Kristin Beebe

Affinity-based proteomics reveal cancer-specific networks coordinated by Hsp90

Most cancers are characterized by multiple molecular alterations, but identification of the key proteins involved in these signaling pathways is currently beyond reach. We show that the inhibitor PU-H71 preferentially targets tumor-enriched Hsp90 complexes and affinity captures Hsp90-dependent oncogenic client proteins. We have used PU-H71 affinity capture to design a proteomic approach that, when combined with bioinformatic pathway analysis, identifies dysregulated signaling networks and key oncoproteins in chronic myeloid leukemia. The identified interactome overlaps with the well-characterized altered proteome in this cancer, indicating that this method can provide global insights into the biology of individual tumors, including primary patient specimens. In addition, we show that this approach can be used to identify previously uncharacterized oncoproteins and mechanisms, potentially leading to new targeted therapies. We further show that the abundance of the PU-H71-enriched Hsp90 species, which is not dictated by Hsp90 expression alone, is predictive of the cells sensitivity to Hsp90 inhibition.

Molecular chaperone TRAP1 regulates a metabolic switch between mitochondrial respiration and aerobic glycolysis.

Significance TNF receptor-associated protein (TRAP1) is found predominantly in mitochondria. A possible direct impact of TRAP1 on mitochondrial metabolism remains unexplored. We used TRAP1-null cells and transient TRAP1 silencing/overexpression to show that TRAP1 regulates a metabolic switch between oxidative phosphorylation and aerobic glycolysis in immortalized mouse fibroblasts and in human tumor cells. TRAP1 deficiency promotes increased mitochondrial respiration, fatty acid oxidation, tricarboxylic acid cycle intermediates, ATP and reactive oxygen species, while concomitantly suppressing glucose metabolism. TRAP1 deficiency also results in strikingly enhanced cell motility and invasiveness. TRAP1 interaction with and regulation of mitochondrial c-Src provide a mechanistic basis for these phenotypes. TRAP1 (TNF receptor-associated protein), a member of the HSP90 chaperone family, is found predominantly in mitochondria. TRAP1 is broadly considered to be an anticancer molecular target. However, current inhibitors cannot distinguish between HSP90 and TRAP1, making their utility as probes of TRAP1-specific function questionable. Some cancers express less TRAP1 than do their normal tissue counterparts, suggesting that TRAP1 function in mitochondria of normal and transformed cells is more complex than previously appreciated. We have used TRAP1-null cells and transient TRAP1 silencing/overexpression to show that TRAP1 regulates a metabolic switch between oxidative phosphorylation and aerobic glycolysis in immortalized mouse fibroblasts and in human tumor cells. TRAP1-deficiency promotes an increase in mitochondrial respiration and fatty acid oxidation, and in cellular accumulation of tricarboxylic acid cycle intermediates, ATP and reactive oxygen species. At the same time, glucose metabolism is suppressed. TRAP1-deficient cells also display strikingly enhanced invasiveness. TRAP1 interaction with and regulation of mitochondrial c-Src provide a mechanistic basis for these phenotypes. Taken together with the observation that TRAP1 expression is inversely correlated with tumor grade in several cancers, these data suggest that, in some settings, this mitochondrial molecular chaperone may act as a tumor suppressor.

Integration of Gene Dosage and Gene Expression in Non-Small Cell Lung Cancer, Identification of HSP90 as Potential Target

Background Lung cancer causes approximately 1.2 million deaths per year worldwide, and non-small cell lung cancer (NSCLC) represents 85% of all lung cancers. Understanding the molecular events in non-small cell lung cancer (NSCLC) is essential to improve early diagnosis and treatment for this disease. Methodology and Principal Findings In an attempt to identify novel NSCLC related genes, we performed a genome-wide screening of chromosomal copy number changes affecting gene expression using microarray based comparative genomic hybridization and gene expression arrays on 32 radically resected tumor samples from stage I and II NSCLC patients. An integrative analysis tool was applied to determine whether chromosomal copy number affects gene expression. We identified a deletion on 14q32.2-33 as a common alteration in NSCLC (44%), which significantly influenced gene expression for HSP90, residing on 14q32. This deletion was correlated with better overall survival (P = 0.008), survival was also longer in patients whose tumors had low expression levels of HSP90. We extended the analysis to three independent validation sets of NSCLC patients, and confirmed low HSP90 expression to be related with longer overall survival (P = 0.003, P = 0.07 and P = 0.04). Furthermore, in vitro treatment with an HSP90 inhibitor had potent antiproliferative activity in NSCLC cell lines. Conclusions We suggest that targeting HSP90 will have clinical impact for NSCLC patients.

Dynamic tyrosine phosphorylation modulates cycling of the HSP90-P50(CDC37)-AHA1 chaperone machine.

