Heat Shock Protein 90 Inhibitor NVP-AUY922 Research Articles

Heat shock protein 90 (HSP90) inhibitors in gastrointestinal cancer: where do we currently stand?-A systematic review.

Dysregulated expression of heat shock proteins (HSP) plays a fundamental role in tumor development and progression. Consequently, HSP90 may be an effective tumor target in oncology, including the treatment of gastrointestinal cancers. We carried out a systematic review of data extracted from clinicaltrials.gov and pubmed.gov, which included all studies available until January 1st, 2022. The published data was evaluated using primary and secondary endpoints, particularly with focus on overall survival, progression-free survival, and rate of stable disease. Twenty trials used HSP90 inhibitors in GI cancers, ranging from phase I to III clinical trials. Most studies assessed HSP90 inhibitors as a second line treatment. Seventeen of the 20 studies were performed prior to 2015 and only few studies have results pending. Several studies were terminated prematurely, due to insufficient efficacy or toxicity. Thus far, the data suggests that HSP90 inhibitor NVP-AUY922 might improve outcome for colorectal cancer and gastrointestinal stromal tumors. It currently remains unclear which subgroup of patients might benefit from HSP90 inhibitors and at what time point these inhibitors may be beneficial. There are only few new or ongoing studies initiated during the last decade.

Abstract 2188: Comparing effects of small molecular weight compounds on proliferation and chemotaxis of pancreatic cancer cells

Abstract Background & Aims: The new therapy for pancreatic cancer which has the poorest prognosis among malignant tumors is high demand nowadays. The overall 5-year survival rate in all stages of this type of cancer is less than 5%, even with the multimodality therapy, including surgery, chemotherapy and radiotherapy. Although gemcitabine (GEM) is a key drug in the treatment of advanced pancreatic cancer, the therapeutic effect is limited because of drug resistance. Heat shock protein 90 (HSP90) is known to be overexpressed in several types of cancer cells, and its inhibition has shown promise in the treatment of solid malignancies. Src kinases work as a signal switch for multiple molecular signal transduction pathways. The objective of this study is to evaluate an antitumor effect of three kinds of small molecular weight compounds (antimetabolite Gemcitabine, HSP90 inhibitor NVP-AUY922, and Src kinase inhibitor Dasatinib) on tumor growth and chemotaxis in pancreatic cancer cell lines. Methods: Pancreatic cancer cell lines were treated with each of the 3 compounds to examine the effect on proliferation assay, apoptosis assay, Western blotting, and chemotaxis assay. Results: Of these compounds, HSP90 inhibitor inhibited cell proliferation in pancreatic cancer cells best in a dose- and time-dependent manner. Growth inhibitory effect of HSP90 inhibitor for pancreatic cancer cells were highest of the three. Further, HSP90 inhibitor inhibited the growth of gemcitabine sensitive and resistant pancreatic cancer cell lines. With flowcytometry and immunostaining, HSP90 inhibitor had higher apoptosis-inducing effect than gemcitabine. In the chemotaxis assay, HSP90 inhibitor inhibited the migration of pancreatic cancer cell best in the three compounds. In western blotting, HSP90 inhibitor suppressed the expression of multiple receptor tyrosine kinase proteins as well as the signal transduction in PI3K-Akt and RAS-MAPK pathways. Conclusion: In this study, HSP90 inhibitor was the best of 3 small molecular compounds and has proven effective in gemcitabine sensitive and resistant pancreatic cancer. The HSP90 inhibitor is useful as an antitumor drug by inhibiting proliferation and chemotaxis of pancreatic cancer cells through multiple kinase pathways. Citation Format: Masahiro Yamamura, Akira Yamauchi, Naoki Katase, Masakiyo Sakaguchi, Yosuke Katata, Hiroaki Tanioka, Makoto Okawaki, Takeshi Nagasaka, Yoshiyuki Yamaguchi. Comparing effects of small molecular weight compounds on proliferation and chemotaxis of pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2188.

