Combination Of 17-AAG Research Articles

Smart Nanofiber Mesh with Locally Sustained Drug Release Enabled Synergistic Combination Therapy for Glioblastoma.

This study aims to propose a new treatment model for glioblastoma (GBM). The combination of chemotherapy, molecular targeted therapy and radiotherapy has been achieved in a highly simultaneous manner through the application of a safe, non-toxic, locally sustained drug-releasing composite Nanofiber mesh (NFM). The NFM consisted of biodegradable poly(ε-caprolactone) with temozolomide (TMZ) and 17-allylamino-17-demethoxygeldanamycin (17AAG), which was used in radiation treatment. TMZ and 17AAG combination showed a synergistic cytotoxicity effect in the T98G cell model. TMZ and 17AAG induced a radiation-sensitization effect, respectively. The NFM containing 17AAG or TMZ, known as 17AAG-NFM and TMZ-NFM, enabled cumulative drug release of 34.1% and 39.7% within 35 days. Moreover, 17AAG+TMZ-NFM containing both drugs revealed a synergistic effect in relation to the NFM of a single agent. When combined with radiation, 17AAG+TMZ-NFM induced in an extremely powerful cytotoxic effect. These results confirmed the application of NFM can simultaneously allow multiple treatments to T98G cells. Each modality achieved a significant synergistic effect with the other, leading to a cascading amplification of the therapeutic effect. Due to the superior advantage of sustained drug release over a long period of time, NFM has the promise of clinically addressing the challenge of high recurrence of GBM post-operatively.

Lamin-A interacting protein Hsp90 is required for DNA damage repair and chemoresistance of ovarian cancer cells

Ovarian cancer is the most malignant gynecologic cancer. Previous studies found that lamin-A was associated with DNA damage repair proteins but the underlying mechanism remains unclear. We speculate that this may be related to its interacting proteins, such as Hsp90. The aim of this study is to investigate the effects of Hsp90 on DNA damage repair and chemoresistance of ovarian cancer cells. In our research, co-immunoprecipitation (co-IP) and mass spectrometry (MS) were used to identify proteins interacting with lamin-A and the interaction domain. Next, the relationship between lamin-A and Hsp90 was explored by Western blotting (WB) and immunofluorescence staining. Then, effect of Hsp90 inhibition on DNA damage repair was assessed through detecting Rad50 and Ku80 by WB. Furthermore, to test the roles of 17-AAG on cell chemosensitivity, CCK-8 and colony formation assay were carried out. Meanwhile, IC50 of cells were calculated, followed by immunofluorescence to detect DNA damage. At last, the mouse xenograft model was used in determining the capacity of 17-AAG and DDP to suppress tumor growth and metastatic potential. The results showed that lamin-A could interact with Hsp90 via the domain of lamin-A1-430. Besides, the distribution of Hsp90 could be affected by lamin-A. After lamin-A knockdown, Hsp90 decreased in the cytoplasm and increased in the nucleus, suggesting that the interaction between lamin-A and Hsp90 may be related to the nucleocytoplasmic transport of Hsp90. Moreover, inhibition of Hsp90 led to an obvious decrease in the expression of DSBs (DNA double-strand break) repair proteins, as well as cell proliferation ability upon DDP treatment and IC50 of DDP, causing more serious DNA damage. In addition, the combination of 17-AAG and DDP restrained the growth of ovarian cancer efficiently in vivo and prolonged the survival time of tumor-bearing mice.

Effect of HSP90AB1 and CC domain interaction on Bcr-Abl protein cytoplasm localization and function in chronic myeloid leukemia cells

BackgroundThe fusion oncoprotein Bcr-Abl is mostly located in the cytoplasm, which causes chronic myeloid leukemia (CML). After moving into the nucleus, the fusion protein can induce apoptosis of CML cells. The coiled-coil domain (CC domain) of Bcr-Abl protein plays a central role in the subcellular localization. However, how CC domain affects subcellular localization of Bcr-Abl remains unclear.MethodsHerein, the key proteins interacting with the Bcr-Abl CC domain were screened by immunoprecipitation binding mass spectrometry. The specific site of Bcr-Abl CC domain binding to target protein was predicted by Deep Viewer. Immunoprecipitation assay was used to confirmed the specific sites of protein binding. IF and western blot were used to observe the subcellular localization of target protein. Western blot was used to examine the protein changes. CCK-8, clonal formation test and FCM cycle detection were used to observe the effect of inhibitor on the proliferation ability of CML cells. FCM apoptosis detection was used to observe the level of cells apoptosis.ResultsHSP90AB1 interacts with Bcr-Abl CC domain via N-terminal domain (NTD), preventing the transport of Bcr-Abl protein to the nucleus and maintaining the activation of Bcr-Abl tyrosine kinase. The nucleus-entrapped Bcr-Abl markedly inhibits the proliferation and induces apoptosis of CML cells by activating p73 and repressing the expression of cytoplasmic oncogenic signaling pathways mediated by Bcr-Abl. Moreover, the combination of 17AAG (Tanespimycin) with Leptomycin B (LMB) considerably decreased the proliferation of CML cells.ConclusionOur study provides evidence that it is feasible to transport Bcr-Abl into the nucleus as an alternative strategy for the treatment of CML, and targeting the NTD of HSP90AB1 to inhibit the interaction with Bcr-Abl is more accurate for the development and application of HSP90 inhibitor in the treatment of CML and other Bcr-Abl-addicted malignancies.F7yTRqYkfkXw_k-2eiVFmgVideo abstract

Combination of HSP90 and autophagy inhibitors promotes hepatocellular carcinoma apoptosis following incomplete thermal ablation.

