Modulation of ER Stress/Unfolded Protein Response (UPR) Pathways in Multiple Myeloma Cells by Inhibition of Hsp90 and Serine-Threonine Kinase CK2.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3840-3840
Author(s):  
Francesco Piazza ◽  
Sabrina Manni ◽  
Carmela Gurrieri ◽  
Anna Colpo ◽  
Laura Quotti Tubi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Er Stress ◽  
Unfolded Protein Response ◽  
Hsp90 Inhibitors ◽  
Hsp90 Inhibition ◽  
Protein Levels ◽  
Unfolded Protein ◽  
Protein Response ◽  
Kinase Ck2

Abstract Abstract 3840 Poster Board III-776 Hsp90 is an essential chaperone molecule that helps in the maturation and folding of a number of cellular client proteins. Hsp90 function is essential for malignant plasma cell survival, since its inhibition in multiple myeloma (MM) cells results in cell death and activation of apoptosis. Clinical trials using Hsp90 inhibitors are currently ongoing in MM patients. Hsp90 inactivation in MM cells causes perturbation of the endoplasmic reticulum (ER) stress/unfolded protein response (UPR), eventually triggering the apoptotic cascades. Protein kinase CK2 critically regulates the activity of the chaperone complex formed by the Cdc37 and Hsp90 proteins. We already described that CK2 is over-expressed in a fraction of MM patients and is an essential MM pro-survival molecule. We have here investigated its role in the ER stress/UPR pathways and in Hsp90 inhibition-induced apoptosis in MM cells. Down-regulation of the catalytic CK2 alpha subunit with selective chemical inhibitors or RNA interference resulted in significant modifications of the main UPR regulating signaling cascades: 1) a marked reduction of IRE1alpha protein levels; 2) a reduction of BiP/GRP78 and Hsp70 chaperone protein levels; 3) an increase of PERK activity and phospho eIF2alpha levels. When UPR was triggered by thapsigargin in CK2-inactivated cells, we observed that the IRE1alpha-dependent axis of the UPR was greatly impaired, as XBP-1short isoform generation and the levels of some induced chaperones were reduced. Interestingly, thapsigargin was able to induce CK2 kinase activity. Remarkably, treatment of CK2-silenced MM cells with Hsp90 inhibitors geldanamycin or its derivative 17-AAG (17-(demethoxy)-17-allylamino geldanamycin) resulted in 1) an even more pronounced reduction of IRE1 alpha protein levels; 2) a marked inhibition of GA or 17-AAG-triggered BiP/GRP78 protein level raise; 3) a more evident increase of eIF2 alpha phosphorylation. Of note, CK2 plus Hsp90 inhibition was followed by apoptotic cell death to a much greater extent than that obtained with the single inhibition of the two molecules. Noteworthy, these effects were also reproduced upon modelling the MM bone marrow (BM) microenvironment by co-culturing MM cells with BM stromal cells. These data suggest that CK2-mediated signaling regulates the ER stress/UPR pathways and modulates the threshold to apoptosis of ER stressed MM cells. CK2 interacts with Hsp90, since its inhibition synergizes with GA or 17-AAG treatments in terms of induction of apoptosis and shift of the ER stress/UPR pathways towards the terminal phase. These results might be useful to set the groundwork in designing novel combination treatments for MM patients. Disclosures: No relevant conflicts of interest to declare.

Hsp90 Inhibitors Deliver a Death Signal Via the Endoplasmic Reticulum Stress Pathway in Myeloma.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 848-848
Author(s):  
Emma L. Davenport ◽  
Hannah Moore ◽  
Alan Dunlop ◽  
Paul Workman ◽  
Gareth J. Morgan ◽  
...  
Keyword(s):  
Cell Death ◽  
Er Stress ◽  
Unfolded Protein Response ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Misfolded Protein ◽  
Hsp90 Inhibitors ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response

