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.

Wnt3 Signaling Augments integrinβ1-Dependent Adhesion-Mediated Drug Resistance of Multiple Myeloma in Autocrine and Paracrine Manner.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1698-1698
Author(s):  
Masayoshi Kobune ◽  
Kazuyuki Murase ◽  
Satoshi Iyama ◽  
Tsutomu Sato ◽  
Yutaka Kawano ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Cell Adhesion ◽  
Wnt Signaling ◽  
Cell Lines ◽  
Stromal Cells ◽  
Critical Role ◽  
Specific Inhibitor ◽  
Myeloma Cell ◽  
Beta Catenin ◽  
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 MM. 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 beta-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, 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 integrinβ1- mediated attachment to stromal cells is induced by Wnt/Rho pathway signal. This CAM-DR for doxorubicin was also significantly reduced by Wnt3 siRNA transfer to KMS-5. On the other hand, the cell adhesion of MM1S was dramatically augmented by addition of Wnt3 containing conditioned medium (CM) and suppressed by integrinα4 or β1 antibody (VLA4)(Fig 1). Furthermore, CAM-DR of MM1S was significantly augmented by Wnt3 CM or adhesion of mesenchymal stem cells which expressed Wnt3, but not BM stromal cells which did not express Wnt3 mRNA. These results suggest that adhesion of myeloma cells on stromal cells was regulated by Wnt signaling in autocrine or paracrine manner. The Wnt3 signaling pathway could be a promising molecular target to overcome CAM-DR.

Bortezomib Overcomes Cell Adhesion-Mediated Drug Resistance Via Down-Regulation of VLA-4 Expression in Multiple Myeloma.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1634-1634 ◽  
Author(s):  
Kaoru Hatano ◽  
Jiro Kikuchi ◽  
Masaaki Takatoku ◽  
Rumi Shimizu ◽  
Taeko Wada ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Cell Adhesion ◽  
Critical Role ◽  
Myeloma Cell ◽  
Functional Screening ◽  
Myeloma Cells ◽  
A Subunit ◽  
Bona Fide ◽  
Blocking Antibodies

Abstract Multiple myeloma (MM) is incurable, mainly because of cell adhesion-mediated drug resistance (CAM-DR). In this study, we performed functional screening using shRNA to define the molecule(s) responsible for CAM-DR of MM. Using 4 bona fide myeloma cell lines (KHM-1B, KMS12-BM, RPMI8226, and U266) and primary myeloma cells, we identified CD29 (b1-integrin), CD44, CD49d (a4-integrin, a subunit of VLA-4), CD54 (ICAM-1), CD138 (syndecan-1) and CD184 (CXCR4) as major adhesion molecules expressed on MM. Short hairpin RNA-mediated knockdown of CD49d but not CD44, CD54, CD138, and CD184 significantly reversed CAM-DR of myeloma cells to bortezomib, vincristine, doxorubicin, and dexamethasone. Experiments using blocking antibodies yielded almost identical results. Bortezomib was relatively resistant to CAM-DR because of its ability to specifically down-regulate CD49d expression. This property was unique to bortezomib and was not observed in other anti-myeloma drugs. Pretreatment with bortezomib was able to ameliorate CAM-DR of myeloma cells to vincristine and dexamethasone. These results suggest that VLA-4 plays a critical role in CAM-DR of MM cells. The combination of bortezomib with conventional anti-myeloma drugs may be effective in overcoming CAM-DR of MM.

Wnt3 Augments Adhesion Mediated Drug Resistance of Myeloma Cells Via Wnt/RhoA Pathway.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5075-5075 ◽  
Author(s):  
Hiroki Chiba ◽  
Masayoshi Kobune ◽  
Kazunori Kato ◽  
Kiminori Nakamura ◽  
Yutaka Kawano ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Cell Adhesion ◽  
Wnt Signaling ◽  
Cell Lines ◽  
Stromal Cells ◽  
Myeloma Cell ◽  
Canonical Pathway ◽  
Beta Catenin ◽  
Myeloma Cells ◽  
Rhoa Signaling

