Pharmacological Inhibition Of Hsp90 Research Articles

Human herpesvirus 6A nuclear matrix protein U37 interacts with heat shock transcription factor 1 and activates the heat shock response.

Nascent nucleocapsids of herpesviruses acquire a primary envelope during their nuclear export by budding through the inner nuclear membrane into the perinuclear space between the inner and outer nuclear membranes. This process is mediated by a conserved viral heterodimeric complex designated the nuclear egress complex, which consists of the nuclear matrix protein and the nuclear membrane protein. In addition to its essential roles during nuclear egress, the nuclear matrix protein has been shown to interact with intracellular signaling pathway molecules including NF-κB and IFN-β to affect viral or cellular gene expression. The human herpesvirus 6A (HHV-6A) U37 gene encodes a nuclear matrix protein, the role of which has not been analyzed. Here, we show that HHV-6A U37 activates the heat shock element promoter and induces the accumulation of the molecular chaperone Hsp90. Mechanistically, HHV-6A U37 interacts with heat shock transcription factor 1 (HSF1) and induces its phosphorylation at Ser-326. We report that pharmacological inhibition of HSF1, Hsp70, or Hsp90 decreases viral protein accumulation and viral replication. Taken together, our results lead us to propose a model in which HHV-6A U37 activates the heat shock response to support viral gene expression and replication. IMPORTANCE Human herpesvirus 6A (HHV-6A) is a dsDNA virus belonging to the Roseolovirus genus within the Betaherpesvirinae subfamily. It is frequently found in patients with neuroinflammatory disease, although its pathogenetic role, if any, awaits elucidation. The heat shock response is important for cell survival under stressful conditions that disrupt homeostasis. Our results indicate that HHV-6A U37 activates the heat shock element promoter and leads to the accumulation of heat shock proteins. Next, we show that the heat shock response is important for viral replication. Overall, our findings provide new insights into the function of HHV-6A U37 in host cell signaling and identify potential cellular targets involved in HHV-6A pathogenesis and replication.

Heat shock protein 90 facilitates SARS-CoV-2 structural protein-mediated virion assembly and promotes virus-induced pyroptosis

Inhibition of heat shock protein 90 (Hsp90), a prominent molecular chaperone, effectively limits severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection but little is known about any interaction between Hsp90 and SARS-CoV-2 proteins. Here, we systematically analyzed the effects of the chaperone isoforms Hsp90α and Hsp90β on individual SARS-CoV-2 viral proteins. Five SARS-CoV-2 proteins, namely nucleocapsid (N), membrane (M), and accessory proteins Orf3, Orf7a, and Orf7b were found to be novel clients of Hsp90β in particular. Pharmacological inhibition of Hsp90 with 17-DMAG results in N protein proteasome-dependent degradation. Hsp90 depletion-induced N protein degradation is independent of CHIP, a ubiquitin E3 ligase previously identified for Hsp90 client proteins, but alleviated by FBXO10, an E3 ligase identified by subsequent siRNA screening. We also provide evidence that Hsp90 depletion may suppress SARS-CoV-2 assembly partially through induced M or N degradation. Additionally, we found that GSDMD-mediated pyroptotic cell death triggered by SARS-CoV-2 was mitigated by inhibition of Hsp90. These findings collectively highlight a beneficial role for targeting of Hsp90 during SARS-CoV-2 infection, directly inhibiting virion production and reducing inflammatory injury by preventing the pyroptosis that contributes to severe SARS-CoV-2 disease.

Role of heat shock protein 90 as an antiviral target for swine enteric coronaviruses

A variety of swine enteric coronaviruses (SECoVs) have emerged and are prevalent in pig populations, including porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome (SADS)-CoV, a newly identified bat-origin CoV with zoonotic potential. Unfortunately, available traditional, inactivated and attenuated SECoV vaccines are of limited efficacy against the variants currently circulating in most pig populations. In this study, we evaluated the role of host factor heat shock protein 90 (Hsp90) as an antiviral target against SECoVs, exemplified by SADS-CoV. Pharmacological inhibition of Hsp90 diminished SADS-CoV replication significantly in porcine and human cell lines, and also decreased replication of SADS-CoV in a porcine intestinal enteroid model. Further mechanistic experiments revealed that both porcine and human isoforms of Hsp90 interact with the SADS-CoV nucleocapsid (N) protein, and inhibition of Hsp90 resulted in autophagic degradation of N protein. Moreover, we linked Hsp90 to virus-induced cellular pyroptosis, as SADS-CoV was found to trigger caspase-1/gasdermin-d-mediated pyroptotic cell death, which was mitigated by inhibition of Hsp90. Finally, we demonstrated that Hsp90 also associated with N proteins and was involved in propagation of PEDV, PDCoV and TGEV. This study thus extends our understanding of immune responses to SADS-CoV infection and offers a new potential therapeutic option against four SECoVs.