The present study evaluated the effect of combining inhibitors (17-AAG) of heat shock protein 90 (HSP90) and autophagy (3-MA) on apoptosis using an incomplete thermal ablation animal model. A total of 28 orthotopic mice with hepatocellular carcinoma were randomly divided into 4 groups to receive different drug interventions. Following palliative laser ablation, changes in autophagy, apoptosis and Akt/mTOR expression levels were assessed in tumors. Compared with the controls, the 17-AAG-treated mice exhibited significantly decreased expression levels of phosphorylated (p)-Akt and p-mTOR with enhanced autophagy and apoptosis; no marked increases in the expression levels of p-Akt and p-mTOR were observed in the 3-MA-treated mice, with no significant changes in autophagy; however, apoptosis was enhanced. No significant decreases in p-Akt and p-mTOR or any increase in autophagy were observed in the mice receiving a combination of 17-AAG and 3-MA, but they did exhibit a marked increase in apoptosis. Compared with 17-AAG alone, the combination of 17-AAG and 3-MA resulted in a marked increase in apoptosis without enhanced autophagy. In the incomplete ablation model, the effects of autophagy and apoptosis are antagonistic. The combined use of 17-AAG and 3-MA can significantly promote apoptosis and is worthy of further study.

A molecular basis for the synergy between 17‑allylamino‑17‑demethoxy geldanamycin with Capecitabine and Irinotecan in human colorectal cancer cells through VEFG and MMP-9 gene expression

Anti-proliferative, anti-metastatic and anti-angiogenic effects of 17‑allylamino‑17‑demethoxy geldanamycin (17-AAG) were studied alone and in combination with Capecitabine (Cap) and/or Irinotecan (IR) on HT-29 human colorectal carcinoma cells. Expression of MMP-9 (matrix metalloproteinase‑9) and VEGF (vascular endothelial growth factor) mRNA was analyzed by real-time PCR method. The study was further followed by wound scratch assay for migration assessment. Nitric oxide content, Malondialdehyde generation and total anti-oxidant capacity were also assessed. Results showed significant differences between mono- and double therapy (p < 0.05). Combination of 17-AAG with IR or Cap resulted in synergistic effect (Combination Index < 1). Among double combination groups only Cap/17-AAG showed significant differences in MMP-9 and VEGF genes expression and wound healing assay. Moreover, a significant decrease of wound area in our triple combination group was obtained, indicating the antagonistic effect. IR/17-AAG and IR/Cap double combination groups resulted in down-regulation of MMP-9 and VEGF mRNA expression, respectively. Significant generation of MDA and decrease in TAC values have been observed in all our tested groups, however, the IR/17-AAG combination was the only group that could elevate NO concentration, significantly. Our findings demonstrated potent anti-angiogenesis and anti-metastatic effects for 17-AAG when it is provided in double combination especially with Cap, suggesting a new protocol in colorectal cancer combination therapy. These findings may indicate that down-regulation of VEGF and MMP-9 genes is directly related to angiogenesis and metastasis.

17-AAG inhibits vemurafenib-associated MAP kinase activation and is synergistic with cellular immunotherapy in a murine melanoma model.

Heat shock protein 90 (HSP90) is a molecular chaperone which stabilizes client proteins with important roles in tumor growth. 17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of HSP90 ATPase activity, occupies the ATP binding site of HSP90 causing a conformational change which destabilizes client proteins and directs them towards proteosomal degradation. Malignant melanomas have active RAF-MEK-ERK signaling which can occur either through an activating mutation in BRAF (BRAFV600E) or through activation of signal transduction upstream of BRAF. Prior work showed that 17-AAG inhibits cell growth in BRAFV600E and BRAF wildtype (BRAFWT) melanomas, although there were conflicting reports about the dependence of BRAFV600E and BRAFWT upon HSP90 activity for stability. Here, we demonstrate that BRAFWT and CRAF are bound by HSP90 in BRAFWT, NRAS mutant melanoma cells. HSP90 inhibition by 17-AAG inhibits ERK signaling and cell growth by destabilizing CRAF but not BRAFWT in the majority of NRAS mutant melanoma cells. The highly-selective BRAFV600E inhibitor, PLX4032 (vemurafenib), inhibits ERK signaling and cell growth in mutant BRAF melanoma cells, but paradoxically enhances signaling in cells with wild-type BRAF. In our study, we examined whether 17-AAG could inhibit PLX4032-enhanced ERK signaling in BRAFWT melanoma cells. As expected, PLX4032 alone enhanced ERK signaling in the BRAFWT melanoma cell lines Mel-Juso, SK-Mel-2, and SK-Mel-30, and inhibited signaling and cell growth in BRAFV600E A375 cells. However, HSP90 inhibition by 17-AAG inhibited PLX4032-enhanced ERK signaling and inhibited cell growth by destabilizing CRAF. Surprisingly, 17-AAG also stimulated melanin production in SK-Mel-30 cells and enhanced TYRP1 and DCT expression without stimulating TYR production in all three BRAFWT cell lines studied as well as in B16F10 mouse melanoma cells. In vivo, the combination of 17-AAG and cellular immunotherapy directed against Tyrp1 enhanced the inhibition of tumor growth compared to either therapy alone. Our studies support a role for 17-AAG and HSP90 inhibition in enhancing cellular immunotherapy for melanoma.

