17-Allylamino-demethoxygeldanamycin (17-AAG), currently in phase I and II clinical trials as an anticancer agent, binds to the ATP pocket of heat shock protein (Hsp90).

Purpose: To determine the maximum tolerated dose (MTD), dose-limiting toxicity, and pharmacokinetics of 17-allylamino-demethoxy-geldanamycin (17-AAG) administered on days 1, 4, 8, and 11 every 21 days and to examine the effect of 17-AAG on the levels of chaperone and client proteins. Experimental Design: A phase I dose escalating trial in patients with advanced solid tumors was done. Toxicity and tumor responses were evaluated by standard criteria. Pharmacokinetics were done and level of target proteins was measured at various points during cycle one. Results: Thirteen patients were enrolled in the study. MTD was defined as 220 mg/m 2 . Dose-limiting toxicities were as follows: dehydration, diarrhea, hyperglycemia, and liver toxicity. At the MTD, the mean clearance of 17-AAG was 18.7 L/h/m 2 .There was a significant decrease in integrin-linked kinase at 6 hours after infusion on day 1 but not at 25 hours in peripheral blood mononuclear cells. Treatment with 17-AAG on day 1significantly increased pretreatment levels of heat shock protein (HSP) 70 on day 4, which is consistent with the induction of a stress response. In vitro induction of a stress response and up-regulation of HSP70 resulted in an increased resistance to HSP90-targeted therapy in A549 cells. Conclusions:The MTD of 17-AAG on a twice-weekly schedule was 220 mg/m 2 . Treatment at this dose level resulted in significant changes of target proteins and also resulted in a prolonged increase in HSP70. This raises the possibility that HSP70 induction as part of the stress response may contribute to resistance to 17-AAG.Heat shock protein (HSP) 90 is part of a chaperone complex for multiple client proteins involved in cell signaling, proliferation, and survival (1 -5). HSP90 function can be disrupted by geldanamycin (6), which results in the dissociation and degradation of client proteins, such as HER-2, RAF, mutant p53, cyclin-dependent kinase 4, Src, focal adhesion kinase, AKT, nuclear factor-nB, and insulin-like growth factor receptor 1 (7 -9). The binding of geldanamycin and its analogues to HSP90 also induces a stress response, which is manifested in part by increased levels of cochaperone and other stress proteins, such as HSP70 (10). In fact, cells deficient in HSF1 do not induce a stress response and are more sensitive to geldanamycin (11). Geldanamycin caused hepatotoxicity in dogs in preclinical studies, so further development was terminated (12). The geldanamycin analogue 17-allylaminodemethoxy-geldanamycin (17-AAG, NSC 330507) was shown to be less toxic than geldanamycin and shown activity in mouse xenograft models (13 -17). 17-AAG is metabolized to 17-aminogeldanamycin (17-AG) by cytochrome P450 3A4/5 and is widely distributed in body tissues but not in the central nervous system (18,19). The 17-AAG metabolite, 17-AG, also binds to HSP90, disrupting its ability to chaperone client proteins.Goetz et al. (20) reported recently results of the phase I study of 17-AAG administered on a weekly schedule to patients with advanced cancer. The m...

Purpose: To evaluate the effects of combining the multiple receptor tyrosine kinase inhibitor AEE788 and histone deacetylase (HDAC) inhibitors on cytotoxicity in a broad spectrum of cancer cell lines, including cisplatin-resistant ovarian adenocarcinoma cells. Experimental Design: Multiple cancer cell lines were treated in vitro using AEE788 and HDAC inhibitors (LBH589, LAQ824, and trichostatin A), either alone or in combination. Effects on cytotoxicity were determined by growth and morphologic assays. Effects of the combination on cell signaling pathways were determined by Western blotting, and the results were confirmed using pathway-specific inhibitors and transfection of constitutively active proteins. Results: Cell treatment with AEE788 and HDAC inhibitors (LBH589, LAQ824, and trichostatin A) in combination resulted in synergistic induction of apoptosis in non^small cell lung cancer (MV522, A549), ovarian cancer (SKOV-3), and leukemia (K562, Jurkat, and ML-1) cells and in OV202hp cisplatin-resistant human ovarian cancer cells. AEE788 alone or in combination with LBH589 inactivated mitogen-activated protein kinase (MAPK) and Akt cascades. Inhibition of either MAPK and/or Akt enhanced LBH589-induced apoptosis. In contrast, constitutively active MAPK or Akt attenuated LBH589 or LBH589 + AEE788^induced apoptosis. Increased apoptosis was correlated with enhanced reactive oxygen species (ROS) generation. The free radical scavenger N-acetyl-L-cysteine not only substantially suppressed the ROS accumulation but also blocked the induction of apoptosis mediated by cotreatment with AEE788 and LBH589. Conclusion: Collectively, these results show that MAPK and Akt inactivation along with ROS generation contribute to the synergistic cytotoxicity of the combination of AEE788 and HDAC inhibitors in a variety of human cancer cell types. This combination regimen warrants further preclinical and possible clinical study for a broad spectrum of cancers.

Despite studies that show the antitumor activity of Hsp90 inhibitors, such as geldanamycin (GA) and its derivative 17-allylamino-demethoxygeldanamycin (17-AAG), recent reports indicate that these inhibitors lack significant singleagent clinical activity. Resistance to Hsp90 inhibitors has been previously linked to expression of P-glycoprotein (P-gp) and the multidrug resistant (MDR) phenotype. However, the stress response induced by GA treatment can also cause resistance to Hsp90-targeted therapy. Therefore, we chose to further investigate the relative importance of P-gp and the stress response in 17-AAG resistance. Colony-forming assays revealed that high expression of P-gp could increase the 17-AAG IC 50

Benzoquinone ansamycin antibiotics such as geldanamycin (GA) bind to the N-terminal ATP binding domain of Hsp90 and inhibit its chaperone functions. Despite in vitro and in vivo studies indicating promising antitumor activity, derivatives of GA, including 17-AAG have demonstrated little clinical efficacy as single agents. Thus, combination studies of 17-AAG and several cancer chemotherapeutics, including cisplatin (CDDP), have begun. In colony-forming assays, the combination of CDDP and GA or 17-AAG was synergistic, and caused increased apoptosis compared to each agent alone. One measurable response that results from treatment with Hsp90-targeted agents is the induction of an HSF-1 heat shock response. Treatment with GA + CDDP revealed that CDDP suppresses upregulation of HSF-1 transcription, causing decreased levels of stress-inducible proteins such as Hsp27 and Hsp70. However, CDDP treatment did not prevent trimerization and nuclear localization of HSF-1, but inhibited DNA binding of HSF-1 as demonstrated by chromatin immunoprecipitation. Melphalan, but not camptothecin, caused similar inhibition of GA-induced HSF-1-mediated Hsp70 upregulation. MTS cell survival assays revealed that deletion of Hsp70 caused increased sensitivity to GA (Hsp70+/+ IC50=63.7±14.9 nM and Hsp70−/− IC50=4.3±2.9 nM), which confirmed that a stress response plays a critical role in decreasing GA sensitivity. Our results suggest that the synergy of GA + CDDP is due, in part, to CDDP-mediated abrogation of the heat shock response through inhibition of HSF-1 activity. Clinical modulation of the HSF-1-mediated heat shock response may enhance the efficacy of Hsp90-directed therapy.