scholarly journals Resistance to Nutrient Deprivation Via Metabolic Re-Programming As Mechanism of 'Metastasis' Promotion in Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3218-3218
Author(s):  
Ioanna Savvidou ◽  
Sahan Chandrasekara ◽  
Maoshan Chenn ◽  
Tiffany Khong ◽  
Andrew Spencer
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Lines ◽  
Research Funding ◽  
Fold Increase ◽  
Autophagic Vacuole ◽  
Metabolic Reprogramming ◽  
Nutrient Deprivation ◽  
Autophagic Flux ◽  
Tumor Resistance

Abstract Aim: Multiple Myeloma (MM) is a disease of the intra-medullary (IM) compartment but with progression myeloma cells can survive in nutrient deprived extramedullary (EM) sites. Autophagy mediates the degradation and re-cycling of intra-cellular proteins and may promote tumor resistance to metabolic stressors, including nutrient deprivation. Using the MM cell lines TK1 and TK2, contemporaneously propagated from bone marrow (IM) and peripheral blood (EM), respectively, of a MM patient we investigated the role of autophagy in resistance to glutamine (Gln) deprivation. Methods & Results:TK2 but not TK1 exhibited up-regulated autophagy under both basal and Gln deprived conditions with higher LC3BII/I turnover on immunoblotting and autophagic vacuole formation on electron microscopy following chloroquine (CQ) exposure. Under conditions of Gln deprivation, TK2 was able to proliferate until day 14, whereas TK1 stopped proliferating at day 3. This proliferative advantage under Gln deprivation was abrogated by autophagy inhibitors (CQ or 3-MA) as determined by both viable cell enumeration (p=0.008 at day 7, inhibitor vs no inhibitor) and Ki67 expression (p=0.0017) only in TK2. Modulation of Gln concentration demonstrated an inverse correlation with the viability of TK1 but not TK2 - TK1 cell death increased from 36% at Gln 8mM to 56% at Gln 0mM, irrespective of autophagy inhibition. In contrast, while Gln deprivation had no significant effect on the viability of TK2, under Gln deprivation CQ exposure induced a 2-fold increase cell death measured by annexin V/PI positivity and 2.5-fold increase of cPARP expression (p=0.0045). RNAseq and GSEA analysis showed enrichment of the Krige Amino Acid Deprivation Response (AADR) pathway (ES=0.083) for TK1 with up-regulated expression of Gln transporters (SLC7A5, SLC1A5, SLC38A5 and SLC7A11). In contrast, Glutamine Synthetase (GS) expression, which is responsible for Gln synthesis, was up-regulated in TK2 cells. Interestingly, immunoblotting analysis revealed that Gln deprivation was able to induce the expression of GS while further inducing autophagic flux (2.66 fold increase of LC3II expression at Gln=0mM) only in TK2, whereas it inhibited mTOR activity in both cell lines. Additionally, GS chemical inhibition under Gln deprivation induced cell death in TK2 but not TK1 (32.56% vs 2.5% increase, respectively) demonstrating that Gln synthesis enables TK2 to overcome Gln deprivation. Conclusion. Metabolic reprogramming mitigating against nutrient deprivation may promote a more 'metastatic' phenotype in advanced MM. Disclosures Spencer: Celgene: Honoraria, Research Funding, Speakers Bureau; Janssen-Cilag: Honoraria, Research Funding, Speakers Bureau; Amgen: Honoraria, Research Funding; BMS: Research Funding; Takeda: Honoraria, Research Funding, Speakers Bureau; STA: Honoraria.

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.

