Activity of the Heat Shock Protein 90 Inhibitor Ganetespib in Melanoma

Heat shock protein 90 (HSP90) is involved in the regulation of diverse biological processes such as cell signaling, proliferation and survival, and has been recently recognized as a potential target for cancer therapy. Ganetespib is a potent ATP competitive inhibitor of HSP90. Ganetespib downregulated the expression of multiple signal transducing molecules including EGFR, IGF-1R, c-Met, Akt, B-RAF and C-RAF, resulting in pronounced decrease in phosphorylation of Akt and Erk1/2 in a panel of five cutaneous melanoma cell lines including those harboring B-RAF and N-RAS mutations. Ganetespib exhibited potent antiproliferative activity on all five of these cell lines, with IC50 values between 37.5 and 84 nM. Importantly, Ganetespib is active on B-RAF mutated melanoma cells that have acquired resistance to B-RAF inhibition. Ganetespib induced apoptosis and cell cycle arrest at G1 and/or G2/M phase. Ganetespib induced cell cycle arrest was accompanied by altered expression of cyclin-dependent kinase inhibitor (CDKI) p21^Cip1 and p27^Kip1, cyclins B1, D1 and E, and/or cyclin-dependent kinases 1, 2 and 4. HSP90 is functionally important for melanoma cells and HSP90 inhibitors such as ganetespib could potentially be effective therapeutics for melanoma with various genetic mutations and acquired resistance to B-RAF inhibition.     

The HSP-RTK-Akt axis mediates acquired resistance to Ganetespib in HER2-positive breast cancer

Breast cancer is the leading cause of cancer-related death in women worldwide. Human epidermal growth factor receptor 2 (HER2)-positive subtype comprises 20% of sporadic breast cancers and is an aggressive disease. While targeted therapies have greatly improved its management, primary and acquired resistance remain a major roadblock to making it a curable malignancy. Ganetespib, an Hsp90 (Heat shock protein 90) small molecule inhibitor, shows preferential efficacy in HER2-positive breast cancer, including therapy-refractory cases, and has an excellent safety profile in ongoing clinical trials (38 in total, six on breast cancer). However, Ganetespib itself evokes acquired resistance, which is a significant obstacle to its clinical advancement. Here, we show that Ganetespib potently, albeit temporarily, suppresses HER2-positive breast cancer in genetic mouse models, but the animals eventually succumb via acquired resistance. We found that Ganetespib-resistant tumors upregulate several compensatory HSPs, as well as a wide network of phospho-activated receptor tyrosine kinases (RTKs), many of which are HSP clients. Downstream of p-RTKs, the MAPK pathway remains suppressed in the resistant tumors, as is HER2 itself. In contrast, the p-RTK effector Akt is stabilized and phospho-activated. Notably, pharmacological inhibition of Akt significantly delays acquired Ganetespib resistance, by 50%. These data establish Akt as a unifying actionable node downstream of the broadly upregulated HSP/p-RTK resistance program and suggests that Akt co-targeting with Ganetespib may be a superior therapeutic strategy in the clinic.Subject terms: Breast cancer, Cancer therapeutic resistance     

Radiosynthesis,biological evaluation and preliminary microPET study of 18F-labeled 5-resorcinolic triazolone derivative based on ganetespib targeting HSP90