HSP90 inhibitor NVP-AUY922 induces cell apoptosis by disruption of the survivin in papillary thyroid carcinoma cells

Heat shock protein 90 (HSP90) is a molecular chaperone required for maintaining the stability and function of signal proteins that plays an important role in promoting the growth and survival of cancer cells. The incidence of papillary thyroid carcinoma (PTC) has been increasing in recent years. The effect of the novel non-geldanamycin HSP90 inhibitor NVP-AUY922 on apoptosis of papillary thyroid carcinoma cells has not been investigated. The influence of AUY922 on the survival of PTC cell lines K1 and IHH4 was evaluated. Cell viability was determined by cell counting kit method. Cell apoptosis was assessed by flow cytometry and western blotting and the potential mechanism was evaluated by western blotting and immunoprecipitation. Overexpression plasmid was transfected by Lipofectamine 2000 method. In K1 and IHH4 cell lines, after the treatment of AUY922, cell viability decreased, and the proportion of apoptosis cells increased. AUY922 caused the cleavage of PARP and caspase-3 proteins, and altered expression of survivin, which was a client protein of HSP90. In AUY922-treated cells, overexpression of survivin attenuated growth inhibition and cell apoptosis. The results indicate that AUY922 induces apoptotic cell death in PTC cells. Moreover, our findings demonstrate that AUY922 induced apoptosis by downregulating the expression of survivin protein in PTC cells.

Highlights of This Issue

Tumor adaptive resistance to therapeutic radiation remains a barrier for further improvement of local cancer control. SIRT3, a member of the sirtuin family of protein deacetylases, prevents cell aging by promoting mitochondrial metabolic homeostasis. Liu and colleagues discovered that SIRT3 was transcriptionally regulated by NF-κB upon radiation, and its enzymatic activity was further enhanced via phosphorylation by mitochondria-localized Cyclin B1–CDK1 complex. Phosphorylation of SIRT3 by CDK1 was required for mitochondrial functions as well as cell survival upon therapeutic radiation shown by in vitro and in vivo radiation tests. Thus, targeting CDK1-mediated SIRT3 phosphorylation is a potential effective approach to enhance tumor response to radiotherapy.Cholangiocarcinoma is a highly malignant carcinoma with poor prognosis. The molecular chaperone, heat shock protein 90 (HSP90), plays an important role in the posttranslational maturation and activation of many critical oncogenic proteins. Here, Shirota and colleagues discovered that the expression of HSP90 was significantly associated with 5-year survival for intrahepatic and extrahepatic cholangiocarcinoma. Furthermore, HSP90 inhibitor NVP-AUY922 showed potent anti- cholangiocarcinoma effect in vitro and in vivo. These data indicate that HSP90 expression is an independent prognostic factor, and that NVP-AUY922 may be an effective treatment option for a subset of patients with cholangiocarcinoma.There is compelling clinical rationale to identify molecular targeting agents that enhance tumor response to radiation. Agents that target MDM2 and p53 are excellent candidates to modulate radiosensitivity. Werner, Huang, and colleauges demonstrate that AMG 232, a potent small molecule MDM2 inhibitor, augments radiation response across a spectrum of human tumors using in vitro and in vivo model systems. Combined AMG 232 and radiation treatment resulted in inhibition of DNA damage repair and induction of senescence, apoptosis, and autophagy. This work previews a promising combination treatment approach for tumors harboring functional p53.Increasing evidence implicates EGFR and HER3 in the modulation of tumor cell response to radiation. In this study, Li, Huang, and colleagues investigate the effect of a dual-specific antibody (MEHD7945A) that simultaneously targets EGFR and HER3 to radiation response in head and neck and lung cancers. The authors found that MEHD7945A enhanced radiation response in both in vitro and in vivo model systems, possibly through an increase of γ-H2AX associated with DNA double-strand breaks and a significant reduction of markers related to tumor proliferation and vasculature. These data suggest the potential value of MEHD7945A as a radiation sensitizer in future clinical trial designs.

