A Novel Irreversible TEAD Inhibitor, SWTX-143, Blocks Hippo Pathway Transcriptional Output and Causes Tumor Regression in Preclinical Mesothelioma Models.

The Hippo pathway and its downstream effectors, the YAP and TAZ transcriptional coactivators, are deregulated in multiple different types of human cancer and are required for cancer cell phenotypes in vitro and in vivo, while largely dispensable for tissue homeostasis in adult mice. YAP/TAZ and their main partner transcription factors, the TEAD1-4 factors, are therefore promising anticancer targets. Because of frequent YAP/TAZ hyperactivation caused by mutations in the Hippo pathway components NF2 and LATS2, mesothelioma is one of the prime cancer types predicted to be responsive to YAP/TAZ-TEAD inhibitor treatment. Mesothelioma is a devastating disease for which currently no effective treatment options exist. Here, we describe a novel covalent YAP/TAZ-TEAD inhibitor, SWTX-143, that binds to the palmitoylation pocket of all four TEAD isoforms. SWTX-143 caused irreversible and specific inhibition of the transcriptional activity of YAP/TAZ-TEAD in Hippo-mutant tumor cell lines. More importantly, YAP/TAZ-TEAD inhibitor treatment caused strong mesothelioma regression in subcutaneous xenograft models with human cells and in an orthotopic mesothelioma mouse model. Finally, SWTX-143 also selectively impaired the growth of NF2-mutant kidney cancer cell lines, suggesting that the sensitivity of mesothelioma models to these YAP/TAZ-TEAD inhibitors can be extended to other tumor types with aberrations in Hippo signaling. In brief, we describe a novel and specific YAP/TAZ-TEAD inhibitor that has potential to treat multiple Hippo-mutant solid tumor types.

Principles of cargo attachment to cytoplasmic motor proteins.

The attachment of cytoskeletal motor proteins to cargo-laden vesicles is crucial for efficient transport in intracellular membrane trafficking pathways. Recent studies have identified specific kinesin-binding and dynein-binding proteins that could serve as membrane-associated 'receptor' proteins for the respective motors. New insights have also emerged about the cargo-binding domains of the motor proteins, and the regulation of motor binding to cargoes.

Molecular characterization of type I IFN-induced cytotoxicity in bladder cancer cells reveals biomarkers of resistance.

Although anti-tumor activities of type I interferons (IFNs) have been recognized for decades, the molecular mechanisms contributing to clinical response remain poorly understood. The complex functions of these pleiotropic cytokines include stimulation of innate and adaptive immune responses against tumors as well as direct inhibition of tumor cells. In high-grade, Bacillus Calmette-Guérin (BCG)-unresponsive non-muscle-invasive bladder cancer, nadofaragene firadenovec, a non-replicating adenovirus administered locally to express the IFNα2b transgene, embodies a novel approach to deploy the therapeutic activity of type I IFNs while minimizing systemic toxicities. Deciphering which functions of type I IFN are required for clinical activity will bolster efforts to maximize the efficacy of nadofaragene firadenovec and other type I IFN-based therapies, and inform strategies to address resistance. As such, we characterized the phenotypic and molecular response of human bladder cancer cell lines to IFNα delivered in multiple contexts, including adenoviral delivery. We found that constitutive activation of the type I IFN signaling pathway is a biomarker for resistance to both transcriptional response and direct cytotoxic effects of IFNα. We present several genes that discriminate between sensitive and resistant tumor cells, suggesting they should be explored for utility as biomarkers in future clinical trials of type I IFN-based anti-tumor therapies.

The high-affinity HSP90-CHIP complex recognizes and selectively degrades phosphorylated tau client proteins.

