Chaperone Heat Shock Protein Research Articles

HSP70 In triple negative breast cancer: Prognostic value and clinical significance.

Triple-negative breast cancer (TNBC) has the worst prognosis and the highest immunogenic potential of all breast cancer subtypes. It is characterized by a lack of estrogen and progesterone receptors as well as HER2. A major component of the tumor microenvironment (TME) of TNBC is tumor-infiltrating lymphocytes (TILs). A chaperone heat shock protein 70 (HSP70) is involved in several pathways that enable tumour growth and progression, as well as in immune modulation. Immunohistochemical analysis of HSP70 expression in immune cells, as well as expression of immunosuppressive markers CTLA4 and PD-L1 and major TILs components: CD8, CD4 and Tregs were analyzed in the superficial and deep tumor layer of primary TNBC and compared with established clinicopathological parameters. Clinical data and surgical tissue samples from 68 TNBC patients who underwent initial surgery were included in the analysis and 36 control samples from benign breast tissue biopsies. A higher expression of TILs, CD4, CD8 and PD-L1 was found in the invasive tumor front (ITF), as compared to the tumor center (TC) (p<0001). HSP70 positive immune cells (HSP70(+) IC) in TC were associated with adverse clinical and pathological markers: higher stage of disease (P=0.013), higher grade (P=0.05) and a higher pN status (P<0.001). In addition, higher expression of HSP70(+) IC from TC was correlated with the higher expression of FOXP3(+)T cells both in ITF (N=61, rho=0.42, p<0.001) and in metastatic tissue from the draining lymph nodes (N=13, rho=0.61, P=0.026). Correlations between HSP70 immune cells expression and individual TILs components support the hypothesis of its active role in inducing immunosuppression and tumor progression. Routine determination of HSP70 expression, in immune cells of TC, may be of added value in the clinical decision-making process concerning axillary surgery.

In silico analysis of the HSP90 chaperone system from the African trypanosome, Trypanosoma brucei.

African trypanosomiasis is a neglected tropical disease caused by Trypanosoma brucei (T. brucei) and spread by the tsetse fly in sub-Saharan Africa. The trypanosome relies on heat shock proteins for survival in the insect vector and mammalian host. Heat shock protein 90 (HSP90) plays a crucial role in the stress response at the cellular level. Inhibition of its interactions with chaperones and co-chaperones is being explored as a potential therapeutic target for numerous diseases. This study provides an in silico overview of HSP90 and its co-chaperones in both T. brucei brucei and T. brucei gambiense in relation to human and other trypanosomal species, including non-parasitic Bodo saltans and the insect infecting Crithidia fasciculata. A structural analysis of T. brucei HSP90 revealed differences in the orientation of the linker and C-terminal domain in comparison to human HSP90. Phylogenetic analysis displayed the T. brucei HSP90 proteins clustering into three distinct groups based on subcellular localizations, namely, cytosol, mitochondria, and endoplasmic reticulum. Syntenic analysis of cytosolic HSP90 genes revealed that T. b. brucei encoded for 10 tandem copies, while T. b. gambiense encoded for three tandem copies; Leishmania major (L. major) had the highest gene copy number with 17 tandem copies. The updated information on HSP90 from recently published proteomics on T. brucei was examined for different life cycle stages and subcellular localizations. The results show a difference between T. b. brucei and T. b. gambiense with T. b. brucei encoding a total of twelve putative HSP90 genes, while T. b. gambiense encodes five HSP90 genes. Eighteen putative co-chaperones were identified with one notable absence being cell division cycle 37 (Cdc37). These results provide an updated framework on approaching HSP90 and its interactions as drug targets in the African trypanosome.

Contrasting genome patterns of two pseudomonas strains isolated from the date palm rhizosphere to assess survival in a hot arid environment.