Many critical protein kinases rely on the Hsp90 chaperone machinery for stability and function. After initially forming a ternary complex with kinase client and the cochaperone p50(Cdc37), Hsp90 proceeds through a cycle of conformational changes facilitated by ATP binding and hydrolysis. Progression through the chaperone cycle requires release of p50(Cdc37) and recruitment of the ATPase activating cochaperone AHA1, but the molecular regulation of this complex process at the cellular level is poorly understood. We demonstrate that a series of tyrosine phosphorylation events, involving both p50(Cdc37) and Hsp90, are minimally sufficient to provide directionality to the chaperone cycle. p50(Cdc37) phosphorylation on Y4 and Y298 disrupts client-p50(Cdc37) association, while Hsp90 phosphorylation on Y197 dissociates p50(Cdc37) from Hsp90. Hsp90 phosphorylation on Y313 promotes recruitment of AHA1, which stimulates Hsp90 ATPase activity, furthering the chaperoning process. Finally, at completion of the chaperone cycle, Hsp90 Y627 phosphorylation induces dissociation of the client and remaining cochaperones.

Asymmetric Hsp90 N Domain SUMOylation Recruits Aha1 and ATP-Competitive Inhibitors

The stability and activity of numerous signaling proteins in both normal and cancer cells depends on the dimeric molecular chaperone heat shock protein 90 (Hsp90). Hsp90s function is coupled to ATP binding and hydrolysis and requires a series of conformational changes that are regulated by cochaperones and numerous posttranslational modifications (PTMs). SUMOylation is one of the least-understood Hsp90 PTMs. Here, we show that asymmetric SUMOylation of a conserved lysine residue in the N domain of both yeast (K178) and human (K191) Hsp90 facilitates both recruitment of the adenosine triphosphatase (ATPase)-activating cochaperone Aha1 and, unexpectedly, the binding of Hsp90 inhibitors, suggesting that these drugs associate preferentially with Hsp90 proteins that are actively engaged in the chaperone cycle. Importantly, cellular transformation is accompanied by elevated steady-state N domain SUMOylation, and increased Hsp90 SUMOylation sensitizes yeast and mammalian cells to Hsp90 inhibitors, providing a mechanism to explain the sensitivity of cancer cells to these drugs.

Loss of Hsp90 association up-regulates Src-dependent ErbB2 activity.

ABSTRACT The receptor tyrosine kinase ErbB2 plays a crucial role in tumorigenesis. We showed previously that the molecular chaperone Hsp90 protects ErbB2 from proteasome-mediated degradation by binding to a short loop structure in the N-lobe of the kinase domain. Here we show that loss of Hsp90 binding correlates with enhanced ErbB2 kinase activity and its transactivating potential, concomitant with constitutively increased phosphorylation of Tyr877, located in the activation loop of the kinase domain. We show further that Tyr877 phosphorylation is mediated by Src and that it is necessary for the enhanced kinase activity of ErbB2. Finally, computer modeling of the kinase domain suggests a phosphorylation-dependent reorientation of the activation loop, denoting the importance of Tyr877 phosphorylation for ErbB2 activity. These findings suggest that Hsp90 binding to ErbB2 participates in regulation of kinase activity as well as kinase stability.

Divergent Synthesis of a Pochonin Library Targeting HSP90 and In Vivo Efficacy of an Identified Inhibitor

