Detecting Posttranslational Modifications of Hsp90 Isoforms.

The molecular chaperone heat shock protein 90 (Hsp90) is essential in eukaryotes. Hsp90 chaperones proteins that are important determinants of multistep carcinogenesis. There are multiple Hsp90 isoforms including the cytosolic Hsp90α and Hsp90ß as well as GRP94 located in the endoplasmic reticulum and TRAP1 in the mitochondria. The chaperone function of Hsp90 is linked to its ability to bind and hydrolyze ATP. Co-chaperones and posttranslational modifications (such as phosphorylation, SUMOylation, and ubiquitination) are important for Hsp90 stability and regulation of its ATPase activity. Both mammalian and yeast cells can be used to express and purify Hsp90 and TRAP1 and also detect post-translational modifications by immunoblotting.

Structural and functional complexity of HSP90 in cellular homeostasis and disease.

Heat shock protein 90 (HSP90) is a chaperone with vital roles in regulating proteostasis, long recognized for its function in protein folding and maturation. A view is emerging that identifies HSP90 not as one protein that is structurally and functionally homogeneous but, rather, as a protein that is shaped by its environment. In this Review, we discuss evidence of multiple structural forms of HSP90 in health and disease, including homo-oligomers and hetero-oligomers, also termed epichaperomes, and examine the impact of stress, post-translational modifications and co-chaperones on their formation. We describe how these variations influence context-dependent functions of HSP90 as well as its interaction with other chaperones, co-chaperones and proteins, and how this structural complexity of HSP90 impacts and is impacted by its interaction with small molecule modulators. We close by discussing recent developments regarding the use of HSP90 inhibitors in cancer and how our new appreciation of the structural and functional heterogeneity of HSP90 invites a re-evaluation of how we discover and implement HSP90 therapeutics for disease treatment.

Second Virtual International Symposium on Cellular and Organismal Stress Responses, September 8-9, 2022.

The Second International Symposium on Cellular and Organismal Stress Responses took place virtually on September 8-9, 2022. This meeting was supported by the Cell Stress Society International (CSSI) and organized by Patricija Van Oosten-Hawle and Andrew Truman (University of North Carolina at Charlotte, USA) and Mehdi Mollapour (SUNY Upstate Medical University, USA). The goal of this symposium was to continue the theme from the initial meeting in 2020 by providing a platform for established researchers, new investigators, postdoctoral fellows, and students to present and exchange ideas on various topics on cellular stress and chaperones. We will summarize the highlights of the meeting here and recognize those that received recognition from the CSSI.

The Mitochondrial HSP90 Paralog TRAP1: Structural Dynamics, Interactome, Role in Metabolic Regulation, and Inhibitors.

The HSP90 paralog TRAP1 was discovered more than 20 years ago; yet, a detailed understanding of the function of this mitochondrial molecular chaperone remains elusive. The dispensable nature of TRAP1 in vitro and in vivo further complicates an understanding of its role in mitochondrial biology. TRAP1 is more homologous to the bacterial HSP90, HtpG, than to eukaryotic HSP90. Lacking co-chaperones, the unique structural features of TRAP1 likely regulate its temperature-sensitive ATPase activity and shed light on the alternative mechanisms driving the chaperone's nucleotide-dependent cycle in a defined environment whose physiological temperature approaches 50 °C. TRAP1 appears to be an important bioregulator of mitochondrial respiration, mediating the balance between oxidative phosphorylation and glycolysis, while at the same time promoting mitochondrial homeostasis and displaying cytoprotective activity. Inactivation/loss of TRAP1 has been observed in several neurodegenerative diseases while TRAP1 expression is reported to be elevated in multiple cancers and, as with HSP90, evidence of addiction to TRAP1 has been observed. In this review, we summarize what is currently known about this unique HSP90 paralog and why a better understanding of TRAP1 structure, function, and regulation is likely to enhance our understanding of the mechanistic basis of mitochondrial homeostasis.

Inhibition of HSP 90 is associated with potent anti-tumor activity in Papillary Renal Cell Carcinoma.