The plant growth-promoting rhizobacteria (PGPRs) improve plant growth and fitness by multiple direct (nitrogen fixation and phosphate solubilization) and indirect (inducing systematic resistance against phytopathogens, soil nutrient stabilization, and maintenance) mechanisms. Nevertheless, the mechanisms by which PGPRs promote plant growth in hot and arid environments remain poorly recorded. In this study, a comparative genome analysis of two phosphate solubilizing bacteria, Pseudomonas atacamensis SM1 and Pseudomonas toyotomiensis SM2, isolated from the rhizosphere of date palm was performed. The abundance of genes conferring stress tolerance (chaperones, heat shock genes, and chemotaxis) and supporting plant growth (plant growth hormone, root colonization, nitrogen fixation, and phosphate solubilization) were compared among the two isolates. This study further evaluated their functions, metabolic pathways, and evolutionary relationship. Results show that both bacterial strains have gene clusters required for plant growth promotion (phosphate solubilization and root colonization), but it is more abundant in P. atacamensis SM1 than in P. toyotomiensis SM2. Genes involved in stress tolerance (mcp, rbs, wsp, and mot), heat shock, and chaperones (hslJ and hslR) were also more common in P. atacamensis SM1. These findings suggest that P. atacamensis SM1could have better adaptability to the hot and arid environment owing to a higher abundance of chaperone genes and heat shock proteins. It may promote plant growth owing to a higher load of root colonization and phosphate solubilization genes and warrants further in vitro study.

Flavonol glycoside complanatoside A requires FOXO/DAF-16, NRF2/SKN-1, and HSF-1 to improve stress resistances and extend the life span of Caenorhabditis elegans.

Aging is associated with the increased risk of most age-related diseases in humans. Complanatoside A (CA) is a flavonoid compound isolated from the herbal medicine Semen Astragali Complanati. CA was reported to have potential anti-inflammatory and anti-oxidative activities. In this study, we investigated whether CA could increase the stress resistance capability and life span of Caenorhabditis elegans. Our results showed that CA could extend the longevity of C. elegans in a dosage-dependent manner, while 50 μM of CA has the best effect and increased the life span of C. elegans by about 16.87%. CA also improved the physiological functions in aging worms, such as enhanced locomotor capacity, and reduced the accumulation of the aging pigment. CA could also reduce the accumulation of toxic proteins (α-synuclein and β-amyloid) and delay the onset of neurodegenerative disorders, such as Alzheimer’s disease and Parkinson’s disease, in models of C. elegans. Further investigation has revealed that CA requires DAF-16/FOXO, SKN-1, and HSF-1 to extend the life span of C. elegans. CA could increase the antioxidation and detoxification activities regulated by transcription factor SKN-1 and the heat resistance by activating HSF-1 that mediated the expression of the chaperone heat shock proteins. Our results suggest that CA is a potential antiaging agent worth further research for its pharmacological mechanism and development for pharmaceutical applications.

Targeting extracellular Hsp90: A unique frontier against cancer.

The molecular chaperone Heat Shock Protein-90 (Hsp90) is known to interact with over 300 client proteins as well as regulatory factors (eg. nucleotide and proteins) that facilitate execution of its role as a chaperone and, ultimately, client protein activation. Hsp90 associates transiently with these molecular modulators during an eventful chaperone cycle, resulting in acquisition of flexible structural conformations, perfectly customized to the needs of each one of its client proteins. Due to the plethora and diverse nature of proteins it supports, the Hsp90 chaperone machinery is critical for normal cellular function particularly in response to stress. In diseases such as cancer, the Hsp90 chaperone machinery is hijacked for processes which encompass many of the hallmarks of cancer, including cell growth, survival, immune response evasion, migration, invasion, and angiogenesis. Elevated levels of extracellular Hsp90 (eHsp90) enhance tumorigenesis and the potential for metastasis. eHsp90 has been considered one of the new targets in the development of anti-cancer drugs as there are various stages of cancer progression where eHsp90 function could be targeted. Our limited understanding of the regulation of the eHsp90 chaperone machinery is a major drawback for designing successful Hsp90-targeted therapies, and more research is still warranted.

Regulation of Protein Transport Pathways by the Cytosolic Hsp90s.