Two natural products, radicicol and geldanamycin (1 and 2, Figure 1), were instrumental in understanding the role of HSP90 in oncogenic processes and its therapeutic potential.[13–15] However, neither natural product has acceptable pharmacological properties for clinical application. Structure-based design and high throughput screening have led to discovery of novel scaffold such as purines[16, 17] and pyrazoles[18] however, improving the pharmacological properties and potency of the natural pharmacophores remains important. Indeed, the most advanced clinical candidate is a semisynthetic derivative of geldanamycin, 17AAG (3, Figure 1), which is currently in multiple phase II studies.[19] Another semisynthetic derivative with a dimethoxyhydroquinone functionality has recently been reported to have better pharmacological properties than 17AAG while acting as a prodrug.[20] Radicicol, while having a higher affinity than does geldanamycin for HSP90, suffers from two limitations: a strained and highly sensitive epoxide and a conjugate diene which functions as a Michael acceptor. Indeed, the inactivity of radicicol in animals models has been attributed to a conjugate addition of thiol nucleophiles at the C13 position.[21] Akinaga and coworkers overcame this limitation by converting radicicol to an oxime which showed significant anti-tumor activity (reduction in tumor growth) in animal models.[21–23] Mindful of the labile epoxide, Danishefsky and coworkers reported a cyclopropyl analogue of radicicol which was nearly as effective in cellular assays; however its efficacy in animals has not been reported.[24, 25] More recently, Moody and coworkers reported the synthesis of radicicol related resorcylides, exploring the importance of the macrocyle ring size.[26] Figure 1 Structure of radicicol, geldanamycin, 17AAG, pochonin D and general structure of the pochonin library (5). We have previously suggested that radicicol’s epoxide moiety is important as a conformational bias which favors the bioactive conformation of the macrocycle and shown that another natural product, pochonin D (4, Figure 1), was also a good ligand for HSP90.[27] Furthermore, we have reported the use of polymer-supported reagents to synthesize a library extending the diversity of the pochonins (5, Figure 1).[28] Screening this library for HSP90 affinity and down-regulation of HSP90’s client proteins revealed important structure-activity relationships and pointed to the ketone moiety as the most favorable position for improving activity. Herein, we report the structure activity relationship of a focused library of this important pharmacophore, an analogue of pochonin D having 100 fold improvements in its cellular activity as well as its efficacy in a breast tumor xenograft (BT-474). While some of the simpler resorcylides such as pochonin D had good affinity for HSP90, their cellular activity was disappointing in comparison to 17AAG. The ambiguous correlation between 17AAG’s HSP90 affinity and cellular activity remains a subject of intense investigation[29–31] but can be rationalized by the kinetics of binding.[31] Similar discrepancies between HSP90 affinity measured by a fluorescence polarization assay and ATPase inhibition have been noted for inhibitors based on the resorcylides motif.[26] Based on the observation that oxime substitutions were tolerated for HSP90 activity in the pochonin series and inspired by previous success with radicicol, [21–23] we develop a divergent synthesis providing rapid access to this class of compounds. Readily available intermediate 6 was deprotonated with LDA (Scheme 1) and reacted with Weinreb amide 7. Quenching the reaction with benzoic acid resin sequestered the amine byproducts and afford 8 with sufficient purity to be engaged directly in the oxime formation with aminooxyacetic acid. After evaporation of the solvent, the crude mixture was treated with an acidic resin which removed the excess hydroxyl amine and some byproducts stemming from conjugate addition to afford 9. Intermediate 9 was then loaded on 2-chlorotrityl resin and the carboxylate group was deprotected under the action of TBAF thus revealing the acid which was engaged in a Mitsunobu esterification with homoallylic alcohols 10 to obtain polymer-bound intermediates 11. It is important to note that this reaction sequence is not possible in the absence of the oxime functionality as it leads to coumarin byproducts.[32] The resins were than treated to the second generation Grubbs’ catalyst under microwave irradiation to obtain the macrocycles 12 in excellent yield and purity after cleavage from the resin with hexafluoropropanol (HFIP). Contrarily to TFA, these mild cleavage conditions were found to leave the EOMs intact thus enabling a selective esterification or amidation. For this purpose, we used an immobilized carbodiimide reagent followed by a sulfonic acid resin treatment to obtain a library of pochonin oximes 13. Scheme 1 Reagents and conditions: a) LDA (2.0 equiv), THF, −78 °C, 5 min; 7 (1.0 equiv), 10 min; PS-COOH (5.0 equiv), −78 to 23 °C, 20 min, ~50%; b) NH2OCH2CHO2H (5.0 equiv), Py/AcOH 5:1, 40 °C, 24 h, 45–95%; c) ... The library was then screened for affinity to HSP90α,[33] Her-2 (Hsp90 client) degradation[34] and cytotoxicity against SKBrS and HCC1954, two breast cancer cell lines which overexpress Her-2 (Figure 2). The most potent inhibitors were compound 13a and b containing the piperidine amide moiety. It is interesting to note that the simplified analogue lacking the chiral methyl group is as active that the parent compound 13c and that while the chlorine atom is important for the activity of both radicicol and pochonin D, it is not for the activity of compound 13b. The structure activity data suggests that the piperidine amide has a relatively good fit in a lipophilic pocket as the morpholino analogue (13d), piperazine analogues (13e) or simple methyl amide (13i) loose significant activity. A cyclohexyl amide (13k) or benzyl amide (13j) on the other hand were also good ligands. Consistent with the previous radicicol oxime,[22] it is interesting to note that there is a significant difference of activity between the E and the Z isomer, with the E isomer having higher activity (13a vs 13f and 13b vs 13g). Based on the potent activity of 13a, this compound was further evaluated in vivo. Figure 2 Biological activity (μM) of pochonin-oxime derivatives (* 1:1 mixture of E:Z oximes). Treatment of CB17/SCID mice with 13a at 100mg/kg for five consecutive days was well tolerated with minimal weigh loss. To investigate the in vivo efficacy of 13a, a xenograft bearing BT-474 (breast tumor cell line) was used as this tumorgenic cell line has been shown to respond to HSP90 inhibitors[35] in an animal model. Based on the cellular potency of 13a, two schedules of 100 mg every other day (q2d) or every four days (q4d) during 28 days were investigated. Gratifyingly, the treatment with 13a resulted in a dose-dependent inhibition of the tumor growth with an 18% regression in tumor volume using the q2d schedule (Figure 3a). In neither schedule was a significant weight loss observed (Figure 3b). Histologic examination of tumors removed from animals receiving either vehicle (DMSO) or drug for 28 days following the q2d schedule revealed a dramatic loss of cellularity in tumors obtained from drug-treated animals. Nuclei of remaining cells were uniformly condensed, suggested the occurrence of massive apoptosis (Figure 4, top panels). This was confirmed by the high degree of nuclear TUNEL staining seen in tumors excised from drug-treated animals (Figure 4, bottom panels). These data suggest that tumor regression in animals treated for 28 days with the q2d schedule may be more dramatic than estimated with tumor volume measurements as depicted in Figure 3, since few to no viable cells can be identified at the end of the treatment period. Figure 3 Tumor volume (BT474) and animal weight following treatment with 13a or control vehicle. Figure 4 Tumor histology and apoptosis in mock- and drug-treated animals. Top panels represent hematoxylin & eosin (H & E) stained paraffin sections. Nuclei appear blue in color. The dark blue condensed nuclei in drug-treated tumors (right) are ... In conclusion, pochoximes 13a and 13b have a higher affinity for HSP90 and are more active in reducing HSP90’s client proteins than radicicol. This is the first report of an HSP90 inhibitor based on the resorcylic macrolide scaffold to show a regression in tumor size and their effectiveness at doses below the maximum tolerated dose suggest a meaningful therapeutic window. The use of polymer bound reagents[36] and solid phase chemistry facilitates the synthesis of new analogues and set a successful precedent for the rapid elaboration of natural product libraries.

