Using a Modified Proximity Ligation Protocol to Study the Interaction Between Chaperones and Associated Proteins.

Molecular chaperones can interact with multiple proteins to form large networks. Understanding these interactions may shed light on the complexity of the chaperone functions. Here we developed a protocol for a modified proximity ligation-based methodology (PLA) for the detection of protein-protein interactions in order to understand how the Hsp70-Bag3 complex interacts with components of the Hippo signaling pathway. These experiments helped to elucidate the mechanisms of transmission of the proteotoxic stress signal to the Hippo pathway. The modified PLA technology has many advantages compared to co-immunoprecipitation protocols. It has higher sensitivity, is quantitative, and can be done in a 96-well format.

Evolution of Resistance to Irinotecan in Cancer Cells Involves Generation of Topoisomerase-Guided Mutations in Non-Coding Genome That Reduce the Chances of DNA Breaks.

Resistance to chemotherapy is a leading cause of treatment failure. Drug resistance mechanisms involve mutations in specific proteins or changes in their expression levels. It is commonly understood that resistance mutations happen randomly prior to treatment and are selected during the treatment. However, the selection of drug-resistant mutants in culture could be achieved by multiple drug exposures of cloned genetically identical cells and thus cannot result from the selection of pre-existent mutations. Accordingly, adaptation must involve the generation of mutations de novo upon drug treatment. Here we explored the origin of resistance mutations to a widely used Top1 inhibitor, irinotecan, which triggers DNA breaks, causing cytotoxicity. The resistance mechanism involved the gradual accumulation of recurrent mutations in non-coding regions of DNA at Top1-cleavage sites. Surprisingly, cancer cells had a higher number of such sites than the reference genome, which may define their increased sensitivity to irinotecan. Homologous recombination repairs of DNA double-strand breaks at these sites following initial drug exposures gradually reverted cleavage-sensitive "cancer" sequences back to cleavage-resistant "normal" sequences. These mutations reduced the generation of DNA breaks upon subsequent exposures, thus gradually increasing drug resistance. Together, large target sizes for mutations and their Top1-guided generation lead to their gradual and rapid accumulation, synergistically accelerating the development of resistance.

Resistance to TOP-1 Inhibitors: Good Old Drugs Still Can Surprise Us.

Irinotecan (SN-38) is a potent and broad-spectrum anticancer drug that targets DNA topoisomerase I (Top1). It exerts its cytotoxic effects by binding to the Top1-DNA complex and preventing the re-ligation of the DNA strand, leading to the formation of lethal DNA breaks. Following the initial response to irinotecan, secondary resistance is acquired relatively rapidly, compromising its efficacy. There are several mechanisms contributing to the resistance, which affect the irinotecan metabolism or the target protein. In addition, we have demonstrated a major resistance mechanism associated with the elimination of hundreds of thousands of Top1 binding sites on DNA that can arise from the repair of prior Top1-dependent DNA cleavages. Here, we outline the major mechanisms of irinotecan resistance and highlight recent advancements in the field. We discuss the impact of resistance mechanisms on clinical outcomes and the potential strategies to overcome resistance to irinotecan. The elucidation of the underlying mechanisms of irinotecan resistance can provide valuable insights for the development of effective therapeutic strategies.

HSF1 activates the FOXO3a-ΔNp63α-CDK4 axis to promote head and neck squamous cell carcinoma cell proliferation and tumour growth.

Head and neck squamous cell carcinoma (HNSCC) is one of the most prevalent cancers worldwide. Heat shock factor 1 (HSF1) is a conserved transcriptional factor that plays a critical role in maintaining cellular proteostasis. However, the role of HSF1 in HNSCC development remains largely unclear. Here, we report that HSF1 promotes forkhead box protein O3a (FOXO3a)-dependent transcription of ΔNp63α (p63 isoform in the p53 family; inhibits cell migration, invasion, and metastasis), which leads to upregulation of cyclin-dependent kinase 4 expression and HNSCC tumour growth. Ablation of HSF1 or treatment with KRIBB11, a specific pharmacological inhibitor of HSF1, significantly suppresses ΔNp63α expression and HNSCC tumour growth. Clinically, the expression of HSF1 is positively correlated with the expression of ΔNp63α in HNSCC tumours. Together, this study demonstrates that the HSF1-ΔNp63α pathway is critically important for HNSCC tumour growth.

