCI-H460 and A549. To establish the optimal schedule for this combination, cells were radiated before, after, or simultaneously with DMAG, and survival was scored by clonogenic assay. The sequence of DMAG administration was critical for synergy with radiation, and pretreatment for 16 h led to maximal synergy. Similar radiosensitization was observed in isogenic cells in which expression of wild-type p53 was silenced by RNA interference, although p53 loss rendered cells overall less radiosensitive. The mechanistic basis for synergy was studied by Western blotting, cell cycle analysis, alkaline comet assay, and direct measurement of the activities of key base excision repair enzymes. Regardless of schedule of administration, DMAG led to degradation of proteins involved in activation of cell survival pathways after radiation, which did not explain the differences in the schedule of administration observed in clonogenic assays. In addition to previously reported decrease in activation of ATM, pretreatment with DMAG blocked activation of base excision repair machinery and activity of key enzymes, apurinic/apyrimidinic endonuclease, and DNA polymerase-B.Similarly, pretreatment with specific apurinic/apyrimidinic endonuclease inhibitor, CRT0044876, reproduced the effects of DMAG. Thus, administration of HSP90 inhibitors before radiation is critical for optimizing their use as radiosensitizers. [Mol Cancer Ther 2008;7(7):1985 -92]

Purpose: Loss of p53 function impairs apoptosis induced by DNA-damaging agents used for cancer therapy. Here, we examined the effect of the heat shock protein 90 (HSP90) inhibitor 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (DMAG) on doxorubicin-induced apoptosis in lymphoma. We aimed to establish the optimal schedule for administration of both drugs in combination and the molecular basis for their interaction. Experimental Design: Isogenic lymphoblastoid and nonisogenic lymphoma cell lines differing in p53 status were exposed to each drug or combination. Drug effects were examined using Annexin V, active caspase-3, cell cycle, and cytotoxicity assays. Synergy was evaluated by median effect/combination index. Protein expression and kinase inhibition provided insight into the molecular mechanisms of drug interaction. Results: Presence of mutant p53 conferred increased survival to single agents. Nevertheless, DMAG showed synergistic toxicity with doxorubicin independently of p53 status. Synergy required exposure to doxorubicin before DMAG. DMAG-mediated down-regulation of CHK1, a known HSP90 client, forced doxorubicin-treated cells into premature mitosis followed by apoptosis. A CHK1 inhibitor, SB-218078, reproduced the effect of DMAG. Administration of DMAG before doxorubicin resulted in G 1 -S arrest and protection from apoptosis, leading to additive or antagonistic interactions that were exacerbated by p53 mutation. Conclusions: Administration of DMAG to doxorubicin-primed cells induced premature mitosis and had a synergistic effect on apoptosis regardless of p53 status. These observations provide a rationale for prospective clinical trials and stress the need to consider schedule of exposure as a critical determinant of the overall response when DMAG is combined with chemotherapeutic agents for the treatment of patients with relapsed/refractory disease.

Supplementary Figure Legends 1-3 from Schedule-Dependent Synergy between the Heat Shock Protein 90 Inhibitor 17-(Dimethylaminoethylamino)-17-Demethoxygeldanamycin and Doxorubicin Restores Apoptosis to p53-Mutant Lymphoma Cell Lines

Supplementary Figure 1 from Schedule-Dependent Synergy between the Heat Shock Protein 90 Inhibitor 17-(Dimethylaminoethylamino)-17-Demethoxygeldanamycin and Doxorubicin Restores Apoptosis to p53-Mutant Lymphoma Cell Lines

Supplementary Figure 3 from Schedule-Dependent Synergy between the Heat Shock Protein 90 Inhibitor 17-(Dimethylaminoethylamino)-17-Demethoxygeldanamycin and Doxorubicin Restores Apoptosis to p53-Mutant Lymphoma Cell Lines