BACKGROUND: There is no universally accepted treatment for patients with advanced papillary renal cell carcinoma (PRCC). The presence of activating mutations in MET, as well as gain of chromosome 7, where the MET gene is located, are the most common genetic alterations associated with PRCC, leading to the clinical evaluation of MET tyrosine kinase inhibitors (TKIs) in this cancer. However, TKIs targeting MET selectively, as well as multitargeted TKIs with activity against MET demonstrate modest efficacy in PRCC and primary and secondary treatment failure is common; other approaches are urgently needed to improve outcomes in these patients. METHODS: High throughput screening with small molecule libraries identified HSP90 inhibitors as agents of interest based on antitumor activity against patient derived PRCC cell lines. We investigated the activity of the orally available HSP90 inhibitor, SNX2112 in vitro, using 2D/3D PRCC cell culture models and in vivo, in mice tumor xenograft models. The molecular pathways mediating antitumor activity of SNX2112 were assessed by Western blot analysis, Flow cytometry, RNA-seq analysis, Real Time qPCR and imaging approaches. RESULTS: SNX2112 significantly inhibited cellular proliferation, induced G2/M cell cycle arrest and apoptosis in PRCC lines overexpressing MET. In contrast to TKIs targeting MET, SNX2112 inhibited both MET and known downstream mediators of MET activity (AKT, pAKT1/2 and pERK1/2) in PRCC cell lines. RNAi silencing of AKT1/2 or ERK1/2 expression significantly inhibited growth in PRCC cells. Furthermore, SNX2112 inhibited a unique set of E2F and MYC targets and G2M-associated genes. Interestingly, interrogation of the TCGA papillary RCC cohort revealed that these genes were overexpressed in PRCC and portend a poor prognosis. Finally, SNX-2112 demonstrated strong antitumor activity in vivo and prolonged survival of mice bearing human PRCC xenograft. CONCLUSIONS: These results demonstrate that HSP90 inhibition is associated with potent activity in PRCC, and implicate the PI3K/AKT and MEK/ERK1/2 pathways as important mediators of tumorigenesis. These data also provide the impetus for further clinical evaluation of HSP90, AKT, MEK or E2F pathway inhibitors in PRCC.

Selective vulnerabilities in the proteostasis network of castration-resistant prostate cancer.

Castration-resistant prostate cancer (CRPC) is associated with an increased reliance on heat shock protein 70 (HSP70), but it is not clear what other protein homeostasis (proteostasis) factors might be involved. To address this question, we performed functional and synthetic lethal screens in four prostate cancer cell lines. These screens confirmed key roles for HSP70, HSP90, and their co-chaperones, but also suggested that the mitochondrial chaperone, HSP60/HSPD1, is selectively required in CRPC cell lines. Knockdown of HSP60 does not impact the stability of androgen receptor (AR) or its variants; rather, it is associated with loss of mitochondrial spare respiratory capacity, partly owing to increased proton leakage. Finally, transcriptional data revealed a correlation between HSP60 levels and poor survival of prostate cancer patients. These findings suggest that re-wiring of the proteostasis network is associated with CRPC, creating selective vulnerabilities that might be targeted to treat the disease.

The XIth International Online Symposium on Heat Shock Proteins in Biology and Medicine : Organizers: Stuart K. Calderwood, Elizabeth Repasky & Len Neckers October 27-29, 2021 IN MEMORIAM: Claudina Rodrigues-Pousada (1941-2021).

Single cell and multicellular organisms encounter physical stress from their environment as well as behavioral stress experienced in more complex organisms. As these stresses can present an existential threat, organisms respond with a coordinated response at the tissue and cellular level, the heat shock response (HSR) and this was the major theme of the symposium. Much of the meeting was concentrated on the heat shock proteins (HSPs), the effector molecules of the response. The balance between the potency of the HSR and the experience of stress naturally plays a key role in the etiology of many disease. Roles in cancer, the immune response, cell metabolism and aging were discussed at length at the meeting. Finally, a major goal of this field is to enhance the HSR in pathological conditions where it becomes inadequate or over stimulated and important findings regarding pharmacological approaches to modulating the HSR were discussed.

