Proteotoxic Stress Response Research Articles

Abstract A50: Modeling and targeting of oncogenic proteotoxic stress in drug-resistant breast cancer

Abstract ERBB2/HER2-positive breast cancers are associated with poor prognosis, and resistance to HER2-targeted therapy (trastuzumab and lapatinib) presents a significant clinical hurdle. There are no treatment options for metastatic HER2+ BCs that have progressed on HER2-targeted therapy. At present, most of the research on therapeutic strategies in therapy-refractory Her2+ BCs focuses on resensitizing tumor cells to HER2-targeting agents. We have shown that targeting of nononcogenic vulnerabilities, specifically adaptive proteotoxic stress response, is a promising therapeutic approach in this subset of BCs (Singh N. et al, HER2-mTOR signaling-driven breast cancer cells require ER-associated degradation to survive. Science Signaling 2015). We showed that Her2+ BCs have specific dependence on the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway to suppress the hyperactive Her2/mTOR pathway-driven proteotoxic stress. Here, we explore the kinetics of protein homeostatic changes during the course of Her2-targeted drug therapy, and identify optimal therapeutic windows for the targeting of ERAD in the therapy-refractory Her2+ BCs. Using an integrated systems approach, we have modeled the dynamics of signaling and protein homeostasis changes in Her2+ cells during remission, resistance, and relapse phases of Her2-targeted therapy. We found that the resistance and relapse phases of anti-Her2 drug (lapatinib and trastuzumab) treatment are associated with Her2-independent acquisition of mTOR signaling, as previously reported. However, these signaling pathway changes also resulted in heightened proteotoxic burden on the ER due to increased protein synthesis rates, which was further exacerbated upon the release of cells from anti-Her2 treatment. Accordingly, cells in the relapse phase, and even more so upon further drug withdrawal, showed significantly heightened sensitivity to the inhibition of ERAD. This study reveals the kinetics of protein homeostasis associated with the signaling pathway changes during acquired resistance to anti-Her2 therapy, and suggests optimal therapeutic windows for the targeting of ERAD in therapy-refractory Her2+ BCs. Citation Format: Navneet Singh, Vishnu Modur, Belal Muhammad, Kakajan Komurov. Modeling and targeting of oncogenic proteotoxic stress in drug-resistant breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A50.

Abstract 1859: Inhibition of proteasome deubiquitinase activity prevents cytoprotective aggresome formation in cancer cells

Abstract Proteasome inhibitors kill cancer cells by inducing an acute proteotoxic stress response characterized by the cytosolic accumulation of poly-ubiquitinated proteins, ER stress and the production of reactive oxygen species (ROS). The aggresome pathway has been described as an escape mechanism from proteasome inhibitor-induced cytotoxicity that functions by sequestering the accumulated protein. We show that b-AP15, an established small molecule inhibitor of proteasome deubiquitinase activity, induces the accumulation of cytosolic poly-ubiquitin in the absence of aggresome formation. In contrast to the clinically used proteasome inhibitor bortezomib, the accumulated poly-ubiquitin complexes induced by b-AP15 remain localized in the cytoplasm and fail to form typical aggresome structures. Inhibition of aggresome formation is dependent on ubiquitin but independent of tubulin dynamics, and may result in the accumulation of a range of substrates of the intracellular transport system on microtubules. Finally, b-AP15 induces hyper-ubiquitination of the aggresome chaperone HDAC6, as well as altering HDAC6 association with the proteasome. This suggests a potential mechanism for the present findings, which will require further investigation. Our data suggests that b-AP15 inhibition of aggresome formation may circumvent a potential resistance pathway following cytotoxicity due to proteasome inhibition via the blocking of microtubule transport. Citation Format: Ellin-Kristina Hillert, Slavica Brjnic, Magdalena Mazurkiewicz, Rolf Larsson, Mårten Fryknäs, Lisa Swanton, Stephen High, Stig Linder, Padraig B. D'Arcy. Inhibition of proteasome deubiquitinase activity prevents cytoprotective aggresome formation in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1859.

Targeting HSF1: A Prime Integrator of Proteotoxic Stress Response in Myeloma.

The HSF1 transcription factor is an integrator of the cellular stress response, and its expression has demonstrated poor prognosis in multiple myeloma. The novel anti-HSF1 small-molecule inhibitors CCT251236 and KRIB11 demonstrate in vitro and in vivo antimyeloma activity, representing a novel approach for targeting the heat shock response in myeloma. Clin Cancer Res; 24(10); 2237-8. ©2018 AACRSee related article by Fok et al., p. 2395.