HSP90 inhibitor NVP-AUY922 enhances TRAIL-induced apoptosis by suppressing the JAK2-STAT3-Mcl-1 signal transduction pathway in colorectal cancer cells

TRAIL has been shown to induce apoptosis in cancer cells, but in some cases, certain cancer cells are resistant to this ligand. In this study, we explored the ability of representative HSP90 (heat shock protein 90) inhibitor NVP-AUY922 to overcome TRAIL resistance by increasing apoptosis in colorectal cancer (CRC) cells. The combination of TRAIL and NVP-AUY922 induced synergistic cytotoxicity and apoptosis, which was mediated through an increase in caspase activation. The treatment of NVP-AUY922 dephosphorylated JAK2 and STAT3 and decreased Mcl-1, which resulted in facilitating cytochrome c release. NVP-AUY922-mediated inhibition of JAK2/STAT3 signaling and down-regulation of their target gene, Mcl-1, occurred in a dose and time-dependent manner. Knock down of Mcl-1, STAT3 inhibitor or JAK2 inhibitor synergistically enhanced TRAIL-induced apoptosis. Taken together, our results suggest the involvement of the JAK2-STAT3-Mcl-1 signal transduction pathway in response to NVP-AUY922 treatment, which may play a key role in NVP-AUY922-mediated sensitization to TRAIL. By contrast, the effect of the combination treatments in non-transformed colon cells was minimal. We provide a clinical rationale that combining HSP90 inhibitor with TRAIL enhances therapeutic efficacy without increasing normal tissue toxicity in CRC patients.

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

The HSP90 inhibitor NVP-AUY922 potently inhibits non-small cell lung cancer growth.

Heat shock protein 90 (HSP90) is involved in protein folding and functions as a chaperone for numerous client proteins, many of which are important in non-small cell lung cancer (NSCLC) pathogenesis. We sought to define preclinical effects of the HSP90 inhibitor NVP-AUY922 and identify predictors of response. We assessed in vitro effects of NVP-AUY922 on proliferation and protein expression in NSCLC cell lines. We evaluated gene expression changes induced by NVP-AUY922 exposure. Xenograft models were evaluated for tumor control and biological effects. NVP-AUY922 potently inhibited in vitro growth in all 41 NSCLC cell lines evaluated with IC50 < 100 nmol/L. IC100 (complete inhibition of proliferation) < 40 nmol/L was seen in 36 of 41 lines. Consistent gene expression changes after NVP-AUY922 exposure involved a wide range of cellular functions, including consistently decreased dihydrofolate reductase after exposure. NVP-AUY922 slowed growth of A549 (KRAS-mutant) xenografts and achieved tumor stability and decreased EGF receptor (EGFR) protein expression in H1975 xenografts, a model harboring a sensitizing and a resistance mutation for EGFR-tyrosine kinase inhibitors in the EGFR gene. These data will help inform the evaluation of correlative data from a recently completed phase II NSCLC trial and a planned phase IB trial of NVP-AUY922 in combination with pemetrexed in NSCLCs.

Novel HSP90 Inhibitor NVP-AUY922 Enhances the Anti-tumor Effect of Temsirolimus Against Oral Squamous Cell Carcinoma

Heat shock protein 90 (HSP90) and mammalian target of rapamycin (mTOR) are involved in the molecular pathogenesis of advanced oral squamous cell carcinoma. HSP90 inhibitors are capable of effectively interfering with multiple signaling pathways, including the mTOR signaling pathway. However, the combined effects of HSP90 and mTOR inhibitors on oral squamous cell carcinoma are still unknown. In this study, we investigated the dual treatment of the novel HSP90 inhibitor NVP-AUY922 and temsirolimus against oral squamous cell carcinoma. The effect of the combination of NVP-AUY922 and temsirolimus on oral squamous cell carcinoma in vitro and in vivo was determined by MTS assay and mouse xenograft models. The effect of the combination on angiogenesis was determined by tube formation assay and angioreactor. The combination treatment of NVP-AUY922 and temsirolimus significantly inhibited the proliferation of SAS oral squamous cell carcinoma cells in vitro and suppressed the growth of oral squamous cell carcinoma xenografts in vivo. We have clearly shown that the combination treatment of NVP-AUY922 and temsirolimus inhibited vascular formation both in vitro and in vivo. Moreover, the combination treatment of NVP-AUY922 and temsirolimus prolonged the survival rate in mice xenografted with oral squamous cell carcinoma. Here, we showed the activity of a combination of mTOR and HSP90 inhibitors for the treatment of advanced oral squamous carcinoma.