HSP90 inhibitors reduce cholesterol storage in Niemann-Pick type C1 mutant fibroblasts.

Niemann-Pick type C1 (NPC1) disease is a lysosomal lipid storage disorder caused by mutations of the NPC1 gene. More than 300 disease-associated mutations are reported in patients, resulting in abnormal accumulation of unesterified cholesterol, glycosphingolipids, and other lipids in late endosomes and lysosomes (LE/Ly) of many cell types. Previously, we showed that treatment of many different NPC1 mutant fibroblasts with histone deacetylase inhibitors resulted in reduction of cholesterol storage, and we found that this was associated with enhanced exit of the NPC1 protein from the endoplasmic reticulum and delivery to LE/Ly. This suggested that histone deacetylase inhibitors may work through changes in protein chaperones to enhance the folding of NPC1 mutants, allowing them to be delivered to LE/Ly. In this study, we evaluated the effect of several HSP90 inhibitors on NPC1I1061T skin fibroblasts. We found that HSP90 inhibition resulted in clearance of cholesterol from LE/Ly, and this was associated with enhanced delivery of the mutant NPC1I1061T protein to LE/Ly. We also observed that inhibition of HSP90 increased the expression of HSP70, and overexpression of HSP70 also reduced cholesterol storage in NPC1I1061T fibroblasts. However, we did not see correction of cholesterol storage by arimoclomol, a drug that is reported to increase HSP70 expression, at doses up to 0.5 mM. The increase in other chaperones as a consequence of HSP90 improves folding of NPC1 protein and relieves cholesterol accumulation in NPC1 mutant fibroblasts.

Phase 1 study of the HSP90 inhibitor onalespib in combination with AT7519, a pan-CDK inhibitor, in patients with advanced solid tumors.

PURPOSE: We conducted a phase 1 trial of the HSP90 inhibitor onalespib in combination with the CDK inhibitor AT7519, in patients with advanced solid tumors to determine the safety profile and maximally tolerated dose, pharmacokinetics, preliminary antitumor activity, and to assess the pharmacodynamic (PD) effects on HSP70 expression in patient-derived PBMCs and plasma. METHODS: This study followed a 3 + 3 trial design with 1 week of intravenous (IV) onalespib alone, followed by onalespib/AT7519 (IV) on days 1, 4, 8, and 11 of a 21-days cycle. PK and PD samples were collected at baseline, after onalespib alone, and following combination therapy. RESULTS: Twenty-eight patients were treated with the demonstration of downstream target engagement of HSP70 expression in plasma and PBMCs. The maximally tolerated dose was onalespib 80 mg/m2 IV + AT7519 21 mg/m2 IV. Most common drug-related adverse events included Grade 1/2 diarrhea (79%), fatigue (54%), mucositis (57%), nausea (46%), and vomiting (50%). Partial responses were seen in a palate adenocarcinoma and Sertoli-Leydig tumor; a colorectal and an endometrial cancer patient both remained on study for ten cycles with stable disease as the best response. There were no clinically relevant PK interactions for either drug. CONCLUSIONS: Combined onalespib and AT7519 is tolerable, though below monotherapy RP2D. Promising preliminary clinical activity was seen. Further benefit may be seen with the incorporation of molecular signature pre-selection. Further biomarker development will require the assessment of the on-target impact on relevant client proteins in tumor tissue.

The mitochondrial HSP90 paralog TRAP1 forms an OXPHOS-regulated tetramer and is involved in mitochondrial metabolic homeostasis.