A primary pathologic component of Alzheimer's disease (AD) is the formation of neurofibrillary tangles composed of hyperphosphorylated tau (p-tau). Expediting the removal of these p-tau species may be a relevant therapeutic strategy. Here we report that inhibition of Hsp90 led to decreases in p-tau levels independent of heat shock factor 1 (HSF1) activation. A critical mediator of this mechanism was carboxy terminus of Hsp70-interacting protein (CHIP), a tau ubiquitin ligase. Cochaperones were also involved in Hsp90-mediated removal of p-tau, while those of the mature Hsp90 refolding complex prevented this effect. This is the first demonstration to our knowledge that blockade of the refolding pathway promotes p-tau turnover through degradation. We also show that peripheral administration of a novel Hsp90 inhibitor promoted selective decreases in p-tau species in a mouse model of tauopathy, further suggesting a central role for the Hsp90 complex in the pathogenesis of tauopathies. When taken in the context of known high-affinity Hsp90 complexes in affected regions of the AD brain, these data implicate a central role for Hsp90 in the development of AD and other tauopathies and may provide a rationale for the development of novel Hsp90-based therapeutic strategies.

A high-affinity conformation of Hsp90 confers tumour selectivity on Hsp90 inhibitors.

Heat shock protein 90 (Hsp90) is a molecular chaperone that plays a key role in the conformational maturation of oncogenic signalling proteins, including HER-2/ErbB2, Akt, Raf-1, Bcr-Abl and mutated p53. Hsp90 inhibitors bind to Hsp90, and induce the proteasomal degradation of Hsp90 client proteins. Although Hsp90 is highly expressed in most cells, Hsp90 inhibitors selectively kill cancer cells compared to normal cells, and the Hsp90 inhibitor 17-allylaminogeldanamycin (17-AAG) is currently in phase I clinical trials. However, the molecular basis of the tumour selectivity of Hsp90 inhibitors is unknown. Here we report that Hsp90 derived from tumour cells has a 100-fold higher binding affinity for 17-AAG than does Hsp90 from normal cells. Tumour Hsp90 is present entirely in multi-chaperone complexes with high ATPase activity, whereas Hsp90 from normal tissues is in a latent, uncomplexed state. In vitro reconstitution of chaperone complexes with Hsp90 resulted in increased binding affinity to 17-AAG, and increased ATPase activity. These results suggest that tumour cells contain Hsp90 complexes in an activated, high-affinity conformation that facilitates malignant progression, and that may represent a unique target for cancer therapeutics.

BIIB021, an orally available, fully synthetic small-molecule inhibitor of the heat shock protein Hsp90.

Inhibition of heat shock protein 90 (Hsp90) results in the degradation of oncoproteins that drive malignant progression, inducing cell death, making Hsp90 a target of substantial interest for cancer therapy. BIIB021 is a novel, fully synthetic inhibitor of Hsp90 that binds competitively with geldanamycin in the ATP-binding pocket of Hsp90. In tumor cells, BIIB021 induced the degradation of Hsp90 client proteins including HER-2, AKT, and Raf-1 and up-regulated expression of the heat shock proteins Hsp70 and Hsp27. BIIB021 treatment resulted in growth inhibition and cell death in cell lines from a variety of tumor types at nanomolar concentrations. Oral administration of BIIB021 led to the degradation of Hsp90 client proteins measured in tumor tissue and resulted in the inhibition of tumor growth in several human tumor xenograft models. Studies to investigate the antitumor effects of BIIB021 showed activity on both daily and intermittent dosing schedules, providing dose schedule flexibility for clinical studies. Assays measuring the HER-2 protein in tumor tissue and the HER-2 extracellular domain in plasma were used to show interdiction of the Hsp90 pathway and utility as potential biomarkers in clinical trials for BIIB021. Together, these data show that BIIB021 is a promising new oral inhibitor of Hsp90 with antitumor activity in preclinical models.

Pharmacological induction of the heat shock response improves myelination in a neuropathic model.

