scholarly journals Biologic sequelae of IκB kinase (IKK) inhibition in multiple myeloma: therapeutic implications

Blood ◽  
2009 ◽  
Vol 113 (21) ◽  
pp. 5228-5236 ◽  
Author(s):  
Teru Hideshima ◽  
Dharminder Chauhan ◽  
Tanyel Kiziltepe ◽  
Hiroshi Ikeda ◽  
Yutaka Okawa ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Growth Inhibition ◽  
Cell Lines ◽  
Nuclear Factor Κb ◽  
Down Regulation ◽  
Growth And Survival ◽  
Therapeutic Implications ◽  
Iκb Kinase ◽  
Canonical Pathways ◽  
Promising Treatment

Abstract Nuclear factor-κB (NF-κB) has an important role in multiple myeloma (MM) cell pathogenesis in the context of the bone marrow (BM) microenvironment. In NF-κB signaling cascades, IκB kinase α (IKKα) and IKKβ are key molecules that predominantly mediate noncanonical and canonical pathways, respectively. In this study, we examined the biologic sequelae of the inhibition of IKKα versus IKKβ in MM cell lines. All MM cell lines have constitutive canonical NF-κB activity, and a subset of MM cell lines shows noncanonical NF-κB activity. Adhesion to BM stromal cells further activates both canonical and noncanonical NF-κB activity. IKKβ inhibitor MLN120B blocks canonical pathway and growth of MM cell lines but does not inhibit the noncanonical NF-κB pathway. Although IKKα knockdown induces significant growth inhibition in the cell lines with both canonical and noncanonical pathways, it does not inhibit NF-κB activation. Importantly, IKKα down-regulation decreases expression of β-catenin and aurora-A, which are known to mediate MM cell growth and survival. Finally, IKKβ inhibitor enhances the growth inhibition triggered by IKKα down-regulation in MM cells with both canonical and noncanonical NF-κB activity. Combination therapy targeting these kinases therefore represents a promising treatment strategy in MM.

scholarly journals Bortezomib induces canonical nuclear factor-κB activation in multiple myeloma cells

Blood ◽  
2009 ◽  
Vol 114 (5) ◽  
pp. 1046-1052 ◽  
Author(s):  
Teru Hideshima ◽  
Hiroshi Ikeda ◽  
Dharminder Chauhan ◽  
Yutaka Okawa ◽  
Noopur Raje ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Molecular Mechanisms ◽  
Nuclear Translocation ◽  
Proteasome Inhibitors ◽  
Xenograft Model ◽  
Nuclear Factor Κb ◽  
Nuclear Factor ◽  
Down Regulation ◽  
Interacting Protein ◽  
Iκb Kinase

Bortezomib is a proteasome inhibitor with remarkable preclinical and clinical antitumor activity in multiple myeloma (MM) patients. The initial rationale for its use in MM was inhibition of nuclear factor (NF)-κB activity by blocking proteasomal degradation of inhibitor of κBα (IκBα). Bortezomib inhibits inducible NF-κB activity; however, its impact on constitutive NF-κB activity in MM cells has not yet been defined. In this study, we demonstrate that bortezomib significantly down-regulated IκBα expression and triggered NF-κB activation in MM cell lines and primary tumor cells from MM patients. Importantly, no inhibition of p65 (RelA) nuclear translocation was recognized after bortezomib treatment in a murine xenograft model bearing human MM cells. Bortezomib-induced NF-κB activation was mediated via the canonical pathway. Moreover, other classes of proteasome inhibitors also induced IκBα down-regulation associated with NF-κB activation. Molecular mechanisms whereby bortezomib induced IκBα down-regulation were further examined. Bortezomib triggered phosphorylation of IκB kinase (IKKβ) and its upstream receptor-interacting protein 2, whereas IKKβ inhibitor MLN120B blocked bortezomib-induced IκBα down-regulation and NF-κB activation, indicating receptor-interacting protein 2/IKKβ signaling plays crucial role in bortezomib-induced NF-κB activation. Moreover, IKKβ inhibitors enhanced bortezomib-induced cytotoxicity. Our studies therefore suggest that bortezomib-induced cytotoxicity cannot be fully attributed to inhibition of canonical NF-κB activity in MM cells.