Antiproliferative effect of the HSP90 inhibitor NVP-AUY922 is determined by the expression of PTEN in esophageal cancer

Heat shock protein90 (HSP90), a molecular chaperone, has provoked great interest as a promising molecular target for cancer treatment, due to its involvement in regulating the conformation, stability and functions of key oncogenic proteins. At present, a variety of chemical compounds targeting HSP90 have been developed and have shown convincing anti-neoplastic activity in various preclinical tumor models. The aim of our study was to evaluate the antitumor effects of a novel HSP90 inhibitor, NVP-AUY922, in esophageal squamous cancer cells (ESCC). Four ESCC cell lines (TE-1, TE-4, TE-8, TE-10) were examined. NVP-AUY922 potently inhibited the proliferation of ESCC, particularly in PTEN-null TE-4cells with a 2-3 times lower IC50 than the other three cell lines. Western blot analysis showed that PTEN-null TE-4cells exhibited higher AKT and ERK activity, which contribute to cell proliferation and survival. NVP-AUY922 significantly suppressed the activity of AKT and ERK in TE-4 but not in PTEN-proficient TE-10cells. Genetic modification experiments demonstrated that the sensitivity to NVP-AUY922 was decreased by exogenous transduction of PTEN in TE-4 and increased by silencing PTEN expression in intact PTEN-expressing TE-10, suggesting that the expression of PTEN may be associated with cell sensitivity in HSP90 inhibition. Furthermore, the enhanced activity of AKT in PTEN-silenced TE-10 was more easily suppressed by NVP-AUY922. Collectively, NVP-AUY922 exhibits a strong antiproliferative effect, revealing its potential as a novel therapeutic alternative to current ESCC treatment. The effect may be improved further by impeding PTEN expression.

The HSP90 Inhibitor NVP-AUY922 Radiosensitizes by Abrogation of Homologous Recombination Resulting in Mitotic Entry with Unresolved DNA Damage

BackgroundHeat shock protein 90 (HSP90) is a molecular chaperone responsible for the conformational maintenance of a number of client proteins that play key roles in cell cycle arrest, DNA damage repair and apoptosis following radiation. HSP90 inhibitors exhibit antitumor activity by modulating the stabilisation and activation of HSP90 client proteins. We sought to evaluate NVP-AUY922, the most potent HSP90 inhibitor yet reported, in preclinical radiosensitization studies.Principal FindingsNVP-AUY922 potently radiosensitized cells in vitro at low nanomolar concentrations with a concurrent depletion of radioresistance-linked client proteins. Radiosensitization by NVP-AUY922 was verified for the first time in vivo in a human head and neck squamous cell carcinoma xenograft model in athymic mice, as measured by delayed tumor growth and increased surrogate end-point survival (p = <0.0001). NVP-AUY922 was shown to ubiquitously inhibit resolution of dsDNA damage repair correlating to delayed Rad51 foci formation in all cell lines tested. Additionally, NVP-AUY922 induced a stalled mitotic phenotype, in a cell line-dependent manner, in HeLa and HN5 cell lines irrespective of radiation exposure. Cell cycle analysis indicated that NVP-AUY922 induced aberrant mitotic entry in all cell lines tested in the presence of radiation-induced DNA damage due to ubiquitous CHK1 depletion, but resultant downstream cell cycle effects were cell line dependent.ConclusionsThese results identify NVP-AUY922 as the most potent HSP90-mediated radiosensitizer yet reported in vitro, and for the first time validate it in a clinically relevant in vivo model. Mechanistic analysis at clinically achievable concentrations demonstrated that radiosensitization is mediated by the combinatorial inhibition of cell growth and survival pathways, ubiquitous delay in Rad51-mediated homologous recombination and CHK1-mediated G2/M arrest, but that the contribution of cell cycle perturbation to radiosensitization may be cell line specific.