Abstract Despite the tremendous effort in developing of conventional chemotherapy and molecular targeting drugs for patients with multiple myeloma (MM), it has been proven difficult to completely abrogate neoplastic cells from bone marrow (BM). Hence, patients with refractory disease still experience poor outcome due to disease progression. Principle obstacle in the treatment of this disease is a chemo-resistance which is mainly caused by the interaction of myeloma cells with BM stromal cells. However, little is known about the molecular mechanism of cell adhesion mediated drug resistance (CAM-DR) in MM. 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, HS-sultan and MM1S) by reverse transcription (RT)-polymerase chain reaction (PCR) 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. RPMI8226, ARH77 and KMS-5 which highly expressed Wnt3 protein, tightly adhered to human BM stromal cells and accumulation of beta-catenin and GTP-bounded RhoA was observed in these myeloma cell lines. This cell adhesion was augmented by addition of Wnt3 containing conditioned medium (CM) and suppressed by Wnt-receptor competitor, secreted Frizzled related protein (sFRP)-1, but not by specific inhibitor of canonical pathway (DKK-1). These results suggest that adhesion of myeloma cells was regulated by non-canonical pathway of Wnt signaling. We further examined whether the Wnt3 mediated adhesion to stromal cells involved in CAM-DR. The drug resistance of ARH-77 for doxorubicin was 1.8 folds enhanced by adhesion to stromal cells in comparison with stroma-free condition. This CAM-DR for doxorubicin was further augmented (2.6 folds) by addition of Wnt3 CM via enhancement of adhesion to stromal cells. Moreover, although the doxorubicin sensitivity of ARH-77 in coculture with stromal cells was significantly reduced by sFRP-1, this effect was not observed in stroma-free culture, indicating that Wnt3-mediated CAM-DR is dependent on attachment with stromal cells. Additionally, CAM-DR was completely restrained by addition of Rho kinase inhibitor Y27632. These results indicate that Wnt3 augments myeloma CAM-DR by enhancement of adhesion to human BM stromal cells via Wnt/RhoA signaling. Thus, Wnt/RhoA signaling pathway could be a promising molecular target to overcome CAM-DR.

scholarly journals An inhibitor of the EGF receptor family blocks myeloma cell growth factor activity of HB-EGF and potentiates dexamethasone or anti–IL-6 antibody-induced apoptosis

Blood ◽  
2004 ◽  
Vol 103 (5) ◽  
pp. 1829-1837 ◽  
Author(s):  
Karène Mahtouk ◽  
Michel Jourdan ◽  
John De Vos ◽  
Catherine Hertogh ◽  
Geneviève Fiol ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Growth Factor ◽  
Cell Growth ◽  
Cell Lines ◽  
Stromal Cells ◽  
Egf Receptor ◽  
Myeloma Cell ◽  
Factor Activity ◽  
Myeloma Cells ◽  
Induced Apoptosis

Abstract We previously found that some myeloma cell lines express the heparin-binding epidermal growth factor–like growth factor (HB-EGF) gene. As the proteoglycan syndecan-1 is an HB-EGF coreceptor as well as a hallmark of plasma cell differentiation and a marker of myeloma cells, we studied the role of HB-EGF on myeloma cell growth. The HB-EGF gene was expressed by bone marrow mononuclear cells in 8 of 8 patients with myeloma, particularly by monocytes and stromal cells, but not by purified primary myeloma cells. Six of 9 myeloma cell lines and 9 of 9 purified primary myeloma cells expressed ErbB1 or ErbB4 genes coding for HB-EGF receptor. In the presence of a low interleukin-6 (IL-6) concentration, HB-EGF stimulated the proliferation of the 6 ErbB1+ or ErbB4+ cell lines, through the phosphatidylinositol 3-kinase/AKT (PI-3K/AKT) pathway. A pan-ErbB inhibitor blocked the myeloma cell growth factor activity and the signaling induced by HB-EGF. This inhibitor induced apoptosis of patients'myeloma cells cultured with their tumor environment. It also increased patients' myeloma cell apoptosis induced by an anti–IL-6 antibody or dexamethasone. The ErbB inhibitor had no effect on the interaction between multiple myeloma cells and stromal cells. It was not toxic for nonmyeloma cells present in patients' bone marrow cultures or for the growth of hematopoietic progenitors. Altogether, these data identify ErbB receptors as putative therapeutic targets in multiple myeloma.

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.