Curcumin (diferuloylmethane) down-regulates the constitutive activation of nuclear factor–κB and IκBα kinase in human multiple myeloma cells, leading to suppression of proliferation and induction of apoptosis

Blood ◽  
2003 ◽  
Vol 101 (3) ◽  
pp. 1053-1062 ◽  
Author(s):  
Alok C. Bharti ◽  
Nicholas Donato ◽  
Sujay Singh ◽  
Bharat B. Aggarwal
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Nuclear Factor Κb ◽  
Parp Cleavage ◽  
Nuclear Factor ◽  
Nuclear Retention ◽  
Iκb Kinase ◽  
Human Multiple Myeloma ◽  
Induction Of Apoptosis ◽  
Iκbα Phosphorylation

Abstract Because of the central role of the transcription factor nuclear factor–κB (NF-κB) in cell survival and proliferation in human multiple myeloma (MM), we explored the possibility of using it as a target for MM treatment by using curcumin (diferuloylmethane), an agent known to have very little or no toxicity in humans. We found that NF-κB was constitutively active in all human MM cell lines examined and that curcumin, a chemopreventive agent, down-regulated NF-κB in all cell lines as indicated by electrophoretic mobility gel shift assay and prevented the nuclear retention of p65 as shown by immunocytochemistry. All MM cell lines showed consitutively active IκB kinase (IKK) and IκBα phosphorylation. Curcumin suppressed the constitutive IκBα phosphorylation through the inhibition of IKK activity. Curcumin also down-regulated the expression of NF-κB–regulated gene products, including IκBα, Bcl-2, Bcl-xL, cyclin D1, and interleukin-6. This led to the suppression of proliferation and arrest of cells at the G1/S phase of the cell cycle. Suppression of NF-κB complex by IKKγ/NF-κB essential modulator-binding domain peptide also suppressed the proliferation of MM cells. Curcumin also activated caspase-7 and caspase-9 and induced polyadenosine-5′-diphosphate-ribose polymerase (PARP) cleavage. Curcumin-induced down-regulation of NF-κB, a factor that has been implicated in chemoresistance, also induced chemosensitivity to vincristine and melphalan. Overall, our results indicate that curcumin down-regulates NF-κB in human MM cells, leading to the suppression of proliferation and induction of apoptosis, thus providing the molecular basis for the treatment of MM patients with this pharmacologically safe agent.

scholarly journals Role of Aiolos and Ikaros in the Antitumor and Immunomodulatory Activity of IMiDs in Multiple Myeloma: Better to Lose Than to Find Them

2021 ◽  
Vol 22 (3) ◽  
pp. 1103
Author(s):  
Marco Cippitelli ◽  
Helena Stabile ◽  
Andrea Kosta ◽  
Sara Petillo ◽  
Angela Gismondi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Plasma Cells ◽  
Immunomodulatory Activity ◽  
Cancer Cell Growth ◽  
Cytotoxic Effects ◽  
Growth And Survival ◽  
Therapeutic Implications ◽  
Development And Differentiation ◽  
Innate Lymphocytes

The Ikaros zing-finger family transcription factors (IKZF TFs) are important regulators of lymphocyte development and differentiation and are also highly expressed in B cell malignancies, including Multiple Myeloma (MM), where they are required for cancer cell growth and survival. Moreover, IKZF TFs negatively control the functional properties of many immune cells. Thus, the targeting of these proteins has relevant therapeutic implications in cancer. Indeed, accumulating evidence demonstrated that downregulation of Ikaros and Aiolos, two members of the IKZF family, in malignant plasma cells as well as in adaptative and innate lymphocytes, is key for the anti-myeloma activity of Immunomodulatory drugs (IMiDs). This review is focused on IKZF TF-related pathways in MM. In particular, we will address how the depletion of IKZF TFs exerts cytotoxic effects on MM cells, by reducing their survival and proliferation, and concomitantly potentiates the antitumor immune response, thus contributing to therapeutic efficacy of IMiDs, a cornerstone in the treatment of this neoplasia.

