Marizomib irreversibly inhibits proteasome to overcome compensatory hyperactivation in multiple myeloma and solid tumour patients.

Proteasome inhibitors (PIs) are highly active in multiple myeloma (MM) but resistance is commonly observed. All clinical stage PIs effectively inhibit chymotrypsin-like (CT-L) activity; one possible mechanism of resistance is compensatory hyperactivation of caspase-like (C-L) and trypsin-like (T-L) subunits, in response to CT-L blockade. Marizomib (MRZ), an irreversible PI that potently inhibits all three 20S proteasome subunits with a specificity distinct from other PIs, is currently in development for treatment of MM and malignant glioma. The pan-proteasome pharmacodynamic activity in packed whole blood and peripheral blood mononuclear cells was measured in two studies in patients with advanced solid tumours and haematological malignancies. Functional inhibition of all proteasome subunits was achieved with once- or twice-weekly MRZ dosing; 100% inhibition of CT-L was frequently achieved within one cycle at therapeutic doses. Concomitantly, C-L and T-L activities were either unaffected or increased, suggesting compensatory hyperactivation of these subunits. Importantly, this response was overcome by continued administration of MRZ, with robust inhibition of T-L and C-L (up to 80% and 50%, respectively) by the end of Cycle 2 and maintained thereafter. This enhanced proteasome inhibition was independent of tumour type and may underlie the clinical activity of MRZ in patients resistant to other PIs.

Epithelial-mesenchymal transition increases tumor sensitivity to COX-2 inhibition by apricoxib.

Although cyclooxygenase-2 (COX-2) inhibitors, such as the late stage development drug apricoxib, exhibit antitumor activity, their mechanisms of action have not been fully defined. In this study, we characterized the mechanisms of action of apricoxib in HT29 colorectal carcinoma. Apricoxib was weakly cytotoxic toward naive HT29 cells in vitro but inhibited tumor growth markedly in vivo. Pharmacokinetic analyses revealed that in vivo drug levels peaked at 2-4 µM and remained sufficient to completely inhibit prostaglandin E(2) production, but failed to reach concentrations cytotoxic for HT29 cells in monolayer culture. Despite this, apricoxib significantly inhibited tumor cell proliferation and induced apoptosis without affecting blood vessel density, although it did promote vascular normalization. Strikingly, apricoxib treatment induced a dose-dependent reversal of epithelial-mesenchymal transition (EMT), as shown by robust upregulation of E-cadherin and the virtual disappearance of vimentin and ZEB1 protein expression. In vitro, either anchorage-independent growth conditions or forced EMT sensitized HT29 and non-small cell lung cancer cells to apricoxib by 50-fold, suggesting that the occurrence of EMT may actually increase the dependence of colon and lung carcinoma cells on COX-2. Taken together, these data suggest that acquisition of mesenchymal characteristics sensitizes carcinoma cells to apricoxib resulting in significant single-agent antitumor activity.

CXCL12 in Pancreatic Cancer: Its Function and Potential as a Therapeutic Drug Target.

Pancreatic ductal adenocarcinoma (PDAC) is a disease with limited therapeutic options and dismal long-term survival. The unique tumor environment of PDAC, consisting of desmoplastic stroma, immune suppressive cells, and activated fibroblasts, contributes to its resistance to therapy. Activated fibroblasts (cancer-associated fibroblasts and pancreatic stellate cells) secrete chemokines and growth factors that support PDAC growth, spread, chemoresistance, and immune evasion. In this review, we focus on one such chemokine, CXCL12, secreted by the cancer-associated fibroblasts and discuss its contribution to several of the classical hallmarks of PDAC and other tumors. We review the various therapeutic approaches in development to target CXCL12 signaling in PDAC. Finally, we propose an unconventional use of tipifarnib, a farnesyl transferase inhibitor, to inhibit CXCL12 production in PDAC.

A high-affinity conformation of Hsp90 confers tumour selectivity on Hsp90 inhibitors.

