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.

Survival Signals, Growth Cytokines, Immunosuppressive Factors and Their Cellular Perpetrators in the Myeloma Tumor Microenvironment.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1664-1664
Author(s):  
Jayakumar R Nair ◽  
Louise M Carlson ◽  
Noreen Ersing ◽  
Asher Alban Chanan-Khan ◽  
Kelvin P. Lee
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Tumor Microenvironment ◽  
Cell Lines ◽  
Plasma Cells ◽  
Immune Escape ◽  
Human Monocyte ◽  
Fold Increase ◽  
Myeloma Cell ◽  
Myeloma Cells

Abstract Multiple myeloma (MM) is an incurable neoplasia of terminally differentiated plasma cells in the bone marrow. Essential interactions of MM cells with host bone marrow stromal cells (BMSC) induce growth factors essential for MM progression and pathogenesis, as well as induce an immunosuppressive environment that inhibits endogenous and therapeutically-induced immune responses against the MM cells. However, despite their importance, little is known about the identity of these BMSC cells or the molecular basis of their interaction with myeloma cells. A potential MM surface protein that could be involved in these interactions is CD28, based on its known pro-survival role in T cells. Clinical studies have shown that expression of CD28 in multiple myeloma highly correlates (p=0.006) with myeloma disease progression. Moreover, CD28+ MM cells invariably express the CD28 ligand CD86. A survival role for MM-CD28 might involve interactions with cellular partners that express the B7 (CD80/CD86) ligands. Potential candidates would include CD86+ myeloma cells themselves or B7+ dendritic cells (DC) that are known to be closely associated with myeloma cells in the patient bone marrow. When myeloma-myeloma interactions were disrupted by using the high affinity CD80/CD86 blocker CTLA4Ig (Abatacept®), increased sensitivity to arsenic trioxide (ATO) and melphalan (MEL) was observed in all the three MM cell lines U266, RPMI8226 and MM1S. For U266 viability was 93% in media alone, 84% with CTLA4Ig (100 μg/ml) alone, 86% with 2 μM ATO alone and was significantly reduced to 36% with CTLA4Ig + ATO. Similar drops in viability were observed with 25 μM MEL in combination with CTLA4Ig (33% as opposed to 71–74 % with CTLA4Ig or MEL alone). Our data suggests that this does not involve the downregulation of anti-apoptotic proteins Bcl-2, Bcl-xL or Mcl-1, commonly associated with drug resistance in myeloma. In the second part of the study, we demonstrate that myeloma cell lines or primary CD138+ myeloma cells can enhance via direct contact the ability of human monocyte derived immature DC to produce the immunosuppressive tryptophan depleting enzyme indoleamine 2,3 dioxygenase (IDO, as estimated by kynurenine (Kyn) (a tryptophan catabolite) levels in the supernatant) and also the pro-plasma cell survival cytokine IL-6. In co-cultures of IFNg treated immature DCs with either MM cell lines or with primary CD138+ myeloma cells from patient BM aspirates, the activity of IDO was enhanced ~ 2–8 fold (81 mM kyn with U266 and 20–43mM with primary cells) over that observed in control IFNg-treated DCs (9.7 mM Kyn). Western analysis also demonstrated increased IDO expression relative to IFNg activated DC controls. Blocking MM-CD28 with (Fab)2 fragments of anti-hCD28 mAb 9.3 downregulated IDO activity (9.3 mM) close to that of control, demonstrating the involvement of MM-CD28 in these interactions. We also demonstrated a significant up-regulation of the pro-myeloma survival cytokine IL-6 when immature DCs were co-cultured with CD28+ MM1S (90–300 pg/ml), a 4–9 fold increase over that of DC only control (25 – 35 pg/ml). This was further enhanced when immature DCs cultured with IL-10 (+ GM-CSF + IL-4) was used in co-cultures with MM-1S (800 – 1300 pg/ml), or with primary CD138+ myeloma cells from patient bone marrow aspirates (128–1142 pg/ml). In conclusion, our data demonstrates that blocking myeloma-CD28 - myeloma-CD86 “autocrine” interaction can enhance drug cytotoxicity, while interactions with DCs produce the essential growth cytokines IL-6 and immunosuppressive enzyme IDO with potential implications in MM survival and immune escape. Use of clinically approved agents (e.g. Abatacept®) to block myeloma-CD28 binding to its B7 ligands (increase chemotherapeutic efficacy), 1-MT to inhibit IDO and targeting DCs in the microenvironment to disrupt the tumor microenvironment could be viable therapeutic strategies for the future treatment of multiple myeloma.

