Small Molecule Inhibitor Of Heat Shock Protein 90 Research Articles

BIIB021, an orally available and small-molecule inhibitor of HSP90, activates intrinsic apoptotic pathway in human cervical adenocarcinoma cell line (HeLa).

Heat shock protein 90 (HSP90) is a highly conserved ATP-dependent chaperone protein that plays a vital role in tumorigenesis. This study aims to investigate the apoptosis inducer role of BIIB021 (orally available HSP90 inhibitors) compound via inhibition of HSP90 activity in the human cervical cancer cell line (HeLa). The anticancer potential of BIIB021 was determined by XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)] cell proliferation assay against the human cervical cancer cell line (HeLa). ATPase and luciferase aggregation assays were carried out to detect the HSP90 inhibitor potential of BIIB021. To determine the antiproliferative mechanism of the BIIB021, the expression level of the pro-apoptotic and antiapoptotic markers was determined by reverse transcription polymerase chain reaction (RT-PCR) and ELISA experiments. BIIB021 exhibited a cytotoxic effect on HeLa cell proliferation and the inhibitory concentration (IC)50 dose of BIIB021 was found to be 14.79 nM at 48 h. BIIB021 decreased the ATP hydrolysis rate of HSP90 and blocked the refolding of the desaturated luciferase in the presence of ATP. To understand the antiproliferative mechanism of the BIIB021 in HeLa cells, the mRNA and protein expression levels of the apoptotic markers [BCL-2 associated X (BAX), B-cell lymphoma 2 (BCL-2), cytochrome-c (CYT-c), and caspase-3 (CAS-3)] were determined by RT-PCR and ELISA experiments. The results obtained indicated that BIIB021 decreased BCL-2 levels and increased BAX, CYT-c, and CAS-3 levels in human cervical cancer cells. These results confirmed that BIIB021 inhibited the chaperone activity of HSP90, resulting in anti-proliferating effects in cervical cancer cells via the induction of the intrinsic apoptotic pathways.

Dose-escalation trial of combination dabrafenib, trametinib, and AT13387 in patients with BRAF-mutant solid tumors.

Combination BRAF and MEK inhibitor therapy is an active regimen in patients who have BRAF V600E-mutated tumors; however, the clinical efficacy of this therapy is limited by resistance. Preclinically, the addition of heat shock protein 90 (HSP90) inhibition improves the efficacy of BRAF inhibitor therapy in both BRAF inhibitor-sensitive and BRAF inhibitor-resistant mutant cell lines. Cancer Therapy Evaluation Program study 9557 (ClinicalTrials.gov identifier NCT02097225) is a phase 1 study that was designed to assess the safety and efficacy of the small-molecule HSP90 inhibitor, AT13387, in combination with dabrafenib and trametinib in BRAF V600E/K-mutant solid tumors. Correlative analyses evaluated the expression of HSP90 client proteins and chaperones. Twenty-two patients with metastatic, BRAF V600E-mutant solid tumors were enrolled using a 3+3 design at four dose levels, and 21 patients were evaluable for efficacy assessment. The most common tumor type was colorectal cancer (N=12). Dose-limiting toxicities occurred in one patient at dose level 3 and in one patient at dose level 4; specifically, myelosuppression and fatigue, respectively. The maximum tolerated dose was oral dabafenib 150mg twice daily, oral trametinib 2mg once daily, and intravenous AT13387 260mg/m2 on days 1, 8, and 15. The best response was a partial response in two patients and stable disease in eight patients, with an overall response rate of 9.5% (90% exact confidence interval [CI], 2%-27%), a disease control rate of 47.6% (90% CI, 29%-67%), and a median overall survival of 5.1months (90% CI, 3.4-7.6 months). There were no consistent proteomic changes associated with response or resistance, although responders did have reductions in BRAF expression, and epidermal growth factor receptor downregulation using HSP90 inhibition was observed in one patient who had colorectal cancer. HSP90 inhibition combined with BRAF/MEK inhibition was safe and produced evidence of modest disease control in a heavily pretreated population. Additional translational work may identify tumor types and resistance mechanisms that are most sensitive to this approach.

