scholarly journals Overcoming Resistance to Apoptosis in Multiple Myeloma By Simultaneous Inhibition of Bcl2 and IAP Families of Anti-Apoptotic Proteins

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2088-2088
Author(s):  
Marcus Gomez ◽  
Vijay G. Ramakrishnan ◽  
Vivek Prasad ◽  
Teresa K. Kimlinger ◽  
Utkarsh Painuly ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Death ◽  
Er Stress ◽  
Cell Lines ◽  
Bcl2 Family ◽  
Er Stress Response ◽  
Simultaneous Inhibition ◽  
Apoptotic Proteins ◽  
Upr Pathway

Abstract Background: Multiple myeloma (MM) cells evade apoptosis through multiple mechanisms thus enabling it to evade therapy. The Bcl2 family of anti-apoptotic proteins is aberrantly expressed in MM cell lines and patient cells. Yet, pharmacological intervention of this family appears to have significant activity only in molecular subgroups of MM patients. This clearly suggests alternate mechanisms of overcoming apoptotic signals in MM cells in addition to the Bcl2 family, through proteins such as IAPs. We have previously shown that simultaneous inhibition of the three major IAP proteins, namely cIAP1, cIAP2 and XIAP is required to induce pronounced apoptosis in MM cells. However, IAP inhibition results in apoptosis in only some MM cell lines and patient cells. Given that levels of Bcl2 family proteins are unaffected by IAP inhibition, we hypothesized that combined inhibition of the IAP proteins using a SMAC mimetic LCL161 and the Bcl2 family proteins using a pan-Bcl2 inhibitor obatoclax (OBX) will lead to more pronounced and synergistic cell death in a broader subgroup of MM patients. Methods: LCL161 was synthesized by Novartis Inc. (Basel, Switzerland). OBX was purchased from Selleckchem (Houston, USA). Stock solutions were made in DMSO, and subsequently diluted in RPMI-1640 medium for use. MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum (20% serum for primary patient cells) supplemented with L-Glutamine, penicillin, and streptomycin. Cytotoxicity was measured using the MTT viability assay and proliferation using thymidine uptake. Apoptosis was measured using flow cytometry upon cell staining with Annexin V-FITC and propidium iodide (PI) for cell lines and patient cells. Immunoblotting was done on cell extracts at various time points following incubation with the drugs in order to study the cell signaling pathways and a Results: LCL161/OBX combination induced synergistic cytotoxicity and anti-proliferative effects on a broad range of human MM cell lines, including drug resistant cell lines like DOX40 and MM1R. Components of the bone marrow microenvironment including bone marrow stromal cells and tumor promoting cytokines (VEGF, IGF and IL6) were unable to protect MM cells from the effects of the drug combination. We saw a time dependent increase in apoptosis, with the combination inducing significantly more apoptosis than either of the single agents alone. Examining the mechanism of action of the drug combination showed clear inhibition of the IAP proteins, activation of caspases 9, 8, 3 and Bid by LCL161 and the combination and up regulation of the pro-apoptotic proteins Bim, Bid, Puma and Noxa and accumulation of LC3-II by OBX and the combination. Using chloroquine along with the OBX, we were able to demonstrate that OBX induced protective autophagy and the addition of LCL161 was able to overcome this protective effect induced after single agent OBX treatment. Since protective autophagy can be induced by the ER stress response, we then examined the expression levels of proteins involved in this pathway. We observed clear induction of ER stress mediated UPR pathway by both the drugs. However, LCL161 and OBX induced different branches of the UPR pathway. OBX activated the ATF6 and pErk/peif2α/ATF4 branches of the UPR, both of which have been implicated in cell survival during ER stress. ATF4 under irrecoverable ER stress can lead to increase in transcription of CHOP and cause apoptosis. We therefore examined levels of CHOP and observed no induction of CHOP post treatment with either of the drugs or the combination. LCL161, however differentially modulated the IRE1 branch of the UPR by down regulating Xbp-1 splicing, which is a pro survival activity of IREI and up regulating pJNK, which indicated a pro-apoptotic activity induced by IRE1 post irrecoverable ER stress This indicated that the ER stress induced apoptosis is triggered by LCL161, which might be important to overcome the ER induced protective effects induced by OBX. Conclusion: Taken together, our studies indicate that LCL161/OBX combination induces synergistic cell death through modulation of apoptosis, authophagy and the ER stress response. Disclosures No relevant conflicts of interest to declare.

Targeting Proteinkinase C Alters ER-Stress and b-Catenin Signaling in Multiple Myeloma: Therapeutic Implications.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 258-258
Author(s):  
Marc S. Raab ◽  
Klaus Podar ◽  
Jing Zhang ◽  
Giovanni Tonon ◽  
Johannes H. Fruehauf ◽  
...  
Keyword(s):  
Cell Death ◽  
Stress Response ◽  
Growth Inhibition ◽  
Er Stress ◽  
Cell Lines ◽  
Wnt Pathway ◽  
Dependent Manner ◽  
P53 Family ◽  
Er Stress Response

