Reduced Response of IRE1α/Xbp-1 Signaling Pathway to Bortezomib Contributes to Drug Resistance in Multiple Myeloma Cells

2016 ◽  
Vol 103 (3) ◽  
pp. 261-267 ◽  
Author(s):  
Xiaoxuan Xu ◽  
Junru Liu ◽  
Beihui Huang ◽  
Meilan Chen ◽  
Shiwen Yuan ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Resistance Mechanism ◽  
Proteasome Inhibition ◽  
Myeloma Cells ◽  
Potential Marker ◽  
Rpmi 8226 ◽  
Basic Level

Purpose Proteasome inhibition with bortezomib eliminates multiple myeloma (MM) cells by partly disrupting unfolded protein response (UPR). However, the development of drug resistance limits its utility and resistance mechanism remains controversial. We aimed to investigate the role of IRE1α/Xbp-1 mediated branch of the UPR in bortezomib resistance. Methods The expression level of Xbp-1s was measured in 4 MM cell lines and correlated with sensitivity to bortezomib. LP1 and MY5 cells with different Xbp-1s level were treated with bortezomib; then pivotal UPR regulators were compared by immunoblotting. RPMI 8226 cells were transfected with plasmid pEX4-Xbp-1s and exposed to bortezomib; then apoptosis was determined by immunoblotting and flow cytometry. Bortezomib-resistant myeloma cells JJN3.BR were developed and the effect on UPR signaling pathway was determined. Results By analyzing 4 MM cell lines, we found little correlation between Xbp-1s basic level and bortezomib sensitivity. Bortezomib induced endoplasmic reticulum stress-initiated apoptosis via inhibiting IRE1α/Xbp-1 pathway regardless of Xbp-1s basic level. Exogenous Xbp-1s reduced cellular sensitivity to bortezomib, suggesting the change of Xbp-1s expression, not its basic level, is a potential marker of response to bortezomib in MM cells. Furthermore, sustained activation of IRE1α/Xbp-1 signaling pathway in JJN3.BR cells was identified. Conclusions Our data indicate that reduced response of IRE1α/Xbp-1 signaling pathway to bortezomib may contribute to drug resistance in myeloma cells.

Wnt3/RhoA/ROCK Signaling Pathway Is Involved in VLA6-Dependent Adhesion-Mediated Drug Resistance of Multiple Myeloma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2518-2518
Author(s):  
Masayoshi Kobune ◽  
Yutaka Kawano ◽  
Rishu Takimoto ◽  
Takuya Matsunaga ◽  
Junji Kato ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Cell Adhesion ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Stromal Cells ◽  
Critical Role ◽  
Rho Kinase ◽  
Myeloma Cell ◽  
Myeloma Cells

Abstract Adhesion of myeloma cells to BM stromal cells is now considered to play a critical role in chemo-resistance. However, little is known about the molecular mechanism of cell adhesion mediated drug resistance (CAM-DR) in multiple myeloma. In this study, we focused on relationship between drug resistance and expression of Wnts, the factor regulating the cell adhesion and proliferation, in myeloma cells. To gain insight into involvement of Wnt signaling in CAM-DR, we first screened the expression of Wnt family in myeloma cell lines (RPMI8226, ARH77, KMS-5 and MM1S) by reverse transcription-polymerase chain reaction analysis. Although the mRNAs of Wnt2b, Wnt7a and Wnt10b were variably expressed in some of myeloma cell lines, Wnt3 mRNA was detected in all the myeloma cells examined. KMS-5 and ARH77, which highly expressed Wnt3 protein, tightly adhered to human BM stromal cells and accumulation of β-catenin and GTP-bounded RhoA was observed in these myeloma cell lines. Conversely, RPMI8226 and MM1S, which modestly expressed Wnt3 protein, rather weakly adhered to human BM stromal cells. We then examined the relevance of Wnt3 expression to adhesive property to stromal cells and to CAM-DR of myeloma cells. KMS-5 and ARH-77 exhibited apparent CAM-DR against Doxorubicin. This CAM-DR was significantly reduced by anti-integrinβ1 antibody, anti- integrinα6 antibody and a Wnt-receptor competitor, secreted Frizzled related protein-1 and Rho kinase inhibitor (Y27632 and OH-fasudil), but not by the specific inhibitor of canonical signaling (DKK-1), indicating that Wnt-mediated CAM-DR which is dependent on integrinα6/β1 (VLA-6)-mediated attachment to stromal cells is induced by Wnt/RhoA-Rho kinase (ROCK) pathway signal. This CAM-DR for doxorubicin was also significantly reduced by Wnt3 siRNA transfer to KMS-5 and further augmented by addition of Wnt3 conditioned medium. These results indicate that Wnt3 contributes to VLA-6-mediated CAM-DR via the Wnt/RhoA/ROCK pathway of myeloma cells. Thus, the Wnt3/RhoA/ROCK signaling pathway could be a promising molecular target to overcome CAM-DR.

