scholarly journals Identification of Toyocamycin, an agent cytotoxic for multiple myeloma cells, as a potent inhibitor of ER stress-induced XBP1 mRNA splicing

2012 ◽  
Vol 2 (7) ◽  
pp. e79-e79 ◽  
Author(s):  
M Ri ◽  
E Tashiro ◽  
D Oikawa ◽  
S Shinjo ◽  
M Tokuda ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Er Stress ◽  
Potent Inhibitor ◽  
Mrna Splicing ◽  
Myeloma Cells ◽  
Xbp1 Mrna

scholarly journals SGK Kinase Activity in Multiple Myeloma Cells Protects against ER Stress Apoptosis via a SEK-Dependent Mechanism

2016 ◽  
Vol 14 (4) ◽  
pp. 397-407 ◽  
Author(s):  
Bao Hoang ◽  
Yijiang Shi ◽  
Patrick J. Frost ◽  
Veena Mysore ◽  
Carolyne Bardeleben ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Er Stress ◽  
Kinase Activity ◽  
Myeloma Cells ◽  
Dependent Mechanism

scholarly journals Targeting the integrated networks of aggresome formation, proteasome, and autophagy potentiates ER stress-mediated cell death in multiple myeloma cells

2014 ◽  
Vol 46 (2) ◽  
pp. 474-486 ◽  
Author(s):  
SHOTA MORIYA ◽  
SEIICHIRO KOMATSU ◽  
KAHO YAMASAKI ◽  
YUSUKE KAWAI ◽  
HIROKO KOKUBA ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Er Stress ◽  
Integrated Networks ◽  
Myeloma Cells ◽  
Aggresome Formation

scholarly journals Therapeutic potential of targeting IRES-dependent c-myc translation in multiple myeloma cells during ER stress

Oncogene ◽  
2015 ◽  
Vol 35 (8) ◽  
pp. 1015-1024 ◽  
Author(s):  
Y Shi ◽  
Y Yang ◽  
B Hoang ◽  
C Bardeleben ◽  
B Holmes ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Er Stress ◽  
Therapeutic Potential ◽  
Myeloma Cells

scholarly journals A Molecular Mechanism for Turning off IRE1α Signaling During Endoplasmic Reticulum Stress

2020 ◽  
Author(s):  
Xia Li ◽  
Sha Sun ◽  
Suhila Appathurai ◽  
Arunkumar Sundaram ◽  
Rachel Plumb ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Mammalian Cells ◽  
Mrna Splicing ◽  
Xbp1 Mrna ◽  
Long Time ◽  
A Subunit ◽  
Active Transcription ◽  
J Domain ◽  
Sustained Activation

SummaryMisfolded proteins in the endoplasmic reticulum (ER) activate IRE1α endoribonuclease in mammalian cells, which mediates XBP1 mRNA splicing to produce an active transcription factor. This promotes the expression of specific genes to alleviate ER stress and thereby attenuating IRE1α. Although sustained activation of IRE1α is linked to human diseases, it is not clear how IRE1α is attenuated during ER stress. Here, we identify that Sec63 is a subunit of the previously identified IRE1α/Sec61 translocon complex. We find that Sec63 recruits and activates BiP ATPase through its luminal J-domain to bind onto IRE1α. This leads to inhibition of higher-order oligomerization and attenuation of IRE1α RNase activity during prolonged ER stress. In Sec63 deficient cells, IRE1α remains activated for a long time despite the presence of excess BiP in the ER. Thus, our data suggest that the Sec61 translocon bridges IRE1α with Sec63/BiP to regulate the dynamics of IRE1α signaling in cells.

