Upregulation Of Unfolded Protein Response Research Articles

Altered secretion patterns and cell wall organization caused by loss of PodB function in the filamentous fungus Aspergillus nidulans

Filamentous fungi are widely used in the production of a variety of industrially relevant enzymes and proteins as they have the unique ability to secrete tremendous amounts of proteins. However, the secretory pathways in filamentous fungi are not completely understood. Here, we investigated the role of a mutation in the POlarity Defective (podB) gene on growth, protein secretion, and cell wall organization in Aspergillus nidulans using a temperature sensitive (Ts) mutant. At restrictive temperature, the mutation resulted in lack of biomass accumulation, but led to a significant increase in specific protein productivity. Proteomic analysis of the secretome showed that the relative abundance of 584 (out of 747 identified) proteins was altered due to the mutation. Of these, 517 were secreted at higher levels. Other phenotypic differences observed in the mutant include up-regulation of unfolded protein response (UPR), deformation of Golgi apparatus and uneven cell wall thickness. Furthermore, proteomic analysis of cell wall components in the mutant revealed the presence of intracellular proteins in higher abundance accompanied by lower levels of most cell wall proteins. Taken together, results from this study suggest the importance of PodB as a target when engineering fungal strains for enhanced secretion of valuable biomolecules.

P-187 - Nox 5 overexpression induces the activation of the unfolded protein response in human aortic endothelial cells

Oxidative stress is a critical mechanism that underlies the pathophysiology of cardiovascular diseases. At the vascular level, an enhanced NADPH oxidase-mediated superoxide production associates with endothelial dysfunction, metalloprotease secretion, cellular senescence, and more recently activation of the unfolded protein response (UPR). Human aortic endothelial cells (HAEC) were infected with Nox 5 or GFP adenovirus, RNA was extracted 12, 18 and 24 hours after infection and used to determine mRNA expression by a Gene Chip Human Transcriptome Array 2.0 (Affymetrix). Nox 5 overexpression after adenoviral infection in HAEC resulted in a significant enhancement of extracellular superoxide and hydrogen peroxide production and led to a significant increase in cellular apoptosis by caspases 3 and 7. The ontological analysis of the array showed an upregulation of UPR. Infection of HAEC with Nox 5 adenovirus activated the UPR pathway, increasing Ire1α and CHOP mRNA expression, GRP78 protein levels and reducing calnexin protein expression. Additionally, Nox 5 overexpression significantly reduced Nox 4 and NOS3 mRNA expression. Nox 5 overexpression promotes a situation of oxidative stress in HAEC, which alters correct protein folding and activates the UPR pathway.

Physical Exercise Reduces Cytotoxicity and Up-Regulates Nrf2 and UPR Expression in Circulating Cells of Peripheral Artery Disease Patients: An Hypoxic Adaptation?

Aim: Ischemia-reperfusion (I-R) produces reactive oxygen species (ROS) that damage cells and favour cytotoxicity and apoptosis in peripheral artery disease (PAD) patients. Since brief episodes of I-R (ischemic conditioning) protect cells against ischemic harms, we evaluated whether a short-course of supervised treadmill training, characterized by repeated episodes of I-R, makes peripheral blood mononuclear cells (PBMCs) from PAD patients with intermittent claudication more resistant to I-R injuries by reducing oxidative stress and by inducing an adaptative response of unfolded protein response (UPR) and nuclear factor-E2-related factor (Nrf2) pathway expression.Methods: 24 PAD patients underwent 21 sessions of treadmill training and a treadmill test as indicator of acute response to I-R.Results: Maximal and pain free walking distance improved (p < 0.01), whereas LDH leakage and apoptosis of PBMCs decreased (p < 0.01); plasma malondialdehyde and ROS generation in PBMCs declined, while plasma glutathione augmented (p < 0.01). Moreover we demonstrated an up-regulation of UPR and Nrf2 expression in PBMCs (p < 0.01). To understand whether treadmill training may act as a trigger of ischemic conditioning, we examined the effect of repeated episodes of I-R on adaptative response in PBMCs derived from the patients. We showed an up-regulation of UPR and Nrf2 gene expression (p < 0.01), while oxidative stress and cytotoxicity, after an initial increase, declined (p < 0.01). This positive effect on cytotoxicity was reduced after inhibition of UPR and Nrf2 pathways.Conclusions: Treadmill training in PAD patients through UPR and Nrf2 up-regulation may trigger hypoxic adaptation similar to conditioning, thus modifying cell survival.

