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Recent insights regarding mechanisms mediating stemness, heterogeneity, and metastatic potential of lung cancers have yet to be fully translated to effective regimens for the treatment of these malignancies. This study sought to identify novel targets for lung cancer therapy.

The 90 kD heat shock proteins (Hsp90) are molecular chaperones that are responsible for the folding of select proteins, many of which are directly associated with cancer progression. Consequently, inhibition of the Hsp90 protein folding machinery results in a combinatorial attack on numerous oncogenic pathways. Seventeen small-molecule inhibitors of Hsp90 have entered clinical trials for the treatment of cancer, all of which bind the Hsp90 N-terminus and exhibit pan-inhibitory activity against all four Hsp90 isoforms, which may lead to adverse effects. The development of Hsp90 isoform-selective inhibitors represents an alternative approach toward the treatment of cancer and may limit some of these detriments. Described herein, is a structure-based approach to develop isoform-selective inhibitors of Hsp90β, which induces the degradation of select Hsp90 clients without concomitant induction of Hsp90 levels. Together, these initial studies support the development of Hsp90β-selective inhibitors as a method for overcoming the detriments associated with pan-inhibition.

Platelets play a pivotal role in hemostasis, wound healing, and inflammation, and are thus implicated in a variety of diseases, including cancer. Platelet function is associated with release of granule content, cellular shape change, and upregulation of receptors that promote establishment of a thrombus and maintenance of hemostasis.

Hepatocyte growth factor (HGF) receptor Met and hypoxia-inducible factor-1 (HIF-1) signaling pathways are commonly activated in aggressive tumors and promote progression. Since both Met and HIF-1α proteins are heat shock protein (Hsp) 90 clients, Hsp90 inhibitors might be expected to positively impact tumor progression. Here, we systematically evaluated the inhibitory effects of the prototypical Hsp90 inhibitor geldanamycin (GA) on cellular processes involved in invasion and angiogenesis in T24 bladder cancer cells stimulated with HGF and chemical hypoxia. First, we demonstrated the positive feedback loop between Met and HIF-1 pathways, which serves to sustain and amplifies their signaling in T24 cells. GA down-regulated Met by inhibiting new protein maturation, thereby dampening HGF signaling. HGF and chemical hypoxia with CoCl2 cooperatively promoted in vitro invasion and vascular endothelial growth factor (VEGF) secretion, while CoCl2 but not HGF activated urokinase-type plasminogen activator and matrix metalloproteinase 2, both of which promote invasion and angiogenesis. Low dose GA (100 nmol/L) inhibited these processes by suppressing both HGF and HIF-1 pathways. Notably, brief GA pretreatment inhibited in vitro invasion and VEGF secretion induced by HGF as effectively as did continuous treatment. Moreover, we found that GA inhibited activation of focal adhesion kinase, focal adhesion assembly, and actin reorganization induced by HGF and integrin engagement by extracellular matrix. Thus, GA widely suppresses extrinsic stimuli-induced signaling that contribute to tumor invasion and angiogenesis in this bladder carcinoma model, suggesting the utility of Hsp90 inhibitors in preventing tumor progression and metastasis.

HIF-1α is a normally labile proangiogenic transcription factor that is stabilized and activated in hypoxia. Although the von Hippel Lindau (VHL) gene product, the ubiquitin ligase responsible for regulating HIF-1α protein levels, efficiently targets HIF-1α for rapid proteasome-dependent degradation under normoxia, HIF-1α is resistant to the destabilizing effects of VHL under hypoxia. HIF-1α also associates with the molecular chaperone Hsp90. To examine the role of Hsp90 in HIF-1α function, we used renal carcinoma cell (RCC) lines that lack functional VHL and express stable HIF-1α protein under normoxia. Geldanamycin (GA), an Hsp90 antagonist, promoted efficient ubiquitination and proteasome-mediated degradation of HIF-1α in RCC in both normoxia and hypoxia. Furthermore, HIF-1α point mutations that block VHL association did not protect HIF-1α from GA-induced destabilization. Hsp90 antagonists also inhibited HIF-1α transcriptional activity and dramatically reduced both hypoxia-induced accumulation of VEGF mRNA and hypoxia-dependent angiogenic activity. These findings demonstrate that disruption of Hsp90 function 1) promotes HIF-1α degradation via a novel, oxygen-independent E3 ubiquitin ligase and 2) diminishes HIF-1α transcriptional activity. Existence of an Hsp90-dependent pathway for elimination of HIF-1α predicts that Hsp90 antagonists may be hypoxic cell sensitizers and possess antiangiogenic activityin vivo, thus extending the utility of these drugs as therapeutic anticancer agents.