Misexpression and intracellular retention of peripheral myelin protein 22 (PMP22) is associated with hereditary neuropathies in humans, including Charcot-Marie-Tooth disease type 1A (CMT1A). Mice expressing extra copies of the human PMP22, termed C22, display morphologic and behavioral characteristics of CMT1A. In neuropathic Schwann cells, the turnover of the newly-synthesized PMP22 is decreased, leading to the formation of cytosolic protein aggregates. To aid the processing of PMP22 and alleviate the associated myelin defects, we pharmacologically stimulated the expression of protein chaperones by synthetic small-molecule inhibitors of heat shock protein 90 (HSP90). The exposure of Schwann cells to these compounds enhanced the levels of cytosolic chaperones in a time- and dose-dependent manner, with minimal cytotoxicity. Treatment of dorsal root ganglion (DRG) explants from neuropathic mice improved myelin formation and the processing of PMP22. These results warrant further studies with HSP90 inhibitors as potential therapeutic candidates for hereditary demyelinating neuropathies.

Additive effect of simultaneous continuous administration of degarelix and TAK-448 on LH suppression in a castrated rat model.

Gonadotropin releasing hormone (GnRH) analogs have long been used in androgen deprivation therapy (ADT) in the treatment of prostate cancer. Chronic administration of either GnRH agonists or antagonists leads to suppression of testosterone production in the testes via either downregulation or direct blockade of the GnRH receptor in the pituitary, respectively. Chronic administration of kisspeptin analogs has more recently been shown to lead to testosterone suppression via desensitization of GnRH neurons in the hypothalamus and an optimized kisspeptin analog, TAK-448, was proven effective in a small phase 1 trial. The current study explored the hypothesis that co-administration of TAK-448 and the GnRH antagonist, degarelix, would have an additive effect on hormonal suppression, as a result of simultaneous intervention in separate steps in the same pathway. TAK-448 or degarelix were first administered individually to castrated rats in order to identify low doses capable of partial or no suppression of luteinizing hormone (LH). In the second step, combinations of the low doses of TAK-448 and degarelix were assessed in a 14 day study and compared to the drugs administered separately. The results showed that simultaneous intervention at the kisspeptin and GnRH receptors caused a more pronounced LH suppression than either drug alone, demonstrating an additive or potentiating effect. These results suggest that such a drug combination may hold promise as novel forms of androgen deprivation therapy in the treatment of prostate cancer.

Rationally designed high-affinity 2-amino-6-halopurine heat shock protein 90 inhibitors that exhibit potent antitumor activity.

Heat shock protein 90 (Hsp90) is a molecular chaperone protein implicated in stabilizing the conformation and maintaining the function of many cell-signaling proteins. Many oncogenic proteins are more dependent on Hsp90 in maintaining their conformation, stability, and maturation than their normal counterparts. Furthermore, recent data show that Hsp90 exists in an activated form in malignant cells but in a latent inactive form in normal tissues, suggesting that inhibitors selective for the activated form could provide a high therapeutic index. Hence, Hsp90 is emerging as an exciting new target for the treatment of cancer. We now report on a novel series of 2-amino-6-halopurine Hsp90 inhibitors exemplified by 2-amino-6-chloro-9-(4-iodo-3,5-dimethylpyridin-2-ylmethyl)purine (30). These highly potent inhibitors (IC50 of 30 = 0.009 microM in a HER-2 degradation assay) also display excellent antiproliferative activity against various tumor cell lines (IC50 of 30 = 0.03 microM in MCF7 cells). Moreover, this class of inhibitors shows higher affinity for the activated form of Hsp90 compared to our earlier 8-sulfanylpurine Hsp90 inhibitor series. When administered orally to mice, these compounds exhibited potent tumor growth inhibition (>80%) in an N87 xenograft model, similar to that observed with 17-allylamino-17-desmethoxygeldanamycin (17-AAG), which is a compound currently in phase I/II clinical trials.

HSP induction mediates selective clearance of tau phosphorylated at proline-directed Ser/Thr sites but not KXGS (MARK) sites.

