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Targeted inhibition of the molecular chaperone Hsp90 results in the simultaneous blockade of multiple oncogenic signaling pathways and has, thus, emerged as an attractive strategy for the development of novel cancer therapeutics. Ganetespib (formerly known as STA-9090) is a unique resorcinolic triazolone inhibitor of Hsp90 that is currently in clinical trials for a number of human cancers. In the present study, we showed that ganetespib exhibits potent in vitro cytotoxicity in a range of solid and hematologic tumor cell lines, including those that express mutated kinases that confer resistance to small-molecule tyrosine kinase inhibitors. Ganetespib treatment rapidly induced the degradation of known Hsp90 client proteins, displayed superior potency to the ansamycin inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG), and exhibited sustained activity even with short exposure times. In vivo, ganetespib showed potent antitumor efficacy in solid and hematologic xenograft models of oncogene addiction, as evidenced by significant growth inhibition and/or regressions. Notably, evaluation of the microregional activity of ganetespib in tumor xenografts showed that ganetespib was efficiently distributed throughout tumor tissue, including hypoxic regions >150 μm from the microvasculature, to inhibit proliferation and induce apoptosis. Importantly, ganetespib showed no evidence of cardiac or liver toxicity. Taken together, this preclinical activity profile indicates that ganetespib may have broad application for a variety of human malignancies, and with select mechanistic and safety advantages over other first- and second-generation Hsp90 inhibitors. Mol Cancer Ther; 11(2); 475–84. ©2011 AACR.

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Supplementary Table S1 from Elesclomol induces cancer cell apoptosis through oxidative stress

Elesclomol (formerly STA-4783) is a novel small molecule undergoing clinical evaluation in a pivotal phase III melanoma trial (SYMMETRY). In a phase II randomized, double-blinded, controlled, multi-center trial in 81 patients with stage IV metastatic melanoma, treatment with elesclomol plus paclitaxel showed a statistically significant doubling of progression-free survival time compared with treatment with paclitaxel alone. Although elesclomol displays significant therapeutic activity in the clinic, the mechanism underlying its anticancer activity has not been defined previously. Here, we show that elesclomol induces apoptosis in cancer cells through the induction of oxidative stress. Treatment of cancer cells in vitro with elesclomol resulted in the rapid generation of reactive oxygen species (ROS) and the induction of a transcriptional gene profile characteristic of an oxidative stress response. Inhibition of oxidative stress by the antioxidant N-acetylcysteine blocked the induction of gene transcription by elesclomol. In addition, N-acetylcysteine blocked drug-induced apoptosis, indicating that ROS generation is the primary mechanism responsible for the proapoptotic activity of elesclomol. Excessive ROS production and elevated levels of oxidative stress are critical biochemical alterations that contribute to cancer cell growth. Thus, the induction of oxidative stress by elesclomol exploits this unique characteristic of cancer cells by increasing ROS levels beyond a threshold that triggers cell death. [Mol Cancer Ther 2008;7(8):2319–27]

PDF file, 560K, Comparison of ganetespib and 17-AAG efficacy in NSCLC cell lines.

Purpose: We describe the anticancer activity of ganetespib, a novel non-geldanamycin heat shock protein 90 (HSP90) inhibitor, in non-small cell lung cancer (NSCLC) models.Experimental Design: The activity of ganetespib was compared with that of the geldanamycin 17-AAG in biochemical assays, cell lines, and xenografts, and evaluated in an ERBB2 YVMA-driven mouse lung adenocarcinoma model.Results: Ganetespib blocked the ability of HSP90 to bind to biotinylated geldanamycin and disrupted the association of HSP90 with its cochaperone, p23, more potently than 17-AAG. In genomically defined NSCLC cell lines, ganetespib caused depletion of receptor tyrosine kinases, extinguishing of downstream signaling, inhibition of proliferation and induction of apoptosis with IC50 values ranging 2 to 30 nmol/L, substantially lower than those required for 17-AAG (20–3,500 nmol/L). Ganetespib was also approximately 20-fold more potent in isogenic Ba/F3 pro-B cells rendered IL-3 independent by expression of EGFR and ERBB2 mutants. In mice bearing NCI-H1975 (EGFR L858R/T790M) xenografts, ganetespib was rapidly eliminated from plasma and normal tissues but was maintained in tumor with t1/2 58.3 hours, supporting once-weekly dosing experiments, in which ganetespib produced greater tumor growth inhibition than 17-AAG. However, after a single dose, reexpression of mutant EGFR occurred by 72 hours, correlating with reversal of antiproliferative and proapoptotic effects. Consecutive day dosing resulted in xenograft regressions, accompanied by more sustained pharmacodynamic effects. Ganetespib also showed activity against mouse lung adenocarcinomas driven by oncogenic ERBB2 YVMA.Conclusions: Ganetespib has greater potency than 17-AAG and potential efficacy against several NSCLC subsets, including those harboring EGFR or ERBB2 mutation. Clin Cancer Res; 18(18); 4973–85. ©2012 AACR.