Proteomic characterization of Withaferin A-targeted protein networks for the treatment of monoclonal myeloma gammopathies

Withaferin A (WA), a natural steroid lactone from the plant Withania somnifera, is often studied because of its antitumor properties. Although many in vitro and in vivo studies have been performed, the identification of Withaferin A protein targets and its mechanism of antitumor action remain incomplete. We used quantitative chemoproteomics and differential protein expression analysis to characterize the WA antitumor effects on a multiple myeloma cell model. Identified relevant targets were further validated by Ingenuity Pathway Analysis and Western blot and indicate that WA targets protein networks that are specific for monoclonal gammopathy of undetermined significance (MGUS) and other closely related disorders, such as multiple myeloma (MM) and Waldenström macroglobulinemia (WM). By blocking the PSMB10 proteasome subunit, downregulation of ANXA4, potential association with HDAC6 and upregulation of HMOX1, WA puts a massive blockage on both proteotoxic and oxidative stress responses pathways, leaving cancer cells defenseless against WA induced stresses. These results indicate that WA mediated apoptosis is preceded by simultaneous targeting of cellular stress response pathways like proteasome degradation, autophagy and unfolded protein stress response and thus suggests that WA can be used as an effective treatment for MGUS and other closely related disorders. SignificanceMultifunctional antitumor compounds are of great potential since they reduce the risk of multidrug resistance in chemotherapy. Unfortunately, characterization of all protein targets of a multifunctional compound is lacking. Therefore, we optimized an SILAC quantitative chemoproteomics workflow to identify the potential protein targets of Withaferin A (WA), a natural multifunctional compound with promising antitumor properties. To further understand the antitumor mechanisms of WA, we performed a differential protein expression analysis and combined the altered expression data with chemoproteome WA target data in the highly curated Ingenuity Pathway database. We provide a first global overview on how WA kills multiple myeloma cancer cells and serve as a starting point for further in depth experiments. Furthermore, the combined approach can be used for other types of cancer and/or other promising multifunctional compounds, thereby increasing the potential development of new antitumor therapies.

TAK-243 Is a Selective UBA1 Inhibitor That Displays Preclinical Activity in Acute Myeloid Leukemia (AML)