Abstract Immunoglobulin production by plasma cells both defines them functionally and also provides a differential target for the therapy of their malignant counterparts. Plasma cells producing high levels of paraprotein are dependent upon the unfolded protein response (UPR) and chaperone proteins to ensure correct protein folding and cell survival. We have evaluated a strategy aimed at manipulating the UPR response to deliver a novel death signal in myeloma. In order to study the apoptotic effects of the disruption of the UPR, a panel of myeloma cell lines were treated with the ER stress inducers (thapsigargin (TG) a Ca2+-ATPase inhibitor and tunicamycin (TM) an N-linked glycosylation inhibitor); the HSP90 inhibitors, 17-AAG and radicicol; and the proteasome inhibitor bortezomib, which disrupts misfolded protein disposal. The presence of misfolded proteins in the ER is detected by a complex of proteins embedded within the membrane comprised of BiP, PERK, IRE-1 and ATF6, each of which has distinct downstream effects that are mediated by their release from the complex and activation of the UPR. Treatment with TG and TM led to the activation of all three branches of the UPR as demonstrated by early splicing of XBP1 to XBP1s indicative of IRE1 activation, PERK activation as measured by transcriptional upregulation of CHOP (7-22 fold) and ATF6 splicing. 17-AAG was also capable of inducing splicing of XBP1 and the induction of CHOP (30 fold) whereas bortezomib resulted in induction of CHOP (25 fold) and minimal, late onset splicing of XBP1. Following treatment with all the drugs expression levels of BiP mRNA were upregulated (3-10 fold), however due to high basal levels of BiP protein we were unable to detect any further rises in this protein. The ER-resident HSP90 analogue, Grp94, underwent minimal transcriptional upregulation (2–3 fold) in response to HSP90 inhibitors but larger responses were noted following treatment with TM and TG (3–5 fold). HSP90 protein expression remained constant following exposure to all drugs. In contrast a time-dependent upregulation of the anti-apoptotic HSP70 was noted in response to 17-AAG, radicicol and bortezomib treatment; TG and TM failed to affect levels of HSP70. Levels of the transcript of EDEM1, an ER stress inducible membrane protein that accelerates the degradation of misfolded protein in the ER by strengthening the ERAD machinery, were induced by all drugs with an increase in the transcript levels of between 2 and 4 fold after 24 hours. All drugs were capable of inhibiting proliferation as demonstrated by MTT assay. In addition TG, bortezomib, 17AAG and radicicol also induced myeloma cell death as demonstrated by trypan blue and Annexin V/PI staining. A distinct pattern of activation of caspases in response to the drugs was also established. Bortezomib activated both the intrinsic (caspases 9 and 3) and the extrinsic (caspases 8 and 3) caspase pathways, whilst 17-AAG appears to mediate cell death via the intrinsic pathway alone. In contrast, TM and TG failed to activate either the extrinsic or intrinsic pathways within 24 hours and within this time frame appeared to induce cell death by a caspase-independent mechanism. In conclusion as well as inducing apoptosis via the intrinsic caspase death pathway, HSP90 inhibitors also induce myeloma cell death via ER stress and the UPR death pathway. Our results confirm that the unfolded protein response is an exciting new pathway that can be therapeutically targeted in myeloma.

scholarly journals A Novel Therapeutic Strategy for Preferential Elimination of Multiple Myeloma Cells By Targeting Protein Disulfide Isomerase

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 32-33
Author(s):  
Metis Hasipek ◽  
Dale Grabowski ◽  
Yihong Guan ◽  
Anand D. Tiwari ◽  
Xiaorong Gu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Endoplasmic Reticulum ◽  
Cell Death ◽  
Er Stress ◽  
Secreted Proteins ◽  
Unfolded Protein ◽  
Protein Response ◽  
Protein Disulfide