The aqueous tuber extract of Pueraria tuberosa (Willd.) D.C. caused cytotoxic effect on HT 29 cell lines with down regulation of nuclear factor-kappa B (NF-κB)

2017 ◽  
Vol 16 (4) ◽  
Author(s):  
Adeolu Alex Adedapo ◽  
Olusegun A Fagbohun ◽  
Christianah Dawurung ◽  
Ademola Adetokunbo Oyagbemi ◽  
Temidayo Olutayo Omobowale ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cytotoxic Effect ◽  
Nuclear Factor Κb ◽  
Male Rats ◽  
Root Extract ◽  
Nuclear Factor ◽  
Down Regulation ◽  
Time Points ◽  
Pueraria Tuberosa ◽  
Ht 29

Abstract Background Pueraria tuberosa (Willd) D.C. (Fabaceae) tubers are already used in traditional medicine by Ayurvedic physicians for the management of fertility disorders, general weakness, and also as anti-ageing therapies. Other known pharmacological properties include: anti-hyperglycemics, hepatoprotective, anti-hyperlipidemic, diuretic, nutritive, and anti-fertility agents in male rats. Methods The anti-proliferative effect of the aqueous tuberous root extract of Pueraria tuberosa on vascular smooth muscle cells (VSMCs) and Human Colorectal Adenocarcinoma Cell lines (HT-29) was investigated using the Cell Titer 96 MTT Proliferation Assay where the viable cells were seeded at a density of 5 × 104 (100 µL/well). For VSMC, log concentrations of the extract at 200 and 800 µg/mL were added and incubated for 24 and 48 h time points. Incubation of the extract in the presence of vascular endothelial growth factor (VEGF) and ET-1 was also conducted at different times. Concentrations of the extract (200, 400 and 700 µg/mL) were also added and incubated with the HT 29 cell lines for 24, 48 and 72 h time points. The effect of the tuber aqueous extract of the plant on nuclear factor-κB (NF-κB) expression after 2 h was also carried out using immunoblotting technique. Results The result showed that after 24 h, the effect of the extract in the presence of the mitogens and on the VSMC was more of proliferation. However, at 48 h, the 200 µg/mL dose, both alone and in the presence of VEGF caused 11.1% and 25.9% decreases respectively, in cell proliferation. In the HT 29 cytotoxic study the 200 µg/mL concentration caused the greatest cytotoxic effect at 77.1% cell inhibition followed by 400 µg/mL concentration at 71.4% after 72 h. The immunoblotting assay showed a down regulation of NF-κB expressions with 0.7 µg/mL concentration showing the greatest effect. NF-κB, a pro-inflammatory agent is increasingly recognized as a crucial player in many steps of cancer initiation and progression. Conclusions It could therefore be concluded that the aqueous root extract of Pueraria tuberosa possesses cytotoxic effect and could serve as a lead compound for anticancer and anti-inflammatory agents.

scholarly journals Chemoresistance in H-Ferritin Silenced Cells: The Role of NF-κB

2018 ◽  
Vol 19 (10) ◽  
pp. 2969 ◽  
Author(s):  
Ilenia Aversa ◽  
Roberta Chirillo ◽  
Emanuela Chiarella ◽  
Fabiana Zolea ◽  
Maddalena Di Sanzo ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
Nuclear Factor Κb ◽  
Cancer Cell Lines ◽  
Nuclear Accumulation ◽  
Therapeutic Implications ◽  
Ferritin Heavy Chain ◽  
Additional Layer ◽  
Ros Scavenger