Neurofibrillary tangles (NFTs) are a characteristic neuropathological feature of Alzheimer's disease (AD), and molecular chaperones appear to be involved in the removal of disease-associated hyperphosphorylated tau, a primary component of NFTs. Here, novel HSP90 inhibitors were used to examine the impact of chaperone elevation on clearance of different tau species in transfected cells using a unique quantitative assay. The HSP90 inhibitors reduced levels of tau phosphorylated at proline-directed Ser/Thr sites (pS202/T205, pS396/S404) and conformationally altered (MC-1) tau species, an epitope that is immeasurable by standard Western blot techniques. The selective clearance of these phospho-tau species and MC-1 tau was mediated via the proteasome, while lysosomal-mediated tau degradation seems to lack specificity for certain tau species, suggesting a more general role in total tau removal. Interestingly, tau phosphorylated at S262/S356 within the tau microtubule binding domain was minimally affected by chaperone induction. Overall, our data show that chaperone induction results in the selective clearance of specific phospho-tau and conformationally altered tau species mediated by the proteasome; however, the apparent stability of pS262/S356 tau may also explain why MARK is able to regulate normal tau function yet still be linked to the initiation of pathogenic tau hyperphosphorylation in AD.

Preclinical Evaluation of MEDI0641, a Pyrrolobenzodiazepine-Conjugated Antibody-Drug Conjugate Targeting 5T4.

Antibody-drug conjugates (ADC) are used to selectively deliver cytotoxic agents to tumors and have the potential for increased clinical benefit to cancer patients. 5T4 is an oncofetal antigen overexpressed on the cell surface in many carcinomas on both bulk tumor cells as well as cancer stem cells (CSC), has very limited normal tissue expression, and can internalize when bound by an antibody. An anti-5T4 antibody was identified and optimized for efficient binding and internalization in a target-specific manner, and engineered cysteines were incorporated into the molecule for site-specific conjugation. ADCs targeting 5T4 were constructed by site-specifically conjugating the antibody with payloads that possess different mechanisms of action, either a DNA cross-linking pyrrolobenzodiazepine (PBD) dimer or a microtubule-destabilizing tubulysin, so that each ADC had a drug:antibody ratio of 2. The resulting ADCs demonstrated significant target-dependent activity in vitro and in vivo; however, the ADC conjugated with a PBD payload (5T4-PBD) elicited more durable antitumor responses in vivo than the tubulysin conjugate in xenograft models. Likewise, the 5T4-PBD more potently inhibited the growth of 5T4-positive CSCs in vivo, which likely contributed to its superior antitumor activity. Given that the 5T4-PBD possessed both potent antitumor activity as well as anti-CSC activity, and thus could potentially target bulk tumor cells and CSCs in target-positive indications, it was further evaluated in non-GLP rat toxicology studies that demonstrated excellent in vivo stability with an acceptable safety profile. Taken together, these preclinical data support further development of 5T4-PBD, also known as MEDI0641, against 5T4+ cancer indications. Mol Cancer Ther; 16(8); 1576-87. ©2017 AACR.

Orally active purine-based inhibitors of the heat shock protein 90.

Orally active Hsp90 inhibitors are of interest as potential chemotherapeutic agents. Recently, fully synthetic 8-benzyladenines and 8-sulfanyladenines such as 4 were disclosed as Hsp90 inhibitors, but these compounds are not water soluble and consequently have unacceptably low oral bioavailabilities. We now report that water-solubility can be achieved by inserting an amino functionality in the N(9) side chain. This results in compounds that are potent, soluble in aqueous media, and orally bioavailable. In an HER-2 degradation assay, the highest potency was achieved with the neopentylamine 42 (HER-2 IC(50) = 90 nM). In a murine tumor xenograft model (using the gastric cancer cell line N87), the H(3)PO(4) salts of the amines 38, 39, and 42 induced tumor growth inhibition when administered orally at 200 mg/kg/day. The amines 38, 39, and 42 are the first Hsp90 inhibitors shown to inhibit tumor growth upon oral dosage.

7'-substituted benzothiazolothio- and pyridinothiazolothio-purines as potent heat shock protein 90 inhibitors.

We report on the discovery of benzo- and pyridino- thiazolothiopurines as potent heat shock protein 90 inhibitors. The benzothiazole moiety is exceptionally sensitive to substitutions on the aromatic ring with a 7'-substituent essential for activity. Some of these compounds exhibit low nanomolar inhibition activity in a Her-2 degradation assay (28-150 nM), good aqueous solubility, and oral bioavailability profiles in mice. In vivo efficacy experiments demonstrate that compounds of this class inhibit tumor growth in an N87 human colon cancer xenograft model via oral administration as shown with compound 37 (8-(7-chlorobenzothiazol-2-ylsulfanyl)-9-(2-cyclopropylamino-ethyl)-9H- purin-6-ylamine).

