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Supplementary Figure Legends 1-2 from P-Glycoprotein–Mediated Resistance to Hsp90-Directed Therapy Is Eclipsed by the Heat Shock Response

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 single-agent 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 IC50 6-fold in cells transfected with P-gp compared with parent cells. A549 cells selected for resistance to GA overexpressed P-gp, but verapamil did not reverse the resistance. These cells also overexpressed Hsp27, and Hsp70 was induced with 17-AAG treatment. When the GA and 17-AAG resistant cells were transfected with Hsp27 and/or Hsp70 small interfering RNA (siRNA), the 17-AAG IC50 decreased 10-fold compared with control transfected cells. Transfection with siRNA directed against Hsp27, Hsp70, or Hsp27 and Hsp70 also increased sensitivity to EC78, a purine scaffold-based Hsp90 inhibitor that is not a P-gp substrate. We conclude that P-gp may contribute, in part, to resistance to 17-AAG, but induction of stress response proteins, such as Hsp27 and Hsp70, by Hsp90-targeted therapy plays a larger role. Taken together, our results indicate that targeting of Hsp27 and Hsp70 should be exploited to increase the clinical efficacy of Hsp90-directed therapy. [Cancer Res 2008;68(18):7419–27]

Supplementary Figure 2 from P-Glycoprotein–Mediated Resistance to Hsp90-Directed Therapy Is Eclipsed by the Heat Shock Response

Supplementary Figure 1 from P-Glycoprotein–Mediated Resistance to Hsp90-Directed Therapy Is Eclipsed by the Heat Shock Response

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 single-agent 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 IC50 6-fold in cells transfected with P-gp compared with parent cells. A549 cells selected for resistance to GA overexpressed P-gp, but verapamil did not reverse the resistance. These cells also overexpressed Hsp27, and Hsp70 was induced with 17-AAG treatment. When the GA and 17-AAG resistant cells were transfected with Hsp27 and/or Hsp70 small interfering RNA (siRNA), the 17-AAG IC50 decreased 10-fold compared with control transfected cells. Transfection with siRNA directed against Hsp27, Hsp70, or Hsp27 and Hsp70 also increased sensitivity to EC78, a purine scaffold-based Hsp90 inhibitor that is not a P-gp substrate. We conclude that P-gp may contribute, in part, to resistance to 17-AAG, but induction of stress response proteins, such as Hsp27 and Hsp70, by Hsp90-targeted therapy plays a larger role. Taken together, our results indicate that targeting of Hsp27 and Hsp70 should be exploited to increase the clinical efficacy of Hsp90-directed therapy. [Cancer Res 2008;68(18):7419–27]

Runx2 and Axin2 regulate craniofacial development and skeletal maintenance. Runx2 is essential for calvarial bone development, as Runx2 haploinsufficiency causes cleidocranial dysplasia. In contrast, Axin2-deficient mice develop craniosynostosis because of high β-catenin activity. Axin2 levels are elevated in Runx2(-/-) calvarial cells, and Runx2 represses transcription of Axin2 mRNA, suggesting a direct relationship between these factors in vivo. Here we demonstrate that Runx2 binds several regions of the Axin2 promoter and that Runx2-mediated repression of Axin2 transcription depends on Hdac3. To determine whether Runx2 contributes to the etiology of Axin2 deficiency-induced craniosynostosis, we generated Axin2(-/-):Runx2(+/-) mice. These double mutant mice had longer skulls than Axin2(-/-) mice, indicating that Runx2 haploinsufficiency rescued the craniosynostosis phenotype of Axin2(-/-) mice. Together, these studies identify a key mechanistic pathway for regulating intramembranous bone development within the skull that involves Runx2- and Hdac3-mediated suppression of Axin2 to prevent the untimely closure of the calvarial sutures.

To better understand the assembly mechanism for the progesterone receptor (PR), we have developed cell-free systems for studying interactions of PR, hsp90, and other associated proteins. When PR is incubated in rabbit reticulocyte lysate, its association with hsp90, hsp70, the three immunophilins FKBP54, FKBP52 and CyP-40, and with p23 is observed. These interactions require ATP Mg 2+ and when ATP is limiting the PR complex is altered to one containing the proteins p60 and p48, but lacking immunophilins and p23. We have studied two pre-formed hsp90 complexes that may participate in the assembly of PR complexes. One contains hsp90 bound to hsp70 and p60 and this complex forms spontaneously in the absence of ATP. A second complex contains hsp90 bound to p23 plus the three immunophilins and some hsp70. The formation of this complex requires ATP. In further studies we have shown that purified hsp90 can bind to purified p23 and this interaction requires both ATP and molybdate. This explains, in part, the known effects of ATP and molybdate on assembly of PR complexes.

