Degradation Of Client Proteins Research Articles

PU-H71 (NSC 750424): a molecular masterpiece that targets HSP90 in cancer and beyond.

Heat shock protein 90 (HSP90) is a pivotal molecular chaperone with multifaceted roles in cellular health and disease. Herein, we explore how HSP90 orchestrates cellular stress responses, particularly through its partnership with heat shock factor 1 (HSF-1). PU-H71, a selective inhibitor of HSP90, demonstrates significant potential in cancer therapy by targeting a wide array of oncogenic pathways. By inducing the degradation of multiple client proteins, PU-H71 disrupts critical signaling pathways such as MAPK, PI3K/Akt, JAK/STAT, EGFR, and mTOR, which are essential for cancer cell survival, proliferation, and metastasis. We examined its impact on combating triple-negative breast cancer and enhancing the effectiveness of carbon-ion beam therapy, offering new avenues for cancer treatment. Furthermore, the dual inhibition of HSP90A and HSP90B1 by PU-H71 proves highly effective in the context of myeloma, providing fresh hope for patients with this challenging malignancy. We delve into its potential to induce apoptosis in B-cell lymphomas that rely on Bcl6 for survival, highlighting its relevance in the realm of hematologic cancers. Shifting our focus to hepatocellular carcinoma, we explore innovative approaches to chemotherapy. Moreover, the current review elucidates the potential capacity of PU-H71 to suppress glial cell activation paving the way for developing novel therapeutic strategies for neuroinflammatory disorders. Additionally, the present report also suggests the promising role of PU-H71 in JAK2-dependent myeloproliferative neoplasms. Eventually, our report sheds more light on the multiple functions of HSP90 protein as well as the potential therapeutic benefit of its selective inhibitor PU-H71 in the context of an array of diseases, laying the foundations for the development of novel therapeutic approaches that could achieve better treatment outcomes.

EP4 receptor agonist CAY10598 upregulates ROS-dependent Hsp90 cleavage in colorectal cancer cells

Prostaglandin E2 (PGE2) interacts with four specific G protein-coupled receptors, namely EP1, EP2, EP3, and EP4, playing a pivotal role in determining the fate of cells. Our previous findings highlighted that stimulating the EP4 receptor with its agonist, CAY10598, triggers apoptosis in colon cancer HCT116 cells via the production of reactive oxygen species (ROS). This process also reduces the phosphorylation of the oncogenic protein JAK2 and leads to its degradation in these cells. In this study, our goal was to explore the pathways through which CAY10598 leads to JAK2 degradation. We focused on Hsp90, a heat shock protein family member known for its role as a molecular chaperone maintaining the stability of several key proteins including EGFR, MET, Akt, and JAK2. Our results show that CAY10598 decreases the levels of client proteins of Hsp90 in HCT116 cells, an effect reversible by pretreatment with the ROS scavenger N-acetyl cysteine (NAC) or the proteasome inhibitor MG132, indicating that the degradation is likely driven by ROS. Furthermore, we observed that CAY10598 cleaves both α and β isoforms of Hsp90, the process inhibited by NAC. Inhibition of EP4 with the antagonist GW627368x not only prevented the degradation of Hsp90 client proteins but also the cleavage of Hsp90 itself in CAY10598-treated HCT116 cells. Additionally, CAY10598 suppressed the growth of HCT116 cells implanted in mice. Our findings reveal that CAY10598 induces apoptosis in cancer cells by a novel mechanism involving the ROS-dependent cleavage of Hsp90, thereby inhibiting the function of crucial Hsp90 client proteins.

Quantitative proteomic analysis reveals unique Hsp90 cycle-dependent client interactions.