Introduction:Ubiquitin-like Modifier Activating Enzyme 1 (UBA1; UAE) is the initiating enzyme in the ubiquitylation cascade in which proteins are tagged with ubiquitin moieties to regulate their degradation or function. Compared to normal hematopoietic cells, AML cell lines and primary AML cells have equal levels of UBA1 protein, but increased requirement for this enzyme. TAK-243 is a potent and selective inhibitor of UBA1 and we determined the preclinical activity, biological effects and mechanisms of resistance to the drug in AML.Results:TAK-243 reduced growth and viability of human AML cell lines (OCI-AML2, TEX, U937 and NB4) in a concentration- and time-dependent manner with IC50's ranging from 15-40 nM after treatment for 48 hours. In primary AML samples, most (n=18/21) were sensitive to TAK-243 with an IC50<75 nM at 48 hours of incubation. These samples included patients with high-risk cytogenetics, FLT3 mutations, and patients refractory to induction chemotherapy. We also compared the effects of TAK-243 on primary AML cells (n=6) and normal hematopoietic cells (n=6) and demonstrated that TAK-243 preferentially inhibited the clonogenic growth of AML cells over normal (19-fold reduction CFU-leukemia vs CFU-GM (normal), p ≤ 0.01).Binding of TAK-243 to UBA1 and related E1 enzymes was measured in intact AML cells using the cellular thermal shift assay (CETSA). In AML cell lines and primary AML samples, TAK-243 bound UBA1 at concentrations associated with cell death, but bound other E1 enzymes UBA3 and UBA6 only at much higher concentrations.Next, we evaluated the biological effects of UBA1 inhibition by TAK-243. At concentrations associated with cell death, TAK-243 decreased the abundance of poly- and mono-ubiquitylated proteins in OCI-AML2 cells and primary AML samples. In addition, TAK-243 treatment increased PERK phosphorylation, CHOP, XBP1s and ATF4 which are markers of proteotoxic stress and unfolded protein response. TAK-243 inhibited DNA double strand break (DSB) repair as evidenced by reduced recruitment of 53BP1 to DSBs and sustained γH2AX foci after 3 Gy of irradiation.We assessed the preclinical efficacy and toxicity of TAK-243 in mouse models of AML. OCI-AML2 cells were injected subcutaneously (sc) into SCID mice, and when tumors were palpable, mice were treated with TAK-243 (20 mg/kg sc twice weekly). TAK-243 significantly delayed tumor growth in mice (T/C=0.02) with no toxicity as evidenced by no changes in mouse body weight, serum chemistry, or organ histology. In tumors and organs isolated from the above treated mice, TAK-243 preferentially reduced levels of mono- and poly-ubiquitylated proteins in tumors over normal tissues. As an additional model, primary AML cells from 2 patients were injected into the femurs of NOD-SCID mice. Two weeks after injection, mice were treated with TAK-243 (20 mg/kg sc twice weekly). After 3 weeks of treatment, mice were sacrificed, and AML engraftment in the non-injected femur was measured by flow cytometry. TAK-243 reduced primary AML tumor burden in both tested samples without toxicity. Using secondary transplantations, we demonstrated that TAK-243 had targeted the leukemic stem cells.To understand mechanisms of resistance to TAK-243, we selected a population of TAK-243-resistant OCI-AML2 by culturing cells with increasing concentrations of the drug. Persisting cells were 33-fold more resistant to TAK-243 compared to wild-type cells (IC50 757 vs 23 nM), but had a normal rate of proliferation and remained equally sensitive to bortezomib, daunorubicin, mitoxantrone and the NEDD8-activating enzyme inhibitor pevonedistat. Using CETSA, we showed reduced binding of TAK-243 to UBA1 in the resistant cells. We sequenced UBA1 exons 12-16 and 23-24 that span the adenylation domain. Resistant cells had a missense mutation in exon 16 resulting in substitution of tyrosine with cysteine at codon 583 (Y583C). Y583 in human UBA1 corresponds to Y551 in yeast Uba1, which makes a favorable interaction with TAK-243 in its Uba1 binding site. Therefore, Y583C substitution is predicted to interfere with TAK-243 binding to UBA1.Conclusions:TAK-243 is a potent and selective UBA1 inhibitor that displays preferential activity towards AML cells over normal hematopoietic cells. Acquired mutations affect drug binding and may be a clinically relevant mechanism of resistance. These data support conducting a clinical trial of TAK-243 in patients with AML. DisclosuresHyer:Takeda Pharmaceuticals International Co.: Employment. Berger:Takeda Pharmaceuticals International Co.: Employment. Traore:Takeda Pharmaceuticals International Co.: Employment. Sintchak:Takeda Pharmaceuticals International Co.: Employment. Milhollen:Takeda Pharmaceuticals International Co.: Employment. Schimmer:Takeda Pharmaceuticals: Research Funding; Medivir: Research Funding; Novartis Pharmaceuticals: Honoraria.

Hikeshi modulates the proteotoxic stress response in human cells: Implication for the importance of the nuclear function of HSP70s.

Hikeshi mediates the heat stress-induced nuclear import of heat-shock protein 70 (HSP70s: HSP70/HSC70). Dysfunction of Hikeshi causes some serious effects in humans; however, the cellular function of Hikeshi is largely unknown. Here, we investigated the effects of Hikeshi depletion on the survival of human cells after proteotoxic stress and found opposite effects in HeLa and hTERT-RPE1 (RPE) cells; depletion of Hikeshi reduced the survival of HeLa cells, but increased the survival of RPE cells in response to proteotoxic stress. Hikeshi depletion sustained heat-shock transcription factor 1 (HSF1) activation in HeLa cells after recovery from stress, but introduction of a nuclear localization signal-tagged HSC70 in Hikeshi-depleted HeLa cells down-regulated HSF1 activity. In RPE cells, the HSF1 was efficiently activated, but the activated HSF1 was not sustained after recovery from stress, as in HeLa cells. Additionally, we found that p53 and subsequent up-regulation of p21 were higher in the Hikeshi-depleted RPE cells than in the wild-type cells. Our results indicate that depletion of Hikeshi renders HeLa cells proteotoxic stress-sensitive through the abrogation of the nuclear function of HSP70s required for HSF1 regulation. Moreover, Hikeshi depletion up-regulates p21 in RPE cells, which could be a cause of its proteotoxic stress resistant.