PDF file, 1349K, Depletion of receptor tyrosine kinases and suppression of downstream signaling in HCC827 cells in response to 17-AAG or ganetespib-mediated HSP90 inhibition.

PDF file, 365K, Activity of ganetespib in HCC827 xenografts.

PDF file, 776K, Effects of 17-AAG and ganetespib on cell viability and mutant ERBB2 expression in ERBB2-dependent Ba/F3 cells.

PDF file, 136K, Median IC50's for NSCLC cell lines (with ganetespib and 17-AAG) with different genotypic subtypes.

PDF file, 361K, Comparison of EGFR TKI and HSP90 inhibitor efficacy in Ba/F3 cells ectopically expressing different EGFR mutations.

PDF file, 1170K, Depletion of oncogenic drivers in NSCLC cell lines.

PDF file, 719K, Comparison of once-weekly and consecutive day dosing schedules among individual mice bearing NCI-H1975 xenografts.

PDF file, 49K, Supplementary Immunohistochemical and Imaging Methods.

PDF file, 294K, Ganetespib Biodistribution and Pharmacokinetic Parameters.

Significance Autosomal recessive mutations or deletions of the gene Survival Motor Neuron 1 ( SMN1 ) cause spinal muscular atrophy, a neurodegenerative disorder. Transcriptional up-regulation of a nearly identical gene, SMN2 , can functionally compensate for the loss of SMN1 , resulting in increased SMN protein to ameliorate the disease severity. Here we demonstrate that the repressed state of SMN2 is reversible by interrupting the recruitment of a repressive epigenetic complex in disease-relevant cell types. Using chemically modified oligonucleotides to bind at a site of interaction on a long noncoding RNA that recruits the repressive complex, SMN2 is epigenetically altered to create a transcriptionally permissive state.

The aberrant overexpression of Wilms tumor 1 (WT1) in myeloid leukemia plays an important role in blast cell survival and resistance to chemotherapy. High expression of WT1 is also associated with relapse and shortened disease-free survival in patients. However, the mechanisms by which WT1 expression is regulated in leukemia remain unclear. Here, we report that heat shock protein 90 (Hsp90), which plays a critical role in the folding and maturation of several oncogenic proteins, associates with WT1 protein and stabilizes its expression. Pharmacologic inhibition of Hsp90 resulted in ubiquitination and subsequent proteasome-dependant degradation of WT1. RNAi-mediated silencing of WT1 reduced the survival of leukemia cells and increased the sensitivity of these cells to chemotherapy and Hsp90 inhibition. Furthermore, Hsp90 inhibitors 17-AAG [17-(allylamino)-17-demethoxygeldanamycin] and STA-9090 significantly reduced the growth of myeloid leukemia xenografts in vivo and effectively down-regulated the expression of WT1 and its downstream target proteins, c-Myc and Bcl-2. Collectively, our studies identify WT1 as a novel Hsp90 client and support the crucial role for the WT1–Hsp90 interaction in maintaining leukemia cell survival. These findings have significant implications for developing effective therapies for myeloid leukemias and offer a strategy to inhibit the oncogenic func-tions of WT1 by clinically available Hsp90 inhibitors.

Elesclomol is a small-molecule investigational agent that selectively induces apoptosis in cancer cells by increasing oxidative stress. Elesclomol plus paclitaxel was shown to prolong progression-free survival compared with paclitaxel alone in a phase II clinical trial in patients with metastatic melanoma. However, the therapeutic potential of elesclomol in human breast cancer is unknown, and the signaling mechanism underlying the elesclomol effect is unclear. Here, we show that elesclomol alone modestly inhibited the growth of human breast cancer cells but not normal breast epithelial cells. Elesclomol potentiated doxorubicin- or paclitaxel-induced apoptosis and suppression of breast cancer cell growth. While both c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase were activated by elesclomol, elesclomol-induced apoptosis was only in part mediated by JNK1. The additive effect of elesclomol on chemotherapy drug-induced apoptosis was associated with increases in cleaved caspase-3, p21(Cip1), and p27(Kip1) and decreases in the Inhibitor of Apoptosis Protein levels and NF-kappaB activity. We also found that Akt/Hsp70 survival signaling was induced by elesclomol, which may reflect a cellular feedback mechanism. Blockade of Akt activation using a small-molecule inhibitor enhanced elesclomol-elicited apoptosis, while expression of a hyperactive Akt abolished the elesclomol effect. These data suggest that elesclomol's interaction with conventional chemotherapeutic and Akt-targeting agents may be exploited to induce apoptosis in breast cancer cells, and clinical trials of combined treatment of elesclomol and chemotherapy drugs or Akt-targeting agents in breast cancer patients, especially the estrogen receptor negative subgroup, may be warranted.