Reolysin Combined with Carfilzomib for Treatment of Relapsed Multiple Myeloma Patients

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1835-1835 ◽  
Author(s):  
Douglas W Sborov ◽  
Flavia Pichiorri ◽  
Gerard J Nuovo ◽  
Don M Benson ◽  
Yvonne A. Efebera ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Er Stress ◽  
Dose Level ◽  
Combination Treatment ◽  
Objective Response ◽  
Low Grade ◽  
Bone Marrow Biopsies ◽  
Myeloma Cells ◽  
Monoclonal Protein

Abstract Introduction. The viral oncolytic agent, Reolysin (RV), is a promising novel therapeutic that selectively proliferates in myeloma cells. Our group conducted a phase 1 clinical trial of single agent RV in patients with relapsed and refractory multiple myeloma (MM), and reported that treatment was well tolerated and associated with prolonged disease stability in 25% of patients. Objective responses were not evident, likely because the viral RNA present in the myeloma cells was not producing infectious viral particles. Proteasome inhibitors can lead to myeloma cell death due to increased endoplasmic reticulum (ER) stress and induction of ER-stress related apoptosis (Kelly, Oncogene, 2012). We confirmed this effect preclinically with Carfilzomib (CFZ), and hypothesized that the addition of CFZ to RV would increase viral proliferation and MM cell death sufficiently to obtain objective response in patients with relapsed MM. Methods. For this pilot trial, patients were required to have relapsed myeloma with IMWG-defined measurable disease, ANC ≥ 1,000/uL, platelet count ≥ 50,000/uL, with no creatinine requirements. Cohorts of 6 patients each were planned. Cohort 1 included patients who were CFZ na•ve or had not progressed on a CFZ containing regimen. Intravenous CFZ (20 mg/m2 days 1 and 2 of cycle 1 and 27 mg/m2 thereafter), Reolysin (3 x 1010 TCID50/day), and dexamethasone (20 mg) were administered on days 1, 2, 8, 9, 15, and 16 of a 28-day cycle (Table 1). In situ based methodologies were used to examine the distribution of CD138, CD8, NK cells (CD117 and IL-22), CD 68, PD L1, reoviral capsid protein, and reoviral RNA in bone marrow biopsies performed prior to treatment on days 1 and 9 of cycle 1. Results. Seven patients have been enrolled, four are male, and all are Caucasian. Patients have a median age of 64, and have received on average 2.4 prior lines of therapy and 4.4 prior treatments. All patients were previously exposed to Revlimid and Velcade, and 4 patients were Velcade refractory. One patient was previously treated with CFZ but was deemed to be CFZ sensitive, one patient has dialysis-dependent CKD, and all but one patient had evidence of high-risk cytogenetics on CD138-selected FISH at the time of enrollment. 6/7 patients suffered myalgias and fever after the first two doses of Reolysin, but these symptoms did not recur in any subsequent doses. Treatment has been well tolerated in 5 patients, but 2 patients were removed from study after 2 doses of combination therapy, one for congestive heart failure, and the other for gastrointestinal bleed in the setting of grade 4 thrombocytopenia and an arteriovenous malformation. Due to these 2 DLTs, patient 7 was enrolled at dose level -1 (Carfilzomib 20 mg/m2 and Reolysin 3 x 109 TCID50/day on days 1, 2, 8, 9, 15, and 16 of a 28 day cycle). Within the first 14 days following the initiation of treatment, the mean decrease in platelets for the 7 evaluable patients was 79 (50 - 139), and this included grade 4 (N = 1), and asymptomatic grade 2 (N = 3), and grade 1 (N = 3) events. All patients have had a reduction of the monoclonal protein, 5 patients remain on study, and the longest duration of response is currently 8 cycles. Responses are VGPR (N = 2), PR (N = 3), MR (N = 1), and SD (N = 1) (Figure 1). Intracellular viral replication will be reported at the meeting. Conclusion. This 3-drug regimen is relatively well tolerated in heavily treated patients with relapsed MM. Most patients experience low grade fever and myalgias after the first two doses, and patients have evidence of thrombocytopenia in cycle 1. Combination treatment is associated with reduction of the monoclonal protein in all patients, and 86% (6/7) CFZ-sensitive patients have evidence of objective response. Table 1. Combination treatment dose levels Dose level Dexamethasone (IVP) Carfilzomib (IVPB) Reolysin (IVPB) -1 20 mg/day 20 mg/m2 /day 3 x 109 TCID50/day 1 (starting dose) 20 mg/day C1 Day 1 & 2 - 20 mg/m2 /dayC1 Day 8 & onward - 27 mg/m2 /day 3 x 1010 TCID50/day Figure 1. Waterfall plot representing response of 7 patients with relapsed MM Figure 1. Waterfall plot representing response of 7 patients with relapsed MM Disclosures Off Label Use: Reolysin - oncolytic viral, anti-cancer agent.