Search for Synergistic Drug Combinations to Treat Chronic Lymphocytic Leukemia.

Finding synergistic drug combinations is an important area of cancer research. Here, we sought to rationally design synergistic drug combinations with an inhibitor of BTK kinase, ibrutinib, which is used for the treatment of several types of leukemia. We (a) used a pooled shRNA screen to identify genes that protect cells from the drug, (b) identified protective pathways via bioinformatics analysis of these gene sets, and (c) identified drugs that inhibit these pathways. Based on this analysis, we established that inhibitors of proteasome and mTORC1 could synergize with ibrutinib both in vitro and in vivo. We suggest that FDA-approved inhibitors of these pathways could be effectively combined with ibrutinib for the treatment of chronic lymphocytic leukemia (CLL).

Hsp70-Bag3 Module Regulates Macrophage Motility and Tumor Infiltration via Transcription Factor LITAF and CSF1.

The molecular chaperone Hsp70 has been implicated in multiple stages of cancer development. In these processes, a co-chaperone Bag3 links Hsp70 with signaling pathways that control cancer development. Recently, we showed that besides affecting cancer cells, Hsp70 can also regulate the motility of macrophages and their tumor infiltration. However, the mechanisms of these effects have not been explored. Here, we demonstrated that the Hsp70-bound co-chaperone Bag3 associates with a transcription factor LITAF that can regulate the expression of inflammatory cytokines and chemokines in macrophages. Via this interaction, the Hsp70-Bag3 complex regulates expression levels of LITAF by controlling its proteasome-dependent and chaperone-mediated autophagy-dependent degradation. In turn, LITAF regulates the expression of the major chemokine CSF1, and adding this chemokine to the culture medium reversed the effects of Bag3 or LITAF silencing on the macrophage motility. Together, these findings uncover the Hsp70-Bag3-LITAF-CSF1 pathway that controls macrophage motility and tumor infiltration.

Integration of the Connectivity Map and Pathway Analysis to Predict Plant Extract's Medicinal Properties-The Study Case of Sarcopoterium spinosum L.

Medicinal properties of plants are usually identified based on knowledge of traditional medicine or using low-throughput screens for specific pharmacological activities. The former is very biased since it requires prior knowledge of plants' properties, while the latter depends on a specific screening system and will miss medicinal activities not covered by the screen. We sought to enrich our understanding of the biological activities of Sarcopoterium spinosum L. root extract based on transcriptome changes to uncover a plurality of possible pharmacological effects without the need for prior knowledge or functional screening. We integrated Gene Set Enrichment Analysis of the RNAseq data to identify pathways affected by the treatment of cells with the extract and perturbational signatures in the CMAP database to enhance the validity of the results. Activities of signaling pathways were measured using immunoblotting with phospho-specific antibodies. Mitochondrial membrane potential was assessed using JC-1 staining. SARS-CoV-2-induced cell killing was assessed in Vero E6 and A549 cells using an MTT assay. Here, we identified transcriptome changes following exposure of cultured cells to the medicinal plant Sarcopoterium spinosum L. root extract. By integrating algorithms of GSEA and CMAP, we confirmed known anti-cancer activities of the extract and predicted novel biological effects on oxidative phosphorylation and interferon pathways. Experimental validation of these pathways uncovered strong activation of autophagy, including mitophagy, and excellent protection from SARS-CoV-2 infection. Our study shows that gene expression analysis alone is insufficient for predicting biological effects since some of the changes reflect compensatory effects, and additional biochemical tests provide necessary corrections. This study defines the advantages and limitations of transcriptome analysis in predicting the biological and medicinal effects of the Sarcopoterium spinosum L. extract. Such analysis could be used as a general approach for predicting the medicinal properties of plants.

Validation of a Mathematical Model Describing the Dynamics of Chemotherapy for Chronic Lymphocytic Leukemia In Vivo.