Heat-shock protein 90 (HSP90) is a molecular chaperone that activates oncogenic transformation in several solid tumors, including lung and breast cancers. Ganetespib, a most promising candidate among several HSP90 inhibitors under clinical trials, has entered Phase III clinical trials for cancer therapy. Despite numerous evidences validating HSP90 as a target of anticancer, there are few studies on PET agents targeting oncogenic HSP90. In this study, we synthesized and biologically evaluated a novel ¹⁸F-labeled 5-resorcinolic triazolone derivative (1, [¹⁸F]PTP-Ganetespib) based on ganetespib. [¹⁸F]PTP-Ganetespib was labeled by click chemistry of Ganetespib-PEG-Alkyne (10) and [¹⁸F]PEG-N₃ (11) with 37.3?±?5.11% of radiochemical yield and 99.7?±?0.09% of radiochemical purity. [¹⁸F]PTP-Ganetespib showed proper LogP (0.96?±?0.06) and good stability in human serum over 97% for 2?h. [¹⁸F]PTP-Ganetespib showed high uptakes in breast cancer cells containing triple negative breast cancer (TNBC) MDA-MB-231 and Her2-negative MCF-7 cells, which are target breast cancer cell lines of HSP90 inhibitor, ganetespib, as an anticancer. Blocking of HSP90 by the pretreatment of ganetespib exhibited significantly decreased accumulation of [¹⁸F]PTP-Ganetespib in MDA-MB-231 and MCF-7 cells, indicating the specific binding of [¹⁸F]PTP-Ganetespib to MDA-MB-231 and MCF-7 cells with high HSP90 expression. In the biodistribution and microPET imaging studies, the initial uptake into tumor was weaker than in other thoracic and abdominal organs, but [¹⁸F]PTP-Ganetespib was retained relatively longer in the tumor than other organs. The uptake of [¹⁸F]PTP-Ganetespib in tumors was not sufficient for further development as a tumor-specific PET imaging agent by itself, but this preliminary PET imaging study of [¹⁸F]PTP-Ganetespib can be basis for developing new PET imaging agents based on HSP90 inhibitor, ganetespib.     

The HSP90 Inhibitor Ganetespib Alleviates Disease Progression and Augments Intermittent Cyclophosphamide Therapy in the MRL/lpr Mouse Model of Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a complex, systemic autoimmune disease with a diverse range of immunological and clinical manifestations. The introduction of broad spectrum immunosuppressive therapies and better management of acute disease exacerbations have improved outcomes for lupus patients over recent years. However, these regimens are burdened by substantial toxicities and confer significantly higher risks of infection, thus there remains a significant and unmet medical need for alternative treatment options, particularly those with improved safety profiles. Heat shock protein 90 (HSP90) is a ubiquitously expressed molecular chaperone that acts as an important modulator of multiple innate and adaptive inflammatory processes. Of note, accumulating clinical and experimental evidence has implicated a role for HSP90 in the pathogenesis of SLE. Here we evaluated the potential of HSP90 as a therapeutic target for this disease using the selective small molecule inhibitor ganetespib in the well-characterized MRL/lpr autoimmune mouse model. In both the prophylactic and therapeutic dosing settings, ganetespib treatment promoted dramatic symptomatic improvements in multiple disease parameters, including suppression of autoantibody production and the preservation of renal tissue integrity and function. In addition, ganetespib exerted profound inhibitory effects on disease-related lymphadenopathy and splenomegaly, and reduced pathogenic T and B cell lineage populations in the spleen. Ganetespib monotherapy was found to be equally efficacious and tolerable when compared to an effective weekly dosing regimen of the standard-of-care immunosuppressive agent cyclophosphamide. Importantly, co-treatment of ganetespib with a sub-optimal, intermittent dosing schedule of cyclophosphamide resulted in superior therapeutic indices and maximal disease control. These findings highlight the potential of HSP90 inhibition as an alternative, and potentially complementary, strategy for therapeutic intervention in SLE. Such approaches may have important implications for disease management, particularly for limiting or preventing treatment-related toxicities, a major confounding factor in current SLE therapy.     

Proteomic interrogation of HSP90 and insights for medical research

Introduction: Heat shock protein 90 (HSP90) regulates protein homeostasis in eukaryotes. As a ‘professional interactor’, HSP90 binds to and chaperones many proteins and has both housekeeping and disease-related functions but its regulation remains in part elusive. HSP90 complexes are a target for therapy, notably against cancer, and several inhibitors are currently in clinical trials. Proteomic studies have revealed the vast interaction network of HSP90 and, in doing so, the extent of cellular processes the chaperone takes part in, especially in yeast and human cells. Furthermore, small-molecule inhibitors were used to probe the global impact of its inhibition on the proteome.

Areas covered: We review here recent HSP90-related interactomics and total proteome studies and their relevance for research on cancer, neurodegenerative and pathogen diseases.

Expert commentary: Proteomics experiments are our best chance to identify the context-dependent global proteome of HSP90 and thus uncover and understand its disease-specific biology. However, understanding the complexity of HSP90 will require multiple complementary, quantitative approaches and novel bioinformatics to translate interactions into ordered functional networks and pathways. Developing therapies will necessitate more knowledge on HSP90 complexes and networks with disease relevance and on total proteome changes induced by their perturbation. Most work has been done in cancer, thus a lot remains to be done in the context of other diseases.     