Hsp90 is an abundant and essential molecular chaperone that mediates the folding and activation of client proteins in a nucleotide-dependent cycle. Hsp90 inhibition directly or indirectly impacts the function of 10-15% of all proteins due to degradation of client proteins or indirect downstream effects. Due to its role in chaperoning oncogenic proteins, Hsp90 is an important drug target. However, compounds that occupy the ATP-binding pocket and broadly inhibit function have not achieved widespread use due to negative effects. More selective inhibitors are needed; however, it is unclear how to achieve selective inhibition. We conducted a quantitative proteomic analysis of soluble proteins in yeast strains expressing wild-type Hsp90 or mutants that disrupt different steps in the client folding pathway. Out of 2,482 proteins in our sample set (approximately 38% of yeast proteins), we observed statistically significant changes in abundance of 350 (14%) of those proteins (log2 fold change ≥ 1.5). Of these, 257/350 (∼73%) with the strongest differences in abundance were previously connected to Hsp90 function. Principal component analysis of the entire dataset revealed that the effects of the mutants could be separated into 3 primary clusters. As evidence that Hsp90 mutants affect different pools of clients, simultaneous co-expression of 2 mutants in different clusters restored wild-type growth. Our data suggest that the ability of Hsp90 to sample a wide range of conformations allows the chaperone to mediate folding of a broad array of clients and that disruption of conformational flexibility results in client defects dependent on those states.

Inhibition of PRMT5/MEP50 Arginine Methyltransferase Activity Causes Cancer Vulnerability in NDRG2low Adult T-Cell Leukemia/Lymphoma.

N-myc downstream-regulated gene 2 (NDRG2), which is a tumour suppressor, is frequently lost in many types of tumours, including adult T-cell leukaemia/lymphoma (ATL). The downregulation of NDRG2 expression is involved in tumour progression through the aberrant phosphorylation of several important signalling molecules. We observed that the downregulation of NDRG2 induced the translocation of protein arginine methyltransferase 5 (PRMT5) from the nucleus to the cytoplasm via the increased phosphorylation of PRMT5 at Serine 335. In NDRG2low ATL, cytoplasmic PRMT5 enhanced HSP90A chaperone activity via arginine methylation, leading to tumour progression and the maintenance of oncogenic client proteins. Therefore, we examined whether the inhibition of PRMT5 activity is a drug target in NDRG2low tumours. The knockdown of PRMT5 and binding partner methylsome protein 50 (MEP50) expression significantly demonstrated the suppression of cell proliferation via the degradation of AKT and NEMO in NDRG2low ATL cells, whereas NDRG2-expressing cells did not impair the stability of client proteins. We suggest that the relationship between PRMT5/MEP50 and the downregulation of NDRG2 may exhibit a novel vulnerability and a therapeutic target. Treatment with the PRMT5-specific inhibitors CMP5 and HLCL61 was more sensitive in NDRG2low cancer cells than in NDRG2-expressing cells via the inhibition of HSP90 arginine methylation, along with the degradation of client proteins. Thus, interference with PRMT5 activity has become a feasible and effective strategy for promoting cancer vulnerability in NDRG2low ATL.

Ginsenoside Rg5's Enhancement of Radiosensitivity of Lung Adenocarcinoma via Reducing HSP90 CDC37 Interaction and Promoting Client Protein Degradation

Ginsenoside Rg5 is a rare ginsenoside showing promising tumor-suppressive effects. This study aimed to explore its radio-sensitizing effects and the underlying mechanisms. Human lung adenocarcinoma cell lines A549 and H460 were used for in vitro and in vivo analysis. Bioinformatic molecular docking prediction and following validation by surface plasmon resonance (SPR) technology and cellular thermal shift assay (CETSA) were conducted to explore the binding between ginsenoside Rg5 and HSP90α. The effects of ginsenoside Rg5 on HSP90-CDC37 interaction, the client protein stability and the downstream regulations were further explored. ginsenoside Rg5 can induce cell cycle arrest at the G1 phase and enhance radiation-induced cell apoptosis. Ginsenoside Rg5 could bind to HSP90α with a high affinity. ginsenoside Rg5 might interfere in the HSP90-CDC37-client interaction. Co-IP assays confirmed that ginsenoside Rg5 weakened the HSP90-CDC37 interaction in a dose-dependent manner. Ginsenoside Rg5 reduced irradiation-induced upregulation of the HSP90-CDC37 client proteins, including SRC, CDK4, RAF1 and ULK1 in A549 cell derived xenograft (CDX) tumors. Ginsenoside Rg5 or MRT67307 pre-treatment suppressed irradiation-induced elevation of the LC3-II/β ratio and restored irradiation-induced downregulation of p62 expression. In A549 CDX tumors, ginsenoside Rg5 treatment suppressed LC3 expression and enhanced irradiation-induced DNA damage. ginsenoside Rg5 may be a potential radiosensitizer for lung adenocarcinoma. It interacts with HSP90 and reduces the binding between HSP90 and CDC37, thereby increasing the ubiquitin-mediated proteasomal degradation of the HSP90-CDC37 client proteins.