Investigation of anticancer activities of STA-9090 (ganetespib) as a second generation HSP90 inhibitor in Saos-2 osteosarcoma cells

Osteosarcoma is common childhood tumour type of the bone. Chemotherapy is the most important step in treatment of osteosarcoma. Despite advanced diagnosis methods and target specific cancer therapeutics, osteosarcoma has still a high mortality rate and a tendency to metastasize. Therefore, new therapeutic strategies are evaluated in osteosarcoma treatment in pre-clinical and clinical studies. In the last ten years, heat shock protein 90 (HSP90) has been important biological target to design target specific cancer drugs. HSP90 play vital roles in proper folding, stabilization and maintenance of oncogenic client proteins in tumorigenesis. Therefore, inhibition of HSP90 has been significant therapeutic aspects in cancer drug design. STA-9090 (ganetespib) is a second generation small molecule HSP90 inhibitor which blocks tumurogenesis in cancer cells. STA-9090 inhibited ATP hydrolysis and protein folding process of HSP90. In this study, STA-9090 decreased Saos-2 cell proliferation and IC50 dose of STA-9090 was found out as 18.71 µM and 10.25 µM at 24 h and 48 h, respectively. STA-9090 inhibited HSP90 ATPase function and disrupted oncogenic client protein folding activity. Also, STA-9090 decreased protein level of the HSP90 in osteosarcoma cells. Expression analysis of osteosarcoma and bone metabolism related genes was performed by RT2 Profiler PCR Array. This study has found the down-regulation of the expression levels of oncogenic genes: DKK1, TWIST1, WNT10B, WNT3A, RANK, RANKL, PTH, FGFR1, FGFR2, LTBP2, IL6, TGFβ1, MMP2 and SPARC genes, in STA-9090 treated Saso-2 cells. Furthermore, expression levels of osteosarcoma related genes, OPG, ERα, ERβ, IL15, BMP2 and BMP7, were found to have increased significantly. Biological activities of STA-9090 on Saos-2 cell line show its potential as a target specific drug to inhibit osteosarcoma and its metastasis.

Post-treatment with a heat shock protein 90 inhibitor prevents chronic lung injury and pulmonary fibrosis, following acute exposure of mice to HCl

Aim/Purpose: Exposure to high levels of hydrochloric acid (HCl) is associated with severe lung injury including both acute inflammation and chronic lung disease, which leads to the development of pulmonary fibrosis. Currently, there are no specific therapeutic agents for HCl-induced lung injury. Heat shock protein 90 (HSP90) has been implicated in the pathogenesis of pulmonary fibrosis. Thus, we have used a murine model of intra-tracheal acid instillation to investigate the antidotal effects of AUY-922, a small molecule HSP90 inhibitor, already in clinical trials for various types of cancer, against HCl-induced chronic lung injury and pulmonary fibrosis.Methods: HCl (0.1 N, 2 μl/g body weight) was instilled into male C57Bl/6J mice at day 0. After 24 h, mice began receiving 1 mg/kg AUY-922, 2x/week for 15 or 30 days.Results: AUY-922 suppressed the HCl-induced sustained inflammation, as reflected in the reduction of leukocyte and protein concentrations in bronchoalveolar lavage fluid, and inhibited the activation of pro-fibrotic biomarkers, ERK and HSP90. Furthermore, AUY-922 improved lung function, decreased the overexpression and accumulation of extracellular matrix proteins and dramatically reduced histologic evidence of fibrosis in the lungs of mice exposed to HCl.Conclusions: We conclude that AUY-922, and possibly other HSP90 inhibitors, successfully block the adverse effects associated with acute exposures to HCl and may represent an effective antidote against HCl-induced chronic lung injury and fibrosis.

Heat Shock Protein 90 Is a Potential Therapeutic Target in Cholangiocarcinoma.