The wild type p53 tumor suppressor protein is rapidly degraded in normal cells by MDM2, the ubiquitin ligase that serves as the key regulator of p53 function by modulating protein stability. Cellular exposure to genotoxic stress triggers the stabilization of p53 by multiple pathways that converge upon interference with MDM2 function. In this study, we first investigated the ability of HDM2 (MDM2 human homologue) to degrade endogenous p53 in neuroblastoma (NB). Although the p53 protein in NB has been reported to be constitutively stabilized, we find that HDM2 in NB is functional and facilitates the rapid turnover of p53 in nonstressed cells via the proteasome pathway. Second, we examined the relationship between p53 and HDM2 in the adriamycin-mediated stabilization of p53 in NB. We demonstrate that while p53 stabilization depends neither upon the phosphorylation of specific N-terminal sites nor upon dissociation from HDM2, it requires inactivation of functional HDM2. In support of this notion, p53 stabilization following adriamycin resulted in an inhibition of both p53 ubiquitination and HDM2 ligase activity. Taken together, these data implicate a requirement for enzymatic inactivation of HDM2 as a novel mechanism for p53 stabilization in the DNA damage response pathway.

Heat shock protein 90 (Hsp90), one of the most abundant chaperones in eukaryotes, participates in folding and stabilization of signal-transducing molecules including steroid hormone receptors and protein kinases. The amino terminus of Hsp90 contains a non-conventional nucleotide-binding site, related to the ATP-binding motif of bacterial DNA gyrase. The anti-tumor agents geldanamycin and radicicol bind specifically at this site and induce destabilization of Hsp90-dependent client proteins. We recently demonstrated that the gyrase inhibitor novobiocin also interacts with Hsp90, altering the affinity of the chaperone for geldanamycin and radicicol and causing in vitroand in vivodepletion of key regulatory Hsp90-dependent kinases including v-Src, Raf-1, and p185ErbB2. In the present study we used deletion/mutation analysis to identify the site of interaction of novobiocin with Hsp90, and we demonstrate that the novobiocin-binding site resides in the carboxyl terminus of the chaperone. Surprisingly, this motif also recognizes ATP, and ATP and novobiocin efficiently compete with each other for binding to this region of Hsp90. Novobiocin interferes with association of the co-chaperones Hsc70 and p23 with Hsp90. These results identify a second site on Hsp90 where the binding of small molecule inhibitors can significantly impact the function of this chaperone, and they support the hypothesis that both amino- and carboxyl-terminal domains of Hsp90 interact to modulate chaperone activity.

Chronic myelogenous leukemia (CML) is a clonal disorder of a pluripotent hematopoietic stem cells characterized by a chimericbcr-abl gene giving rise to a p210Bcr-Ablprotein with dysregulated tyrosine kinase activity. Radicicol, a macrocyclic antifungal antibiotic, binds to the N-terminal of heat shock protein 90 (Hsp90) and destabilizes Hsp90-associated proteins such as Raf-1. This study investigated the effect of radicicol, novel oxime derivatives of radicicol (KF25706 and KF58333), and herbimycin A (HA), a benzoquinoid ansamycin antibiotic, on the growth and differentiation of human K562 CML cells. Although KF25706 and KF58333 induced the expression of glycophorin A in K562 cells, radicicol and HA caused erythroid differentiation transiently. Cell cycle analysis showed that G1 phase accumulation was observed in K562 cells treated with KF58333. KF58333 treatment depleted p210Bcr-Abl, Raf-1, and cellular tyrosine phosphorylated proteins in K562 cells, whereas radicicol and HA showed transient depletion of these proteins. KF58333 also down-regulated the level of cell cycle–dependent kinases 4 and 6 and up-regulated cell cycle–dependent kinase inhibitor p27Kip1protein without an effect on the level of Erk and Hsp90 proteins. Immunoprecipitation analysis showed that p210Bcr-Abl formed multiple complexes with Hsp90, some containing p23 and others Hsp70; KF58333 treatment dissociated p210Bcr-Abl from Hsp90/p23 chaperone complexes. Furthermore, KF58333 induced apoptosis in K562 cells and administration of KF58333 prolonged the survival time of SCID mice inoculated with K562 cells. These results suggest that KF58333 may have therapeutic potential for the treatment of CML that involves abnormal cellular proliferation induced by p210Bcr-Abl.