Ginsenoside Rg5 enhances the radiosensitivity of lung adenocarcinoma via reducing HSP90-CDC37 interaction and promoting client protein degradation

Ginsenoside Rg5 is a rare ginsenoside showing promising tumor-suppressive effects. This study aimed to explore its radio-sensitizing effects and the underlying mechanisms. Human lung adenocarcinoma cell lines A549 and Calu-3 were used for in vitro and in vivo analysis. Bioinformatic molecular docking prediction and following validation by surface plasmon resonance (SPR) technology, cellular thermal shift assay (CETSA), and isothermal titration calorimetry (ITC) were conducted to explore the binding between ginsenoside Rg5 and 90 kD heat shock protein alpha (HSP90α). The effects of ginsenoside Rg5 on HSP90-cell division cycle 37 (CDC37) interaction, the client protein stability, and the downstream regulations were further explored. Results showed that ginsenoside Rg5 could induce cell-cycle arrest at the G1 phase and enhance irradiation-induced cell apoptosis. It could bind to HSP90α with a high affinity, but the affinity was drastically decreased by HSP90α Y61A mutation. Co-immunoprecipitation (Co-IP) and ITC assays confirmed that ginsenoside Rg5 disrupts the HSP90-CDC37 interaction in a dose-dependent manner. It reduced irradiation-induced upregulation of the HSP90-CDC37 client proteins, including SRC, CDK4, RAF1, and ULK1 in A549 cell-derived xenograft (CDX) tumors. Ginsenoside Rg5 or MRT67307 (an IKKε/TBK1 inhibitor) pretreatment suppressed irradiation-induced elevation of the LC3-II/β ratio and restored irradiation-induced downregulation of p62 expression. In A549 CDX tumors, ginsenoside Rg5 treatment suppressed LC3 expression and enhanced irradiation-induced DNA damage. In conclusion, ginsenoside Rg5 may be a potential radiosensitizer for lung adenocarcinoma. It interacts with HSP90α and reduces the binding between HSP90 and CDC37, thereby increasing the ubiquitin-mediated proteasomal degradation of the HSP90-CDC37 client proteins.

Elucidation of novel TRAP1-Selective inhibitors that regulate mitochondrial processes

Hsp90 isoform-selective inhibitors represent a new paradigm for novel anti-cancer drugs as each of the four isoforms have specific cellular localization, function, and client proteins. The mitochondrial isoform, TRAP1, is the least understood member of the Hsp90 family due to the lack of small molecule tools to study its biological function. Herein, we report novel TRAP1-selective inhibitors used to interrogate TRAP1's biological function along with co-crystal structures of such compounds bound to the N-terminus of TRAP1. Solution of the co-crystal structure allowed for a structure-based approach that resulted in compound 36, which is a 40 nM inhibitor with >250-fold TRAP1 selectivity over Grp94, the isoform with the highest structural similarity to TRAP1 within the N-terminal ATP binding site. Lead compounds 35 and 36 were found to selectively induce TRAP1 client protein degradation without inducing the heat shock response or disrupting Hsp90-cytosolic clients. They were also shown to inhibit OXPHOS, alter cellular metabolism towards glycolysis, disrupt TRAP1 tetramer stability, and disrupt the mitochondrial membrane potential.

Alvespimycin Inhibits Heat Shock Protein 90 and Overcomes Imatinib Resistance in Chronic Myeloid Leukemia Cell Lines.