A Topical Zinc Ionophore Blocks Tumorigenic Progression in UV‐exposed SKH‐1 High‐risk Mouse Skin

Nonmelanoma skin cancer (NMSC) is the most common malignancy in the United States representing a considerable public health burden. Pharmacological suppression of skin photocarcinogenesis has shown promise in preclinical and clinical studies, but more efficacious photochemopreventive agents are needed. Here, we tested feasibility of harnessing pharmacological disruption of intracellular zinc homeostasis for photochemoprevention in vitro and in vivo. Employing the zinc ionophore and FDA-approved microbicidal agent zinc pyrithione (ZnPT), used worldwide in over-the-counter (OTC) topical consumer products, we first demonstrated feasibility of achieving ZnPT-based intracellular Zn2+ overload in cultured malignant keratinocytes (HaCaT-ras II-4; SCC-25) employing membrane-permeable fluorescent probes. Zinc overload was accompanied by induction of intracellular oxidative stress, associated with mitochondrial superoxide release as substantiated by MitoSOX Red™ fluorescence microscopy. ZnPT-induced cell death observable in malignant keratinocytes was preceded by induction of metal (MT2A), proteotoxic (HSPA6, HSPA1A, DDIT3, HMOX1) and genotoxic stress response (GADD45A, XRCC2) gene expression at the mRNA and protein levels. Comet analysis revealed introduction of formamidopyrimidine-DNA glycosylase (Fpg)-sensitive oxidative DNA lesions. In a photocarcinogenesis model (UV-exposed SKH-1 high-risk mouse skin), topical ZnPT administration post-UV caused epidermal zinc overload and stress response gene expression with pronounced blockade of tumorigenesis. Taken together, these data suggest feasibility of repurposing a topical OTC drug for zinc-directed photochemoprevention of solar UV-induced NMSC.

Differential adiponectin signalling couples ER stress with lipid metabolism to modulate ageing in C. elegans

The metabolic and endocrine functions of adipose tissue and the ability of organisms to cope with cellular stress have a direct impact on physiological ageing and the aetiology of various diseases such as obesity-related pathologies and cancer. The endocrine effects of adipose tissue are mediated by secreted adipokines, which modulate metabolic processes and influence related maladies. Although a plethora of molecules and signaling pathways associate ageing with proteotoxic stress and cellular metabolism, our understanding of how these pathways interconnect to coordinate organismal physiology remains limited. We dissected the mechanisms linking adiponectin signalling pathways and endoplasmic reticulum (ER) proteotoxic stress responses that individually or synergistically affect longevity in C. elegans. Animals deficient for the adiponectin receptor PAQR-1 respond to ER stress, by rapidly activating the canonical ER unfolded protein response (UPRER) pathway, which is primed in these animals under physiological conditions by specific stress defence transcription factors. PAQR-1 loss enhances survival and promotes longevity under ER stress and reduced insulin/IGF-1 signalling. PAQR-1 engages UPRER, autophagy and lipase activity to modulate lipid metabolism during ageing. Our findings demonstrate that moderating adiponectin receptor -1 activity extends lifespan under stress, and directly implicate adiponectin signalling as a coupler between proteostasis and lipid metabolism during ageing.

Abstract 4985: Understanding the vulnerabilities in cancer cells upon inhibition of glutathione synthesis

Abstract A hallmark of cancer is the rewiring of cellular metabolic pathways, including those that mediate oxidative stress. Cancer initiation and progression, which demands increased production of ATP and synthesis of proteins, also results in elevated generation of toxic by-products such as reactive oxygen species (ROS). Cancer cells counteract ROS by increasing the production of antioxidants, which convert ROS into benign species. It has been shown that production of glutathione (GSH), the most abundant intracellular antioxidant, is necessary for many cancers to initiate malignant transformation. Whether inhibition of GSH synthesis can impact the survival of established malignant tumors remains poorly understood. In this study we investigated the extent to which GSH is required in cancer cells and across different subtypes of breast cancer. Profiling of an extensive panel of breast cancer cell lines demonstrated that the majority of cells are resistant to death after acute GSH depletion, while only luminal breast cancer cells are sensitive to chronic GSH depletion. To understand how most tumor cells are resistant to GSH depletion and oxidative stress, we conducted a small-molecule high-throughput screen interrogating more than 500 compounds in a 10-point dilution format. Importantly, screening plates were imaged and cell numbers quantified to avoid any false positives/negatives potentially associated with interrogating cellular metabolism using classical high throughput techniques, such as CellTiter-Glo. This screen revealed that depletion of glutathione sensitized breast cancer cells to inhibition of deubiquitinases, resulting in a drastic induction of a proteotoxic stress response. Furthermore, this sensitivity was found to be specific to malignant cells, as non-transformed cells were resistant to the combined treatments. It has been hypothesized that GSH, acting as an abundant thiol group, non-enzymatically reacts with ubiquitin and limits ubiquitination of proteins. Loss of glutathione, in combination with deubiquitinase inhibitors, leads to deregulation of ubiquitinated protein pools, and cancer cells undergo proteotoxic stress and cell death. These findings elucidate a novel vulnerability in cancers cells and shed light on an unexpected relationship between cancer metabolism and protein homeostasis. Citation Format: Isaac S. Harris, Jennifer E. Endress, Joan S. Brugge. Understanding the vulnerabilities in cancer cells upon inhibition of glutathione synthesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4985. doi:10.1158/1538-7445.AM2017-4985