Heat shock protein 90 (Hsp90) is a molecular chaperone that plays a key role in the conformational maturation of oncogenic signalling proteins, including HER-2/ErbB2, Akt, Raf-1, Bcr-Abl and mutated p53. Hsp90 inhibitors bind to Hsp90, and induce the proteasomal degradation of Hsp90 client proteins. Although Hsp90 is highly expressed in most cells, Hsp90 inhibitors selectively kill cancer cells compared to normal cells, and the Hsp90 inhibitor 17-allylaminogeldanamycin (17-AAG) is currently in phase I clinical trials. However, the molecular basis of the tumour selectivity of Hsp90 inhibitors is unknown. Here we report that Hsp90 derived from tumour cells has a 100-fold higher binding affinity for 17-AAG than does Hsp90 from normal cells. Tumour Hsp90 is present entirely in multi-chaperone complexes with high ATPase activity, whereas Hsp90 from normal tissues is in a latent, uncomplexed state. In vitro reconstitution of chaperone complexes with Hsp90 resulted in increased binding affinity to 17-AAG, and increased ATPase activity. These results suggest that tumour cells contain Hsp90 complexes in an activated, high-affinity conformation that facilitates malignant progression, and that may represent a unique target for cancer therapeutics.

BIIB021, an orally available, fully synthetic small-molecule inhibitor of the heat shock protein Hsp90.

Inhibition of heat shock protein 90 (Hsp90) results in the degradation of oncoproteins that drive malignant progression, inducing cell death, making Hsp90 a target of substantial interest for cancer therapy. BIIB021 is a novel, fully synthetic inhibitor of Hsp90 that binds competitively with geldanamycin in the ATP-binding pocket of Hsp90. In tumor cells, BIIB021 induced the degradation of Hsp90 client proteins including HER-2, AKT, and Raf-1 and up-regulated expression of the heat shock proteins Hsp70 and Hsp27. BIIB021 treatment resulted in growth inhibition and cell death in cell lines from a variety of tumor types at nanomolar concentrations. Oral administration of BIIB021 led to the degradation of Hsp90 client proteins measured in tumor tissue and resulted in the inhibition of tumor growth in several human tumor xenograft models. Studies to investigate the antitumor effects of BIIB021 showed activity on both daily and intermittent dosing schedules, providing dose schedule flexibility for clinical studies. Assays measuring the HER-2 protein in tumor tissue and the HER-2 extracellular domain in plasma were used to show interdiction of the Hsp90 pathway and utility as potential biomarkers in clinical trials for BIIB021. Together, these data show that BIIB021 is a promising new oral inhibitor of Hsp90 with antitumor activity in preclinical models.

Cyclooxygenase-2 Inhibition Potentiates the Efficacy of Vascular Endothelial Growth Factor Blockade and Promotes an Immune Stimulatory Microenvironment in Preclinical Models of Pancreatic Cancer.

Resistance to standard therapy remains a major challenge in the treatment of pancreatic ductal adenocarcinoma (PDA). Although anti-VEGF therapy delays PDA progression, therapy-induced hypoxia results in a less differentiated mesenchymal-like tumor cell phenotype, which reinforces the need for effective companion therapies. COX-2 inhibition has been shown to promote tumor cell differentiation and improve standard therapy response in PDA. Here, we evaluate the efficacy of COX-2 inhibition and VEGF blockade in preclinical models of PDA. In vivo, the combination therapy was more effective in limiting tumor growth and metastasis than single-agent therapy. Combination therapy also reversed anti-VEGF-induced epithelial-mesenchymal transition and collagen deposition and altered the immune landscape by increasing tumor-associated CD8+ T cells while reducing FoxP3+ T cells and FasL expression on the tumor endothelium. IMPLICATIONS: Together, these findings demonstrate that COX-2 inhibition enhances the efficacy of anti-VEGF therapy by reducing hypoxia-induced epithelial plasticity and promoting an immune landscape that might facilitate immune activation.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/17/2/348/F1.large.jpg.

Rationally designed high-affinity 2-amino-6-halopurine heat shock protein 90 inhibitors that exhibit potent antitumor activity.