Heat shock protein 90 (HSP90) facilitates folding and stability and prevents the degradation of multiple client proteins. One of these HSP90 clients is BCR-ABL, the oncoprotein characteristic of chronic myeloid leukemia (CML) and the target of tyrosine kinase inhibitors, such as imatinib. Alvespimycin is an HSP90 inhibitor with better pharmacokinetic properties and fewer side effects than other similar drugs, but its role in overcoming imatinib resistance is not yet clarified. This work studied the therapeutic potential of alvespimycin in imatinib-sensitive (K562) and imatinib-resistant (K562-RC and K562-RD) CML cell lines. Metabolic activity was determined by the resazurin assay. Cell death, caspase activity, mitochondrial membrane potential, and cell cycle were evaluated by means of flow cytometry. Cell death was also analyzed by optical microscopy. HSPs expression levels were assessed by western blotting. Alvespimycin reduced metabolic activity in a time-, dose-, and cell line-dependent manner. Resistant cells were more sensitive to alvespimycin with an IC50 of 31 nM for K562-RC and 44 nM for K562-RD, compared to 50 nM for K562. This drug induced apoptosis via the mitochondrial pathway. In K562 cells, alvespimycin induced cell cycle arrest in G0/G1. As a marker of HSP90 inhibition, a significant increase in HSP70 expression was observed. Our results suggest that alvespimycin might be a new therapeutic approach to CML treatment, even in cases of resistance to imatinib.

CHIP: A Co-chaperone for Degradation by the Proteasome and Lysosome.

Protein homeostasis relies on a balance between protein folding and protein degradation. Molecular chaperones like Hsp70 and Hsp90 fulfill well-defined roles in protein folding and conformational stability via ATP-dependent reaction cycles. These folding cycles are controlled by associations with a cohort of non-client protein co-chaperones, such as Hop, p23, and Aha1. Pro-folding co-chaperones facilitate the transit of the client protein through the chaperone-mediated folding process. However, chaperones are also involved in proteasomal and lysosomal degradation of client proteins. Like folding complexes, the ability of chaperones to mediate protein degradation is regulated by co-chaperones, such as the C-terminal Hsp70-binding protein (CHIP/STUB1). CHIP binds to Hsp70 and Hsp90 chaperones through its tetratricopeptide repeat (TPR) domain and functions as an E3 ubiquitin ligase using a modified RING finger domain (U-box). This unique combination of domains effectively allows CHIP to network chaperone complexes to the ubiquitin-proteasome and autophagosome-lysosome systems. This chapter reviews the current understanding of CHIP as a co-chaperone that switches Hsp70/Hsp90 chaperone complexes from protein folding to protein degradation.

Structural Characterization of Human Heat Shock Protein 90 N-Terminal Domain and Its Variants K112R and K112A in Complex with a Potent 1,2,3-Triazole-Based Inhibitor.

Heat shock protein 90 (Hsp90) is a ubiquitous molecular chaperone that stabilizes client proteins in a folded and functional state. It is composed of two identical and symmetrical subunits and each monomer consists of three domains, the N-terminal (NTD), the middle (MD), and the C-terminal domain (CTD). Since the chaperone activity requires ATP hydrolysis, molecules able to occupy the ATP-binding pocket in the NTD act as Hsp90 inhibitors, leading to client protein degradation and cell death. Therefore, human Hsp90 represents a validated target for developing new anticancer drugs. Since protozoan parasites use their Hsp90 to trigger important transitions between different stages of their life cycle, this protein also represents a profitable target in anti-parasite drug discovery. Nevertheless, the development of molecules able to selectively target the ATP-binding site of protozoan Hsp90 is challenging due to the high homology with the human Hsp90 NTD (hHsp90-NTD). In a previous work, a series of potent Hsp90 inhibitors based on a 1,4,5-trisubstituted 1,2,3-triazole scaffold was developed. The most promising inhibitor of the series, JMC31, showed potent Hsp90 binding and antiproliferative activity in NCI-H460 cells in the low-nanomolar range. In this work, we present the structural characterization of hHsp90-NTD in complex with JMC31 through X-ray crystallography. In addition, to elucidate the role of residue 112 on the ligand binding and its exploitability for the development of selective inhibitors, we investigated the crystal structures of hHsp90-NTD variants (K112R and K112A) in complex with JMC31.

The Therapeutic Potential of Targeting Hsp90-Cdc37 Interactions in Several Diseases.