HSP90 modulates actin dynamics: Inhibition of HSP90 leads to decreased cell motility and impairs invasion

HSP90, a major molecular chaperone, plays an essential role in the maintenance of several signaling molecules. Inhibition of HSP90 by inhibitors such as 17-allylamino-demethoxy-geldanamycin (17AAG) is known to induce apoptosis in various cancer cells by decreasing the activation or expression of pro-survival molecules such as protein kinase B (Akt). While we did not observe either decrease in expression or activation of pro-survival signaling molecules in human breast cancer cells upon inhibiting HSP90 with 17AAG, we did observe a decrease in cell motility of transformed cells, and cell motility and invasion of cancer cells. We found a significant decrease in the number of filopodia and lamellipodia, and in the F-actin bundles upon HSP90 inhibition. Our results show no change in the active forms or total levels of FAK and Pax, or in the activation of Rac-1 and Cdc-42; however increased levels of HSP90, HSP90α and HSP70 were observed upon HSP90 inhibition. Co-immuno-precipitation of HSP90 reveals interaction of HSP90 with G-actin, which increases upon HSP90 inhibition. FRET results show a significant decrease in interaction between actin monomers, leading to decreased actin polymerization upon HSP90 inhibition. We observed a decrease in the invasion of human breast cancer cells in the matrigel assay upon HSP90 inhibition. Over-expression of αB-crystallin, known to be involved in actin dynamics, did not abrogate the effect of HSP90 inhibition. Our work provides the molecular mechanism by which HSP90 inhibition delays cell migration and should be useful in developing cancer treatment strategies with known anti-cancer drugs such as cisplatin in combination with HSP90 inhibitors.     

Mechanism of simvastatin on induction of heat shock protein in osteoblasts

It has recently been reported that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) stimulate bone formation. However, the mechanism of stimulation of bone metabolism by statins is not precisely clarified. In this study, we investigated whether simvastatin induces heat shock protein (HSP) 27, HSP70, and HSP90 in osteoblast-like MC3T3-E1 cells. Simvastatin increased the levels of HSP27 while having little effect on the levels of HSP70 or HSP90. The effect of simvastatin on HSP27 accumulation was dose dependent. Cycloheximide reduced the accumulation. Simvastatin induced an increase in the levels of mRNA for HSP27. Actinomycin D suppressed the mRNA levels. Simvastatin induced the phosphorylation of p38 mitogen-activated protein (MAP) kinase among the MAP kinase superfamily. SB203580 and PD169316, inhibitors of p38 MAP kinase, suppressed the HSP27 accumulation by simvastatin while SB202474, a negative control of p38 MAP kinase inhibitor, had no effect. SB203580 reduced the simvastatin-increased mRNA levels for HSP27. Lovastatin, another statin, also induced the HSP27 accumulation and SB203580 suppressed the HSP27 accumulation. These results strongly suggest that statins such as simvastatin do not stimulate the induction of HSP70 and HSP90, but do stimulate the induction of HSP27 in osteoblasts and that p38 MAP kinase plays a role in this induction.     

Induction of Premature Senescence by Hsp90 Inhibition in Small Cell Lung Cancer

Background

The molecular chaperone Hsp90 is a promising new target in cancer therapy and selective Hsp90 inhibitors are currently in clinical trials. Previously these inhibitors have been reported to induce either cell cycle arrest or cell death in cancer cells. Whether the cell cycle arrest is reversible or irreversible has not generally been assessed. Here we have examined in detail the cell cycle arrest and cell death responses of human small cell lung cancer cell lines to Hsp90 inhibition.

Methodology/Principal Findings

In MTT assays, small cell lung cancer cells showed a biphasic response to the Hsp90 inhibitors geldanamycin and radicicol, with low concentrations causing proliferation arrest and high concentrations causing cell death. Assessment of Hsp90 intracellular activity using loss of client protein expression showed that geldanamycin concentrations that inhibited Hsp90 correlated closely with those causing proliferation arrest but not cell death. The proliferation arrest induced by low concentrations of geldanamycin was not reversed for a period of over thirty days following drug removal and showed features of senescence. Rare populations of variant small cell lung cancer cells could be isolated that had additional genetic alterations and no longer underwent irreversible proliferation arrest in response to Hsp90 inhibitors.