Methyljasmonate, An Inhibitor of Glycolytic Energy Production, Displays Pre-Clinical Activity against Multiple Myeloma Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1741-1741
Author(s):  
Steffen Klippel ◽  
Jana Jakubikova ◽  
Jake Delmore ◽  
Melissa G. Ooi ◽  
Douglas McMillin ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Death ◽  
Cancer Cells ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Energy Production ◽  
Advisory Committees ◽  
Normal Cells

Abstract Abstract 1741 Poster Board I-767 Background In contrast to most normal cells, cancer cells typically produce energy predominantly by glycolysis as demonstrated by O. Warburg more than 50 years ago. Methyljasmonate (MJ), a hormone produced by plants in response to biotic & abiotic stresses such as herbivory and wounding, has been shown to prevent the interaction of hexokinase (Hxk) and voltage dependent anion channels (VDACs), thereby significantly impacting the onset of glycolytic energy production. This may explain promising preclinical results observed with MJ against a variety of cancer cells, including myeloid leukemia and B-cell lymphoma cell lines. Methods and Results We tested the potential of MJ against Multiple Myeloma (MM) cells. We first evaluated the response of 16 different MM cell lines to 24 h of exposure to MJ concentrations of 0.5 – 3.5 mM using MTT assays. 15/16 of the MM cell lines tested displayed an IC50 of < 1.5 mM. In contrast, HS-5 stroma cells and peripheral blood mononuclear cells (PBMCs) did not respond to that MJ concentration, and even at a concentration of 2.5 mM MJ showed a maximal reduction of cell viability of 40%. Similarly to MM cell lines, purified CD138+ primary tumor cells of 3 MM patients displayed an IC50 of < 1.5 mM, suggesting that the differential sensitivity of MM vs. normal cells to MJ is not restricted to cell lines, but is also observed with primary tumor cells. Importantly, neither co-culture with HS-5 stroma nor IL-6 protected MM cells against MJ. Cell death commitment assays revealed that 1h exposure of 1.5 mM MJ induced cell death. Annexin V/PI FACS analysis of MJ-exposed MM cells showed that the cell death is mainly driven by apoptosis, evidenced by cleavage of caspases 3, 8 and 9 as well as of PARP. However, pre-incubation of MM cells with specific caspase inhibitors such as 10 mM of AC-DEVD-CHO, Z-IETD-fmk, Z-LEHD-fmk or 50 mM of Z-VAD only minimally protects the cancer cells from MJ exposure. Therefore, the impact of the MJ is not solely due to caspase triggered proteolytic cascades. Measurements of cellular ATP content by cell titer glow (CTG; Promega, Madison, WI) assay showed rapid depletion of ATP triggered by MJ action in sensitive MM cell lines. Additionally, we observed that 1 h exposure to 2 mM MJ modulated signaling pathways including IRS1/PI3K/AKT, MEK1/2, as well as Stat3 and JNK. FACS-based cell cycle analysis after propidium iodide staining did not show cell cycle arrest, but rather a rapid transition of cells to G0/G1 No correlation of sensitivity of MM cell lines and the number of mitochondria per cancer cell, as determined by Mitotracker Green (Invitrogen, Carlsbad, CA) -based flow analysis, was observed. We next examined if MJ exhibits either significant antagonism or synergy with established or novel anti-MM agents, including Bortezomib, Lenalidomide, Doxorubicin, Rapamycin or Dexamethasone, but discovered neither. However, MJ displayed synergy when combined with 2-Deoxyglucose. Finally, MJ was tested in vivo in scid/nod mice irradiated with 150 rads, injected with 1× 106 MM1S cells, and then, treated at 500 mg/kg by IP administration on a 5 days on / 2 days off schedule starting two weeks after tumor cell injection, There was an overall survival advantage of MJ-treated animals over the respective controls, with all treated mice (n=10) still alive but 6/10 control mice dead after 27 d. Conclusions Based on its rapidity of anti-MM action, favorable safety profile in preclinical models, distinct pattern of molecular sequelae, and compatibility with established anti-MM agents, MJ represents a promising investigational anti-MM agent. Disclosures Laubach: Novartis: Consultancy, Honoraria. Richardson:Millennium: (Speakers Bureau up to 7/1/09), Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: (Speakers Bureau up to 7/1/09), Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Anderson:Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Mitsiades:Novartis Pharmaceuticals: Consultancy, Honoraria; Milllennium: Consultancy, Honoraria; Bristol-Myers Squibb : Consultancy, Honoraria; Merck &Co.: Consultancy, Honoraria; Kosan Pharmaceuticals : Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; PharmaMar: Patents & Royalties; Amgen: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis Pharmaceuticals: Research Funding.