Abstract 2771: HSP90 inhibitor NVP-AUY922 exhibits the potent antiproliferative effect in gastrointestinal cancer cells with a lack of PTEN expression

Abstract Background: The heat shock protein 90 (HSP90), a molecular chaperone, has provoked great interest as a promising molecular target for cancer treatment, since it is involved in regulating the conformation, stability and functions of key oncogenic proteins. At present, a variety of chemical compounds targeting HSP90 have been developed and have shown convincing anti-neoplastic activity in various preclinical tumor models. Several inhibitors of HSP90 have been reported exhibiting more antitumor efficacy in PTEN-null tumor xenograft. The aim of our study was to evaluate the antitumor effects of a novel HSP90 inhibitor, NVP-AUY922, in human malignant cells, especially in PTEN-null cancer cells. Materials and Methods: Human esophageal cancer (TE-1, TE-4, TE-8, TE-10), gastric cancer (NUGC-3, NUGC-3/IL), pancreatic cancer (PANC-1, MIAPACA-2, Bxpc3) and mutant c-kit-driven lymphocytes (Ba/F3-KE11, Ba/F3-K642, Ba/F3-820Mz), mimicking gastrointestinal stromal tumor cells, were examined. WST assay and/or trypan blue exclusion assay were conducted to assess the anti-proliferation effects of NVP-AUY922 in those cells mentioned above. The activity of cell growth-related molecules such as AKT, ERK, and PTEN were investigated by western blot analysis. Genetic modification using siRNA and expression vector were applied for silencing and overexpression of PTEN, respectively, in cancer cells. Results: Cell viability assays showed that NVP-AUY922 potently inhibits the proliferation of human cancer cell lines, regardless of 5-FU resistance or c-Kit mutation, by which the sensitivity to imatinib can be affected. PTEN-null TE-4 cells exhibited more sensitivity to NVP-AUY922. Interestingly, The sensitivity to NVP-AUY922 was increased by silencing PTEN expression in intact PTEN-expressing TE-10 cells and decreased by exogenous transduction of PTEN in TE-4 cells. Furthermore, NVP-AUY922 significantly inhibited the activity of AKT and ERK in TE-4 cells, but not in TE-10 cells. Conclusion: Inhibition of Hsp90 function by NVP-AUY922 exhibited strong cytotoxicity against most of the cancer cells, including some anticancer drug-resistant cells, revealing its potential for a novel therapeutic alternative to cancer treatment. In addition, this study suggests that the expression status of PTEN determines the sensitivity to HSP90 inhibitor for cancer treatment. 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 2771. doi:1538-7445.AM2012-2771

Abstract 4749: Insights into the molecular mechanism of HSP90 binding of methoxy-substituted resorcinylic isoxazole amide inhibitors reveal different isoform selectivity profiles