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.

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.

PYK2 Mediates Microenvironment-Specific Myeloma Survival

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4443-4443
Author(s):  
Mark Meads ◽  
Bin Fang ◽  
Linda Mathews ◽  
Jennifer Gemmer ◽  
Liang Nong ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Drug Resistance ◽  
Cell Adhesion ◽  
Focal Adhesion ◽  
Myeloma Cell ◽  
P Value ◽  
Myeloma Cells ◽  
Cycle Arrest ◽  
Beta1 Integrin ◽  
Proteomic Screening

Multiple myeloma remains an incurable bone marrow resident malignancy due to the eventual development of drug resistance and minimal residual disease linked to both acquired genetic changes and the dynamic influences of the microenvironment. We and others have shown that adhesion of tumor cells to the extracellular matrix component fibronectin (FN) via integrins leads to cell cycle arrest and protection from chemotherapy or Cell Adhesion-Mediated Drug Resistance (CAM-DR). The pleiotropic cytokine IL-6 has also been shown to mediate drug resistance in both solid and hematopoietic tumors [1]. The influence of these two effectors on drug resistance has primarily been studied separately; however, within the context of the bone marrow, myeloma cells are influenced by both soluble and physical effectors simultaneously. Using a reductionist model of the multivariate microenvironment, we demonstrated that unique collaborative signaling between FN-adhered cells and IL-6 leads to increased proliferation of protected tumor cells and a more malignant phenotype [2]. Our results demonstrate the FN-adhesion mediated cell-cycle arrest of myeloma cells was reversed following stimulation of adhered cells with IL-6 and drug resistance was maintained. This phenotype was associated with a novel amplification of IL-6-induced STAT3 activation in adhered cells [2]. We have since validated the FN-adhesion-dependent amplification of IL-6-induced STAT3 phosphorylation in myeloma patient specimens by flow cytometry. To further characterize post-translational events induced by beta1 integrin mediated adhesion and under multivariate conditions, we performed immune-affinity phosphotyrosine proteomic screening in RMPI8226 myeloma cells maintained in suspension or adhered to FN with or without IL-6 stimulation. Screening identified 179 differentially tyrosine phosphorylated peptides among four conditions (Suspension or FN, +/- IL-6). Among these were proteins involved in signal transduction, cytoskeleton assembly, survival, and metabolic pathways. Among 67 peptides upregulated by adhesion to FN alone, proline-rich tYrosine Kinase 2 (PYK2/FAK2) was highly phosphorylated at Y580 in RPMI8226 myeloma cells, and focal adhesion formation in adhered cells was validated by phosphorylation of paxillin at pY118 and p130CAS at pY128/pY249. PYK2 is a downstream intermediate of integrin signaling and has been demonstrated to amplify EGFR and cSrc-induced STAT3 activation [3]. PYK2 has also been linked to downstream signaling events of JAK-dependent cytokine receptors, but not to a STAT3 amplification mechanism within the Type I or Type II cytokine receptor families [4-7]. As such, we hypothesized that PYK2 may be an important modulator of the enhanced STAT3 activation following multivariate signaling between beta1 integrin and the IL-6 signal transducer, gp130. In addition to enhanced phosphorylation of PYK2 at Y580 identified by proteomic screening, western blot analysis demonstrated PYK2 autophosphorylation of Y402 following myeloma cell adhesion to FN correlating with the amplification of IL-6-induced STAT3 and JAK1. Targeting PYK2 with RNA interference attenuated the adhesion-associated amplification of STAT3 and JAK1 phosphorylation, but did not influence the limited STAT3 activation in cells grown in suspension. PYK2 siRNA did not inhibit myeloma cell adhesion (n=4, p-value >0.05). We have also demonstrated that STAT3 activation is markedly enhanced in myeloma cell lines adhered to patient bone marrow stroma (BMS), but not cells grown in transwell coculture (no myeloma cell-BMS contact). The protective advantage afforded myeloma cells in coculture was attenuated knockdown of PYK2 or STAT3 expression in myeloma cell lines and patient specimens using novel antisense oligonucleotides (ISIS Pharmaceuticals, n=4, p-value < 0.05). Critically, inhibition of PYK2 with nanomolar amounts of the focal adhesion kinase inhibitor PF562.271 had a similar impact only on myeloma cells directly adhered to BMS, but did not impact BMS viability. These data demonstrate a novel PYK2-mediated JAK1/STAT3 signaling cascade within the context of multivariate stimulation. Moreover these data identify a novel PYK2-mediated survival pathway activated only within the context of multivariate stimulation in the bone marrow microenvironment, and suggests that PYK2 represents a MRD-specific target. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The type III transforming growth factor-β receptor inhibits proliferation, migration, and adhesion in human myeloma cells

