Hsp90 is a protein chaperone that promotes the maturation and conformational stabilization of a subset of cellular proteins important in transducing proliferation and survival signals. Hsp90 clients include protein kinases (e.g., HER2, Raf-1, Akt, and Cdk4), steroid receptors (e.g., androgen receptor and estrogen receptor), and transcription factors (e.g., Hif1α; refs. 1
). Given the critical roles played by these Hsp90 clients in tumor growth and maintenance, inhibition of Hsp90 has emerged as a possible strategy for the treatment of advanced cancers. Several mutant oncoproteins, including v-Src, mutant EGFR, and mutant B-Raf, are also Hsp90 clients whereas their wild-type counterparts are either not dependent or only weakly dependent on Hsp90 chaperone function (8
). The dependence of these gain-of-function mutants on Hsp90 suggests that Hsp90 may be permissive for the development of tumors that express these oncogenes.
In this study, we characterized the antitumor effects of SNX-2112, a novel compound that binds selectively to the NH2-terminal ATP pocket of Hsp90. The SNX-2112 scaffold was identified by screening the purine-binding proteome for non-quinone- and nonpurine-containing scaffolds that bind selectively to Hsp90. This compound is pan-selective for the Hsp90 family in that it binds to Hsp90α, Hsp90β, Grp94, and Trap-1. To determine whether binding of SNX-2112 to Hsp90 resulted in inhibition of Hsp90 chaperone activity, we compared the effects of SNX-2112 to those of the geldanamycin derivative 17-AAG using a panel of breast, ovarian, and lung cancer cell lines. We found that SNX-2112 potently down-regulated HER2 expression and inhibited Akt and Erk pathway activity in breast cancer cells with HER2 amplification. These effects occurred with a kinetics and potency similar to that of 17-AAG. We further showed that treatment of breast cancer cells in vitro with SNX-2112, like 17-AAG, resulted in marked growth inhibition and other hallmarks of the natural product Hsp90 inhibitors, including a Rb-dependent G1 growth arrest and morphologic differentiation in selected models. These data suggest that the drugs have the same target activity (antagonizing Hsp90 activity) and comparable antitumor activities in vitro.
One notable exception, however, was the breast cancer cell line MDA-468. In this model, SNX-2112 was markedly more potent than 17-AAG. Previous work has shown that 17-AAG is metabolized by DT-diaphorase to the more potent hydroquinone 17-AAGH2
). MDA-468 cells are resistant to 17-AAG because the gene encoding for this activity, NQO1
, is mutated in MDA-468 cells. Transfection of NQO1
into MDA-468 cells, however, restores sensitivity of this model to 17-AAG, confirming that loss of DT-diaphorase expression can confer 17-AAG resistance (15
). Our data thus suggest that SNX-2112 activity is independent of NQO1
activity and that SNX-2112 may therefore have a broader spectrum of antitumor activity than 17-AAG. Given that the purine-based synthetic Hsp90 inhibitor PU24FCl has a similar activity profile to SNX-2112 in these cells, we hypothesize that the dependence of MDA-468 cells on Hsp90 function is not accurately reflected by the lack of activity of 17-AAG in this model.
SNX-2112 has variable oral bioavailability and therefore with the goal of testing the utility of this compound in mice, several prodrugs of SNX-2112 were developed that display improved solubility and pharmacologic properties. We show that a single dose of SNX-5542, a water-soluble prodrug of SNX-2112, was sufficient to induce the degradation of HER2 in tumor-bearing xenografts. Following oral administration of SNX-5542, the drug was rapidly converted to SNX-2112 where it preferentially accumulated in tumor tissues. Notably, recovery of HER2 expression and the activity of its downstream effector pathways were observed at late time points (24 and 48 h) despite the continued presence in the tumor of SNX-2112 concentrations greater than those necessary to inhibit Hsp90 in vitro. We speculate that this may be due to either intracellular compartmentalization of the drug or induction of Hsp70 expression. In experiments where we rechallenged with daily doses of SNX-5542 in vivo, we did observe similar kinetics of client degradation and signal deactivation after a third consecutive dose. Thus, at least for three consecutive doses, we did not find significant tachyphylaxis to the effects of SNX-2112.
In mice with established BT474 (HER2-amplified) xenografts, daily oral administration of SNX-5542 resulted in partial tumor regressions. These data were comparable if not superior to the effects of i.p. administration of 17-AAG in this model system using intermittent dosing schedules (three times per week or days 1–5 every 2 weeks). In prior studies of 17-AAG, daily dosing was not, however, feasible in either mice or in patients due to hepatotoxicity (31
). For this reason, 17-AAG is currently being tested in phase 2 trials using only intermittent dosing schedules: either weekly or days 1, 4, 8, and 11 every 21 days. Notably, in a pilot dog study, no significant hepatotoxicity was observed after 13 days of twice-daily dosing of SNX-5542 at the highest dose tested, 10 mg/kg. These data suggest that non-ansamycin Hsp90 inhibitors such as SNX-2112 may have a more favorable toxicity profile than 17-AAG, although human clinical trials will be necessary to test this hypothesis.
In addition to 17-AAG, several novel ansamycins are now in clinical development. These include the water-soluble and orally bioavailable geldanamycin derivative 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin and IPI-504, a prodrug of 17-AAG with improved solubility and oral bioavailability (36
). Although these agents have superior pharmacologic profiles to 17-AAG in terms of solubility and oral bioavailability, they also contain the quinone species found in 17-AAG and would therefore be predicted to retain the hepatotoxicity characteristic of this class of agents. As a nonquinone-based Hsp90 inhibitor, SNX-2112 may therefore have potential toxicologic advantages over these geldanamycin derivatives.
Finally, it is important to note that Hsp90 inhibition has shown provocative activity in a variety of cancer types. In this report, we show that SNX-5542 has activity in a model of EGFR mutant non–small cell lung cancer. We find that SNX-5542 is superior to 17-AAG in this model. In contrast to the effects of SNX-5542 in mice with established BT-474 tumors, monotherapy with SNX-5542 was insufficient to induce complete growth inhibition in mice with established H1650 xenografts. It is unclear if the greater resistance of this tumor to SNX-5542 relates to intrinsic properties of the tumor model or deficiencies in target inhibition in this system. For example, H1650 cells contain not only an EGFR deletion mutant but also are PTEN deficient. Although tumor regression was not observed in this model with SNX-5542 alone, we have recently shown that the combination of 17-AAG and paclitaxel is synergistic in this model. Therefore, despite its advantages over 17-AAG, the use of combination strategies will likely still prove necessary in some systems despite the presence of a sensitive Hsp90 client oncoprotein. Nevertheless, given the potential advantages of the small-molecule platform, these studies underscore the impetus for the clinical testing of SNX-5542, an Hsp90 inhibitor with superior pharmacologic properties.