Heat Shock Protein 90 Inhibitor Research Articles

Heat shock protein 90 promotes RNA helicase DDX5 accumulation and exacerbates hepatocellular carcinoma by inhibiting autophagy.

Objective:Hepatocellular carcinoma (HCC), the main type of liver cancer, has a high morbidity and mortality, and a poor prognosis. RNA helicase DDX5, which acts as a transcriptional co-regulator, is overexpressed in most malignant tumors and promotes cancer cell growth. Heat shock protein 90 (HSP90) is an important molecular chaperone in the conformational maturation and stabilization of numerous proteins involved in cell growth or survival.Methods:DDX5 mRNA and protein expression in surgically resected HCC tissues from 24 Asian patients were detected by quantitative real-time PCR and Western blot, respectively. The interaction of DDX5-HSP90 was determined by molecular docking, immunoprecipitation, and laser scanning confocal microscopy. The autophagy signal was detected by Western blot. The cell functions and signaling pathways of DDX5 were determined in 2 HCC cell lines. Two different murine HCC xenograft models were used to determine the function of DDX5 and the therapeutic effect of an HSP90 inhibitor.Results:HSP90 interacted directly with DDX5 and inhibited DDX5 protein degradation in the AMPK/ULK1-regulated autophagy pathway. The subsequent accumulation of DDX5 protein induced the malignant phenotype of HCC by activating the β-catenin signaling pathway. The silencing of DDX5 or treatment with HSP90 inhibitor both blocked in vivo tumor growth in a murine HCC xenograft model. High levels of HSP90 and DDX5 protein were associated with poor prognoses.Conclusions:HSP90 interacted with DDX5 protein and subsequently protected DDX5 protein from AMPK/ULK1-regulated autophagic degradation. DDX5 and HSP90 are therefore potential therapeutic targets for HCC.

SU086, a New Inhibitor of HSP90, Impairs Glycolysis and Represents a New Treatment Strategy for Advanced Prostate Cancer

Among men, prostate cancer is the second leading cause of cancer-associated mortality, with advanced disease remaining a major clinical challenge. We describe a small molecule, SU086, as a new therapeutic strategy for advanced prostate cancer. We demonstrate that SU086 inhibits the growth of prostate cancer cells in vitro, cell line and patient-derived xenografts in vivo, and ex vivo prostate cancer patient specimens. Furthermore, SU086 in combination with standard of care second-generation anti-androgen therapies displayed increased impairment of prostate cancer cell and tumor growth in vitro and in vivo. Cellular thermal shift assay (CETSA) revealed that SU086 binds to heat shock protein 90 (HSP90) and leads to a decrease in HSP90 levels. Proteomic and metabolomic profiling further revealed that the SU086 decreases HSP90 and leads to perturbation of glycolysis. Our study identifies SU086 as a new treatment for advanced prostate cancer as a single agent or when combined with second-generation anti-androgens.

Effect of Geldanamycin Binding on the Thr90 Phosphorylation Site of Hsp90

Prostate cancer is hormone-dependent and the androgen receptor (AR) is involved in its development. AR is a transcription factor that is activated by ligand binding, result in its translocation into the nucleus, where it initiates gene transcription. In an inactive state in cytoplasm AR exists as a complex with heat shock protein 90 (HSP90) and some other proteins. When the agonist binds, a conformational change in AR occurs, resulting in HSP90 and other chaperones dissociating. Recently it has been shown that for the dissociation of the HSP90-AR complex and the translocation of the latter into the nucleus, phosphorylation of the Thr-90 residue of the N-terminal domain of HSP90 is necessary. In this work, the effect of the HSP90 inhibitor, geldanamycin, interacting with the ATP-binding site, on the Thr90 phosphorylation site was investigated by molecular modeling methods. It has been shown that inhibitor binding slightly affects the size and mobility of cavity around Thr90. It is suggested that inhibitor binding to HSP90 does not result in changing the protein structure and does not influence on protein phosphorylation, and partially explains low effectiveness of such type of drugs in the therapy of prostate cancer.