Protein Kinase D3 (PKD3) Requires Hsp90 for Stability and Promotion of Prostate Cancer Cell Migration.

Prostate cancer metastasis is a significant cause of mortality in men. PKD3 facilitates tumor growth and metastasis, however, its regulation is largely unclear. The Hsp90 chaperone stabilizes an array of signaling client proteins, thus is an enabler of the malignant phenotype. Here, using different prostate cancer cell lines, we report that Hsp90 ensures PKD3 conformational stability and function to promote cancer cell migration. We found that pharmacological inhibition of either PKDs or Hsp90 dose-dependently abrogated the migration of DU145 and PC3 metastatic prostate cancer cells. Hsp90 inhibition by ganetespib caused a dose-dependent depletion of PKD2, PKD3, and Akt, which are all involved in metastasis formation. Proximity ligation assay and immunoprecipitation experiments demonstrated a physical interaction between Hsp90 and PKD3. Inhibition of the chaperone-client interaction induced misfolding and proteasomal degradation of PKD3. PKD3 siRNA combined with ganetespib treatment demonstrated a specific involvement of PKD3 in DU145 and PC3 cell migration, which was entirely dependent on Hsp90. Finally, ectopic expression of PKD3 enhanced migration of non-metastatic LNCaP cells in an Hsp90-dependent manner. Altogether, our findings identify PKD3 as an Hsp90 client and uncover a potential mechanism of Hsp90 in prostate cancer metastasis. The molecular interaction revealed here may regulate other biological and pathological functions.

TauLUM, an in vivo Drosophila sensor of tau multimerization, identifies neuroprotective interventions in tauopathy

SummaryTau protein aggregates are a defining neuropathological feature of “tauopathies,” a group of neurodegenerative disorders that include Alzheimer’s disease. In the current study, we develop a Drosophila split-luciferase-based sensor of tau-tau interaction. This model, which we term “tauLUM,” allows investigators to quantify tau multimerization at individual time points or longitudinally in adult, living animals housed in a 96-well plate. TauLUM causes cell death in the adult Drosophila brain and responds to both pharmacological and genetic interventions. We find that transgenic expression of an anti-tau intrabody or pharmacological inhibition of HSP90 reduces tau multimerization and cell death in tauLUM flies, establishing the suitability of this system for future drug and genetic modifier screening. Overall, our studies position tauLUM as a powerful in vivo discovery platform that leverages the advantages of the Drosophila model organism to better understand tau multimerization.

Aha1 Is an Autonomous Chaperone for SULT1A1.

The cochaperone Aha1 activates HSP90 ATPase to promote the folding of its client proteins; however, very few client proteins of Aha1 are known. With the use of an ascorbate peroxidase (APEX)-based proximity labeling method, we identified SULT1A1 as a proximity protein of HSP90 that is modulated by genetic depletion of Aha1. Immunoprecipitation followed by Western blot analysis showed the interaction of SULT1A1 with Aha1, but not HSP90. We also observed a reduced level of SULT1A1 protein upon genetic depletion of Aha1 but not upon pharmacological inhibition of HSP90, suggesting that the SULT1A1 protein level is regulated by Aha1 alone. Maturation-dependent interaction assay results showed that Aha1, but not HSP90, binds preferentially to newly synthesized SULT1A1. Reconstitution of Aha1-depleted cells with wild-type Aha1 and its E67K mutant, which is deficient in interacting with HSP90, restored SULT1A1 protein to the same level. Nonetheless, complementation of Aha1-depleted cells with an Aha1 mutant lacking the first 20 amino acids, which disrupts its autonomous chaperone function, was unable to rescue the SULT1A1 protein level. Together, our study revealed, for the first time, Aha1 as an autonomous chaperone in regulating SULT1A1. SULT1A1 is a phase-II metabolic enzyme, where it adds sulfate groups to hydroxyl functionalities in endogenous hormones and xenobiotic chemicals to improve their solubilities and promote their excretion. Thus, our work suggests the role of Aha1 cochaperone in modulating the detoxification of endogenous and environmental chemicals.