Identification of Toyocamycin, an Agent Cytotoxic for Multiple Myeloma Cells, As a Potent Inhibitor of ER Stress-Induced XBP1 mRNA Splicing

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 5034-5034
Author(s):  
Masaki Ri ◽  
Etsu Tashiro ◽  
Daisuke Oikawa ◽  
Satoko Shinjo ◽  
Mio Tokuda ◽  
...  
Keyword(s):  
Er Stress ◽  
Cell Lines ◽  
Inhibitory Effect ◽  
Mrna Splicing ◽  
Cellular Viability ◽  
Unfolded Protein ◽  
Xbp1 Mrna ◽  
Dose Dependent ◽  
Adenosine Analog

Abstract Abstract 5034 Introduction: The IRE1α-XBP1 pathway, a key component of the endoplasmic reticulum (ER) stress response, is considered to be a critical regulator for survival of multiple myeloma (MM) cells. Because of the production of abundant immunoglobulins and cytokines, MM cells need to survive under chronic ER stress. In addition, MM cells are located in the bone marrow milieu, which is usually considered hypoxic compared to other organs. Therefore, MM cells need to possess mechanisms to protect against ER stress. Among the unfolded protein responses in MM cells, the IRE1α-XBP1 pathway has been implicated in the proliferation and survival of MM cells to a greater extent than in those of monoclonal gammopathy of undetermined significance or normal plasma cells. It has been reported to be a prognostic factor and could be a target for immunotherapy or chemotherapy. Based on previous reports, it is proposed that an inhibitor of IRE1α-XBP1 activation should be a potent therapeutic agent for MM. Therefore, the availability of small molecule inhibitors targeting this pathway would offer a new therapeutic strategy for MM. Here, we screened small molecule inhibitors of ER stress-induced XBP1 activation, and identified toyocamycin from a culture broth of an Actinomycete strain. Materials & Methods: First, we evaluated the mechanism of toyocamycin-induced inhibition of IREα activity, with focused on its kinase activity, endonuclease activity, and other unfolded protein responses. Next, the activity of toyocamycin was evaluated on MM cell lines and other tumor cells about IRE1α activity and cytotoxicity. Similarly, 9 primary MM cells were tested. Finally, the in vivo efficacy of toyocamycin was evaluated in a human MM xenograft model. Results & Discussion: Toyocamycin was shown to suppress thapsigargin-, tunicamycin- and 2-deoxyglucose-induced XBP1 mRNA splicing in HeLa cells without affecting ATF6 and PERK activation. Furthermore, although toyocamycin was unable to inhibit IRE1 a phosphorylation, it prevented IRE1α-induced XBP1 mRNA cleavage in vitro. Thus, toyocamycin is an inhibitor of IRE1α-induced XBP1 mRNA cleavage. Next, we examined the effect of toyocamycin on MM cells. Most MM cell lines have activated XBP1 protein expression, represented as the overexpression of spliced XBP1 isoform, whereas non-MM cells including other hematological and solid tumor cells have little activation of XBP1. Toyocamycin inhibited constitutive activation of XBP1 in MM cell lines without affecting IRE1α phosphorylation. This inhibition occurred within 6 hours after exposure to 30 nM toyocamycin. We then evaluated the growth inhibitory effect of toyocamycin on 7 MM cell lines with high spliced-XBP1 expression, 3 MM cell lines with low spliced-XBP1 expression, and 4 non-MM cell lines as assessed by MTS assay. All MM cells with high spliced-XBP1 expression showed remarkable decline in cellular viability at 30 nM or higher concentrations of toyocamycin than other MM cells with low spliced-XBP1 expression, and non-MM cell lines showed little reduction in cellular viability. MM cell lines expressing high spliced-XBP1 showed robust dose-dependent apoptosis after exposure to various concentrations of toyocamycin for 24 hours, as assessed by the number of Annexin V-positive cells. Toyocamycin also induces marked apoptosis on two bortezomib (BTZ)-resistant MM cells at nM concentration. It also inhibited constitutive activation of XBP1 expression in primary MM cells derived from patients, showing dose-dependent reduced viability without any cytotoxicity to PBMCs from healthy donors. Toyocamycin also showed synergistic effects with bortezomib, and induced apoptosis of primary MM cells from patients including bortezomib-resistant cases at nano-molar levels in a dose-dependent manner. It also inhibited growth of xenografts in an in vivo model of human MM, and showed enhanced growth inhibition when combined with bortezomib. Taken together, we found that adenosine analog toyocamycin has a potent IRE1α-XBP1 inhibitory effect on MM cells with excessive ER-stress. It triggers dose-dependent apoptosis in MM cells. These results suggest toyocamycin can be a lead compound for developing novel anti-MM therapy, and also provide a preclinical rationale for conducting clinical trials using toyocamycin or other adenosine analog alone or in combination with BTZ for treating MM. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Crispr Sgrnas Genome-Wide Screen Identifies the Proteasome Regulatory Subunit PSMC6 As a Bortezomib Resistance Gene in Human Multiple Myeloma Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 450-450 ◽  
Author(s):  
Chang-Xin Shi ◽  
Klaus M Kortum ◽  
Yuan Xiao Zhu ◽  
Patrick Jedlowski ◽  
Esteban Braggio ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Next Generation Sequencing ◽  
Er Stress ◽  
Cell Line ◽  
Cell Lines ◽  
Myeloma Cells ◽  
Human Multiple Myeloma ◽  
Rpmi 8226 ◽  
Generation Sequencing ◽  
Specific Inhibitors