Heat Shock Protein-90 Inhibition Alters Activation of Pancreatic Stellate Cells and Enhances the Efficacy of PD-1 Blockade in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) has a prominent fibrotic stroma, which is a result of interactions between tumor, immune and pancreatic stellate cells (PSC), or cancer-associated fibroblasts (CAF). Targeting inflammatory pathways present within the stroma may improve access of effector immune cells to PDAC and response to immunotherapy. Heat shock protein-90 (Hsp90) is a chaperone protein and a versatile target in pancreatic cancer. Hsp90 regulates a diverse array of cellular processes of relevance to both the tumor and the immune system. However, to date the role of Hsp90 in PSC/CAF has not been explored in detail. We hypothesized that Hsp90 inhibition would limit inflammatory signals, thereby reprogramming the PDAC tumor microenvironment to enhance sensitivity to PD-1 blockade. Treatment of immortalized and primary patient PSC/CAF with the Hsp90 inhibitor XL888 decreased IL6, a key cytokine that orchestrates immune changes in PDAC at the transcript and protein level in vitro XL888 directly limited PSC/CAF growth and reduced Jak/STAT and MAPK signaling intermediates and alpha-SMA expression as determined via immunoblot. Combined therapy with XL888 and anti-PD-1 was efficacious in C57BL/6 mice bearing syngeneic subcutaneous (Panc02) or orthotopic (KPC-Luc) tumors. Tumors from mice treated with both XL888 and anti-PD-1 had a significantly increased CD8+ and CD4+ T-cell infiltrate and a unique transcriptional profile characterized by upregulation of genes associated with immune response and chemotaxis. These data demonstrate that Hsp90 inhibition directly affects PSC/CAF in vitro and enhances the efficacy of anti-PD-1 blockade in vivo.

Antimyeloma activity of heat shock protein-90 inhibition

AbstractWe show that multiple myeloma (MM), the second most commonly diagnosed hematologic malignancy, is responsive to hsp90 inhibitors in vitro and in a clinically relevant orthotopic in vivo model, even though this disease does not depend on HER2/neu, bcr/abl, androgen or estrogen receptors, or other hsp90 chaperoning clients which are hallmarks of tumor types traditionally viewed as attractive clinical settings for use of hsp90 inhibitors, such as the geldanamycin analog 17-AAG. This class of agents simultaneously suppresses in MM cells the expression and/or function of multiple levels of insulin-like growth factor receptor (IGF-1R) and interleukin-6 receptor (IL-6R) signaling (eg, IKK/NF-κB, PI-3K/Akt, and Raf/MAPK) and downstream effectors (eg, proteasome, telomerase, and HIF-1α activities). These pleiotropic proapoptotic effects allow hsp90 inhibitors to abrogate bone marrow stromal cell-derived protection on MM tumor cells, and sensitize them to other anticancer agents, including cytotoxic chemotherapy and the proteasome inhibitor bortezomib. These results indicate that hsp90 can be targeted therapeutically in neoplasias that may not express or depend on molecules previously considered to be the main hsp90 client proteins. This suggests a more general role for hsp90 in chaperoning tumor- or tissue-type-specific constellations of client proteins with critical involvement in proliferative and antiapoptotic cellular responses, and paves the way for more extensive future therapeutic applications of hsp90 inhibition in diverse neoplasias, including MM.

Inhibition of Embryonic HSP 90 Function Promotes Variation of Cold Tolerance in Zebrafish.

Accumulating evidence indicates that heat shock protein 90 (HSP90) plays essential roles in modulation of phenotypic plasticity in vertebrate development, however, the roles of HSP90 in modulation of cold tolerance capacity in fish are still unclear. In the present study, we showed that transient inhibition of embryonic HSP90 function by a chemical inhibitor or low conductivity stress promoted variation of cold tolerance capacity in adult zebrafish. Further work showed that embryonic HSP90 inhibition enhanced cold tolerance in adult zebrafish could be transmitted to their offspring. RNA-seq data showed that embryonic HSP90 inhibition enhanced cold tolerance involves variation of gene expression related to proteasome, lysosome, autophagy, and ribosome. Experiments with zebrafish ZF4 cells showed that two differentially expressed genes atg9b and psmd12 were up-regulated by radicicol treatment and provided protective roles for cells under cold stress, indicating that up-regulation of autophagy and proteasome function contributes to enhanced cold tolerance. The present work sheds a light on the roles of HSP90 in regulation of phenotypic plasticity associated with thermal adaptation in fish.

Heat shock protein90 as a molecular target for therapy in oral squamous cell carcinoma: Inhibitory effects of 17‑DMAG and ganetespib on tumor cells.