Abstract We have previously shown that the novel orally available small molecule inhibitor of PKC enzastaurin (Eli Lilly and Company) inhibits MM cell growth, survival and angiogenesis both in vitro and in vivo. To date, however, the downstream effects contributing to growth inhibition and cell death remain to be determined. Here, we performed global gene expression profiling on enzastaurin treated MM cells and identified 200 Genes to be differentially regulated with a > 2-fold cut off. Strikingly, two major groups of up-regulated probe sets were associated with either of two pathways - endoplasmatic reticulum (ER)-stress response or WNT-signaling. Importantly, MM cells, producing high levels of paraprotein, are highly susceptible to perturbation of ER function and protein folding. Moreover, PKC isoforms have been reported to directly regulate the canonical WNT pathway via phosphorylation of b-catenin (CAT), leading to its ubiquination and proteasomal degradation. Specifically, we fist evaluated the role of enzastaurin in mediating ER-stress in MM cells. The transcriptional up-regulation of genes involved in ER-stress (GADD153/CHOP, GADD34, ATF3), triggered by enzastaurin at 3h, was confirmed by western blot analysis, accompanied by induction of the molecular ER chaperone BiP/grp78, phosphorylation of eIF2a consistent with PERK activation, and up-regulation of p21. These events were preceded by an early (1h) increase of intracellular calcium levels, a hallmark of ER-stress, assessed by FLUO4 staining. These data suggest an important role of ER-stress response in the early growth inhibition of MM cells caused by enzastaurin. Second, we delineated effects of enzastaurin on WNT pathway in MM and other tumor cell lines. Upon enzastaurin treatment, CAT was dephosphorylated at Ser33, 37, 41 in a dose- and time-dependent manner in all cell lines tested (10 MM, 3 colon cancer, HeLa, as well as human embryonic kidney 293 cells). Consequently, accumulation of CAT occurred in both cytosolic and nuclear fractions of treated MM cells, associated with activated TOPflash LUC-reporter system, confirming nuclear transactivating activity. Specific inhibition of CAT by siRNA partially rescued HeLa, HEK 293, and MM cells from cell death induced by enzastaurin. Analysis of downstream target molecules revealed a CAT-dependent up-regulation of c-Jun, but not of c-Myc or Cyclin D1. c-Jun has been reported to stabilize p73, a pro-apoptotic p53-family member; CAT induction by enzastaurin led to p73 (but not p53) activation and was also abrogated by CAT-specific siRNA. In turn, specific knockdown of p73 by siRNA rescued cells from enzastaurin-induced apoptosis. Finally, ectopic overexpression of CAT in HeLa and MM cells induced c-Jun expression and p73 activation, followed by apoptotic cell death. Our studies therefore indicate that ER-stress response contributes to the immediate inhibition of proliferation by enzastaurin, followed by CAT accumulation leading to p73 activation, contributing to enzastaurin-mediated cell death. These findings provide a novel link between CAT and p53-family members. Moreover p73, which is only rarely mutated in human cancers, represents a novel therapeutic target in MM.

Nocodazole Induces Myeloma Cell Death by Disrupting the Microtubular Network, Resulting in G2/M Arrest

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3673-3673
Author(s):  
Rentian Feng ◽  
Jorge A Rios ◽  
Markus Mapara ◽  
Suzanne Lentzsch
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Death ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Caspase 8 ◽  
Inhibition Rate ◽  
Growth And Survival ◽  
Microtubular Network

Abstract Patients with relapsed multiple myeloma (MM) previously treated with bortezomib and lenalidomide often fail to respond to further therapies. To identify potential new treatment approaches for MM, we used Luminex technology to screen a library of 1,120 compounds provided by the Multiple Myeloma Research Foundation. By multiplex cytokine array, we identified benzimidazoles including the anthelmintics mebendazole, fenbendazole, albendazole, nocodazole and pyrvinium pamoate, as inhibiting the production of cytokines essential for MM cell growth and survival, such as IL-6 (inhibition rate 40–70%), MIP-1α (inhibition rate 65–75%), VEGF (inhibition rate 75%), and soluble IL-6R (inhibition rate 40–52%). Consequently, these anthelmintics demonstrated dose-dependent inhibition of myeloma cell (RPMI-8226, H929, U266 and MM1S) proliferation. The lead compound, nocodazole, caused nuclear fragmentation and caspase-8 activation in MM cell lines and primary CD138+ cells in dose- and time-dependent fashion (IC50: 30–60 nM). Importantly, growth and survival signals provided by bone marrow stromal cells in bone marrow co-cultures failed to protect MM cells from nocodazole-induced cell death. In the apoptotic cells, caspase-8 was more activated than caspase-9, suggesting that mitochondrial signaling is not a major apoptotic pathway. Cell cycle analysis indicated that G2/M cell cycle arrest reached a peak at 17 hr. Sub-G1 proportion was strongly increased after treatment for 24 hr in all tested cell lines. Electron microscope (EM) and nuclear staining studies consistently showed the accumulation of metaphase cells, and morphologic elongation at 7 hr, at which time G2/M arrest was obvious. Most of the elongated cells had only one nucleus, suggesting that they failed to progress to mitosis due to overall microtubular network disarray. We conclude that nocodazole exposure induced microtubular network disarray with cell elongation, and G2/M arrest with a late stage mitotic block resulting in cell death. Benzimidazoles including nocodazole, traditionally used as antihelmintic drugs, have shown antitumor activity against hepatocellular, lung and adrenocortical carcinoma, and melanoma. In our study, we identified the anthelmintic compound nocodazole as a new anti-myeloma agent. Nocodazole warrants further investigation for its anti-MM effects in vitro and in vivo.