Inhibition of neuroblastoma xenograft growth by Hsp90 inhibitors.

BACKGROUND: Advanced-stage neuroblastomas are often resistant to chemotherapy. Heat shock protein (Hsp) 90 is a molecular chaperone that maintains the stability of important signal transduction proteins. We have previously reported that geldanamycin (GA), an Hsp90 inhibitor, decreases Raf-1 and Akt protein expressions and induces apoptosis in neuroblastoma cells. We sought to determine the in vivo effects of Hsp90 inhibitor compounds on human neuroblastomas. MATERIALS AND METHODS: Human neuroblastoma (LAN-1 and SK-N-SH) xenografts (4-mm3 tumor implants) were placed in the flanks of athymic nude mice. The mice received either Hsp90 inhibitors (17-AAG or EC5) or vehicle (control). The tumor dimensions were measured twice weekly. Proteins were extracted for Western immunoblotting. RESULTS: Hsp90 inhibitor compounds significantly blocked both LAN-1 and SK-N-SH neuroblastoma growth in vivo. Drug-treated tumors showed decreases in Raf-1 and cleaved PARP expressions. CONCLUSION: Hsp90 inhibitors may prove to be important novel therapeutic agents for patients with advanced-stage neuroblastoma who fail to respond to current treatment regimens.

Structure-Cytotoxicity Relationships of Analogues of N14-Desacetoxytubulysin H.

Herein we report structure-cytotoxicity relationships for analogues of N14-desacetoxytubulyisn H 1. A novel synthetic approach toward 1 enabled the discovery of compounds with a range of activity. Calculated basicity of the N-terminus of tubulysins was shown to be a good predictor of cytotoxicity. The impact of structural modifications at the C-terminus of 1 upon cytotoxicity is also described. These findings will facilitate the development of new tubulysin analogues for the treatment of cancer.

Rational design, biophysical and biological characterization of site-specific antibody-tubulysin conjugates with improved stability, efficacy and pharmacokinetics.

Antibody-drug conjugates (ADCs) are among the most promising empowered biologics for cancer treatment. ADCs are commonly prepared by chemical conjugation of small molecule cytotoxic anti-cancer drugs to antibodies through either lysine side chains or cysteine thiols generated by the reduction of interchain disulfide bonds. Both methods yield heterogeneous conjugates with complex biophysical properties and suboptimal serum stability, efficacy, and pharmacokinetics. To limit the complexity of cysteine-based ADCs, we have engineered and characterized in vitro and in vivo antibody cysteine variants that allow precise control of both site of conjugation and drug load per antibody molecule. We demonstrate that the chemically-defined cysteine-engineered antibody-tubulysin conjugates have improved ex vivo and in vivo stability, efficacy, and pharmacokinetics when compared to conventional cysteine-based ADCs with similar drug-to-antibody ratios. In addition, to limit the non-target FcγRs mediated uptake of the ADCs by cells of the innate immune system, which may result in off-target toxicities, the ADCs have been engineered to lack Fc-receptor binding. The strategies described herein are broadly applicable to any full-length IgG or Fc-based ADC and have been incorporated into an ADC that is in phase I clinical development.

Therapeutic and diagnostic implications of Hsp90 activation.

The molecular chaperone heat-shock protein 90 (Hsp90) is involved in the stabilization and conformational maturation of many signaling proteins that are deregulated in cancers. Hsp90 inhibition results in the proteasomal degradation of these client proteins and leads to potent antitumor activity. The Hsp90 inhibitor 17-allylaminogeldanamycin (17-AAG) is presently in clinical trials. Recent work has identified the role of Hsp90 in multiple signal transduction pathways and revealed that the molecular mechanism of tumor selectivity by Hsp90 inhibitors is the result of an activated, high-affinity conformation of Hsp90 in tumors. This review discusses these recent advances in the understanding of tumor Hsp90 for the treatment and diagnosis of cancer. In addition, the role of Hsp90 in non-oncological diseases will also be discussed.