Heat shock protein90 (HSP90) expression is upregulated in numerous types of cancer. However, its role as a candidate for molecular targeted therapy in oral squamous cell carcinoma (OSCC) cells is poorly understood. In the present study, a common upstream search was performed using molecular network analysis software for proteins with expression abnormalities that were found in a proteomic analysis of six OSCC cell lines. HSP90 was identified as a target protein. In clinical samples, high frequencies of HSP90‑high expression were detected via immunohistochemistry (26/58;45%). Furthermore, the HSP90 expression status was associated with cervical lymph node metastasis (P=0.015). Furthermore, the potential of HSP90 as a candidate for molecular targeted therapy in OSCC cells was investigated using the HSP90 inhibitors 17‑dimethylaminoethylamino‑17‑demethoxygeldanamycin (17‑DMAG) and ganetespib. KON cells, which strongly express HSP90, were treated with the HSP90 inhibitors. The numbers of living cells in the 17‑DMAG and ganetespib‑treated groups were lower than those in the non‑treated group. The cells treated with the inhibitors demonstrated reduced cell viability and migration, and this was associated with markedly decreased levels of the HSP90 target proteins EGFR, phospho‑EGFR, phospho‑MEK and phospho‑MAPK in the treated groups compared with the non‑treated group. To the best of our knowledge, this was the first study to investigate the effects of 17‑DMAG and ganetespib on OSCC cells. The present results indicated the potential of HSP90 as a useful candidate for molecular targeted therapy in OSCC. However, additional studies with larger sample sizes are required to confirm these findings.

Priming with HDAC Inhibitors Sensitizes Ovarian Cancer Cells to Treatment with Cisplatin and HSP90 Inhibitors.

Ovarian cancer is the fifth leading cause of cancer deaths. Chemoresistance, particularly against platinum compounds, contributes to a poor prognosis. Histone deacetylase inhibitors (HDACi) and heat shock protein 90 inhibitors (HSP90i) are known to modulate pathways involved in chemoresistance. This study investigated the effects of HDACi (panobinostat, LMK235) and HSP90i (luminespib, HSP990) on the potency of cisplatin in ovarian cancer cell lines (A2780, CaOV3, OVCAR3 and cisplatin-resistant sub-clones). Preincubation with HDACi increased the cytotoxic potency of HSP90i, whereas preincubation with HSP90i had no effect. Preincubation with HSP90i or HDACi 48h prior to cisplatin enhanced the cisplatin potency significantly in all cell lines via apoptosis induction and affected the expression of apoptosis-relevant genes and proteins. For CaOV3CisR and A2780CisR, a preincubation with HDACi for 48–72 h led to complete reversal of cisplatin resistance. Furthermore, permanent presence of HDACi in sub-cytotoxic concentrations prevented the development of cisplatin resistance in A2780. However, triple combinations of HDACi, HSP90i and cisplatin were not superior to dual combinations. Overall, priming with HDACi sensitizes ovarian cancer cells to treatment with HSP90i or cisplatin and has an influence on the development of cisplatin resistance, both of which may contribute to an improved ovarian cancer treatment.

Improving the Efficacy of Checkpoint Blockade Inhibitors in Breast Cancer by Combining with Radiation Therapy