scholarly journals PIM Kinase Inhibition Decreases the Proangiogenic Properties of Multiple Myeloma Cells and Affects the Metabolic State of the Vascular Endothelium

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 16-17
Author(s):  
Filip Garbicz ◽  
Anna Szumera-Ciećkiewicz ◽  
Joanna Barankiewicz ◽  
Dorota Komar ◽  
Michał Pawlak ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Endothelial Cells ◽  
Cell Death ◽  
Tumor Cell ◽  
Cell Lines ◽  
Fold Increase ◽  
Pim Kinases ◽  
Rpmi 8226

The development and progression of multiple myeloma (MM) depend on the formation and perpetual evolution of an immunosuppressive and hypervascular bone marrow microenvironment. MM undergoes an angiogenic switch during its early progression stages and initiates the secretion of proangiogenic proteins, such as VEGFA and Galectin-1. Following their engagement with the VEGF receptor 2 on the surface of the endothelium, quiescent endothelial cells (ECs) rapidly switch to an activated state, thus gaining the ability to create sprouts, migrate and proliferate. However, chronic angiogenic stimulation results in the formation of a dense and leaky network of pathological vessels, which in the case of MM also serves as a major source of prosurvival paracrine signals. Since PIM kinases are known modulators of cytokine signaling, owing to their ability to activate NFκB, JAK/STAT and mTOR pathways, we analyzed the expression pattern of PIM1, PIM2 and PIM3 in multiple myeloma bone marrow samples using immunohistochemistry. We found that both MM cells as well as myeloma-associated ECs exhibit a significantly higher PIM3 expression than their normal bone marrow counterparts. Since the role of PIM kinases in the vascular compartment of the tumor microenvironment is currently unknown, we decided to explore the proangiogenic functions of PIM kinases using in vitro MM and EC model cell lines. 3 MM cell lines (RPMI 8226, MM1.s, U266), immortalized bone marrow ECs (HBMEC-60) and human umbilical vein ECs (HUVECs) were used for the experiments. Primary MM cells were obtained from MACS-separated bone marrow aspirates. Chemical blockade of PIM kinase activity was achieved using the pan-PIM inhibitor SEL24/MEN1703. The compound decreased the viability of MM cell lines with IC50 in the submicromolar range, induced G2 cell cycle arrest and apoptosis. Moreover, SEL24/MEN1703 induced apoptosis in primary MM cells, even when cocultured with the CD138- bone marrow fraction. PIM inhibitor treatment inhibited the phosphorylation of mTOR substrates S6 and 4EBP1, STAT3/5, as well as RelA/p65. Consequently, we observed markedly decreased VEGFA and Gal-1 levels in SEL24/MEN1703-treated MM cells. When cultured together, separated by a permeable transwell membrane, both RPMI 8226 cells, as well as ECs, exhibited a 2-fold increase in proliferation rate. This effect was completely blocked by a 2-day treatment with a PIM inhibitor. Exposure of ECs to recombinant VEGFA (10ng/ul) or MM supernatant resulted in an increase in VEGFR2 Y1175 phosphorylation level and induction of PIM3 expression. Increased MYC activity is a hallmark of VEGF-dependent endothelial activation and is necessary to support the creation of new vessels. Since the PIM3 promoter region contains putative MYC-binding sites (E-boxes), we checked if PIM3 induction depends on MYC in ECs. MYC silencing using siRNA resulted in an 88% lower PIM3 expression than the non-targeting siRNA. One of MYC's main tasks during angiogenesis is the stimulation of cellular ATP synthesis to meet the energy demands created by the dynamic remodeling of the actin cytoskeleton. Surprisingly, PIM inhibition decreased the total ATP content in ECs by 25%, thus disrupting the energetic homeostasis, as evidenced by a 9.6-fold increase in phosphorylated AMPK T172 levels. Furthermore, SEL24/MEN1703-treated ECs were depleted of higher-order actin structures necessary for efficient angiogenesis, such as actin stress fibers, membrane ruffles and lamellipodia. In consequence, PIM kinase inhibition decreased proliferation, migration and formation of new vessel-like structures in Matrigel by ECs. Collectively, our data demonstrate that PIM inhibition induces MM cell death and abolishes important tumor cell-ECs interactions. In addition, we show that PIM3 is overexpressed in MM tumor endothelial cells and PIM inhibition disrupts the activation state in in vitro cultured ECs. Hence, targeting PIM kinases may represent an efficient approach to induce tumor cell death and to block angiogenesis in MM. RNA-sequencing studies on the downstream effectors of PIM3 are currently ongoing in order to unravel the molecular mechanism behind the observed effects. Figure Disclosures Brzózka: Ryvu Therapeutics: Current Employment. Rzymski:Ryvu Therapeutics: Current Employment. Tomirotti:Menarini Ricerche: Current Employment. Lech-Marańda:Roche, Novartis, Takeda, Janssen-Cilag, Amgen, Gilead, AbbVie, Sanofi: Consultancy; Roche, Amgen, Gilead: Speakers Bureau. Juszczynski:Ryvu Therapeutics: Other: member of advisory board.