Abstract 3232: Combined inhibition of mTOR and HSP90 potentiates cisplatin antitumor effect and reverts cisplatin resistance in vitro and in vivo models of epithelial ovarian cancer by modulating HSF1-dependent transactivation

Abstract INTRODUCTION: Epithelial ovarian cancer (EOC) represents the most lethal gynecological disease, with a 5-year relative survival rate of 46% after the diagnosis. The standard treatment of advanced EOC is based on surgery, followed by platinum (Pt)-based chemotherapy. However, the development of platinum resistant disease could occur and strongly impact on the survival of EOC patients for whom we still do not have valid therapeutic options. By using a proteomic approach followed by a bioinformatic analysis, we previously validated the role of HSP90 in the mechanism of platinum-resistance. Here, we propose a novel therapeutic strategy based on the combined pharmacologic inhibition of HSP90 and mTOR to further potentiate Pt-based chemotherapy and to revert Pt-resistance in EOC and non-small-cell lung cancer (NSCLC) models. METHODS: TOV-112D parental and Pt-resistant cells were characterized by phosphoproteomics followed by functional analysis and proteins validation by western blot. Synergistic anti-tumor effect was evaluated in Pt-sensitive and Pt-resistant EOC and NSCLC cell lines by calculating combination indexes (CI), colony formation assay, apoptosis and DNA damage, as well as on 3D in vitro microtissues obtained by co-culturing cancer cells with normal fibroblasts and in vivo EOC and NSCLC xenograft models. RESULTS: 542 differentially phosphorylated expressed proteins were identified in Pt-resistant TOV-112D compared with parental cells, and mTOR and the transcription factor HSF1 emerged as the most enriched pathways. The up-regulation of the phosphorylated form of PDK1, AKT, mTOR and rpS6 was observed in Pt-resistant compared to parental cells. Moreover, we also demonstrated the up-regulation of the activity of HSF1 along with the elevation of its targets such as heat shock proteins HSP90, HSP70 and HSP40, crucial components of chaperone complex machinery. Interestingly, among the differentially expressed proteins, we identified the kinase DYRK2 able to phosphorylate HSF1, supporting its transactivation. Accordingly, the combination of HSP90 inhibitor ganetespib and the mTOR inhibitor temsirolimus plus cisplatin, synergistically reduced colony formation, cancer cells and microtissues cell growth in vitro by increasing DNA-damage and apoptosis and in vivo by enhancing mouse survival. Mechanistically, the triple combination treatment, impaired the proteins involved in mTOR signalling and HSF1 transactivation. Notably, all these data were confirmed in Pt-resistant NSCLC models, supporting the possibility that the same mechanism is present in different tumor types. CONCLUSIONS: Our findings identify a promising new antitumor strategy based on the combination of HSP90 and mTOR inhibitors to revert Pt-resistance that that warrant further clinical evaluation. Citation Format: Rita Lombardi, Laura Addi, Biagio Pucci, Maura Sonego, Maria Serena Roca, Francesca Capone, Federica Iannelli, Francesca Bruzzese, Gustavo Baldassarre, Alfredo Budillon. Combined inhibition of mTOR and HSP90 potentiates cisplatin antitumor effect and reverts cisplatin resistance in vitro and in vivo models of epithelial ovarian cancer by modulating HSF1-dependent transactivation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3232.

Inhibition of Hsp90 regulates fluconazole resistance in the pathogenic fungus Cryptococcus neoformans

Cryptococcus neoformans is an encapsulated yeast, found in the lungs of immunocompromised hosts. In the human host, Cryptococcusis subject to multiple forms of environmental stress, including heat stress. Heat shock protein 90 (Hsp90) is an essential protein that has been shown to protect fungal cells against extreme temperature in the human host and to assist in proper cell division. Hsp90 has also been implicated in resistance to the anti‐fungal drug fluconazole. To better characterize the role of Hsp90 in fluconazole resistance in Cryptococcuswe addressed two questions: 1. Does concomitant partial inhibition of Hsp90 and treatment with fluconazole lead to increased sensitivity to fluconazole and 2. Does prolonged partial inhibition of Hsp90 prior to treatment with fluconazole lead to subsequent increase in development of fluconazole resistance. Using growth assays, disk assays, and a standardized e‐test to determine the Minimum Inhibitory Concentration (MIC) of fluconazole, we found that indeed, pharmacological partial inhibition of Hsp90 with radicicol, concomitant with fluconazole treatment leads to increased sensitivity to fluconazole. However, initial pre‐treatment of Cryptococcuswith sub‐inhibitory concentration of radicicol followed by exposure to fluconazole leads to development of fluconazole resistant Cryptococcuscolonies. We characterized the resistant colonies, which show a lower MIC to fluconazole compared to cells that were never exposed to radicicol and fluconazole. Cells derived from the resistant colonies exhibit slower growth on drug‐free media, and lose resistance to the drug after passaging in drug‐free media.Based on the fact that resistance to fluconazole is lost when fluconazole is removed from the environment, we hypothesize that compromising Hsp90 function during the pre‐treatment with radicicol triggers aneuploidy in Cryptococcus neoformans. Furthermore, we hypothesize that the resistant colonies are aneuploids that possess an altered chromosome number to maintain drug resistance in response to an environmental insult, such as presence of fluconazole.