Histone deacetylase (Hdac) inhibitors are used clinically to treat cancer and epilepsy. Although Hdac inhibition accelerates osteoblast maturation and suppresses osteoclast maturation in vitro, the effects of Hdac inhibitors on the skeleton are not understood. The purpose of this study was to determine how the pan-Hdac inhibitor, suberoylanilide hydroxamic acid (SAHA; a.k.a. vorinostat or Zolinza™) affects bone mass and remodeling in vivo. Male C57BL/6 mice received daily SAHA (100 mg/kg) or vehicle injections for three to four weeks. SAHA decreased trabecular bone volume fraction and trabecular number in the distal femur. Cortical bone at the femoral midshaft was not affected. SAHA reduced serum levels of P1NP, a bone formation marker, and also suppressed tibial mRNA levels of type I collagen, osteocalcin and osteopontin, but did not alter Runx2 or osterix transcripts. SAHA decreased histological measures of osteoblast number but interestingly increased indices of osteoblast activity including mineral apposition rate and bone formation rate. Neither serum (TRAcP 5b) nor histological markers of bone resorption were affected by SAHA. P1NP levels returned to baseline in animals which were allowed to recover for four weeks after four weeks of daily SAHA injections, but bone density remained low. In vitro, SAHA suppressed osteogenic colony formation, decreased osteoblastic gene expression, induced cell cycle arrest, and caused DNA damage in bone marrow-derived adherent cells. Collectively, these data demonstrate that bone loss following treatment with SAHA is primarily due to a reduction in osteoblast number. Moreover, these decreases in osteoblast number can be attributed to the deleterious effects of SAHA on immature osteoblasts, even while mature osteoblasts are resistant to the harmful effects and demonstrate increased activity in vivo, indicating that the response of osteoblasts to SAHA is dependent upon their differentiation state. These studies suggest that clinical use of SAHA and other Hdac inhibitors to treat cancer, epilepsy or other conditions may potentially compromise skeletal structure and function.

Histone deacetylase (Hdac)3 is a nuclear enzyme that contributes to epigenetic programming and is required for embryonic development. To determine the role of Hdac3 in bone formation, we crossed mice harboring loxP sites around exon 7 of Hdac3 with mice expressing Cre recombinase under the control of the osterix promoter. The resulting Hdac3 conditional knockout (CKO) mice were runted and had severe deficits in intramembranous and endochondral bone formation. Calvarial bones were significantly thinner and trabecular bone volume in the distal femur was decreased 75% in the Hdac3 CKO mice due to a substantial reduction in trabecular number. Hdac3-CKO mice had fewer osteoblasts and more bone marrow adipocytes as a proportion of tissue area than their wildtype or heterozygous littermates. Bone formation rates were depressed in both the cortical and trabecular regions of Hdac3 CKO femurs. Microarray analyses revealed that numerous developmental signaling pathways were affected by Hdac3-deficiency. Thus, Hdac3 depletion in osterix-expressing progenitor cells interferes with bone formation and promotes bone marrow adipocyte differentiation. These results demonstrate that Hdac3 inhibition is detrimental to skeletal health.

To better understand assembly mechanisms of progesterone receptor (PR) complexes, we have developed a cell-free system for studying PR interactions with the 90- and 70-kDa heat shock proteins (hsp90 and hsp70), and we have used this system to examine requirements for hsp90 binding to PR. Purified chick PR, free of hsp90 and immobilized on an antibody affinity resin, will rebind hsp90 in rabbit reticulocyte lysate when several conditions are met. These include: 1) absence of progesterone, 2) elevated temperature (30 degrees C), 3) presence of ATP, and 4) presence of Mg2+. We have obtained maximal hsp90 binding to receptor when lysate is supplemented with 3 mM MgCl2 and an ATP-regenerating system. ATP depletion of lysate by dialysis or by enzymatic means blocks hsp90 binding to PR; likewise, addition of EDTA to lysate blocks hsp90 binding, but binding is restored by the addition of excess Mg2+. Addition to lysate of monoclonal antibody against hsp70 inhibits hsp90 binding to PR and destabilizes preformed complexes. Stabilization of hsp90-receptor complexes also requires ATP, indicating that ATP and hsp70 are needed to form and to maintain hsp90 complexes. Hormone-dependent activation of reconstituted receptor complexes was also examined. The addition of progesterone to the reticulocyte lysate promotes dissociation of hsp90 and hsp70 from the receptor. This also appears to require ATP and dissociation is most efficient in the presence of an ATP-regenerating system. In conclusion, these studies indicate that PR-hsp90 complexes do not self-assemble; instead, assembly is probably a multistep process requiring ATP and other cellular factors.