The highly conserved molecular chaperone heat shock protein 90 (Hsp90) is well-known for maintaining metastable proteins and mediating various aspects of intracellular protein dynamics. Intriguingly, high-throughput interactome studies suggest that Hsp90 is associated with a variety of other pathways. Here, we will highlight the potential impact of Hsp90 in protein transport. Currently, a limited number of studies have defined a few mechanistic contributions of Hsp90 to protein transport, yet the relevance of hundreds of additional connections between Hsp90 and factors known to aide this process remains unresolved. These interactors broadly support transport pathways including endocytic and exocytic vesicular transport, the transfer of polypeptides across membranes, or unconventional protein secretion. In resolving how Hsp90 contributes to the protein transport process, new therapeutic targets will likely be obtained for the treatment of numerous human health issues, including bacterial infection, cancer metastasis, and neurodegeneration.

Immunohistochemical Demonstration of the pGlu79 α-Synuclein Fragment in Alzheimer's Disease and Its Tg2576 Mouse Model.

The deposition of β-amyloid peptides and of α-synuclein proteins is a neuropathological hallmark in the brains of Alzheimer’s disease (AD) and Parkinson’s disease (PD) subjects, respectively. However, there is accumulative evidence that both proteins are not exclusive for their clinical entity but instead co-exist and interact with each other. Here, we investigated the presence of a newly identified, pyroglutamate79-modified α-synuclein variant (pGlu79-aSyn)—along with the enzyme matrix metalloproteinase-3 (MMP-3) and glutaminyl cyclase (QC) implicated in its formation—in AD and in the transgenic Tg2576 AD mouse model. In the human brain, pGlu79-aSyn was detected in cortical pyramidal neurons, with more distinct labeling in AD compared to control brain tissue. Using immunohistochemical double and triple labelings and confocal laser scanning microscopy, we demonstrate an association of pGlu79-aSyn, MMP-3 and QC with β-amyloid plaques. In addition, pGlu79-aSyn and QC were present in amyloid plaque-associated reactive astrocytes that were also immunoreactive for the chaperone heat shock protein 27 (HSP27). Our data are consistent for the transgenic mouse model and the human clinical condition. We conclude that pGlu79-aSyn can be generated extracellularly or within reactive astrocytes, accumulates in proximity to β-amyloid plaques and induces an astrocytic protein unfolding mechanism involving HSP27.

Profibrotic Role of Inducible Heat Shock Protein 90α Isoform in Systemic Sclerosis.

Systemic sclerosis (SSc) is an autoimmune disease that affects skin and multiple internal organs. TGF-β, a central trigger of cutaneous fibrosis, activates fibroblasts with the involvement of the stress-inducible chaperone heat shock protein 90 isoform α (Hsp90α). Available evidence supports overexpression and secretion of Hsp90α as a feature in profibrotic pathological conditions. The aim of this work is to investigate the expression and function of Hsp90α in experimental models of skin fibrosis such as human fibroblasts, C57BL/6 mice, and in human SSc. For this purpose, we generated a new experimental model based on doxorubicin administration with improved characteristics with respect to the bleomycin model. We visualized disease progression in vivo by fluorescence imaging. In this work, we obtained Hsp90α mRNA overexpression in human skin fibroblasts, in bleomycin- and doxorubicin-induced mouse fibrotic skin, and in lungs of bleomycin- and doxorubicin-treated mice. Hsp90α-deficient mice showed significantly decreased skin thickness compared with wild-type mice in both animal models. In SSc patients, serum Hsp90α levels were increased in patients with lung involvement and in patients with the diffuse form of SSc (dSSc) compared with patients with the limited form of SSc. The serum Hsp90α levels of patients dSSc were correlated with the Rodnan score and the forced vital capacity variable. These results provide new supportive evidence of the contribution of the Hsp90α isoform in the development of skin fibrosis. In SSc, these results indicated that higher serum levels were associated with dSSc and lung fibrosis.

Novel Drugs with High Efficacy against Tumor Angiogenesis.