Heat shock protein 90 (Hsp90) is a molecular chaperone protein implicated in stabilizing the conformation and maintaining the function of many cell-signaling proteins. Many oncogenic proteins are more dependent on Hsp90 in maintaining their conformation, stability, and maturation than their normal counterparts. Furthermore, recent data show that Hsp90 exists in an activated form in malignant cells but in a latent inactive form in normal tissues, suggesting that inhibitors selective for the activated form could provide a high therapeutic index. Hence, Hsp90 is emerging as an exciting new target for the treatment of cancer. We now report on a novel series of 2-amino-6-halopurine Hsp90 inhibitors exemplified by 2-amino-6-chloro-9-(4-iodo-3,5-dimethylpyridin-2-ylmethyl)purine (30). These highly potent inhibitors (IC50 of 30 = 0.009 microM in a HER-2 degradation assay) also display excellent antiproliferative activity against various tumor cell lines (IC50 of 30 = 0.03 microM in MCF7 cells). Moreover, this class of inhibitors shows higher affinity for the activated form of Hsp90 compared to our earlier 8-sulfanylpurine Hsp90 inhibitor series. When administered orally to mice, these compounds exhibited potent tumor growth inhibition (>80%) in an N87 xenograft model, similar to that observed with 17-allylamino-17-desmethoxygeldanamycin (17-AAG), which is a compound currently in phase I/II clinical trials.

Orally active purine-based inhibitors of the heat shock protein 90.

Orally active Hsp90 inhibitors are of interest as potential chemotherapeutic agents. Recently, fully synthetic 8-benzyladenines and 8-sulfanyladenines such as 4 were disclosed as Hsp90 inhibitors, but these compounds are not water soluble and consequently have unacceptably low oral bioavailabilities. We now report that water-solubility can be achieved by inserting an amino functionality in the N(9) side chain. This results in compounds that are potent, soluble in aqueous media, and orally bioavailable. In an HER-2 degradation assay, the highest potency was achieved with the neopentylamine 42 (HER-2 IC(50) = 90 nM). In a murine tumor xenograft model (using the gastric cancer cell line N87), the H(3)PO(4) salts of the amines 38, 39, and 42 induced tumor growth inhibition when administered orally at 200 mg/kg/day. The amines 38, 39, and 42 are the first Hsp90 inhibitors shown to inhibit tumor growth upon oral dosage.

7'-substituted benzothiazolothio- and pyridinothiazolothio-purines as potent heat shock protein 90 inhibitors.

We report on the discovery of benzo- and pyridino- thiazolothiopurines as potent heat shock protein 90 inhibitors. The benzothiazole moiety is exceptionally sensitive to substitutions on the aromatic ring with a 7'-substituent essential for activity. Some of these compounds exhibit low nanomolar inhibition activity in a Her-2 degradation assay (28-150 nM), good aqueous solubility, and oral bioavailability profiles in mice. In vivo efficacy experiments demonstrate that compounds of this class inhibit tumor growth in an N87 human colon cancer xenograft model via oral administration as shown with compound 37 (8-(7-chlorobenzothiazol-2-ylsulfanyl)-9-(2-cyclopropylamino-ethyl)-9H- purin-6-ylamine).

Inhibition of neuroblastoma xenograft growth by Hsp90 inhibitors.

BACKGROUND: Advanced-stage neuroblastomas are often resistant to chemotherapy. Heat shock protein (Hsp) 90 is a molecular chaperone that maintains the stability of important signal transduction proteins. We have previously reported that geldanamycin (GA), an Hsp90 inhibitor, decreases Raf-1 and Akt protein expressions and induces apoptosis in neuroblastoma cells. We sought to determine the in vivo effects of Hsp90 inhibitor compounds on human neuroblastomas. MATERIALS AND METHODS: Human neuroblastoma (LAN-1 and SK-N-SH) xenografts (4-mm3 tumor implants) were placed in the flanks of athymic nude mice. The mice received either Hsp90 inhibitors (17-AAG or EC5) or vehicle (control). The tumor dimensions were measured twice weekly. Proteins were extracted for Western immunoblotting. RESULTS: Hsp90 inhibitor compounds significantly blocked both LAN-1 and SK-N-SH neuroblastoma growth in vivo. Drug-treated tumors showed decreases in Raf-1 and cleaved PARP expressions. CONCLUSION: Hsp90 inhibitors may prove to be important novel therapeutic agents for patients with advanced-stage neuroblastoma who fail to respond to current treatment regimens.