Lectin, Galactoside-Binding Soluble 3 Binding Protein Promotes 17-N-Allylamino-17-demethoxygeldanamycin Resistance through PI3K/Akt Pathway in Lung Cancer Cell Line.

Heat shock protein 90 (HSP90) stabilizing oncoproteins has been an attractive target in cancer therapy. 17-N-Allylamino-17-demethoxygeldanamycin (17-AAG), an HSP90 inhibitor, was tested in phase II/III clinical trials, but due to lack of efficacy, clinical evaluation of 17-AAG has achieved limited success, which led to resistance to 17-AAG. However, the mechanism of 17-AAG resistance has not clearly been identified. Here, we identified LGALS3BP (Lectin, galactoside-binding soluble 3 binding protein), a secretory glycoprotein, as a 17-AAG resistance factor. In the clinical reports, it was suggested that LGALS3BP was associated with low survival rate, development of cancer progression, and enhancement of metastasis in human cancers. As we confirmed that the LGALS3BP level was increased in 17-AAG-resistant cells (H1299_17R) compared with that of the parental cell line (H1299_17P), knockdown of LGALS3BP expression increased sensitivity to 17-AAG in H1299_17R cells. Overexpression of LGALS3BP also augmented PI3K/Akt and ERK signaling pathways. Furthermore, we determined that the PI3K/Akt signaling pathway was involved in LGALS3BP-mediated 17-AAG resistance in vitro and in vivo, demonstrating that LGALS3BP mediates the resistance against 17-AAG through PI3K/Akt activation rather than ERK activation. These findings suggest that LGALS3BP would be a target to overcome resistance to 17-AAG in lung cancer. For example, the combination of 17-AAG and PI3K/Akt inhibitor would effectively suppress acquired resistance to 17-AAG. In conclusion, targeting of LGALS3BP-mediated-specific survival signaling pathway in resistant cells may provide a novel therapeutic model for the cancer therapy. Mol Cancer Ther; 16(7); 1355-65. ©2017 AACR.

HSP90 inhibitor enhances anti-proliferative and apoptotic effects of celecoxib on HT-29 colorectal cancer cells via increasing BAX/BCL-2 ratio.

Due to the high prevalence and mortality rate of colorectal cancer (CRC), new treatment approaches like combination therapy seem to be necessary. The relationship between chronic inflammation and colorectal cancer development and progression has been shown to be important. Celecoxib, a selective COX-2 inhibitor, is the only non-steroidal anti-inflammatory drug (NSAID) that has been approved for cancer therapy and prevention. Because of cardiovascular side effects of COX-2 inhibitors, combination therapy may improve the therapeutic profile. 17-Demethoxy-17-allylamino geldanamycin (17-AAG), a heat shock protein 90 (HSP90) inhibitor, shows anti-inflammatory effects via down-regulation of the key mediators of inflammation such as Nuclear Factor κB (NF-kB), JAK/Signal Transducer and Activator of Transcription (JAK/STAT). Thus, we studied the effect(s) of combination of 17-AAG and celecoxib on HT-29 cells viability and apoptosis induction. Based on MTT results, we showed an increase in the inhibitory effect of celecoxib when combined with 17-AAG, especially at low a concentration of celecoxib. Flow cytometry analysis demonstrated that apoptosis induction is probably the mechanism of additive inhibitory effects of 17-AAG and celecoxib combination. To explore the possible mechanism of apoptosis induction by 17-AAG and celecoxib combination, levels of BAX and BCL-2 proteins were determined by western blotting. The BAX/BCL-2 ratio in the combination group was increased compared to 17-AAG or celecoxib alone, mainly via decreasing BCL-2 levels. In conclusion, 17-AAG, increased inhibitory effects of celecoxib on HT-29 cells, probably by induction of apoptosis.

17-Allylamino-17-Demethoxygeldanamycin and the Enhancement of PS-341-Induced Lung Cancer Cell Death by Blocking the NF-κB and PI3K/Akt Pathways.

PS-341 is a highly selective and potent proteasome inhibitor that is cytotoxic to various types of cancer. However, no objective response was seen in a clinical trial with PS-341 as a single agent in non-small cell lung cancer. Its antitumor activity is limited by the simultaneously activated antiapoptosis pathway. Recently, PS-341-induced NF-κB activation via IκBα degradation has been suggested to be one of its antiapoptotic effects. In this study, we investigated the effects of a combined application of the heat shock protein (Hsp) 90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) with PS-341 in lung cancer cells. Hsp90 inhibition with 17-AAG was effective in enhancing PS-341-induced lung cancer cell death in vitro and in vivo. 17-AAG pretreatment induced the degradation of upstream regulators of IκB, IL-1R-associated kinase-1 (IRAK-1), and IκB kinases (IKKs), dose and time dependently, which resulted in blocking of PS-341-induced IκBα degradation, p65 nuclear translocation, transcriptional activity, and NF-κB-regulated antiapoptotic gene expressions such as COX-2. The concentrations of 17-AAG used for combinatorial treatment with PS-341 did not change cell viability or the activity of proteasome complex. Moreover, 17-AAG pretreatment decreased the level of phsophorylated Akt at serine 473 residue and suppressed active Akt-dependent inactivation of glycogen synthase kinase 3β. 17-AAG mediated the dissociation of its client proteins (IRAK-1, IKKs, and Akt) from the Hsp90 complex. As a result, it induced degradation of target proteins. Our results suggest that the combination of 17-AAG and PS-341 could be an effective anticancer therapy that overcomes the limited effects of PS-341.