Cholangiocarcinoma is an aggressive malignancy with a poor prognosis, with no effective therapy other than surgical resection. Heat shock protein 90 (HSP90) is a key component of a multichaperone complex involved in the posttranslational folding of a number of client proteins, many of which play essential roles in tumorigenesis. Here, we attempted to clarify its prognostic significance and potential utility as a therapeutic target in cholangiocarcinoma. Immunohistochemical expression of HSP90 was assessed retrospectively in 399 cholangiocarcinoma cases and 17 human cholangiocarcinoma cell lines, along with the effect of a small-molecule HSP90 inhibitor (NVP-AUY922) on cholangiocarcinoma tumor growth and angiogenesis in human cholangiocarcinoma cell lines and xenografts. The positivity of HSP90 was 44.6% in intrahepatic cholangiocarcinoma (IHCC) and 32.8% in extrahepatic cholangiocarcinoma (EHCC), respectively. HSP90 expression was significantly associated with the 5-year survival rate for IHCC (P < 0.001) and EHCC (P < 0.001). HSP90 inhibition showed potent antiproliferative activity and reduced growth-associated signaling in human cholangiocarcinoma cells in vitro. Furthermore, treatment of cholangiocarcinoma xenograft-bearing mice with NVP-AUY922 significantly inhibited growth at doses far below the maximum-tolerated dose. HSP90 overexpression is a prognostic marker for cholangiocarcinoma. HSP90-targeted therapy may be an option for a subset of cholangiocarcinoma.

Ganetespib: research and clinical development.

Under stressful conditions, the heat shock protein 90 (HSP90) molecular chaperone protects cellular proteins (client proteins) from degradation via the ubiquitin-proteasome pathway. HSP90 expression is upregulated in cancers, and this contributes to the malignant phenotype of increased proliferation and decreased apoptosis and maintenance of metastatic potential via conservation of its client proteins, including human epidermal growth factor receptor 2, anaplastic lymphoma kinase, androgen receptor, estrogen receptor, Akt, Raf-1, cell cycle proteins, and B-cell lymphoma 2 among others. Hence, inhibition of HSP90 leads to the simultaneous degradation of its many clients, thereby disrupting multiple oncogenic signaling cascades. This has sparked tremendous interest in the development of HSP90 inhibitors as an innovative anticancer strategy. Based on the wealth of compelling data from preclinical studies, a number of HSP90 inhibitors have entered into clinical testing. However, despite enormous promise and anticancer activity reported to date, none of the HSP90 inhibitors in development has been approved for cancer therapy, and the full potential of this class of agents is yet to be realized. This article provides a review on ganetespib, a small molecule HSP90 inhibitor that is currently under evaluation in a broad range of cancer types in combination with other therapeutic agents with the hope of further enhancing its efficacy and overcoming drug resistance. Based on our current understanding of the complex HSP90 machinery combined with the emerging data from these key clinical trials, ganetespib has the potential to be the first-in-class HSP90 inhibitor to be approved as a new anticancer therapy.

586 Phase I study of pan-histone deacetylase inhibitor abexinostat in combination with cisplatin in patients with advanced solid tumors

been linked to more aggressive disease and poor clinical outcomes. HER2 is particularly reliant on the chaperoning activity of heat shock protein 90 (HSP90) for its stability and function. Pharmacological blockade of HSP90 results in cellular depletion and loss of its client proteins, in turn providing a means to simultaneously disrupt multiple oncogenic pathways. Ganetespib is a clinically advanced small molecule inhibitor of HSP90. Here, we have evaluated the antitumor activity and efficacy of ganetespib, both as a single agent and in combination with clinically approved drugs, in models of human gastric cancer. Material and Methods: Cell viability was determined by CellTiterGlo. The anticancer effect of ganetespib as single agent or coupled with other chemotherapeutic agents was examined via immunoblot, immunofluorescence, flow cytometry and xenograft models in SCID mice. Results: Ganetespib demonstrated potent in vitro cytotoxic activity in a panel of 22 gastric cancer cell lines in vitro, with an average IC50 value 20 nM. The panel included 3 HER2-overexpressing (HER2+) lines and antiproliferative activity was independent of p53 status. At the molecular level, ganetespib treatment promoted destabilization of multiple HSP90 client proteins and effectors, including HER2, EGFR, IGF-IR, cMET, cKit, P-AKT, P-ERK1/2 and a number of cell cycle modulators, which ultimately resulted in apoptosis. These effects of ganetespib were durable, with kinetic analysis showing client suppression was maintained for over 48 hours following a 1 hour drug exposure. Combinations of ganetespib with chemotherapeutic agents (doxorubicin, docetaxel, cisplatin) as well as the dual HER2/EGFR kinase inhibitor lapatinib promoted significant increases in total cell death in comparison to monotherapy. Synergistic effects were also observed when ganetespib was combined with the PI3K/mTOR inhibitor BEZ235, due in part to ganetespibs’ ability to overcome the rebound in AKT activity that arises from PI3K/mTOR inhibition. The combinatorial benefit observed in vitro translated to improved efficacy in vivo, where combinations of ganetespib with capecitabine or docetaxel induced tumor regressions in NCI-N87 xenografts. Conclusions: Taken together, these data highlight the potent antitumor activity of ganetespib for gastric cancer cells, including HER2+ lines, both in vitro and in animal models. The findings support the potential therapeutic value of ganetespib, particularly in combination with standardof-care agents, for the treatment of patients with gastric cancer.