scholarly journals A Role for Syntenin-1 in Multiple Myeloma Cell Survival

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1008-1008
Author(s):  
Tyler Moser-Katz ◽  
Catherine M. Gavile ◽  
Benjamin G Barwick ◽  
Sagar Lonial ◽  
Lawrence H. Boise
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Death ◽  
Cell Survival ◽  
Cell Lines ◽  
Plasma Cells ◽  
Surface Expression ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Rpmi 8226

Abstract Multiple myeloma is the second most common hematological malignancy in the U.S. with an estimated 30,700 new diagnoses in 2018. It is a clonal disease of plasma cells that, despite recent therapeutic advances, remains incurable. Myeloma cells retain numerous characteristics of normal plasma cells including reliance on survival signals in the bone marrow for long term viability. However, malignant transformation of plasma cells imparts the ability to proliferate, causing harmful bone lesions in patients, and in advanced stages independence of the bone-marrow microenvironment. Therefore, we are investigating the molecular mechanisms of myeloma cell survival that allow them to become extramedullary. We identified syntenin-1 (SDCBP) as a protein involved in myeloma cell survival and a potential therapeutic target. Syntenin-1 is an adapter protein that has been shown to regulate surface expression of several transmembrane proteins by binding with membrane phospholipids and mediating vesicular trafficking of proteins throughout the cell. Syntenin-1 regulates the surface expression of CD138, a plasma/myeloma cell marker. Syntenin-1 has been shown to regulate apoptosis in numerous cancer cell lines including breast cancer, glioma, and pancreatic cancer but its role in multiple myeloma survival has not been studied. To determine if syntenin-1 expression has an effect on myeloma cell survival, we utilized the CoMMpass dataset (IA12), a longitudinal study of myeloma patients that includes transcriptomic analysis throughout treatment. We found that patients with the highest expression of syntenin-1 mRNA (top quartile) had significantly worse overall survival, progression-free survival, and a shorter response duration than those in the bottom quartile of expression. To determine if syntenin-1 has a role in myeloma cell survival, we used short hairpin RNA to knock down syntenin-1 (shsyn) in RPMI 8226 and MM1.s myeloma cell lines. We then determined the amount of cell death using Annexin-V staining flow cytometry four days following lentiviral infection. We found increased cell death in syntenin-1-silenced cells compared to our empty vector control in both RPMI 8226 (control=42.17%, shsyn=71.53%, p=0.04) and MM1.s cell lines (control=8.57%, shsyn=29.9%, p=0.04) suggesting that syntenin-1 is important for myeloma cell survival. Syntenin-1 contains two PDZ domains that allow it to bind to receptor proteins via their corresponding PDZ-binding motifs. We therefore wanted to look at correlation of syntenin-1 expression with CD138 and CD86, two PDZ-binding domain containing proteins expressed on the surface of myeloma cells. Using the CoMMpass dataset, we found patients with high expression of syntenin-1 had a median expression of CD86 that was twice as high as the total population (P<0.0001) while syntenin-1-low patients expressed CD86 at levels that were half as much as the population (P<0.0001). In contrast, there was no clear relationship between syntenin-1 and CD138 mRNA expression. Indeed if one takes into account all patients, there is a positive correlation between CD86 and syntenin-1 expression (r=0.228, P<0.0001) while there is a negative correlation between CD138 and syntenin-1 (r=-0.1923, P<0.0001). The correlation with CD86 but not CD138 suggests a previously undescribed role for syntenin-1 in myeloma cells. Our lab has previously shown that expression of CD86 is necessary for myeloma cell survival, and signals via its cytoplasmic domain to confer drug resistance. Silencing syntenin-1 results in a decrease in CD86 surface expression. However, there is no change in CD86 transcript or total cellular CD86 protein levels in our shsyn treated cells. Moreover, knockdown of CD86 resulted in increased protein expression and transcript levels of syntenin-1. Taken together, these data suggest that syntenin-1 may regulate CD86 expression on the cell surface. Our data supports a novel role for syntenin-1 in myeloma cell viability and as a potential regulator of CD86 surface expression. The role of syntenin-1 has not previously been explored in multiple myeloma and determining its molecular function is warranted as it may be an attractive target for therapeutic treatment of the disease. Disclosures Lonial: Amgen: Research Funding. Boise:AstraZeneca: Honoraria; Abbvie: Consultancy.

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 A New Therapeutic Hypothesis: Nonsense-Mediated Decay Is an Exploitable Target in Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1937-1937
Author(s):  
Alexander Leeksma ◽  
Ingrid A.M. Derks ◽  
Brett Garrick ◽  
Torsten Trowe ◽  
Aldo Jongejan ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Er Stress ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Protein Production ◽  
Splicing Factors ◽  
Advisory Committees ◽  
Nonsense Mediated Decay