Abstract LB-014: Targeting p53 aggregation in ovarian cancer chemoresistant cells

Abstract Background: About half of patients diagnosed with ovarian cancer develop chemoresistance and succumb to the disease. The underlying mechanisms that lead to the development of chemoresistance are poorly understood. We previously demonstrated that p53 protein aggregation inhibited p53 pro-apoptotic activities consequently leading to platinum resistance in ovarian cancer cells with cancer stem cell properties. Since heat shock protein 90 (HSP90), a molecular chaperone, can sustain the accumulation of protein aggregates, the purpose of this study is to determine if HSP90 inhibitors can inhibit the accumulation of p53 aggregates, reactivate p53 pro-apoptotic function, and sensitize ovarian cancer stem cells (OCSCs) to carboplatin. Method: Chemoresistant CD44+/MyD88+ OCSCs were treated with the HSP90 inhibitor, 17-AAG, carboplatin, or the combination of 17-AAG and carboplatin. Cell viability was monitored by IncuCyte ZOOM live-cell imaging system. Caspase activation was determined by Caspase-Glo assay. p53 aggregation was detected by non-denaturing gel and western blot. The interaction between p53 and HSP90 proteins was determined by co-immunoprecipitation (co-IP). Chromatin immunoprecipitation (CHIP) of p53 and RT-QPCR of p53 targets (PUMA, BAX, et al.) were performed to demonstrate p53 transcriptional activation. Result: OCSCs were resistant to single treatment with Carboplatin. In line with results from our previous studies, carboplatin neither decreased cell viability nor induced caspase activation in these cells. Interestingly, carboplatin enhanced the levels of aggregated p53 and hence failed to upregulate the expression of p53-related pro-apoptotic genes. Co-IP results demonstrated that 17-AAG blocked the interaction between p53 and HSP90. More importantly, 17-AAG was able to sensitize OCSCs to carboplatin. The combination therapy effectively induced the transcriptional activity of p53, upregulated pro-apoptotic genes, and stimulated caspase activity and cell death in the carboplatin-resistant OCSCs. Conclusion: The HSP90 inhibitor, 17-AAG can inhibit the formation of p53 aggregates by blocking the interaction between p53 and HSP90. By releasing p53 from the aggregates, 17-AAG reactivates the ability of p53 to bind to DNA and to upregulate the expression of target genes, which leads to the apoptosis of OCSCs. 17-AAG sensitizes chemoresistant OCSCs to carboplatin treatment. Using HSP90 inhibitors to target p53 aggregation and sensitize chemoresistant cells with protein aggregates may significantly improve the response of ovarian cancer to conventional chemotherapies. Citation Format: Yang Yang-Hartwich, Carlos Cardenas, Mary Pitruzzello, Eydis Lima, Ayesha B. Alvero, Gil Mor. Targeting p53 aggregation in ovarian cancer chemoresistant cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-014. doi:10.1158/1538-7445.AM2015-LB-014

Dual targeting of heat shock proteins 90 and 70 promotes cell death and enhances the anticancer effect of chemotherapeutic agents in bladder cancer

Heat shock proteins (HSPs), which are molecular chaperones that stabilize numerous vital proteins, may be attractive targets for cancer therapy. The aim of the present study was to investigate the possible anticancer effect of single or dual targeting of HSP90 and HSP70 and the combination treatment with HSP inhibitors and chemotherapeutic agents in bladder cancer cells. The expression of HSP90 and the anticancer effect of the HSP90 inhibitor 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) coupled with cisplatin, docetaxel, or gemcitabine were examined using immunohistochemistry, quantitative real-time PCR, cell growth, flow cytometry, immunoblots and caspase-3/7 assays. The expression of HSP70 under HSP90 inhibition and the additive effect of HSP70 inhibitor pifithrin-μ (PFT-μ) were examined by the same assays and transmission electron microscopy. HSP90 was highly expressed in bladder cancer tissues and cell lines. 17-AAG enhanced the antiproliferative and apoptotic effects of each chemotherapeutic agent. 17-AAG also suppressed Akt activity but induced the upregulation of HSP70. PFT-μ enhanced the effect of 17-AAG or chemotherapeutic agents; the triple combination of 17-AAG, PFT-μ and a chemotherapeutic agent showed the most significant anticancer effect on the T24 cell line. The combination of 17-AAG and PFT-μ markedly suppressed Akt and Bad activities. With HSP90 suppression, HSP70 overexpression possibly contributes to the avoidance of cell death and HSP70 may be a key molecule for overcoming resistance to the HSP90 inhibitor. The dual targeting of these two chaperones and the combination with conventional anticancer drugs could be a promising therapeutic option for patients with advanced bladder cancer.

