The selective heat shock protein 90 (HSP90) inhibitor 17-allyamino-17-demethoxygeldanamycin (17-AAG) is currently in phase I/II clinical studies at numerous institutions. Heretofore, the biomarkers to detect 17-AAG bioactivity (Hsp70, Raf-1, and cyclin-dependent kinase 4) had to be analyzed by Western blot of cellular samples, either from tumor biopsies or peripheral blood leukocytes, a method that is both laborious and invasive. We have identified two new biomarkers [insulin-like growth factor binding protein-2 (IGFBP2) and HER-2 extracellular domain] that can be readily detected in patient sera by ELISA. Both secreted proteins are derived from or regulated by Hsp90 client proteins, raising hopes that they might be sensitive serum markers of HSP90 inhibitor activity. Several structurally unrelated HSP90 inhibitors dose-dependently decreased secretion of both IGFBP-2 and HER-2 extracellular domain into culture medium, and both proteins were more sensitive to HSP90 inhibitors than previously identified biomarkers. In sera from BT474 tumor-bearing mice, both IGFBP-2 and HER-2 extracellular domain were downregulated by 17-AAG in a time-dependent and dosedependent manner, coincident with the degradation of HER-2 and attenuation of AKT activity in the tumors. Furthermore, IGFBP-2 levels at the end of treatment correlated with residual tumor load, suggesting that IGFBP-2 might serve as an early indicator of therapeutic response. In addition, we also found that both IGFBP-2 and HER-2 extracellular domain levels are elevated in patient sera from several cancer types, suggesting that these novel secreted biomarkers could be valuable pharmacodynamic tools in clinical trials of HSP90 inhibitors.

The geldanamycin derivative 17-allyamino-17-demethoxygeldanamycin (17-AAG) is a clinical stage ATP-competitive HSP90 inhibitor that induces degradation of HSP90 client proteins. 17-AAG contains 1 ansamycin moiety and is highly potent in conventional cell killing assays. Since active Hsp90 exists as a dimer, we hypothesized that dimeric compounds containing 2 ansamycin pharmacophores might inhibit Hsp90 function more efficiently than 17-AAG. Here, we show that monomeric and dimeric ansamycins exert their activity in distinct ways. Under conditions of continuous exposure, 17-AAG induced client degradation and cell growth inhibition more readily than the dimeric drugs CF237 and CF483. By contrast, 24 hr treatment of various tumor cells with 17-AAG followed by drug washout caused temporary client degradation and cell cycle arrest but minimal cell death, whereas both dimers induced massive apoptosis. CF237 remained bound to Hsp90 for days after drug withdrawal and, while both monomeric and dimeric compounds caused accumulation of the inactive intermediate Hsp90 complex, this effect disappeared following washout of 17-AAG but not CF237. The dimer was also retained for longer in tumor xenografts and displayed superior antitumor activity in vivo. These results indicate that monomeric and dimeric Hsp90 inhibitors have distinct biological profiles and work differentially toward target inhibition. ' 2006 Wiley-Liss, Inc.Key words: Hsp90; 17-AAG; dimeric inhibitor; cancer therapy Heat shock protein 90 (HSP90) is a conserved molecular chaperone that mediates the maturation and stability of a set of cancerassociated proteins, referred to as ÔclientsÕ. These include steroid receptors, EGFR family members, MET, Raf-1 kinase, AKT, Bcrabl, mutant p53, CDK4 and many other oncogenic molecules. [1][2][3] Hsp90 functions as a super-chaperone complex in association with various cochaperone proteins. Hsp90 mainly exists in 2 types of multiprotein complexes, referred to as ÔintermediateÕ and ÔmatureÕ. 3,4In the intermediate complex, which is the ADP-binding form, the major cochaperones are Hsp70, Hsp40, HOP and HIP. Upon ATP binding, cdc37, p23 and immunophilins replace the original cochaperones to assist the conformational maturation of the client proteins and maintain those proteins in an active state to exert their function. 4 It is thought that Hsp90 inhibitors bind to and stabilize the intermediate complex, leading to recruitment of ubiquitin ligases and degradation of client proteins in the proteasome. 5,6 Ansamycin antibiotics such as geldanamycin (GM) are natural products that bind to the N-terminal ATP/ADP binding pocket of HSP90.5-8 Exposure of cells to these compounds induces the degradation of a range of HSP90 clients and results in cell cycle arrest followed in some cases by apoptotic cell death. 9 The GM derivative 17-allylaminogeldanamycin (17-AAG) was the first HSP90 inhibitor to enter clinical trials. The drug is well tolerated, despite the fact that it simultaneously targets many intracellular signaling proteins. 10,1...