Development of a high throughput drug screening assay for the detection of changes in tau levels -- proof of concept with HSP90 inhibitors.

Therapeutic development for Alzheimer's disease has largely focused on the removal of beta amyloid because of its suggested role in the primary agent in initiating the disease process. However, with the recent discovery of mutations that result as pathologic buildup of tau in the absence of amyloid pathology, tau is beginning to be recognized as a potential target for drug discovery. We have developed a high-throughput drug screening method that allows for direct intracellular quantitation of tau protein species, enabling the fast, reliable detection of these changes. We have identified a family of small, blood brain barrier penetrant heat shock protein 90 inhibitors that significantly reduce tau protein levels in vitro. Western blot analysis demonstrated a clear inverse correlation between the tau levels and the increase in HSP27, HSP40 and HSP90. Modifications to this assay will further allow the specific analysis of pathologically relevant species. Using this assay, we have demonstrated that a class of HSP90 inhibitors is able to significantly lower intracellular tau levels most likely through induction of a heat shock response.

Synthesis and biological evaluation of a new class of geldanamycin derivatives as potent inhibitors of Hsp90.

The heat shock protein Hsp90 has increasingly become an important therapeutic target especially for treatment of cancers. Inhibition of the ATPase activity of Hsp90 by natural products (e.g., 17-allylaminogeldanamycin or radicicol) leads to the ubiquitination of oncogenic client proteins such as Her-2, Raf-1, and p-Akt followed by their proteasomal degradation. Hsp90 inhibitors simultaneously target multiple oncogenic proteins and provide an advantage for cancer therapy due to the potential for increased efficacy and overcoming drug resistance. In an effort to convert geldanamycin into a druglike compound with better pharmacokinetic properties and efficacy in human tumor xenograft models, geldanamycin was derivatized on the 17-position to prepare new analogues such as 17-geldanamycin amides, carbamates, and ureas and 17-arylgeldanamycins. All the compounds were first evaluated ex vivo using a cell-based Her-2 degradation assay and in vitro using biochemical assays that measure recombinant Hsp90 (rHsp90) competitive binding and changes in rHsp90 conformation. In addition, we confirmed the selectivity of geldanamycin analogues for Hsp90 derived from tumor cells using a novel cell lysate binding assay.

Novel role for NFAT3 in ERK-mediated regulation of CXCR4.

The G-protein coupled chemokine (C-X-C motif) receptor CXCR4 is linked to cancer, HIV, and WHIM (Warts, Hypogammaglobulinemia, Infections, and Myelokathexis) syndrome. While CXCR4 is reported to be overexpressed in multiple human cancer types and many hematological cancer cell lines, we have observed poor in vitro cell surface expression of CXCR4 in many solid tumor cell lines. We explore further the possible factors and pathways involved in regulating CXCR4 expression. Here, we showed that MEK-ERK signaling pathway and NFAT3 transcriptional factor plays a novel role in regulating CXCR4 expression. When cultured as 3D spheroids, HeyA8 ovarian tumor cells showed a dramatic increase in surface CXCR4 protein levels as well as mRNA transcripts. Furthermore, HeyA8 3D spheroids showed a decrease in phospho-ERK levels when compared to adherent cells. The treatment of adherent HeyA8 cells with an inhibitor of the MEK-ERK pathway, U0126, resulted in a significant increase in surface CXCR4 expression. Additional investigation using the PCR array assay comparing adherent to 3D spheroid showed a wide range of transcription factors being up-regulated, most notably a > 20 fold increase in NFAT3 transcription factor mRNA. Finally, chromatin immunoprecipitation (ChIP) analysis showed that direct binding of NFAT3 on the CXCR4 promoter corresponds to increased CXCR4 expression in HeyA8 ovarian cell line. Taken together, our results suggest that high phospho-ERK levels and NFAT3 expression plays a novel role in regulating CXCR4 expression.