Abstract 3339: HSP90 inhibitor 17-allylamino-geldanamycin enhances sensitivity to double-strand DNA break-inducing agents (platinum and PARP inhibitors) in epithelial ovarian cancer.

Abstract Double-strand DNA break (DSB)-inducing agents such as platinum analogues and poly (ADP-ribose) polymerase inhibitors (PARPis) exhibit significant antitumor activity in epithelial ovarian cancer (EOC). Approximately 50% of patients with EOC harbor genetic or epigenetic alterations of the homologous recombination (HR) DNA repair pathway and are highly responsive to these agents. However, resistance to DSB-inducing agents develops in the majority of patients and represents a major limitation for successful treatment of EOC. We recently defined a gene expression profile of “BRCAness” that correlates with responsiveness to double-strand DNA break-inducing agents (platinum and PARPis) in EOC. We applied this profile to the Connectivity Map to identify candidate compounds that might be able to enhance sensitivity to these agents. This analysis showed that the top-performing compound was the heat shock protein 90 (HSP90) inhibitor 17-allylamino-geldanamycin (17-AAG) with very high connectivity scores (&amp;gt;0.8) across several cell lines and concentrations (permutation p value &amp;lt; 0.00001). We then evaluated the ability of 17-AAG to enhance sensitivity to double-strand DNA break-inducing agents in a panel of ovarian cancer cell lines. Exposure to increasing concentrations of 17-AAG (0.02-0.1uM) was associated with increased sensitivity of 36M2 and OVCAR5 cells to carboplatin in a dose response manner. Similarly, exposure to increasing concentrations of 17-AAG (0.02-0.1uM) was associated with increased sensitivity of 36M2 and OVCAR5 cells to PARPi olaparib in a dose response manner. Exposure of these cell lines to combination of 17-AAG with carboplatin induced increased phosphorylation of H2AX (gamma H2AX) - a surrogate of DNA double-strand breaks - compared to carboplatin alone, as assessed by Western blotting. In conclusion, the HSP90 inhibitor 17-AAG enhances sensitivity of ovarian cancer cell lines to carboplatin and olaparib. The mechanism of this previously unknown, off-target effect, seems to be due to increasing DNA double strand breaks. Citation Format: Chiara Battelli, Alexander Morse, Hai Hu, Elena Levantini, Gerburg Wulf, Panagiotis A. Konstantinopoulos. HSP90 inhibitor 17-allylamino-geldanamycin enhances sensitivity to double-strand DNA break-inducing agents (platinum and PARP inhibitors) in epithelial ovarian cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3339. doi:10.1158/1538-7445.AM2013-3339

Abstract 2057: In vitro effect of AZD1208 (Pim Kinase Inhibitor) and 17-AAG (HSP90 Inhibitor) combination in CLL.

Abstract Heat shock protein 90 (HSP90) is a molecular chaperone that regulates the functional stability of client oncoproteins, which is driven by the binding and hydrolysis of ATP. The geldanamycin analog, 17-AAG, binds to the ATP pocket of HSP90 leading to the degradation of client proteins such as Akt, p53, STAT3, Bcr-Abl, and Pim-1. Pim (Pim-1, -2, -3) kinases are constitutively active serine/threonine kinases overexpressed in hematological malignancies such as chronic lymphocytic leukemia (CLL). Pim kinases have several substrates which are involved in gene transcription, protein translation, cell proliferation, and apoptosis. AZD1208 is a small molecule Pim kinase inhibitor currently in clinical trials. The PI3K-dependent activation of AKT (a client protein of HSP90) and the JAK/STAT-dependent induction of PIM are partially overlapping kinase pathways. Therefore, we hypothesized that the mechanism-based combination of 17-AAG (to decrease AKT levels) with AZD1208 (to inhibit Pim kinases) will result in an increase in cytotoxicity in CLL cells. An AZD1208 dose-response experiment was performed in 4 CLL patient samples to determine the optimal cytotoxic concentrations. AZD1208 concentrations of 3 μM and 10 μM were selected to treat 22 additional CLL patient samples for 24 hours. Cell death ranged from 0-30% and 2-45%, respectively, as determined by Annexin-V/PI staining after subtraction of endogenous cell death. All CLL patient samples were sensitive to AZD1208 regardless of their prognostic markers such as IgVH mutation and ZAP70 positivity or cytogenetics such as 17p del (p53 locus) or 11q del (ATM locus). Similarly, prior treatment history or Rai stage did not influence the biological response of AZD1208 in CLL cells. Based on published data, 0.5 μM and 2 μM 17-AAG were selected for the combination treatments with AZD1208 for 24 and 48 hours. Cytotoxicity for these combinations was assessed in 13 CLL blood samples, resulting in additive (n=8) or more than additive (n=3) cell death when using the fractional two-drug combination analysis. Immunoblot analysis was performed in 3 CLL samples probing for HSP90, HSP70, Akt, Pim-1, Mcl-1, Bcl-2, Phospho-4E-BP1 (Thr37/46), 4E-BP1, and β-actin. In agreement with previous work, 17-AAG treatment resulted in an increase of HSP90 and HSP70 protein levels while Akt levels were markedly decreased. In these preliminary immunoblots, Pim-1 and Bcl-2 levels remained unchanged; however, Mcl-1 protein levels decreased in the combination treatments at 24 and 48 hours. Levels for total and phospho-4E-BP1 also declined. Taken together, these preliminary data indicate that AZD1208 (at 3 and 10 μM) has a modest cytotoxic effect in CLL cells. However, when combined with 17-AAG for 24 and 48 hours there was an additive or more than additive effect in majority of CLL samples, with the possible mechanisms of action being through the Akt and 4E-BP1 pathways. Citation Format: Fabiola Gomez, Lisa Chen, William Wierda, Varsha Gandhi. In vitro effect of AZD1208 (Pim Kinase Inhibitor) and 17-AAG (HSP90 Inhibitor) combination in CLL. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2057. doi:10.1158/1538-7445.AM2013-2057