Heat shock protein (Hsp) 90 is an ATP-dependent chaperone and plays a vital role in the folding, maturation, and stability of a protein. Hsp90 and its client proteins have become targets of various diseases through the regulation of disease-related proteins. Inhibition of Hsp90 production and activity prevents ATP hydrolysis, resulting in the ubiquitination and proteasome degradation of client proteins. However, the Hsp90 inhibitor has obvious toxic side effects and the inevitable heat shock response. Cell division cycle 37 (Cdc37) is a crucial Hsp90 kinase-specific co-chaperone, which forms a complex with Hsp90 to regulate kinase and non-kinase client's activities, cell communication, and signal transduction. The Hsp90-Cdc37 complex maintains cell survival by stabilizing abnormal client proteins and regulating cell growth signals. The abnormal activation of Hsp90-Cdc37 protein-protein interaction (PPI) often leads to the aggravation of diseases, such as cancer and neurodegenerative diseases. Compared with ATP competitive Hsp90 inhibitors, blocking Hsp90-Cdc37 PPI has higher selectivity, fewer toxic side effects, and better application prospects. This review detailed the biological characteristics of Hsp90-Cdc37 PPI and its role in several human diseases. Besides, the latest research progress in inhibitors is summarized and discussed to guide further research and clinical application.

ACY-1215 suppresses the proliferation and induces apoptosis of chronic myeloid leukemia cells via the ROS/PTEN/Akt pathway.

Chronic myeloid leukemia (CML) is a hematological tumor marked by the bcr-abl fusion gene formed by t (9;22) (q34; q11), which translated into the BCR-ABL protein. Tyrosine kinase inhibitors (TKIs) have been widely used to cure CML patients. Nevertheless, the emergence of TKI resistance has become the problem to the outcome of CML patients. Histone deacetylase 6 (HDAC6), a kind of Hsp90α deacetylase, was detected to be overexpressed in chronic myeloid leukemia stem cells. Besides, the loss of HDAC6 enzymatic activity can result in the degradation of Hsp90α's client proteins, such as BCR-ABL, the oncoprotein of CML. Here, we explored the expression of HDAC6 and discovered that it was upregulated compared with control in CML. Then we explored the effect of Rocilinostat (ACY-1215), a specific HDAC6 inhibitor, on CML cells. Our results proved that ACY-1215 could induce apoptosis and cell cycle arrest in a ROS-dependent manner. Moreover, we detected a downregulation of the BCR-ABL signaling pathway in the ACY-1215 treatment group. Mechanistically, we noted that the upregulation of PTEN was induced after being treated by ACY-1215 and its downstream protein p-Akt was decreased. The Akt activator SC79 can partially reverse the influence of ACY-1215 on CML cells. Besides, our results also proved that ACY-1215 can synergize with imatinib to suppress chronic myeloid leukemia in vitro and in vivo. On the whole, our study revealed that HDAC6 is a possible therapeutic target in CML, and the combination therapy of TKI and HDAC6 inhibitor may improve the outcome of CML patients.

Anti-Cancer Properties of Ginkgolic Acids in Human Nasopharyngeal Carcinoma CNE-2Z Cells via Inhibition of Heat Shock Protein 90.

Ginkgo biloba L. has been used in traditional Chinese medicine (TCM) for thousands of years. However, the anti-cancer properties of ginkgolic acids (GAS) isolated from G. biloba have not been investigated in human nasopharyngeal carcinoma cells. In this study, GAS exhibited an inhibitory effect on the ATPase activity of heat shock protein 90 (Hsp90) and anti-proliferative activities against four human cancer cell lines, with IC50 values ranging from 14.91 to 23.81 μg·mL−1. In vivo experiments confirmed that GAS inhibited tumor growth in CNE-2Z cell-xenografted nude mice with low hepatotoxicity. We further demonstrated that GAS suppressed migration and invasion and induced the apoptosis of CNE-2Z cells by inducing the degradation of Hsp90 client proteins (MMP-2, MMP-9, Her-2, c-Raf, Akt, and Bcl-2). Together, GAS are new Hsp90 inhibitors by binding to Hsp90 (hydrogen bond and hydrophobic interaction). Thus, GAS from G. biloba might represent promising Hsp90 inhibitors for the development of anti-nasopharyngeal carcinoma agents.

Oncogenic HSP90 Facilitates Metabolic Alterations in Aggressive B-cell Lymphomas.