Large cohorts of recent clinical studies have established that increased levels of tumor-infiltrating lymphocytes (TILs) in patients with the triple-negative breast cancer (TNBC) subtype predicted better clinical outcomes compared to other subtypes. These observations led to the hypothesis that women with TNBC may respond to checkpoint blockade therapy. However, early results from these trials using checkpoint inhibitors alone or with chemotherapy has shown very little promise in breast cancer patients, despite the remarkable long-lasting responses in other hard to treat malignancies such as non-small cell lung and melanoma. Although the outcome falls short of the expectation, we hypothesized that radiation therapy can be utilized to increase the efficacy of check point blockade inhibitors of the stress-induced heat shock protein 70 (HSP70) in order to reverse immunosuppression and induce neo-antigens to improve the outcomes of TNBC. To test the effectiveness of a combination of checkpoint blockade inhibitors and radiation therapy, this study utilized 6-week old female BALB/c 4T1 tumor-bearing mice as a model for TNBC. Combination treatment of HSP70 inhibitors and targeted irradiation was performed prior to tumor resection and data collection. Specifically, Radiation Therapy doses were planned and delivered using a Small Animal Radiation Platform (SARRP) equipped with on-board cone-beam CT. A single fraction of 17 Gy (Two fractions of 11.5 Gy) doses were delivered based on EQD2 calculation (alpha Beta ratio = 4). To achieve highly localized dose delivery, an x-ray collimator was used to focus the broad beam from a 225 kVp x-ray tube. Our preliminary data provide evidence that targeting HSP70 in combination with radiation therapy has dual activity on the tumor and the immunosuppressive microenvironment. Furthermore, reversal of HSP70 blockade significantly reduces the immunosuppressive MDSC infiltration in lungs by more than 50% compared to lungs from control mice. This also led to increased tumor infiltration of effector T cells from 1-2% in controls to 12% in lungs from mice treated with HSP70 inhibitor in combination with radiation. Interestingly, T cell infiltration is further increased to 20% when check point blockade inhibitor is combined with HSP70 inhibitor and radiation. Therefore, we showed that HSP70 regulates three fundamental processes; i) protecting tumor cells from cytotoxic cell death, ii) serving as a powerful tumor antigen attracting TILs, and iii) modulating immunosuppressive MDSCs. In summary, our studies elucidate the molecular mechanism by which HSP70 cytoprotects tumor cells from cytotoxic agents while regulating the immunosuppressive MDSCs which limits the anti-tumor activity of TILs. They also provide evidence that blocking HSP70 potentiates the efficacy of the checkpoint blockade particularly when combined with radiation in syngeneic mouse models representing TNBC subtype.

Target-Mediated Brain Tissue Binding for Small Molecule Inhibitors of Heat Shock Protein 90.

Drug distribution in the brain is generally associated with an affinity for fatty brain tissues and therefore known to be species- and concentration-independent. We report here the effect of target affinity on brain tissue binding for 10 small molecules designed to inhibit brain heat shock protein 90 (HSP90), a widespread protein whose expression is 1–2% of total cytosolic proteins in eucaryotes. Our results show that increasing the test item concentrations from 0.3 to 100 µM increased the unbound fraction 32-fold for the most potent molecules, with no change for the inactive one (1.1 fold change). Saturation of HSP90 led to normal concentration-independent brain tissue binding. In vivo pharmacokinetics performed in rats showed that the overall volume of distribution of compounds is correlated with their affinity for HSP90. The in vitro binding and in vivo pharmacokinetics (PK) performed in rats showed that small molecule HSP90 inhibitors followed the principle of target-mediated drug disposition. We demonstrate that assessing unbound fractions in brain homogenate was subject to HSP90 target interference; this may challenge the process of linking systemic-free drug concentrations to central nervous system unbound concentrations necessary to establish the proper pharmacokinetics/pharmacodynamics (PK/PD) relation needed for human dose prediction.

Sulfoxythiocarbamate S-4 inhibits HSP90 in human cutaneous squamous cell carcinoma cells

Cancer cells rely heavily on molecular chaperones, such as heat shock protein 90 (HSP90), and their co-chaperones. The development of HSP90 inhibitors is an attractive therapeutic approach that has the potential to affect multiple hallmarks of cancer. Such approach is particularly needed for tumors that carry large mutational burdens, including cutaneous squamous cell carcinomas (cSCC). We previously identified sulfoxythiocarbamate S-4 as an HSP90 inhibitor. In this study, we investigated the mechanism(s) by which S-4 compromises the viability of human cSCC cells. S-4 inhibits HSP90 and causes depletion of its clients HER2, a tyrosine kinase oncoprotein, and Bcl-2, an anti-apoptotic protein. The decrease in Bcl-2 is accompanied by cytochrome c release from mitochondria into the cytoplasm, suggesting apoptosis. In the surviving cells, depletion of the HSP90 clients cyclin D and CDK4 by S-4 prevents phosphorylation of the retinoblastoma protein Rb and the release of transcription factor E2F, inhibiting G1-S cell cycle progression and cell division. These findings illustrate the comprehensive effectiveness of S-4 and encourage future development of compounds of this type for cancer prevention and treatment.

The HSP90 Inhibitor, 17-AAG, Influences the Activation and Proliferation of T Lymphocytes via AKT/GSK3β Signaling in MRL/lpr Mice.