Expression analysis of protein homeostasis pathways in the peripheral blood mononuclear cells of sporadic amyotrophic lateral sclerosis patients

Misfolded protein aggregates are the hallmark of Amyotrophic Lateral Sclerosis (ALS) which suggests involvement of protein homeostasis pathways in etiology of ALS. However, status of protein homeostasis in peripheral blood of ALS is not well established. We analyzed expression levels of key genes of proteostasis pathways in peripheral blood mononuclear cells (PBMCs) of sporadic ALS (sALS) patients and healthy controls. Increased protein carbonylation was observed in patients reflecting oxidative damage in PBMCs. We observed increased transcript and protein levels of GRP78 suggesting Endoplasmic reticulum (ER) insult to cells. Further, significant upregulation of spliced XBP1 and two stress sensors: IRE1α/ERN1 and ATF6 indicated induction of unfolded protein response (UPR). Genes involved in autophagosome initiation (ULK1, ULK2, ATG13); nucleation and elongation (BECLIN1, ATG7, ATG16L1, ATG5, ATG10) and vesicular trafficking genes were significantly increased in patients. Increased lipidation of LC3 validated induction of autophagy. Accumulation of low molecular weight ubiquitinated proteins in patients suggested deregulation of proteasome (UPS) pathway. In addition, cytosolic chaperones (HSP70 and HSP27) and HSF1 were elevated in patients. Increased TDP43 indicated role of TDP43 in disease pathology. Our findings suggest that there is oxidative insult and upregulation of UPR, vesicular trafficking and autophagy in PBMCs of sALS patients.

Sex-Specific Modulation of Fetal Adipogenesis by Gestational Bisphenol A and Bisphenol S Exposure.

The endocrine-disrupting chemical bisphenol A (BPA) increases adipose tissue mass in vivo and promotes adipogenesis in vitro; however, mechanisms explaining BPA's obesogenic effect remain unknown. We investigated the effects of gestational BPA and its analog, bisphenol S (BPS), exposure on the adipogenic differentiation ability of fetal preadipocytes and the role of endoplasmic reticulum stress in regulating this process. Pregnant sheep (n = 7 to 8 per group) mated to the same male were exposed to BPA or BPS from days 30 to 100 of gestation; pregnancies were terminated 20 days later. Adipose tissue was harvested and fetal preadipocytes isolated. Adipose tissue gene expression, adipocyte size, preadipocyte gene expression, adipogenic differentiation, and dynamic expression of genes involved in adipogenesis and endoplasmic reticulum stress were assessed. Gestational BPA enhanced adipogenic differentiation in female, but not male, preadipocytes. The unfolded protein response (UPR) pathway was upregulated in BPA-exposed female preadipocytes supportive of a higher endoplasmic reticulum stress. Increased expression of estradiol receptor 1 and glucocorticoid receptor in female preadipocytes suggests that this may be a potential cause behind the sex-specific effects observed upon BPA exposure. Gestational BPS affected adipogenic terminal differentiation gene expression in male preadipocytes, but not adipogenic differentiation potential. We demonstrate that gestational BPA exposure can modulate the differentiation ability of fetal preadipocytes. UPR upregulation in gestationally BPA-exposed female preadipocytes may contribute to the increased preadipocyte's adipogenic ability. The marked sex-specific effect of BPA highlights higher susceptibility of females to bisphenol A and potentially, a higher risk to develop obesity in adulthood.