BACKGROUND: The molecular chaperone TRAP1, the mitochondrial isoform of cytosolic HSP90, remains poorly understood with respect to its pivotal role in the regulation of mitochondrial metabolism. Most studies have found it to be an inhibitor of mitochondrial oxidative phosphorylation (OXPHOS) and an inducer of the Warburg phenotype of cancer cells. However, others have reported the opposite, and there is no consensus on the relevant TRAP1 interactors. This calls for a more comprehensive analysis of the TRAP1 interactome and of how TRAP1 and mitochondrial metabolism mutually affect each other. RESULTS: We show that the disruption of the gene for TRAP1 in a panel of cell lines dysregulates OXPHOS by a metabolic rewiring that induces the anaplerotic utilization of glutamine metabolism to replenish TCA cycle intermediates. Restoration of wild-type levels of OXPHOS requires full-length TRAP1. Whereas the TRAP1 ATPase activity is dispensable for this function, it modulates the interactions of TRAP1 with various mitochondrial proteins. Quantitatively by far, the major interactors of TRAP1 are the mitochondrial chaperones mtHSP70 and HSP60. However, we find that the most stable stoichiometric TRAP1 complex is a TRAP1 tetramer, whose levels change in response to both a decline and an increase in OXPHOS. CONCLUSIONS: Our work provides a roadmap for further investigations of how TRAP1 and its interactors such as the ATP synthase regulate cellular energy metabolism. Our results highlight that TRAP1 function in metabolism and cancer cannot be understood without a focus on TRAP1 tetramers as potentially the most relevant functional entity.

Proteasome inhibition disrupts the metabolism of fumarate hydratase- deficient tumors by downregulating p62 and c-Myc.

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is characterized by germline mutations of the FH gene that encodes for the TCA cycle enzyme, fumarate hydratase. HLRCC patients are at risk for the development of an aggressive form of type 2 papillary renal cell carcinoma. By studying the mechanism of action of marizomib, a proteasome inhibitor able to cross the blood-brain barrier, we found that it modulates the metabolism of HLRCC cells. Marizomib decreased glycolysis in vitro and in vivo by downregulating p62 and c-Myc. C-Myc downregulation decreased the expression of lactate dehydrogenase A, the enzyme catalyzing the conversion of pyruvate to lactate. In addition, proteasomal inhibition lowered the expression of the glutaminases GLS and GLS2, which support glutamine metabolism and the maintenance of the redox balance. Thus, in HLRCC cells, proteasome inhibition disrupts glucose and glutamine metabolism, restricting nutrients and lowering the cells' anti-oxidant response capacity. Although the cytotoxicity induced by proteasome inhibitors is complex, the understanding of their metabolic effects in HLRCC may lead to the development of effective therapeutic strategies or to the development of markers of efficacy.

Hsp90 middle domain phosphorylation initiates a complex conformational program to recruit the ATPase-stimulating cochaperone Aha1.

Complex conformational dynamics are essential for function of the dimeric molecular chaperone heat shock protein 90 (Hsp90), including transient, ATP-biased N-domain dimerization that is necessary to attain ATPase competence. The intrinsic, but weak, ATP hydrolyzing activity of human Hsp90 is markedly enhanced by the co-chaperone Aha1. However, the cellular concentration of Aha1 is substoichiometric relative to Hsp90. Here we report that initial recruitment of this cochaperone to Hsp90 is markedly enhanced by phosphorylation of a highly conserved tyrosine (Y313 in Hsp90α) in the Hsp90 middle domain. Importantly, phosphomimetic mutation of Y313 promotes formation of a transient complex in which both N- and C-domains of Aha1 bind to distinct surfaces of the middle domains of opposing Hsp90 protomers prior to ATP-directed N-domain dimerization. Thus, Y313 represents a phosphorylation-sensitive conformational switch, engaged early after client loading, that affects both local and long-range conformational dynamics to facilitate initial recruitment of Aha1 to Hsp90.

Heat shock factor 1 (HSF1)-targeted anticancer therapeutics: overview of current preclinical progress.