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.

Inhibition of Constitutively Activated JAK/STAT3 Pathway in Multiple Myeloma Cells Leads to Induction of Protective Autophagy.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4914-4914
Author(s):  
Huihui Ma ◽  
Caisheng Lu ◽  
Judith Ziegler ◽  
Rentian Feng ◽  
Suzanne Lentzsch ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Lines ◽  
Research Funding ◽  
Specific Inhibitor ◽  
Janus Kinase ◽  
Signal Pathways ◽  
Myeloma Cells

Abstract Abstract 4914 Constitutive activation of Janus Kinase-2 (JAK-2)/signal transducer and activator of transcription-3 (STAT3) has been implicated to play a crucial role in the pathogenesis of Multiple Myeloma (MM). Therefore, targeted inhibition of STAT3 is an attractive therapy for MM patients. JSI-124 is a natural product isolated from various plant families and has been recently described as a specific inhibitor of JAK2/STAT3. It has been shown to exert strong apoptosis-inducing effects against STAT3+ tumor cell lines in vitro and in vivo. However, there is little information available about the anti-tumor effects of JSI-124 on myeloma cells. We therefore tested the role of JSI-124 as a potential novel anti-MM compound and were able to determine that JSI-124 has potent anti-myeloma properties against human MM cell lines. Our results revealed that JSI-124 could inhibit both constitutively expressed and IL-6 induced phosphorylation of Tyr705 STAT3 as well as other important cell signal pathways that regulate cell proliferation in MM cell lines. However, the anti-myeloma effects were not restricted to STAT3+ MM cells. In STAT3+ cells JSI-124 induced cell death, which was predominantly caspase dependent. In contrast, JSI-124 induced both caspase-dependent and caspase-independent cell death in STAT3-MM cells. Furthermore, JSI-124 induced DNA damage only in STAT3- MM cells. Surprisingly, STAT3- cell lines were more sensitive to JSI-124-induced growth inhibition and induction of cell death than STAT3+ MM cell lines. Further analysis revealed that JSI-124 led to induction of autophagy only in STAT3+ MM cells as determined by upregulation of LC3B. A dramatic increase of JSI-124-dependent apoptosis was observed when the autophagy pathway was blocked by Chloroquine suggesting that STAT3-inhibition in STAT3+ MM cells leads to protective autophagy, which can be overcome by Chlorquine treatment. Interestingly, inhibition of autophagy by Chloroquine or 3-mA in STAT3+ cell lines U266 or ARH 77 resulted in AIF release and well as caspase-independent cell death. The results in this study suggest that JSI-124 could be an effective anti-myeloma agent regardless of STAT3 activation status and that combination with Chloroquine may enhance its anti-MM effect. Disclosures Lentzsch: celgene: Consultancy, Honoraria, Research Funding; cephalon: Consultancy, Research Funding. Mapara:Resolvyx: Consultancy, Honoraria, Research Funding; Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees; GENTIUM: STOCK OWNERSHIP.

Synergistic Induction of Cell Death By Combined Inhibition of PIM and AKT Kinases in Cytogenetically Defined Standard and High-Risk Multiple Myeloma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4723-4723 ◽  
Author(s):  
Koremu Meja ◽  
Dean Smith ◽  
Laura Percy ◽  
Dennis Huszar ◽  
Barry R Davies ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Lines ◽  
Research Funding ◽  
Risk Group ◽  
Akt Inhibitor ◽  
Employment Equity ◽  
Synergistic Induction ◽  
Equity Ownership ◽  
Standard Risk