Abstract 2933: HSP90 inhibiting anti-cancer therapeutics enhance bone loss by increasing osteoclast formation: Mechanism of action.

Abstract Breast cancer cells metastasize to bone where they cause bone destruction. Here, cancer cells promote bone loss through the recruitment of osteoclasts, specialised macrophage-like cells that resorb bone. Bone resorption releases bone matrix factors that increase tumor growth, resulting in vicious cycle of bone resorption and tumor progression. Previously we found 17-AAG, an Hsp90 inhibitor and anti-cancer therapeutic significantly increases MDA-MB-231 breast cancer cell growth in bone following intracardiac inoculation in nude mice. 17-AAG also increases osteoclast formation in vitro and in vivo even though Hsp90 client proteins are important in osteoclast formation. In addition to degradation of Hsp90 client proteins, inhibition by 17-AAG also causes cell stress responses through the activation of Heat Shock Factor 1 (HSF1). Therefore we sought to characterize 17-AAG and new HSP90 inhibitors, to examine the role of HSF1 in their effects and to determine the molecular mechanism of 17-AAG. 17-AAG and new, but structurally unrelated, Hsp90 blockers CCT018159 and NVP-AUY922 dose-dependently enhanced osteoclast formation from mouse bone marrow and RAW264.7 cells. Ablating HSF1 action by shRNA transfection and by pharmacological inhibition using KNK437 inhibited 17-AAG effects upon osteoclast formation. Osteoclast formation from HSF1 knockout mouse bone marrow were unresponsive to 17-AAG, but wild type cells did respond. To determine the mechanism of how 17-AAG is increasing osteoclast formation, the effect of 17-AAG upon critical transcription factors in the osteoclast formation pathway was observed. 17-AAG treatment did not affect transcriptional activity of NFκB, AP-1 or NFATc1. Moreover 17-AAG did not increase NFATc1 or cFos protein expression over time. In contrast, 17-AAG treatment increased protein expression of MITF,in a dose and time dependent manner. RAW264.7 cells were then transiently transfected with a luciferase reporter construct containing the promoter region of a MITF target gene v-ATPase-d2. 17-AAG treatment increased the transcriptional activity of this promoter. RANKL and 17-AAG combination treatment increased reporter activity of the v-ATPase-d2 promoter in an additive manner. The stress induced kinase p38 is known to be important in osteoclast formation. Our results show inhibiting p38 with SB203580 decreases MITF protein expression. These data indicate that Hsp90 inhibition causes an increase in osteoclast formation in an HSF1-dependent manner. The data provides a mechanism of action, whereby 17-AAG is increasing osteoclast numbers, through the increased protein expression of Mitf. Citation Format: A. Gabrielle J. van der Kraan, Ryan R. Chai, Michelle M. Kouspou, Ben J. Lang, Preetinder P. Singh, Jiake Xu, Damien Eeles, Matthew T. Gillespie, Julian M. Quinn, John T. Price. HSP90 inhibiting anti-cancer therapeutics enhance bone loss by increasing osteoclast formation: Mechanism of action. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2933. doi:10.1158/1538-7445.AM2013-2933

Pharmacokinetics and dose escalation of the heat shock protein inhibitor 17-allyamino-17-demethoxygeldanamycin in combination with bortezomib in relapsed or refractory acute myeloid leukemia

This phase I study was conducted to determine the maximum tolerated dose (MTD) and dose limiting toxicities (DLTs) of the heat shock protein 90 (HSP90) inhibitor 17-allyamino-17-demethoxygeldanamycin (17-AAG) in combination with bortezomib, and to provide pharmacokinetic data in relapsed or refractory acute myeloid leukemia (AML). Eleven patients were enrolled. The MTD was 17-AAG 150 mg/m2 and bortezomib 0.7 mg/m2. Hepatic toxicity and cardiac toxicity were dose limiting. Co-administration on day 4 led to a decrease in clearance (p = 0.005) and increase in AUC (p = 0.032) of 17-amino-17-demethoxygeldanamycin (17-AG), not observed when 17-AAG was administered alone. Pharmacokinetic parameters of patients who developed toxicities and those who did not were not different. The combination of 17-AAG and bortezomib led to toxicity without measurable response in patients with relapsed or refractory AML. Pharmacokinetic data provide insight for studies of related agents in AML. Next-generation HSP90 inhibitors are appealing for further development in this area.