Overexpression of BRD4 Is an Adverse Prognostic Factor in Acute Myeloid Leukemia

Background:Bromodomain containing protein 4 (BRD4), a member of the bromodomain and extra terminal domain (BET) protein family, has been implicated in the pathogenesis of a number of hematological and solid tumors. BRD4 is an epigenetic reader that plays a central role in transcriptional regulation, cellular growth control, and cell cycle progression. As a consequence, BET inhibitors elicit anticancer activity in numerous malignant contexts. While deregulation of BRD4 activity has been shown to be essential for the maintenance of AML, its expression profile and prognostic value in AML have not been investigated. Additionally, how BRD4 is regulated in cancer cells remains largely unknown.Methodology: We therefore evaluated the level of BRD4 expression using Reverse Phase Protein Array (RPPA) technology in marrow and blood samples from 511 newly diagnosed AML patients, and determined a correlation between BRD4 expression and clinical characteristics and overall survival in AML patients. We also correlated expression of BRD4 with 232 other proteins (encompassing signal transduction, metabolism, apoptosis and cellular proliferation pathways) that were included in RPPA. Potential role of molecular chaperone heat shock protein 90 (Hsp90) in regulating BRD4 protein stability was also investigated.Results: BRD4 expression was similar in peripheral blood and bone marrow and expression was found to be higher at diagnosis than at relapse (P = .035). Compared with normal bone marrow derived CD34+ cells, BRD4 expression in AML was higher in 28% of patients and that higher BRD4 levels significantly predicted shorter overall survival (P = .002). There was no significant difference in BRD4 expression on the basis of age, sex, or performance status, antecedent malignancies, and previous chemotherapy and/or radiotherapy. BRD4 expression levels were not strongly correlated with French-American-British classification (FAB), although levels were slightly higher in M5 subtype. BRD4 levels were found to be significantly higher in patients with NPM1 mutation (P = .003) or FLT3-ITD (P = .057) or between those with both FLT3-D835F and NPM1 mutation (P = .01). BRD4 levels were not associated with the attainment of complete remission (CR), although rates were somewhat lower for those with high. However, patients with high levels of BRD4 had significantly inferior remission duration (25.9 vs 50 weeks; P = 0.03) compared with those with normal levels of BRD4. The overall survival (OS) of patients with higher levels of BRD4 was significantly inferior compared with those with normal levels of BRD4 (28.3 vs 55.3 weeks; P = .002). Patients with unfavorable cytogenetics and above-normal levels of BRD4 had inferior survival compared with those with normal levels of BRD4 (22.4 vs 38.9 weeks; P = .0043; Figure 3C). BRD4 expression was found to be strongly correlated (R > 0.2 and P < 0.0001 or 0.00001) either positively (n = 48) or negatively (n = 14) with proteins known to be important in AML. BRD4 expression was positively correlated with various cell proliferation-related proteins (Hsp90, pSTAT5, nucleolin and mTOR), anti-apoptotic protein (Bcl-2), oncoproteins (FLI1, CREB and ELK1), proteins important for stem cell functions (Ash2L and LSD1), hematopoietic transcription factors (ERG and PU.1/Spi1), and Smad proteins, signal transducers for the members of the transforming growth factor-β (TGF-β) superfamily. The stability, activity and function of several mutated, chimeric, and overexpressed signaling proteins crucial for oncogenesis are often maintained by Hsp90. Our study also demonstrates that BRD4 stability is dependent upon Hsp90 activity and pharmacologic inhibition of Hsp90 promoted ubiquitination followed by proteasome-dependent degradation of BRD4 protein.Conclusions: These findings suggest BRD4 as a prognostic biomarker for unfavorable outcomes and supports the clinical rational for using BRD4 inhibitors in AML. Future studies could identify mechanistic insights into BRD4-associated proteins as biomarkers for response to BET inhibition. DisclosuresKarnad:ADC Therapeutics: Research Funding. Weitman:Tolero Pharmaceuticals, Inc.: Employment. Iyer:Takeda: Research Funding; Genentech: Research Funding.