Abstract 3693: Preclinical antitumor activity of SST0116CL1, a novel heat shock protein 90 (HSP90) inhibitor

Abstract Hsp90 is a component of a molecular chaperone complex, involved in the folding, maturation and stabilisation of key signaling molecules which control cell proliferation, survival and transformation. It works by a modulation of a set of cancer-associated proteins, collectively referred to as ‘‘clients’'. Inhibition of Hsp90 causes simultaneous destabilization and eventual degradation of client proteins that result in suppression of tumor growth, and a number of novel synthetic HSP90 inhibitors are currently under oncology clinical investigations for the treatment of a wide variety of tumor types. SST0116CL1 (property of sigma-tau Research Switzerland S.A.) is a potent, second generation, small-molecule HSP90 inhibitor, with a chemical structure (4-amino substituted resorcino-isoxazole) unrelated to the first generation ansamycin family of HSP90 inhibitors. SST0116CL1 binds to the ATP binding pocket of HSP90, and interferes with HSP90 chaperone function thus resulting in client protein degradation and tumor growth inhibition. In in vitro studies, SST0116CL1 was shown to inhibit recombinant HSP90α and to induce the degradation of the oncogenic Her2 tyrosine kinase in BT-474 human breast cancer cells. Moreover, it was able to induce the destabilization and depletion of different client proteins, often overexpressed and constitutively activated in numerous types of hematological or solid human tumors. These results correlated well with those obtained from the cell proliferation assay as well as with the HSP90 competitive binding assay, and clearly suggested that target modulation of HSP90 was achieved. In preclinical in vivo studies, in different solid and hematological tumor xenograft models, such as A431 epidermoid carcinoma with EGFR overexpression, GTL-16 gastric carcinoma with c-Met overexpression, MV4;11 B-myelomonocytic leukemia with flt3 mutation, A2780/Dx multidrug resistant ovarian carcinoma with P-glycoprotein overexpression, SST0116CL1 delivered intravenously or intraperitoneally revealed a potency at least comparable or higher than that found with the HSP90 inhibitor NVP-AUY922, under Phase II clinical trials investigation, or chemotherapeutic drugs currently used in cancer therapy. SST0116CL1 showed to be a manageable compound when given according to different schedules (qd5x/w; q4d/w; q2dx3/w), since it was well-tolerated and active to inhibit the tumor growth. A modulation of PD biomarkers in terms of down-regulation of EGFR, AKT and CDK4 was reached in tumor lesions of A431 tumor bearing mice treated with SST0116CL1 and a down-regulation of c-Met, AKT and CDK4 was observed in tumors collected from GTL-16 tumor bearing mice. PK parameters demonstrated a rapid clearance of SST0116CL1 from normal tissues and a blood compartment with prolonged retention in tumors. On the complex, these results strongly encourage the selection of SST0116CL1 as a promising candidate for clinical trial. Citation Format: Loredana Vesci, Ferdinando Milazzo, Valeria Carollo, Silvia Pace, Giuseppe Giannini. Preclinical antitumor activity of SST0116CL1, a novel heat shock protein 90 (HSP90) inhibitor. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3693. doi:10.1158/1538-7445.AM2014-3693

Ganetespib and HSP90: Translating Preclinical Hypotheses into Clinical Promise

As with many physiologic processes that become subverted during tumorigenesis, the chaperoning activity of heat shock protein 90 (HSP90) is often exploited by cancer cells to confer aberrant proliferative, survival, and/or metastatic potential. Functional inhibition of HSP90 results in the degradation of its client proteins, in turn providing a means to concomitantly disrupt multiple oncogenic signaling cascades through one molecular target. Pharmacologic blockade of HSP90 has, therefore, emerged as an innovative and multifaceted approach for the development of new antineoplastic agents. However, no HSP90 inhibitors are currently approved for cancer therapy and the full promise of this class of agents is yet to be realized. This review focuses on the preclinical activity profile of ganetespib, a potent small-molecule inhibitor of HSP90, the characterization of which has provided important frameworks for the optimal design and application of HSP90 inhibitor-based strategies in a variety of cancer types. Beyond client protein-driven tumors, ganetespib can also potentiate the effects of other molecularly targeted and standard-of-care therapeutics while simultaneously overcoming drug resistance in multiple tumor types, thereby positioning this compound as the leading HSP90 inhibitor currently under clinical development.

