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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.

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.

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.

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.

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.

Expression of two types of transactivation-defective E2F1 mutants in human Rb-/- tumor cells led to an increase in the proportion of cells in the G1 phase of the cell cycle as determined by FACS analysis. Experiments revealed two different mechanisms of action. One mutant type induced a G1 arrest after the restriction point, with cells phenotypically at a cell cycle stage later than G1. The action of this mutant was, at least in part, dependent on specific DNA binding and was over-ridden by co-expression of its wild-type counterpart. The other mutant type, which is defective in DNA binding, slowed the G1 progression and restored a checkpoint for cell cycle withdrawal. The G1 phase withdrawal of these tumor cells allowed the initiation of skeletal muscle cell differentiation. Thus, E2F1 appears to have two different functions before and after the cell cycle restriction point. This report also may provide a basis for a gene therapy approach for certain human cancers.

We have previously shown that the tat protein of HIV-1 can be used as a carrier to promote the intracellular delivery of heterologous proteins. Here we have tested if the tat-delivery technology can be used to direct MHC class I presentation of native protein, using ovalbumin (OVA) as a model system. We show that a tat-ovalbumin conjugate (tatOVA) can be delivered into cells and that subsequent processing and presentation occurs, resulting in effective and specific killing of these target cells by an OVA specific cytotoxic T-lymphocyte (CTL) line. Comparison with the E.G7 line that expresses the OVA gene indicates that tat-mediated delivery is as efficient as endogenous expression in this system. Tat-mediated antigenic protein delivery may be useful both as a research technique and, potentially, as a therapeutic or prophylactic vaccine.

The human immunodeficiency virus-1 transactivator protein (tat) was codelivered efficiently with a reporter gene under the control of a tat-responsive DNA element using different formulations of cationic liposomes. Expression of a tat-responsive reporter gene was induced by incubating cells with a mixture of purified recombinant tat protein, reporter DNA, and liposomes. Different cell lines were tested successfully as targets for the codelivery. Tat was shown to trans-activate the codelivered virus promoter specifically in the cells tested. Codelivery of tat with DNA is a useful model for studying the function of trans-acting factors and their cis-acting DNA elements. The currently available methods such as foot-printing only reveal the binding, but not the functional consequence of the binding, of the factor with the element. In addition, this system may prove useful as a model for high level and regulated transgene expression in target cells.

Papillomaviruses are the causative agents of benign and malignant epithelial tumors of the skin and mucosa. They encode a DNA-binding protein, E2, that regulates viral transcription and replication, making it an important therapeutic target. By deleting the amino-terminal trans-activation domain of human papillomavirus type 16 (HPV-16) E2 while retaining its carboxy-terminal DNA binding and dimerization domain, an E2 repressor (E2R) that efficiently inhibits transcriptional activation by full-length HPV E2 was generated. To deliver this repressor protein into animal cells, we have utilized the human immunodeficiency virus type 1 (HIV-1) Tat protein which itself is taken up efficiently into intact cells. Chimeras of E2R and the cellular uptake domain of Tat specifically inhibited E2-dependent reporter gene expression in COS-7 cells. Treatment of cervical intraepithelial neoplasia cells having episomally replicating HPV-31 DNA with this Tat-E2R protein led to a dose-dependent loss of HPV DNA copies and inhibition of cell growth. Tat-mediated delivery can be a valuable tool for assessing protein function and may allow the development of novel therapeutic proteins having intracellular targets.

The Tat protein of human immunodeficiency virus 1 (HIV-1) can enter cells efficiently when added exogenously in tissue culture. To assess if Tat can carry other molecules into cells, we chemically cross-linked Tat peptides (residues 1-72 or 37-72) to beta-galactosidase, horseradish peroxidase, RNase A, and domain III of Pseudomonas exotoxin A (PE) and monitored uptake colorimetrically or by cytotoxicity. The Tat chimeras were effective on all cell types tested, with staining showing uptake into all cells in each experiment. In mice, treatment with Tat-beta-galactosidase chimeras resulted in delivery to several tissues, with high levels in heart, liver, and spleen, low-to-moderate levels in lung and skeletal muscle, and little or no activity in kidney and brain. The primary target within these tissues was the cells surrounding the blood vessels, suggesting endothelial cells, Kupffer cells, and/or splenic macrophages. Tat-mediated uptake may allow the therapeutic delivery of macromolecules previously thought to be impermeable to living cells.