Marizomib activity as a single agent in malignant gliomas: ability to cross the blood-brain barrier.

BACKGROUND: The proteasome plays a vital role in the physiology of glioblastoma (GBM), and proteasome inhibition can be used as a strategy for treating GBM. Marizomib is a second-generation, irreversible proteasome inhibitor with a more lipophilic structure that suggests the potential for penetrating the blood-brain barrier. While bortezomib and carfilzomib, the 2 proteasome inhibitors approved for treatment of multiple myeloma, have little activity against malignant gliomas in vivo, marizomib could be a novel therapeutic strategy for primary brain tumors. METHODS: The in-vitro antitumor activity of marizomib was studied in glioma cell lines U-251 and D-54. The ability of marizomib to cross the blood-brain barrier and regulate proteasome activities was evaluated in cynomolgus monkeys and rats. The antitumor effect of marizomib in vivo was tested in an orthotopic xenograft model of human GBM. RESULTS: Marizomib inhibited the proteasome activity, proliferation, and invasion of glioma cells. Meanwhile, free radical production and apoptosis induced by marizomib could be blocked by antioxidant N-acetyl cysteine. In animal studies, marizomib distributed into the brain at 30% of blood levels in rats and significantly inhibited (>30%) baseline chymotrypsin-like proteasome activity in brain tissue of monkeys. Encouragingly, the immunocompromised mice, intracranially implanted with glioma xenografts, survived significantly longer than the control animals (P < .05) when treated with marizomib. CONCLUSIONS: These preclinical studies demonstrated that marizomib can cross the blood-brain barrier and inhibit proteasome activity in rodent and nonhuman primate brain and elicit a significant antitumor effect in a rodent intracranial model of malignant glioma.

Therapeutic and diagnostic implications of Hsp90 activation.

The molecular chaperone heat-shock protein 90 (Hsp90) is involved in the stabilization and conformational maturation of many signaling proteins that are deregulated in cancers. Hsp90 inhibition results in the proteasomal degradation of these client proteins and leads to potent antitumor activity. The Hsp90 inhibitor 17-allylaminogeldanamycin (17-AAG) is presently in clinical trials. Recent work has identified the role of Hsp90 in multiple signal transduction pathways and revealed that the molecular mechanism of tumor selectivity by Hsp90 inhibitors is the result of an activated, high-affinity conformation of Hsp90 in tumors. This review discusses these recent advances in the understanding of tumor Hsp90 for the treatment and diagnosis of cancer. In addition, the role of Hsp90 in non-oncological diseases will also be discussed.

Purified herpes simplex virus thymidine kinase retroviral particles: III. Characterization of bystander killing mechanisms in transfected tumor cells.