Abstract Background Nonsense-mediated decay (NMD) is a cellular quality control system that degrades mRNAs containing premature termination codons (PTCs) as well as ~10% of normal mRNAs (Kurosaki and Maquat, 2016). NMD thus prevents translation of misfolded proteins, and potential activation of the unfolded protein response (UPR). Mutations in splicing factors such as SF3B1, SRSF2, U2AF1 and ZRSR2 found in hematological as well as solid tumors, can lead to generation of aberrant mRNAs that contain PTCs. Aberrant splicing patterns in cancer cells can possibly result in increased pressure on the NMD machinery. CC-115, a potent inhibitor of mTOR kinase (TORK) and of DNA-dependent protein kinase, (DNA-PK; Mortensen et al., 2015; Tsuji et al., 2017), is in clinical development for the treatment of solid and hematologic malignancies (Thijssen et al., 2016). Preclinical data revealed an additional target of CC-115 and its differential effect on NMD. Our hypothesis was that a subset of tumor cells, especially hematologic tumors with high protein production and/or splicing factor mutations, would be susceptible to NMD inhibition by CC-115. Methods In total, 141 cell lines were screened for sensitivity to CC-115-mediated inhibition of proliferation and induction of cell death, in comparison to specific inhibition of TORK (CC-223). Isogenic DNA-PK knockout cell lines HCT116/HCT116 DNA-PK-/- and M059K/M059J DNA-PK-/- were treated with CC-115 and CC-223. Activity on NMD in vivo was tested using HCT-116 xenograft tumors treated with Vehicle or CC-115. Dependence on CC-115 sensitivity was determined using CRISPR/Cas9 technology of apoptosis or UPR genes in various MM cell lines. RNA sequencing was used for identification of potential targets in sensitive and resistant cell lines. Results A subset of cancer cell lines underwent cell death at sub-micromolar concentrations of CC-115 due to inhibition of NMD, but this was independent of mutations in splicing factors such as SF3B1. We next focused on MM cells as these generally produce high levels of (immunoglobulin) proteins and are prone to ER stress, and therefore potentially susceptible to NMD inhibition. Indeed, treatment with CC-115 resulted in activation of the UPR independent of TORK and DNA-PK inhibition, and cell death in 11/12 MM cell lines. Activity of CC-115 correlated strongly with cell death by the known ER-stress inducer, thapsigargin. Cell death by CC-115 occurred by the mitochondrial pathway of apoptosis, as it depended on caspase activity and the presence of Bax-Bak. Analysis of RNA sequencing data is ongoing and has indicated potential targets dictating sensitivity to CC-115-mediated cell death. Conclusions We describe that hematologic tumors with high protein production are specifically sensitive to CC-115, a novel and clinically exploitable inhibitor of NMD. This might lead to application in malignancies that depend on NMD to avoid excessive protein stress, such as multiple myeloma. Disclosures Garrick: Celgene: Employment. Trowe:Celgene: Employment. Kater:Acerta: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche/Genentech: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. Eldering:Celgene: Research Funding. Filvaroff:Celgene: Employment.

Survivin in Multiple Myeloma: Prognostic and Therapeutic Implications

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 137-137
Author(s):  
Verena Wagner ◽  
Dirk Hose ◽  
Anja Seckinger ◽  
Ludmila Weitz ◽  
Tobias Meißner ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Stress Response ◽  
Cell Lines ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Survivin Expression ◽  
Marrow Stromal Cells ◽  
Er Stress Response

Abstract Abstract 137 The IAP (inhibitor-of-apoptosis) family member, survivin, is one of the most significantly over-expressed genes in malignant cells. Survivin has been reported to inhibit apoptosis and regulate mitosis as well as cytokinesis. We therefore first determined the expression of survivin in CD138-purified multiple myeloma (MM) cells from previously untreated patients at our centers' trial group (TG) (n=246) and the UAMS Arkansas (n=345) validation group (VG). Using the PANP algorithm, survivin is aberrantly expressed in 27% (TG) of MM cell samples. It is not expressed in normal bone marrow plasma cell samples (n=7) while expression increases significantly from MM stage I-III (P<.001). Survivin expression correlates with proliferation as assessed by gene expression- (r=.8, P<.001) or propidium iodide (r=.7, P<.001). Presence of survivin expression correlates with inferior event-free and overall survival in patients undergoing high-dose chemotherapy in the TG (22.6 vs. 35.4 months, P<.001, 52.9 vs. n.r., P=.002) as well as in the VG (12.3 vs. 54.1 months, P<.001, and 17.4 vs. n.r., respectively). These results support the further evaluation of survivin as a therapeutic target in MM. We next assessed the effects of siRNA-mediated knock-down of survivin in vitro. Suppression of survivin by siRNA induced cell cycle arrest and apoptosis in MM cell lines. The small molecule suppressant of survivin, YM155, is currently in clinical development for the treatment of solid tumors. Here, we investigated YM155 for its anti-MM activity. YM155 abrogated proliferation and induced apoptosis in a panel of 10 human MM cell lines and MM cells isolated from multidrug-resistant patients at an IC50 of 4–50nM while the IC50 was not reached in primary bone marrow stromal cells up to 500nM. YM155 was also able to overcome the protective effect of IL-6, IGF-1 and the presence of bone marrow stromal cells, respectively. The induction of apoptosis by YM155 closely correlated with down-regulation of intracellular survivin protein expression within 24 to 36h of treatment with 50–100nM of YM155. However, inhibition of cell proliferation is already detectable at 12h at 5–10nM, suggesting two different dose- and time-dependent mechanisms of action. We therefore performed gene expression and protein profiling on YM155-treated MM cells. Strikingly, these data revealed early up-regulation of the ER stress response (PERK, phospho-eIF2a, ATF4, ATF3) followed by increased CHOP expression and a profound abrogation of proliferation. This appeared to be independent of cellular survivin levels, indicating that the early proliferation arrest at very low nanomolar concentrations is mediated primarily by the ER stress response. Moreover, gene signatures regulated by the IL-6/STAT pathway (CCND1, BCL2L1, MCL1, BIRC5A) were markedly altered upon YM155 treatment. Importantly, IL-6 profoundly sensitized IL-6 responsive MM cell lines to treatment with YM155. We therefore hypothesized that YM155 might abrogate upstream regulatory signaling pathways of survivin expression. Indeed, YM155 abrogated constitutive as well as IL-6 induced phosphorylation of STAT3, an important transcription factor for survivin expression in MM cells. In contrast, phosphorylation of ERK1/2 and AKT remained unchanged. Dephosphorylation of STAT3 closely correlated with the loss of intracellular survivin. In conclusion, we have demonstrated the prognostic significance of survivin expression and a potential therapeutic role for the small molecule suppressant of survivin YM155 in MM. Disclosures: No relevant conflicts of interest to declare.