2011 ◽  
Vol 22 (9) ◽  
pp. 1463-1472 ◽  
Author(s):  
Kathleen E. Lambert ◽  
Huang Huang ◽  
Karthikeyan Mythreye ◽  
Gerard C. Blobe
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Adhesion ◽  
Growth Factor ◽  
Cell Growth ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Β Receptor

Transforming growth factor-β (TGF-β) plays an important role in regulating hematopoiesis, inhibiting proliferation while stimulating differentiation when appropriate. We previously demonstrated that the type III TGF-β receptor (TβRIII, or betaglycan) serves as a novel suppressor of cancer progression in epithelial tumors; however, its role in hematologic malignancies is unknown. Here we demonstrate that TβRIII protein expression is decreased or lost in the majority of human multiple myeloma specimens. Functionally, restoring TβRIII expression in myeloma cells significantly inhibited cell growth, proliferation, and motility, largely independent of its ligand presentation role. In a reciprocal fashion, shRNA-mediated silencing of endogenous TβRIII expression enhanced cell growth, proliferation, and motility. Although apoptosis was not affected, TβRIII inhibited proliferation through induction of the cyclin-dependent kinase inhibitors p21 and p27. TβRIII further regulated myeloma cell adhesion, increasing homotypic myeloma cell adhesion while decreasing myeloma heterotropic adhesion to bone marrow stromal cells. Mechanistically, live cell imaging of myeloma and stroma cell cocultures revealed that TβRIII-mediated inhibition of heterotropic adhesion was associated with decreased duration of myeloma/bone marrow stromal cell interaction. These results suggest that loss of TβRIII expression during multiple myeloma progression contributes to disease progression through its functional effects on increased cell growth, proliferation, motility, and adhesion.

Pyk2 Mediates Enhanced STAT3 Phosphorylation Following Collaborative Signaling Between gp130 and beta1 Integrins in Adhered Myeloma Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4021-4021
Author(s):  
Kenneth Shain ◽  
Mark Meads ◽  
Liang Nong ◽  
Jennifer Gemmer ◽  
Linda Mathews ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Drug Resistance ◽  
Cell Adhesion ◽  
Tumor Cells ◽  
Myeloma Cell ◽  
P Value ◽  
Myeloma Cells ◽  
Cycle Arrest ◽  
Stat3 Signaling ◽  
Stat3 Phosphorylation