Abstract The molecular chaperone Heat Shock Protein 90 (HSP90) regulates the conformation, stability and activity of several oncogenic client proteins (eg ERBB2, ALK, BRAF, CRAF and AKT). Inhibition of HSP90 results in the degradation of these clients, leading to cell cycle arrest and apoptosis. The resorcinylic isoxazole amide HSP90 inhibitor NVP-AUY922 is currently in Phase I/II clinical trials and other related agents are in development. We previously reported that one or both of the phenolic groups on the resorcinol ring are important for HSP90 inhibitory activity. To further characterise the importance of the phenol groups on protein binding and biological activity, a series of mono- and dimethoxy-substituted resorcinylic 5′-ethyl isoxazole amides were synthesized and compared with the 2′,4′-dihydroxy compound by X-ray crystal structure and biological techniques. The most potent HSP90 inhibitors in terms of binding to the HSP90α target was the 2′,4′-dihydroxy-5′-ethyl isoxazole CCT239215 (Kd=11nM), followed by the 2′-hydroxy-4′-methoxy analogue CCT078722 (Kd=36nM). In contrast, the 2′-methoxy-4′-hydroxy and 2′,4′-dimethoxy derivatives showed no binding to HSP90. X-ray crystal structures showed that for CCT078722, the 4′-methoxy group of the resorcinol ring disrupts the hydrogen bonding that is seen with 2′,4′-dihydroxy CCT239215 and NVP-AUY922, and instead is involved in van der Waals contacts. CCT239215 also showed the greatest cellular growth inhibitory potency (GI50=5-16nM in a panel of human cancer cell lines), followed by CCT078722 (GI50=59-588nM). As predicted, CCT078723 and CCT078721 were inactive in the cells (GI50&amp;gt;10µM). This structure-activity relationship was also maintained with respect to biomarkers of HSP90 target inhibition, as measured by depletion of HSP90 client proteins and induction of HSP72. In HCT116 human colon cells, CCT239215 and CCT078722 caused a loss of HSP90 from immunoprecipitates of the HSP90 co-chaperone, demonstrating dissociation between P23 and HSP90, further confirming their on-target effects. No such dissociation was observed with the inactive analogs. These results explain how the hydroxy group at the 2′-position on the resorcinol ring is crucial for HSP90 inhibitory activity. Interestingly, the 2′-hydroxy-4′-methoxy-5′-ethyl isoxazole CCT078722 exhibited 55-fold less affinity for GRP94 when compared to HSP90α/α, indicating that these compounds show potential for different isoform selectivity profiles. Modelling studies suggest that IIe247 in GRP94 negatively impacts on the binding of CCT078722. Our findings offer insights into the molecular mechanism of HSP90 binding by resorcinylic isoxazole inhibitors, provide active/inactive probe compounds for mechanistic studies and suggest potential approaches to isoform selectivity. 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 4749. doi:1538-7445.AM2012-4749

HSP90 Inhibition Targets JAK2 and Is Highly Effective in CRLF2-Rearranged Acute Lymphoblastic Leukemia

