Pathways In Multiple Myeloma Cells Research Articles

Decursin and Doxorubicin Are in Synergy for the Induction of Apoptosis via STAT3 and/or mTOR Pathways in Human Multiple Myeloma Cells

Background. Combination cancer therapy is one of the attractive approaches to overcome drug resistance of cancer cells. In the present study, we investigated the synergistic effect of decursin from Angelica gigas and doxorubicin on the induction of apoptosis in three human multiple myeloma cells. Methodology/Principal Findings. Combined treatment of decursin and doxorubicin significantly exerted significant cytotoxicity compared to doxorubicin or decursin in U266, RPMI8226, and MM.1S cells. Furthermore, the combination treatment enhanced the activation of caspase-9 and -3, the cleavage of PARP, and the sub G1 population compared to either drug alone in three multiple myeloma cells. In addition, the combined treatment downregulated the phosphorylation of mTOR and its downstream S6K1 and activated the phosphorylation of ERK in three multiple myeloma cells. Furthermore, the combined treatment reduced mitochondrial membrane potential, suppressed the phosphorylation of JAK2, STAT3, and Src, activated SHP-2, and attenuated the expression of cyclind-D1 and survivin in U266 cells. Conversely, tyrosine phosphatase inhibitor pervanadate reversed STAT3 inactivation and also PARP cleavage and caspase-3 activation induced by combined treatment of doxorubicin and decursin in U266 cells. Conclusions/Significance. Overall, the combination treatment of decursin and doxorubicin can enhance apoptotic activity via mTOR and/or STAT3 signaling pathway in multiple myeloma cells.

Membrane Lipid Biosynthesis As a Potential Therapeutic Target in Multiple Myeloma

AbstractAbstract 1836Phosphatidylcholine (PC) is the most prominent phospholipid in mammalian endoplasmic reticulum (ER) membranes. The rate-limiting step in PC synthesis through the Kennedy pathway is the conversion of phosphocholine + cytidine triphosphate (CTP) to cytidine diphosphocholine, (CDP)-choline, via the enzyme CTP:phosphocholine cytidylyltransferase (CCT) (see figure). [Display omitted] Multiple myeloma (MM) cells may be particularly dependent on this biosynthetic reaction because of their high consistent level of ER stress and requirement to continuously replenish ER membranes. Indeed, CCT-null mice have a defect in differentiation of B lymphocytes to plasma cells and deficiencies in Ig synthesis. To test whether this pathway remains critical in survival of malignant MM cells, we exposed MM cell lines to an inhibitor shown to inhibit CCT activity, HexPC. HexPC induced apoptosis in all MM cell lines in a concentration- and time-dependent manner. The addition of lysophosphatidylcholine (LPC), presumably converted to PC independently of the Kennedy pathway, completely rescued MM cell apoptosis. In contrast, similar concentrations of LPC in the same cell lines could not rescue apoptosis induced by bortezomib. An additional intervention to inhibit phosphatidylcholine synthesis, namely inducing pyrimidine starvation, also resulted in MM cell apoptosis and down-regulation of CDP-choline levels. Apoptosis of MM cells induced by HexPC was associated with induction of ER stress as shown by enhanced phosphorylation of IRE1 and eIF-2alpha. This ER stress was also prevented when LPC was added to HexPC although LPC could not prevent similar ER stress induced by bortezomib. These results underscore the importance of this phosphatidylcholine synthesis pathway in MM cells and provide new targets for future therapy. Disclosures:No relevant conflicts of interest to declare.

CXCR4 Monoclonal Antibody, BMS-936564 (MDX-1338), Modulates Epithelial to Mesenchymal Transition (EMT) in Multiple Myeloma Cells