Inhibition of Hsp90 Counteracts the Established Experimental Dermal Fibrosis Induced by Bleomycin.

Our previous study demonstrated that heat shock protein 90 (Hsp90) is overexpressed in the involved skin of patients with systemic sclerosis (SSc) and in experimental dermal fibrosis. Pharmacological inhibition of Hsp90 prevented the stimulatory effects of transforming growth factor-beta on collagen synthesis and the development of dermal fibrosis in three preclinical models of SSc. In the next step of the preclinical analysis, herein, we aimed to evaluate the efficacy of an Hsp90 inhibitor, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), in the treatment of established experimental dermal fibrosis induced by bleomycin. Treatment with 17-DMAG demonstrated potent antifibrotic and anti-inflammatory properties: it decreased dermal thickening, collagen content, myofibroblast count, expression of transforming growth factor beta receptors, and pSmad3-positive cell counts, as well as leukocyte infiltration and systemic levels of crucial cytokines/chemokines involved in the pathogenesis of SSc, compared to vehicle-treated mice. 17-DMAG effectively prevented further progression and may induce regression of established bleomycin-induced dermal fibrosis to an extent comparable to nintedanib. These findings provide further evidence of the vital role of Hsp90 in the pathophysiology of SSc and characterize it as a potential target for the treatment of fibrosis with translational implications due to the availability of several Hsp90 inhibitors in clinical trials for other indications.

Hsp90-stabilized MIF supports tumor progression via macrophage recruitment and angiogenesis in colorectal cancer

Macrophage migration inhibitory factor (MIF) is an upstream regulator of innate immunity, but its expression is increased in some cancers via stabilization with HSP90-associated chaperones. Here, we show that MIF stabilization is tumor-specific in an acute colitis-associated colorectal cancer (CRC) mouse model, leading to tumor-specific functions and selective therapeutic vulnerabilities. Therefore, we demonstrate that a Mif deletion reduced CRC tumor growth. Further, we define a dual role for MIF in CRC tumor progression. Mif deletion protects mice from inflammation-associated tumor initiation, confirming the action of MIF on host inflammatory pathways; however, macrophage recruitment, neoangiogenesis, and proliferative responses are reduced in Mif-deficient tumors once the tumors are established. Thus, during neoplastic transformation, the function of MIF switches from a proinflammatory cytokine to an angiogenesis promoting factor within our experimental model. Mechanistically, Mif-containing tumor cells regulate angiogenic gene expression via a MIF/CD74/MAPK axis in vitro. Clinical correlation studies of CRC patients show the shortest overall survival for patients with high MIF levels in combination with CD74 expression. Pharmacological inhibition of HSP90 to reduce MIF levels decreased tumor growth in vivo, and selectively reduced the growth of organoids derived from murine and human tumors without affecting organoids derived from healthy epithelial cells. Therefore, novel, clinically relevant Hsp90 inhibitors provide therapeutic selectivity by interfering with tumorigenic MIF in tumor epithelial cells but not in normal cells. Furthermore, Mif-depleted colonic tumor organoids showed growth defects compared to wild-type organoids and were less susceptible toward HSP90 inhibitor treatment. Our data support that tumor-specific stabilization of MIF promotes CRC progression and allows MIF to become a potential and selective therapeutic target in CRC.

HSP90 identified by a proteomic approach as druggable target to reverse platinum resistance in ovarian cancer.