An important consequence of the suicide gene therapeutic paradigm is the phenomenon of bystander cell killing, the death of adjacent tumor cells not transduced with the thymidine kinase (TK) gene from herpes simplex virus (HSV) after treatment with the antiviral drug, ganciclovir (GCV). Evidence from quantitative in vitro assays of glioma cell lines suggest that both murine and human gliomas are similar in expressing high sensitivity to the bystander effect. In five of six glial tumors examined, the presence of only 5% of HSV-TK-expressing transduced cells in the culture resulted in >90% tumor cell death/stasis after addition of GCV. Several lines of evidence support gap junction intercellular communication (GJIC) as important in the bystander effect. In vitro metabolic assays, performed with GCV in the medium, indicated that more tumor burden was reduced when culture conditions supported cell-cell contact of parental and HSV-TK-transduced cells. Additionally, a double dye transfer assay showed that cell communication through the gap junction is greatest for glioma, less for melanoma, and much less for colorectal carcinoma cell lines. In vitro metabolic assays with mixtures of TK+/TK- homologous tumor cells confirmed that glioma cell lines were more susceptible to bystander killing than melanomas. Assays with chimeric tumor mixtures of TK+/TK - cells showed that the level of the bystander killing obtained was characteristic of the TK-bystander cells. The in vitro findings were confirmed in vivo with GCV-treated homologous and chimeric tumors composed of TK+/TK- cells. Day 21 mean tumor volumes (MTVs) indicated the growths obtained were characteristic of the bystander activity reflective of the nontransduced cell population. Furthermore, nontransduced, high-GJIC cells in a chimeric tumor mass appeared to effectively bridge between transduced tumor cells and poorly communicating nontransduced cells. Finally, the importance of a gap junction protein, such as connexin-43, in facilitating the bystander effect was demonstrated with the HT29 low-GJIC cell line. When the TK-nontransduced cell population expressed connexin-43, a better bystander kill was achieved compared to the parental counterpart.

Therapeutic potential of ERK5 targeting in triple negative breast cancer.

Triple negative breast cancers (TNBCs) account for 15% of all breast cancers, and represent one of the most aggressive forms of the disease, exhibiting short relapse-free survival. In contrast to other breast cancer subtypes, the absence of knowledge about the etiopathogenic alterations that cause TNBCs force the use of chemotherapeutics to treat these tumors. Because of this, efforts have been devoted with the aim of incorporating novel therapies into the clinical setting. Kinases play important roles in the pathophysiology of several tumors, including TNBC. Since expression of the MAP kinase ERK5 has been linked to patient outcome in breast cancer, we analyzed the potential value of its targeting in TNBC. ERK5 was frequently overexpressed and active in samples from patients with TNBC, as well as in explants from mice carrying genetically-defined TNBC tumors. Moreover, expression of ERK5 was linked to a worse prognosis in TNBC patients. Knockdown experiments demonstrated that ERK5 supported proliferation of TNBC cells. Pharmacological inhibition of ERK5 with TG02, a clinical stage inhibitor which targets ERK5 and other kinases, inhibited cell proliferation by blocking passage of cells through G1 and G2, and also triggered apoptosis in certain TNBC cell lines. TG02 had significant antitumor activity in a TNBC xenograft model in vivo, and also augmented the activity of chemotherapeutic agents commonly used to treat TNBC. Together, these data indicate that ERK5 targeting may represent a valid strategy against TNBC, and support the development of trials aimed at evaluating the clinical effectiveness of drugs that block this kinase.

Potent antimyeloma activity of a novel ERK5/CDK inhibitor.

PURPOSE: To analyze the antimyeloma potential of TG02, an ERK5/CDK inhibitory drug. EXPERIMENTAL DESIGN: Utilizing different multiple myeloma cell lines we determined the effect of TG02 over viability by MTT assays. The apoptotic effect over multiple myeloma patient samples was studied ex vivo by cytometry. The mechanism of action of TG02 was analyzed in the cell line MM1S, studying its effect on the cell cycle, the induction of apoptosis, and the loss of mitochondrial membrane potential by cytometry and Western blot. Two models of multiple myeloma xenograft were utilized to study the in vivo action of TG02. RESULTS: TG02 potently inhibited proliferation and survival of multiple myeloma cell lines, even under protective bone marrow niche conditions, and selectively induced apoptosis of primary patient-derived malignant plasma cells. TG02 displayed significant single-agent activity in two multiple myeloma xenograft models, and enhanced the in vivo activity of bortezomib and lenalidomide. Signaling analyses revealed that the drug simultaneously blocked the activity of CDKs 1, 2, and 9 as well as the MAP kinase ERK5 in MM1S cells, leading to cell-cycle arrest and rapid commitment to apoptosis. TG02 induced robust activation of both the intrinsic and extrinsic pathways of apoptosis, and depletion of XIAP and the key multiple myeloma survival protein Mcl-1. CONCLUSIONS: TG02 is a promising new antimyeloma agent that is currently in phase I clinical trials in leukemia and multiple myeloma patients.