AbstractAbstract 4009 Background.The SDF1/CXCR4 axis plays a major role in homing and trafficking of multiple myeloma (MM) to the bone marrow (BM), and disruption of the interaction of tumor cells with the BM leads to enhanced sensitivity to therapeutic agents. Also, hypoxia leads to EMT activation as well as CXCR4 up-regulation in MM cells. We therefore hypothesized that CXCR4 may represent a crucial regulator of EMT in MM and an important target for preventing MM disease dissemination. Methods.Primary MM cells (CD138+); MM cell lines (MM.1S, RPMI.8226); and primary MM bone marrow stromal cells (BMSCs) were used. Dissemination of MM.1S/GFP+ cells to distant bone marrow niches was evaluated in vivo, by using in vivo confocal microscopy. CXCR4-loss of function studies were performed by transfecting MM cells with either a scrambled probe or CXCR4-siRNA. A novel HuMAb anti-CXCR4 (BMS-936564; Bristol Myers Squibb, NY) was used. Migration towards SDF-1 and BMSCs was evaluated. Cytotoxicity and DNA synthesis were measured by MTT and 3H-thymidine uptake, respectively. Cell signaling, apoptotic- and EMT-related pathways were studied by Western Blot. Synergism was calculated by using the Chou-Talalay method. In vivo, MM tumor growth was evaluated by using xenograft mouse models and a melanoma xenograft mouse model was used to validate the effect of anti-CXCR4 antibody on modulating tumor cell metastasis. Results.We demonstrated down-regulation of Twist, Snail and Slug, together with up-regulation of E-Cadherin in CXCR4-siRNA-transfected cells, compared to scrambled probe-transfected cells. These findings were next validated by using the new selective CXCR4 antibody (BMS-936564); and confirmed that BMS-936564-dependent inhibition of CXCR4 led to inhibition of Twist, Snail, and Slug; with up-regulation of E-Cadherin. These data were further corroborated in vivo, by using in vivo confocal microscopy: mice treated with BMS-936564 presented with less MM cell dissemination to distant bone marrow niches, compared to vehicle-treated mice, supporting the hypothesis that CXCR4 may represent a crucial modulator of tumor cell dissemination. These data were also confirmed in vivo, by using a xenograft melanoma model, where BMS-936564-treated mice presented with a reduced number of metastasis, compared to vehicle-treated mice. These in vivo data were supported by in vitro evidence showing the ability of BMS-936564 to functionally target MM cells in terms of migration, adhesion and survival. BMS-936564 inhibited migration of MM cells towards SDF-1a and primary MM BMSCs, in a dose-dependent manner. In addition, survival and adhesion of primary MM cells to BMSCs were inhibited by BMS-936564 in a dose-dependent manner. BMS-936564 targeted MM cells in the context of BM milieu, by overcoming BMSCs-induced proliferation of tumor cells. Moreover, BMS-936564 synergistically enhanced bortezomib-induced cytotoxicity in MM cells. BMS-936564-dependent activation of apoptotic pathways in MM cells was documented, as shown by cleavage of caspase-9 and PARP. SDF-1a-induced ERK-, Akt-, and Src-phosphorylation were inhibited by BMS-936564 in a dose-dependent manner. Importantly, BMS936564 inhibited MM cell proliferation in vivo in xenograft mouse models. Conclusion.These findings indicate that CXCR4 represents a valid therapeutic target due to its ability to modulate EMT, and that BMS-936564 functionally targets MM cell migration, adhesion and survival; thus providing evidence for using the anti-CXCR4 antibody, BMS-936564, as a therapeutic modality for MM. Disclosures:Kuhne:Bristol-Myers Squibb: Employment. Cohen:Bristol-Myers Squibb: Employment. Cardarelli:Bristol-Myers Squibb: Employment. Ghobrial:Novartis: Advisory Board Other; Onyx: Advisory Board, Advisory Board Other; Millennium: Advisory Board, Advisory Board Other; Bristol Myers Squibb: Advisory Board, Advisory Board Other.

Dual Inhibition of Canonical and Non-Canonical NF-KB Pathway Demonstrates Significant Anti-Tumor Activities in Multiple Myeloma (MM)

Abstract 2902NF-kB plays a crucial role in the pathogenesis of multiple myeloma (MM). In MM cells, NF-kB pathway is constitutively activated and regulates transcription of genes whose protein products mediate proliferation, survival and drug resistance. In the context of the bone marrow (BM) microenvironment, NF-kB modulates the expression of cytokines (ie, IL6, TNFalpha) and adhesion molecules (ie, ICAM-1). Importantly, these cytokines and adhesion to BM stromal cells (BMSCs) further activate NF-kB pathway. Previous studies have shown that both canonical and non-canonical pathways contribute to total NF-kB activity in MM cells. Therefore inhibition of both pathways is necessary to target NF-kB. However, current therapeutic strategies can only inhibit the canonical, but not the non-canonical pathway. In this study, we examined the biologic impact of dual inhibition of both canonical and non-canonical pathways in MM cells using a novel small molecule inhibitor PBS-1086 (Profectus BioSciences) which selectively inhibits binding of Rel family member proteins to DNA. Importantly, the binding activity of all Rel family member proteins (RelA, RelB, NF-kB1, NF-kB2, cRel) to DNA was inhibited by PBS-1086, confirming that PBS-1086 blocks both canonical and non-canonical pathways in MM cell lines. We first investigated growth inhibitory effect of PBS-1086 in vitro. PBS-1086 potently inhibited the growth of MM cell lines (MM1S, MM1R, INA6, LR5, Dox40, KMS18, RPMI-8226 and U266) in a dose-dependent fashion with IC50 ranges of 0.15–5 μM. In contrast, PBS-1086 showed modest cytotoxicity on normal peripheral blood mononuclear cells from healthy volunteers. Similar growth inhibitory effect were observed in CD138+ primary tumor cells derived from MM patients. PBS-1086 induced apoptosis in MM1S cell line in a time-dependent manner, evidenced by annexin V-PI staining by flow cytometry and cleaved caspase 8, 9, 3 and PARP, suggesting that PBS-1086 activates both extrinsic and intrinsic apoptotic pathways. Importantly, PBS-1086 can overcome the proliferative and anti-apoptotic effects of BMSCs, associated with inhibition of NF-kB activity. We next examined the combination effect of PBS-1086 with other agents. PBS-1086 with bortezomib synergistically enhanced anti-MM activity even in bortezomib-resistant cell lines (Dox40, ANBL6-VR5) and primary tumor cells from MM patients. Finally, we investigated the effect of PBS-1086 in vivo in a murine xenograft model of human MM cells. Tumor-bearing mice were divided into 6 groups: non-treated, vehicle control, PBS-1086 (7.5 mg/kg ip daily), bortezomib (0.5 mg/kg IV, twice weekly) and the combination of PBS-1086 (either at 2.5 mg/kg or 7.5 mg/kg) with bortezomib. PBS-1086 showed significant anti-MM activity in combination (2.5 and 7.5 mg/kg) groups versus control group (p =0.00039 and p =0.00084, respectively). Combination groups also had significantly (p < 0.05) prolonged survival compared to single agent treatment group (PBS-1086 or bortezomib). In conclusion, our preclinical studies show that PBS-1086 is a promising novel therapeutic agent and our data supports further clinical evaluation of this agent in combination with bortezomib for the treatment of MM. Disclosures:Bobb:Profectus BioSciences: Employment; Rel-MD: Employment. Zhang:Profectus BioSciences: Employment; Rel-MD: Employment. Meshulam:Profectus BioSciences: Employment; Rel-MD: Employment. Mitsiades:Millennium: Consultancy, Honoraria. Richardson:Millennium: ; Celgene: ; Johnson & Johnson: ; Novartis: ; Bristol Myers Squibb:. Hideshima:Acetylon: Consultancy. Anderson:Celgene: Membership on an entity’s Board of Directors or advisory committees; Millennium: Membership on an entity’s Board of Directors or advisory committees; Onyx: Membership on an entity’s Board of Directors or advisory committees; Merck: Membership on an entity’s Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity’s Board of Directors or advisory committees; Novartis: Membership on an entity’s Board of Directors or advisory committees; Acetylon: Membership on an entity’s Board of Directors or advisory committees.