Nuclear Factor-κB (NF-κB) is frequently activated in tumor cells contributing to aggressive tumor growth and resistance to chemotherapy. Here we demonstrate that Ferritin Heavy Chain (FHC) protein expression inversely correlates with NF-κB activation in cancer cell lines. In fact, FHC silencing in K562 and SKOV3 cancer cell lines induced p65 nuclear accumulation, whereas FHC overexpression correlated with p65 nuclear depletion in the same cell lines. In FHC-silenced cells, the p65 nuclear accumulation was reverted by treatment with the reactive oxygen species (ROS) scavenger, indicating that NF-κB activation was an indirect effect of FHC on redox metabolism. Finally, FHC knock-down in K562 and SKOV3 cancer cell lines resulted in an improved cell viability following doxorubicin or cisplatin treatment, being counteracted by the transient expression of inhibitory of NF-κB, IκBα. Our results provide an additional layer of information on the complex interplay of FHC with cellular metabolism, and highlight a novel scenario of NF-κB-mediated chemoresistance triggered by the downregulation of FHC with potential therapeutic implications.

A Fully Human Antagonist Anti-CD40 Antibody Triggers Significant Antitumor Activity Against Human Multiple Myeloma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2414-2414
Author(s):  
Yu-Tzu Tai ◽  
Xian-Feng Li ◽  
Xia Tong2 ◽  
Laurence Catley ◽  
Daniel Santos ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Dependent Manner ◽  
Peripheral Blood Mononuclear ◽  
Specific Lysis ◽  
Growth And Survival ◽  
Human Multiple Myeloma ◽  
Dose Dependent

Abstract We previously demonstrated that CHIR-12.12, a fully human anti-CD40 mAb (IgG1) generated in XenoMouseÒ mice (Abgenix, Inc), blocks CD40/CD40 ligand (CD40L) interactions and has more potent anti-lymphoma activity than Rituximab both in vivo and in vitro (abstract #2386, ASH, San Diego, Dec. 2003). In this study, we assess the efficacy of CHIR-12.12 against human multiple myeloma (MM) using CD40-expressing MM cell lines and purified CD138+ patient cells. CHIR-12.12 binds to purified CD138+ MM cells in >80% (10/12) of patient samples, as measured by flow cytometry: the mean fluorescence intensity (MFI) range was 1 to 20 for CHIR-12.12 vs 0.2–0.9 for control human IgG1. We next examined the antagonist activity of CHIR-12.12 in MM cells. CHIR-12.12 blocked CD40L-mediated proliferation of CD40-expressing MM lines and purified CD138+ patient cells from 2 MM patients in a dose-response manner. In contrast, CHIR-12.12 alone did not alter constitutive MM cell proliferation. Immunoblotting analysis demonstrated that PI3-K/AKT, NF-kB, and ERK activation induced by hCD40L in the 12BM MM cell line was significantly inhibited by CHIR-12.12 (5 μg/ml). Adhesion of MM cells to bone marrow stromal cells (BMSCs) confers growth and survival benefit for tumor cells. Since CD40 activation, either by stimulatory mouse anti-CD40 mAb G28.5 or formaldehyde-fixed CHO cells expressing hCD40L, induces MM cell adhesion to fibronectin (FN) or BMSCs, we next asked whether antagonist CHI12.12 abrogates this process. CHIR-12.12 inhibited CD40L-induced adhesion of MM cell lines to FN in a dose dependent manner (0.001-10 μg/ml), whereas control human IgG did not. Moreover, CHIR-12.12 (1 μg/ml) blocked hCD40L-induced adhesion of freshly isolated patient MM cells to BMSCs. Adhesion of MM cells to BMSCs induces IL-6 secretion, an important growth and survival cytokine for MM cells, and treatment of MM cells with hCD40L further augmented adhesion-induced IL-6 secretion. Conversely, pretreatment of CD40-expressing MM cell lines with CHIR-12.12 significantly decreased IL-6 secretion triggered by coculture of MM cells with BMSCs. We next examined whether CHIR-12.12 stimulates antibody-dependent cellular cytotoxicity (ADCC) against CD40-expressing MM cells. Human peripheral blood mononuclear cells and purified NK cells (CD56+CD3−) were used as effector cells. CHIR-12.12 triggered MM cell lysis in a dose dependent manner, as measured in CD40-expressing MM cell lines. The maximum specific lysis of 20–70 % was achieved at 10 μg/ml concentration of CHIR-12.12. CHIR-12.12 mediated lysis was specific to CD40-expressing MM cells, as CHIR-12.12 did not induce ADCC against CD40-negative MM cells. Importantly, CHIR-12.12 induced ADCC against CD138+ cells isolated from 2 MM patients. These results provide preclinical rationale for clinical evaluation of CHIR-12.12 with the goal of improving patient outcome in MM.