Abstract 576Enzymatic inhibitors of Janus kinase 2 (JAK2) are in clinical development for the treatment of B-cell acute lymphoblastic leukemia (B-ALL) with rearrangements of the cytokine receptor subunit CRLF2, myeloproliferative neoplasms, and other tumors with constitutive JAK2 signaling. In addition, JAK2 is a client of heat shock protein 90 (HSP90) and HSP90 inhibition results in JAK2 degradation. To explore the utility of blocking JAK2 in CRLF2-rearranged B-ALL, we exposed the MHH-CALL4 and MUTZ-5 cell lines, which both have CRLF2/IGH rearrangements and activating JAK2 mutations to a panel of JAK2 inhibitors (JAK inhibitor-1, INCB18424, tofacitinib, NVP-BSK805, NVP-BVB808, TG101348) and the HSP90 inhibitor NVP-AUY922. Both MUTZ-5 and MHH-CALL4 were highly sensitive to AUY922 (GI50, 25–26 nM), with 50- to >1,000-fold superior potency compared with the panel of JAK2 enzymatic inhibitors. AUY922 and the structurally divergent HSP90 inhibitors 17-AAG, PU-H71 and HSP990 were all potently active against a panel of Ba/F3 lines dependent on CRLF2 and JAK2 signaling (GI50, 1–11 nM). Treatment of MUTZ-5 and MHH-CALL4 cells with JAK inhibitor-1 reduced but did not eliminate phospho (P-) STAT5 and P-ERK1/2 in both lines but promoted increases in P-AKT in MUTZ-5 and P-JAK2 in MHH-CALL4. In contrast, AUY922 treatment more extensively reduced or eliminated phosphorylation of all the targets. The combination of AUY922+JAK inhibitor-1 had little or no additional effect on target phosphorylation compared with AUY922 alone and pairwise dose-response studies with isobologram analysis failed to identify synergistic effects. We performed transcriptional profiling on MUTZ-5 and MHH-CALL cells treated with either vehicle (DMSO), JAK inhibitor-1, AUY922 or JAK inhibitor-1+AUY922. Unsupervised hierarchical clustering distinguished samples treated with AUY922 (or combination) from those treated with JAK inhibitor-1 or vehicle. To formally assess whether AUY922 targets the same genes as JAK inhibitor-1, we defined a ‘JAK inhibitor signature’ from the top/bottom 250 most differentially expressed genes following treatment with JAK inhibitor-1. Using gene set enrichment analysis (GSEA), the ‘JAK inhibitor signature’ was highly enriched upon treatment with AUY922 (p=0.003). GSEA also demonstrated that STAT5A signatures were enriched upon treatment with JAK inhibitor-1, AUY922, or JAK inhibitor-1+AUY922. To identify additional targets of HSP90 inhibition beyond the inhibition of JAK2, we used the C3 database from the MsigDB compendium to perform a transcription factor binding site enrichment analysis on the most differentially expressed genes between JAK inhibitor-1 and AUY922. The top 5 hits were all heat-shock factors (HSF, FDR<0.05). GSEA revealed that an HSF1 signature was only enriched upon treatment with AUY922 or AUY922+JAKinh-1, but not after JAKinh-1 alone. To extend our findings to in vivo treatment of human B-ALL, we established primary B-ALL xenografts from CRLF2-rearranged, patient-derived bone marrow samples in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. To stringently assay established disease in vivo, we waited until bone marrow leukemia burden exceeded 30% and then initiated treatment with BVB808 50mg/kg twice daily, AUY922 50mg/kg thrice weekly, BVB808+AUY922 or vehicle. After 5 days of treatment, spleens from mice treated with vehicle or BVB808 had nearly complete effacement by B-ALL, while AUY922 or BVB808+AUY922 treatment resulted in visible islands of hematopoiesis. Based on immunohistochemistry, mice receiving AUY922 or BVB808+AUY922, but not BVB808 or vehicle, had nearly complete loss of P-STAT5 as well as upregulation of the pharmacodynamic marker HSP70. Immunoblotting of spleens from treated mice demonstrated reductions in P-STAT5, P-JAK2, and total JAK2 in AUY922- or BVB808+AUY922-treated mice. In contrast, treatment with single-agent BVB808 only modestly suppressed P-STAT5. Treatment with either BVB808 or AUY922 prolonged overall survival compared to vehicle (p=0.01 for both xenografts). Treatment with AUY922 further prolonged overall survival compared to BVB808 (p<0.01 for both xenografts), while the combination of BVB808 and AUY922 had no additional benefit compared to AUY922 alone. In conclusion, HSP90 is a promising therapeutic target in CRLF2-rearranged B-ALL and merits clinical evaluation. Disclosures:Romanet:Novartis Pharma AG: Employment. Murakami:Novartis Pharma AG: Employment. Sallan:Enzon Pharmaceuticals: Honoraria. Kung:Novartis Pharmaceuticals: Consultancy, Research Funding. Radimerski:Novartis Pharma AG: Employment. Weinstock:Novartis: Consultancy, Research Funding.

Mechanistic Evaluation of the Novel HSP90 Inhibitor NVP-AUY922 in Adult and Pediatric Glioblastoma

The dismal prognosis of glioblastoma (GB) indicates the urgent need for new therapies for these tumors. Heat shock protein 90 (HSP90) inhibitors induce the proteasome-mediated degradation of many oncogenic client proteins involved in all of the hallmark characteristics of cancer. Here, we explored the mechanistic potential of the potent synthetic diarylisoxazole amide resorcinol HSP90 inhibitor, NVP-AUY922, in adult and pediatric GB. In vitro antiproliferative potency (nanomolar range) was seen in both adult and pediatric human GB cell lines with different molecular pathologies. A cytostatic effect was observed in all GB lines; more apoptosis was observed at lower concentrations in the SF188 pediatric GB line and at 144 hours in the slower growing KNS42 pediatric GB line, as compared with the adult GB lines U87MG and SF268. In vitro combination studies with inhibitors of phosphoinositide 3-kinase/mammalian target of rapamycin (PI-103) or mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (PD-0325901) supported the hypothesis that sustained inhibition of ERK up to 72 hours and at least temporary inhibition of AKT were necessary to induce apoptosis in GB lines. In athymic mice bearing established s.c U87MG GB xenografts, NVP-AUY922 (50 mg/kg i.p x 3 days) caused the inhibition of ERK1/2 and AKT phosphorylation and induced apoptosis, whereas 17-AAG used at maximum tolerated dose was less effective. NVP-AUY922 antitumor activity with objective tumor regression resulted from antiproliferative, proapoptotic, and antiangiogenic effects, the latter shown by decreased microvessel density and HIF1alpha levels. Our results have established a mechanistic proof of concept for the potential of novel synthetic HSP90 inhibitors in adult and pediatric GB, alone or in combination with phosphoinositide 3-kinase/mammalian target of rapamycin and mitogen-activated protein/ERK kinase inhibitors.