There is accumulating evidence that dysregulated JAK signaling occurs in a wide variety of cancer types. In particular, mutations in JAK2 can result in the constitutive activation of STAT transcription factors and lead to oncogenic growth. JAK kinases are established Hsp90 client proteins and here we show that the novel small molecule Hsp90 inhibitor ganetespib (formerly STA-9090) exhibits potent in vitro and in vivo activity in a range of solid and hematological tumor cells that are dependent on JAK2 activity for growth and survival. Of note, ganetespib treatment results in sustained depletion of JAK2, including the constitutively active JAK2(V617F) mutant, with subsequent loss of STAT activity and reduced STAT-target gene expression. In contrast, treatment with the pan-JAK inhibitor P6 results in only transient effects on these processes. Further differentiating these modes of intervention, RNA and protein expression studies show that ganetespib additionally modulates cell cycle regulatory proteins, while P6 does not. The concomitant impact of ganetespib on both cell growth and cell division signaling translates to potent antitumor efficacy in mouse models of xenografts and disseminated JAK/STAT-driven leukemia. Overall, our findings support Hsp90 inhibition as a novel therapeutic approach for combating diseases dependent on JAK/STAT signaling, with the multimodal action of ganetespib demonstrating advantages over JAK-specific inhibitors.

Heat shock protein 90 (Hsp90) is a molecular chaperone that regulates the post-translational folding of its protein substrates (“client proteins”). Cancer cells contain elevated levels of active Hsp90 and, because many client proteins play critical oncogenic roles, cancer cells are especially sensitive to Hsp90 inhibition. Here we report on the initial characterization of STA-9090, a highly potent Hsp90 inhibitor that is currently in multiple Phase 1/2 clinical trials in solid tumor and hematological malignancies. STA-9090 is a small molecule drug that is structurally unrelated to the ansamycin Hsp90 inhibitor 17-AAG and binds the N-terminal ATP-binding pocket of the chaperone. In vitro, treatment with STA-9090 rapidly induced the degradation of known Hsp90 client proteins, such as HER2 and KIT, and growth inhibition IC50 values typically ranged from 1 to 100 nM. STA-9090 demonstrated, on average, ∼30-fold greater potency than 17-AAG for the ∼60 hematological and solid tumor cell lines tested. STA-9090 also retained its potency against cell lines expressing mutated kinases that confer resistance to kinase inhibitors such as erlotinib and imatinib. In vivo, STA-9090 demonstrated single-agent activity in a wide variety of human tumor cell line subcutaneous xenograft models in mice, including those representing solid tumor malignancies such as gastric carcinoma, non-small cell lung cancer, prostate carcinoma and melanoma, and hematological malignancies such as acute myeloid leukemia, B-cell lymphoma, chronic myeloid leukemia and multiple myeloma. In a mouse leukemia model in which wild type BCR-ABL or imatinib/dasatinib-resistant BCR-ABLT315I was introduced into mouse bone marrow cells and then transplanted into host mice to induce the development of B-cell acute lymphoblastic leukemia, STA- 9090 prolonged average survival from 27 to 37 days for BCR-ABL and from 29 to 57 days for BCR-ABLT315I. STA-9090 also accumulated in tumors, with a half-life of 58 hr in tumors versus 3–5 hr in plasma and non-tumor tissues. To examine the potential for therapeutic synergy, STA-9090 was combined with the microtubule stabilizer paclitaxel. STA-9090 dramatically synergized with paclitaxel in in vitro cytotoxicity assays, particularly when paclitaxel treatment preceded treatment with STA-9090. Similarly, STA-9090 enhanced the activity of paclitaxel in the erlotinib-resistant NCI-H1975 lung cancer xenograft model, although this enhancement depended less on the order of dosing than was observed in vitro. No significant pharmacokinetic interactions were observed between the two agents. In conclusion, STA-9090 is a highly potent Hsp90 inhibitor that in vitro and in vivo rapidly induces the degradation of Hsp90 client proteins and apoptosis in a broad range of cancer types, including those resistant to targeted kinase inhibitors. It also demonstrates synergy with paclitaxel. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B199.