Acquired resistance to platinum (Pt)‐based therapies is an urgent unmet need in the management of epithelial ovarian cancer (EOC) patients. Here, we characterized by an unbiased proteomics method three isogenic EOC models of acquired Pt resistance (TOV‐112D, OVSAHO, and MDAH‐2774). Using this approach, we identified several differentially expressed proteins in Pt‐resistant (Pt‐res) compared to parental cells and the chaperone HSP90 as a central hub of these protein networks. Accordingly, up‐regulation of HSP90 was observed in all Pt‐res cells and heat‐shock protein 90 alpha isoform knockout resensitizes Pt‐res cells to cisplatin (CDDP) treatment. Moreover, pharmacological HSP90 inhibition using two different inhibitors [17‐(allylamino)‐17‐demethoxygeldanamycin (17AAG) and ganetespib] synergizes with CDDP in killing Pt‐res cells in all tested models. Mechanistically, genetic or pharmacological HSP90 inhibition plus CDDP ‐induced apoptosis and increased DNA damage, particularly in Pt‐res cells. Importantly, the antitumor activities of HSP90 inhibitors (HSP90i) were confirmed both ex vivo in primary cultures derived from Pt‐res EOC patients ascites and in vivo in a xenograft model. Collectively, our data suggest an innovative antitumor strategy, based on Pt compounds plus HSP90i, to rechallenge Pt‐res EOC patients that might warrant further clinical evaluation.

Hsp90/C terminal Hsc70-interacting protein regulates the stability of Ikaros in acute myeloid leukemia cells.

The stability of Ikaros family zinc finger protein 1 (Ikaros), a critical hematopoietic transcription factor, can be regulated by cereblon (CRBN) ubiquitin ligase stimulated by immunomodulatory drugs in multiple myeloma. However, other stabilization mechanisms of Ikaros have yet to be elucidated. In this study, we show that the pharmacologic inhibition or knockdown of Hsp90 downregulates Ikaros in acute myeloid leukemia (AML) cells. Proteasome inhibitor MG132 but not autophagy inhibitor chloroquine could suppress the Hsp90 inhibitor STA-9090-induced reduction of Ikaros, which is accompanied with the increased ubiquitination of Ikaros. Moreover, Ikaros interacts with E3 ubiquitin-ligase C terminal Hsc70 binding protein (CHIP), which mediates the STA-9090-induced ubiquitination of Ikaros. In addition, the knockdown of Ikaros effectively inhibits the proliferation of leukemia cells, but this phenomenon could be rescued by Ikaros overexpression. Collectively, our findings indicate that the interplay between HSP90 and CHIP regulates the stability of Ikaros in AML cells, which provides a novel strategy for AML treatment through targeting the HSP90/Ikaros/CHIP axis.

Hsp90 regulates HDAC3-dependent gene transcription while HDAC3 regulates the functions of Hsp90

Deregulated DNA methylation and post-translational histone modifications are majorly associated with cancer progression. Histone modification regulates the gene expression patterns that contribute to the emergence of sporadic cancers. Histone deacetylases (HDACs) act as erasers of acetylation marks, and their functions are often deregulated in cancer. Since non-histone proteins can also act as substrates for HDACs, identifying their involvement in vital regulatory molecules contributing to cancer progression is essential. Hsp90 is a cancer chaperone that contributes to kinase evolution and, thus, cellular adaptations. Acetylated Hsp90 loses its chaperoning functions and client protein interactions. Robust cell proliferation is one of the hallmarks of cancer. However, Hsp90 involvement in cancer promoting gene transcription is less understood. Using human breast cancer cells, we demonstrate that nuclear Hsp90 functions are regulated by HDAC3, while Hsp90 regulates HDAC3 nuclear translocation. Pharmacological inhibition of Hsp90 decreased the HDAC3 nuclear translocation and increased the gene expression relevant to epithelial to mesenchymal transition. Further, inhibition of HDAC3 resulted in the nuclear accumulation of acetylated Hsp90. Additionally, Hsp90 inhibition affected the global histone acetylation and methylation patterns, whereas HDAC3 inhibition exhibited less impact. Our results display a novel regulatory mechanism mediated by Hsp90 and HDAC3 in tumor cells. Considering that Hsp90 and histone deacetylase inhibitors are emerging as novel anticancer agents, our findings may have clinical relevance.

Phenotypic Screen with TSC-Deficient Neurons Reveals Heat-Shock Machinery as a Druggable Pathway for mTORC1 and Reduced Cilia

SUMMARYTuberous sclerosis complex (TSC) is a neurogenetic disorder that leads to elevated mechanistic targeting of rapamycin complex 1 (mTORC1) activity. Cilia can be affected by mTORC1 signaling, and ciliary deficits are associated with neurodevelopmental disorders. Here, we examine whether neuronal cilia are affected in TSC. We show that cortical tubers from TSC patients and mutant mouse brains have fewer cilia. Using high-content image-based assays, we demonstrate that mTORC1 activity inversely correlates with ciliation in TSC1/2-deficientneurons.To investigate the mechanistic relationship between mTORC1 and cilia, we perform a phenotypic screen for mTORC1 inhibitors with TSC1/2-deficient neurons. We identify inhibitors ofthe heat shock protein 90 (Hsp90) that suppress mTORC1 through regulation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling. Pharmacological inhibition of Hsp90 rescues ciliation through downregulation of Hsp27. Our study uncovers the heat-shock machinery as a druggable signaling node to restore mTORC1 activity and cilia due to loss of TSC1/2, and it provides broadly applicable platforms for studying TSC-related neuronal dysfunction.