Anti-Tumor Activity of a Novel Immunosuppressant FTY720 in Multiple Myeloma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3456-3456
Author(s):  
Hiroshi Yasui ◽  
Teru Hideshima ◽  
Aldo M. Roccaro ◽  
Norihiko Shiraishi ◽  
Makoto Hamasaki ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Growth ◽  
Growth Inhibition ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Human Cancer ◽  
Human Peripheral Blood ◽  
Parp Cleavage ◽  
Igf I ◽  
Sphingosine 1 Phosphate

Abstract Sphingosine and its metabolites are bioactive sphingolipids involved in lipid biosynthesis, signal transduction and apoptosis. FTY720, a synthetic sphingosine analogue of myriocine derived from culture filtrates of Isaria sinclairii, has been reported to interact with the sphingosine-1-phosphate specific G protein-linked receptors (S1P1, 3, 4 and 5) (Mandala S et al. Science, 2002) and alter the migration and homing of lymphocytes, thereby inhibiting the immune response (Matloubian M et al. Nature, 2004). Recent studies have also shown that FTY720 induces growth inhibition and/or apoptosis in human cancer cells in vitro as well as in vivo murine model (Azuma H et al. Cancer Research, 2002). To date, however, the biologic sequelae of inhibiting sphingosine-1-phosphate activity on multiple myeloma (MM) cells have not been demonstrated. In the present study, we examined whether FTY720 triggers anti MM activity. FTY720 induced potent cytotoxicity against MM cell lines including MM.1S, U266, RPMI8226, with IC50 at 24 h of 3.0 – 7.0 mM, assessed by trypan-blue exclusion and MTT assays. FTY720 also inhibited growth of doxorubicin (Dox)-resistant RPMI8226-Dox40 and dexamethasone (Dex)-resistant MM.1R cell lines, with IC50 values similar to the parental drug-sensitive cell lines. In contrast, no cytotoxicity of FTY720 was recognized against human peripheral blood mononuclear cells from normal healthy donors. The combination of Dex with FTY720 demonstrated enhanced cytotoxicity compared to either agent alone. Importantly, neither interleukin-6 (IL-6) nor insulin like growth factor-I (IGF-I), which induces MM cell growth and protection against Dex-induced apoptosis, protected against FTY720-induced growth inhibition. The anti-MM mechanisms of action of FTY720 were next studied, and FTY720 induced caspase-dependent apoptosis in MM cell lines: FTY720 triggers caspase−8, −9 and −3 cleavage, followed by PARP cleavage and DNA fragmentation, as confirmed by Western blotting and agarose gel electrophoresis, respectively. Moreover, FTY720 abrogated both IL-6 mediated phosphorylation of Akt-1, STAT3 and p42/44MAPK, and IGF-I mediated Akt-1 phosphorylation. Importantly, paracrine MM cell growth with bone marrow stromal cells was strongly inhibited by FTY720. These results suggest that FTY720 overcomes drug resistance in MM cells and, providing the rationale for its clinical evaluation to improve patient outcome in MM.