Phospholipase Lpl1 links lipid droplet function with quality control protein degradation.

Protein misfolding is toxic to cells and is believed to underlie many human diseases, including many neurodegenerative diseases. Accordingly, cells have developed stress responses to deal with misfolded proteins. The transcription factor Rpn4 mediates one such response and is best known for regulating the abundance of the proteasome, the complex multisubunit protease that destroys proteins. Here we identify Lpl1 as an unexpected target of the Rpn4 response. Lpl1 is a phospholipase and a component of the lipid droplet. Lpl1 has dual functions: it is required for both efficient proteasome-mediated protein degradation and the dynamic regulation of lipid droplets. Lpl1 shows a synthetic genetic interaction with Hac1, the master regulator of a second proteotoxic stress response, the unfolded protein response (UPR). The UPR has long been known to regulate phospholipid metabolism, and Lpl1's relationship with Hac1 appears to reflect Hac1's role in stimulating phospholipid synthesis under stress. Thus two distinct proteotoxic stress responses control phospholipid metabolism. Furthermore, these results provide a direct link between the lipid droplet and proteasomal protein degradation and suggest that dynamic regulation of lipid droplets is a key aspect of some proteotoxic stress responses.

Clinical Significance of Ck2 (CSNK2) and C-Myc Expression in Childhood Acute Lymphoblastic Leukemia