HSP90 is critical for maintenance of the cellular proteostasis. In cancer cells, HSP90 also becomes a nucleating site for the stabilization of multiprotein complexes including signaling pathways and transcription complexes. Here we described the role of this HSP90 form, referred to as oncogenic HSP90, in the regulation of cytosolic metabolic pathways in proliferating B-cell lymphoma cells. Oncogenic HSP90 assisted in the organization of metabolic enzymes into non-membrane-bound functional compartments. Under experimental conditions that conserved cellular proteostasis, oncogenic HSP90 coordinated and sustained multiple metabolic pathways required for energy production and maintenance of cellular biomass as well as for secretion of extracellular metabolites. Conversely, inhibition of oncogenic HSP90, in absence of apparent client protein degradation, decreased the efficiency of MYC-driven metabolic reprogramming. This study reveals that oncogenic HSP90 supports metabolism in B-cell lymphoma cells and patients with diffuse large B-cell lymphoma, providing a novel mechanism of activity for HSP90 inhibitors. SIGNIFICANCE: The oncogenic form of HSP90 organizes and maintains functional multienzymatic metabolic hubs in cancer cells, suggesting the potential of repurposing oncogenic HSP90 selective inhibitors to disrupt metabolism in lymphoma cells.

Structure-Activity Relationships of Benzothiazole-Based Hsp90 C-Terminal-Domain Inhibitors.

Heat shock protein 90 (Hsp90) is a chaperone responsible for the maturation of many cancer-related proteins, and is therefore an important target for the design of new anticancer agents. Several Hsp90 N-terminal domain inhibitors have been evaluated in clinical trials, but none have been approved as cancer therapies. This is partly due to induction of the heat shock response, which can be avoided using Hsp90 C-terminal-domain (CTD) inhibition. Several structural features have been shown to be useful in the design of Hsp90 CTD inhibitors, including an aromatic ring, a cationic center and the benzothiazole moiety. This study established a previously unknown link between these structural motifs. Using ligand-based design methodologies and structure-based pharmacophore models, a library of 29 benzothiazole-based Hsp90 CTD inhibitors was prepared, and their antiproliferative activities were evaluated in MCF-7 breast cancer cells. Several showed low-micromolar IC50, with the most potent being compounds 5g and 9i (IC50, 2.8 ± 0.1, 3.9 ± 0.1 μM, respectively). Based on these results, a ligand-based structure–activity relationship model was built, and molecular dynamics simulation was performed to elaborate the binding mode of compound 9i. Moreover, compound 9i showed degradation of Hsp90 client proteins and no induction of the heat shock response.

CircSTK40 contributes to recurrent implantation failure via modulating the HSP90/AKT/FOXO1 axis

Increasing evidence has revealed a close relationship between non-coding RNAs and recurrent implantation failure (RIF). However, the role of circular RNAs (circRNAs) in RIF pathogenesis remains largely unknown. Microarray analyses were used to identify the differentially expressed circRNA-circSTK40. Functional experiments, including decidualization induction and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay, were performed to determine the effects of circSTK40 on human endometrial stromal cells (ESCs). The interactions between circSTK40 and proteins were investigated by RNA pull-down, RNA immunoprecipitation, and co-immunoprecipitation (coIP) assays. We observed that circSTK40 expression was upregulated in the RIF midluteal-phase endometrial samples. circSTK40 overexpression in ESCs inhibited the decidualization process but concurrently enhanced cell survival during stress. Mechanistically, circSTK40 directly bound to HSP90 and CLU, thus functioning as a scaffold to block their interactions and hinder the proteasomal degradation of HSP90. The resulting high levels of HSP90 led to the activation of the AKT pathway and downregulation of FOXO1 expression. Inhibitors of AKT (MK-2206) and HSP90 (17AAG) both abolished the effects of circSTK40 overexpression in ESCs and increased the decidualization levels in a dose-dependent manner. Our findings indicate a novel epigenetic mechanism for RIF pathogenesis involving circSTK40 activity and provide a foundation for targeted treatments in patients with low endometrial receptivity.