In recent years, mathematical models have developed into an important tool for cancer research, combining quantitative analysis and natural processes. We have focused on Chronic Lymphocytic Leukemia (CLL), since it is one of the most common adult leukemias, which remains incurable. As the first step toward the mathematical prediction of in vivo drug efficacy, we first found that logistic growth best described the proliferation of fluorescently labeled murine A20 leukemic cells injected in immunocompetent Balb/c mice. Then, we tested the cytotoxic efficacy of Ibrutinib (Ibr) and Cytarabine (Cyt) in A20-bearing mice. The results afforded calculation of the killing rate of the A20 cells as a function of therapy. The experimental data were compared with the simulation model to validate the latter's applicability. On the basis of these results, we developed a new ordinary differential equations (ODEs) model and provided its sensitivity and stability analysis. There was excellent accordance between numerical simulations of the model and results from in vivo experiments. We found that simulations of our model could predict that the combination of Cyt and Ibr would lead to approximately 95% killing of A20 cells. In its current format, the model can be used as a tool for mathematical prediction of in vivo drug efficacy, and could form the basis of software for prediction of personalized chemotherapy.

Cytoplasmic proteotoxicity regulates HRI-dependent phosphorylation of eIF2α via the Hsp70-Bag3 module.

The major heat shock protein Hsp70 forms a complex with a scaffold protein Bag3 that links it to components of signaling pathways. Via these interactions, the Hsp70-Bag3 module functions as a proteotoxicity sensor that controls cell signaling. Here, to search for pathways regulated by the complex, we utilized JG-98, an allosteric inhibitor of Hsp70 that blocks its interaction with Bag3. RNAseq followed by the pathway analysis indicated that several signaling pathways including UPR were activated by JG-98. Surprisingly, only the eIF2α-associated branch of the UPR was activated, while other UPR branches were not induced, suggesting that the response was unrelated to the ER proteotoxicity and ER-associated kinase PERK1. Indeed, induction of the UPR genes under these conditions was driven by a distinct eIF2α kinase HRI. Hsp70-Bag3 directly interacted with HRI and regulated eIF2α phosphorylation upon cytoplasmic proteotoxicity. Therefore, cytosolic proteotoxicity can activate certain UPR genes via Hsp70-Bag3-HRI-eIF2α axis.

A Cell Double-Barcoding System for Quantitative Evaluation of Primary Tumors and Metastasis in Animals That Uncovers Clonal-Specific Anti-Cancer Drug Effects.

Imaging in monitoring metastasis in mouse models has low sensitivity and is not quantitative. Cell DNA barcoding, demonstrating high sensitivity and resolution, allows monitoring effects of drugs on the number of tumor and metastatic clones. However, this technology is not suitable for comparison of sizes of metastatic clones in different animals, for example, drug treated and untreated, due to high biological and technical variability upon tumor and metastatic growth and isolation of barcodes from tissue DNA. However, both numbers of clones and their sizes are critical parameters for analysis of drug effects. Here we developed a modification of the barcoding approach for monitoring drug effects on tumors and metastasis that is quantitative, highly sensitive and highly reproducible. This novel cell double-barcoding system allows simultaneously following the fate of two or more cell variants or cell lines in xenograft models in vivo, and also following the fates of individual clones within each of these populations. This system allows comparing effects of drugs on different cell populations and thus normalizing drug effects by drug-resistant lines, which corrects for both biological and technical variabilities and significantly increases the reproducibility of results. Using this barcoding system, we uncovered that effects of a novel DYRK1B kinase inhibitor FX9847 on primary tumors and metastasis is clone-dependent, while a distinct drug osimertinib demonstrated clone-independent effects on cancer cell populations. Overall, a cell double-barcoding approach can significantly enrich our understanding of drug effects in basic research and preclinical studies.

The role of Bag3 in cell signaling.