Abstract 4507: Upregulation of unfolded protein response (UPR): A novel activity of the tumor suppressor klotho in colorectal cancer

Abstract Klotho is a transmembrane protein, which can be shed and act as a circulating hormone. Klotho-deficient mice manifest a syndrome resembling accelerated aging, while klotho overexpression extends life span. We previously identified klotho as a breast and pancreatic tumor suppressor and as an inhibitor of the IGF-1 pathway. Recent data indicate klotho as a tumor suppressor in an array of malignancies, including colorectal cancer (CRC). We aimed to decipher the role of klotho as a tumor suppressor in CRC. Analysis of a public database (Oncomine) indicated reduced expression of klotho in CRC. Two models were employed to study the effect of klotho in vivo. Using the azoxymethane inflammation model, we discovered that klotho inhibited colon polyp formation. Using an orthotopic model consisting of direct injection of MC38 cells to the mice colons, we found that klotho inhibited colonic obstruction and tumor formation. Similarly, klotho inhibited colony formation and proliferation of CRC cell lines. Aberrant activation of the canonical Wnt pathway is implicated in CRC development and progression. β-catenin, the key effector of this pathway, functions with T-cell factor/lymphoid-enhancer-factor (TCF/LEF) to activate expression of Wnt target genes. While we did not see an effect of klotho on the IGF-1 pathway, it reduced Wnt3A and β-catenin levels as measured by Western blotting, and inhibited TFC/LEF transcriptional activation in luciferase assay. As transcriptional inhibition was abrogated by a constitutively active β-catenin, we suspected that klotho inhibited the pathway upstream of β-catenin. Indeed, co-immunoprecipitation analyses indicated direct interaction between klotho and Wnt3A. Yet, the inhibitory effect of klotho on CRC cell colony formation was only partially rescued by a constitutively active β-catenin. These data indicate on additional mechanisms involved in the TS activity of klotho. Thus, we conducted an RNA expression array and observed involvement of klotho in endoplasmic reticulum (ER) stress and unfolded protein response (UPR). Further studies showed that klotho induced elevation in XBP1 RNA levels, GRP78 protein levels, and eIF2α phosphorylation, all indicative of UPR regulation. Importantly, pharmacologic inhibition of ER stress and UPR overcame the growth suppression effect of klotho in CRC cells. Our data indicate klotho as a potent tumor suppressor in CRC, and suggest its role at very early stages of tumor formation, already at polyp formation. Our data also indicate, for the first time, klotho as a regulator of ER stress and UPR in cancer. These findings may pave the way for the development of novel therapeutic strategies based on enhancement of UPR. Citation Format: Tal Etan, Tammi Rubinstein, Tami Rubinek, Shiri Shahmoon, Chen Varol, Ehud Zigmond, Rina Rosin-Arbesfeld, Iris Barshack, Ido Wolf. Upregulation of unfolded protein response (UPR): A novel activity of the tumor suppressor klotho in colorectal cancer [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 4507. doi:10.1158/1538-7445.AM2017-4507

Reduced Sleep During Social Isolation Leads to Cellular Stress and Induction of the Unfolded Protein Response.

Social isolation has a multitude of negative consequences on human health including the ability to endure challenges to the immune system, sleep amount and efficiency, and general morbidity and mortality. These adverse health outcomes are conserved in other social species. In the fruit fly Drosophila melanogaster, social isolation leads to increased aggression, impaired memory, and reduced amounts of daytime sleep. There is a correlation between molecules affected by social isolation and those implicated in sleep in Drosophila. We previously demonstrated that acute sleep loss in flies and mice induced the unfolded protein response (UPR), an adaptive signaling pathway. One mechanism indicating UPR upregulation is elevated levels of the endoplasmic reticular chaperone BiP/GRP78. We previously showed that BiP overexpression in Drosophila led to increased sleep rebound. Increased rebound sleep has also been demonstrated in socially isolated (SI) flies. D. melanogaster were used to study the effect of social isolation on cellular stress. SI flies displayed an increase in UPR markers; there were higher BiP levels, increased phosphorylation of the translation initiation factor eIF2α, and increased splicing of xbp1. These are all indicators of UPR activation. In addition, the effects of isolation on the UPR were reversible; pharmacologically and genetically altering sleep in the flies modulated the UPR. The reduction in sleep observed in SI flies is a cellular stressor that results in UPR induction.