Biochemical Basis for Interactions Between Thalidomide and Inhibitors of the Isoprenoid Biosynthetic Pathway in Multiple Myeloma Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2635-2635
Author(s):  
Sarah A. Holstein ◽  
Huaxiang Tong ◽  
Raymond J. Hohl
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Annexin V ◽  
Differential Sensitivity ◽  
Parp Cleavage ◽  
Myeloma Cells ◽  
Protein Isoprenylation ◽  
Geranylgeranyl Transferase ◽  
Synthase Inhibitor ◽  
Rpmi 8226

Abstract Introduction: The isoprenoid biosynthetic pathway (IBP) is responsible for the production of key sterol and nonsterol species, including farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) which serve as substrates for protein isoprenylation reactions. Several agents known to target the IBP have been observed to have cytotoxic effects in multiple myeloma cells. Thalidomide (Thal) has emerged as an effective agent for treating multiple myeloma. While Thal has been noted to have a variety of direct and indirect effects on myeloma cells, the precise mechanism of action remains unknown. Aim: We examined interactions between inhibitors of the IBP and Thal in multiple myeloma cells. The mechanisms underlying the observed differential sensitivity to these agents were explored. Methods: Studies were performed in three human multiple myeloma cell lines (RPMI-8226, U266, H929). Cytotoxicity was assessed via MTT assays, while apoptosis induction was determined by Annexin V staining and evaluation of PARP cleavage. Western blot analysis was used to evaluate inhibition of protein isoprenylation. Intracellular FPP and GGPP levels were measured via enzymatic coupling to fluorescently-tagged peptides, HPLC fractionation and fluorescence detection. Pharmacologic manipulation of the IBP was achieved with the following agents: lovastatin (Lov) as an HMG-CoA reductase inhibitor, zoledronic acid (ZA) as a FPP synthase inhibitor, digeranyl bisphosphonate (DGBP) as a GGPP synthase inhibitor, FTI-277 as a farnesyl transferase inhibitor (FTI), and GGTI-286 as a geranylgeranyl transferase I inhibitor (GGTI). Results: Addition of Thal to Lov (at both 24 & 48h), zoledronic acid (at 48h), or DGBP (at 24 & 48h) in RPMI-8266 cells results in marked enhancement in cytotoxicity. Isobologram analysis could not be performed as Thal by itself does induce cytotoxicity in MTT assays. Although Lov induces cytotoxicity in a concentration- and time-dependent manner in the U266 and H929 cells, the addition of Thal did not result in increased cytotoxicity. Neither ZA nor DGBP induced cytotoxicity in the U266 cell line, while the H929 cell line showed effects only at 48 hours. Addition of Thal to FTI or GGTI did not result in enhanced cytotoxicity in tested cell lines. Annexin V experiments confirmed enhanced induction of apoptosis in RPMI-8226 cells incubated with the combination of Thal/Lov or Thal/DGBP. Add-back experiments revealed that the enhanced cytotoxicity/induction of apoptosis observed with the addition of Thal could be prevented with the addition of mevalonate or GGPP in Lov-treated cells or GGPP in DGBP-treated cells. PARP cleavage was demonstrated in RPMI-8226 and H929 cells treated with Lov or DGBP (with or without Thal) and in U266 cells treated with Lov. As expected, Lov resulted in the accumulation of unmodified forms of proteins normally farnesylated (Ras) and geranylgeranylated (Rap1a and Rab6) in these cells. Interestingly however, while DGBP led to accumulation of unmodified Rap1a and Rab6 in RPMI-8226 and H929 cells, no effect was seen in the U266 line. Examination of intracellular levels of FPP and GGPP revealed that the U266 line has markedly larger pools of FPP (8.5-fold) and GGPP (2.7-fold) compared to RPMI-8226 and that treatment with DGBP only partially depletes U266 cells of GGPP. Conclusions: These studies demonstrate an interaction between thalidomide and IBP inhibitors in multiple myeloma cells. These effects appear dependent on depletion of GGPP. Since treatment with a geranylgeranyl transferase-I inhibitor does not produce similar results, this suggests that substrates of geranylgeranyl transferase-II, such as the Rab proteins, may play critical roles in myeloma pathophysiology. The finding that intracellular levels of FPP and GGPP vary markedly amongst cell lines explains differential sensitivity of these cells to pharmacologic manipulation of the IBP and may also influence sensitivity to chemotherapeutic agents. Further studies will determine the extent to which isoprenoid pool sizes vary in primary samples and may ultimately allow for the identification of multiple myeloma patients who would benefit from the addition of an IBP inhibitor to their treatment plan. Figure Figure