Angiogenesis is involved in physiological and pathological processes in the body. Tumor angiogenesis is a key factor associated with tumor growth, progression, and metastasis. Therefore, there is great interest in developing antiangiogenic strategies. Hypoxia is the basic initiating factor of tumor angiogenesis, which leads to the increase of vascular endothelial growth factor (VEGF), angiopoietin (Ang), hypoxia-inducible factor (HIF-1), etc. in hypoxic cells. The pathways of VEGF and Ang are considered to be critical steps in tumor angiogenesis. A number of antiangiogenic drugs targeting VEGF/VEGFR (VEGF receptor) or ANG/Tie2, or both, are currently being used for cancer treatment, or are still in various stages of clinical development or preclinical evaluation. This article aims to review the mechanisms of angiogenesis and tumor angiogenesis and to focus on new drugs and strategies for the treatment of antiangiogenesis. However, antitumor angiogenic drugs alone may not be sufficient to eradicate tumors. The molecular chaperone heat shock protein 90 (HSP90) is considered a promising molecular target. The VEGFR system and its downstream signaling molecules depend on the function of HSP90. This article also briefly introduces the role of HSP90 in angiogenesis and some HSP90 inhibitors.

Unravelling the suitability of Branchinecta gaini as a potential biomonitor of contaminants of emerging concern in the Antarctic Peninsula region

AbstractThe occurrence and impact of contaminants of emerging concerns (CECs) have been investigated in Antarctica much less than in other parts of the world. Although legacy anthropogenic pollutants can reach Antarctica via long-range transport, CECs mainly originate from local sources. Here, we investigated the ability of a freshwater crustacean, the Antarctic fairy shrimp Branchinecta gaini, to cope with nanoscale titanium dioxide (n-TiO2), a widely used pigment in consumer products (e.g. paintings), including those for personal care (e.g. sunscreens). An in vivo acute short-term exposure study (9 h, n-TiO2 concentration range 50–200 μg ml-1) was performed and the expression levels of several genes involved in stress response were evaluated. No effect on the expression of heat-shock protein chaperone genes was found, with the exception of Hsp70a, which was significantly upregulated at 200 μg ml-1 n-TiO2. Similarly, cytochrome P450 was upregulated at 100 and 200 μg ml-1 n-TiO2, while the expression levels of cathepsin L and of antioxidant genes such as superoxide dismutase and glutathione peroxidase were significantly reduced with increasing concentrations of n-TiO2. This study shows for the first time the responsiveness and sensitivity of an Antarctic freshwater crustacean to n-TiO2 exposure and supports its suitability as a biomonitor of CECs in Antarctica.

A Computational Method for the Visualization of Nitrated Hsp90 Distribution in 3D Culture Models

Protein tyrosine (Y) nitration is an oxidative post‐translational modification that occurs in pathological conditions but is absent in normal tissue. We previously showed that nitrated proteins support tumor cell survival and migration, and recently identified the chaperone Heat Shock Protein 90 (Hsp90) as the first protein that, when nitrated, acquires a novel tumorigenic activity in glioblastoma multiforme (GBM). While Hsp90 is expressed in all cells, we showed that nitration of Hsp90 on Y33 and/or Y56 induces a pathological gain‐of‐function. Nitration on Y33 (Hsp90NY33) decreases mitochondrial metabolism by inhibiting complex IV activity, while nitration on Y56 (Hsp90NY56) activates the P2X7 receptor (P2X7R), which is linked to increased glycolysis in tumors. Together, these metabolic changes are characteristics of the Warburg effect, a metabolic phenotype common amongst tumors, suggesting that nitrated Hsp90 may play a role as a metabolic switch driving tumor progression. Here, we developed a computational approach to visualize the subcellular and spatial distribution of differentially nitrated Hsp90 in 3D cell culture models of GBM (tumoroids) using confocal Z‐Stack images. Our automated, high‐throughput method takes Z‐stack outputs and generates a 3D heatmap of a differentially localized protein of interest, normalized to a housekeeping fluorescence signal. These normalized heatmaps can be generated from any fluorescence image and serve as an unbiased visualization tool to use in conjunction with fluorescence microscopy images. GBM tumoroids were fluorescently labelled for Hsp90NY33or Hsp90NY56using specific and selective in‐house monoclonal antibodies, and for mitochondrial complex IV and P2X7R. DAPI was used as nuclear stain and tumoroids were imaged using confocal laser scanning microscopy. We observed a ~50% loss in fluorescence signal across the depth of tumoroid Z‐stacks, a common problem related to tissue penetration of laser light that leads to an underrepresented fluorescent signal at the core and bottom of imaged tumoroids. To overcome this problem, Python was used to analyze each focal plane within a confocal Z‐stack, normalizing the signal corresponding to the protein of interest by the mean DNA content (DAPI signal) at each focal plane. This normalized signal was used to generate a 3D heatmap of the protein of interest within the tumoroids. Method‐generated heatmaps confirmed the intracellular localization of Hsp90NY33in mitochondria and cytosol, and a distinct localization in the tumoroid periphery. In contrast, Hsp90NY56 was also localized in the nucleus, and was homogeneously distributed throughout the tumoroid. P2X7R showed a clear cell membrane localization, with increased expression in the tumoroid periphery. This suggests that Hsp90NY33may reduce aerobic respiration in the periphery of the tumoroid, allowing oxygen diffusion into the core, while Hsp90NY56 supports glycolysis throughout the tumoroid. Our results provide a novel fluorescence image analysis tool with potential to be expanded for several other 2D and 3D imaging modalities.