Translational Focus on Targeting HDAC6 with Ricolinostat Confirms Potent Activity in Preclinical Models of Lymphoma and a Favorable Toxicity Profile in Patients with Relapsed or Refractory Lymphoma

Pan-class HDAC inhibitors are active agents for select subtypes of lymphoma but are often associated with toxicities such as fatigue, diarrhea/constipation, cardiac arrhythmias and myelosuppression. HDAC6 plays a critical role in protein homeostasis by managing misfolded proteins and inducing the unfolded protein response (UPR). Ricolinostat is an isoform selective HDAC6 inhibitor. We evaluated the preclinical effects of ricolinostat and translated the findings into a multicenter Phase 1b study in patients with lymphoma (NCT 02091063). Cytotoxicity was evaluated in 16 lymphoma cell lines (8 DBCL, 4 MCL, 4 TCL) by Cell TiterGlo and Annexin V. Ricolinostat has activity with IC50 values ranging from 0.9-4.7 uM range, which is in the range observed in clinical PK studies. Ricolinostat displayed marked synergy with bortezomib, PI3K inhibitors, disatinib, alisertib, and ibrutinib with synergy coeficients of 0.24, 0.29, 0.64, 0.07, 0.16 respectively (values In an effort to translate these findings, a multi-center Phase Ib open-label, single agent study designed to study the safety of 2 dose schedules of ricolinostat in patients with relapsed or refractory lymphoid malignancies was initiated. Patients were treated with 160 mg of oral ricolinostat continuously on a 28 day cycle. Patients were accrued sequentially to 2 dose cohorts: 3 patients were enrolled on single daily dosing (QD) schedule, and following safety assessment, patients were enrolled to twice daily dosing (BID). Samples were collected for PK and PD analysis. As of July 25th 2015, 11 patients were enrolled on the phase Ib study and were evaluated for safety. The median age was 36 yrs (24-86) and 7 (64%) were male. Sixty-four percent had 5 prior treatments (range: 1-15). Histologies included HL (N=3) and NHL (N=8) including DLBCL=3, MZL=2, FL=1, PTCL=1, PTLD=1. Three patients were enrolled in QD dosing and 8 patients in the BID schedule. There were no DLTs at either of the dosing schedules, and the maximum administrable dose was determined on the BID dosing schedule. The median number of cycles completed was 2 and ranged from 1 through 12+ (currently ongoing). The only grade 3 toxicities reported were anemia (27%), hyponatremia (9%), hypercalcemia (9%) and hypotension (9%), none thought to be attributed to the study drug. Other treatment emergent events included nausea (45%), diarrhea (36%), fatigue (36%) and vomiting (27%) which were all grade 1 and 2. Three patients achieved stable disease, all of whom were treated on the BID schedule (3 of 8, 37.5%), no complete or partial responses have been observed to date. One patient with refractory HL with 14 prior lines of therapy achieved stable disease (SD) and had symptomatic improvement corresponding to an increase of hemoglobin after 9 cycles, as well as resolution of B symptoms. Another patient with PTCL had immediate resolution of B symptoms such as fevers and pruritus after starting QD dosing. This is the first reported experience with an isoform selective HDAC inhibitor to be studied for the treatment of patients with relapsed or refractory lymphoid malignancies. Preclinical studies support the notion that ricolinostat mediates its effects through the UPR and potently synergizes with complementary agents. Ricolinostat is well tolerated with no DLTs. Thirty-seven percent of patients achieved SD on the BID schedule. PK and PD data will be evaluated for acetylation of alpha-tubulin and modulation of the UPR in an effort to confirm preclinical findings. Efficacy will be evaluated in a phase II study. We believe that ricolinostat in rational combinations will lead to effective treatment for patients with relapsed lymphoid malignancies. Disclosures Amengual:Acetylon Pharmaceuticals, INC: Consultancy, Research Funding. Off Label Use: Ricolinostat is being studied for the treatment of lymphoma. Neylon:Seattle Genetics: Speakers Bureau; Celgene: Speakers Bureau; Genentech: Speakers Bureau; Gilead: Speakers Bureau. Deng:TG Therapeutics: Research Funding; Gilead: Research Funding; Amgen/Onyx: Research Funding. Sawas:Seattle Genetics: Research Funding; Gilead Sciences: Honoraria. Shah:Pharmacyclics: Speakers Bureau; NCCN: Consultancy; SWOG: Consultancy; Rosetta Genomics: Research Funding; Acetylon Pharmaceuticals, INC: Membership on an entity9s Board of Directors or advisory committees; Plexus Communications: Honoraria; Spectrum: Speakers Bureau; Bayer: Honoraria; Celgene: Consultancy, Membership on an entity9s Board of Directors or advisory committees, Speakers Bureau; DeBartolo Institute for Personlaized Medicine: Research Funding; Seattle Genetics: Research Funding; Janssen: Speakers Bureau. Wheeler:Acetylon Pharmaceuticals, INC: Employment. Jones:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. O9Connor:Spectrum Pharmaceuticals: Consultancy, Honoraria, Research Funding; Takeda Millennium: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Research Funding; Bristol-Myers Squibb Company: Consultancy; Novartis: Consultancy, Honoraria; Seattle Genetics: Consultancy; Bayer: Consultancy, Honoraria; Acetylon Pharmaceuticals, INC: Consultancy; Mundipharma: Consultancy, Honoraria, Research Funding.