Blocking of Transient Receptor Potential Vanilloid1 (TRPV1) Promotes Lysosomal Destabilization and Enhances Bortezomib-Induced ER Stress and Cell Death Via HSP70 and LAMP3 Down-Regulation: Novel Therapeutic Target for Multiple Myeloma

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 804-804
Author(s):  
Katia Beider ◽  
Valeria Voevoda ◽  
Hanna Bitner ◽  
Evgenia Rosenberg ◽  
Yaarit Sirovsky ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Er Stress ◽  
Cell Lines ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Acidic Vesicles ◽  
Trpv1 Antagonist ◽  
Transient Receptor ◽  
Apoptotic Factors

Abstract Introduction: Multiple myeloma (MM) is a neoplastic disorder that is characterized by clonal proliferation of plasma cells in the bone marrow (BM). Acquired or de novo resistance to current anti-MM therapy remains a major treatment obstacle. Novel new therapies are thus in need. Recent data have highlighted the contribution of Ca2+channels in the regulation of cell proliferation, chemo-resistance, migration and invasion. Transient Receptor Potential Vanilloid type-1 (TRPV1) is a calcium-permeable ion channel that has been demonstrated to be expressed in solid tumors. As no data is available evaluating TRPV1 in MM, the aim of the current study was to evaluate its possible role in MM. Results: Elevated levels of TRPV1 transcript was detected in MM cell lines (n=8) and BM aspirates from MM patients (n=24) in comparison to normal BM (n=5). AMG9810 a specific antagonist of TRPV1, significantly reduced the viability of MM cell lines (n=8) and primary CD138+ cells (n=6),in a time- and dose-dependent manner (p&lt;0.01) and induced apoptosis manifested by phosphatidylserine externalization, loss of mitochondrial membrane potential (ψm), caspase 3 cleavage and DNA fragmentation. AMG9810-triggered apoptosis could be partially blocked by inhibition of calpains and cathepsins, indicating the role of lysosomal rapture in AMG9810-mediated cell death. Indeed, treatment with TRPV1 antagonist induced rapid lysosomal acidification and increased the number of acidic vesicles (detected by acridine orange stain). The acidic vesicles appeared as early as 1 hour post exposure to AMG9810 preceding the mitochondrial destabilization and apoptosis, thus suggesting that TRPV1 blockade induces lysosomal-induced cell death in MM. Furthermore, TRPV1 inhibition with AMG9810 completely suppressed the pro-survival AKT/mTOR pathway and significantly reduced the levels of anti-apoptotic factors BCL-2 and BCL-XL. Combining AMG9810 with the proteasome inhibitor bortezomib (Bort) induced synergistic cell death in both native and Bort-resistant cells (CI&lt;0.4). Moreover, TRPV1 inhibition successfully overcame the CXCR4-mediated protection from Bort provided by BM stromal cells. This finding suggests that the TRPV1 channel may regulate the activity of CXCR4 chemokine receptor in MM cells affecting the MM-microenvironment interactions. In accordance, the TRPV1 antagonist AMG8910 prevented the responsiveness of CXCR4-expressing MM cells to CXCL12 stimulation, decreased the phosphorylation of signaling mediators like Erk1/2 and AKT and suppressed cell migration, while TRPV1 activator capsaicin promoted the CXCR4-mediated signaling and migration. Gene and protein expression analysis were next performed to delineate the molecular mechanisms underlying the observed synergism between Bort and AMG9810. Bort treatment resulted in robust induction of endoplasmic reticulum (ER) stress genes including the increase in pro-apoptotic factors ATF4, CHOP and GADD34. Compensatory unfolded protein response (UPR) was activated as well, with increase in chaperons HSP27, HSP70, HSP90, and lysosomal chaperon LAMP3 known to stabilize lysosome, protecting cells against lysosomal membrane permeabilization (LMP) and subsequent cell death. AMG9810 further increased ER stress, elevating CHOP and GADD34 expression, while significantly reducing both basal and Bort-increased levels of HSP70 and LAMP3, thus overcoming the protective response to Bort treatment and prompting lethal LMP. Finally, combining Bort with AMG9810 resulted in significantly reduced ROS that was correlated with impaired mitochondria and increased MM apoptosis, suggesting that dissipation of intracellular ROS may be involved in AMG9810-promoted cytotoxicity. Conclusions: Altogether, our data indicate that TRPV1 is implicated in MM cell survival, proliferation, migration, microenvironment interactions and stress response. TRPV1 inhibition by AMG9810 inhibits CXCR4-mediated migration and stromal protection, synergizes with Bort, amplifies ER stress, targets cytoprotective HSP70 and LAMP3, destabilizes lysosome, impairs mitochondria and promotes MM cell death. These results unravel the mechanism mediating the strong synergistic anti-MM activity of Bort in combination with TRPV1 inhibition which may be translated into the clinic. Disclosures Peled: Biokine: Consultancy; Biosight: Consultancy.