ObjectiveTo explore the molecular mechanism of 17-AAG in the treatment of systemic lupus erythematosus (SLE), and the effects of the heat shock protein 90 (HSP90) inhibitor 17-AAG on the activation and proliferation of lymphocytes and the AKT/GSK3β signaling pathway in MRL/lpr mice were detected.MethodsMRL/lpr mice were randomly divided into the control group and the experimental group. The experimental group was injected intraperitoneally with 17-AAG, and T lymphocytes were separated by magnetic beads. Lymphocyte proliferation was detected by MTT and flow cytometry (FCM), and the expression of the HSP90 protein and PI3K/AKT signaling pathway-related proteins was detected by Western blotting. Renal histopathology and immune complex deposition were also observed in both groups.ResultsImmune complex deposition and inflammation decreased in kidneys from MRL/lpr mice in the experimental group. HSP90 protein expression, T lymphocyte proliferation and phosphorylated AKT and GSK3β levels also decreased in the experimental group.Conclusion17-AAG can inhibit the activation and proliferation of T lymphocytes and downregulate the AKT/GSK3β signaling pathway, which may be relevant for the treatment of SLE.

HSP90 Inhibitor, 17-DMAG, Alone and in Combination with Lapatinib Attenuates Acquired Lapatinib-Resistance in ER-positive, HER2-Overexpressing Breast Cancer Cell Line.

Simple SummaryLapatinib is a tyrosine kinase inhibitor widely used as a treatment for a Human Epidermal growth factor Receptor 2 (HER2) (+) breast cancer patients. However, when resistance is acquired through continued exposure, and it is associated with a poor prognosis for patients. In this study, we identified HSP90 as a common node for acquired resistance to lapatinib in two lapatinib resistant cell lines using proteomic analysis. Notably, in vitro and in vivo studies demonstrated synergy effect between lapatinib and an HSP90 inhibitor were observed in the estrogen receptor (+) HER2 (+) breast cancer cell only. These results could be a potential strategy for future clinical trials for HSP90 inhibitors in treatment—refractory HER2 (+) metastatic cancer patientsLapatinib, a Human Epidermal growth factor Receptor 2 (HER2)-targeting therapy in HER2-overexpressing breast cancer, has been widely used clinically, but the prognosis is still poor because most patients acquire resistance. Therefore, we investigated mechanisms related to lapatinib resistance to evaluate new therapeutic targets that may overcome resistance. Lapatinib-resistant cell lines were established using SKBR3 and BT474 cells. We evaluated cell viability and cell signal changes, gene expression and protein changes. In the xenograft mouse model, anti-tumor effects were evaluated using drugs. Analysis of the protein interaction network in two resistant cell lines with different lapatinib resistance mechanisms showed that HSP90 protein was commonly increased. When Heat Shock Protein 90 (HSP90) inhibitors were administered alone to both resistant cell lines, cell proliferation and protein expression were effectively inhibited. However, inhibition of cell proliferation and protein expression with a combination of lapatinib and HSP90 inhibitors showed a more synergistic effect in the LR-BT474 cell line than the LR-SKBR3 cell line, and the same result was exhibited with the xenograft model. These results suggest that HSP90 inhibitors in patients with lapatinib-resistant Estrogen Receptor (ER) (+) HER2 (+) breast cancer are promising therapeutics for future clinical trials.

Priming of Anti-tumor Immune Mechanisms by Radiotherapy Is Augmented by Inhibition of Heat Shock Protein 90.

Radiotherapy is an essential part of multi-modal cancer therapy. Nevertheless, for certain cancer entities such as colorectal cancer (CRC) the indications of radiotherapy are limited due to anatomical peculiarities and high radiosensitivity of the surrounding normal tissue. The development of molecularly targeted, combined modality approaches may help to overcome these limitations. Preferably, such strategies should not only enhance radiation-induced tumor cell killing and the abrogation of tumor cell clonogenicity, but should also support the stimulation of anti-tumor immune mechanisms – a phenomenon which moved into the center of interest of preclinical and clinical research in radiation oncology within the last decade. The present study focuses on inhibition of heat shock protein 90 (HSP90) whose combination with radiotherapy has previously been reported to exhibit convincing therapeutic synergism in different preclinical cancer models. By employing in vitro and in vivo analyses, we examined if this therapeutic synergism also applies to the priming of anti-tumor immune mechanisms in model systems of CRC. Our results indicate that the combination of HSP90 inhibitor treatment and ionizing irradiation induced apoptosis in colorectal cancer cells with accelerated transit into secondary necrosis in a hyperactive Kras-dependent manner. During secondary necrosis, dying cancer cells released different classes of damage-associated molecular patterns (DAMPs) that stimulated migration and recruitment of monocytic cells in vitro and in vivo. Additionally, these dying cancer cell-derived DAMPs enforced the differentiation of a monocyte-derived antigen presenting cell (APC) phenotype which potently triggered the priming of allogeneic T cell responses in vitro. In summary, HSP90 inhibition – apart from its radiosensitizing potential – obviously enables and supports the initial steps of anti-tumor immune priming upon radiotherapy and thus represents a promising partner for combined modality approaches. The therapeutic performance of such strategies requires further in-depth analyses, especially for but not only limited to CRC.