Objective Mutations of the receptor tyrosine kinase Kit occur in several human and canine cancers. While Kit inhibitors have activity in the clinical setting, they possess variable efficacy against particular forms of mutant Kit and drug resistance often develops over time. Inhibitors of heat shock protein 90 (HSP90), a chaperone for which Kit is a client protein, have demonstrated activity against human cancers and evidence suggests they downregulate several mutated and imatinib-resistant forms of Kit. The purpose of this study was to evaluate a novel HSP90 inhibitor, STA-9090, against wild-type (WT) and mutant Kit in canine bone marrow–derived cultured mast cells (BMCMCs), malignant mast cell lines, and fresh malignant mast cells. Materials and Methods BMCMCs, cell lines, and fresh malignant mast cells were treated with STA-9090, 17-AAG, and SU11654 and evaluated for loss in cell viability, cell death, alterations in HSP90 and Kit expression/signaling, and Kit mutation. STA-9090 activity was tested in a canine mastocytoma xenograft model. Results Treatment of BMCMCs, cell lines, and fresh malignant cells with STA-9090 induced growth inhibition, apoptosis that was caspase-3/7–dependent, and downregulation of phospho/total Kit and Akt, but not extracellular signal-regulated kinase (ERK) or phosphoinositide-3 kinase (PI-3K). Loss of Kit cell-surface expression was also observed. Furthermore, STA-9090 exhibited superior activity to 17-AAG and SU11654, and was effective against malignant mast cells expressing either WT or mutant Kit. Lastly, STA-9090 inhibited tumor growth in a canine mastocytoma mouse xenograft model. Conclusions STA-9090 exhibits broad activity against mast cells expressing WT or mutant Kit, suggesting it may be an effective agent in the clinical setting against mast cell malignancies.

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Previously, we provided evidence that adenovirus-mediated interferon-beta (IFN-beta) gene therapy inhibits tumor formation and causes dramatic regression of established tumors in immunodeficient mice. We suggested that local IFN-beta gene therapy with adenoviral vectors could be an effective treatment for cancer. In this report, the actions of murine IFN-beta (MuIFN-beta) gene delivery on both subcutaneous and metastatic tumors were evaluated in syngeneic immunocompetent mice. We found that the antitumor response mediated by MuIFN-beta gene delivery relied on CD8(+) T cells but was completely independent of CD4(+) T cells. In fact, depletion of CD4(+) T cells appeared to enhance the effect on tumor inhibition and animal survival induced by adenovirus-MuIFN-beta gene delivery. Therefore, adenovirus-MuIFN-beta gene therapy can bypass CD4(+) T helper (Th) cells and activate an effective CD8(+) T cell-dependent antitumor immunity in immunocompetent mice. Furthermore, we found that depletion of macrophages but not natural killer (NK) cells suppressed the antitumor response induced by MuIFN-beta gene therapy. These data, together with our previous results, suggest that in the clinical setting, local adenovirus-mediated IFN-beta gene therapy may lead to an efficient and long-lasting eradication of tumors by a direct antitumor effect and via activation of the innate and the adoptive immune systems.

Most patients succumbing to colorectal cancer fail with liver-predominant metastases. To make a clinical impact in this disease, a systemic or whole-liver therapy may be required, whereas most cancer gene therapy approaches are limited in their ability to treat beyond local disease. As a preclinical model for cancer gene therapy, recombinant adenovirus containing the human IFN-beta (hIFN-beta) cDNA was delivered systemically in nude mouse xenograft models of human colorectal cancer liver metastases. The vector targeted hepatocytes that produced high levels of hIFN-beta in the liver, resulting in a profound apoptotic response in the tumors and significant tumor regression. hIFN-beta gene therapy not only resulted in improved survival and long-term cure in a micrometastatic model, but provided similar benefits in a clinically relevant gross disease model. A similar recombinant adenovirus containing the murine IFN-beta (mIFN-beta) cDNA also resulted in a therapeutic response and improved survival in syngeneic mouse models of colorectal cancer liver metastases. Depletion studies demonstrate a contribution of natural killer cells to this therapeutic response. The toxicity of an adenoviral vector expressing murine IFN-beta in a syngeneic model is also presented. These encouraging results warrant further investigation of the use of cancer gene therapy for targeting metastatic disease.