Abstract 5601: Overcoming acquired resistance to BRAF inhibitors in melanoma with the HSP90 inhibitor ganetespib

Abstract Activating BRAF(V600E) kinase mutations occur in 50% of melanoma, 45% of papillary thyroid cancer and 10% of colon cancer. BRAF inhibitors have elicited significant clinical response in melanomas but acquired resistance frequently develops after initial responses. Therefore, consideration of new treatment strategies to block activity of mutant BRAF and deter therapeutic resistance is warranted. Heat shock protein 90 (HSP90) is a molecular chaperone required for the stability of hundreds of client proteins, including the RAF family of kinases, and has emerged as a highly relevant anticancer target. Ganetespib is a second generation, small molecule HSP90 inhibitor currently being evaluated in multiple clinical trials. To investigate the potential for ganetespib in the treatment of melanoma we evaluated its preclinical activity as monotherapy, and in combination with targeted agents, in cells sensitive or resistant to BRAF inhibitors. Ganetespib displayed potent anticancer activity in a large panel of BRAF(V600E) mutant melanoma, thyroid and colon cancer cell lines, with IC50 between 4 and 40 nM. Further analysis showed that ganetespib disrupted oncogenic signaling by inducing the destabilization of mutant BRAF and subsequent inactivation of MEK and ERK. Levels of wild-type BRAF were only slightly reduced in response to ganetespib treatment, implying that the stability of activated BRAF is more reliant on HSP90 and that ganetespib may selectively deplete oncogenic forms of BRAF. Notably, ganetespib treatment also led to depletion of CRAF, which is implicated in driving therapeutic resistance to BRAF inhibitors. In an effort to maximize antitumor activity, we combined low levels of ganetespib with BRAF or MEK inhibitors in vitro and observed increased cytotoxicity compared to single agent treatment. Both the BRAF and MEK inhibitors were cytostatic on their own, which may be due to continued PI3K/mTOR signaling. We subsequently assayed the combination of ganetespib with a PI3K/mTOR inhibitor and observed synergistic increases in cell death. Importantly, ganetespib was capable of blocking the feedback activation on MAPKK induced by a MEK inhibitor as well as the activation of ERK by PI3K/mTOR inhibitors. To determine if these results translated in vivo, similar combinations were performed in BRAF mutant human melanoma xenografts. Co-administration of ganetespib with either a BRAF or PI3K/mTOR inhibitor suppressed in vivo tumor growth to a greater extent compared to monotherapy. Finally, we show that ganetespib effectively blocked MAPK signaling and killed RPMI-7951 melanoma cells (IC50 = 4 nM), which are resistant to BRAF and MEK inhibitors due to the overexpression of COT. In conclusion, targeting HSP90 with ganetespib represents a promising approach for the treatment of BRAF(V600E)-driven cancers and may be especially relevant in the context of acquired resistance to BRAF inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5601. doi:1538-7445.AM2012-5601

Abstract 2770: Antitumor activity of heat shock protein 90 (HSP90) inhibitor AT13387 in head and neck squamous cell carcinoma