scholarly journals Inhibition of WIP1 Phosphatase in Multiple Myeloma Overcomes Bortezomib Resistance and Promotes Cell Death Via ER Stress-Induced Apoptotic JNK/c-Jun Signaling and Downregulation of Inhibitors of Apoptosis Proteins (IAPs)

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1366-1366
Author(s):  
Katia Beider ◽  
Evgenia Rosenberg ◽  
Valeria Voevoda ◽  
Hanna Bitner ◽  
Yaarit Sirovsky ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Stress Response ◽  
Er Stress ◽  
Cell Lines ◽  
Potential Role ◽  
Cyclin B1 ◽  
Parp Cleavage ◽  
Induced Apoptosis

Abstract Acquired or de novo resistance to the traditional and novel anti-multiple myeloma (MM) agents remains a major treatment obstacle, therefore novel therapies are in need. Wild-type p53-induced phosphatase 1 (WIP1) is an oncogenic serine/threonine phosphatase implicated in silencing of cellular responses to genotoxic stress. WIP1 overexpression was documented in various solid cancers in correlation with aggressive features and poor prognosis. Thus, we studied WIP1 in MM addressing its potential role in mediating resistance and aggressive phenotype. Increased expression of WIP1 was detected in MM cell lines (n=8) and primary samples (n=18) at both mRNA and protein level as compared with normal PBMCs (n=5). Furthermore, a positive correlation between WIP1 and CXCR4 levels (p<0.02, R2=0.5) was revealed. The latter is a well-known oncogenic receptor in MM. WIP1 expression levels were significantly up-regulated following bortezomib (Bort) treatment. Using MM cell lines with acquired resistance to Bort (RPMI8226BortRes and CAGBortRes), a higher induction of WIP1 upon Bort exposure could be demonstrated, suggesting a possible role for WIP1 in the acquisition of MM drug resistance to proteasome inhibitors. WIP1 was also upregulated in MM cells cultured on human BM stroma (BMSC) known to protect the tumor cells from Bort-induced apoptosis, further supporting its function in mediating resistance. GSK2830371 (GSK), a novel allosteric inhibitor of WIP1, significantly suppressed MM cells proliferation (p<0.01) and induced apoptosis, as demonstrated by phosphatidylserine externalization, mitochondrial depolarization (ψm), caspase 3 and PARP cleavage, and DNA fragmentation. Moreover, combined treatment with GSK and Bort synergistically potentiated cell death in both Bort-sensitive and resistant MM cells and overcame BMSC protection (CI<0.5). The robust apoptosis induced by Bort/GSK treatment was accompanied by increased mitochondrial ROS accumulation, subsequent mitochondrial destabilization and extensive DNA damage. GSK treatment resulted in a reduction of WIP1 basal expression and abrogated WIP1 induction upon Bort treatment. Thus, we defined that GSK can regulate WIP1 expression in MM cells. To determine the molecular mechanism of Bort/GSK synergism we performed gene and protein expression analysis. Combination of both agents significantly reduced expression of anti-apoptotic proteins such as cIAP1, cIAP2, XIAP and Survivin. Previous studies indicate that maintaining IAPs expression is part of an adaptive unfolded protein response that promotes MM survival upon Bort-induced endoplasmic reticulum (ER) stress. Therefore, it is conceivable that targeting IAPs upon WIP1 inhibition may overcome protective responses, inducing unresolved ER stress and MM cell death. Indeed, we found that combination of Bort and GSK significantly enhanced ER stress, as indicated by increase in the pro-apoptotic factors ATF4, CHOP and GADD34. Concomitantly, mitosis-inducing factors Cyclin B1, CDK1 and PLK1 were prominently reduced upon Bort/GSK treatment. To assess the potential role of p53 activation in GSK-mediated effects, p53-stabilizing agents nutlin3a and PRIMA1 were applied in combination with WIP1 inhibition. We observed a significant (p<0.01) increase in the responsiveness of both p53WT and p53mut MM cells to GSK-mediated apoptosis. Consistently, combined GSK/Bort treatment upregulated p53 targets, including PUMA, NOXA, GADD45A and p21 genes. These data suggest that p53 may potentiate the WIP1 inhibition mediated stress induction. Finally, we assessed the signaling pathways that may be involved in WIP1 mediated cessation of stress response. GSK profoundly augmented Bort-induced phosphorylation of JNK and c-Jun, without affecting p38 phosphorylation. Accordingly, JNK inhibitor SP600125 successfully reverted both the apoptosis and the downregulation of IAPs induced by Bort/GSK treatment. Altogether, these results identify pro-apoptotic JNK/c-Jun signaling being preferential target of WIP1 in the process of dampening Bort-induced stress response. To conclude, we disclose the role of WIP1 in blunting stress response and promoting resistance to bortezomib. Collectively, WIP1 suppression prevents MM cell adaptation and recovery upon ER stress. These findings may provide the scientific basis for a novel combinatorial anti-myeloma therapy. Disclosures Peled: Cellect Biotherapeutics Ltd: Consultancy.