Systemic administration of a recombinant adenovirus encoding the human interferon-beta gene (H5.110CMVhIFN-beta) results in transduction of hepatocytes and detectable circulating levels of IFN-beta protein. In preclinical studies in mice, we noticed a distinctly nonlinear dose response, with low levels of virus (1-3 x 10(10) viral particles) yielding barely detectable levels of IFN-beta but with a higher viral dose (1 x 10(11) particles) resulting in disproportionately high IFN-beta levels. Further studies showed that transgene expression levels from low viral doses could be dramatically enhanced by coadministering an unrelated recombinant adenovirus (H5.110CMVlacZ), suggesting that there was a viral dose threshold effect for efficient viral transduction and/or IFN-beta expression. This enhancement of reporter expression by a nonreporter adenovirus, effective upon coadministration, was further enhanced by preadministration of H5.110CMVlacZ (up to 8 h), but was ineffective if the helper virus was administered as little as 5 min after the H5.110CMVhIFN-beta reporter virus. Our data suggest that the reticuloendothelial system plays a role in this threshold effect, such that low doses of virus are efficiently taken up by the RES/Kupffer cells without leading to appreciable transgene expression, whereas high doses saturate these cells and are able to productively transduce hepatocytes. A better understanding of this phenomenon could have an impact on gene therapy clinical trial safety and efficacy.

Despite the potential of type 1 interferons (IFNs) for the treatment of cancer, clinical experience with IFN protein therapy of solid tumors has been disappointing. IFN-β has potent antiproliferative activity against most human tumor cellsin vitroin addition to its known immunomodulatory activities. The antiproliferative effect, however, relies on IFN-β concentrations that cannot be achieved by parenteral protein administration because of rapid protein clearance and systemic toxicities. We demonstrate here thatex vivoIFN-β gene transduction by a replication-defective adenovirus in as few as 1% of implanted cells blocked tumor formation. Directin vivoIFN-β gene delivery into established tumors generated high local concentrations of IFN-β, inhibited tumor growth, and in many cases caused complete tumor regression. Because the mice were immune-deficient, it is likely that the anti-tumor effect was primarily through direct inhibition of tumor cell proliferation and survival. Based on these studies, we argue that local IFN-β gene therapy with replication-defective adenoviral vectors might be an effective treatment for some solid tumors.

Baculovirus vectors recently have been shown to be capable of efficient transduction of human hepatoma cells and primary hepatocytes in culture. This paper describes the generation of a novel recombinant baculovirus (VGZ3) in which the vesicular stomatitis virus glycoprotein G (VSV G) is present in the viral envelope. The gene encoding VSV G was inserted into the baculovirus genome under the control of the polyhedrin promoter such that it was expressed at very high levels in infected insect cells but not in mammalian cells. Expression of the lacZ reporter gene was driven by a promoter that is functional in mammalian cells (the Rous sarcoma virus long terminal repeat). We show by Western analysis that VSV G protein was present in purified baculovirus preparations. A VSV G monoclonal antibody blocked transduction of mammalian cells by VGZ3. This virus was morphologically distinct from baculovirus lacking VSV G, with virions adopting an oval rather than rod-shaped morphology. VGZ3 transduced human hepatoma cells in vitro at an efficiency roughly 10-fold greater than baculovirus lacking VSV G (the virus Z4). VGZ3 was also capable of transducing cell lines that could not be transduced efficiently by Z4. We provide evidence that VSV G protein may enhance transduction by increasing the efficiency of escape of baculovirus from intracellular vesicles rather than by increasing cell binding or uptake of the virus. The possible use of this and related baculoviruses in gene therapy is discussed.

We have compared a series of molecular constructs that contain the minimal DNA-binding and dimerization domain of bovine papillomavirus type 1 (BPV-1) E2 alone or this binding domain plus the adjacent 16 or 40 amino acids to test the role of the flanking sequences in E2 function. The presence of these sequences resulted in an up to eightfold increase in the affinity of E2 for its target DNA and stabilized the protein against denaturation both in the absence of DNA and in the form of DNA-protein complexes. In addition, an aspartic acid-to-tyrosine mutation within the flanking region blocked DNA binding and function. These data demonstrate that sequences flanking the core domain contribute to E2 function and are, in fact, an integral part of the DNA-binding domain of BPV-1 E2.

The papillomavirus E2 protein contains an amino-terminal region thought necessary and sufficient to support transcriptional activation and a carboxy-terminal region shown to direct sequence-specific DNA binding and dimerization. A cysteine residue in the center of the E2 DNA recognition helix is highly conserved among papillomavirus E2 proteins. Mutations of this cysteine in bovine papillomavirus type 1 E2 to serine and glycine resulted in proteins which failed to activate E2dependent promoters in mammalian cells. These E2 mutants were DNA-binding competent, dimeric, and nuclear. When fused to the VP16 transactivation domain, C-terminal regions of E2 containing the mutations at 340 supported transcriptional activation, indicating that the heterologous trans-activation domain did not require cysteine in the DNA-binding helix as did the full-length E2 transactivating protein. Although cysteine-340 was required for transcriptional activation it was not required for DNA replication in vivo. Together, these results suggest that the E2 DNA-binding domain may directly contribute to functions of transcriptional activation previously thought limited to the N-terminal domain. q 1996 Academic Press, Inc.