Abstract 1259: Identification of p97 as a prime target to inhibit cancer growth and stemness

Abstract Purpose: The unfolded protein response (UPR) in the endoplasmic reticulum (ER) is an adaptive mechanism for cancer cell survival manifested in many different tumors. In UPR, p97 is an AAA ATPase essential for the degradation of the misfolded proteins, a process also known as the ER-associated protein degradation (ERAD). Previous studies have shown that small molecule inhibitors targeting the key players of UPR and ERAD can inhibit tumor growth. However, the efficacy of these inhibitors has not been compared, and it is unknown whether they affect the population of cancer stem cells (CSC). The purpose of this study is to systematically study the effect of these inhibitors on the growth and differentiation of multiple tumors. Methods: Immunohistochemistry was performed to verify the upregulation of UPR and ERAD proteins in multiple cancers. Immunoblotting and reserve transcription PCR were used to examine the protein expression and the splicing of XBP-1. MTS, Annexin V-PI and Transwell assays were used to measure the proliferation, apoptosis and invasion of cancer cells in vitro. The sphere culture and flow cytometry were used to evaluate the percentage of mammary CSCs. Xenograft tumor model in nude mice was used to evaluate the growth and differentiation of breast cancer cells in vivo. Results: We found that p97, together with IRE1α, BiP/GRP78, ATF6α and ubiquitin, is up-regulated in multiple cancers when compared with the surronding non-cancerous tissues, and these proteins are expressed to different extent in eight solid and four blood tumor cell lines, which may reflect different degrees of UPR activation in these tumors. PERK and eIF2α are also phosphorylated in several tumor cells under normal culture condition, although no splicing of XBP-1 was detected. When cultured in low glucose condition, UPR activation is significantly increased. The inhibitors against IRE1α, PERK, ATF6α and p97 retard the proliferation of different tumor cells with different efficacy, while only p97 inhibitor induces significant apoptosis under normal culture condition. When combined with cisplatin or bortezomib, p97 inhibitor significantly increases cytotoxicity. Among all the inhibitors targeting UPR and ERAD, only p97 inhibitor significantly decreases the percentage of mammary CSC subpopulation of in vitro cultured MDA-MB-231 cells. P97 inhibitor also decreases tumor growth in vivo and reduces the CSC percentage in the tumor isolated from nude mice. Mechanistically, p97 inhibitor impairs the mammosphere forming and the cancer cell invasion through the inactivation of NF-κB signaling. Conclusion: Our comprehensive study demonstrates that the p97 inhibitor not only significantly reduces the growth of multiple tumors but also the CSC subpopulation, the major cause of tumor metastasis, recurrence and chemotherapy resistance, thus establishing p97 as a prime druggable target in the UPR pathway for interventional cancer therapy. Citation Format: Chuang Li, Meng Nie, Hongyang Quan, Qianqian Fan, Nan Zhang, Quancai Cui, Lin Wang. Identification of p97 as a prime target to inhibit cancer growth and stemness. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1259. doi:10.1158/1538-7445.AM2015-1259