Background. Acute lymphoblastic leukemia (ALL) is the most common malignancy of childhood and a major cause of childhood cancer-related mortality. Although the cure rate now approaches 90%, certain pediatric ALL subgroups present subsequent relapse. For this reason, analyses of cell signaling pathways will help to identify new markers and/or targets for tailored therapy. PI3K/AKT/mTOR activation is frequently found in both B-ALL and T-ALL. Protein kinase Ck2 (CSNK2) activity in pediatric ALL was increased and its inhibition restored PTEN phosphatase activity with subsequent inactivation of AKT. Moreover, Ck2 may serve the activity of oncogenes such as BCR-ABL and c-MYC, control the activation of other critical signaling cascades (JAK-STAT), and sustain multiple cellular stress-elicited pathway such as the proteotoxic stress, unfolded protein and DNA-damage responses. Ck2 has also been shown to have an essential role in tuning signals derived from the stromal tumor microenvironment (Piazza F et al, Oncogene2016).Material and Methods. We analyzed cDNA collected from 46 patients with B-ALL [19 High Risk (HR) for Minimal Residual Disease (MRD) and 27 NON-HR] and 25 with T-ALL (8 HR and 17 NON-HR), respectively, diagnosed in our Center from 2000 to 2012. The latter subgroup was screened fro PTEN-Exon7 mutations and TXL3 rearrangements. We evaluated the gene expression of Ck2 and c-Myc genes using RQ-PCR with Sybr-Green and a relative quantification method (ΔΔCt method), comparing gene’s expression from patients with samples from 6 healty donors (HDs). In order to demonstrate the correlation between genetic alteration and signaling transduction, specifically in HR patients , we analyzed some phosphoproteins by Phospho-flow approach. We profiled 5 proteins (STAT3, STAT5, CREB, PTEN and pS6) in 4 T-ALL cases (3 with PTEN-Exon 7 mutation).Results. We observed a significant difference of Ck2 expression in T-ALL NON-HR patients vs HDs (Mean Ck2 Fold-Changes 3.494 vs 1.17, p=0.0315) and in T-ALL HR patients (6.384 vs 1.17, p=0.0219) vs HDs (Fig 1A and B). Comparing NON-HR vs HR cases, we found a statistically significant difference (p<0.0001) (Fig 1C). c-Myc mean expression was very similar between the two T-ALL subgroups. Moreover, among T-ALL cases, we identified 5 patients with PTEN-Exon7 mutations and 6 with TLX3 rearrangements. We observed that cases with PTEN-Exon7 mutation showed lower c-Myc expression than cases with PTEN-Exon7 wild-type (mean c-Myc 8.550 vs 1.920) whereas patients with TLX3 rearrangements showed higher c-Myc expression than TLX3 negative (mean c-Myc 18.260 vs 5.502) (p<0.005) (Fig 2A and B). We did not observe any correlation between these rearrangements and Ck2 expression. We also performed Ck2 and c-Myc expression analysis in B-ALL (NON-HR and HR) subgroups. We surprisingly observed a Ck2 overexpression in both NON-HR and HR B-ALLs compared to HDs. On the other side, we did not observed significant difference about c-Myc expression in cases with B-ALL vs HDs; whilst we observed an overexpression of c-Myc in HR vs NON-HR patients with B-ALL (mean 7.075 vs 2.095, respectively)(p<0.0004). Phospho-Flow analysis, in 3 cases with PTEN-Exon7 mutation (1 Ck2+/normal Myc, 1 normal Ck2/normal Myc, 1 Ck2-/normal Myc) showed PTEN null, very lower pS6 basal level and higher CREB basal level than in case with PTEN-Exon7 wild-type. Moreover, we observed that the latter patient, presented with a TLX3 rearrangements (Ck2+/Myc+) with higher c-Myc expression, showed higher STAT3 basal state level confirming that STAT3 induces the expression of c-Myc.Conclusions. Based on our preliminary findings, Ck2 could be considered as a marker and /or a potential candidate for targeted therapy, specifically in HR-ALL, as confirmed by the use of CK2 inhibitor (CX-4945) in ongoing clinical trials. c-Myc overexpression confirmed its association with HR features. The potential role as markers of both genes needs to be demonstrated in a larger population study. Combined application of genomic and phosphoproteomic strategies will lead us to better profile diagnostic samples of HR-ALL, addressing future tailored treatments. DisclosuresNo relevant conflicts of interest to declare.

Azadiradione ameliorates polyglutamine expansion disease inDrosophilaby potentiating DNA binding activity of heat shock factor 1

Aggregation of proteins with the expansion of polyglutamine tracts in the brain underlies progressive genetic neurodegenerative diseases (NDs) like Huntington's disease and spinocerebellar ataxias (SCA). An insensitive cellular proteotoxic stress response to non-native protein oligomers is common in such conditions. Indeed, upregulation of heat shock factor 1 (HSF1) function and its target protein chaperone expression has shown promising results in animal models of NDs. Using an HSF1 sensitive cell based reporter screening, we have isolated azadiradione (AZD) from the methanolic extract of seeds of Azadirachta indica, a plant known for its multifarious medicinal properties. We show that AZD ameliorates toxicity due to protein aggregation in cell and fly models of polyglutamine expansion diseases to a great extent. All these effects are correlated with activation of HSF1 function and expression of its target protein chaperone genes. Notably, HSF1 activation by AZD is independent of cellular HSP90 or proteasome function. Furthermore, we show that AZD directly interacts with purified human HSF1 with high specificity, and facilitates binding of HSF1 to its recognition sequence with higher affinity. These unique findings qualify AZD as an ideal lead molecule for consideration for drug development against NDs that affect millions worldwide.

Oral Supplementation of Glucosamine Fails to Alleviate Acute Kidney Injury in Renal Ischemia-Reperfusion Damage.