Abstract Heat shock protein 90 (HSP90) has been shown to be overexpressed in a wide range of cancers, and plays a critical role in tumor cell growth and survival by stabilizing aberrant signaling proteins and by interfering with apoptosis. Several natural and synthetic inhibitors of HSP90 have been shown to have antitumor effects, but single agent development has been limited by poor solubility, poor availability and off-target toxicities (hepatotoxicity, retinal toxicity). The aim of this study is to investigate the biological activity of the small-molecule HSP90 inhibitor, AT13387 (Astex Pharmaceuticals) for antitumor activity in head and neck squamous cell carcinoma (HNSCC). Human HNSCC cell lines, UMSCC −1 (wild-type p53) and −46 (mutant p53) were treated with AT13387. The inhibitory concentrations (IC[[Unsupported Character - Codename &amp;shy;]]50) of AT13387 on HNSCC cell lines were determined using MTT proliferation assay. AT13387 (55 mg/kg two consecutive days every week) antitumor activity, toxicity and survival were evaluated in the UMSCC −1 and −46 xenograft model in BALB/c SCID mice. AT13387 inhibited the proliferation of both UMSCC cell lines in vitro with IC50 levels of 0.05-.10 µM by MTT assay. In vivo, AT13387, at 55 mg/kg showed significant antitumor effect and improved survival in both wtTP53 UMSCC-1 and mtTP53 UMSCC-46 xenograft models. The treatment was well-tolerated and no significant body weight loss observed. These data suggest that AT13387 may possess potent antitumor activity in human HNSCC, regardless of the p53 mutation status. The potential of AT13387 as a chemosensitizer for radiotherapy is being investigated. These preclinical data suggest that AT13387 holds promise as a potential chemotherapeutic agent for HNSCC clinical trials. (Supported by NIDCD project ZIA-DC-000073 and AT13387 provided to NCI by Astex Pharmaceuticals) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2770. doi:1538-7445.AM2012-2770

Abstract P6-15-13: Biologic Activity of a Novel Heat Shock Protein 90 Inhibitor, PF-4928473, in Human Breast Cancer Cell Lines

Abstract Background: Heat shock protein 90 (HSP90) is a key molecular chaperone involved with the post-translational folding and maturation of various cellular proteins, including Raf, Src, ER, EGFR, HER2/ErbB2, IGFR, and VEGFR. Therefore, targeting HSP90 represents an attractive strategy for modulating aberrant oncogenic signal transduction in human malignancies. In the present study, we investigate the anti-cancer activity of PF-4928473, a novel small molecule inhibitor of HSP90, in a diverse panel of human breast cancer cell lines in vitro. Methods: 49 breast cancer cell lines (representing 10 luminal, 18 HER2- amplified, 18 non-luminal, 3 immortalized subtypes), including two HER2- amplified lines with conditioned trastuzumab and lapatinib resistance (SKBR3 parental), were assessed. Cell proliferation assays with two-fold drug dilutions over 9 concentrations were performed over 6 days to determine growth-adjusted IC50 and IC100 values, the latter as a surrogate of in vitro drug lethality. Flow-cytometry was performed in a selected subset of 21 cell lines to assess effects on cell cycle and apoptosis after 48 hours and 5 days of drug exposure, respectively. Western blot analysis evaluated the biochemical effects of HSP90 drug inhibition. Results: In our panel, IC50s ranged from 8 (±1) nM to 525 (±2) nM, with 45 cell lines exhibiting IC50s ≥50 nM. Differential drug sensitivity was observed with HER2-amplified lines, with 15 of 18 demonstrating IC50s ≥28 nM. For SKBR3-trastuzumab and -lapatinib-resistant lines, IC50s were 13 (±1) nM and 21 (±2) nM, respectively. IC100s ranged from 9 (±3) nM to &amp;gt; 1000 nM. 17 of 18 HER2-amplified lines had IC100s between 9 (±3) nM and 69 (±1) nM, including SKBR3-trastuzumab and -lapatinib-resistant lines at 31 (±2) nM and 66 (±2) nM, respectively. Flow cytometry with 50 nM of PF-4928473 revealed moderate early apoptotic effects in cell lines with IC50s &amp;lt; 30 nM, encompassing the majority of HER2-amplified lines. Mild effects on cell cycle, primarily G0/G1 arrest, were observed in 8 lines at 50 and/or 100 nM. Western blots of representative cell lines at 50 and 100 nM of PF-4928473 identified downregulation of p-AKT and p-ERK in the HER2-amplified SKBR3 line (IC50: 19 ±4 nM), vs. relative lack of effect for the non-luminal MDA-MB-468 (IC50: 24 ±5 nM) and luminal KPL-1 (IC50: 68 ±2 nM) lines. Upregulation of HSP70, a known biomarker associated with HSP90 inhibition, was observed at 12 hours post-drug exposure regardless of cell sensitivity. Further biochemical studies are ongoing. Conclusions: PF-4928473 demonstrated low nanomolar in vitro anti-proliferative activity, lethality, and apoptosis in a wide panel of human breast cancer cell lines, including trastuzumab and lapatinib-resistant lines. Biological effects were most pronounced in HER2-amplified lines, in keeping with HER2 as a known protein client of HSP90. Based on these results, further translational evaluation of HSP90 inhibitors in this population merits consideration. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-15-13.