We have explored the use of cationic liposomes to deliver the human immunodeficiency virus-1 trans-activator protein tat using a reporter gene expression assay. The human epidermoid carcinoma cell A431 stably transfected with a reporter gene under the control of human immunodeficiency virus-1 promoter was used as a target cell. Phosphatidylcholine-containing cationic liposomes had no detectable tat delivery activity. In contrast, delivery of tat was enhanced by up to 150-fold using cationic liposomes enriched with dioleoyl phosphatidylethanolamine (DOPE), a lipid which readily transforms a bilayer into a nonbilayer structure. Enhanced delivery of tat by DOPE-containing liposomes was most likely the result of the endosomolytic activity of the liposome. This phospholipid-rich formulation showed no toxicity at concentrations sufficient for maximal delivery of tat. A variety of cationic liposome formulations which contain DOPE were tested successfully for tat delivery.

Vascular disrupting agents (VDAs) are an emerging class of therapeutics targeting the existing vascular network of solid tumors. However, their clinical progression has been hampered because of limited single-agent efficacy, primarily caused by the persistence of surviving cells at the well perfused "viable rim" of tumors, which allows rapid tumor regrowth to occur. In addition, off-target adverse events, including cardiovascular toxicities, underscore a need for compounds with improved safety profiles. Here, we characterize the mechanism of action, antitumor efficacy, and cardiovascular safety profile of (S)-2-amino-N-(2-methoxy-5-(5-(3,4,5-trimethoxyphenyl)isoxazol-4-yl)phenyl)-3-phenylpropanamide hydrochloride (STA-9584), a novel tubulin-binding VDA. In vitro, 2-methoxy-5-(5-(3,4,5-trimethoxyphenyl)isoxazol-4-yl)aniline (STA-9122) (active metabolite of STA-9584) displayed increased potency relative to other tubulin-binding agents and was highly cytotoxic to tumor cells. STA-9584 induced significant tumor regressions in prostate and breast xenograft models in vivo and, in an aggressive syngeneic model, demonstrated superior tumor growth inhibition and a positive therapeutic index relative to combretastatin A-4 phosphate (CA4P). It is noteworthy that histological analysis revealed that STA-9584 disrupted microvasculature at both the center and periphery of tumors. Compared with CA4P, STA-9584 induced a 73% increase in central necrotic area, 77% decrease in microvasculature, and 7-fold increase in tumor cell apoptosis in the remaining viable rim 24 h post-treatment. Ultrasound imaging confirmed that STA-9584 rapidly and efficiently blocked blood flow in highly perfused tumor regions. Moreover, cardiovascular effects were evaluated in the Langendorff assay and telemetered dogs, and cardiovascular toxicity was not predicted to be dose-limiting. This bioactivity profile distinguishes STA-9584 from the combretastatin class and identifies the compound as a promising new therapeutic VDA candidate.

Purpose: We describe the anticancer activity of ganetespib, a novel non-geldanamycin heat shock protein 90 (HSP90) inhibitor, in non-small cell lung cancer (NSCLC) models. Experimental Design: The activity of ganetespib was compared with that of the geldanamycin 17-AAG in biochemical assays, cell lines, and xenografts, and evaluated in an ERBB2 YVMA-driven mouse lung adenocarcinoma model. Results: Ganetespib blocked the ability of HSP90 to bind to biotinylated geldanamycin and disrupted the association of HSP90 with its cochaperone, p23, more potently than 17-AAG. In genomically defined NSCLC cell lines, ganetespib caused depletion of receptor tyrosine kinases, extinguishing of downstream signaling, inhibition of proliferation and induction of apoptosis with IC50 values ranging 2 to 30 nmol/L, substantially lower than those required for 17-AAG (20–3,500 nmol/L). Ganetespib was also approximately 20-fold more potent in isogenic Ba/F3 pro-B cells rendered IL-3 independent by expression of EGFR and ERBB2 mutants. In mice bearing NCI-H1975 (EGFR L858R/T790M) xenografts, ganetespib was rapidly eliminated from plasma and normal tissues but was maintained in tumor with t1/2 58.3 hours, supporting once-weekly dosing experiments, in which ganetespib produced greater tumor growth inhibition than 17-AAG. However, after a single dose, reexpression of mutant EGFR occurred by 72 hours, correlating with reversal of antiproliferative and proapoptotic effects. Consecutive day dosing resulted in xenograft regressions, accompanied by more sustained pharmacodynamic effects. Ganetespib also showed activity against mouse lung adenocarcinomas driven by oncogenic ERBB2 YVMA. Conclusions: Ganetespib has greater potency than 17-AAG and potential efficacy against several NSCLC subsets, including those harboring EGFR or ERBB2 mutation. Clin Cancer Res; 18(18); 4973–85. ©2012 AACR.