Sphingolipids As a Novel Target For The Treatment Of Multiple Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3163-3163 ◽  
Author(s):  
Jagadish Kummetha Venkata ◽  
Robert K Stuart ◽  
Luciano J Costa ◽  
Ningfei An ◽  
Houjian Cai ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Growth ◽  
Cell Lines ◽  
Stromal Cells ◽  
Myeloma Cell ◽  
Sphingolipid Metabolism ◽  
Myeloma Cells ◽  
S1p Receptors

Abstract Introduction Multiple Myeloma (MM) is the second most common hematological malignancy in the United States and accounts for ∼10,600 deaths annually. MM remains an incurable disease and almost all patients will eventually relapse and become refractory to currently available therapeutic agents. There is an unmet need for better understanding of the disease’s molecular pathways and identifying novel therapeutic targets. Sphingolipid metabolism is being increasingly recognized as a key pathway in cancer biology. In particular, sphingosine kinases (SK1 and SK2) provide a potential site for manipulation of the ceramide / sphingosine 1-phosphate (S1P) rheostat that regulates the balance between tumor cell proliferation and apoptosis, as well as tumor sensitivity to drugs. Currently, very little is known about sphingolipid metabolism in MM. We herein for the first time provide a detailed analysis of sphingolipid metabolism in MM and demonstrate the potential of targeting SK2 for the treatment of MM. Methods We first quantified sphingolipid metabolites and sphingolipid metabolizing genes in myeloma cell lines, in freshly isolated human primary CD138+ myeloma cells, and in a publically available gene expression dataset from MM patients. We then tested the anti-myeloma activity of SK2-specific shRNA and determined the efficacy of a selective SK2 inhibitor (ABC294640) in killing myeloma cell lines and primary human myeloma cells in vitro. The mechanistic pathway of apoptosis was analyzed by immunoblotting and flowcytometry. MM cell lines stably expressing luciferase and eGFP were generated for xenograft experiments and for in vitro co-cultures with stromal cells. Results From the publically available GSE6477 microarray data set, we found that one third of the genes involved in sphingolipid metabolism were significantly different in CD138+ MM cells from newly diagnosed MM patients compared to normal individuals, including SK2 and S1P receptors. In 5 MM cell lines compared to immortalized B cells (IBC), 19 key sphingolipid metabolites were measured, and we found that ceramides were significantly reduced whereas S1P was significantly increased. mRNA analyses of 11 sphingolipid metabolizing genes including S1P receptors in 7 MMs showed that SK1, SK2, and alkaline ceramidases were significantly increased compared to IBC. Furthermore, we isolated CD138+ myeloma cells from 21 MM patients and found that 13 of the patients had higher SK2 expression in CD138+ MM cells compared to CD138-cells. These data demonstrated abnormal sphingolipid metabolism and dys-regulated SK2 in myeloma cells. We generated SK2-specific shRNA and found that SK2 shRNA down-regulated SK2 mRNA, inhibited proliferation, and induced death in myeloma cells, suggesting that SK2 is important in myeloma cell survival. We then tested the efficacy of ABC294640 (the most-advanced, non-lipid SK2 inhibitor) in 6 MM cell lines. ABC294640 inhibited myeloma cell growth with an IC50s of ∼30 μM, including steroid-resistant and doxorubicin-resistant myeloma cells. ABC294640 inhibited MM cell growth as early as 6 hours after exposure and induced apoptotic cell death as demonstrated by Annexin V staining, PARP cleavage and caspase 9 activation. ABC294640 inhibited primary human CD138+MM cells with the same efficacy as with MM cell lines, demonstrating the potential of ABC294640 for the treatment of MM. Additionally, we found that blocking S1P receptors with FTY720 (a S1PR agonist with receptor degradation) induced apoptosis in MM cells. We performed extensive mechanistic and signaling pathway analyses and found that ABC294640 inhibited Mcl-1 and C-Myc expression, but had no effects on Bcl2. Furthermore, ABC294640 induced cell death by directing Mcl-1 to proteosomal degradation. MM is dependent on the bone marrow niche microenvironment for survival and progression. We found that ABC294640 was effective in inducing apoptosis in MM cells even in the presence of stromal cells. Finally, we are currently testing the in vivo effect of ABC294640 alone and in combination with bortezomib, thalidomide and dexamethasone in MM xenograft model transplanted with MM cells stably expressing luciferase. Our early preliminary results were encouraging. Conclusion Our data demonstrate that sphingolipid metabolism is abnormal and provides an attractive target in the treatment of refractory/relapsed MM. Disclosures: Costa: Otsuka: Research Funding.