Potent Triazolothione Inhibitor of Heat‐Shock Protein‐90

Heat-shock protein-90 is an attractive target for anticancer drugs, as heat-shock protein-90 blockers such as the ansamycin 17-(allylamino)-17-demethoxygeldanamycin greatly reduce the expression of many signaling molecules that are disregulated in cancer cells and are key drivers of tumor growth and metastasis. While 17-(allylamino)-17-demethoxygeldanamycin has shown promise in clinical trials, this compound class has significant template-related drawbacks. In this paper, we describe a new, potent non-ansamycin small-molecule inhibitor of heat-shock protein-90, BX-2819, containing resorcinol and triazolothione rings. Structural studies demonstrate binding of BX-2819 to the ADP/ATP-binding pocket of heat-shock protein-90. The compound blocked expression of heat-shock protein-90 client proteins in cancer cell lines and inhibited cell growth with a potency similar to 17-(allylamino)-17-demethoxygeldanamycin. In a panel of four cancer cell lines, BX-2819 blocked growth with an average IC(50) value of 32 nM (range of 7-72 nM). Efficacy studies demonstrated that treatment with BX-2819 significantly inhibited the growth of NCI-N87 and HT-29 tumors in nude mice, consistent with pharmacodynamic studies showing inhibition of heat-shock protein-90 client protein expression in tumors for greater than 16 h after dosing. These data support further studies to assess the potential of BX-2819 and related analogs for the treatment of cancer.

The HSP90 inhibitor 17-AAG synergizes with doxorubicin and U0126 in anaplastic large cell lymphoma irrespective of ALK expression

Heat shock protein 90 (HSP90) chaperones and maintains the molecular integrity of a variety of signal transduction proteins, including the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) oncogenic protein, a genetic abnormality that is frequently observed in anaplastic large cell lymphoma (ALCL) cells. Here we demonstrate that HSP90 is overexpressed in primary and cultured ALK-positive and ALK-negative ALCL cells, and we evaluate the potential role of the small molecule inhibitor of HSP90, 17-allylamino-17-demethoxygeldanamycin (17-AAG) in treating ALCL. The antiproliferative effect of 17-AAG-cultured cells was determined by MTS assay. Apoptosis and cell-cycle arrest were determined by Annexin-V/propidium iodide and propidium iodide staining, respectively, and fluorescein-activated cell sorting analysis. Expression of HSP90 was evaluated by immunohistochemistry, and molecular changes were determined by Western blot. Treatment of cultured ALCL cells with 17-AAG induced cell-cycle arrest and apoptosis, irrespective of ALK expression. At the molecular level, 17-AAG induced degradation of ALK and Akt proteins, dephosphorylated extracellular signal-regulated kinase, and degraded the cell-cycle regulatory protein cyclin D1 and its cyclin-dependent kinases, CDK4 and CDK6, but had a differential effect on p27 and p53 proteins. Inhibition of extracellular signal-regulated kinase phosphorylation by the mitogen activated protein kinase inhibitor U0126 induced cell death in all ALCL cell lines, and sublethal concentration 17-AAG showed synergistic antiproliferative effects when combined with U0126 or doxorubicin. Our data demonstrate that targeting HSP90 function by 17-AAG may offer a novel therapeutic strategy for ALCL, either as single-agent activity or by combining 17-AAG with conventional or targeted therapeutic schemes.

Heat Shock Protein 90 (HSP90) Is Overexpressed in Primary and Cultured Hodgkin Lymphoma (HL) Cells: Role of Therapeutic Targeting Using the Small Molecule Inhibitor 17-AAG.