Apricoxib, a novel inhibitor of COX-2, markedly improves standard therapy response in molecularly defined models of pancreatic cancer.

PURPOSE: COX-2 is expressed highly in pancreatic cancer and implicated in tumor progression. COX-2 inhibition can reduce tumor growth and augment therapy. The precise function of COX-2 in tumors remains poorly understood, but it is implicated in tumor angiogenesis, evasion of apoptosis, and induction of epithelial-to-mesenchymal transition (EMT). Current therapeutic regimens for pancreatic cancer are minimally effective, highlighting the need for novel treatment strategies. Here, we report that apricoxib, a novel COX-2 inhibitor in phase II clinical trials, significantly enhances the efficacy of gemcitabine/erlotinib in preclinical models of pancreatic cancer. EXPERIMENTAL DESIGN: Human pancreatic cell lines were evaluated in vitro and in vivo for response to apricoxib ± standard-of-care therapy (gemcitabine + erlotinib). Tumor tissue underwent posttreatment analysis for cell proliferation, viability, and EMT phenotype. Vascular parameters were also determined. RESULTS: COX-2 inhibition reduced the IC(50) of gemcitabine ± erlotinib in six pancreatic cancer cell lines tested in vitro. Furthermore, apricoxib increased the antitumor efficacy of standard combination therapy in several orthotopic xenograft models. In vivo apricoxib combination therapy was only effective at reducing tumor growth and metastasis in tumors with elevated COX-2 activity. In each model examined, treatment with apricoxib resulted in vascular normalization without a decrease in microvessel density and promotion of an epithelial phenotype by tumor cells regardless of basal COX-2 expression. CONCLUSIONS: Apricoxib robustly reverses EMT and augments standard therapy without reducing microvessel density and warrants further clinical evaluation in patients with pancreatic cancer.

EC144 is a potent inhibitor of the heat shock protein 90.

Alkyne 40, 5-(2-amino-4-chloro-7-((4-methoxy-3,5-dimethylpyridin-2-yl)methyl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-2-methylpent-4-yn-2-ol (EC144), is a second generation inhibitor of heat shock protein 90 (Hsp90) and is substantially more potent in vitro and in vivo than the first generation inhibitor 14 (BIIB021) that completed phase II clinical trials. Alkyne 40 is more potent than 14 in an Hsp90α binding assay (IC(50) = 1.1 vs 5.1 nM) as well as in its ability to degrade Her-2 in MCF-7 cells (EC(50) = 14 vs 38 nM). In a mouse model of gastric tumors (N87), 40 stops tumor growth at 5 mg/kg and causes partial tumor regressions at 10 mg/kg (po, qd × 5). Under the same conditions, 14 stops tumor growth only at 120 mg/kg, and does not induce partial regressions. Thus, alkyne 40 is approximately 20-fold more efficacious than 14 in mice.

Heat shock protein 90 inhibitor BIIB021 (CNF2024) depletes NF-kappaB and sensitizes Hodgkin's lymphoma cells for natural killer cell-mediated cytotoxicity.

PURPOSE: In Hodgkin's lymphoma, constitutive activation of NF-kappaB promotes tumor cell survival and proliferation. The molecular chaperone heat shock protein 90 (HSP90) has immune regulatory activity and supports the activation of NF-kappaB in Hodgkin's lymphoma cells. EXPERIMENTAL DESIGN: We analyzed the effect of HSP90 inhibition on viability and NF-kappaB activity in Hodgkin's lymphoma cells and the consequences for their recognition and killing through natural killer (NK) cells. RESULTS: The novel orally administrable HSP90 inhibitor BIIB021 (CNF2024) inhibited Hodgkin's lymphoma cell viability at low nanomolar concentrations in synergy with doxorubicin and gemcitabine. Annexin V/7-aminoactinomycin D binding assay revealed that BIIB021 selectively induced cell death in Hodgkin's lymphoma cells but not in lymphocytes from healthy individuals. We observed that BIIB021 inhibited the constitutive activity of NF-kappaB and this was independent of IkappaB mutations. Furthermore, we analyzed the effect of HSP90 inhibition on NK cell-mediated cytotoxicity. BIIB021 induced the expression of ligands for the activating NK cell receptor NKG2D on Hodgkin's lymphoma cells resulting in an increased susceptibility to NK cell-mediated killing. In a xenograft model of Hodgkin's lymphoma, HSP90 inhibition significantly delayed tumor growth. CONCLUSIONS: HSP90 inhibition has direct antitumor activity in Hodgkin's lymphoma in vitro and in vivo. Moreover, HSP90 inhibition may sensitize Hodgkin's lymphoma cells for NK cell-mediated killing via up-regulation of ligands engaging activating NK cell receptors.