Abstract The PIM and PI3K/Akt pathways have been studied extensively for their effects on cell growth, proliferation and survival and shown to have both distinct and overlapping functions. The PIM proteins (PIM1, 2 and 3) are serine/threonine kinases that are constitutively active, with control of PIM signaling largely at the level of transcriptional regulation. Recent studies have shown PIM2 mRNA to be highly expressed in multiple myeloma (MM) and Akt has previously been shown to be constitutively active in primary MM cells in at least 50% of samples. As results of targeting single signaling pathways can be compromised by compensatory mechanisms, we have investigated the effects of the pan-PIM inhibitor AZD1897 and the Akt inhibitor AZD5363 alone and in combination in MM, utilising a panel of cell lines representing the major MM translocation subtypes and primary patient CD138+ samples. Western blotting of a panel of human myeloma cell lines (HMCL) and primary MM samples showed that PIM2 is the major PIM isoform expressed. PIM2 protein levels were highest in HMCL with the (4;14) translocation. This finding was confirmed in primary samples where levels were quantified by densitometry (corrected for GAPDH expression) – relative levels in cases with t(4;14) 3.6±1, t(11;14) 1±0.4 and hyperdiploidy 0.7±0.3 (n=5, 6 and 4 respectively; p=0.02 by ANOVA). PIM and Akt inhibitors reduced proliferation in HMCL but had modest effects on cell death when used on their own. Combined PIM+Akt inhibition led to a more marked reduction in proliferation and produced synergistic cytotoxicity with induction of >50% cell death at 72 hours in 4 of 4 cell lines with t(4;14), 2 of 3 with t(14;16) and 2 of 4 with t(11;14). Chou-Talalay analysis confirmed synergistic effects with combination indices ranging from 0.1 to 0.8. Direct comparison of inhibitors of Akt, PI3K (GDC0941) and mTORC1/2 (KU-0063794) in combination with AZD1897 showed that the PIM+Akt inhibitor combination was most effective at inducing cell death in 8 out of 12 HMCL. As Akt is downstream of PI3K, this result may appear surprising – however, AZD5363 inhibits both Akt and has additional direct activity against p70S6K/S6K1. As previous studies have shown a PI3K-independent component to S6K signaling in some MM cells, this may at least partly explain this phenomenon. Investigation of downstream signaling showed that PIM inhibition alone led to decreased BAD S112 phosphorylation and of Akt alone to reduced FOXO3 and PRAS40 phosphorylation, confirming blockade of these validated targets at the concentrations employed. Combined PIM+Akt inhibition had enhanced effects compared with either agent alone on mTORC1 outputs, including phosphorylation of 4EBP1 and S6. Importantly, combined inhibition synergised to significantly reduce MCL1 levels in cells that showed marked cell death in response to AZD1897+AZD5363, but not in those that did not, suggesting a mechanistic basis for the cytotoxicity observed. Next we investigated the effect of PIM and Akt inhibition in 23 primary MM samples, subdivided by FISH analysis into standard risk [t(11;14), hyperdiploidy, normal] or adverse risk [t(4;14), t(14;16), gain of 1q, TP53 deleted] groups. Viable CD138+ cell survival after 72 hours in the standard risk group (n=13) was 74±4% of control for AZD1897(PIM inhibitor), 65±4% for AZD5363(Akt inhibitor) and 50±4% for the combination. The equivalent figures for the adverse risk group (n=10) were 70±4%, 57±8% and 38±6%, indicating that dual PIM and Akt targeting is effective across all clinical risk categories. We also assessed the effect of the combination of individual PIM or Akt inhibitors with other anti-MM agents. In HMCL treated with dexamethasone, enhanced effects were seen particularly for the AZD5363+Dex combination eg H929 cells, AZD5363 54% survival, Dex (10nM) 95%, both 15%; KMS28BM 59%, 90%, 28% respectively. Dual PIM+Akt inhibition could also enhance the effects of bortezomib in some HMCL eg H929 PIM+Akt 81% survival, BTZ (25nM) 73%, combination 28%. In conclusion, combined inhibition of PIM (AZD1897) and Akt (AZD5363) kinases resulted in significant anti-MM activity across all cytogenetically defined clinical risk groups, with synergistic induction of cell death. Biomarkers of activity included mTORC1 targets such as p4EBP1 and pS6 and also MCL1 levels. These results provide a rationale for clinical studies of combined PIM and Akt inhibition in MM. Disclosures Meja: AstraZeneca: Research Funding. Huszar:AstraZeneca: Employment, Equity Ownership. Davies:AstraZeneca: Employment, Equity Ownership. Khwaja:AstraZeneca: Research Funding.