Abstract Background: The proteasome inhibitor Bortezomib (BTZ) is an efficient treatment option for both, newly diagnosed and relapsed/refractory multiple myeloma (MM). Despite the effectiveness, most patients eventually acquire drug resistance for reasons not fully understood Materials and methods: To better understand BTZ resistance mechanism we used a CRISPR library (GeCKO V2) targeting 19052 human genes, trying to identify genes responsible for BTZ resistance. CRISPR sgRNAs targeting the ERN1-XBP1 pathway were used as positive controls. We first infected RPMI 8226 myeloma cell line expressing Cas9 with the CRISPR library packaged into lenti-vectors and selected for resistance to BTZ. Surviving cells were subjected to next generation sequencing. Based on the initial screen results we constructed a second CRISPR sgRNA library including 31 genes, each gene targeted with four sgRNAs. After the second round screening and subsequent sequencing, we selected the top 20 genes for individual validation. Results: Proteasome regulatory gene PSMC6 was identified as the only reproducible gene conferring BTZ resistance. Interestingly, the same gene was independently found by a second group using a CRISPR approach (Sheffer M et al. ASH Abstract 273, 2014). Resistance was reproducible using a PSMC6 knockout by three individual CRISPR sgRNAs targeting exonic regions and one pair of SgRNA targeting intron region flanking exon for the deletion of exon one. PSMC6 knockout was verified by PCR and Sanger sequencing. Sensitivity to BTZ was rescued by over expression of PSMC6 cDNA in RPMI 8226 cells harboring a deletion of PSMC6 exon 1. MM cells lacking PSMC6 also developed resistance against Carfilzomib. Resistance was reproduced on a second MM cell line, KMS11. We did not see any significant difference of toxicity in PSMC6 deleted cells for other chemicals tested (tunicamycin, staurosporine, dexamethasone and melphalan). We demonstrated that the sensitivity of chymotrypsin-like activity of proteasome against BTZ was significantly reduced in cells lacking PSMC6. Consequently, MM cells without the PSMC6 gene were relatively resistant to apoptosis induced by BTZ, which was verified by Western blot for caspase 8 degradation and luminescent assay for caspase 3/7 activities. Clinically we could not correlate the PSMC6 expression level with the outcome of BTZ treatment in BTZ naive patients using publically available gene expression data. We initially used CRISPR sgRNAs targeting ER stress pathway (ERN1 and XBP1) as a positive control. However we could not derive any resistant cells from the experiment. We also could not identify any sgRNAs targeting the ERN1-XBP1 pathway from our whole exome screen and next generation sequencing. Since this contradicts published data, we decided to knockout ERN1 and XBP1 genes individually. Clones of cells with successful knockout of ERN1 in three cell lines (RPMI 8226, KMS11 and JJN3) and XBP1 in two cell lines (RPMI 8226 and KMS11) were tested for response to BTZ and Carfilzomib , however we did not find any drug response difference between the knockout and parent cells. We also found that the ERN1-XBP1 knockout cells did not show difference in response to ERN1 specific inhibitors (4u8C and STF-038010) and ER stress inducers (tunicamycin and thapsigargin) compared to parental cells. Conclusions: Human multiple myeloma cells lacking the PSMC6 gene develop significant resistance to apoptosis induced by BTZ. We have however not found a correlation of PSMC6 expression levels with outcome to BTZ treatment in BTZ naïve patients. We are therefore currently investigating the PSMC6 mutation rate in relapsed MM patients after proteasome treatment. In contrast to previous reports showing that progenitor MM cells lacing XBP-1 or ERN-1 invoked BTZ resistance, we were not able to demonstrate a change in sensitivity after full CRISPR knock out of either ERN1 or XBP1. It has long been believed that ERN1-XBP1 pathway plays an important role for MM treatment, leading to the development of ERN1 specific inhibitors. However, we demonstrated that the toxicity of two ERN1-specific inhibitors appears independent of the ERN1-XBP1 pathway. We also demonstrated that the toxicity of two important ER stress inducers, tunicamycin and thapsigargin, is independent of the ERN1-XBP1 pathway. Disclosures Stewart: Celgene: Consultancy; Oncospire Inc.: Equity Ownership; BMS: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy.