Insect herbivory antagonizes leaf cooling responses to elevated temperature in tomato

As global climate change brings elevated average temperatures and more frequent and extreme weather events, pressure from biotic stresses will become increasingly compounded by harsh abiotic stress conditions. The plant hormone jasmonate (JA) promotes resilience to many environmental stresses, including attack by arthropod herbivores whose feeding activity is often stimulated by rising temperatures. How wound-induced JA signaling affects plant adaptive responses to elevated temperature (ET), however, remains largely unknown. In this study, we used the commercially important crop plant Solanum lycopersicum (cultivated tomato) to investigate the interaction between simulated heat waves and wound-inducible JA responses. We provide evidence that the heat shock protein HSP90 enhances wound responses at ET by increasing the accumulation of the JA receptor, COI1. Wound-induced JA responses directly interfered with short-term adaptation to ET by blocking leaf hyponasty and evaporative cooling. Specifically, leaf damage inflicted by insect herbivory or mechanical wounding at ET resulted in COI1-dependent stomatal closure, leading to increased leaf temperature, lower photosynthetic carbon assimilation rate, and growth inhibition. Pharmacological inhibition of HSP90 reversed these effects to recapitulate the phenotype of a JA-insensitive mutant lacking the COI1 receptor. As climate change is predicted to compound biotic stress with larger and more voracious arthropod pest populations, our results suggest that antagonistic responses resulting from a combination of insect herbivory and moderate heat stress may exacerbate crop losses.

Heat Shock Protein 90 Chaperone Regulates the E3 Ubiquitin-Ligase Hakai Protein Stability.

The E3 ubiquitin-ligase Hakai binds to several tyrosine-phosphorylated Src substrates, including the hallmark of the epithelial-to-mesenchymal transition E-cadherin, and signals for degradation of its specific targets. Hakai is highly expressed in several human cancers, including colon cancer, and is considered as a drug target for cancer therapy. Here, we report a link between Hakai and the heat shock protein 90 (Hsp90) chaperone complex. Hsp90 participates in the correct folding of its client proteins, allowing them to maintain their stability and activity. Hsp90 inhibitors specifically interfere with the association with its Hsp90 client proteins, and exhibit potent anti-cancer properties. By immunoprecipitation, we present evidence that Hakai interacts with Hsp90 chaperone complex in several epithelial cells and demonstrate that is a novel Hsp90 client protein. Interestingly, by overexpressing and knocking-down experiments with Hakai, we identified Annexin A2 as a Hakai-regulated protein. Pharmacological inhibition of Hsp90 with geldanamycin results in the degradation of Hakai in a lysosome-dependent manner. Interestingly, geldanamycin-induced Hakai degradation is accompanied by an increased expression of E-cadherin and Annexin A2. We also show that geldanamycin suppresses cell motility at least in part through its action on Hakai expression. Taken together, our results identify Hakai as a novel Hsp90 client protein and shed light on the regulation of Hakai stability. Our results open the possibility to the potential use of Hsp90 inhibitors for colorectal cancer therapy through its action on Hakai client protein of Hsp90.

281P - Selective depletion of tumour-associated SAMHD1 by HSP90 inhibitors enhances the anti-AML effect of cytarabine