Proteasome Maturation Protein (pomp) Is Associated With Proteasome Inhibitor Resistance In Myeloma, and Its Suppression Enhances The Activity Of Bortezomib and Carfilzomib

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 280-280 ◽  
Author(s):  
Bingzong Li ◽  
Hua Wang ◽  
Robert Z. Orlowski
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Xenograft Model ◽  
Proteasome Inhibition ◽  
Over Expression ◽  
Murine Xenograft Model ◽  
Drug Naïve ◽  
Inhibitor Resistance ◽  
Rpmi 8226

Abstract Background Proteasome inhibition with bortezomib has revolutionized the treatment of multiple myeloma, but the vast majority of patients eventually develop clinical bortezomib resistance through poorly understood mechanisms. One of the most conserved cellular responses to proteasome inhibition is to up-regulate proteasome subunit expression, presumably with the goal of enhancing proteasome activity and restoring intracellular protein homeostasis. We therefore hypothesized that proteasome inhibitor resistance could be associated with enhanced proteasome assembly, and that suppression of this assembly process could help restore drug sensitivity. The current studies focused on POMP, which is involved in addition of catalytically active b subunits to the hemiproteasome ring initially formed by structural a subunits. Methods We studied ANBL-6, KAS-6/1, OPM-2, and RPMI 8226 multiple myeloma cell lines which had acquired bortezomib resistance through prolonged exposure to increasing drug concentrations, and compared them to their drug-naïve, vehicle-treated counterparts. In addition, we evaluated primary cells derived from patients with myeloma, and examined an in vivo murine xenograft model of human myeloma. Results Bortezomib-resistant (V10R) ANBL-6, KAS-6/1, OPM-2, and RPMI 8226 cell lines showed enhanced levels of POMP mRNA by quantitative (q) PCR compared to their drug-sensitive counterparts, which was associated with higher POMP protein levels seen by immunoblotting. Exogenous over-expression of POMP in drug-naïve OPM-2 and KAS-6/1 cells was sufficient by itself to induce resistance to both bortezomib and carfilzomib. Conversely, suppression of POMP with one of two different Lentiviral small hairpin (sh) RNAs restored sensitivity in OPM-2 and KAS-6/1 V10R cells to bortezomib and carfilzomib. Since no known pharmaceuticals directly target POMP, we examined its promoter region, and found a consensus binding site for nuclear factor (erythroid-derived 2)-like (NRF) 2. Consistent with a role of NRF2 in POMP expression, NRF2 mRNA and protein were increased in V10R myeloma cells, and in drug-naïve cells treated with bortezomib. Moreover, transfection of cells with NRF2 cDNA activated a POMP promoter reporter, while chromatin immunoprecipitation with anti-NRF2 antibodies preferentially precipitated sequences near the POMP promoter. Also, NRF2 over-expression induced POMP and enhanced proteasome chymotrypsin-like activity, while its suppression had the opposite effects. All trans-retinoic acid (ATRA) blocked nuclear accumulation of NRF2 in OPM-2 and KAS-6/1 V10R cells, and reduced expression of POMP. Combinations of bortezomib with ATRA showed enhanced activity against these drug-resistant cell lines in association with greater proteasome inhibition, and were synergistic in drug-naïve cells. In primary samples, ATRA with bortezomib induced a greater reduction in viability than did either treatment alone. Finally, in a murine xenograft model with OPM-2 V10R cells, neither ATRA nor bortezomib showed substantial activity, while the combination regimen, by comparison, retained efficacy. Conclusions Taken together, our data support the hypotheses that NRF-2-influenced POMP over-expression contributes to proteasome inhibitor resistance in multiple myeloma, while approaches targeting POMP hold promise in overcoming resistance. Moreover, they provide a framework for translation of proteasome inhibitors with ATRA to the clinic to enhance activity, and to overcome resistance to this important class of anti-myeloma agents. Disclosures: No relevant conflicts of interest to declare.