scholarly journals Modulation of CD38 Expression Levels on Multiple Myeloma Tumor Cells By All-Trans Retinoic Acid Improves the Efficacy of the Anti-CD38 Monoclonal Antibody Daratumumab

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2096-2096 ◽  
Author(s):  
Inger S. Nijhof ◽  
Henk M. Lokhorst ◽  
Berris van Kessel ◽  
Richard W.J. Groen ◽  
Anton C. Martens ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Monoclonal Antibody ◽  
Retinoic Acid ◽  
Cell Lines ◽  
Research Funding ◽  
Fold Increase ◽  
Expression Levels ◽  
Cd38 Expression ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid

Abstract INTRODUCTION Daratumumab is an anti-CD38 monoclonal antibody (mAb) with lytic activity against multiple myeloma (MM) cells, including ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity). In current clinical phase I/II trials, daratumumab induced anti-MM activity; however, the depth of the response varied between patients. Up till now it is unknown what determines the intrinsic sensitivity of MM cells towards daratumumab-mediated ADCC and CDC. We examined potential determinants of daratumumab sensitivity including CD38 levels, the frequency of effector cells, and expression levels of the complement inhibitory proteins, CD46, CD55, and CD59, which interfere with the different steps of complement activation. RESULTS In bone marrow samples from MM patients, we observed a significant correlation between CD38 expression and daratumumab-mediated ADCC (127 patients; R = 0.428; P < 0.0001) as well as CDC (56 patients; R = 0.338; P = 0.011). Similarly, experiments with isogenic MM cell lines expressing different levels of CD38, revealed that the level of CD38 expression correlates with the extent of daratumumab-mediated ADCC and CDC. Other determinants of daratumumab susceptibility include the effector:target ratio for ADCC, and levels of the complement-inhibitory proteins CD55 and CD59 for CDC. Our data suggest that upregulation of CD38 expression may improve the anti-MM activity of daratumumab. Since, interaction of all-trans retinoic acid (ATRA) with nuclear retinoic acid receptors results in altered expression of target genes including induction of CD38 expression, we evaluated the combination of ATRA and daratumumab. As little as 10 nM ATRA was sufficient to induce a 1.9 – 4.4-fold increase in CD38 expression on the MM cell lines RPMI8226, UM9, and XG1, which resulted in a significant improvement of daratumumab-mediated ADCC and CDC. Importantly, 10 nM ATRA alone resulted in no or only a minimal increase in MM cell death. In addition, ATRA induced a 1.0 – 26.5 (median 1.7) fold increase in CD38 expression on primary MM cells from 26 patients. Also in these primary MM cells, pretreatment with ATRA resulted in a significant increase in their susceptibility to daratumumab-mediated CDC in 13 out of 16 patients as well as ADCC in 8 out of 11 patients. ATRA also enhanced the efficacy of daratumumab in MM cells which are completely resistant to daratumumab-mediated CDC and/or ADCC. Pooled results of these patients show that ATRA improved CDC mediated by 10 µg/mL daratumumab from 16.1 % to 43.9 % (P < 0.0001), and ADCC from 25.1 % to 39.5 % (P = 0.0315). Importantly, expression levels of CD55 and CD59 on MM cells were also significantly reduced by ATRA, which may explain that ATRA improves CDC to a higher extent than ADCC. CONCLUSION Our results provide evidence that CD38 expression levels may predict response to daratumumab. Furthermore, we show that ATRA increases CD38 expression on MM cells, resulting in enhanced daratumumab-mediated lysis of MM cells. Our results provide the preclinical rationale for further evaluation of daratumumab combined with ATRA in MM patients. Disclosures Lokhorst: Celgene: Research Funding; J&J: Research Funding; Genmab: Research Funding. Martens:J&J: Research Funding. Doshi:Janssen R&D: Employment. Mutis:Genmab BV: Research Funding; J&J: Research Funding; Celgene: Research Funding. Sasser:Janssen R&D: Employment. van de Donk:Genmab BV: Research Funding; J&J: Research Funding; Celgene: Research Funding.