17-allylamino-17-demethoxygeldanamycin and ritonavir inhibit renal cancer growth by inhibiting the expression of heat shock factor-1

Our previous study showed that the combination of a histone deacetylase (HDAC) inhibitor and an HIV protease inhibitor is effective against renal cancer cells. Because HDAC inhibition disrupts the chaperon function of heat shock protein (HSP) 90, we hypothesized that the combination of 17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of HSP90, and the HIV protease inhibitor ritonavir would also act against renal cancer. The combination of 17-AAG and ritonavir induced apoptosis and inhibited the proliferation of renal cancer cells effectively. It also suppressed the expression of cyclin-dependent kinase 4 and cyclin D1, leading to the accumulation of the cells in the sub-G1 fraction. The expression of HSPs 27, 70 and 90 was increased by 17-AAG alone but reduced by 17-AAG combined with ritonavir. The combination decreased the expression of heat shock factor-1 (HSF-1), an HSP transcription factor, and this might be one of the mechanisms of the effect of the combination. We have also found that silencing of HSF-1 by siRNA inhibited the proliferation of renal cancer cells and that in surgically resected specimens the levels of HSF-1 expression in renal cancer tissue are higher than those in normal parenchyma. This is the first study showing the beneficial effect of combining 17-AAG and ritonavir and our data suggest that HSF-1 may be a novel therapeutic target in the treatment of renal cancer.

Abstract 5303: Serial HER2-PET imaging with 89Zr-trastuzumab-F(ab’)2 of lapatinib and 17AAG treated mice bearing SKBR3 xenografts

Abstract Background: Understanding molecular responses involved in human epidermal growth factor receptor-2 (HER2) targeted drugs may improve treatment of HER2 positive breast cancer. In vivo assessment of effects on HER2 expression is of value in this setting. The EGFR/HER2 tyrosine kinase inhibitor lapatinib inhibits HER2 signaling and stabilizes HER2 at the cell membrane, while the heat shock protein-90 (HSP90) inhibitor 17AAG degrades HER2/EGFR. We therefore evaluated the effect on HER2 dynamics in vivo with 89Zr-trastuzumab-F(ab’)2 HER2-PET imaging in human tumor bearing mice treated with lapatinib and 17AAG. Materials and methods: Effects of lapatinib and 17AAG on EGFR and HER2 membrane expression were evaluated in vitro in the human breast cancer cell line SKBR3 using flow cytometry. In vivo effects on HER2 expression were evaluated with 89Zr-trastuzumab-F(ab’)2 μPET imaging in SKBR3 xenograft bearing mice. 89Zr-trastuzumab-F(ab’)2 (5 MBq; 20 μg) was injected two times with a 7 day interval and μPET imaging was performed 24 hours after tracer injection. Lapatinib was dosed 100 mg/kg/day orally for 6 days, starting 1 day after the first μPET scan. 17AAG was dosed 3×50 mg intraperitoneal in the 24 hours prior to the second tracer injection. A non-treated group of mice served as control and for validation of 89Zr-trastuzumab-F(ab’)2 μPET imaging. Imaging data were compared with ex vivo biodistribution analysis, immunohistochemistry, and with an in vitro HER2 internalization experiment. Results: On flow cytometry, lapatinib induced upregulation of EGFR (+70%; P = 0.0011), but not HER2 (+19%; P = 0.1275). 17AAG treatment lowered both EGFR (−41%; P = 0.016) and HER2 (−76%; P = 0.022) expression. This EGFR/HER2 reduction by 17AAG was inhibited by lapatinib. In vivo, 89Zr-trastuzumab-F(ab’)2 tumor uptake on the second μPET scan was lowered by lapatinib (−32%; P = 0.00004), 17AAG (−34%; P = 0.0022) and the lapatinib plus 17AAG combination (−53%; P = 0.011). 89Zr-trastuzumab-F(ab’)2 tumor uptake in mice treated with lapatinib plus 17AAG was lower than with either lapatinib or 17AAG alone (P = 0.0043 and P = 0.0022, respectively). In vitro, 89Zr-trastuzumab-F(ab’)2 internalization was reduced by lapatinib (−25%; P = 0.0022), explaining the lower 89Zr-trastuzumab-F(ab’)2 tumor uptake after lapatinib. Conclusion: 89Zr-trastuzumab-F(ab’)2 μPET visualized treatment induced impact on HER2 expression and -dynamics. Lapatinib inhibited HER2 internalization, and thereby decreased 89Zr-trastuzumab-F(ab’)2 tumor uptake. HSP90 inhibition lowered HER2 membrane expression and thereby 89Zr-trastuzumab-F(ab’)2 tumor uptake. This knowledge on HER2 dynamics ameliorates understanding of efficacy of several combinations of HER2 targeted agents. Supported by grant 2007-3739 of the Dutch Cancer Society. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5303. doi:10.1158/1538-7445.AM2011-5303

Hsp90 inhibition induces destabilization of actin cytoskeleton in tumor cells: functional significance of Hsp90 interaction with F–actin