INTRODUCTION: The heat shock factor 1 (HSF1) plays a pivotal role in guarding proteome stability or proteostasis by induction of heat shock proteins (HSPs). While HSF1 remains mostly latent in unstressed normal cells, it is constitutively active in malignant cells, rendering them addicted to HSF1 for their growth and survival. HSF1 affects tumorigenesis, cancer progression, and treatment resistance by preserving cancer proteostasis, thus suggesting disruption of HSF1 activity as a potential anticancer strategy. Areas covered: In this review, we focus on the HSF1 activation cycle and its interaction with HSPs, the role of HSF1 in oncogenesis, and development of HSF1-targeted drugs as a potential anticancer therapy for disrupting cancer proteostasis. Expert opinion: HSF1 systematically maintains proteostasis in malignant cancer cells. Although genomic instability is widely accepted as a hallmark of cancer, little is known about the role of proteostasis in cancer. Unveiling the complicated mechanism of HSF1 regulation, particularly in cancer cells, will enable further development of proteostasis-targeted anticancer therapy. ABBREVIATIONS: AMPK: AMP-activated protein kinase; DBD: DNA-binding domain; HR-A/B; HR-C: heptad repeats; HSE: heat shock elements; HSF1: heat shock factor; HSPs: heat shock proteins; HSR: heat shock response; MEK: mitogen-activated protein kinase kinase; mTOR: mammalian target of rapamycin; NF1: neurofibromatosis type 1; P-TEFb: positive transcription elongation factor b; RD: regulatory domain; RNAi: RNA interference; TAD: transactivation domain; TRiC: TCP-1 ring complex.

The IXth CSSI international symposium on heat shock proteins in biology and medicine: stress responses in health and disease : Alexandria Old Town, Alexandria, Virginia, November 10-13, 2018.

The stress response has been studied now for over 50 years and is known to have significance in the survival of organisms in a challenging environment and in the healthy development of all known descendants of the last common universal ancestor (LUCA). This meeting was concentrated mostly on the responses of cells and organisms to environmental and cell stress including the impact of thermal stress, which was a major theme throughout this meeting. One emphasis was on the deployment of the heat shock response that permits damage to proteins to be detected and responded to by the abundant synthesis of heat shock proteins (HSPs). Speakers and presenters of posters responded to the questions of how are the HSPs rapidly induced by stressors? By which mechanisms are they are regulated in the cell by protein-protein interactions or posttranslational modification? And, what are the consequences when these abundantly expressed proteins escape the confines of the cell and influence the extracellular microenvironment? Key among the questions was how does stress influence longevity and aging and what happens in terms of disease control (malignant, neurodegenerative) when stress responses become compromised? In this context, many presenters addressed the question of pharmacologically modifying the heat shock response and HSP functions and thus improving responses to a range of disease types.

Exploration of Benzothiazole Rhodacyanines as Allosteric Inhibitors of Protein-Protein Interactions with Heat Shock Protein 70 (Hsp70).

Cancer cells rely on the chaperone heat shock protein 70 (Hsp70) for survival and proliferation. Recently, benzothiazole rhodacyanines have been shown to bind an allosteric site on Hsp70, interrupting its binding to nucleotide-exchange factors (NEFs) and promoting cell death in breast cancer cell lines. However, proof-of-concept molecules, such as JG-98, have relatively modest potency (EC50 ≈ 0.7-0.4 µM) and are rapidly metabolized in animals. Here, we explored this chemical series through structure- and property-based design of ∼300 analogs, showing that the most potent had >10-fold improved EC50 values (∼0.05 to 0.03 µM) against two breast cancer cells. Biomarkers and whole genome CRISPRi screens confirmed members of the Hsp70 family as cellular targets. On the basis of these results, JG-231 was found to reduce tumor burden in an MDA-MB-231 xenograft model (4 mg/kg, ip). Together, these studies support the hypothesis that Hsp70 may be a promising target for anticancer therapeutics.

HSP90 inhibitors disrupt a transient HSP90-HSF1 interaction and identify a noncanonical model of HSP90-mediated HSF1 regulation.

Heat shock factor 1 (HSF1) initiates a broad transcriptional response to proteotoxic stress while also mediating a cancer-specific transcriptional program. HSF1 is thought to be regulated by molecular chaperones, including Heat Shock Protein 90 (HSP90). HSP90 is proposed to sequester HSF1 in unstressed cells, but visualization of this interaction in vivo requires protein crosslinking. In this report, we show that HSP90 binding to HSF1 depends on HSP90 conformation and is only readily visualized for the ATP-dependent, N-domain dimerized chaperone, a conformation only rarely sampled by mammalian HSP90. We have used this mutationally fixed conformation to map HSP90 binding sites on HSF1. Further, we show that ATP-competitive, N-domain targeted HSP90 inhibitors disrupt this interaction, resulting in the increased duration of HSF1 occupancy of the hsp70 promoter and significant prolongation of both the constitutive and heat-induced HSF1 transcriptional activity. While our data do not support a role for HSP90 in sequestering HSF1 monomers to suppress HSF1 transcriptional activity, our findings do identify a noncanonical role for HSP90 in providing dynamic modulation of HSF1 activity by participating in removal of HSF1 trimers from heat shock elements in DNA, thus terminating the heat shock response.