Nitration of Hsp90 Affects its Spatial Distribution and Promotes Schwannoma Cell Proliferation

Neurofibromatosis Type 2 is a genetic disorder characterized by the development of schwannomas throughout the nervous system caused by loss of merlin tumor suppressor activity. Solid tumors develop in an oxidative environment due to production of oxidants such as peroxynitrite by tumor and infiltrating immune cells. We discovered peroxynitrite production and subsequent protein tyrosine (Y) nitration support schwannoma cell survival/proliferation. Further, Y nitration induces a metabolic reprogramming characterized by decreased oxidative phosphorylation activity, and increased glycolysis and glutaminolysis. Here, we identified the first nitrated protein supporting schwannoma cell proliferation, the molecular chaperone heat shock protein 90 (Hsp90). Out of five Y residues prone to nitration in Hsp90, we showed nitration at Y33 down‐regulates mitochondrial metabolism, while nitration at Y56 activates the ATP‐gated receptor P2X7, which increases glycolysis in tumor cells. We found schwannomas contain endogenous levels of Hsp90 nitrated at either position. Therefore, we hypothesized that nitrated Hsp90 (Hsp90NY) promotes schwannoma cell proliferation by regulating their metabolism. To investigate the role of Hsp90NY in proliferation, we increased the intracellular concentration of Hsp90NY in schwannoma cells by delivering Hsp90NY intracellularly at concentrations like those endogenously present in these cells (~6.5% of cellular Hsp90, calculated by quantitative dot blot). Doubling the intracellular concentration of Hsp90NYbut not wild‐type Hsp90 significantly increased cell proliferation 24 and 48 h post‐delivery. To establish the metabolic role of Hsp90NY, we delivered wild‐type or Hsp90NY into wild‐type Schwann cells (WT‐SC) and studied the metabolic changes by extracellular flux analysis 24 h post‐delivery. Hsp90NY decreased all parameters of mitochondrial activity, with no effect on the glycolytic rate. To test if Y33 and/or Y56 were responsible for the tumorigenic activity of Hsp90NY, we intracellularly delivered to WT‐SC either wild‐type Hsp90, or different site‐specific nitrated Hsp90 proteins produced by genetic code expansion: Hsp90 nitrated at Y33 (Hsp90NY33), at Y56 (Hsp90NY56), or at Y33 and Y56 simultaneously (Hsp90NY33+56). All three forms of Hsp90NY significantly increased WT‐SC proliferation at 24 and 48 h post‐delivery, suggesting Y33 and Y56 induce schwannoma cell proliferation independently. Using a three‐dimensional schwannoma cell culture model (tumoroids), we discovered Hsp90NY33colocalized with mitochondria and remained in the tumoroid periphery, while Hsp90NY56was also found in nuclei with a homogenous distribution throughout the tumoroid. Collectively, our results show that Hsp90, when nitrated at Y33 or Y56, gains a tumorigenic activity that supports schwannoma cell proliferation. In addition, residue‐specific nitration results in distinct localization, and potentially function, of the protein. This is the first nitrated protein shown to support tumor cell proliferation, and a potential tumor‐directed target for therapeutic development.