Senescence may mediate conversion of tau phosphorylation-induced apoptotic escape to neurodegeneration

Neurodegeneration is the characteristic pathology in the brains of Alzheimer's disease (AD). However, the nature and molecular mechanism leading to the degeneration are not clarified. Given that only the neurons filled with neurofibrillary tangles survive to the end stage of the disease and the major component of the tangles is the hyperphosphorylated tau proteins, it is conceivable that tau hyperphosphorylation must play a crucial role in AD neurodegeneration. We have demonstrated that tau hyperphosphorylation renders the cells more resistant to the acute apoptosis. The molecular mechanisms involve substrate competition of tau and β-catenin for glycogen synthase kinase 3β (GSK-3β); activation of Akt; preservation of Bcl-2 and suppression of Bax, cytosolic cytochrome-c, and caspase-3 activity; and upregulation of unfolded protein response (UPR), i.e., up-regulating phosphorylation of PERK, eIF2 and IRE1 with an increased cleavage of ATF6 and ATF4. On the other hand, tau hyperphosphorylation promotes its intracellular accumulation and disrupts axonal transport; hyperphosphorylated tau also impairs cholinergic function and inhibits proteasome activity. These findings indicate that tau hyperphosphorylation and its intracellular accumulation play dual role in the evolution of AD. We speculate that transient tau phosphorylation helps cells abort from an acute apoptosis, while persistent tau hyperphosphorylation/accumulation may trigger cell senescence that eventually causes a chronic neurodegeneration. Therefore, the nature of “AD neurodegeneration” may represent a new type of tau-regulated chronic neuron death; and the stage of cell senescence may provide a broad window for the intervention of AD.

Dual Targeting With The Selective Histone Deacetylase (HDAC) 6 Inhibitor, ACY-1215, and Bortezomib (BOR) Leads To Marked Disruption Of Protein Degradation Pathways and Apoptosis In Preclinical Models Of Lymphoma