Acute kidney injury is a leading contributor to morbidity and mortality in the ageing population. Proteotoxic stress response pathways have been suggested to contribute to the development of acute renal injury. Recent evidence suggests that increased synthesis of N-glycan precursors in the hexosamine pathway as well as feeding of animals with aminosugars produced in the hexosamine pathway may increase stress resistance through reducing proteotoxic stress and alleviate pathology in model organisms. As feeding of the hexosamine pathway metabolite glucosamine to aged mice increased their life expectancy we tested whether supplementation of this aminosugar may also protect mice from acute kidney injury after renal ischemia and reperfusion. Animals were fed for 4 weeks ad libitum with standard chow or standard chow supplemented with 0.5% N-acetylglucosamine. Preconditioning with caloric restriction for four weeks prior to surgery served as a positive control for protective dietary effects. Whereas caloric restriction demonstrated the known protective effect both on renal function as well as survival in the treated animals, glucosamine supplementation failed to promote any protection from ischemia-reperfusion injury. These data show that although hexosamine pathway metabolites have a proven role in enhancing protein quality control and survival in model organisms oral glucosamine supplementation at moderate doses that would be amenable to humans does not promote protection from ischemia-reperfusion injury of the kidney.

The bromodomain protein BRD4 regulates splicing during heat shock.

The cellular response to heat stress is an ancient and evolutionarily highly conserved defence mechanism characterised by the transcriptional up-regulation of cyto-protective genes and a partial inhibition of splicing. These features closely resemble the proteotoxic stress response during tumor development. The bromodomain protein BRD4 has been identified as an integral member of the oxidative stress as well as of the inflammatory response, mainly due to its role in the transcriptional regulation process. In addition, there are also several lines of evidence implicating BRD4 in the splicing process. Using RNA-sequencing we found a significant increase in splicing inhibition, in particular intron retentions (IR), following heat treatment in BRD4-depleted cells. This leads to a decrease of mRNA abundancy of the affected transcripts, most likely due to premature termination codons. Subsequent experiments revealed that BRD4 interacts with the heat shock factor 1 (HSF1) such that under heat stress BRD4 is recruited to nuclear stress bodies and non-coding SatIII RNA transcripts are up-regulated. These findings implicate BRD4 as an important regulator of splicing during heat stress. Our data which links BRD4 to the stress induced splicing process may provide novel mechanisms of BRD4 inhibitors in regard to anti-cancer therapies.

Tmc1 Is a Dynamically Regulated Effector of the Rpn4 Proteotoxic Stress Response

The ubiquitin-proteasome system represents the major pathway of selective intracellular protein degradation in eukaryotes. Misfolded proteins represent an important class of substrates for this pathway, and the failure to destroy misfolded proteins is associated with a number of human diseases. The transcription factor Rpn4 mediates a key proteotoxic stress response whose best known function is to control proteasome abundance by a homeostatic feedback mechanism. Here we identify the uncharacterized zinc finger protein Tmc1 as a dynamically regulated stress-responsive protein. Rpn4 induces TMC1 transcription in response to misfolded proteins. However, this response is counteracted by rapid proteasome-dependent degradation of Tmc1, which serves to normalize Tmc1 protein levels after induction. Precise control of Tmc1 levels is needed in vivo to survive multiple stressors related to proteostasis. Thus, Tmc1 represents a novel effector and substrate of the Rpn4 proteotoxic stress response.

Metabolic control of the proteotoxic stress response: implications in diabetes mellitus and neurodegenerative disorders.

Proteome homeostasis, or proteostasis, is essential to maintain cellular fitness and its disturbance is associated with a broad range of human health conditions and diseases. Cells are constantly challenged by various extrinsic and intrinsic insults, which perturb cellular proteostasis and provoke proteotoxic stress. To counter proteomic perturbations and preserve proteostasis, cells mobilize the proteotoxic stress response (PSR), an evolutionarily conserved transcriptional program mediated by heat shock factor 1 (HSF1). The HSF1-mediated PSR guards the proteome against misfolding and aggregation. In addition to proteotoxic stress, emerging studies reveal that this proteostatic mechanism also responds to cellular energy state. This regulation is mediated bythe key cellular metabolic sensor AMP-activated protein kinase (AMPK). In this review, we present an overview of the maintenance of proteostasis by HSF1, the metabolic regulation of the PSR, particularly focusing on AMPK, and their implications in the two major age-related diseases-diabetes mellitus and neurodegenerative disorders.

HSF1 critically attunes proteotoxic stress sensing by mTORC1 to combat stress and promote growth.