Dual Antitumoral and Bone Antiresorptive Effect Of The Pan-Pim Kinase Inhibitor, LGH447, In Multiple Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4435-4435
Author(s):  
Teresa Paíno ◽  
Antonio Garcia-Gomez ◽  
Lorena González-Méndez ◽  
Laura San-Segundo ◽  
Montserrat Martín-Sánchez ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Annexin V ◽  
Myeloma Cells ◽  
Pim Kinases ◽  
Castilla Y Leon ◽  
Ic50 Values ◽  
Rpmi 8226 ◽  
Resorptive Activity

Introduction Multiple myeloma (MM) is characterized by the accumulation of malignant plasma cells in the bone marrow (BM) and is closely associated with osteolytic lesions, in part due to an increase in the bone-resorptive activity and number of osteoclasts (OCs). The activation of survival pathways in myeloma cells could be the cause of treatment failure rendering the disease incurable. Pim kinases are a family of survival serine/threonine kinases composed of three members (Pim1, Pim2 and Pim3) that are overexpressed in MM cells and may have a role in MM pathogenesis. However, little is known about the role of Pim kinases in OCs and its involvement in myeloma bone disease. Here, we have evaluated the preclinical activity of a new pan-Pim kinase inhibitor, LGH447, on MM cells and OCs. Cell lines, primary samples, material and methods LGH447 was provided by Novartis Pharmaceuticals. The human MM cell lines MM1S, MM1R, RPMI-8226 (or RPMI-8226-luc), RPMI-LR5, MM144, NCI-H929, OPM-2, U266, U266-Dox4 and U266-LR7 were employed. PBMCs from healthy volunteers were used to generate OCs, whereas primary mesenchymal stromal cells (MSCs) were obtained from bone marrow aspirates of MM patients. Cell viability was studied using MTT colorimetric assay or bioluminescence. Apoptosis was measured by annexin-V staining. For cell cycle analysis, propidium iodide staining was used. OC formation was assessed by enumeration of multinucleated (≥3) TRAP-positive cells and OC resorption was assessed on calcium-coated slides. Immunoblotting, quantitative PCR and immunofluorescence were used to further investigate the mechanism of action of LGH447. Results All MM cell lines expressed the three isoforms of Pim kinases with higher levels of Pim2. The dose-response curves to LGH447 after a 48 hour treatment revealed two groups of MM cell lines with regard to sensitivity to this drug: high sensitive, with IC50 values ranging from 0.2 to 3.3 µM (MM1S, MM1R, RPMI-8226, MM144, U266 and NCI-H929); and low sensitive, with IC50 values >7 µM (OPM-2, RPMI-LR5, U266-Dox4 and U266-LR7). Our results indicated that LGH447 promoted apoptosis in myeloma cells as shown by the increase in annexin-V positive cells and by the cleavage of initiator (caspases 8 and 9) and effector caspases (caspases 3 and 7) and of PARP. LGH447 also blocked the cell cycle in MM cells as demonstrated by the increase in G0-G1 and the decrease in S-G2-M phases. Importantly, LGH447 was also able to overcome the growth advantage conferred to RPMI-8226-luc cells by co-culture with MSCs or OCs. Regarding the mechanisms involved in these effects, LGH447 inhibited the mTOR pathway, demonstrated by a decreased phosphorylation of the downstream mTOR effectors, 4EBP1 and S6 in residues Thr37/46 and Ser235/236, respectively. Interestingly, LGH447 also inhibited OC formation and resorption activity. LGH447 treatment of human pre-OCs diminished the expression of key molecules involved in OC differentiation (p-Erk1/2 and NFATc1) and function [CAII (carbonic anhidrase II), CLCN7 (chloride channel 7), ATP6V1A (vacuolar-H+-ATPase catalytic subunit A1) and MMP9 (matrix metalloproteinase 9)] and also disrupted the F-actin ring necessary for OC effective resorption. Conclusion Overall, our results demonstrate that both MM cells and OCs are targets of the pan-Pim kinase inhibitor, LGH447. Therefore, the inhibition of Pim kinases could potentially provide a dual benefit in myeloma patients as a consequence of cytotoxic effects exerted on MM cells and an anti-resorptive activity on bone. This work was supported by funding from the Fundación Española de Hematología y Hemoterapia (AG-G), Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León, the RTICC-Hematology Group (RD12/0036/0058), Spanish FIS (PI12/02591) and the Junta de Castilla y León, Gerencia Regional de Salud (GRS 862/A/13). Disclosures: Off Label Use: LGH447 is a pan-Pim kinase inhibitor (Novartis Pharmaceuticals). It has been used for pre-clinical studies in multiple myeloma.