BackgroundAcute myeloid leukemia (AML) is the most common acute type of leukemia in adults, with a disappointing prognosis and high death rate. Cytarabine (ara-C) is a primary first-line chemotherapy drug used in the treatment of AML, and resistance to it represents a major source of treatment failure. Sterile alpha motif and HD domain-containing protein 1 (SAMHD1), the only known human dNTPase, is known to attenuate ara-C cytotoxicity in AML cells, resulting in treatment failure and a poor prognosis. Therefore, selective inhibition of SAMHD1 in AML should prove a promising strategy for increasing the efficacy of ara-C. Nevertheless, no effective strategy to target SAMHD1 is available yet. MethodsCell culture, Monocyte isolation and differentiation, Cytotoxicity assays, Protein extraction and Western blot analysis, Immunoprecipitation, Immunofluorescent staining, Tissue staining, Mass spectrum system, mRNA analysis, RNA silencing, Flow cytometry, Orthotopic AML animal model, Heterotopic AML animal model. ResultsHere, we report that SAMHD1 is a novel HSP90-dependent protein in various tumor types. Pharmacological inhibition of HSP90 led to SAMHD1 depletion in a wide range of tumor cell types but had no or minimally effect on SAMHD1 of peripheral blood mononuclear cells (PBMCs) or macrophages from healthy donors due to the phosphorylation status on Thr 592. The combination of ara-C and HSP90 inhibitors showed a synergistic effect in killing AML cells. The HSP90 inhibitor also significantly enhanced the therapeutic efficacy of ara-C in both orthotopic and heterotopic models, alleviating tumor burden and prolonging survival. ConclusionsOverall, these data suggest that the addition of an HSP90 inhibitor to ara-C-based chemotherapy represents a potential new treatment strategy for AML patients. Legal entity responsible for the studyXiaofang Yu. FundingNational Natural Science Foundation of China (81772169). DisclosureAll authors have declared no conflicts of interest.

Use of Acid Ceramidase and Sphingosine Kinase Inhibitors as Antiviral Compounds Against Measles Virus Infection of Lymphocytes in vitro.

As structural membrane components and signaling effector molecules sphingolipids influence a plethora of host cell functions, and by doing so also the replication of viruses. Investigating the effects of various inhibitors of sphingolipid metabolism in primary human peripheral blood lymphocytes (PBL) and the human B cell line BJAB we found that not only the sphingosine kinase (SphK) inhibitor SKI-II, but also the acid ceramidase inhibitor ceranib-2 efficiently inhibited measles virus (MV) replication. Virus uptake into the target cells was not grossly altered by the two inhibitors, while titers of newly synthesized MV were reduced by approximately 1 log (90%) in PBL and 70–80% in BJAB cells. Lipidomic analyses revealed that in PBL SKI-II led to increased ceramide levels, whereas in BJAB cells ceranib-2 increased ceramides. SKI-II treatment decreased sphingosine-1-phosphate (S1P) levels in PBL and BJAB cells. Furthermore, we found that MV infection of lymphocytes induced a transient (0.5–6 h) increase in S1P, which was prevented by SKI-II. Investigating the effect of the inhibitors on the metabolic (mTORC1) activity we found that ceranib-2 reduced the phosphorylation of p70 S6K in PBL, and that both inhibitors, ceranib-2 and SKI-II, reduced the phosphorylation of p70 S6K in BJAB cells. As mTORC1 activity is required for efficient MV replication, this effect of the inhibitors is one possible antiviral mechanism. In addition, reduced intracellular S1P levels affect a number of signaling pathways and functions including Hsp90 activity, which was reported to be required for MV replication. Accordingly, we found that pharmacological inhibition of Hsp90 with the inhibitor 17-AAG strongly impaired MV replication in primary PBL. Thus, our data suggest that treatment of lymphocytes with both, acid ceramidase and SphK inhibitors, impair MV replication by affecting a number of cellular activities including mTORC1 and Hsp90, which alter the metabolic state of the cells causing a hostile environment for the virus.

Transcriptomics-Based Screening Identifies Pharmacological Inhibition of Hsp90 as a Means to Defer Aging

SummaryAging strongly influences human morbidity and mortality. Thus, aging-preventive compounds could greatly improve our health and lifespan. Here we screened for such compounds, known as geroprotectors, employing the power of transcriptomics to predict biological age. Using age-stratified human tissue transcriptomes and machine learning, we generated age classifiers and applied these to transcriptomic changes induced by 1,309 different compounds in human cells, ranking these compounds by their ability to induce a “youthful” transcriptional state. Testing the top candidates in C. elegans, we identified two Hsp90 inhibitors, monorden and tanespimycin, which extended the animals’ lifespan and improved their health. Hsp90 inhibition induces expression of heat shock proteins known to improve protein homeostasis. Consistently, monorden treatment improved the survival of C. elegans under proteotoxic stress, and its benefits depended on the cytosolic unfolded protein response-inducing transcription factor HSF-1. Taken together, our method represents an innovative geroprotector screening approach and was able to identify a class that acts by improving protein homeostasis.