Targeting HSP90 sensitizes pancreas carcinoma to PD-1 blockade

ABSTRACT Interferon gamma (IFNG/IFNγ)-induced adaptive immune resistance remains a challenge for tumor therapy. We observed that the chaperone heat shock protein 90 (HSP90) stabilizes the transcription factor signal transducer and activator of transcription 1 (STAT1), resulting in IFNγ-induced expression of immunosuppressive indoleamine 2,3-dioxygenase 1 (IDO1) and programmed death-ligand 1 (PD-L1/CD274). Pharmacological inhibition of HSP90 enhances the efficacy of programmed cell death 1 (PDCD1/PD-1) targeting immunotherapy in suitable mouse models without any toxicity.

Harnessing the Heterogeneity of Prostate Cancer for Target Discovery Using Patient-Derived Explants.

Simple SummaryThere is a widespread push toward more biologically relevant pre-clinical models of prostate cancer that can improve the discovery and translation of new drugs and biomarkers for this disease. Patient-derived explant culture is an innovative pre-clinical model that utilizes surgical prostate cancer specimens in a way that retains the architecture, microenvironment and heterogeneity of prostate tumors—factors that critically influence cell behavior and response to therapy. With increasing tissue complexity comes increasing complexity of analysis. The aim of this study was to provide critical information for the successful application and analysis of the patient-derived prostate cancer explant model.Prostate cancer is a complex and heterogeneous disease, but a small number of cell lines have dominated basic prostate cancer research, representing a major obstacle in the field of drug and biomarker discovery. A growing lack of confidence in cell lines has seen a shift toward more sophisticated pre-clinical cancer models that incorporate patient-derived tumors as xenografts or explants, to more accurately reflect clinical disease. Not only do these models retain critical features of the original tumor, and account for the molecular diversity and cellular heterogeneity of prostate cancer, but they provide a unique opportunity to conduct research in matched tumor samples. The challenge that accompanies these complex tissue models is increased complexity of analysis. With over 10 years of experience working with patient-derived explants (PDEs) of prostate cancer, this study provides guidance on the PDE method, its limitations, and considerations for addressing the heterogeneity of prostate cancer PDEs that are based on statistical modeling. Using inhibitors of the molecular chaperone heat shock protein 90 (Hsp90) as an example of a drug that induces robust proliferative response, we demonstrate how multi-omics analysis in prostate cancer PDEs is both feasible and essential for identification of key biological pathways, with significant potential for novel drug target and biomarker discovery.

Molecular chaperone heat shock protein 70 inhibitors suppress conditioned place preference induced by morphine exposure in male rats.

Previous studies have indicated a role for molecular chaperone heat shock protein 70 (Hsp70) in the development of behavioural sensitization to morphine in rodents, suggesting that Hsp70 expression following morphine exposure is involved in molecular changes that may underlie addiction vulnerability. The current study was carried out to investigate the role of Hsp70 in the positive reinforcing properties of morphine using conditioned place preference (CPP) in male rats. An unbiased CPP procedure of three phases (pre-conditioning: d1-d3; conditioning: d4-d6; and testing: d7) was used. During the conditioning phase, morphine injections (5 mg/kg, subcutaneously) were administered to induce significant place preference. To explore the effect of Hsp70 on the development and expression of morphine CPP, Hsp70 inhibitors (PES, KNK437 and methylene blue) were administered into the lateral ventricle prior to either morphine conditioning sessions or a morphine challenge on the test day. Furthermore, Hsp70 expression within the mesocorticolimbic system was measured after the treatment with KNK437, a transcriptional inhibitor. We found that PES and KNK437, respectively, injected intracerebroventricularly dose-dependently attenuated both the development and expression of morphine CPP. Methylene blue treatment demonstrated an attenuation of the development, but had no effect on the expression of morphine CPP. Following KNK437 treatment, Hsp70 expression was significantly inhibited in the shell of nucleus accumbens (NAc) during both the development and expression of morphine CPP. The findings suggest that Hsp70 in the NAc shell plays an important role in the reinforcing effects of morphine and may be involved in the development of morphine dependence.