Chronic myelogenous leukemia (CML) is a clonal disorder of a pluripotent hematopoietic stem cells characterized by a chimericbcr-ablgene giving rise to a p210Bcr-Ablprotein with dysregulated tyrosine kinase activity. Radicicol, a macrocyclic antifungal antibiotic, binds to the N-terminal of heat shock protein 90 (Hsp90) and destabilizes Hsp90-associated proteins such as Raf-1. This study investigated the effect of radicicol, novel oxime derivatives of radicicol (KF25706 and KF58333), and herbimycin A (HA), a benzoquinoid ansamycin antibiotic, on the growth and differentiation of human K562 CML cells. Although KF25706 and KF58333 induced the expression of glycophorin A in K562 cells, radicicol and HA caused erythroid differentiation transiently. Cell cycle analysis showed that G1phase accumulation was observed in K562 cells treated with KF58333. KF58333 treatment depleted p210Bcr-Abl, Raf-1, and cellular tyrosine phosphorylated proteins in K562 cells, whereas radicicol and HA showed transient depletion of these proteins. KF58333 also down-regulated the level of cell cycle–dependent kinases 4 and 6 and up-regulated cell cycle–dependent kinase inhibitor p27Kip1protein without an effect on the level of Erk and Hsp90 proteins. Immunoprecipitation analysis showed that p210Bcr-Ablformed multiple complexes with Hsp90, some containing p23 and others Hsp70; KF58333 treatment dissociated p210Bcr-Ablfrom Hsp90/p23 chaperone complexes. Furthermore, KF58333 induced apoptosis in K562 cells and administration of KF58333 prolonged the survival time of SCID mice inoculated with K562 cells. These results suggest that KF58333 may have therapeutic potential for the treatment of CML that involves abnormal cellular proliferation induced by p210Bcr-Abl.

• Whole blood serotonin concentrations were studied in 33 chronic schizophrenic patients who previously had computed tomographic (CT) brain scans and in 23 healthy volunteers. The chronic schizophrenic patients had a mean serotonin concentration significantly higher than that of the controls. The patients were subcategorized into a group with abnormal CT scan findings (enlargement of cerebral ventricles, cerebral atrophy, or both) and a group with normal CT scans. The patients with abnormal CT scans had significantly higher serotonin concentrations when compared with schizophrenics with normal CT scans and with controls. The chronic schizophrenic patients with normal CT scans did not have significantly elevated serotonin concentrations compared with controls. Furthermore, ventricular size in the total patient group was significantly correlated with serotonin concentration.

The Raf-1-MEK-MAPK pathway plays an important role in transducing extracellular growth factor signaling into altered nuclear transcription factor function. The benzoquinone ansamycin Geldanamycin (GA) specifically binds to the heat shock protein HSP90 and alters its complex with Raf-1. This leads to a decrease in Raf-1 levels and to disruption of the Raf-1-MEK-MAPK signaling pathway. The enhanced degradation of Raf-1 protein was prevented by inhibitors of the proteasome, while inhibition of lysosomal or other proteases was ineffective. Raf-1 that was protected from GA-induced degradation was of higher molecular weight and showed a laddering pattern consistent with its polyubiquitination. Unlike Raf-1 in untreated cells, the protein was insoluble in Triton X100- or NP40-based buffers. Signaling through this pathway was inhibited by GA, concomitant with loss of Raf-1 protein, but was restored if Raf-1 was protected from GA-induced degradation by proteasome inhibitors.

Abstract: Purpose: Benzoquinone ansamycins are antibiotics with anticancer potential. First described as tyrosine kinase inhibitors, they are now frequently used to target HSP90 chaperone function. While herbimycin A and geldanamycin (GA) have been widely used in preclinical studies, both drugs are poor candidates for clinical trials owing to their in vivo toxicity and lack of stability. We therefore examined the biologic effects of 17-allylamino-17-demethoxygeldanamycin (17-AG), an ansamycin derivative with lower in vivo toxicity than GA. Methods: Binding of 17-AG to HSP90 was studied in vitro using a GA-affinity beads competition assay. We analyzed the drug-induced destabilization of p185erbB2, Raf-1 and mutant p53 in SKBR3 breast cancer cells by Western blotting. The antiproliferative activities of 17-AG and GA were compared using the MTT assay. Results: We found that, in a similar manner to GA itself, 17-AG bound specifically to HSP90. It also led to degradation of the receptor tyrosine kinase p185erbB2, the serine/threonine kinase Raf-1 and mutant p53. Both GA and 17-AG displayed comparable antiproliferative effects in SKBR3 and MCF7 cells. Even though HSP90 binding by 17-AG was weaker than by GA, 17-AG and GA caused biologic effects in tumor cells at similar doses. Conclusion: 17-AG shares the important biologic features of its parent compound GA. Since 17-AG has a better toxicity profile than GA, it is an interesting candidate benzoquinone ansamycin for clinical development.