89Zr-Bevacizumab PET of Early Antiangiogenic Tumor Response to Treatment with HSP90 Inhibitor NVP-AUY922

Angiogenesis is a critical step in tumor development, in which vascular endothelial growth factor (VEGF) is a key growth aspect. Heat shock protein 90 (HSP90), a molecular chaperone, is essential for the activity of key proteins involved in VEGF transcription. Currently, no biomarkers to predict the effect of, or monitor, HSP90 inhibition therapy in individual patients exist. (89)Zr-bevacizumab PET provides a noninvasive tool to monitor tumor VEGF levels. The aim of this study was to investigate (89)Zr-bevacizumab PET for early antiangiogenic tumor response evaluation of treatment with the new HSP90 inhibitor NVP-AUY922. In xenografts of A2780 and its cisplatin-resistant CP70 human ovarian cancer subline, (89)Zr-bevacizumab small-animal PET was performed before and after NVP-AUY922 treatment and verified with histologic response and ex vivo tumor VEGF levels. Compared with pretreatment values, 2 wk of NVP-AUY922 treatment decreased (89)Zr-bevacizumab uptake by 44.4% (P = 0.0003) in A2780 xenografts, whereas tumor uptake was not affected in CP70 xenografts. The same pattern was observed in A2780 and CP70 tumor VEGF levels, measured with enzyme-linked immunosorbent assay, and mean vessel density after NVP-AUY922 treatment. These findings coincided with reduction in the proliferation rate, assessed by Ki67 staining, in A2780 tumor tissue only. (89)Zr-bevacizumab PET was in line with the antiangiogenic response and direct antitumor effects after NVP-AUY922 treatment, supporting the specificity of (89)Zr-bevacizumab PET as a sensitive technique to monitor the antiangiogenic response of HSP90 inhibition in vivo.

Synergistic action of the novel HSP90 inhibitor NVP‐AUY922 with histone deacetylase inhibitors, melphalan, or doxorubicin in multiple myeloma

Heat shock protein 90 (HSP90) is a promising target for tumor therapy. The novel HSP90 inhibitor NVP-AUY922 has preclinical activity in multiple myeloma, however, little is known about effective combination partners to design clinical studies. Multiple myeloma cell lines, OPM-2, RPMI-8226, U-266, LP-1, MM1.S, and primary myeloma cells were exposed to NVP-AUY922 and one of the combination partners histone deacetylase inhibitor NVP-LBH589, suberoylanilide hydroxamic acid (SAHA), melphalan, or doxorubicin, either simultaneously or in sequential patterns. Effects on cell proliferation and apoptosis were determined. Synergistic effects were evaluated using the method of Chou and Talalay. Combined sequential incubation with NVP-AUY922 and SAHA showed that best synergistic effects were achieved with 24 h preincubation with SAHA followed by another 48 h of combination treatment. Combination of NVP-AUY922 with SAHA, NVP-LBH589, melphalan, or doxorubicin resulted in synergistic inhibition of viability, with strong synergy (combination index < 0.3) in the case of melphalan. Importantly, resistance of the RPMI-8226 cell line and relative resistance of some primary myeloma cells against NVP-AUY922 could be overcome by combination treatment. These data show impressive synergistic action of the novel HSP90 inhibitor NVP-AUY922 with melphalan, doxorubicin, NVP-LBH589, and SAHA in multiple myeloma and build the frame work for clinical trials.