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 Polyunsaturated Fatty Acid (PUFA) Signaling Induces Ferroptosis-Mediated Cell-Death in Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3108-3108
Author(s):  
Cristina Panaroni ◽  
Keertik Fulzele ◽  
Rosemary Soucy ◽  
Cherrie Huang ◽  
Kenta Mukaihara ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Multiple Myeloma ◽  
Cell Death ◽  
Fatty Acid ◽  
Signaling Pathways ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Specific Gene ◽  
Myeloma Cells ◽  
Cox Inhibitor

Altered cellular metabolic pathways are the hallmark of tumor cells. Multiple myeloma (MM) is positively correlated with metabolic disorders such as obesity and Gaucher's disease. The local bone marrow (BM) microenvironment (TME) majorly influences the initiation and progression of MM. In a typical MM patient, BM adipocytes make up 70% of the cellular volume. The abundance of adipocyte-secreted free fatty acids (FFA) may shift myeloma cellular metabolism from aerobic glycolysis to more energy-producing fatty acid oxidation. The FFAs are important catalysts of key downstream drug-targetable signaling pathways such as cyclooxygenase (COX), cytochrome P450 (CYP), and lipoxygenase (LOX) pathways. In this study, we hypothesized that altered lipid profile in the local BM TME contributes to MM progression. BM-Fat enriched tissue isolated from BM aspirates of Monoclonal Gammopathy of Undetermined Significance (MGUS) and smoldering MM (SMM) patients showed a significant increase in adipogenic PPARγ gene expression compared to aged-matched healthy donors (N≥3). The BM mesenchymal stem/progenitor cells (BMSCs) from MGUS/SMM patients expressed normal levels of BMSC markers CD271, CD105, CD44, CD106, CD29, CD90, CD49e, and Notch4 but showed significantly increased expression of adipogenic markers including Preadipocyte factor 1, Leptin Receptor, and Perilipin A (N=6). This also translated into significantly increased adipogenic differentiation of patient BMSCs when cultured alone or with the human MM cell-line MM.1S (N≥3). Furthermore, MM.1S showed significantly increased proliferation when co-cultured with BMSCs from MGUS/SMM patients (N=5). These data demonstrate a vicious cycle where adipogenesis is increased in early precursor MM stages that further support the growth of myeloma cells. We performed gas chromatometry based lipidomics analysis on the supernatant of BM aspirates from MGUS, SMM, and newly diagnosed MM (NDMM) patients. The analysis identified significant decreases in key polyunsaturated fatty acids (PUFA) including Arachidonic Acid (AA) and Docosatetraenoic acid (N≥5). Lipid metabolism specific gene array on RNA from adipose tissue fraction of BM aspirates from MGUS, SMM and NDMM patients showed altered changes in genes responsible for fatty acid synthesis and metabolism. PUFA are involved in anti-inflammatory mechanisms in cancer. We hypothesized that increased levels of certain PUFA, such as AA, in the BM TME may decrease MM progression. To test this hypothesis, we treated MM cells with physiological doses of AA. AA dose-dependently decreased proliferation and viability of human MM cell lines, MM1S, H929, and U266, and CD138+ patient myeloma cells. For in vivo studies, humanized MM tumor model was generated in SCID mice by growing MM.1S cells in the intrascapular subcutaneous region for 3-weeks. Mice were then treated with daily localized injections of vehicle, 100µg/g AA, 500µg/g of AA, or IV with 2mg/kg/biweekly Carfilzomib (CFZ), or CFZ with 500µg/g of AA (COMBO). Tumor volume significantly decreased in 500µg/g AA treatment group beginning 10-days and was comparable to the CFZ treatment. Gross examination and flow cytometry analysis of CD138+ myeloma cells showed dramatically increased tumor-cell apoptosis in 500µg/g AA and COMBO treatment groups. To identify the primary apoptosis-inducing AA signaling pathway in MM cells, we used specific inhibitors of each of these signaling pathways including ibuprofen (Cox inhibitor), baicalein (12-LOX inhibitor), BW B70C (5,15-LOX inhibitor), 1-aminobenzotriazole (CYP450 inhibitor), and ferrostatin (Ferroptosis/lipid peroxidation inhibitor). Among these compounds, ferrostatin treatment completely rescued AA induced apoptosis in the human MM.1S cells. Ferroptotic cell death is the result of an accumulation of lipid peroxides which is generally prevented by the enzyme Glutathione peroxidase 4 (GPX4). We, therefore investigated the role of AA on GPX4 and found that all MM cell lines partially or completely lost the expression of GPX4 when exposed to AA and that this effect was completely prevented when cotreated with Ferrostatin. Taken together, we show that BM adipocytes promote myeloma cell proliferation at least in part through secreted FFAs. Therapeutically targeting members of this signaling pathway, such as ferroptosis, is a potential novel treatment strategy for MM especially in the MGUS and SMM stages. Disclosures Raje: Celgene Corporation: Consultancy; Amgen Inc.: Consultancy; Bristol-Myers Squibb: Consultancy; Takeda: Consultancy; Janssen: Consultancy; Merck: Consultancy.