Cell Adhesion Mediated Drug Resistance (CAM-DR): Role of Integrins and Resistance to Apoptosis in Human Myeloma Cell Lines

Blood ◽  
1999 ◽  
Vol 93 (5) ◽  
pp. 1658-1667 ◽  
Author(s):  
Jason S. Damiano ◽  
Anne E. Cress ◽  
Lori A. Hazlehurst ◽  
Alexander A. Shtil ◽  
William S. Dalton
Keyword(s):  
Drug Resistance ◽  
Cell Adhesion ◽  
Cell Survival ◽  
Cell Lines ◽  
Drug Exposure ◽  
Myeloma Cell ◽  
Cytotoxic Drugs ◽  
Survival Advantage ◽  
Drug Resistant ◽  
Myeloma Cells

Abstract Integrin-mediated adhesion influences cell survival and may prevent programmed cell death. Little is known about how drug-sensitive tumor cell lines survive initial exposures to cytotoxic drugs and eventually select for drug-resistant populations. Factors that allow for cell survival following acute cytotoxic drug exposure may differ from drug resistance mechanisms selected for by chronic drug exposure. We show here that drug-sensitive 8226 human myeloma cells, demonstrated to express both VLA-4 (4β1) and VLA-5 (5β1) integrin fibronectin (FN) receptors, are relatively resistant to the apoptotic effects of doxorubicin and melphalan when pre-adhered to FN and compared with cells grown in suspension. This cell adhesion mediated drug resistance, or CAM-DR, was not due to reduced drug accumulation or upregulation of anti-apoptotic Bcl-2 family members. As determined by flow cytometry, myeloma cell lines selected for drug resistance, with either doxorubicin or melphalan, overexpress VLA-4. Functional assays revealed a significant increase in 4-mediated cell adhesion in both drug-resistant variants compared with the drug-sensitive parent line. When removed from selection pressure, drug-resistant cell lines reverted to a drug sensitive and 4-low phenotype. Whether VLA-4–mediated FN adhesion offers a survival advantage over VLA-5–mediated adhesion remains to be determined. In conclusion, we have demonstrated that FN-mediated adhesion confers a survival advantage for myeloma cells acutely exposed to cytotoxic drugs by inhibiting drug-induced apoptosis. This finding may explain how some cells survive initial drug exposure and eventually express classical mechanisms of drug resistance such as MDR1 overexpression.

Influence of Targeted Therapy in Redefining High-Risk Myeloma.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. sci-7-sci-7
Author(s):  
Lori Hazlehurst ◽  
William S. Dalton ◽  
Danielle Yarde ◽  
Yulia Nefedova ◽  
Dmitry Gabrilovich
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Drug Resistance ◽  
Clinical Trials ◽  
Cell Adhesion ◽  
Tumor Microenvironment ◽  
Residual Disease ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Acquired Drug Resistance

Abstract Multiple myeloma is a disease that typically responds to initial treatment; however, the disease is not cured by chemotherapy, and drug resistance ultimately develops. Most studies investigating the problem of drug resistance have focused on acquired resistance or resistance that occurs after response to prior therapy as a result of residual disease. Intrinsic factors, such as reduced drug uptake, enhanced damage response (i.e., DNA repair), altered drug metabolism, or inhibition of programmed cell death pathways are known to contribute to acquired drug resistance. For example, it was recently reported that the acquired melphalan resistant phenotype in myeloma cell lines was associated with over-expression of the Fanconi anemia (FA)/BRCA pathway genes. Enhanced interstrand cross-link (ICL) repair via the FA/BRCA pathway was causally related to melphalan resistance and disruption of this pathway using knock-down techniques reversed drug resistance. Furthermore, bortezomib (Velcade) has been reported to enhance melphalan treatment, and recent pre-clinical data has shown that bortezomib reduces FA/BRCA gene expression and function. Clinical trials are necessary to determine the role of the FA/ BRCA pathway in acquired drug resistance for myeloma patients and whether targeting this pathway enables prevention of or the ability to overcome acquired melphalan resistance in myeloma patients. Conversely, factors that promote tumor cell survival and drug resistance that are external to the tumor cell itself might exist. Evidence supporting the importance of understanding the influence of the tumor microenvironment on drug sensitivity has been reported by several investigators. The tumor microenvironment for hematologic malignancies, including myeloma, is principally the bone marrow. The bone marrow contains candidate components that contribute to reduced drug activity, minimal residual disease, and emergence of drug resistant cells. Cell adhesion molecules expressed by myeloma cells, including the β integrins, bind to fibronectin and other extracellular matrix components of the bone marrow, and this interaction contributes to a reversible, de novo drug resistance phenotype called “cell adhesion mediated drug resistance” or CAMDR. Adhesion via integrins is known to activate a network of signal transduction pathways that influence cell survival, growth, and differentiation. Several targets that are influenced by integrin adhesion and may contribute to CAM-DR include the following: reduced proapoptotic Bim levels, alterations in nuclear topoisomerase II levels, increased p27 kip1 levels, and changes in FLIP1 levels. In addition, myeloma cell adhesion to bone marrow stroma (BMS) involves other adhesion molecules and signaling events that promote CAMDR. For example, Notch1 receptors expressed on multiple myeloma cells when stimulated by Jagged causes growth arrest and protection from drug-induced apoptosis. Recently, approaches to inhibit integrin and Notch signaling associated with CAM-DR have been examined pre-clinically. Clinical trials are necessary to determine if these approaches will prevent or overcome CAM-DR in patients.