Background: Heat shock protein 90 (Hsp90) is a molecular chaperone that is required for the stability and function of many important signal transduction proteins that regulate the growth of cancer cells. Hsp90 inhibition results in ubiquitination and proteasomal degradation of these client proteins, which include clinically validated drug targets such as BCR-ABL, mutant EGFR, HER2, KIT and VEGFR. STA-9090 is a novel small molecule Hsp90 inhibitor that is currently in multiple Phase 1/2 clinical trials in solid tumor and hematological malignancies. STA-9090 is structurally unrelated to the first-generation anasamycin Hsp90 inhibitors 17-AAG and IPI-504 and inhibits Hsp90 by binding to its N-terminal ATP-binding pocket. Although Hsp90 inhibitors such as STA-9090 induce rapid client protein degradation, cell cycle arrest and apoptosis of cancer cells, it is possible that frequent drug dosing in the clinic may be needed to continuously maintain decreased client protein expression and avoid renewed tumor growth. To investigate this possibility, we conducted in vitro and in vivo studies using the human NCI-H1975 non-small cell lung cancer cell line, which expresses the Hsp90 client protein EGFRL858R/T790M, a mutationally activated and erlotinib-resistant form of the epidermal growth factor receptor. Results: In an in vitro cytotoxicity assay using this cell line, STA-9090 and 17-AAG displayed IC50 values of 10 and 40 nM after 72 hr drug exposure, respectively. These results closely correlated with decreased expression of EGFRL858R/T790M and other Hsp90 client proteins. Unexpectedly, exposure to STA-9090 for only 1 hr still resulted in an IC50 of 670 nM, suggesting that even brief drug exposure in vivo may be sufficient to affect tumor growth. Consistent with this, intravenous dosing of 125 mg/kg STA-9090 on a 1X/week × 3 week schedule (∼80–100% of the highest non-severely toxic dose) induced stable disease in a NCI-H1975 xenograft model, whereas 175 mg/kg 17-AAG resulted in progressive disease, with %T/C values of 15 and 50, respectively. Inhibition of tumor growth was correlated with decreased expression of EGFRL858R/T790M and other client proteins, and importantly, these effects persisted in tumors for 3–6 days after a single drug dose. Similarly, histological analysis of tumors indicated that STA-9090 inhibited cell proliferation by 7-fold and induced apoptosis by 9-fold, with maximal effects being observed at 1–3 days after treatment. Consistent with these observations, STA-9090 accumulated in tumors relative to normal tissues, with a tumor half-life of 58 hr versus 3–5 hr in liver, lung and plasma, and the tumor concentration remained 140-fold higher than the in vitro IC50 (72 hr) even 6 days after a single drug dose. Conclusions: Taken together, these results demonstrate that STA-9090 is a highly potent Hsp90 inhibitor that selectively accumulates in tumors and induces long-lasting client protein degradation, cell cycle arrest, increased apoptosis and tumor growth inhibition in a lung cancer xenograft model. Our results suggest that an infrequent dosing schedule may have clinical activity in cancer patients. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C91.

To determine whether HIV-1 tat can transactivate a heterologous promoter lacking HIV sequences other than the TAR element, TAR was placed downstream of the chicken beta-actin promoter. Tat increased expression directed by the actin-TAR promoter to a degree equal to tat induction of the HIV-1 LTR. Optimal transactivation was observed when TAR was positioned downstream of the actin promoter such that the expected cap site of transcripts from this promoter would be the same as in transcripts directed by the HIV-1 LTR. Tat was able to transactivate, though to a lesser extent, a promoter consisting solely of a TATA element fused to TAR. Thus, tat induction does not require HIV-1 LTR promoter sequences other than TAR. Tat, when fused to the DNA binding domain of BPV-1 E2, was able to transactivate a truncated SV40 promoter containing upstream E2 binding sites, indicating that tat may be capable of transactivation when directed by a DNA binding protein to an upstream site in a heterologous promoter lacking all HIV sequences. Substitution of Ala for Lys at position 41 of tat in the tat-E2 fusion, a mutation which dramatically decreases tat transactivation of the HIV-1 LTR, eliminated this transactivation.