Abstract Abstract 4021 Multiple myeloma remains an incurable bone marrow resident malignancy, in part, due to the eventual development of drug resistance linked to both acquired genetic changes and the dynamic influences of the microenvironment. We and others have shown that adhesion of tumor cells to the extracellular matrix component fibronectin (FN) via integrins leads to cell cycle arrest and protection from chemotherapy or Cell Adhesion-Mediated Drug Resistance (CAM-DR). The pleiotropic cytokine IL-6 has also been shown to mediate drug resistance in both solid and hematopoietic tumors [1]. The influence of these two effectors on drug resistance has primarily been studied separately; however, within the context of the bone marrow myeloma cells are influenced by both soluble and physical effectors simultaneously. Using a reductionist model of the multivariant microenvironment, we demonstrated that unique collaborative signaling between FN-adhered cells and IL-6 leads to increased proliferation of protected tumor cells and a more malignant phenotype [2]. Our results demonstrate the FN-adhesion mediated cell-cycle arrest of myeloma cells was reversed following stimulation of adhered cells with IL-6 and drug resistance was maintained. This phenotype was associated with a novel amplification of IL-6-induced STAT3 activation in adhered cells [2]. We have since validated the FN-adhesion-dependent amplification of IL-6-induced STAT3 phosphorylation in myeloma patient specimens by flow cytometry. To further characterize post-translational events induced under multivariant conditions, we performed immune-affinity phosphotyrosine proteomic screening in RMPI8226 myeloma cells maintained in suspension or adhered to FN with or without IL-6 stimulation. Screening identified 338 differentially tyrosine phosphorylated peptides. Among these were proteins involved in signal transduction, cytoskeleton assembly, survival, and metabolic pathways. Proline-rich tYrosine Kinase 2 (Pyk2/FAK2) was highly phosphorylated at Y597 in FN-adhered RPMI8226 myeloma cells. Eighty-three phosphorylation events were up-regulated by adhesion, including paxillin at pY118 and p130CAS at pY249 indicating focal adhesion formation. Pyk2 is a downstream intermediate of integrin signaling and has been demonstrated to amplify EGFR and cSrc-induced STAT3 activation. As such, we hypothesized that Pyk2 may be an important modulator of the enhanced STAT3 activation following multivariant signaling between beta1 integrins and gp130 [3]. In addition to enhanced phosphorylation of Pyk2 at Y579 identified by proteomic screening, western blot analysis demonstrated Pyk2 autophosphorylation of Y402 following myeloma cell adhesion to FN correlating with the amplification of IL-6-induced STAT3 and JAK1. Targeting Pyk2 with RNA interference attenuated the adhesion-associated amplification of STAT3 and JAK1 phosphorylation, but did not influence the limited STAT3 activation in cells grown in suspension. Pyk2 siRNA did not inhibit myeloma cell adhesion (n=4, p-value >0.05). Our previous results demonstrated that that the enhanced STAT3 signaling involved a FN-adhesion specific binding of unphosphorylated STAT3 with gp130 (independent of IL-6 stimulation). To determine if Pyk2 was similarly recruited to gp130 under co-stimulatory conditions we have used proximal ligation assay (PLA). Confocal imaging of the PLA reaction with antisera to Pyk2 and gp130 demonstrated colocalization of the two effectors upon adhesion of patient myeloma cells to FN, but not in those grown in suspension. We have also demonstrated that STAT3 activation is markedly enhanced in myeloma cell lines adhered to patient bone marrow stroma (BMS), but not cells grown in transwell coculture (no myeloma cell-BMS contact). Critically, the protective advantage afforded myeloma cells in coculture was attenuated only by dual knockdown of STAT3 and Pyk2 using novel antisense oligonucleotides (ISIS Pharmaceuticals, n=4, p-value 0.027). These data demonstrate a novel Pyk2-mediated JAK1/STAT3 signaling cascade within the context of multivariant stimulation. Moreover, these data suggest that strategies targeting of both lateral and vertical signaling (Pyk2 & STAT3) may be required to overcome therapy resistance conferred to tumor cells by the multivariant bone marrow niche. Disclosures: No relevant conflicts of interest to declare.

Multiple Myeloma Cells Adhere to Netrin-1 Via Heparin-Sulphate Moieties

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3952-3952
Author(s):  
Jeesun Park ◽  
David R Fooksman ◽  
Amitabha Mazumder ◽  
Michael L Dustin
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Adhesion ◽  
Conflicts Of Interest ◽  
Cell Spreading ◽  
Myeloma Cell ◽  
Axonal Guidance ◽  
Intercellular Adhesion Molecule ◽  
Myeloma Cells ◽  
Guidance Cue

Abstract Abstract 3952 A major obstacle to the treatment of Multiple Myeloma (MM) is the localization of myeloma cells to the bone marrowstroma, enabling drug resistance. The exact mechanisms of adhesion of myeloma cells to the bone marrow are not known, but adhesion molecules and chemokine signals, in particular vascular cell adhesion protein 1 (VCAM-1) and C-X-C chemokine 12 (CXCL12) which control bone marrow tropism, are thought to be the main players. Netrin-1, which acts as an axonal guidance cue, plays a role in leukocyte migration in lymph nodes and in atherosclerotic lesions, but has not been tested as a substrate for myeloma cell adhesion previously. Based on expression of the netrin-1 receptor Deleted in Colorectal Cancer (DCC) on activated human B cells, we tested the ability of myeloma cells to adhere to netrin-1, an axonal guidance cue. Using interference reflection microscopy (IRM) which employs the method of interference of light reflected from nearby surfaces to measure cell-substratum distances and cell-substratum adhesion, we assessed cell adhesion and cell spreading on substrates immobilized on glass. Here, we used this technique to assess myeloma cell adhesion and migration on various substrates and found netrin-1 to be an exceptional adhesion ligand for myeloma cells. We prepared glass substrates coated with the recombinants ligands intercellular adhesion molecule,ICAM-1(50μM), and VCAM-1(50μM), with or without chemokine ligand, CXCL12(0.1mg/mL), which have been implicated in plasma cells and myeloma cell migration, previously. We used freshly purified, fluorescently-labeled primary myeloma cells from newly diagnosed patients, prior to any treatment. Using IRM, we imaged the cell contacting the substrate in order to measure adhesion and differentiate crawling versus fluid flow movement. Based on the IRM image, we could calculate the fraction of cells in the field that were adhered to the substrate, and compared between conditions and for various patient samples. We observed that myeloma cells can adhere and migrate slowly on VCAM-1 in the presence of CXCL12, but are unable to adhere to ICAM-1 with or without chemokines. We tested myeloma cell binding to netrin-1 and saw a strong adhesion 60–90% of cells in 7 out of 9 patients samples tested. The cell spreading on netrin-1 was more than 3 times larger than on VCAM-1 with CXCL12 substrates. Expression of netrin-1 in the bone marrow has not been determined yet nor its role in MM. Heparin-like molecule, SST0001, has been tested in myeloma studies, in an attempt to interfere with heparinase activity and syndecan-1 shedding, and tumor growth. We tested pre-blocking netrin-1 substrates with heparin and observed elimination of greater than 95% of myeloma cell adhesion in all patients samples tested. Treating patients with heparin-like molecules may have additional functions, by blocking binding to netrin-1 and soluble signals that contain heparin-binding domains. Reciprocally, blocking heparin-sulfated groups with netrin-1 may block myeloma cell adhesion and can be used to targeting strategy for chemotherapeutic drugs as well. Disclosures: No relevant conflicts of interest to declare.