Psoriasis is characterized by hyperplasia of the epidermis and infiltration of leukocytes into both the dermis and epidermis. IL-23, a key cytokine that induces T(H)17 cells, has been found to play a critical role in the pathogenesis of psoriasis. Apilimod is a small-molecule compound that selectively suppresses synthesis of IL-12 and IL-23. An open-label clinical study of oral administration of apilimod was conducted in patients with psoriasis. Substantial improvements in histology and clinical measurements were observed in patients receiving 70 mg QD. The expression of IL-23p19 and IL-12/IL-23p40 in skin lesions was significantly reduced in this dose group, with a simultaneous increase in IL-10 observed. A decrease in the levels of T(H)1 and T(H)17 cytokines/chemokines in skin lesions followed these p19 and p40 changes. In parallel, a reduction in skin-infiltrating CD11c(+) dendritic cells and CD3(+) T cells was seen, with a greater decrease in the CD11c(+) population. This was accompanied by increases in T and B cells, and decreases in neutrophils and eosinophils in the periphery. This study demonstrates the immunomodulatory activity of apilimod and provides clinical evidence supporting the inhibition of IL-12/IL-23 synthesis for the treatment of T(H)1- and T(H)17-mediated inflammatory diseases.

Targeted inhibition of the molecular chaperone Hsp90 results in the simultaneous blockade of multiple oncogenic signaling pathways and has, thus, emerged as an attractive strategy for the development of novel cancer therapeutics. Ganetespib (formerly known as STA-9090) is a unique resorcinolic triazolone inhibitor of Hsp90 that is currently in clinical trials for a number of human cancers. In the present study, we showed that ganetespib exhibits potent in vitro cytotoxicity in a range of solid and hematologic tumor cell lines, including those that express mutated kinases that confer resistance to small-molecule tyrosine kinase inhibitors. Ganetespib treatment rapidly induced the degradation of known Hsp90 client proteins, displayed superior potency to the ansamycin inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG), and exhibited sustained activity even with short exposure times. In vivo, ganetespib showed potent antitumor efficacy in solid and hematologic xenograft models of oncogene addiction, as evidenced by significant growth inhibition and/or regressions. Notably, evaluation of the microregional activity of ganetespib in tumor xenografts showed that ganetespib was efficiently distributed throughout tumor tissue, including hypoxic regions >150 μm from the microvasculature, to inhibit proliferation and induce apoptosis. Importantly, ganetespib showed no evidence of cardiac or liver toxicity. Taken together, this preclinical activity profile indicates that ganetespib may have broad application for a variety of human malignancies, and with select mechanistic and safety advantages over other first- and second-generation Hsp90 inhibitors. Mol Cancer Ther; 11(2); 475–84. ©2011 AACR.

Background The novel water soluble compound STA-1474 is metabolized to ganetespib (formerly STA-9090), a potent HSP90 inhibitor previously shown to kill canine tumor cell lines in vitro and inhibit tumor growth in the setting of murine xenografts. The purpose of the following study was to extend these observations and investigate the safety and efficacy of STA-1474 in dogs with spontaneous tumors. Methods and Findings This was a Phase 1 trial in which dogs with spontaneous tumors received STA-1474 under one of three different dosing schemes. Pharmacokinetics, toxicities, biomarker changes, and tumor responses were assessed. Twenty-five dogs with a variety of cancers were enrolled. Toxicities were primarily gastrointestinal in nature consisting of diarrhea, vomiting, inappetence and lethargy. Upregulation of HSP70 protein expression was noted in both tumor specimens and PBMCs within 7 hours following drug administration. Measurable objective responses were observed in dogs with malignant mast cell disease (n = 3), osteosarcoma (n = 1), melanoma (n = 1) and thyroid carcinoma (n = 1), for a response rate of 24% (6/25). Stable disease (>10 weeks) was seen in 3 dogs, for a resultant overall biological activity of 36% (9/25). Conclusions This study provides evidence that STA-1474 exhibits biologic activity in a relevant large animal model of cancer. Given the similarities of canine and human cancers with respect to tumor biology and HSP90 activation, it is likely that STA-1474 and ganetespib will demonstrate comparable anti-cancer activity in human patients.