To cope with proteotoxic stress, cells attenuate protein synthesis. However, the precise mechanisms underlying this fundamental adaptation remain poorly defined. Here we report that mTORC1 acts as an immediate cellular sensor of proteotoxic stress. Surprisingly, the multifaceted stress-responsive kinase JNK constitutively associates with mTORC1 under normal growth conditions. Upon activation by proteotoxic stress, JNK phosphorylates both RAPTOR at Ser863 and mTOR at Ser567, causing partial disintegration of mTORC1 and subsequent translation inhibition. Importantly, HSF1, the central player in the proteotoxic stress response (PSR), preserves mTORC1 integrity and function by inactivating JNK, independently of its canonical transcriptional action. Thereby, HSF1 translationally augments the PSR. Beyond promoting stress resistance, this intricate HSF1-JNK-mTORC1 interplay, strikingly, regulates cell, organ and body sizes. Thus, these results illuminate a unifying mechanism that controls stress adaptation and growth.

Targeting CK2-driven non-oncogene addiction in B-cell tumors.

Genetic mutations of oncogenes often underlie deranged cell growth and altered differentiation pathways leading to malignant transformation of B-lymphocytes. However, addiction to oncogenes is not the only drive to lymphoid tumor pathogenesis. Dependence on non-oncogenes, which act by propelling basic mechanisms of cell proliferation and survival, has also been recognized in the pathobiology of lymphoid leukemias, lymphomas and multiple myeloma. Among the growing number of molecules that may uphold non-oncogene addiction, a key place is increasingly being recognized to the serine-threonine kinase CK2. This enzyme is overexpressed and overactive in B-acute lymphoblastic leukemia, multiple myeloma, chronic lymphocytic leukemia and non-Hodgkin lymphomas, such as mantle cell, follicular, Burkitt's and diffuse large B-cell lymphomas. In these tumors, CK2 may serve the activity of oncogenes, similar to BCR-ABL and c-MYC, control the activation of critical signaling cascades, such as NF-κB (nuclear factor-κB), STAT3 (signal transducer and activator of transcription 3) and PTEN/PI3K/AKT (phosphatase and tensin homolog protein/phosphoinositide 3-kinase/AKR thymoma), and sustain multiple cellular stress-elicited pathways, such as the proteotoxic stress, unfolded protein and DNA-damage responses. CK2 has also been shown to have an essential role in tuning signals derived from the stromal tumor microenvironment. Not surprisingly, targeting CK2 in lymphoid tumor cell lines or mouse xenograft models can boost the cytotoxic effects of both conventional chemotherapeutics and novel agents, similar to heat-shock protein 90, proteasome and tyrosine kinases inhibitors. In this review, we summarize the evidence indicating how CK2 embodies most of the features of a cancer growth-promoting non-oncogene, focusing on lymphoid tumors. We further discuss the preclinical data of the use of small ATP-competitive CK2 inhibitors, which hold the promise to be additional options in novel drug combinations for the therapy of lymphoid and plasmacellular malignancies.

Proteomic Analysis Identifies Ribosome Reduction as an Effective Proteotoxic Stress Response

Stress responses are adaptive cellular programs that identify and mitigate potentially dangerous threats. Misfolded proteins are a ubiquitous and clinically relevant stress. Trivalent metalloids, such as arsenic, have been proposed to cause protein misfolding. Using tandem mass tag-based mass spectrometry, we show that trivalent arsenic results in widespread reorganization of the cell from an anabolic to a catabolic state. Both major pathways of protein degradation, the proteasome and autophagy, show increased abundance of pathway components and increased functional output, and are required for survival. Remarkably, cells also showed a down-regulation of ribosomes at the protein level. That this represented an adaptive response and not an adverse toxic effect was indicated by enhanced survival of ribosome mutants after arsenic exposure. These results suggest that a major source of toxicity of trivalent arsenic derives from misfolding of newly synthesized proteins and identifies ribosome reduction as a rapid, effective, and reversible proteotoxic stress response.

HSF1: Guardian of Proteostasis in Cancer.

Proteomic instability is causally related to human diseases. In guarding proteome stability, the heat shock factor 1 (HSF1)-mediated proteotoxic stress response plays a pivotal role. Contrasting with its beneficial role of enhancing cell survival, recent findings have revealed a compelling pro-oncogenic role for HSF1. However, the mechanisms underlying the persistent activation and function of HSF1 within malignancy remain poorly understood. Emerging evidence reveals that oncogenic signaling mobilizes HSF1 and that cancer cells rely on HSF1 to avert proteomic instability and repress tumor-suppressive amyloidogenesis. In aggregate, these new developments suggest that cancer cells endure chronic proteotoxic stress and that proteomic instability is intrinsically associated with the malignant state, a characteristic that could be exploited to combat cancer.