Objective To examine the role of heat shock protein 90 (Hsp 90) in the maintenance of actin cytoskeleton in human neuroblastoma tumor cells. Methods Co-precipitation experiments were performed to examine Hsp 90 interaction with actin. Hsp 90 and actin interactions were evaluated by protein refolding and acto-myosin motility assays. 17-(Allylamino)-17-demethoxygeldanamycin (17AAG) induced actin-cytoskeleton re-organization was examined by laser scanning confocal microcopy. Results It was shown that inhibition of Hsp 90 by 17AAG accelerates detergent induced cell lysis of neuroblastoma tumor cells through destabilization of actin cytoskeleton. The in vitro co-precipitation experiments showed that functional but not mutant Hsp 90 binds with F-actin. Among biochemical modifications, phopshorylation and oligomerization enhanced Hsp 90 binding with F-actin. F-actin binding to Hsp 90 interfered with Hsp 90 chaperone activity in protein refolding assays, and Hsp 90 binding to F-actin interfered with actin motility on myosin coated flow cell. In the combination treatment, 17AAG irreversibly augmented the effect of cytochalasin D, an inhibitor of actin polymerization. Conclusions It can be concluded that Hsp 90 binds to F-actin in tumor cells and maintains the cellular integrity. The results display a novel element of Hsp 90 inhibition in destabilizing the actin cytoskeleton of tumor cells, therefore suggest that 17AAG combination with cytoskeletal disruptor may be effective in combating cancer.

Abstract 2633: Adenosine analog enhances actions of 17-AAG

Abstract Heat shock protein 90 (HSP90) is a molecular chaperone that regulates the functional stability of client oncoproteins, which is driven by the binding and hydrolysis of ATP. The geldanamycin analog, 17-AAG, binds to the ATP pocket of HSP90 leading to the degradation of client proteins. However, 17-AAG also induces transcription of antiapoptotic HSP70 and HSP27 resulting in cell death resistance. The nucleoside analog 8-Chloro-Adenosine (8-Cl-Ado), currently in clinical trial, is a transcription inhibitor that gets metabolized into its cytotoxic form 8-Cl-ATP causing a parallel decrease of the cellular ATP pool. Based on its mechanisms of action we hypothesize that the combination of 17-AAG and 8-Cl-Ado will result in a favorable effect on cytotoxicity. % Cell Death Annexin V / 7-AAD TreatmentsHrsMM.1S8226-RPMIU2661)17-AAG 0.5 µM85452)17-AAG 0.5 µM202463)8-Cl-Ado 10 µM125324)8-Cl-Ado 10 µM2022825)17-AAG→8-Cl-Ado8→12 = 201511106)8-Cl-Ado→17-AAG12→8 = 205515197)8-Cl-Ado+17-AAG20421434 As shown in the Table, simultaneous or sequential combination (rows 6 and 7) were more than additive in multiple myeloma cell lines. With 17-AAG alone, there was a transcriptional induction of HSP90α, HSP90β, HSP70, HSC70 and HSP27 as measured by real time RT-PCR. With the drug combinations, there was no reduction of the 17-AAG-induced HSPs mRNA levels, rather a several fold increase was observed for the stress-inducible HSP70 and HSP27. In accordance, the protein levels of the HSPs were also increased when treated with 17-AAG but remained the same when 8-Cl-Ado was added. This suggests that the transcription inhibition by 8-Cl-Ado was not able to abrogate 17-AAG-mediated HSP induction. The transcript levels for client proteins (STAT3, Akt and c-Raf) remained at endogenous levels when treated with the drugs alone or in combination. However, the combination treatments resulted in a greater decline of the protein expression of STAT3 (1-60%), Akt (42-78%), and Raf-1 (25-85%). This may be due to the reduction in intracellular ATP levels after 8-Cl-Ado treatment, MM.1S (57%), 8226-RPMI (24%), and U266 (56%). Taken together, the increased cytotoxicity of 17-AAG and 8-Cl-Ado combination is mediated by the actions of 8-Cl-Ado on the cellular ATP pool and not due to its effect on transcription. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2633.

17-(Allylamino)-17-Demethoxygeldanamycin Combination with Curcumin Selectively Targets Mitogen Kinase Pathway in A Human Neuroblastoma Cell Line

Pharmacological inhibition of Hsp90 has emerged as a novel anticancer treatment. In this study we have investigated the effect of Hsp90 inhibitor drug 17AAG combination with curcumin on human neuroblastoma cells. The 17AAG treatment of cells for 18 h induced G1/S cell cycle arrest associated with cyclin D1 down regulation, and degradation of Raf-1 and inactivation of Akt. However, 17AAG treatment activated the mitogen kinase, ERK1, and induced the expression of stress proteins, Hsp70 and p53. The curcumin treatment resulted in G2/M cell cycle arrest and activation of both Raf1 and ERK1 kinases. The drugs in combination induced proteolytic degradation of Raf1 and Akt, and surpassed curcumin induced G2/M arrest. The combination treatment additionally inactivated MEK, inhibited activation and nuclear localization of ERK1, and also inhibited the stress protein induction. EGF stimulation induced re-activation of mitogen signaling with individual drug treatments but not in combination. This study highlights that 17AAG combination with curcumin selectively targets mitogen signal transduction mechanism through ERK1 inactivation. In conclusion, our study proposes the beneficial effects of 17AAG combination with curcumin in combating cancer.