Multiple myeloma (MM) is a genetically complex hematological disease which is characterized by clonal proliferation of plasma cells in the bone marrow and secretion of monoclonal antibodies and cytokines that can damage bone, bone marrow, and kidney function1. MM cells constantly operate at the limit of their unfolded protein response (UPR) in the face of a secretory load of immunoglobin (Ig) and cytokines that is unparalleled by any other mammalian cell 2,3 and microenvironmental factors that aggravate the degree of physiologic misfolding that occurs during synthesis of secreted proteins. The endoplasmic reticulum (ER) resident protein disulfide isomerases (PDIs) are indispensable for folding of secreted proteins that require intramolecular disulfide-bond arrangement 4 like antibodies and many cytokines. As the main PDI family member, near-complete function of PDIA1 is essential for survival of MM cells while its inhibition should be manageable by the UPR in normal cells creating an opportunity for a large therapeutic window for PDI inhibitors in MM. Previously, we discovered and characterized an irreversible PDI inhibitor (CCF642) that induced cell death in MM cells at doses that did not affect survival of normal bone marrow cells. However, CCF642 has poor solubility and suboptimal selectivity precluding clinical translation. Using structure guided medicinal chemistry, we developed and characterized a highly potent and selective PDI inhibitor, with 10-fold higher potency (Fig 1B) and selectivity. CCF642-34 showed remarkable selectivity against PDIA1 and off-target bindings were eliminated when compared to CCF642 (Fig 1C). In addition to improved selectivity and in vitro PDI inhibition, CCF642-34 demonstrated more than 3-fold higher potency compared to CCF642 against MM1.S and bortezomib resistant MM1.S cells remained sensitive to CCF642-34. Importantly, the novel analogue CCF642-34 has 18-fold better potency in restricting the colony forming abilities of RPMI1640 cells while at no effect on the clonogenic potential of CD34+ cells derived from healthy bone marrow was observed at equivalent doses. CCF642-34 induces ER stress in MM1.S cells as observed in dose and time dependent cleavage of XBP1, IRE1α oligomerization and the profound induction of programmed cell death reflected by PARP and caspase 3 cleavage. To further analyze the modes of action of CCF642-34 and CCF642 we performed RNAseq after treatment of MM1.S cells and found exclusive induction of genes associated with UPR and downstream cell cycle and apoptotic responses for CCF642-34 while additional genes affecting were detected after CCF642 treatment. There were 362 and 568 differentially expressed genes in CCF642-34 and CCF-642 (compared to controls) treated MM1.S cells, respectively. Among these differentially expressed genes 87 down regulated and 142 upregulated were common to both, including downregulation of cell division and mitotic cell cycle process, and upregulation of response to ER stress, unfolded protein response, and apoptotic process gene sets. Results confirm that both CCF642 and CCF642-34 treatment act by inducing lethal ER-stress with greater selectivity for CCF642-34. Accordingly, hierarchical clustering showed distinct gene expression profiles in 642-34 and 642 treated MM1S cells (Fig. 2). CCF642-34 is orally bioavailable and highly efficacious in against established multiple myeloma in a syngeneic 5TGM1-luc/C57BL/KaLwRij model of myeloma. All vehicle control animals were dead by 52 days while 3 out of 6 mice lived beyond 6 months with no sign of relapse. In summary, we synthesized and characterized a novel lead PDIA1 inhibitor based on structure-guided medicinal chemistry that has improved pharmacologic properties to act as novel lead for clinical translation. References: 1. Manier S, Salem KZ, Park J, et al. Genomic complexity of multiple myeloma and its clinical implications. Nat. Rev. Clin. Oncol. 2017; 2. Fonseca R, Bergsagel PL, Drach J, et al. International Myeloma Working Group molecular classification of multiple myeloma: Spotlight review. Leukemia. 2009; 3. Wang M, Kaufman RJ. The impact of the endoplasmic reticulum protein-folding environment on cancer development. Nat. Rev. Cancer. 2014; 4. Freedman RB, Hirst TR, Tuite MF. Protein disulphide isomerase: building bridges in protein folding. Trends Biochem. Sci. 1994; Disclosures Valent: Takeda Pharmaceuticals: Other: Teaching, Speakers Bureau; Celgene: Other: Teaching, Speakers Bureau; Amgen Inc.: Other: Teaching, Speakers Bureau.

scholarly journals ATR-101, a Selective and Potent Inhibitor of Acyl-CoA Acyltransferase 1, Induces Apoptosis in H295R Adrenocortical Cells and in the Adrenal Cortex of Dogs

Endocrinology ◽  
2016 ◽  
Vol 157 (5) ◽  
pp. 1775-1788 ◽  
Author(s):  
Christopher R. LaPensee ◽  
Jacqueline E. Mann ◽  
William E. Rainey ◽  
Valentina Crudo ◽  
Stephen W. Hunt ◽  
...  
Keyword(s):  
Cell Death ◽  
Er Stress ◽  
Unfolded Protein Response ◽  
Adrenal Cortex ◽  
Caspase 3 ◽  
Potent Inhibitor ◽  
Adrenocortical Cell ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response

Abstract ATR-101 is a novel, oral drug candidate currently in development for the treatment of adrenocortical cancer. ATR-101 is a selective and potent inhibitor of acyl-coenzyme A:cholesterol O-acyltransferase 1 (ACAT1), an enzyme located in the endoplasmic reticulum (ER) membrane that catalyzes esterification of intracellular free cholesterol (FC). We aimed to identify mechanisms by which ATR-101 induces adrenocortical cell death. In H295R human adrenocortical carcinoma cells, ATR-101 decreases the formation of cholesteryl esters and increases FC levels, demonstrating potent inhibition of ACAT1 activity. Caspase-3/7 levels and terminal deoxynucleotidyl transferase 2′-deoxyuridine 5′-triphosphate nick end labeled-positive cells are increased by ATR-101 treatment, indicating activation of apoptosis. Exogenous cholesterol markedly potentiates the activity of ATR-101, suggesting that excess FC that cannot be adequately esterified increases caspase-3/7 activation and subsequent cell death. Inhibition of calcium release from the ER or the subsequent uptake of calcium by mitochondria reverses apoptosis induced by ATR-101. ATR-101 also activates multiple components of the unfolded protein response, an indicator of ER stress. Targeted knockdown of ACAT1 in an adrenocortical cell line mimicked the effects of ATR-101, suggesting that ACAT1 mediates the cytotoxic effects of ATR-101. Finally, in vivo treatment of dogs with ATR-101 decreased adrenocortical steroid production and induced cellular apoptosis that was restricted to the adrenal cortex. Together, these studies demonstrate that inhibition of ACAT1 by ATR-101 increases FC, resulting in dysregulation of ER calcium stores that result in ER stress, the unfolded protein response, and ultimately apoptosis.