The papillomavirus E2 protein is a DNA binding protein that regulates viral transcription and replication. E2 binds DNA as a dimer. Recent crystallographic data for E2 complexed to DNA revealed that novel peptide structures in E2 mediated dimerization and DNA binding. To identify important features of these motifs we have used limited proteolysis and urea denaturation as biochemical probes for structure, applying these techniques to E2 alone, E2 bound to DNA, cross-linked products, and mutants that were targeted at Trp360, a contact point along the dimer interface. DNA binding stabilized E2 structure, shifting the point at which it denatures from 5 to 7.6 M urea. In contrast, Trp360 mutant proteins, while dimeric, were more sensitive to denaturation by urea when bound to DNA. The most striking results came from uv cross-linking studies in which Trp360 was targeted as the site of cross-linking. Ultraviolet cross-linking dramatically increased the resistance of E2 to proteolysis regardless of the protease tested and with no deleterious effect on the affinity of E2 for DNA. Cross-linking through Cys356 with bismaleimidohexane did not promote stabilization. The ability to stabilize or destabilize E2 by Trp360-targeted modifications demonstrates the importance of the Trp360-Trp360 interaction, which may represent a general feature of the β-barrel motif.

Osteosarcoma (OSA), the most common malignant bone tumor in dogs and children, exhibits a similar clinical presentation and molecular biology in both species. Unfortunately, 30-40% of children and 90% of dogs still die of disease despite aggressive therapy. The purpose of this study was to test the biologic activity of a novel heat shock protein 90 (HSP90) inhibitor, STA-1474, against OSA. Canine and human OSA cell lines and normal canine osteoblasts were treated with STA-1474 and evaluated for effects on proliferation (CyQuant), apoptosis (Annexin V, PARP cleavage, caspase 3/7 activation) and known HSP90 client proteins. HSP90 was immunoprecipitated from normal and malignant osteoblasts and Western blotting for co-chaperones was performed. Mice bearing canine OSA xenografts were treated with STA-1474, and tumors samples were evaluated for caspase-3 activation and loss of p-Akt/Akt. Treatment with STA-1474 promoted loss of cell viability, inhibition of cell proliferation and induction of apoptosis in OSA cell lines. STA-1474 and its active metabolite STA-9090 also demonstrated increased potency compared to 17-AAG. STA-1474 exhibited selectivity for OSA cells versus normal canine osteoblasts, and HSP90 co-precipitated with co-chaperones p23 and Hop in canine OSA cells but not in normal canine osteoblasts. Furthermore, STA-1474 downregulated the expression of p-Met/Met, p-Akt/Akt and p-STAT3. Finally, STA-1474 induced tumor regression, caspase-3 activation and downregulation of p-Met/Met and p-Akt/Akt in OSA xenografts. Together, these data suggest that HSP90 represents a relevant target for therapeutic intervention in OSA.

A new gene transfer protocol has been developed that introduces up to 800 copies of an expression vector into Chinese hamster ovary cells in a single step by electroporation. The DNA typically integrates in tandem repeats so that the restriction endonuclease site used to linearize the input DNA remains intact. This is likely due to ligation of vector DNA via cohesive ends prior to integration. This high-copy-number procedure is far more rapid than the conventional stepwise gene amplification method used to generate stable eukaryotic protein production cell lines. By employing the expression vector pJODtPA, in which the selectable marker dihydrofolate reductase (DHFR) and the human tissue plasminogen activator (tPA) casettes are separated by a spacer and an RNA polymerase II terminator, cell lines secreting as much as 24 pg/cell.day tPA were isolated following electroporation and a single methotrexate selection. Gene copies and expression levels are stable over long periods of growth. A single round of gene amplification was performed following the high-copy-number procedure to yield a clone having a tPA production level of 45 pg/cell.day.

The interleukin-12 (IL-12) cytokine induces the differentiation of naive T cells to the T helper cell type 1 (Th1) phenotype and is integral to the pathogenesis of Th1-mediated immunologic disorders. A more recently discovered IL-12 family member, IL-23, shares the p40 protein subunit with IL-12 and plays a critical role in the generation of effector memory T cells and IL-17–producing T cells. We introduce a novel compound, STA-5326, that down-regulates both IL-12 p35 and IL-12/IL-23 p40 at the transcriptional level, and inhibits the production of both IL-12 and IL-23 cytokines. Oral administration of STA-5326 led to a suppression of the Th1 but not Th2 immune response in mice. In vivo studies using a CD4+CD45Rbhigh T-cell transfer severe combined immunodeficiency (SCID) mouse inflammatory bowel disease model demonstrated that oral administration of STA-5326 markedly reduced inflammatory histopathologic changes in the colon. A striking decrease in interferon-γ (IFN-γ) production was observed in ex vivo culture of lamina propria cells harvested from animals treated with STA-5326, indicating a down-regulation of the Th1 response by STA-5326. These results suggest that STA-5326 has potential for use in the treatment of Th1-related autoimmune or immunologic disorders. STA-5326 currently is being evaluated in phase 2 clinical trials in patients with Crohn disease and rheumatoid arthritis.