CDC37 As a Novel Target for the Treatment of NPM1-ALK Expressing Anaplastic Large Cell Lymphomas

Anaplastic large cell lymphoma (ALCL) is an aggressive CD30+ T-cell lymphoma that accounts for 2-8% and 10-15% of non-Hodgkin lymphomas in adults and children, respectively. The currently used standard therapy for anaplastic lymphoma kinase (ALK, a member of the insulin receptor superfamily) positive ALCL has limited effectiveness, resulting in a substantial percentage of cases with poor outcomes, either failing to enter remission or relapse within a few months after starting treatment. Thus, there is a clear unmet clinical need for developing novel, effective and safer therapeutic strategies for ALCL. Nucleophosmin 1 (NPM1) is a nucleolar phosphoprotein, which functions as a molecular chaperone for proteins and nucleic acids. Approximately 50% of ALCL cases are positive for the NPM1-ALK fusion chimera generated by the t(2;5) chromosomal translocation. The oligomerization domain of NPM1 in the fusion protein NPM1-ALK mediates the ligand-independent dimerization of chimeric protein, which results in constitutive activation of the chimeric tyrosine kinase activity leading to downstream signaling pathways responsible for the oncogenicity. As NPM1-ALK is an established heat shock protein 90 (HSP90) client protein, we hypothesized that disruption of the interaction between master chaperone HSP90 and co-chaperone Cdc37 would be a viable druggable target. Celastrol is a natural compound extracted from the root bark of Chinese medicinal plant Tripterygium wilfordi. Anticancer properties of celastrol are attributed to its action on the interaction of HSP90 and its co-chaperone Cdc37. Hsp90 is an ATP-dependent master molecular chaperone. Cdc37 is a Hsp90 co-chaperone and disruption of Hsp90-Cdc37 interaction will result in the degradation of Hsp90 client proteins. Celastrol disrupts the interaction of Hsp90 and Cdc37 through inhibition of Hsp90 ATPase activity without blockage of ATP binding site as observed with Hsp90 inhibitors. We used NPM1-ALK endogenously expressing Karpas299, SUDHL-1 and ectopically expressing Ba/F3-FG-NPM1-ALK cell lines in our study. Treatment with celastrol (0.1-1 µM) significantly induced apoptosis dose dependently in ALCL cells. Apoptosis was associated with activated caspase 3 and poly (ADP-ribose) polymerase cleavage. We also observed upregulation of proapoptotic protein BAD and downregulation of antiapoptotic proteins survivin and MCL1. Celastrol treatment inhibited NPM1-ALK mediated signaling and downstream signaling effectors STAT3, AKT, and ERK1/2 by degradation of the NPM1-ALK fusion protein. Cell survival was evaluated by clonogenic survival assay and results were clearly indicative that the clonogenic survival is diminished in celastrol treated samples compared to controls. Our immunoprecipitation studies clearly demonstrated that celastrol treatment disrupted the interaction between Hsp90 and Cdc37 and induced degradation of the NPM1-ALK fusion protein. Altogether, targeting Cdc37 using celastrol is a novel therapeutic approach to induce apoptosis in ALCL cells expressing NPM1-ALK and warrants developing future therapeutic intervention strategies. DisclosuresGanguly:Amgen: Other: Advisory Board; Seattle Genetics: Speakers Bureau.

Discovery of a covalent inhibitor of heat shock protein 90 with antitumor activity that blocks the co-chaperone binding via C-terminal modification

Heat shock protein (Hsp90), a critical molecular chaperone that regulates the maturation of a large number of oncogenic client proteins, plays an essential role in the growth of neoplastic cells. Herein, DDO-6600 is identified to covalent modification of Cys598 on Hsp90 from in silico study and is verified by a series of biological assays. We demonstrated that DDO-6600 covalently bound to Cys598 on the Hsp90 C terminus and exhibited antiproliferative activities against multiple tumor cells without inhibiting ATPase activity. Further studies showed that DDO-6600 disrupted the interaction between Hsp90 and Cdc37, which induced the degradation of kinase client proteins in multiple tumor cell lines, promoted apoptosis, and inhibited cell motility. Our findings offer mechanic insights into the covalent modification of Hsp90 and provide an alternative strategy for the development of Hsp90 covalent regulators or chemical probes to explore the therapeutical potential of Hsp90.