Establishment of An HS23 Stromal Cell-Dependent Myeloma Cell Line: Fibronectin and IL-6 Are Critical for Survival

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2949-2949
Author(s):  
Akira Sakai ◽  
Miyo Oda ◽  
Noriaki Yoshida ◽  
Mitsuhiro Itagaki ◽  
Koji Arihiro ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Pleural Effusion ◽  
Cell Line ◽  
Stromal Cells ◽  
Myeloma Cell ◽  
High Dose ◽  
Control Medium ◽  
Myeloma Cell Line ◽  
Myeloma Cells

Abstract Abstract 2949 Adhesion of multiple myeloma (MM) cells to bone marrow stromal cells triggers cytokine-mediated tumor cell growth, survival, and drug resistance. In particular, integrin a4b1, very late antigen 4 (VLA-4)-mediated fibronectin adhesion confers a survival advantage to myeloma cells. One of the problems in treating patients with MM is that it is very hard to eliminate residual myeloma cells, even following high-dose chemotherapy followed by auto-stem cell transplantation. Importantly, cell adhesion-mediated drug resistance (CAM-DR) must be overcome in order to eliminate the minimal residual disease of MM. Here we characterized a multiple myeloma cell line, MSG1, which depends on HS23 stromal cells for its survival, was established from the pleural effusion of a patient with MM who expressed the M-protein of IgA-λ in his serum. During the first two months of culture, the myeloma cells survived on adhesive cells from the pleural effusion and subsequently, they continued to proliferate on HS23 stromal cells but not on HS27A stromal cells (both stromal cells were established by Torok-Storb B, Blood 1995). The phenotype of the established MSG1 cell line was: CD138+, CD38++, CD19−, CD56−, VLA-4+, VEGFR1+, and VEGFR2+. Furthermore, immunohistochemical staining demonstrated expression of IgA and λ chain in the cytoplasm. Karyotype analysis indicated complex chromosomal abnormalities, basically hypertriploidy including the deletion of chromosome 13 and 17, and c-myc translocation. MSG1 cells continued to proliferate, not only when co-cultured with HS23 cells, but also when cultured on fibronectin-coated plates with the supernatant of HS23 cells or RPMI1640 medium supplemented with 10% FBS (control medium) containing IL-6 (10 ng/ml). Notably, MSG1 could not survive in control medium containing IL-6 or in HS23 supernatant unless bound to fibronectin, which was also expressed on HS23 and HS27A cells. IL-6 and VEGF production were detected in the supernatants of both HS23 and HS27A stromal cells (36.8±4.5 pg/ml and 131±5.8 pg/ml; 13.2±1.9 pg/ml and 16664±418 pg/ml, respectively). Next, we analyzed the effect of tocilizumab, an anti-IL-6R antibody, and bevacizumab, an anti-VEGF antibody on MSG1 survival. Tocilizumab (50 μ g/ml) inhibited MSG1 survival when cultured on fibronectin-coated plates in control medium containing IL-6 (10 ng/ml), and tocilizumab (10 μ g/ml) inhibited MSG1 survival when cultured on HS23 stromal cells. However, bevacizumab (500 μ g/ml) did not show such inhibition. Therefore, MSG1 survival depends on HS23 stromal cells: in other words, it depends on binding to fibronectin and IL-6. If these factors induced CAM-DR in myeloma cells, MSG1 may be a unique myeloma cell line that will be useful for analysis of CAM-DR, and tocilizumab might be a useful drug for treatment of MM. Furthermore, since MSG1 could survive on irradiated HS27A cells, and since HS23 and HS27A express similar adhesion molecules (Torok-Storb B et al., Blood 1995), these data suggest that HS27A might secrete factors that are detrimental to MSG1 survival. The identification of such an inhibitory factors could be of interest in terms of the regulation of myeloma proliferation. Disclosures: No relevant conflicts of interest to declare.