scholarly journals Translational and posttranslational regulation of XIAP by eIF2α and ATF4 promotes ER stress–induced cell death during the unfolded protein response

2014 ◽  
Vol 25 (9) ◽  
pp. 1411-1420 ◽  
Author(s):  
Nobuhiko Hiramatsu ◽  
Carissa Messah ◽  
Jaeseok Han ◽  
Matthew M. LaVail ◽  
Randal J. Kaufman ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Death ◽  
Er Stress ◽  
Unfolded Protein Response ◽  
Protein Misfolding ◽  
Down Regulation ◽  
Unfolded Protein ◽  
Activity Loss ◽  
The Unfolded Protein Response ◽  
Protein Response

Endoplasmic reticulum (ER) protein misfolding activates the unfolded protein response (UPR) to help cells cope with ER stress. If ER homeostasis is not restored, UPR promotes cell death. The mechanisms of UPR-mediated cell death are poorly understood. The PKR-like endoplasmic reticulum kinase (PERK) arm of the UPR is implicated in ER stress–induced cell death, in part through up-regulation of proapoptotic CCAAT/enhancer binding protein homologous protein (CHOP). Chop−/− cells are partially resistant to ER stress–induced cell death, and CHOP overexpression alone does not induce cell death. These findings suggest that additional mechanisms regulate cell death downstream of PERK. Here we find dramatic suppression of antiapoptosis XIAP proteins in response to chronic ER stress. We find that PERK down-regulates XIAP synthesis through eIF2α and promotes XIAP degradation through ATF4. Of interest, PERK's down-regulation of XIAP occurs independently of CHOP activity. Loss of XIAP leads to increased cell death, whereas XIAP overexpression significantly enhances resistance to ER stress–induced cell death, even in the absence of CHOP. Our findings define a novel signaling circuit between PERK and XIAP that operates in parallel with PERK to CHOP induction to influence cell survival during ER stress. We propose a “two-hit” model of ER stress–induced cell death involving concomitant CHOP up-regulation and XIAP down-regulation both induced by PERK.

scholarly journals DJ-1 modulates the unfolded protein response and cell death via upregulation of ATF4 following ER stress

2019 ◽  
Vol 10 (2) ◽  
Author(s):  
Jungwoo Yang ◽  
Kwang Soo Kim ◽  
Grace O. Iyirhiaro ◽  
Paul C. Marcogliese ◽  
Steve M. Callaghan ◽  
...  
Keyword(s):  
Cell Death ◽  
Er Stress ◽  
Unfolded Protein Response ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response

scholarly journals Downregulation of miR-17-92 cluster by PERK fine-tunes unfolded protein response mediated apoptosis

2020 ◽  
Author(s):  
Danielle E. Read ◽  
Ananya Gupta ◽  
Karen Cawley ◽  
Laura Fontana ◽  
Patrizia Agostinis ◽  
...  
Keyword(s):  
Cell Death ◽  
Er Stress ◽  
Unfolded Protein Response ◽  
Molecular Mechanisms ◽  
Ectopic Expression ◽  
Proximal Region ◽  
Dependent Manner ◽  
Unfolded Protein ◽  
Promoter Deletion Analysis ◽  
Protein Response

AbstractAn important event in the unfolded protein response (UPR) is the activation of the endoplasmic reticulum kinase PERK (EIF2AK3). The PERK signalling branch first mediates a prosurvival response, which switches into a proapoptotic response upon prolonged ER stress. However, the molecular mechanisms of PERK-mediated cell death are not well understood. Here we show that expression of the primary miR-17-92 transcript and mature miRNAs belonging to miR-17-92 cluster is decreased during UPR. We found that activity of miR-17-92 promoter reporter was reduced during UPR in a PERK-dependent manner. We show that activity of miR-17-92 promoter is repressed by ectopic expression of ATF4 and NRF2. The promoter deletion analysis and ChIP assays mapped the region responding to UPR-mediated repression to site in the proximal region of the miR-17-92 promoter. Hypericin-mediated photo-oxidative ER damage reduced the expression of miRNAs belonging to miR-17-92 cluster in wild-type but not in PERK-deficient cells. Importantly, ER stress-induced apoptosis was inhibited upon miR-17-92 overexpression in SH-SY5Y and H9c2 cells. Our results reveal a novel function for NRF2, where repression of miR-17-92 cluster by NRF2 plays an important role in ER stress-mediated apoptosis. The data presented here provides mechanistic details how sustained PERK signalling via NRF2 mediated repression of miR-17-92 cluster can potentiate cell death.

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