Conclusions/Significance

We conclude that: (1) Hsp90 inhibition primarily induces premature senescence, rather than cell death, in small cell lung cancer cells; (2) small cell lung cancer cells can bypass this senescence through further genetic alterations; (3) Hsp90 inhibitor-induced cell death in small cell lung cancer cells is due to inhibition of a target other than cytosolic Hsp90. These results have implications with regard to how these inhibitors will behave in clinical trials and for the design of future inhibitors in this class.     

Dependence of HSP27 cellular level on protein kinase CK2 discloses novel therapeutic strategies

Background

HSP27 plays a role in various diseases, including neurodegenerative diseases, ischemia, and atherosclerosis. It is particularly important in the regulation of the development, progression and metastasis of cancer as well as cell apoptosis and drug resistance. However, the absence of an ATP binding domain, that is, instead, present in other HSPs such as HSP90 and HSP70, hampers the development of small molecules as inhibitors of HSP27.

Identification of Mixed Lineage Leukemia 1(MLL1) Protein as a Coactivator of Heat Shock Factor 1(HSF1) Protein in Response to Heat Shock Protein 90 (HSP90) Inhibition

Heat shock protein 90 (HSP90) inhibition inhibits cancer cell proliferation through depleting client oncoproteins and shutting down multiple oncogenic pathways. Therefore, it is an attractive strategy for targeting human cancers. Several HSP90 inhibitors, including AUY922 and STA9090, show promising effects in clinical trials. However, the efficacy of HSP90 inhibitors may be limited by heat shock factor 1 (HSF1)-mediated feedback mechanisms. Here, we identify, through an siRNA screen, that the histone H3 lysine 4 methyltransferase MLL1 functions as a coactivator of HSF1 in response to HSP90 inhibition. MLL1 is recruited to the promoters of HSF1 target genes and regulates their expression in response to HSP90 inhibition. In addition, a striking combination effect is observed when MLL1 depletion is combined with HSP90 inhibition in various human cancer cell lines and tumor models. Thus, targeting MLL1 may block a HSF1-mediated feedback mechanism induced by HSP90 inhibition and provide a new avenue to enhance HSP90 inhibitor activity in human cancers.     

Advances in the clinical development of heat shock protein 90 (Hsp90) inhibitors in cancers

Hsp90 is an ATP dependent molecular chaperone protein which integrates multiple oncogenic pathways. As such, Hsp90 inhibition is a promising anti-cancer strategy. Several inhibitors that act on Hsp90 by binding to its N-terminal ATP pocket have entered clinical evaluation. Robust pre-clinical data suggested anti-tumor activity in multiple cancer types. Clinically, encouraging results have been demonstrated in melanoma, acute myeloid leukemia, castrate refractory prostate cancer, non-small cell lung carcinoma and multiple myeloma. In breast cancer, proof-of-concept was demonstrated by first generation Hsp90 inhibitors in combination with trastuzumab mainly in human epidermal growth factor receptor 2 (HER2)+metastatic breast cancer. There are a multitude of second generation Hsp90 inhibitors currently under investigation. To date, however, there is no FDA approved Hsp90 inhibitor nor standardized assay to ascertain Hsp90 inhibition. This review summarizes the current status of both first and second generation Hsp90 inhibitors based on their chemical classification and stage of clinical development. It also discusses the pharmacodynamic assays currently implemented in clinic as well as other novel strategies aimed at enhancing the effectiveness of Hsp90 inhibitors. Ultimately, these efforts will aid in maximizing the full potential of this class of agents. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).     