B cell neoplasms are clonal expansions of B lymphocytes thought to be frozen at various points along the normal B cell differentiation pathway. We studied cell suspensions from lymph nodes involved by follicular (nodular) non-Hodgkin' x2018;s lymphoma to determine the capacity of the malignant B cells to secrete immunoglobulin (Ig). Neoplastic B cells from all 14 follicular lymphomas secreted mono-clonal immunoglobulin in culture when appropriate signals were provided. In most cases, maximal Ig secretion occurred when autologous T cells were removed by E rosette depletion, replaced with allogeneic normal T cells, and the cultures were exposed to 12-O-tetradecanoylphor-bol-13-acetate. Autologous T cells exerted a suppressor effect on Ig secretion in 8/8 cases studied, diminishing the response of the malignant B cells to allogeneic T cells. This suppressor effect did not correlate with the percentage of cells staining with anti-Leu-2a or with “helper-suppressor” (Leu-3a-Leu-2a) ratios of the lymph node T cells. Our findings demonstrate that the arrested differentiation of most follicular lymphomas is reversible and implicate a T cell-mediated host immunoregulatory mechanism affecting Ig secretion in vivo. An additional contribution of our results is the demonstration of a cell culture system for synthesis of sufficient monoclonal Ig for use as an immunogen in production of anti-idiotype antibodies. © 1985 by Grune & Stratton, Inc.

Many functions of the chaperone, heat shock protein 90 (hsp90), are inhibited by the drug geldanamycin that specifically binds hsp90. We have studied an amino-terminal domain of hsp90 whose crystal structure has recently been solved and determined to contain a geldanamycin-binding site. We demonstrate that, in solution, drug binding is exclusive to this domain. This domain also binds ATP linked to Sepharose through the γ-phosphate. Binding is specific for ATP and ADP and is inhibited by geldanamycin. Mutation of four glycine residues within two proposed ATP binding motifs diminishes both geldanamycin binding and the ATP-dependent conversion of hsp90 to a conformation capable of binding the co-chaperone p23. Since p23 binding requires regions outside the 1–221 domain of hsp90, these results indicate a common site for nucleotides and geldanamycin that regulates the conformation of other hsp90 domains.

The Ewing's sarcoma family of tumors (ESFT) is the second most common pediatric malignancy originating in the bone and is characterized by the t(11; 22) translocation. PAX3, a member of the paired box family of genes, is expressed during embryonal development of neural crest cells and is involved in the t(2; 13) translocation found in alveolar rhabdomyosarcoma. Since ESFTs are believed to be derived from neural crest tissue, we screened a series of Ewing's sarcoma and peripheral neuroectodermal tumor cell lines and tumor specimens for expression of PAX3. We found expression of PAX3 in most, but not all, of the specimens analyzed, including cell lines and patient material.

β Plasma phenylalanine, tyrosine, and tryptophan concentrations were measured in chronic schizophrenic patients, normal controls, and heterozygotes for phenylketonuria. Schizophrenic patients'plasma concentrations of these amino acids could not be distinguished from those of normal controls, either when fasting or following oral or intravenous (IV) phenylalanine challenge. No neuroleptic effect was observed. Plasma phenylalanine-tyrosine ratios following IV phenylalanine challenge could easily distinguish heterozygotes from schizophrenic and normal control subjects but could not distinguish schizophrenic subjects from normal control subjects. No overlap between heterozygotes' values and those of the schizophrenic and normal subjects was observed. These studies find no evidence of abnormal phenylalanine metabolism in schizophrenic persons. Phenylalanine challenge did not affect the abstraction or judgment capacities of the subjects.

The expression of the interleukin-2 (IL-2) receptor was studied in neoplastic cells derived from acute leukemias, T-cell lymphoblastic lymphomas, peripheral T-cell lymphomas, chronic lymphocytic leukemias, well-differentiated lymphocytic lymphomas, and established cell lines by both flow cytometric analysis and sodium dodecyl sulfate/ polyacrylamide gel electrophoresis (SDS-PAGE) after affinity crosslinking of radiolabeled IL-2. Cells from most acute leukemias (19 of 22), irrespective of their subtype (T, common or nonlymphoid leukemias), as well as T-cell lymphoblastic lymphomas and peripheral T-cell lymphomas expressed only the p70-75 β subunit of the IL-2 receptor. Cells from the more mature B-cell neoplasms, chronic lymphocytic leukemia, and well-differentiated lymphocytic lymphoma, expressed predominately αβ IL-2 receptors (11 of 14). In contrast to these results, most cell lines established from hematopoietic malignancies do not express either chain of the IL-2 receptor. Further studies are necessary to determine the exact function of the IL-2R p70-75 β subunit in immature hematopoietic cells, but its wide distribution throughout the hematopoietic system suggests that IL-2 may play a role in the early stages of hematopoiesis.© 1989 by Grune & Stratton, Inc. 0006-4971/89/7307-0038$3.00/0