Stew in its Own Juice: Protein Homeostasis Machinery Inhibition Reduces Cell Viability in Multiple Myeloma Cell Lines

2019 ◽  
Vol 19 (2) ◽  
pp. 112-119 ◽  
Author(s):  
Mariana B. de Oliveira ◽  
Luiz F.G. Sanson ◽  
Angela I.P. Eugenio ◽  
Rebecca S.S. Barbosa-Dantas ◽  
Gisele W.B. Colleoni
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Viability ◽  
Cell Lines ◽  
Treatment Options ◽  
Compensatory Mechanism ◽  
Protein Homeostasis ◽  
Protein Response ◽  
Rpmi 8226

Introduction:Multiple myeloma (MM) cells accumulate in the bone marrow and produce enormous quantities of immunoglobulins, causing endoplasmatic reticulum stress and activation of protein handling machinery, such as heat shock protein response, autophagy and unfolded protein response (UPR).Methods:We evaluated cell lines viability after treatment with bortezomib (B) in combination with HSP70 (VER-15508) and autophagy (SBI-0206965) or UPR (STF- 083010) inhibitors.Results:For RPMI-8226, after 72 hours of treatment with B+VER+STF or B+VER+SBI, we observed 15% of viable cells, but treatment with B alone was better (90% of cell death). For U266, treatment with B+VER+STF or with B+VER+SBI for 72 hours resulted in 20% of cell viability and both treatments were better than treatment with B alone (40% of cell death). After both triplet combinations, RPMI-8226 and U266 presented the overexpression of XBP-1 UPR protein, suggesting that it is acting as a compensatory mechanism, in an attempt of the cell to handle the otherwise lethal large amount of immunoglobulin overload.Conclusion:Our in vitro results provide additional evidence that combinations of protein homeostasis inhibitors might be explored as treatment options for MM.

Simvastatin Induces Death of Multiple Myeloma Cell Lines

2004 ◽  
Vol 52 (5) ◽  
pp. 335-344 ◽  
Author(s):  
Naomi Gronich ◽  
Liat Drucker ◽  
Hava Shapiro ◽  
Judith Radnay ◽  
Shai Yarkoni ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Cytoplasmic Calcium ◽  
Multiple Myeloma Cell ◽  
Cycle Arrest ◽  
Rpmi 8226

BackgroundAccumulating reports indicate that statins widely prescribed for hypercholesteromia have antineoplastic activity. We hypothesized that because statins inhibit farnesylation of Ras that is often mutated in multiple myeloma (MM), as well as the production of interleukin (IL)-6, a key cytokine in MM, they may have antiproliferative and/or proapoptotic effects in this malignancy.MethodsU266, RPMI 8226, and ARH77 were treated with simvastatin (0-30 μM) for 5 days. The following aspects were evaluated: viability (IC50), cell cycle, cell death, cytoplasmic calcium ion levels, supernatant IL-6 levels, and tyrosine kinase activity.ResultsExposure of all cell lines to simvastatin resulted in reduced viability with IC50s of 4.5 μM for ARH77, 8 μM for RPMI 8226, and 13 μM for U266. The decreased viability is attributed to cell-cycle arrest (U266, G1; RPMI 8226, G2M) and cell death. ARH77 underwent apoptosis, whereas U266 and RPMI 8226 displayed a more necrotic form of death. Cytoplasmic calcium levels decreased significantly in all treated cell lines. IL-6 secretion from U266 cells was abrogated on treatment with simvastatin, whereas total tyrosine phosphorylation was unaffected.ConclusionsSimvastatin displays significant antimyeloma activity in vitro. Further research is warranted for elucidation of the modulated molecular pathways and clinical relevance.

scholarly journals Induction of Apoptosis, Autophagy and Ferroptosis by Thymus vulgaris and Arctium lappa Extract in Leukemia and Multiple Myeloma Cell Lines