Purpose: To determine the maximum tolerated dose (MTD) and characterize the dose-limiting toxicities (DLT) of 17-AAG, gemcitabine and/or cisplatin. Levels of the proteins Hsp90, Hsp70 and ILK were measured in peripheral blood mononuclear cell (PMBC) lysates to assess the effects of 17-AAG. Experimental design: Phase I dose-escalating trial using a “3 + 3” design performed in patients with advanced solid tumors. Once the MTD of gemcitabine + 17-AAG + cisplatin was determined, dose escalation of 17-AAG with constant doses of gemcitabine and cisplatin was attempted. After significant hematologic toxicity occurred, the protocol was amended to evaluate three cohorts: gemcitabine and 17-AAG; 17-AAG and cisplatin; and gemcitabine, 17-AAG and cisplatin with modified dosing. Results: The 39 patients enrolled were evaluable for toxicity and response. The MTD for cohort A was 154 mg/m2 of 17-AAG, 750 mg/m2 of gemcitabine, and 40 mg/m2 of cisplatin. In cohort A, DLTs were observed at the higher dose level and included neutropenia, hyperbilirubinemia, dehydration, GGT elevation, hyponatremia, nausea, vomiting, and thrombocytopenia. The MTD for cohort C was 154 mg/m2 of 17-AAG and 750 mg/m2 of gemcitabine, with one DLT observed (alkaline phosphatase elevation) observed. In cohort C, DLTs of thrombocytopenia, fever and dyspnea were seen at the higher dose level. The remaining cohorts were closed to accrual due to toxicity. Six patients experienced partial responses. Mean Hsp90 levels were decreased and levels of Hsp70 were increased compared to baseline. Conclusions: 17-AAG in combination with gemcitabine and cisplatin demonstrated antitumor activity, but significant hematologic toxicities were encountered. 17-AAG combined with gemcitabine is tolerable and has demonstrated evidence of activity at the MTD. The recommended phase II dose is defined as 154 mg/m2 of 17-AAG and 750 mg/m2 of gemcitabine, and is currently being investigated in phase II studies in ovarian and pancreatic cancers. There is no recommended phase II dose for the cisplatin-containing combinations.

The chaperone Hsp90 is required for the appropriate regulation of numerous key signaling molecules, including the progesterone receptor (PR). Many important cochaperones bind Hsp90 through their tetratricopeptide repeat (TPR) domains. Two such proteins, GCUNC45 and FKBP52, assist PR chaperoning and are thought to interact sequentially with PR-Hsp90 complexes. TPR proteins bind to the C-terminal MEEVD sequence of Hsp90, but GCUNC45 has been shown also to bind to a novel site near the N-terminus. We now show that FKBP52 is also able to bind to this site, and that these two cochaperones act competitively, through Hsp90, to modulate PR activity. The N-terminal site involves noncontiguous amino acids within or near the ATP binding pocket of Hsp90. TPR interactions at this site are thus strongly regulated by nucleotide binding and Hsp90 conformation. We propose an expanded model for client chaperoning in which the coordinated use of TPR recognition sites at both N- and C-terminal ends of Hsp90 enhances its ability to coordinate interactions with multiple TPR partners.

Checkpoint kinase 1 (Chk1), a serine/threonine kinase that regulates DNA damage checkpoints, is destabilized when heat shock protein 90 (Hsp90) is inhibited, suggesting that Chk1 is an Hsp90 client. In the present work we examined the interplay between Chk1 and Hsp90 in intact cells, identified a source of unchaperoned Chk1, and report the in vitro chaperoning of Chk1 in reticulocyte lysates and with purified chaperones and co-chaperones. We find that bacterially expressed Chk1 is post-translationally chaperoned to an active kinase. This reaction minimally requires Hsp90, Hsp70, Hsp40, Cdc37, and the protein kinase CK2. The co-chaperone Hop, although not essential for the activation of Chk1 in vitro, enhanced the chaperoning process, whereas the co-chaperone p23 did not stimulate the chaperoning reaction. Additionally, we found that the C-terminal regulatory domain of Chk1 affects the association of Chk1 with Hsp90. Collectively these results provide new insights into Hsp90-dependent chaperoning of a client kinase and identify a novel, biochemically tractable model system that will be useful to further dissect the Hsp90-dependent chaperoning of this important and ubiquitous class of Hsp90 clients.