Preclinical in vitro and in vivo studies have demonstrated synergistic interactions between 5-fluorouracil (5-FU) and type I and II IFNs against human colorectal cancer cells. Despite these activities, randomized human trials have failed to identify a clinical benefit for this combination treatment. These limited clinical results may be secondary to the short half-life of recombinant IFN protein and the increased systemic toxicities of 5-FU/IFN combinations. We have previously reported an adenoviral-mediated IFN-β gene therapy strategy, which may circumvent the pitfalls of recombinant IFN therapy. However, a dose-dependent toxicity and acute inflammatory response to systemically administered adenovirus vectors may limit the clinical application of this therapy. The combination of adenoviral-mediated IFN-β gene therapy and 5-FU resulted in tumor regression, apoptosis, and improved survival in an established liver metastases model. These therapeutic effects were observed at a significantly lower vector dose than we had previously reported and with limited toxicity. This approach may allow for an effective clinical application of this therapy and warrants additional investigation.

Type I IFNs are known to inhibit tumor cell growth and stimulate the immune system. However, little is known of the mechanism of type I IFN-induced apoptosis in human cancer cells. In this study, we have IFN-beta treatment of a human colorectal cell line (KM12L4) and a resistant clone of this cell line, L4RIFN. We demonstrate the induction of apoptosis in the parent cell line. This process was associated with the induction of the Jak-Stat signaling pathway, induction of the proapoptotic mediator tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and activation of procaspase-3, -8, -9, and -10. Additionally, we evaluated the role of Stat1 in mediating IFN-beta induction of these proapoptotic signals in a fibrosarcoma cell line (2ftgh) and a Stat1-deficient clone (U3A). Our results demonstrate that IFN-beta induction of apoptosis and the induction of proapoptotic mediator TRAIL is Stat1 dependent. Evaluation of a stable transfectant of the KM12L4 cell line expressing c-FLIP supports the role of TRAIL and the cell-surface death signaling pathways in IFN-beta induction of apoptosis. Studies evaluating the TRAIL promoter indicate induction of TRAIL promoter activity by IFN-beta. These results may represent a novel pathway by which IFN-beta may induce therapeutic effects.

Interferon-beta (IFN-beta) induces aberrant cell cycle progression as well as cytotoxicity and apoptosis. However, the relationship between the cell cycle alteration and the induction of cytotoxicity/apoptosis is unknown. Here, we report the first demonstration that the IFN-beta-induced direct cytotoxic/apoptotic effect can be separated functionally from its cell cycle effect. By using lentiviral transduction, we generated human tumor cells that stably expressed IFN-beta and were resistant to its direct cytotoxic/apoptotic effect. Despite this resistance to apoptosis, these cells showed significant S phase accumulation as measured by both FACS analyses and bromodeoxyuridine (BrdU) incorporation. Although the cells proliferated in the presence of high levels of IFN-beta, they had lost their tumorigenicity in mice. A portion of these cells was observed to undergo a tumor cell-specific senescence. Therefore, our study revealed a direct tumor-suppressor function of IFN-beta. This tumor-suppressor function was independent of IFN-beta-induced direct cytotoxic effect. It was also distinct from the IFN-beta-induced immunologic antitumor response, an indirect effect of IFN-beta. We conclude that the antiproliferative effect on human tumor cells is the collective activities of the direct cytotoxic/apoptotic effect, the cell cycle alteration that occurs as predominantly S phase accumulation, and less frequently other cell cycle effects, such as G(1) arrest, and the promotion of tumor cells into a senescent-like state.

Previously, we suggested that local human interferon-beta (IFN-beta) gene therapy with replication-defective adenoviral vectors can be an effective cancer treatment. Clinical trials to treat cancers with adenovirus expressing the human IFN-beta gene (IFNB1) has been planned. As a continued effort to explore the mechanisms of action of human IFN-beta gene therapy that can occur in the clinical setting, we tested mouse IFN-beta gene therapy in human xenograft tumors in both ex vivo and in vivo models. Delivery of the mouse IFN-beta gene (Ifnb) caused tumor inhibition; this effect was dependent on the indirect anti-tumor activities of IFN-beta, notably a stimulation of natural killer cells. IFN-beta does not show cross-species activity in its anti-proliferative effect and mouse IFN-beta does not cause as significant an anti-proliferative effect on mouse tumor cells as human IFN-beta causes on human tumor cells. Therefore, we believe that mouse models using either human IFN-beta or mouse IFN-beta gene transfer do not capture all aspects of the action of adenovirus-mediated human IFN-beta gene therapy that may be present in the clinical setting. Due to its multiple mechanisms of action, human IFN-beta gene therapy may be effective in treating human cancers that are either sensitive or resistant to the direct anti-proliferative effect of IFN-beta.