MLN4924, An Investigational NAE Inhibitor, Suppresses AKT and mTOR Signaling Pathway Through up Regulating REDD1 in Human Myeloma Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1870-1870
Author(s):  
Yanyan Gu ◽  
Jonathan L. Kaufman ◽  
Lawrence H. Boise ◽  
Sagar Lonial
Keyword(s):  
Multiple Myeloma ◽  
Western Blot ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Research Funding ◽  
Myeloma Cell ◽  
Mtor Pathway ◽  
Mtor Signaling ◽  
Mtor Signaling Pathway ◽  
Myeloma Cells

Abstract Abstract 1870 Introduction: The development and survival of normal plasma cells as well as multiple myeloma cells depend on an elaborately regulated ubiquitin proteasome system (UPS). Proteasome inhibitors such as bortezomib have proved to be highly active in the treatment of multiple myeloma. MLN4924, a newly developed investigational NEDD8 activating enzyme (NAE) inhibitor, exhibits promising anti-tumor effect through both clinical and laboratory observation. We sought to evaluate the individual signaling effects of MLN4924 in multiple myeloma, with the intent of further understanding the mechanism of action and identifying potential combinations. Methods: Human myeloma cell lines (MM.1S, MM.1R and U266) were treated with increasing concentrations of MLN4924 for 24, 48 and 72 hrs respectively or concurrently with bortezomib. Cell viability (MTT), apoptosis, western blot, RT-qPCR and siRNA assays were used to identify the cellular and molecular sequelae of MLN4924 treatment. Results: Single agent studies demonstrate that MLN4924 induces cytotoxicity in all three MM cell lines. Cytotoxicity is associated with increased apoptosis and suppression of AKT and mTOR signaling pathway, as detected by FACS and western blot. MLN4924 suppresses protein turnover of Cullin-ring ligases substrates leading to stabilization of specific proteins, such as p27, CDT1, NRF2. We find that REDD1, a substrate of CUL4 A–DDB 1–ROC1–β-TRCP ubiquitin ligase and negative regulator of mTOR pathway, increases in as early as 2 hours when treated with MLN4924. Knock-down of REDD1 using siRNA alleviates MLN4924 induced AKT, mTOR signaling suppression as well as the growth inhibition, which suggests that MLN4924 inhibits AKT, mTOR signaling through upregulating REDD1 protein leading to cytotoxicity. Combining MLN4924 with bortezomib synergistically enhances the antitumor effect of MLN4924. Western blot confirms the development of significantly increased procaspase and PARP cleavage, as well as substantial down regulation of AKT and mTOR signaling. Neither IL-6 nor IGF-1 is able to abrogate these combination effects. When we silence REDD1 in the combination assay in MM.1R, we find less cell apoptosis and suppression of AKT, mTOR pathway, which further support that REDD1 is an important regulator for MLN4924 induced cytotoxicity in MMs. Conclusion: MLN4924 is a potent investigational antitumor compound suppressing mTOR signaling pathways in myeloma cell lines. Down-regulation of the mTOR signaling pathway is associated with increased expression of REDD1. Combination of MLN4924 with the proteasome inhibitor bortezomib induces synergistic apoptosis in MMs cell lines which can overcome the prosurvival effects of growth factor (IL-6 and IGF-1). These findings could positively impact clinical combination strategies. Disclosures: Kaufman: Millenium: Consultancy; Onyx Pharmaceuticals: Consultancy; Novartis: Consultancy; Keryx: Consultancy; Merck: Research Funding; Celgene: Research Funding. Lonial:Millennium Pharmaceuticals, Inc.: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; Onyx: Consultancy; Merck: Consultancy.

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