EC144, a synthetic inhibitor of heat shock protein 90, blocks innate and adaptive immune responses in models of inflammation and autoimmunity

Heat shock protein 90 (Hsp90) is a molecular chaperone involved in folding and stabilizing multiple intracellular proteins that have roles in cell activation and proliferation. Many Hsp90 client proteins in tumor cells are mutated or overexpressed oncogenic proteins driving cancer cell growth, leading to the acceptance of Hsp90 as a potential therapeutic target for cancer. Because several signal transduction molecules that are dependent on Hsp90 function are also involved in activation of innate and adaptive cells of the immune system, we investigated the mechanism by which inhibiting Hsp90 leads to therapeutic efficacy in rodent models of inflammation and autoimmunity. EC144, a synthetic Hsp90 inhibitor, blocked LPS-induced TLR4 signaling in RAW 264.7 cells by inhibiting activation of ERK1/2, MEK1/2, JNK, and p38 MAPK but not NF-κB. Ex vivo LPS-stimulated CD11b(+) peritoneal exudate cells from EC144-treated mice were blocked from phosphorylating tumor progression locus 2, MEK1/2, and ERK1/2. Consequently, EC144-treated mice were resistant to LPS administration and had suppressed systemic TNF-α release. Inhibiting Hsp90 also blocked in vitro CD4(+) T cell proliferation in mouse and human MLRs. In vivo, semitherapeutic administration of EC144 blocked disease development in rat collagen-induced arthritis by suppressing the inflammatory response. In a mouse collagen-induced arthritis model, EC144 also suppressed disease development, which correlated with a suppressed Ag-specific Ab response and a block in activation of Ag-specific CD4(+) T cells. Our results describe mechanisms by which blocking Hsp90 function may be applicable to treatment of autoimmune diseases involving inflammation and activation of the adaptive immune response.     

A role of Rab7 in stabilizing EGFR-Her2 and in sustaining Akt survival signal

Rab7 plays an important role in regulating endocytic traffic. In view of an emerging role of membrane traffic in signaling and diseases, we have examined the possible role of Rab7 in oncogenesis. The role of Rab7 was investigated using shRNA‐mediated knockdown in A431 and MCF7 cancer cells. To our surprise, Rab7 knockdown effectively suppressed anchorage‐independent growth of cancer cells in soft agar. Anoikis (matrix‐detachment triggered apoptosis) was enhanced, while the level of phosphorylated (active) Akt (which is a key survival factor) was significantly reduced. Also intriguing was the observation that EGFR and Her2 levels were significantly reduced when Rab7 was knocked‐down. More robust reduction of EGFR and Her2 levels was observed when knocked‐down cells were treated with HSP90 inhibitor geldanamycin (GA). Low concentration of GA (50–100 nm)‐induced apoptosis of the Rab7 knocked‐down cells but not control cells, suggesting that Rab7 and HSP90 together contribute to the optimal stability of EGFR and Her2 as well as to protect cancer cells from apoptosis. Rab7 seems to protect EGFR and Her2 from proteosome‐mediated degradation. These results suggest that Rab7 is likely involved in protecting EGFR and Her2 from being degraded by the proteosome and in maintaining optimal Akt survival signal (especially during cell detachment or when HSP90 is inhibited). Rab7 is potentially a novel target for combinatory therapy with Hsp90 inhibitors. J. Cell. Physiol. 227: 2788–2797, 2012. © 2011 Wiley Periodicals, Inc.     

Targeting GRP75 Improves HSP90 Inhibitor Efficacy by Enhancing p53-Mediated Apoptosis in Hepatocellular Carcinoma

Heat shock protein 90 (HSP90) inhibitors are potential drugs for cancer therapy. The inhibition of HSP90 on cancer cell growth largely through degrading client proteins, like Akt and p53, therefore, triggering cancer cell apoptosis. Here, we show that the HSP90 inhibitor 17-AAG can induce the expression of GRP75, a member of heat shock protein 70 (HSP70) family, which, in turn, attenuates the anti-growth effect of HSP90 inhibition on cancer cells. Additionally, 17-AAG enhanced binding of GRP75 and p53, resulting in the retention of p53 in the cytoplasm. Blocking GRP75 with its inhibitor MKT-077 potentiated the anti-tumor effects of 17-AAG by disrupting the formation of GRP75-p53 complexes, thereby facilitating translocation of p53 into the nuclei and leading to the induction of apoptosis-related genes. Finally, dual inhibition of HSP90 and GRP75 was found to significantly inhibit tumor growth in a liver cancer xenograft model. In conclusion, the GRP75 inhibitor MKT-077 enhances 17-AAG-induced apoptosis in HCCs and increases p53-mediated inhibition of tumor growth in vivo. Dual targeting of GRP75 and HSP90 may be a useful strategy for the treatment of HCCs.