Initiation of T-lymphocyte proliferation by mitogen or antigen involves a cascade of gene activation events. Thus, by the time mitogen-activated T cells have reached the G1/S interface, many genes that are transcriptionally silent in GO, like the c-myc, IL-2, IL-2 receptor (IL-2R) and transferrin receptor (TfR) genes, have been transcriptionally activated1–4. To understand the role of the individual genes in the activation process, one must be able to interfere specifically with the expression or function of each particular gene product. In this way, by blocking the IL-2R with an antibody, it has been demonstrated that IL-2/IL-2R interaction is required to induce TfR expression in activated T cells3. When the function or expression of intracellular proteins is to be blocked, however, the need to introduce antibodies into the cytoplasm of viable cells, although possible5–7, is a limiting factor. We have taken another approach, namely the exogenous addition to bulk cell cultures of small antisense oligomers. Sequence-specific anti-sense oligodeoxyribonucleotides have been reported to inhibit intracellular viral replication without interfering with cellular protein synthesis8,9. Similarly, rabbit globin mRNA translation in a cell-free system and in rabbit reticulocytes has been inhibited by oligomers complementary to the globin mRNA initiation codon region10. Recently, a pentadecadeoxyribonucleotide complementary to the initiation codon and four downstream codons of human c-myc mRNA was reported to inhibit the proliferation of the human leukaemic cell line HL-60 specifically11. We report here that the same c-myc complementary oligonuelectide inhibits mitogen-induced c-myc protein expression in human T lymphocytes and prevents S phase entry. Interestingly, c-myc antisense treatment did not inhibit G0to G1traversal as assessed by morphologic blast transformation, transcriptional activation of the IL-2R and TfR genes, or induction of 3H-uridine incorporation.

The abundant, cytoplasmic 90-kDa heat-shock protein associates transiently with the Rous sarcoma virus oncogenic protein tyrosine kinase, pp60v-src, directs its cellular trafficking and negatively regulates its kinase activity. Here we report that the serine/threonine phosphatase inhibitor, okadaic acid, destabilized the heat-shock protein 90-pp60v-srcchaperone complex in v-src-transfected cells. Concomitant with complex destabilization by okadaic acid, phosphoserine was doubled and phosphothreonine was increased 20-fold in the heat-shock protein 90. Although phosphorylation of the total pool of immunoprecipitable pp60v-srcwas unchanged, okadaic acid slightly increased phosphoserine and phosphothreonine levels specifically in pp60v-srcbound to heat-shock protein 90. The low level of tyrosine phosphorylation in the pp60v-srccomplexed with heat-shock protein 90 was further decreased by okadaic acid. Interestingly, okadaic acid-stabilized hyperphosphorylation of the heat-shock protein 90-pp60v-srccomplex lowered the level of pp60v-srcin cell membranes, the functional location for pp60v-src. We suggest that serine/threonine phosphorylation of heat-shock protein 90 and/or pp60v-srcfunctions as a regulatory molecular trigger to release pp60v-srcfrom the chaperone complex at the inner surface of cell membranes.

The reliance of many cancers on aerobic glycolysis has stimulated efforts to develop lactate dehydrogenase (LDH) inhibitors. However, despite significant efforts, LDH inhibitors (LDHi) with sufficient specificity and in vivoactivity to determine whether LDH is a feasible drug target are lacking. We describe an LDHi with potent, on-target, in vivoactivity. Using hyperpolarized magnetic resonance spectroscopic imaging (HP-MRSI), we demonstrate in vivoLDH inhibition in two glycolytic cancer models, MIA PaCa-2 and HT29, and we correlate depth and duration of LDH inhibition with direct anti-tumor activity. HP-MRSI also reveals a metabolic rewiring that occurs in vivowithin 30 min of LDH inhibition, wherein pyruvate in a tumor is redirected toward mitochondrial metabolism. Using HP-MRSI, we show that inhibition of mitochondrial complex 1 rapidly redirects tumor pyruvate toward lactate. Inhibition of both mitochondrial complex 1 and LDH suppresses metabolic plasticity, causing metabolic quiescence in vitroand tumor growth inhibition in vivo.