Bag3 has been implicated in a wide variety of physiological processes from autophagy to aggresome formation and from cell transformation to survival. We argue that involvement of Bag3 in many of these processes is due to its distinct function in cell signaling. The structure of Bag3 suggests that it can serve as a scaffold that links molecular chaperones Hsp70 and small Hsps with components of a variety of signaling pathways. Major protein-protein interaction motifs of Bag3 that recruit components of signaling pathways are WW domain and PXXP motif that interacts with SH3-domain proteins. Furthermore, Hsp70-Bag3 appears to be a sensor of abnormal polypeptides during the proteotoxic stress. It also serves as a sensor of a mechanical force during mechanotransduction. Common feature of these and probably certain other sensory mechanisms is that they represent responses to specific kinds of abnormal proteins, i.e. unfolded filamin A in case of mechanotransduction or stalled translating polypeptides in case of sensing proteasome inhibition. Overall Hsp70-Bag3 module represents a novel signaling node that responds to multiple stimuli and controls multiple physiological processes.

The Hsp70-Bag3 complex modulates the phosphorylation and nuclear translocation of Hippo pathway protein Yap.

Protein abnormalities can accelerate aging causing protein misfolding diseases, and various adaptive responses have evolved to relieve proteotoxicity. To trigger these responses, cells must detect the buildup of aberrant proteins. Previously we demonstrated that the Hsp70-Bag3 (HB) complex senses the accumulation of defective ribosomal products, stimulating signaling pathway proteins, such as stress kinases or the Hippo pathway kinase LATS1. Here, we studied how Bag3 regulates the ability for LATS1 to regulate its key downstream target YAP (also known as YAP1). In naïve cells, Bag3 recruited a complex of LATS1, YAP and the scaffold AmotL2, which links LATS1 and YAP. Upon inhibition of the proteasome, AmotL2 dissociated from Bag3, which prevented phosphorylation of YAP by LATS1, and led to consequent nuclear YAP localization together with Bag3. Mutations in Bag3 that enhanced its translocation into nucleus also facilitated nuclear translocation of YAP. Interestingly, Bag3 also controlled YAP nuclear localization in response to cell density, indicating broader roles beyond proteotoxic signaling responses for Bag3 in the regulation of YAP. These data implicate Bag3 as a regulator of Hippo pathway signaling, and suggest mechanisms by which proteotoxic stress signals are propagated.

Targeted methylation facilitates DNA double strand breaks and enhances cancer suppression: A DNA intercalating/methylating dual-action chimera Amonafidazene.

A DNA intercalating agent Amonafide interferes with topoisomerase 2 (Topo II) activity and prevents re-ligation of DNA strands, leading to double strand breaks (DSB). If DSB repair fails, cells stop dividing and eventually die. In a search of approaches to enhance anti-cancer activities of Topo II inhibitors, we hypothesized that introduction of additional damage in proximity to the DSB may suppress DNA repair and enhance cancer cell killing. Accordingly, chimeric molecules were created that target a DNA alkylating component to the proximity of Topo II-induced DSBs. These chimeras consist of Amonafide or its 4-amino isomer, and DNA methylating methyl triazene moiety Azene protected with a carbamate group, connected via linker. Treatment of cancer cells with the chimeric molecules leads to significantly higher number of DSBs, which were repaired slower compared to Amonafide or monomethyl triazene-treated cells. On the other hand, methyl triazene linked to non-intercalating Amonafide analogs was ineffective. Together, these data strongly support our hypothesis. In line with increased DSBs, the chimeric molecules exhibited significantly higher antiproliferative activity in cancer cell lines compared to Amonafide or monomethyl triazene constituent Azene. We utilized the fluorescent properties of chimera Amonafidazene to develop ''photo-switchable'' reporting system to monitor the prodrug activation. Using this approach, we found that the chimera accumulated and was activated at the tumor sites specifically and demonstrated significantly stronger tumor suppressing activities compared to Amonafide in a xenograft model. Therefore, targeting alkylating groups to the proximity of DSB sites may become an effective approach towards enhancing anti-cancer activities of inhibitors of topoisomerases.

Cross organelle stress response disruption promotes gentamicin-induced proteotoxicity.