CD27-Mediated Apoptosis Is Dependent on Siva-Induced Caspase Activation in Human Multiple Myeloma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3398-3398 ◽  
Author(s):  
Yu-Tzu Tai ◽  
Xian-Feng Li ◽  
Rory Coffey ◽  
Iris Breitkreutz ◽  
Laurence Catley ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Growth ◽  
Growth Inhibition ◽  
Plasma Cells ◽  
Death Domain ◽  
Caspase Activation ◽  
Growth And Survival ◽  
Induced Apoptosis ◽  
Cell Growth And Survival

Abstract CD27, a member of tumor necrosis factor receptor superfamily that lacks a death domain in its cytoplasmic region, and its interaction with its ligand, CD70, is crucial for differentiation into plasma cells. In malignant B cells, aberrant expression and reverse signaling of CD70 might contribute to disease progression. Recent studies showed that CD27 is heterogeneously expressed on multiple myeloma (MM) plasma cells and the expression is reduced with the progression of MM. However, a possible role for the loss of CD27-CD70 interaction in myelomagenesis was never defined. In this study, we identify functional significance of CD27-CD70 interaction in 4 CD27-expressing MM lines and define mechanisms regulating CD27-mediated MM cell death. Using RT-PCR and flow cytometric analysis, we first found that all of MM lines highly express CD70 (n=10) and 4 MM lines 12BM, 12PE, 28BM, 28PE express CD27 on the cell surface. We next evaluated the effect of CD27 ligation, by CD70-transfected NIH3T3 cells (CD70 transfectant), on [3H] thymidine incorporation by CD27-expressing MM lines. CD27 ligation by CD70 transfectants inhibited DNA synthesis in these 4 CD27-expressing MM lines, but not the control transfectants. Conversely, a blocking anti-CD70 mAb blocked CD27-mediated growth inhibition in a dose-dependent manner, indicating induced growth inhibition specific triggered by CD27-CD70 interaction. Using MTT assay, CD27 ligation by CD70 transfectant also inhibited MM cell survival. IL-6 (20 ng/ml) could overcome the inhibitory effect triggered by CD27 ligation on MM cell growth and survival. In addition, CD27 ligation further enhanced Dex-induced MM cell death. Importantly, CD27-mediated MM cell death was also observed in 2 CD27-expressing patient MM cells. Since Siva is a death domain-containing proapoptotic protein identified as an intracellular ligand of CD27, we investigated its role in CD27-mediated apoptosis in MM cells. Overexpression of Siva by transducing adenovirus-expressing Siva (Ad-Siva-GFP) in 12BM MM line is sufficient to induce cell death whereas control adenovirus (Ad-GFP) transduction did not alter 12BM cell growth and survival. CD27 ligation by CD70 transfectants on Siva-overexpressing 12BM cells further enhanced Siva-induced apoptosis, as evidenced by increased subG0 fraction in cell cycle analysis. Thus, the apoptosis triggered by Siva overexpression was related to the CD27-mediated apoptotic pathway. We further determined caspase involvement in the Siva-induced apoptosis in the absence and presence of CD70 transfectants. Caspase 8 and caspase 9 activities were detected 24h following Ad-Siva-GFP transduction in 12BM cells, whereas caspas-3 activity was detected 48h after transduction. Coculture of Ad-Siva-GFP-transduced 12BM cells with CD70 transfectant further enhanced caspase activities. Therefore, overexpression of Siva is sufficient to induce apoptosis and CD27-mediated apoptosis is mediated by Siva-dependent caspase activation in MM. Furthermore, these results suggest that lack of CD27 may lead to evasion of apoptosis in human MM.