Ring-opening of five-membered heterocycles conjugated 4-isopropylresorcinol scaffold-based benzamides as HSP90 inhibitors suppressing tumor growth invitro and invivo.

A series of ring-opened dihydroxybenzamides have been designed and synthesized as heat shock protein 90 inhibitors. One of derivatives, compound 6b ((N-ethyl-2,4-dihydroxy-5-isopropyl-N-(pyridin-3-yl)benzamide)) demonstrated remarkable antiproliferative activity against in human KRAS mutant A549 and EGFR T790M mutant H1975 lung cancer cell lines with GI50 values of 0.07 and 0.05μM, respectively. It is also active against in other cancer cell lines, such as colorectal HCT116 (GI50=0.09μM), liver Hep3B (GI50=0.20μM) and breast MDA-MB-231 (GI50=0.09μM), and shows no evidence of toxicity in normal cell line. Compound 6b has an IC50 of 110.18nM in HSP90α inhibitory activity, slightly better than reference compound 1 (17-AAG, IC50=141.62nM) and achieves the degradation of multiple HSP90 client proteins in a dose- and time-dependent manner and downstream signaling of Akt in a concentration- and time-dependent manner in the human A549 lung cancer cell line. In the Boyden chamber assay, compound 6b can efficiently inhibit the migration of A549cells when compared to the reference compound 1. It also induce significant activity through the apoptotic pathway. Treatment with 6b showed no vision toxicity (IC50>10μM) on 661w photoreceptor cells as compared to AUY922 (3a) with a 0.04μM values of IC50 and has no effect in hERG test. In a bidirectional Caco-2 permeability assay, compound 6b was classified as a highly permeable compound which is not a substrate of efflux transporters. In a pharmacokinetic study in rats, 6b showed an F=17.8% of oral bioavailability. The effect of metabolic stability of compound 6b in human hepatocytes showed a T1/2 of 67.59min. Compound 6b (50mg/kg, po, daily) exhibits antitumor activity with a 72% TGD (tumor growth delay) in human A549 lung xenograft. The combination of 6b and afatinib, orally administered, showed tumor growth suppression with 67.5% of TGI in lung H1975 xenograft model. Thus compound 6b is a lead compound for further development of potential agents to treat lung cancer.

C0818, a novel curcumin derivative, induces ROS-dependent cytotoxicity in human hepatocellular carcinoma cells in vitro via disruption of Hsp90 function.

Heat shock protein 90 (Hsp90) is the most common molecular chaperone that controls the maturation of many oncoproteins critical in tumor development. Hsp90 has been considered as a promising target for cancer treatment, but the clinical significance of Hsp90 and the mechanisms of Hsp90 regulating the tumor-promoting effects in hepatocellular carcinoma (HCC) remain obscure. Previous studies have shown that curcumin, a polyphenol derived from the plant turmeric (Curcuma longa), inhibits tumor growth, which may provide an effective alternative therapy for HCC. Compared to curcumin, a novel derivative of curcumin, 3,5-(E)-Bis(3-methoxy-4-hydroxybenzal)-4-piperidinone hydrochloride (C0818) that is more potent in Hsp90 inhibition and antitumor activity. In this study, we investigated the effect of C0818 on HCC cells in vitro and its relation to Hsp90 inhibition. We showed that C0818 concentration-dependently inhibited the proliferation, the colony formation and induced apoptosis in HepG2 and Sk-Hep-1 cells. C0818 concentration-dependently inhibited DNA synthesis and induced G2/M phase arrest in HepG2 and Sk-Hep-1 cells. We further demonstrated that C0818induced ROS- and caspase-dependent apoptosis in HCC cells through the mitochondrial-mediated pathway. C0818 induced the degradation of Hsp90 client proteins as RAS, C-Raf, P-C-Raf, Erk, P-ERK, MEK, P-MEK, Akt and P-Akt, which led to subsequent inhibition of the RAS/RAF/MEK/ERK and PI3K/AKT pathways. We revealed that C0818 could inhibit the binding of Hsp90 with its clients without affecting their transcription, which subsequently induced the degradation of Hsp90 clients by the proteasome rather than the lysosome. These results are of potential importance for elucidating a novel Hsp90 inhibitor targeting HCC.