scholarly journals Exosomal miR-135b shed from hypoxic multiple myeloma cells enhances angiogenesis by targeting factor-inhibiting HIF-1

Blood ◽  
2014 ◽  
Vol 124 (25) ◽  
pp. 3748-3757 ◽  
Author(s):  
Tomohiro Umezu ◽  
Hiroko Tadokoro ◽  
Kenko Azuma ◽  
Seiichiro Yoshizawa ◽  
Kazuma Ohyashiki ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Tube Formation ◽  
Myeloma Cells ◽  
Endothelial Tube Formation ◽  
Key Points ◽  
Resistant Cells

Key Points We established hypoxia-resistant cells that can mimic in vivo conditions of hypoxic bone marrow. Exosomal miR-135b derived from these cell lines enhanced endothelial tube formation under hypoxia via the HIF-FIH signaling pathway.

Type I MAGE Proteins: Novel Markers of Proliferation in Multiple Myeloma Progenitor Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5115-5115
Author(s):  
Hearn J. Cho ◽  
Otavia Caballero ◽  
Achim A. Jungbluth ◽  
Maurizio DiLiberto ◽  
Ruben Niesvizky ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Molecular Markers ◽  
Cell Line ◽  
Progenitor Cells ◽  
Cell Lines ◽  
Primary Antibody ◽  
Type I ◽  
Color Analysis ◽  
Myeloma Cells ◽  
Rpmi 8226