Conventional chemotherapy is the golden standard for therapy of Hodgkin Lymphoma (HL). Nevertheless, considerable toxicity and secondary malignancies indicate the need for targeted therapy that preferentially kills the malignant cells. The molecular chaperone heat shock protein 90 (HSP90) is expressed in all mammalian cells, but it is overexpressed in malignancy. 17-AAG, a small molecule inhibitor of HSP90, has been shown to induce apoptosis and cell cycle arrest in a variety of tumor types. In the present study we show that HSP90 is overexpressed in the primary Hodgkin and Reed-Sternberg (HRS) cells, as well as in HL derived cells lines. Inhibition of HSP90 17-AAG showed antiproliferative effect in HL derived cell lines in a dose dependent manner. Cell death was due to apoptosis, as determined by Annexin-V staining and FACS analysis. Apoptosis was mediated by the activation of the caspase pathway, especially by caspase 8, 9, and 3. Inhibition of caspase activity by the pancaspase inhibitor Z-VAD-FMK partially reversed the 17-AAG lethal effect. 17-AAG had no significant on the level of the antiapoptotic Bcl-2 family members or the cellular or X-Linked inhibitors of apoptosis. In contrast, there was considerable degradation of cFLIP. Moreover, 17-AAG treatment reduced the intracellular levels of molecules that have been shown to be of key importance in HRS cell survival and proliferation, including AKT and the phosphorylated ERK1/2, but with minimal change in total ERK1/2. Cell cycle arrest was observed at G0/G1 or at G2/M phase, and was mediated by reduction in the levels of MDM2, cyclin D1 with cdk4 and cdk6, and cyclin B1. The potential synergy of 17-AAG with conventional chemotherapy and anti-TRAIL death receptor monoclonal antibody, was explored by the simultaneous incubation of HL derived cells with both doxorubicin or antibodies against TRAIL receptors R1 and R2, respectively. The combination of 17-AAG with doxorubicin or anti-TRAIL antibodies was significantly more effective than either agent alone. Based on these data we are conducting a phase II study of 17-AAG in patients with relapsed classical HL.

The Geldanamycin Analogue Heat Shock Protein 90 (HSP90) Inhibitor 17-AAG in Lymphomas: Molecular Mechanisms of 17-AAG-Induced Cell Death in Hodgkin Disease and Non- Hodgkin Lymphoma Cell Lines.

Heat Shock Proteins (HSP) are stress-induced proteins that act as molecular chaperones in all benign and malignant cells. HSPs are important for maintaining the function of several client proteins involved in transcriptional regulation, signal transduction, and cell-cycle control. HSP90 has been recently identified as a potential therapeutic target in several types of tumors. However, the effect HSP90 inhibition in Hodgkin disease (HD) and non-Hodgkin lymphomas (NHL) remains unknown. The geldanamycin analogue 17-allylamino-17-demethoxy-geldanamycin (17-AAG) is a small molecule inhibitor of HSP90, which interferes with ATP-dependent chaperones activity, with subsequent degredation of client proteins by the proteasome. We studied the activity of 17-AAG in a panel of cell lines of HD (HD-MyZ, HD-LM2, L-428, KM-H2) and NHL (SUDHL1, and Jurkat) origin. HSP90 was expressed in all HD cell lines but not in normal peripheral blood lymphocytes of healthy donors. 17-AAG demonstrated a significant antiproliferative effect in a dose (01 micromolar - 5 micromolar) and time (24 hours to 72 hours) dependent manner in all cells lines, but was most prominant in the HD-LM2 cells. In all HD-derived cell lines, 17-AAG had more than 50% inhibitory effect over control cells. This effect was most prominant after 72 hours of incubation. Subsequently, we examined the molecular mechanisms responsible for this antiproliferative effect. Both cell cycle arrest and induction of apoptosis were observed. In the HD-LM2 cell line, 17-AAG predominantly induced a G0/G1 cell cycle phase arrest, while in the L-428 cell line there was a G2/M phase arrest (as determined by propidium iodide staining and flow-cytometric analysis). This effect was associated with alterations in MDM2 and cyclin D1 proteins. Cell cycle arrest was followed by apoptosis as determined by Annexin-V binding assay, which was predominantly mediated by caspase 9 activation. 17-AAG also reduced the cellular content of Bcl-2, cFLIP, and AKT, but had no significant effect on XIAP or cIAP2. Furthermore, 17-AAG decreased the total ERK1/2 which has been shown to play an important role in HRS cell survival. 17-AAG-induced apoptosis was partially reversed by the pancaspase inhibitor ZVAD-FMK. Preliminary data suggested that 17-AAG did not enhance the effect of doxoorubicin or gemcitabine chemotherapy. These data suggest a first proof-of-principle that inhibition of HSP90 might be of therapeutic value in HD and NHL.