The HSP90 Inhibitor Ganetespib Synergizes with the MET Kinase Inhibitor Crizotinib in both Crizotinib-Sensitive and -Resistant MET-Driven Tumor Models

The proto-oncogene MET is aberrantly activated via overexpression or mutation in numerous cancers, making it a prime anticancer molecular target. However, the clinical success of MET-directed tyrosine kinase inhibitors (TKI) has been limited due, in part, to mutations in the MET kinase domain that confer therapeutic resistance. Circumventing this problem remains a key challenge to improving durable responses in patients receiving MET-targeted therapy. MET is an HSP90-dependent kinase, and in this report we show that HSP90 preferentially interacts with and stabilizes activated MET, regardless of whether the activation is ligand-dependent or is a consequence of kinase domain mutation. In contrast, many MET-TKI show a preference for the inactive form of the kinase, and activating mutations in MET can confer resistance. Combining the HSP90 inhibitor ganetespib with the MET-TKI crizotinib achieves synergistic inhibition of MET, its downstream signaling pathways, and tumor growth in both TKI-sensitive and -resistant MET-driven tumor models. These data suggest that inclusion of an HSP90 inhibitor can partially restore TKI sensitivity to previously resistant MET mutants, and they provide the foundation for clinical evaluation of this therapeutic combination in patients with MET-driven cancers.

Inhibition of HSP90 with pochoximes: SAR and structure-based insights.

The pochoximes, based on the radicicol pharmacophore, are potent inhibitors of heat shock protein 90 (HSP90) that retain their activity in vivo. Herein we report an extended library that broadly explores the structure–activity relationship (SAR) of the pochoximes with four points of diversity. Several modifications were identified that afford improved cellular efficacy, new opportunities for conjugation, and further diversifications. Cocrystal structures of pochoximes A and B with HSP90 show that pochoximes bind to a different conformation of HSP90 than radicicol and provide a rationale for the enhanced affinity of the pochoximes relative to radicicol and the pochonins.

Sensitivity of epidermal growth factor receptor and ErbB2 exon 20 insertion mutants to Hsp90 inhibition

The mature epidermal growth factor receptor (EGFR) neither associates with nor requires the molecular chaperone heat-shock protein 90 (Hsp90). Mutations in EGFR exons 18, 19, and 21 confer Hsp90 chaperone dependence. In non-small cell lung cancer (NSCLC), these mutations are associated with enhanced sensitivity to EGFR inhibitors in vitro and with clinical response in vivo. Although less prevalent, insertions in EGFR exon 20 have also been described in NSCLC. These mutations, however, confer resistance to EGFR inhibitors. In NSCLC, exon 20 insertions have also been identified in the EGFR family member ErbB2. Here, we examined the sensitivity of exon 20 insertion mutants to an Hsp90 inhibitor currently in the clinic. Our data demonstrate that both EGFR and ErbB2 exon 20 insertion mutants retain dependence on Hsp90 for stability and downstream-signalling capability, and remain highly sensitive to Hsp90 inhibition. Use of Hsp90 inhibitors should be considered in NSCLC harbouring exon 20 insertions in either EGFR or ErbB2.