scholarly journals The NEDD8 Activating Enzyme Inhibitor Pevonedistat Induces ER Stress/UPR-Mediated Cell Death and Rebalances Homeostasis of Pro- and Anti-Apoptotic Proteins in Favor of Cell Death in Acute Lymphoblastic Leukemia Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1327-1327
Author(s):  
Gilles M. Leclerc ◽  
Shuhua Zheng ◽  
Guy J. Leclerc ◽  
Joanna DeSalvo ◽  
Ronan T. Swords ◽  
...  
Keyword(s):  
Cell Death ◽  
Protein Synthesis ◽  
Acute Lymphoblastic Leukemia ◽  
Er Stress ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Current Therapy ◽  
Parp Cleavage ◽  
E3 Ligases ◽  
Apoptotic Proteins

Abstract Acute lymphoblastic leukemia (ALL) is the leading cause of cancer-related death in children. Most adults will die from the disease. Hence, current therapy in ALL is inadequate. We previously showed that ALL cells were differentially sensitive to agents inducing endoplasmic reticulum (ER) stress (Mol Cancer Res 2012; 10:969-978; PLoS One 2013; 8:e74420). In this regard, we investigated the anti-leukemic activity of the NEDD8-activating enzyme (NAE) inhibitor pevonedistat (MLN4924). Pevonedistat prevents neddylation of cullin-RING E3 ligases, leading to accumulation of cullin dependent substrates with anti-proliferative effects. We demonstrated that pevonedistat induced dose-dependent cell growth inhibition (IC50 between 159-300 nM) and cell death (EC50 between 317-463 nM) in both T- and B-ALL cells (cell lines and primary patient material). We found that neddylated cullin levels were significantly decreased confirming inhibition of the NEDD8 conjugation pathway by pevonedistat. Pevonedistat induced ER stress and unfolded protein response (UPR) mediated cell death in ALL cell lines and primary cells as evidenced by increased expression of UPR markers (GRP78 and CHOP), and cleaved-PARP. Mechanistically, pevonedistat caused proteotoxic/ER stress from failure to halt protein translation mediated by p-eIF2α (Ser51) de-phosphorylation via up-regulation of the PERK inhibitor p58IPK. We also observed up-regulation of mTOR/p70S6K, further increasing protein synthesis and augmenting proteotoxic/ER stress. Indeed, we demonstrated that pevonedistat induced nascent protein synthesis, and that co-treatment with protein synthesis inhibitors (rapamycin, cycloheximide) rescued ALL cells from pevonedistat induced cytotoxicity (p < 0.01 for combination treatment vs. pevonedistat alone). The observed rescue correlated with decreased UPR markers and P-p70S6K (Thr389) expression, suggesting that proteotoxic stress is a central determinant in pevonedistat-induced ALL cell death. Additional studies into the mechanism of pevonedistat-induced apoptosis revealed that homeostasis of pro- and anti-apoptotic proteins was rebalanced in favor of cell death through decreased Mcl-1 pro-survival activity, via sequestration by NOXA and BIM. Co-immunoprecipitation experiments showed that pevonedistat increased the interaction between NOXA and Mcl-1, and BIM and Mcl-1 proteins, resulting in the reduction of Mcl-1 pro-survival activity. siRNA-mediated down-regulation of NOXA and BIM expression decreased the sensitivity of ALL cells to pevonedistat, supporting the sequestration by NOXA and BIM of Mcl-1's pro-survival activity. Activation of the MEK/ERK/Mcl-1 pathway following pevonedistat-induced cell death was also noted, possibly as a compensatory mechanism. We found that pevonedistat plus a MEK inhibitor (PD98059, selumetinib) induced significant cell death compared to each drug alone (p < 0.0001), which was associated with decreased Mcl-1 expression and increased cleaved-PARP cleavage, supporting a critical role of Mcl-1 in ALL cell survival. In addition, we demonstrated synergy between pevonedistat and effective anti-leukemic agents such as dexamethasone, doxorubicin and cytarabine in ALL cell line models, with CI values of 0.17, 0.46, and 0.23, respectively. More importantly, we showed that NSG mice engrafted with human ALL cells had statistically significant increased survival when treated with pevonedistat (pevo) plus dexamethasone (dex) compared to single agent therapy (p = 0.0076 for pevo+dex vs. dex alone; p = 0.0182 pevo+dex vs. pevo alone), lending support for the use of pevonedistat as part of a multi-agent approach. Taken together, our data demonstrate that the NAE inhibitor pevonedistat alters cellular translational machinery leading to ER stress/UPR-mediated cell death, and suggest that pevonedistat may have a "priming" effect on ALL cells by altering the apoptotic threshold through modulation of Mcl-1's pro-survival activity. Disclosures No relevant conflicts of interest to declare.

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