Gentamicin is a nephrotoxic antibiotic that causes acute kidney injury (AKI) primarily by targeting the proximal tubule epithelial cell. The development of an effective therapy for gentamicin-induced renal cell injury is limited by incomplete mechanistic insight. To address this challenge, we propose that RNAi signal pathway screening could identify a unifying mechanism of gentamicin-induced cell injury and suggest a therapeutic strategy to ameliorate it. Computational analysis of RNAi signal screens in gentamicin-exposed human proximal tubule cells suggested the cross-organelle stress response (CORE), the unfolded protein response (UPR), and cell chaperones as key targets of gentamicin-induced injury. To test this hypothesis, we assessed the effect of gentamicin on the CORE, UPR, and cell chaperone function, and tested the therapeutic efficacy of enhancing cell chaperone content. Early gentamicin exposure disrupted the CORE, evidenced by a rise in the ATP:ADP ratio, mitochondrial-specific H2O2 accumulation, Drp-1-mediated mitochondrial fragmentation, and endoplasmic reticulum-mitochondrial dissociation. CORE disruption preceded measurable increases in whole-cell oxidative stress, misfolded protein content, transcriptional UPR activation, and its untoward downstream effects: CHOP expression, PARP cleavage, and cell death. Geranylgeranylacetone, a therapeutic that increases cell chaperone content, prevented mitochondrial H2O2 accumulation, preserved the CORE, reduced the burden of misfolded proteins and CHOP expression, and significantly improved survival in gentamicin-exposed cells. We identify CORE disruption as an early and remediable cause of gentamicin proteotoxicity that precedes downstream UPR activation and cell death. Preserving the CORE significantly improves renal cell survival likely by reducing organelle-specific proteotoxicity during gentamicin exposure.

A first order phase transition mechanism underlies protein aggregation in mammalian cells.

The formation of misfolded protein aggregates is a hallmark of neurodegenerative diseases. The aggregate formation process exhibits an initial lag phase when precursor clusters spontaneously assemble. However, most experimental assays are blind to this lag phase. We develop a quantitative assay based on super-resolution imaging in fixed cells and light sheet imaging of living cells to study the early steps of aggregation in mammalian cells. We find that even under normal growth conditions mammalian cells have precursor clusters. The cluster size distribution is precisely that expected for a so-called super-saturated system in first order phase transition. This means there exists a nucleation barrier, and a critical size above which clusters grow and mature. Homeostasis is maintained through a Szilard model entailing the preferential clearance of super-critical clusters. We uncover a role for a putative chaperone (RuvBL) in this disassembly of large clusters. The results indicate early aggregates behave like condensates. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

p62 /SQSTM1 coding plasmid prevents age related macular degeneration in a rat model.

P62/SQSTM1, a multi-domain protein that regulates inflammation, apoptosis, and autophagy, has been linked to age-related pathologies. For example, previously we demonstrated that administration of p62/SQSTM1-encoding plasmid reduced chronic inflammation and alleviated osteoporosis and metabolic syndrome in animal models. Herein, we built upon these findings to investigate effect of the p62-encoding plasmid on an age-related macular degeneration (AMD), a progressive neurodegenerative ocular disease, using spontaneous retinopathy in senescence-accelerated OXYS rats as a model. Overall, the p62DNA decreased the incidence and severity of retinopathy. In retinal pigment epithelium (RPE), p62DNA administration slowed down development of the destructive alterations of RPE cells, including loss of regular hexagonal shape, hypertrophy, and multinucleation. In neuroretina, p62DNA prevented gliosis, retinal thinning, and significantly inhibited microglia/macrophages migration to the outer retina, prohibiting their subretinal accumulation. Taken together, our results suggest that the p62DNA has a strong retinoprotective effect in AMD.

Hsp70-Bag3 complex is a hub for proteotoxicity-induced signaling that controls protein aggregation.