The development of isoform selective HDAC inhibitors has opened the opportunity to better define and target pathways germane to lymphoma. Class IIb HDAC6 binds polyubiquinated, misfolded proteins facilitating their transport to the aggresome for proteosome-independent degradation. The aggresome is a key outlet for the unfolded protein response (UPR), a quality control mechanism that promotes survival during endoplasmic reticulum stress and signals CHOP (C/EBP-homologous protein) mediated apoptosis when homeostasis cannot be reestablished. We investigated the impact of the selective HDAC6 inhibitor ACY-1215 (Acetylon Pharmaceuticals, Inc) in preclinical models of lymphoma, its synergistic potential with BOR, its mechanism of action with respect to inhibiting protein degradation pathways, and potential biomarkers for response.Single agent concentration-effect curves were generated for 8 diffuse large B-cell lymphoma (DLBCL) (4 GCB: OCI-Ly1, OCI-Ly7, Su-DHL4, Su-HDL6; 4 ABC: OCI-Ly10, Su-DHL2, HBL-1, RIVA), 5 mantle cell lymphoma (MCL) (Maver, JVM-1, JEKO, Rec-1, HBL-2 ), and 4 T-cell lymphoma cell lines (HH, H9, CCL-119, Sup-T1). Maximal cytotoxicity was observed at 48-72 hr with IC50 ranging between 240-3500 nM. Activity was greatest in DLBCL, with 72 hr IC50 values as low as 240 nM. ACY-1215 began to show irreversible activity after 6 hr exposure. Single agent exposure at the IC50led to minimal apoptosis as analyzed by annexin V/propidium iodide (PI) staining, and caspase 3 and PARP cleavage, but led to G1 cell cycle arrest.The synergistic potential of ACY-1215 in combination with BOR was measured and evaluated for schedule and dose dependency in 6 cell lines. The greatest synergy was observed with simultaneous exposure of ACY-1215 (750 nM) and BOR (2 nM) at 48 hr with synergy coefficients as low as 0.24. Cell death occurred by apoptosis as evaluated by annexin V/PI, and cleavage of caspase 3 and PARP.Treatment with ACY-1215 led to inhibition of the aggresome as evidenced by increased poly-ubiquitinated proteins and acetylation of α-tubulin. Upregulation of UPR was demonstrated by acetylation of GRP78 and subsequent dissociation of PERK, increased p-eIF2a, spliced XBP-1 and CHOP. These effects were enhanced by treatment with BOR, confirm that accumulation of misfolded proteins activates the UPR response triggering apoptosis, and substantiate blocking two protein degradation systems simultaneously. Minimal acetylation of histone was observed following treatment with ACY-1215, and was considerably less relative to cells treated with pan-HDAC inhibitors confirming selectivity of the drug.These findings were validated in an in vivomodel of DLBCL (OCI-Ly10). Mice were treated with ACY-1215 50 mg/kg days 1-5, 8-12, 15-19 and/or BOR 1.5 mg/kg days 1, 8, 11. Treatment was well tolerated with no toxicity related deaths with the combination. One cycle of the combination led to statistically significant tumor growth delay (p=0.001) and extended overall survival (p<0.05) compared to either single agent. Tumors from mice treated with ACY-1215 demonstrated acetylation of α-tubulin at 4-8 hr, and increased GRP78, and XBP-1. These pharmacodynamic effects were enhanced by the addition of BOR. Interestingly, these findings were not observed in the spleen, suggesting a preferential effect in malignant lymphocytes.Establishing biomarkers for response to isoform selective HDAC6 inhibition may lead to the treatment of patients with specific molecular and phenotypic signatures. Immunoblot levels of basal HDAC6 did not correlate with sensitivity to ACY-1215 in 8 cell lines. Resistance was characterized by combined increase in Bcl2 and loss of BIM. All cell lines expressed more GRP78 than normal peripheral blood mononuclear cells. Fifty-one untreated lymphoma patient samples (10 DLBCL, 11 FL, 10 MZL, 7 MCL, 13 TCL) were analyzed for markers of the UPR by immunohistochemistry. HDAC6 had bright expression in 30/41 lymphoma samples compared to 0/11 reactive lymph nodes. A similar pattern was observed for GRP78 (36/46 vs 0/10), and XBP-1 (26/50 vs 2/12).Selective targeting of HDAC6 with ACY-1215 inhibits aggresome mediated protein degradation inducing the UPR and cell cycle arrest. Dual targeting of protein degradation pathways represents a novel and rational approach for the treatment of lymphoma. Plans to study the clinical efficacy of ACY-1215 in patients with relapsed lymphoma are underway. Disclosures:Amengual:Acetylon Pharmaceuticals, INC: Membership on an entity's Board of Directors or advisory committees, Research Funding. Off Label Use: ACY-1215 is not approved for the treatment of lymphoma. Jones:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. O'Connor:Acetylon Pharmaceuticals, INC: Membership on an entity's Board of Directors or advisory committees.

ER stress and a chronic inflammatory response are induced in an Angiotensin II/DOCA salt model of chronic kidney disease.

We have developed a C57BL/6 mouse model of Chronic Kidney Disease (CKD). The mice underwent a uninephrectomy (Unx) or a sham operation. Unx mice received 1% NaCl in the drinking water and a 50 mg 21‐day release deoxycorticosterone (DOCA) pellet with Angiotensin II (AngII) infusion (1.5 ng Ang II/minute/g). Mice were sacrificed at day 21. This model produced CKD characterized by hypertensive proteinuria. We hypothesized that hypertension‐induced proteinuria results in endoplasmic reticulum (ER) stress as microarray studies in patients with established proteinuria showed upregulation of unfolded protein response (UPR) genes. In the model, electron microscopy showed disruption of the podocyte filtration barrier. Light microscopy revealed an increase in PAS‐positive intratubular protein casts. RT‐PCR analysis showed increased ER stress markers: GRP78, spliced XBP1, CHOP and TDAG51. TUNEL staining showed increased apoptosis in the tubular epithelium of Unx animals. An increase in T‐cell and macrophage infiltration confirmed an inflammatory response to CKD accompanied by renal interstitial fibrosis as shown by de novo α‐smooth muscle actin and collagen staining. This response may be due to ER stress induced pro‐inflammatory mediators such as NFκB. Future studies will assess whether the therapeutic inhibition of ER stress could alter the progression of chronic inflammation in CKD. Funding, CIHR OSO‐115895.