Dual Targeting Strategies on Histone Deacetylase 6 (HDAC6) and Heat Shock Protein 90 (Hsp90).

The design of multi-target drugs acting simultaneously on multiple signaling pathways is a growing field in medicinal chemistry, especially for the treatment of complex diseases, such as cancer. Histone deacetylase 6 (HDAC6) is an established anticancer drug target involved in tumor cells transformation. Being an epigenetic enzyme at the interplay of many biological processes, HDAC6 has become an attractive target for polypharmacology studies aimed at improving the therapeutic efficacy of anticancer drugs. For example, the molecular chaperone Heat shock protein 90 (Hsp90) is a substrate of HDAC6 deacetylation, and several lines of evidence demonstrate that simultaneous inhibition of HDAC6 and Hsp90 promotes synergistic antitumor effects on different cancer cell lines, highlighting the potential benefits of developing a single molecule endowed with multi-target activity. This review will summarize the complex interplay between HDAC6 and Hsp90, providing also useful hints for multi-target drug design and discovery approaches in this field. To this end, crystallographic structures of HDAC6 and Hsp90 complexes will be extensively reviewed in light of discussing binding pockets features and pharmacophore requirements and providing useful guidelines for the design of dual inhibitors. The few examples of multi-target inhibitors obtained so far, mostly based on chimeric approaches, will be summarized and put into context. Finally, the main features of HDAC6 and Hsp90 inhibitors will be compared, and ligand- and structure-based strategies potentially useful for the development of small molecular weight dual inhibitors will be proposed and discussed.

Heat Shock Transcription Factor CgHSF1 Is Required for Melanin Biosynthesis, Appressorium Formation, and Pathogenicity in Colletotrichum gloeosporioides.

Heat shock transcription factors (HSFs) are a family of transcription regulators. Although HSFs’ functions in controlling the transcription of the molecular chaperone heat shock proteins and resistance to stresses are well established, their effects on the pathogenicity of plant pathogenic fungi remain unknown. In this study, we analyze the role of CgHSF1 in the pathogenicity of Colletotrichum gloeosporioides and investigate the underlying mechanism. Failure to generate the Cghsf1 knock-out mutant suggested that the gene is essential for the viability of the fungus. Then, genetic depletion of the Cghsf1 was achieved by inserting the repressive promoter of nitrite reductase gene (PniiA) before its coding sequence. The mutant showed significantly decrease in the pathogenicity repression of appressorium formation, and severe defects in melanin biosynthesis. Moreover, four melanin synthetic genes were identified as direct targets of CgHSF1. Taken together, this work highlights the role of CgHSF1 in fungal pathogenicity via the transcriptional activation of melanin biosynthesis. Our study extends the understanding of fungal HSF1 proteins, especially their involvement in pathogenicity.

HSP90-CDC37 functions as a chaperone for the oncogenic FGFR3-TACC3 fusion

The FGFR3-TACC3 (F3-T3) fusion gene was discovered as an oncogenic molecule in glioblastoma and bladder cancers, and has subsequently been found in many cancer types. Notably, F3-T3 was found to be highly expressed in both untreated and matched recurrence glioblastoma under the concurrent radiotherapy and temozolomide (TMZ) treatment, suggesting that targeting F3-T3 is a valid strategy for treatment. Here, we show that the F3-T3 protein is a client of heat shock protein 90 (HSP90), forming a ternary complex with the cell division cycle 37 (CDC37). Deprivation of HSP90 or CDC37 disrupts the formation of the ternary complex, which destabilizes glycosylated F3-T3, and thereby suppresses F3-T3 oncogenic activity. Gliomas harboring F3-T3 are resistant to TMZ chemotherapy. HSP90 inhibitors sensitized F3-T3 glioma cells to TMZ via the inhibition of F3-T3 activation and potentiated TMZ-induced DNA damage. These results demonstrate that F3-T3 oncogenic function is dependent on the HSP90 chaperone system and suggests a new clinical option for targeting this genetic aberration in cancer.