HSP70, a Novel Regulatory Molecule in B Cell-Mediated Suppression of Autoimmune Diseases

B cells have recently emerged as playing regulatory role in autoimmune diseases. We have previously demonstrated that human peripheral blood CD19+CD24hiCD27+ B cells have regulatory function both in healthy donors and in patients with autoimmune disease. However, the mechanism of this regulation is still not fully understood. In this study, microarrays were utilized to compare gene expression of CD19+CD24hiCD27+ B cells (regulatory B cells, Bregs) with CD19+CD24loCD27− B cells (non-Bregs) in human peripheral blood. We found that heat shock protein 70 (HSP70) expression was significantly upregulated in Bregs. In vitro studies explored that HSP70 inhibition impaired the regulatory function of peripheral blood Bregs. In mouse models of autoimmune disease, using HSP70-deficient mice or HSP70 inhibitors, Bregs suppressed effector cells and rescued disease-associated phenotypes that were dependent on HSP70. Mechanistically, Bregs secreted HSP70, directly suppressing effector cells, such as T effect cells. These findings reveal that HSP70 is a novel factor that modulates Breg function and suggest that enhancing Breg-mediated production of HSP70 could be a viable therapy for autoimmune disease.

HDAC6-HSP90 pathway-mediated protein folding and degradation regulates tumor cell proliferation and metastasis

The folding and degradation of proteins in tumor cells are the key mechanisms maintaining their survival, and functions of the proteins involved are often related to the prevailing tumor resistance. With further scientific research, an increasing number of auxiliary proteins involved in protein folding and degradation have been reported in the literature. The chaperone heat shock protein 90 (HSP90) not only promotes protein folding and plays a role in maintaining protein conformation stability but also assists the proteasomal degradation of client proteins. Current research suggests that the function of HSP90 is regulated by post-translational modifications, such as acetylation. Histone deacetylase 6 (HDAC6) can inhibit the over-acetylation of HSP90 and maintain the chaperone function of HSP90 to ensure that the client protein of the chaperone HSP90 can be stably folded and maturely released, which creates conditions for tumor survival, proliferation, and metastasis. Furthermore, it can act as the client protein and autophagy receptor of HSP90. It recruits ubiquitinated proteins and deacetylates and modifies autophagy-related proteins, thus playing important roles in protein degradation and autophagy. Recently, studies revealed HDAC6, a client protein of HSP90, which plays an important role in protein degradation, cell autophagy, and maintenance of the activity of HSP90 by preventing its excessive acetylation. By inhibiting HDAC6 to promote HSP90 over-acetylation, it can effectively inhibit the binding between HSP90 and ATP, thereby affecting the folding and degradation of HSP90 on tumor proliferation and metastasis-related proteins. Simultaneously, HDAC inhibitors can help HSP90 bind more closely to its inhibitors. Therefore, HDAC6 and HSP90 inhibitors have also been widely used in related research on tumor treatment, and studies have shown that the combined application of HDAC6 and HSP90 inhibitors has a significant effect on inhibiting tumor proliferation and metastasis. Whether it is to inhibit HDAC6 alone or HSP90 alone, there are deficiencies in cancer treatment. The use of inhibitors alone may lead to the activation of other pathways related to tumor cell proliferation and survival. HDAC6-HSP90 dual-target inhibition can reduce the activation of compensatory pathways, such as autophagy, and reduce the possibility of off-target effects of HSP90 inhibitors. In this review, we focus on the crosstalk between HDAC6 and HSP90 to further explain how tumor cells initiate proliferation and metastasis signals by regulating protein folding and degradation mechanisms and discuss the research applications of HDAC6 and HSP90 inhibitors. The advantages and disadvantages are assessed to obtain novel strategies for cancer therapy by targeting the HSP90-HDAC6 pathway.