Molecules ◽  
2020 ◽  
Vol 25 (21) ◽  
pp. 5016
Author(s):  
Aveen N. Adham ◽  
Mohamed Elamir F. Hegazy ◽  
Alaadin M. Naqishbandi ◽  
Thomas Efferth
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Death ◽  
Ethyl Acetate ◽  
Cell Cycle Arrest ◽  
Cell Lines ◽  
Thymus Vulgaris ◽  
Arctium Lappa ◽  
Cycle Arrest ◽  
Induction Of Apoptosis

Thymus vulgaris and Arctium lappa have been used as a folk remedy in the Iraqi Kurdistan region to deal with different health problems. The aim of the current study is to investigate the cytotoxicity of T. vulgaris and A. lappa in leukemia and multiple myeloma (MM) cell lines and determine the mode of cell death triggered by the most potent cytotoxic fractions of both plants in MM. Resazurin assay was used to evaluate cytotoxic and ferroptosis activity, apoptosis, and modulation in the cell cycle phase were investigated via Annexin V-FITC/PI dual stain and cell-cycle arrest assays. Furthermore, we used western blotting assay for the determination of autophagy cell death. n-Hexane, chloroform, ethyl acetate, and butanol fractions of T. vulgaris and A. lappa exhibited cytotoxicity in CCRF-CEM and CEM/ADR 5000 cell lines at concentration range 0.001–100 μg/mL with potential activity revealed by chloroform and ethyl acetate fractions. NCI-H929 displayed pronounced sensitivity towards T. vulgaris (TCF) and A. lappa (ACF) chloroform fractions with IC50 values of 6.49 ± 1.48 and 21.9 ± 0.69 μg/mL, respectively. TCF induced apoptosis in NCI-H929 cells with a higher ratio (71%), compared to ACF (50%) at 4 × IC50. ACF demonstrated more potent autophagy activity than TCF. TCF and ACF induced cell cycle arrest and ferroptosis. Apigenin and nobiletin were identified in TCF, while nobiletin, ursolic acid, and lupeol were the main compounds identified in ACF. T. vulgaris and A. lappa could be considered as potential herbal drug candidates, which arrest cancer cell proliferation by induction of apoptosis, autophagic, and ferroptosis.

scholarly journals Iron Causes Lipid Oxidation and Inhibits Proteasome Function in Multiple Myeloma Cells: A Proof of Concept for Novel Combination Therapies

Cancers ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 970 ◽  
Author(s):  
Jessica Bordini ◽  
Federica Morisi ◽  
Fulvia Cerruti ◽  
Paolo Cascio ◽  
Clara Camaschella ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Lines ◽  
Iron Toxicity ◽  
Ammonium Citrate ◽  
Ferric Ammonium Citrate ◽  
Ferric Carboxymaltose ◽  
High Dose ◽  
Iron Loading

Adaptation to import iron for proliferation makes cancer cells potentially sensitive to iron toxicity. Iron loading impairs multiple myeloma (MM) cell proliferation and increases the efficacy of the proteasome inhibitor bortezomib. Here, we defined the mechanisms of iron toxicity in MM.1S, U266, H929, and OPM-2 MM cell lines, and validated this strategy in preclinical studies using Vk*MYC mice as MM model. High-dose ferric ammonium citrate triggered cell death in all cell lines tested, increasing malondialdehyde levels, the by-product of lipid peroxidation and index of ferroptosis. In addition, iron exposure caused dose-dependent accumulation of polyubiquitinated proteins in highly iron-sensitive MM.1S and H929 cells, suggesting that proteasome workload contributes to iron sensitivity. Accordingly, high iron concentrations inhibited the proteasomal chymotrypsin-like activity of 26S particles and of MM cellular extracts in vitro. In all MM cells, bortezomib-iron combination induced persistent lipid damage, exacerbated bortezomib-induced polyubiquitinated proteins accumulation, and triggered cell death more efficiently than individual treatments. In Vk*MYC mice, addition of iron dextran or ferric carboxymaltose to the bortezomib-melphalan-prednisone (VMP) regimen increased the therapeutic response and prolonged remission without causing evident toxicity. We conclude that iron loading interferes both with redox and protein homeostasis, a property that can be exploited to design novel combination strategies including iron supplementation, to increase the efficacy of current MM therapies.

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