Boosting Natural Killer Cell Therapies in Glioblastoma Multiforme Using Supramolecular Cationic Inhibitors of Heat Shock Protein 90.

Allogeneic natural killer (aNK) cell adoptive therapy has the potential to dramatically impact clinical outcomes of glioblastoma multiforme (GBM). However, in order to exert therapeutic activity, NK cells require tumor expression of ligands for activating receptors, such as MHC Class I peptide A/B (MICA/B) and ULBPs. Here, we describe the use of a blood–brain barrier (BBB) permissive supramolecular cationic drug vehicle comprising an inhibitor of the chaperone heat shock protein 90 (Hsp90), which sustains a cytotoxic effect on GBM cells, boosts the expression of MICA/B and ULBPs on the residual population, and augments the activity of clinical-grade aNK cells (GTA002). First, we identify Hsp90 mRNA transcription and gain of function as significantly upregulated in GBM compared to other central nervous system tumors. Through a rational chemical design, we optimize a radicicol supramolecular prodrug containing cationic excipients, SCI-101, which displays >2-fold increase in relative BBB penetration compared to less cationic formulations in organoids, in vitro. Using 2D and 3D biological models, we confirm SCI-101 sustains GBM cytotoxicity 72 h after drug removal and induces cell surface MICA/B protein and ULBP mRNA up to 200% in residual tumor cells compared to the naked drug alone without augmenting the shedding of MICA/B, in vitro. Finally, we generate and test the sequential administration of SCI-101 with a clinical aNK cell therapy, GTA002, differentiated and expanded from healthy umbilical cord blood CD34+ hematopoietic stem cells. Using a longitudinal in vitro model, we demonstrate >350% relative cell killing is achieved in SCI-101–treated cell lines compared to vehicle controls. In summary, these data provide a first-of-its-kind BBB-penetrating, long-acting inhibitor of Hsp90 with monotherapy efficacy, which improves response to aNK cells and thus may rapidly alter the treatment paradigm for patients with GBM.

TAS‐116 (pimitespib), a heat shock protein 90 inhibitor, shows efficacy in preclinical models of adult T‐cell leukemia

Adult T‐cell leukemia/lymphoma (ATL) is a highly chemoresistant malignancy of peripheral T lymphocytes caused by human T‐cell leukemia virus type 1 infection, for which there is an urgent need for more effective therapeutic options. The molecular chaperone heat shock protein 90 (HSP90) plays a crucial role in nuclear factor‐κB (NF‐κB)‐mediated antiapoptosis in ATL cells, and HSP90 inhibitors are new candidate therapeutics for ATL. Accordingly, we investigated the anti‐ATL effects of a novel oral HSP90 inhibitor, TAS‐116 (pimitespib), and the mechanisms involved in ex vivo and in vivo preclinical models. TAS‐116 achieved IC50 values of less than 0.5 μmol/L in 10 ATL‐related cell lines and less than 1 μmol/L in primary peripheral blood cells of nine ATL patients; no toxicity was observed toward CD4+ lymphocytes from healthy donors, indicating the safety of this agent. Given orally, TAS‐116 also showed significant inhibitory effects against tumor cell growth in ATL cell‐xenografted mice. Furthermore, gene expression profiling of TAS‐116‐treated Tax‐positive or ‐negative cell lines and primary ATL cells using DNA microarray and multiple pathway analysis revealed the significant downregulation of the NF‐κB pathway in Tax‐positive cells and cell‐cycle arrest in Tax‐negative cells and primary ATL cells. TAS‐116 suppressed the activator protein‐1 and tumor necrosis factor pathways in all examined cells. These findings strongly indicate the efficacy of TAS‐116, regardless of the stage of ATL progression, and its potential application as a novel clinical anti‐ATL therapeutic agent.