The heat-shock protein 90 inhibitor 17-allylamino-17-demethoxygeldanamycin suppresses glial inflammatory responses and ameliorates experimental autoimmune encephalomyelitis.

The heat-shock response (HSR), a highly conserved cellular response, is characterized by rapid expression of heat-shock proteins (HSPs), and inhibition of other synthetic activities. The HSR can attenuate inflammatory responses, via suppression of transcription factor activation. A HSR can be induced pharmacologically by HSP90 inhibitors, through activation of the transcription factor Heat Shock Factor 1 (HSF1). In the present study we characterized the effects of 17-allylamino-17-demethoxygeldanamycin (17-AAG), a less toxic derivative of the naturally occurring HSP90 inhibitor geldanamycin, on glial inflammatory responses and the development of experimental autoimmune encephalomyelitis. In primary enriched glial cultures, 17-AAG dose dependently reduced lipopolysaccharide-dependent expression and activity of inducible nitric oxide synthase, attenuated interleukin (IL)-1beta expression and release, increased inhibitor of kappaB protein levels, and induced HSP70 expression. 17-AAG administration to mice immunized with myelin oligodendrocyte glycoprotein peptide prevented disease onset when given at an early time, and reduced clinical symptoms when given during ongoing disease. T cells from treated mice showed a reduced response to immunogen re-stimulation, and 17-AAG reduced CD3- and CD28-dependent IL-2 production. Together, these data suggest that HSP90 inhibitors could represent a new approach for therapeutic intervention in autoimmune diseases such as multiple sclerosis.

ZAP-70 is a novel conditional heat shock protein 90 (Hsp90) client: inhibition of Hsp90 leads to ZAP-70 degradation, apoptosis, and impaired signaling in chronic lymphocytic leukemia.

The zeta-associated protein of 70 kDa (ZAP-70) is expressed in patients with aggressive chronic lymphocytic leukemia (CLL). We found that ZAP-70+ CLL cells expressed activated heat-shock protein 90 (Hsp90) with high binding affinity for Hsp90 inhibitors, such as 17-allyl-amino-demethoxy-geldanamycin (17-AAG), whereas normal lymphocytes or ZAP-70- CLL cells expressed nonactivated Hsp90. Activated Hsp90 bound and stabilized ZAP-70, which behaved like an Hsp90 client protein only in CLL cells. Treatment with Hsp90 inhibitors such as 17-AAG and 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) induced ZAP-70 degradation and apoptosis in CLL cells but not in T cells, and also impaired B-cell receptor signaling in leukemia cells. Transduction of ZAP-70- CLL cells with an adenovirus encoding ZAP-70 activated Hsp90 and specifically rendered the leukemia cells sensitive to 17-AAG. These data indicate that Hsp90 is necessary for ZAP-70 expression and activity; that ZAP-70 is unique among Hsp90 clients, in that its chaperone-dependency is conditional on the cell type in which it is expressed; and also that ZAP-70 is required for cell survival and signaling in CLL. Additionally, ZAP-70 expression in CLL cells confers markedly heightened sensitivity to 17-AAG or 17-DMAG, suggesting that these or other Hsp90 inhibitors could be valuable therapeutically in patients with aggressive CLL.