scholarly journals Stress-induced tyrosine phosphorylation of RtcB modulates IRE1 activity and signaling outputs

2020 ◽  
Author(s):  
Alexandra Papaioannou ◽  
Alice Metais ◽  
Marion Maurel ◽  
Luc Negroni ◽  
Matías González-Quiroz ◽  
...  
Keyword(s):  
Er Stress ◽  
Tyrosine Kinases ◽  
Regulatory Network ◽  
Initial Step ◽  
Mrna Splicing ◽  
Rnase Activity ◽  
Unfolded Protein ◽  
Xbp1 Mrna ◽  
The Unfolded Protein Response ◽  
Protein Response

AbstractEndoplasmic Reticulum (ER) stress is a hallmark of various diseases. Cells cope with ER stress through the activation of an adaptive signaling pathway named the Unfolded Protein Response (UPR) which is mediated by three ER-resident sensors. The most evolutionary conserved of the UPR sensors is IRE1α that elicits diverse downstream effects through its kinase and endoribonuclease (RNase) activities. IRE1α RNase activity can either catalyze the initial step of XBP1 mRNA unconventional splicing or degrade a number of RNAs through Regulated IRE1-Dependent Decay (RIDD). The degree of exertion of these two activities plays an instrumental role in cells’ life and death decisions upon ER stress. Until now, the biochemical and biological outputs of IRE1α RNase activity have been well documented, however, the precise mechanisms controlling whether IRE1 signaling is adaptive or pro-death (terminal) remain unclear. This prompted us to further investigate those mechanisms and we hypothesized that XBP1 mRNA splicing and RIDD activity could be co-regulated within the context of the IRE1α RNase regulatory network. We showed that a key nexus in this pathway is the tRNA ligase RtcB which, together with IRE1α, is responsible for XBP1 mRNA splicing. We demonstrated that RtcB is tyrosine phosphorylated by c-Abl and dephosphorylated by PTP1B. Moreover, we identified RtcB Y306 as a key residue which, when phosphorylated, perturbs RtcB interaction with IRE1α, thereby attenuating XBP1 mRNA splicing and favoring RIDD. Our results demonstrate that the IRE1α RNase regulatory network is dynamically fine-tuned by tyrosine kinases and phosphatases upon various stresses and depending on the nature of the stress determines cell adaptive or death outputs.

Abstract B150: Toyocamycin inhibits ER stress‐induced XBP1 mRNA splicing through IRE1α activation

2009 ◽  
Author(s):  
Etsu Tashiro ◽  
Yumi Yokouchi ◽  
Kohta Yamamoto ◽  
Masaya Imoto
Keyword(s):  
Er Stress ◽  
Mrna Splicing ◽  
Xbp1 Mrna
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