Expression of the hyperphosphorylated tau attenuates ER stress-induced apoptosis with upregulation of unfolded protein response

The neural dysfunction in Alzheimer's disease (AD) could arise from endoplasmic reticulum (ER) stress and deficits of the unfolded protein response (UPR). To explore whether tau hyperphosphorylation, a hallmark of AD brain pathologies, plays a role in ER stress-induced alterations of cell viability, we established cell lines with stable expression of human tau (HEK293/tau) or the vector (HEK293/vec) and treated the cells with thapsigargin (TG), an ER stress inducer. We observed that the HEK293/tau cells were more resistant than the HEK293/vec cells to the TG-induced apoptosis, importantly, a time dependent increase of tau phosphorylation at Thr205 and Thr231 sites was positively correlated with the inhibition of apoptosis. We also observed that expression of tau upregulated phosphorylation of PERK, eIF2 and IRE1 with an increased cleavage of ATF6 and ATF4. The potentiation of UPR was also detected in HEK293/tau cells treated with other ER stress inducers, including staurosporine, camptothecin and hydrogen peroxide, in which a suppressed apoptosis was also shown. Our data suggest that tau hyperphosphorylation could attenuate the ER stress-induced apoptosis with the mechanism involving upregulation of UPR system.

Phenylbutyric acid induces the cellular senescence through an Akt/p21WAF1 signaling pathway

It has been well known that three sentinel proteins – PERK, ATF6 and IRE1 – initiate the unfolded protein response (UPR) in the presence of misfolded or unfolded proteins in the ER. Recent studies have demonstrated that upregulation of UPR in cancer cells is required to survive and proliferate. Here, we showed that long exposure to 4-phenylbutyric acid (PBA), a chemical chaperone that can reduce retention of unfolded and misfolded proteins in ER, induced cellular senescence in cancer cells such as MCF7 and HT1080. In addition, we found that treatment with PBA activates Akt, which results in p21WAF1 induction. Interestingly, the depletion of PERK but not ATF6 and IRE1 also induces cellular senescence, which was rescued by additional depletion of Akt. This suggests that Akt pathway is downstream of PERK in PBA induced cellular senescence. Taken together, these results show that PBA induces cellular senescence via activation of the Akt/p21WAF1 pathway by PERK inhibition.

Oxidative stress‐triggered unfolded protein response is upstream of intrinsic cell death evoked by parkinsonian mimetics

Oxidative stress is a key player in a variety of neurodegenerative disorders including Parkinson's disease. Widely used as a parkinsonian mimetic, 6-hydroxydopamine (6-OHDA) generates reactive oxygen species (ROS) as well as coordinated changes in gene transcription associated with the unfolded protein response (UPR) and apoptosis. Whether 6-OHDA-induced UPR activation is dependent on ROS has not yet been determined. The present study used molecular indicators of oxidative stress to place 6-OHDA-generated ROS upstream of the appearance of UPR markers such as activating transcription factor 3 (ATF3) and phosphorylated stress-activated protein kinase (SAPK/JNK) signaling molecules. Antioxidants completely blocked 6-OHDA-mediated UPR activation and rescued cells from toxicity. Moreover, cytochrome c release from mitochondria was observed after the appearance of early UPR markers, suggesting that cellular stress pathways are responsible for its release. Mechanistically, the 6-OHDA-induced UPR was independent of intracellular calcium changes. Rather, evidence of protein oxidation was observed before the expression of UPR markers, suggesting that the rapid accumulation of damaged proteins triggered cell stress/UPR. Taken together, 6-OHDA-mediated cell death in dopaminergic cells proceeds via ROS-dependent UPR up-regulation which leads to an interaction with the intrinsic mitochondrial pathway and downstream caspase activation.