Targeting loss of the Hippo signaling pathway in NF2-deficient papillary kidney cancers.

Papillary renal cell carcinomas (PRCC) are a histologically and genetically heterogeneous group of tumors that represent 15-20% of all kidney neoplasms and may require diverse therapeutic approaches. Alteration of the NF2 tumor suppressor gene, encoding a key regulator of the Hippo signaling pathway, is observed in 22.5% of PRCC. The Hippo signaling pathway controls cell proliferation by regulating the transcriptional activity of Yes-Associated Protein, YAP1. Loss of NF2 results in aberrant YAP1 activation. The Src family kinase member Yes also regulates YAP1 transcriptional activity. This study investigated the importance of YAP and Yes activity in three NF2-deficient PRCC cell lines. NF2-deficency correlated with increased expression of YAP1 transcriptional targets and siRNA-based knockdown of YAP1 and Yes1 downregulated this pathway and dramatically reduced cell viability. Dasatinib and saracatinib have potent inhibitory effects on Yes and treatment with either resulted in downregulation of YAP1 transcription targets, reduced cell viability, and G0-G1 cell cycle arrest. Xenograft models for NF2-deficient PRCC also demonstrated reduced tumor growth in response to dasatinib. Thus, inhibiting Yes and the subsequent transcriptional activity of YAP1 had a substantial anti-tumor cell effect both in vitro and in vivo and may provide a viable therapeutic approach for patients with NF2-deficient PRCC.

Methods to validate Hsp90 inhibitor specificity, to identify off-target effects, and to rethink approaches for further clinical development.

The molecular chaperone Hsp90 is one component of a highly complex and interactive cellular proteostasis network (PN) that participates in protein folding, directs misfolded and damaged proteins for destruction, and participates in regulating cellular transcriptional responses to environmental stress, thus promoting cell and organismal survival. Over the last 20 years, it has become clear that various disease states, including cancer, neurodegeneration, metabolic disorders, and infection by diverse microbes, impact the PN. Among PN components, Hsp90 was among the first to be pharmacologically targeted with small molecules. While the number of Hsp90 inhibitors described in the literature has dramatically increased since the first such small molecule was described in 1994, it has become increasingly apparent that not all of these agents have been sufficiently validated for specificity, mechanism of action, and lack of off-target effects. Given the less than expected activity of Hsp90 inhibitors in cancer-related human clinical trials, a re-evaluation of potentially confounding off-target effects, as well as confidence in target specificity and mechanism of action, is warranted. In this commentary, we provide feasible approaches to achieve these goals and we discuss additional considerations to improve the clinical efficacy of Hsp90 inhibitors in treating cancer and other diseases.

Phosphorylation induced cochaperone unfolding promotes kinase recruitment and client class-specific Hsp90 phosphorylation.

During the Hsp90-mediated chaperoning of protein kinases, the core components of the machinery, Hsp90 and the cochaperone Cdc37, recycle between different phosphorylation states that regulate progression of the chaperone cycle. We show that Cdc37 phosphorylation at Y298 results in partial unfolding of the C-terminal domain and the population of folding intermediates. Unfolding facilitates Hsp90 phosphorylation at Y197 by unmasking a phosphopeptide sequence, which serves as a docking site to recruit non-receptor tyrosine kinases to the chaperone complex via their SH2 domains. In turn, Hsp90 phosphorylation at Y197 specifically regulates its interaction with Cdc37 and thus affects the chaperoning of only protein kinase clients. In summary, we find that by providing client class specificity, Hsp90 cochaperones such as Cdc37 do not merely assist in client recruitment but also shape the post-translational modification landscape of Hsp90 in a client class-specific manner.