Abstract 5686: Engineering multifunctional nanoparticles to selectively target multiple myeloma cells and overcome cell adhesion-mediated drug resistance

2012 ◽  
Author(s):  
Tanyel Kiziltepe ◽  
Jonathan D. Ashley ◽  
Jared F. Stefanick ◽  
Nathan J. Alves ◽  
Micheal W. Handlogten ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Cell Adhesion ◽  
Multifunctional Nanoparticles ◽  
Myeloma Cells

Amphiregulin Is Produced by Myeloma Cells and Is Involved in Tumor-Environment Interactions in Multiple Myeloma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4306-4306
Author(s):  
Karène Mahtouk ◽  
Dirk Hose ◽  
Thierry Reme ◽  
John De Vos ◽  
Michel Jourdan ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Growth Factors ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Erbb Receptors ◽  
Heparin Binding ◽  
Myeloma Cells ◽  
Heparin Binding Growth Factors ◽  
Egf Family

Abstract Multiple myeloma (MM) is characterized by the accumulation of clonal malignant plasma cells in the bone marrow. One of the hallmarks of plasma cells is the expression of the heparan-sulfate proteoglycan syndecan-1. In epithelial cells, syndecan-1 plays a major role as a coreceptor for heparin-binding growth factors and chemokines. This stresses that heparin-binding growth factors may play a major role in the biology of MM cells. Recently we have demonstrated that heparin-binding EGF-like growth factor (HB-EGF), one of the ten members of the Epidermal Growth Factor (EGF) family, is produced by the tumor microenvironment and is able to trigger myeloma cell growth. As amphiregulin (AREG) is another member of the EGF family that also binds heparan-sulphate chains, we investigated its role in MM. We looked for AREG expression on a panel of 7 normal plasmablastic cells (PPCs), 7 normal bone marrow plasma cells (BMPCs), purified MM cells from 65 patients and 20 myeloma cell lines (HMCLs), with Affymetrix U133A+B microarrays. We showed that primary MM cells overexpress AREG compared to normal BMPCs and PPCs. We then investigated the expression of the ErbB receptors with real-time RT-PCR. Myeloma cells variably expressed the 4 ErbB receptors. Normal BMPCs also expressed ErbB1 and ErbB2 unlike PPCs that did not express any ErbB receptors. We demonstrated that the high AREG expression by primary myeloma cells may have a dual effect. On the one hand, AREG stimulated IL-6 production and growth of bone-marrow stromal cells that highly express the AREG ErbB1 receptor. On the other hand, AREG could promote HMCL proliferation, suggesting that a functional autocrine loop involving AREG and ErbB receptors is involved in MM cell growth. Finally, we looked for the effect of ErbB inhibitors on MM cells of 14 patients cultured for 6 days together with their bone marrow environment. A pan-ErbB inhibitor (PD-169540, Pfizer) and an ErbB1-inhibitor (IRESSA, Astrazeneca) induced strong MM cell apoptosis in respectively 71% of patients (10 of 14) and 29% of patients (4 of 14). Of major interest, when PD169540 or IRESSA were combined with dexamethasone, they induced a dramatic myeloma cell death (respectively 92% and 69% inhibition of MM cell survival), while non-myeloma cells were unaffected. Thus ErbB activation is critical to trigger MM-cell survival in short-term culture. In conclusion, our findings provide evidence for a major role of AREG and HB-EGF in the biology of multiple myeloma and identify ErbB receptors as putative therapeutic targets. These data emphasize the interest of clinical evaluation of specific-ErbB-inhibitors in patients with MM, either used alone or in combination with dexamethasone.

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