Abstract 3564: Anti-tumor effect and HIF1α inhibition by combining CDK4 inhibitor with HSP90 inhibitor in various cancer types including Rb-deficient tumor cells

Abstract As solid tumors outgrow their oxygen supply, regions with abnormal vasculature become poorly oxygenated, known as intratumoral hypoxia. Hypoxia-inducible factor 1α (HIF1α) is the main mediator to hypoxia and promotes angiogenesis, cell survival as well as cancer metastases. Overexpression of HIF1α is correlated with poor prognosis in many malignancies such as colorectal cancers. Targeting HIF1α is a potential strategy for cancer therapy. We have previously shown that cyclin-dependent kinase 1 (CDK1) stabilizes HIF1α through direct phosphorylation of its Ser668 residue in a Von Hippel-Lindau (VHL)-independent manner both under hypoxia and at G2/M under normoxia (Warfel et al., 2013). It has been acknowledged that the heat shock protein 90 (HSP90) is also a VHL-independent HIF1α stabilizer (Isaacs JS et al., 2002). We found that CDK1 may contribute to HSP90-mediated HIF1α stabilization. Combination treatment of HSP90 inhibition and CDK1 knockdown reduces HIF1α expression in hypoxia more robustly than either single treatment. Dual inhibition of CDK1 and HSP90 synergistically decreases cell viability in colorectal cancer cells and suppresses colony formation. We have observed that CDK4 plays a role in HIF1α stabilization. Using the FDA-approved CDK4/6 inhibitors (palbociclib & abemaciclib), we observed that dual inhibition of CDK4 and HSP90 synergistically inhibits cancer viability in colorectal cancer cell lines (eg. HCT116, SW480, DLD1) under both normoxia and hypoxia. Multiple HSP90 inhibitors have been tested, including ganetespib, onalespib, XL888 and TAS116, which indicates such combinational inhibition as a class effect. As expected, the combination treatment of CDK4 knockdown/inhibition and HSP90 inhibition is able to reduce the level of HIF1α and suppress cell viability in various cancer cell lines (eg. colorectal, glioblastoma, breast, and prostate cancers). The combination treatment induces PARP cleavage, indicating activated apoptosis. Interestingly, we found that knockdown of RB does not affect the response of HCT116 or SW480 cells to the combination treatment with abemaciclib and TAS116, although RB deficiency contributes to a relative resistance to CDK4/6 inhibitor monotherapy. Combination of abemaciclib plus TAS116 inhibits cell viability in Rb-deficient cells (eg. H1048 and Saos2). Hypoxia sensitizes the Rb-deficient MDA-MB-468 breast cancer cell line to abemaciclib treatment. Our findings suggest a therapeutic potential for utilizing the combination of CDK4 and HSP90 inhibitors in cancer treatment. Citation Format: Shuai Zhao, Lanlan Zhou, David T. Dicker, Wafik S. El-Deiry. Anti-tumor effect and HIF1α inhibition by combining CDK4 inhibitor with HSP90 inhibitor in various cancer types including Rb-deficient tumor cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3564.

Inhibiting Heat Shock Protein 90 Protects Nucleus Pulposus-Derived Stem/Progenitor Cells From Compression-Induced Necroptosis and Apoptosis.

Nucleus pulposus-derived stem/progenitor cells (NPSCs) provide novel prospects for the regeneration of degenerated intervertebral disc (IVD). Nevertheless, with aging and degeneration of IVD, the frequency of NPSCs markedly decreases. Excessive cell death could be the main reason for declined frequency of NPSCs, however, the exact mechanisms remain elusive. Thus, the present study was undertaken to explore the mechanisms of compression-induced NPSCs death, and the effects of heat shock protein 90 (HSP90) on NPSCs survival. Here, we found that compression could trigger receptor-interacting protein kinase 1 (RIPK1)/receptor-interacting protein kinase 3 (RIPK3)/mixed lineage kinase domain-like protein (MLKL)-mediated necroptosis of NPSCs. Furthermore, we found that elevated expression of HSP90 was involved in compression-induced NPSCs death, and inhibiting HSP90 could dramatically attenuate compression-induced necroptosis of NPSCs via regulating the expression and activity of RIPK1/RIPK3/MLKL, and alleviating the mitochondrial dysfunction (mitochondrial membrane potential loss and ATP depletion) and oxidative stress [production of mitochondrial reactive oxygen species (ROS), cellular total ROS and malondialdehyde, and downregulation of superoxide dismutase 2]. Besides necroptosis, compression-induced apoptosis of NPSCs was also attenuated by HSP90 inhibition. In addition, we found that enhanced expression of HSP70 contributed to the cytoprotective effects of inhibiting HSP90. More encouragingly, our results demonstrated that inhibiting HSP90 could also mitigate the exhaustion of NPSCs in vivo. In conclusion, RIPK1/RIPK3/MLKL-mediated necroptosis participates in compression-induced NPSCs death. Furthermore, targeting HSP90 to simultaneously inhibit necroptosis and apoptosis of NPSCs might be an efficient strategy for preventing the death of NPSCs, thus rescuing the endogenous repair capacity of NP tissue.