Purpose We determined the maximum-tolerated dose (MTD) and the dose-limiting toxicities (DLT) of 17-allylamino-17-demethoxygeldanamycin (17-AAG) when infused on days 1, 8, and 15 of a 28-day cycle in advanced solid tumor patients. We also characterized the pharmacokinetics of 17-AAG, its effect on chaperone and client proteins, and whether cytochrome P450 (CYP) 3A5 and NAD(P)H:quinone oxidoreductase 1 (NQO1) polymorphisms affected 17-AAG disposition or toxicity. Patients and Methods An accelerated titration design was used. Biomarkers were measured in peripheral-blood mononuclear cells (PBMCs) at baseline and on days 1 and 15, and pharmacokinetic analysis was performed on day 1 of cycle 1. CYP3A5*3 and NQO1*2 genotypes were determined and correlated with pharmacokinetics and toxicity. Results Twenty-one patients received 52 courses at 11 dose levels. DLTs at 431 mg/m2 were grade 3 bilirubin (n = 1), AST (n = 1), anemia (n = 1), nausea (n = 1), vomiting (n = 1), and myalgias (n = 1). No tumor responses were seen. 17-AAG consistently increased heat shock protein (Hsp) 70 levels in PBMCs. At the MTD, the clearance and half-life (t1/2) of 17-AAG were 11.6 L/h/m2 and 4.15 hours, respectively; whereas the active metabolite 17-aminogeldanamycin had a t1/2 of 7.63 hours. The CYP3A5*3 and NQO1*2 polymorphisms were not associated with 17-AAG toxicity. The CYP3A5*3 polymorphism was associated with higher 17-AAG clearance. Conclusion The MTD of weekly 17-AAG is 308 mg/m2. 17-AAG induced Hsp70 in PBMCs, indicating that Hsp90 has been affected. Further evaluation of 17-AAG is ongoing using a twice-weekly regimen, and this schedule of 17-AAG is being tested in combination with chemotherapy.

Previous studies have demonstrated the ATP-dependent formation of a complex containing the heat shock protein hsp90, the unique hsp90 binding protein p23, and one of three high molecular weight immunophilins. In the present study, hsp90 and p23 are shown to form a complex that requires elevated temperature and ATP/Mg2+. Complex formation is strongly promoted by molybdate and by the nonionic detergent Nonidet P-40. ADP and the benzoquinone ansamycin, geldanamycin, are potent inhibitors of complex formation. The ATP-dependent process alters the state of hsp90, not p23, and influences the ability of hsp90 to bind to phenyl-Sepharose. Conversion of hsp90 to the ATP-bound state lowers its affinity for phenyl-Sepharose. These results show that hsp90 can exist in at least two functional states, one able to bind p23 and the other with a high affinity for hydrophobic resins. A model is presented where these states are dictated by the binding of either ATP or ADP.

3058 Background: 17-AAG, a benzoquinone ansamycin derivative that targets the heat shock protein hsp90, is currently undergoing clinical evaluation in phase I/II trials. We have demonstrated sequen...

The molecular chaperone Hsp90 plays an essential role in the folding and function of important cellular proteins including steroid hormone receptors, protein kinases and proteins controlling the cell cycle and apoptosis. A 15 Å deep pocket region in the N-terminal domain of Hsp90 serves as an ATP/ADP-binding site and has also been shown to bind geldanamycin, the only specific inhibitor of Hsp90 function described to date. We now show that radicicol, a macrocyclic antifungal structurally unrelated to geldanamycin, also specifically binds to Hsp90. Moreover, radicicol competes with geldanamycin for binding to the N-terminal domain of the chaperone, expressed either by in vitro translation or as a purified protein, suggesting that radicicol shares the geldanamycin binding site. Radicicol, as does geldanamycin, also inhibits the binding of the accessory protein p23 to Hsp90, and interferes with assembly of the mature progesterone receptor complex. Radicicol does not deplete cells of Hsp90, but rather increases synthesis as well as the steady-state level of this protein, similar to a stress response. Finally, radicicol depletes SKBR3 cells of p 185erbB2, Raf-1 and mutant p53, similar to geldanamycin. Radicicol thus represents a structurally unique antibiotic, and the first non-benzoquinone ansamycin, capable of binding to Hsp90 and interfering with its function.