Abstract CT7 (MAGE-C1) and MAGE-A3, two members of the type I MAGE family of Cancer-Testis antigens, are commonly expressed at both the mRNA and protein levels in primary tumor specimens from multiple myeloma patients. In previous analyses, tumors with higher percentages of type I MAGE-expressing cells had a positive correlation with abnormally elevated plasma cell proliferation. These data support the hypothesis that type I MAGE proteins are molecular markers of proliferating myeloma progenitor cells (the so-called “myeloma stem cell”) and may play a role in the pathobiology of this disease. To test this hypothesis, we examined the expression of type I MAGE in proliferating myeloma cells by flow cytometry. Human multiple myeloma cell lines U266, RPMI-8226, and KMS-11 were co-cultured for 12 or 24 hours with the nucleoside analog bromodeoxyuridine (BrdU), then fixed, permeabilized, and stained with CT7-33, a monoclonal antibody (mAb) to CT7, or M3H67 (to MAGE-A3), followed by a phycoerythrin (PE)-conjugated secondary mAb. The cells were then treated with DNAse and stained with a fluoroisothiocyanate (FITC)-conjugated mAb against BrdU. Proliferating cells that incorporated BrdU into their DNA exhibited high FITC fluorescence. For mAb M3H67, dual color analysis of this population showed that greater than 99% demonstrated a significant shift in PE fluorescence in all three of these cell lines as measured by Mean Fluorescence Index (MFI= geometric mean fluorescence [specific primary antibody]/mean fluorescence [no primary antibody], table 1). For CT7-33 mAb, greater than 85% demonstrated a shift in two of three lines (U266 and KMS-11), but not in RPMI-8226. For all three of these cell lines, dual color analysis of the BrdU-low population demonstrated less than 65% staining with either type I MAGE mAb. Interestingly, RT-PCR with CT7-specific primers of total RNA from RPMI-8226 revealed a product of lower molecular weight than expected, suggesting that a gene deletion occurred in this cell line possibly resulting in a stop codon, decreased translation, or decreased protein stability. This PCR product is being sequenced to determine the nature of the deletion. These results demonstrate that type I MAGE proteins are expressed in proliferating myeloma cells and are molecular markers of this population. These data suggest that novel therapeutics such as vaccines that target type I MAGE may preferentially eliminate the cycling myeloma cells, resulting in long-term cures. Table 1. Type I MAGE expression in proliferating (BrdU+) myeloma cells Cell line MFI CT7-33 MFI M3H67 U266 65.8 62.5 KMS-11 9.9 48.4 RPMI-8226 3.1 12.3

Autocrine Sonic Hedgehog Protects Multiple Myeloma Cells From Apoptosis and Enhances Drug Resistance

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2904-2904
Author(s):  
Zhiqiang Liu ◽  
Yuhuan Zheng ◽  
Haiyan Li ◽  
Yong Lu ◽  
Donna M. Weber ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Tumor Growth ◽  
Signaling Pathway ◽  
Sonic Hedgehog ◽  
Hedgehog Signaling ◽  
Conflicts Of Interest ◽  
Myeloma Cells ◽  
Apoptosis Rate ◽  
Chemotherapy Drugs

Abstract Abstract 2904 The secreted protein sonic hedgehog (SHH) and the hedgehog signaling are of great importance in proliferation and differentiation of cells in the hematopoietic system, and also play a vital role in oncogenesis of B cell malignance. However, the functions and mechanism of SHH signaling in multiple myeloma (MM) is mostly unknown. Thus far, aberrant activation of the hedgehog signaling in tumor growth promoting and/or survival capabilities as well as a paracrine model of SHH secretion have been demonstrated in MM. In the current study, we demonstrated a new autocrine SHH functioning manner in MM cells. The Shh mRNA and the SHH protein were highly expressed both in the MM cell lines and in purified CD138+ MM cells from patients using real-time PCR, Western Blot and immunohistochemistry analyses, respectively; and the SHH protein was also detected in the culture medium. Accordingly, the Hh ligand receptor PTCH1 and PTCH2 as well as the transcriptional factor GLI1 were all overexpressed in MM cells, indicating the activation of Hh signaling pathway. Autocrine SHH played a role in MM cells survival and protected MM cells from apoptosis in vitro, and autocrine SHH accelerated xenograft tumor growth in myeloma-SCID mouse model in vivo. Moreover, autocrine SHH enhanced drug resistance of MM cells, as SHH overexpressed CAG cells (SHH+CAG) had a significantly low apoptosis rate when treated with chemotherapy drugs dexamethasone or bortezomib, as compared with wild type cells (wt-CAG). On the contrary, SHH knockdown cells (SHH-CAG) had a dramatically higher apoptosis rate. Blocking autocrine SHH ligand and treating cells with dexamethasone or bortezomib significantly improved the drug killing effect. Finally, we found that upregulated BLC2 via SHH-Gli1signaling is the signaling pathway by which MM cells enhanced the drug resistance. Our study provides a new insight into the biologic function of the autocrine SHH in proliferation, survival and the drug resistance in the myeloma cells. Disclosures: No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document