42. IDENTIFICATION OF BRAIN METASTASIS VULNERABILITIES USING METPLATFORM

The diagnosis of brain metastasis involves high morbidity and mortality and remains an unmet clinical need in spite of being the most common tumor in the brain. Exclusion of these cancer patients from clinical trials is a major cause of their limited therapeutic options. In this study, we report a novel drug-screening platform (METPlatform) based on organotypic cultures which allows identifying effective anti-metastasis agents in the presence of the organ microenvironment. We have applied this approach to clinically relevant stages of brain metastasis using both experimental models and human tumor tissue (by performing patient-derived organotypic cultures). We identified heat shock protein 90 (HSP90) as a promising therapeutic target for brain metastasis. Debio-0932, a blood-brain barrier permeable HSP90 inhibitor, shows high potency against mouse and human brain metastases from melanoma, lung and breast adenocarcinoma with distinct oncogenomic profiles at clinically relevant stages of the disease, including a novel model of local relapse after neurosurgery. Furthermore, we have also used METPlatform to perform unbiased proteomics of brain metastases in situ. By applying this analysis to brain metastases treated with the chaperone inhibitor, we uncovered non-canonical clients of HSP90 as potential novel mediators of brain metastasis and actionable mechanisms of resistance driven by autophagy. Combined therapy using HSP90 and autophagy inhibitors showed synergistic effects compared to sublethal concentrations of each monotherapy, demonstrating the potential of METPlatform to design and test rationale combination therapies to target metastasis more effectively. In conclusion, our work validates METPlatform as a potent resource for metastasis research integrating drug-screening and unbiased omic approaches that is fully compatible with human samples and questions the rationale of excluding patients with brain metastasis from clinical trials. We envision that METPlatform will be established as a clinically relevant strategy to personalize the management of metastatic disease in the brain and elsewhere.

Grp94 Regulates the Recruitment of Aneural AChR Clusters for the Assembly of Postsynaptic Specializations by Modulating ADF/Cofilin Activity and Turnover.

Temperature is a physiological factor that affects neuronal growth and synaptic homeostasis at the invertebrate neuromuscular junctions (NMJs); however, whether temperature stress could also regulate the structure and function of the vertebrate NMJs remains unclear. In this study, we use Xenopus laevis primary cultures as a vertebrate model system for investigating the involvement of heat shock protein 90 (HSP90) family of stress proteins in NMJ development. First, cold temperature treatment or HSP90 inhibition attenuates the formation of aneural acetylcholine receptor (AChR) clusters, but increases their stability after they are formed, in cultured muscles. HSP90 inhibition specifically affects the stability of aneural AChR clusters and their associated intracellular scaffolding protein rapsyn, instead of causing a global change in cell metabolism and protein expression in Xenopus muscle cultures. Upon synaptogenic stimulation, a specific HSP90 family member, glucose-regulated protein 94 (Grp94), modulates the phosphorylation and dynamic turnover of actin depolymerizing factor (ADF)/cofilin at aneural AChR clusters, leading to the recruitment of AChR molecules from aneural clusters to the assembly of agrin-induced postsynaptic specializations. Finally, postsynaptic Grp94 knock-down significantly inhibits nerve-induced AChR clustering and postsynaptic activity in nerve-muscle co-cultures as demonstrated by live-cell imaging and electrophysiological recording, respectively. Collectively, this study suggests that temperature-dependent alteration in Grp94 expression and activity inhibits the assembly of postsynaptic specializations through modulating ADF/cofilin phosphorylation and activity at aneural AChR clusters, which prevents AChR molecules from being recruited to the postsynaptic sites via actin-dependent vesicular trafficking, at developing vertebrate NMJs.