Inhibition of ERK1/2 Activity by the MEK1/2 Inhibitor AZD6244 (ARRY-142886) Induces Human Multiple Myeloma Cell Apoptosis in the Bone Marrow Microenvironment: A New Therapeutic Strategy for MM.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3460-3460 ◽  
Author(s):  
Yu-Tzu Tai ◽  
Xian-Feng Li ◽  
Iris Breitkreutz ◽  
Weihua Song ◽  
Peter Burger ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Growth ◽  
Cell Lines ◽  
Caspase 3 ◽  
Parp Cleavage ◽  
Dependent Manner ◽  
Growth And Survival ◽  
Cyclin E1 ◽  
Human Multiple Myeloma

Abstract Activation of the extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (ERK1/2 MAPK) signaling pathway mediates tumor cell growth in many cancers, including human multiple myeloma (MM). Specifically, this pathway mediates MM cell growth and survival induced by cytokines/growth factors (i.e. IL-6, IGF-1, CD40, BAFF) and adhesion to bone marrow stromal cells (BMSCs), thereby conferring resistance to apoptosis in the bone marrow (BM) milieu. In this study, we therefore examined the effect of the MEK1/2 inhibitor AZD6244 (ARRY-142886), on human MM cell lines, freshly isolated patient MM cells and MM cells adhered to BMSCs. AZD6244, inhibits constitutive and cytokine (IL-6, IGF-1, CD40)-stimulated ERK1/2, but not AKT phosphorylation. Importantly, AZD6244 inhibits the proliferation and survival of human MM cell lines, regardless of sensitivity to conventional chemotherapy, as well as freshly isolated patient MM cells. AZD6244 induces apoptosis in patient MM cells even in the presence of BMSCs, as evidenced by caspase 3 activity and PARP cleavage at concentrations as low as 20 nM. AZD6244 overcomes resistance to apoptosis in MM cells conferred by IL-6 and BMSCs, and inhibits IL-6 secretion induced by MM adhesion to BMSCs. AZD6244 suppresses MM cell survival/growth signaling pathways (i.e., STAT3, Bcl-2, cyclin E1, CDK1, CDK3, CDK7, p21/Cdc42/Rac1-activated kinase 1, casein kinase 1e, IRS1, c-maf) and up-regulates proapoptotic cascades (i.e., BAX, BINP3, BIM, BAG1, caspase 3, 8, 6). AZD6244 also upregulates proteins triggering cell cycle arrest (i.e. p16INK4A, p18INK4C, p21/WAF1 [Cdkn1a], p27 [kip1], p57). In addition, AZD6244 inhibits adhesion molecule expression in MM cells (i.e. integrin a4 [VLA-4], integrin b7, ICAM-1, ICAM-2, ICAM-3, catenin a1, c-maf) associated with decreased MM adhesion to BMSCs. These pleiotropic proapoptotic, anti-survival, anti-adhesion and -cytokine secretion effects of AZD6244 abrogate BMSC-derived protection of MM cells, thereby sensitizing them to both conventional (dexamethasone) and novel (perifosine, lenalidomide, and bortezomib) therapies. In contrast, AZD6244 has minimal cytotoxicity in BMSCs and does not inhibit DNA synthesis in CD40 ligand-stimulated CD19 expressing B-cells derived from normal donors at concentrations toxic to MM cells (between 0.02–2 mM). Furthermore, AZD6244 inhibits the expression/secretion of osteoclast (OC)-activating factors (i.e., macrophage inflammatory protein (MIP)-1a, MIP-1b, IL-1b, VEGF) from MM cells. It also downregulates MM growth and survival factors (IL-6, BAFF, APRIL) in OC cultures derived from MM patient peripheral blood mononuclear cells (PBMCs). Significantly, AZD6244 inhibits OC differentiation from MM PBMCs (n=10) in a dose-dependent manner. Together these results provide the preclinical basis for clinical trials with AZD6244 (ARRY-142886) in MM.

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