Sundin and Varshavsky (J. Mol. Biol. 132:535-546, 1979) found that nearly two-thirds of simian virus 40 (SV40) minichromosomes obtained from nuclei of SV40-infected cells become singly nicked or cleaved across both strands after digestion with staphylococcal nuclease at 0 degrees C. The same treatment of SV40 DNA causes complete digestion rather than the limited cleavages produced in minichromosomal DNA. We have explored this novel behavior of the minichromosome and found that the nuclease sensitivity is dependent upon the topology of the DNA. Thus, if minichromosomes are pretreated with wheat germ DNA topoisomerase I, the minichromosomal DNA is completely resistant to subsequent digestion with staphylococcal nuclease at 0 degrees C. If the minichromosome-associated topoisomerase is removed, virtually all of the minichromosomes are cleaved to nicked or linear structures by the nuclease treatment. The cleavage sites are nonrandomly located; instead they occur at discrete loci throughout the SV40 genome. SV40 minichromosomal DNA is also cleaved to nicked circles and full-length linear fragments after treatment with the single strand-specific endonuclease S1; this cleavage is also inhibited by pretreatment with topoisomerase I. Thus, it may be that the nuclease sensitivity of minichromosomes is due to the transient or permanent unwinding of discrete regions of their DNA. Direct comparisons of the extent of negative supercoiling of native and topoisomerase-treated SV40 minichromosomes revealed that approximately two superhelical turns were removed by the topoisomerase treatment. The loss of these extra negative supercoils from the DNA probably accounts for the resistance of the topoisomerase-treated minichromosomes to the staphylococcal and S1 nucleases. These findings suggest that the DNA in SV40 intranuclear minichromosomes is torsionally strained. The functional significance of this finding is discussed.

A new approach was developed for analysis of complex mixtures of staphylococcal nuclease-produced nucleosomes, consisting of two-dimensional electrophoretic fractionation of nucleosomes (low ionic strength electrophoresis of deoxyribo-nucleoproteins in the first dimension followed by DNA electrophoresis in the second dimension), with subsequent electrophoretic transfer of nucleosomal DNA from polyacrylamide gels to DBM † paper for hybridization with specific DNA probes. Hybridization was accompanied by parallel two-dimensional analysis of protein composition of mononucleosomes from HeLa chromatin. The major results are: 1. (1) The H1-lacking mononucleosomes containing proteins HMG 14 and HMG 17 migrate as a broad band in the first dimension, and their DNA fragments form an identifiable subset of the total mononucleosomal DNA pattern. 2. (2) Hybridization of a highly repetitive human DNA fragment to fractionated HeLa mononucleosomal DNA lights up all major mononucleosomal DNA spots, suggesting that transcriptionally inactive regions of HeLa chromatin contain both A24 semihistone and HMG proteins. 3. (3) Hybridization of cloned ribosomal DNA to fractionated HeLa mononucleosomal DNA also lights up all major DNA spots. The regions corresponding to A24- and HMG-containing mononucleosomes, however, appear to be under-represented in the hybridization pattern. 4. (4) A fragment of highly repetitive rat DNA hybridizes to a subset of the total oligonucleosomal DNA pattern from rat 14B chromatin, strongly suggesting that the range of nucleosomal DNA repeat lengths within regions of chromatin homologous to this highly repetitive probe is much narrower than that in bulk chromatin. Our findings indicate that specific interactions of HMG proteins with transcriptionally active regions of chromatin (Weisbrod et al. , 1980) are superimposed over a significant background of apparently non-specific HMG chromatin interactions. We suggest that the process of transcription itself is responsible for “saturation” of a chromatin region with HMG proteins in vitro .