Protein abnormalities in cells are the cause of major pathologies, and a number of adaptive responses have evolved to relieve the toxicity of misfolded polypeptides. To trigger these responses, cells must detect the buildup of aberrant proteins which often associate with proteasome failure, but the sensing mechanism is poorly understood. Here we demonstrate that this mechanism involves the heat shock protein 70-Bcl-2-associated athanogene 3 (Hsp70-Bag3) complex, which upon proteasome suppression responds to the accumulation of defective ribosomal products, preferentially recognizing the stalled polypeptides. Components of the ribosome quality control system LTN1 and VCP and the ribosome-associated chaperone NAC are necessary for the interaction of these species with the Hsp70-Bag3 complex. This complex regulates important signaling pathways, including the Hippo pathway effectors LATS1/2 and the p38 and JNK stress kinases. Furthermore, under proteotoxic stress Hsp70-Bag3-LATS1/2 signaling regulates protein aggregation. We established that the regulated step was the emergence and growth of abnormal protein oligomers containing only a few molecules, indicating that aggregation is regulated at very early stages. The Hsp70-Bag3 complex therefore functions as an important signaling node that senses proteotoxicity and triggers multiple pathways that control cell physiology, including activation of protein aggregation.

Author Correction: Cancer cell responses to Hsp70 inhibitor JG-98: Comparison with Hsp90 inhibitors and finding synergistic drug combinations.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Translation inhibition corrects aberrant localization of mutant alanine-glyoxylate aminotransferase: possible therapeutic approach for hyperoxaluria.

Primary hyperoxaluria type 1 is a severe kidney stone disease caused by abnormalities of the peroxisomal alanine-glyoxylate aminotransferase (AGT). The most frequent mutation G170R results in aberrant mitochondrial localization of the active enzyme. To evaluate the population of peroxisome-localized AGT, we developed a quantitative Glow-AGT assay based on the self-assembly split-GFP approach and used it to identify drugs that can correct mislocalization of the mutant protein. In line with previous reports, the Glow-AGT assay showed that mitochondrial transport inhibitors DECA and monensin increased peroxisomal localization of the mutant. Here, we demonstrate that prolonged treatment with the translation elongation inhibitor emetine, a medicinal alkaloid used in treatment of amoebiasis, corrected G170R-AGT mislocalization. Furthermore, emetine reduced the augmented oxalate level in culture media of patient-derived hepatocytes bearing the G170R mutation. A distinct translation inhibitor GC7 had a similar effect on the mutant Glow-AGT relocalization indicating that mild translation inhibition is a promising therapeutic approach for primary hyperoxaluria type 1 caused by AGT misfolding/mistargeting. KEY MESSAGES: • There is no effective conservative treatment to decrease oxalate production in PH1 patients. • Chemical chaperones rescue mislocalization of mutant AGT and reduce oxalate levels. • We have developed an assay for precise monitoring of the peroxisomal AGT. • Inhibition of translation by emetine reroutes the mutant protein to peroxisome. • Mild translation inhibition is a promising cure for conformational disorders.

Cancer cell responses to Hsp70 inhibitor JG-98: Comparison with Hsp90 inhibitors and finding synergistic drug combinations.

Hsp70 is a promising anti-cancer target. Our JG-98 series of Hsp70 inhibitors show anti-cancer activities affecting both cancer cells and tumor-associated macrophages. They disrupt Hsp70 interaction with a co-chaperone Bag3 and affect signaling pathways important for cancer development. Due to a prior report that depletion of Hsp70 causes similar responses as depletion of Hsp90, interest to Hsp70 inhibitors as drug prototypes is hampered by potential similarity of their effects to effects of Hsp90 inhibitors. Here, using the Connectivity Map platform we demonstrate that physiological effects of JG-98 are dissimilar from effects of Hsp90 inhibitors, thus justifying development of these compounds. Using gene expression and ActivSignal IPAD platform, we identified pathways modulated by JG-98. Some of these pathways were affected by JG-98 in Bag3-dependent (e.g. ERK) and some in Bag3-independent manner (e.g. Akt or c-myc), indicating multiple effects of Hsp70 inhibition. Further, we identified genes that modulate cellular responses to JG-98, developed approaches to predict potent combinations of JG-98 with known drugs, and demonstrated that inhibitors of proteasome, RNApol, Akt and RTK synergize with JG-98. Overall, here we established unique effects of novel Hsp70 inhibitors on cancer cell physiology, and predicted potential drug combinations for pre-clinical development.