A new method for the isolation of simian virus 40 nucleoprotein complexes from nuclei of lytically infected cells is described. The method is based on the addition of a thiol-specific reagent, 5'5'-dithiobis(2-nitrobenzoic acid), to lysis and extraction buffers. By inhibiting an uncoating activity during simian virus 40 extraction, 5'5'-dithiobis (2-nitrobenzoic acid) allows the use of efficient extraction buffers, such as one containing Triton X-100 and EDTA, for the isolation of native simian virus 40 minichromosomes and virion-type structures. Use of the method is illustrated by following encapsidation of simian virus 40 minichromosomes in a pulse-chase experiment. Since 5'5'-dithiobis (2-nitrobenzoic acid) is an inhibitor of many different enzymes, the 5',5'-dithiobis (2-nitrobenzoic acid) extraction technique may be useful for the isolation of not only papovaviruses but also other viruses and possibly cellular chromatin.

Previous work has shown that the presence of a phorbol ester tumor promoter, phorbol 12-myristate 13-acetate (PMA), during a single-step selection for methotrexate (MTX)-resistant mouse 3T6 cells results in an up to 100-fold increase in the incidence of MTX-resistant, colony-forming cells. MTX resistance of most of these cells is due to amplification of the gene for dihydrofolate reductase (DHFR), the target enzyme for MTX. We show here that other active, noncytotoxic phorbol ester tumor promoters, such as phorbol 12, 13-didecanoate and 20-phorbol 12,13-butyrate, at their optimal concentrations (approximately equal to 0.1 microM) are approximately equal to PMA in increasing the incidence of MTX-resistant 3T6 colonies. Mezerein, a potent second-stage tumor promoter, but a weak complete promoter, increases the incidence of MTX resistance up to 350-fold, the strongest effect for any of the agents so far tested. PMA analogs that are inactive as tumor promoters, such as phorbol or phorbol 12,13,20-triacetate, have no effect on the incidence of MTX-resistant 3T6 colonies. Anthralin, a nonphorbol tumor promoter, is approximately equal to 40% as active as PMA in the MTX selection assay. Remarkably, the hormones insulin, arginine vasopressin, and epidermal growth factor, all of which are mitogenic for 3T6 cells, also exert a strong PMA-like effect on the incidence of MTX-resistant 3T6 colonies under conditions of MTX selection. The effect of insulin at its optimal concentration (approximately equal to 1 microgram/ml) is approximately equal to 70% that of PMA. Although the effect of PMA on the incidence of MTX-resistant 3T6 colonies does not significantly depend on the initial density of seeded cells or volume of the medium added, the analogous effect of insulin is strongly influenced by these parameters. Mevalonic acid, arachidonic acid, thymidine, caffeine, and nicotine, all of which are known to influence patterns of DNA synthesis in mammalian cells, were tested at their highest noncytotoxic concentrations and failed to produce any significant effect on the incidence of MTX-resistant 3T6 colonies. We discuss possible mechanisms of hormone- and tumor promoter-facilitated gene amplification in mammalian cells, relationship of mitogenic hormones to tumor promoters, and also implications of our findings for the problem of drug resistance in cancer chemotherapy.

Repression of papillomavirus E2-dependent gene expression was studied by using transient transfections into mouse embryo fibroblast cells. Cotransfection of a gene corresponding to the naturally occurring repressor E2-TR along with the full-length E2 gene resulted in up to 98% repression of E2-dependent reporter gene expression. A series of E2 DNA-binding domain mutants were transferred into the E2-TR form and characterized for their ability to repress E2-dependent transactivation. All mutants which were defective for DNA binding but were dimerization competent repressed E2 transactivation as well or nearly as well as the wild-type repressor. E2 mutants which lacked dimerization activity repressed transactivation poorly or not at all. These results indicate that the E2 repressor can inhibit transcription, in the absence of DNA binding, by forming heterodimers with full-length E2.