The New CXCR4 Inhibitor MDX-1338 Exerts Anti-Tumor Activity in Multiple Myeloma

Abstract 1844 Background.We have previously shown the SDF1/CXCR4 axis plays a major role in homing and trafficking of multiple myeloma (MM) to the bone marrow (BM), and disruption of the interaction of tumor cells with the BM leads to enhanced sensitivity to therapeutic agents. We hypothesize that the novel anti-CXCR4 antibody, BMS936564/MDX-1338, may prevent the homing and adhesion of MM cells to the BM and will sensitize them to therapeutic agents. Methods.Primary MM cells (CD138+); MM cell lines (MM.1S, RPMI.8226); and primary MM bone marrow stromal cells (BMSCs) were used. Migration towards SDF-1 and BMSCs has been evaluated. Cytotoxicity and DNA synthesis were measured by MTT and thymidine uptake, respectively. Cell signaling and apoptotic pathways were studied by Western Blot. Synergism was calculated using the Chou-Talalay method. In vivo MM tumor growth was evaluated with xenograft mouse models. Results.MDX-1338 inhibited migration of MM cells toward SDF-1a and primary MM BMSCs, in a dose-dependent manner. Adhesion of primary MM cells to BMSCs was also inhibited by BMS936564/MDX-1338 in a dose-dependent manner, while also inducing cytotoxicity on primary BM-derived CD138+ cells. BMS936564/MDX-1338 targeted MM cells in the context of BM milieu by overcoming BMSC-induced proliferation of tumor cells. In addition, BMS936564/MDX-1338 synergistically enhanced bortezomib-induced cytotoxicity in MM cells. BMS936564/MDX-1338-dependent activation of apoptotic pathways in MM cells was documented, as shown by cleavage of caspase-9 and PARP. SDF-1a-induced ERK-, Akt-, and Src-phosphorilation was inhibited by BMS936564/MDX-1338 in a dose-dependent manner. Importantly, BMS936564/MDX-1338 inhibited MM cell proliferation in vivo in xenograft mouse models. Conclusion.These studies therefore show that targeting CXCR-4 in MM by using BMS936564/MDX-1338 represents a valid therapeutic strategy in this disease. Disclosures:Roccaro:Roche:. Kuhne:BMS: Employment. Pan:Bristol-Myers Squibb: Employment. Cardarelli:Bristol-Myers Squibb: Employment. Ghobrial:Noxxon: Research Funding; Bristol-Myers Squibb: Research Funding; Millennium: Research Funding; Noxxon:; Millennium:; Celegene:; Novartis:.

Redeployment-based drug screening identifies the anti-helminthic niclosamide as anti-myeloma therapy that also reduces free light chain production

Despite recent therapeutic advancements, multiple myeloma (MM) remains incurable and new therapies are needed, especially for the treatment of elderly and relapsed/refractory patients. We have screened a panel of 100 off-patent licensed oral drugs for anti-myeloma activity and identified niclosamide, an anti-helminthic. Niclosamide, at clinically achievable non-toxic concentrations, killed MM cell lines and primary MM cells as efficiently as or better than standard chemotherapy and existing anti-myeloma drugs individually or in combinations, with little impact on normal donor cells. Cell death was associated with markers of both apoptosis and autophagy. Importantly, niclosamide rapidly reduced free light chain (FLC) production by MM cell lines and primary MM. FLCs are a major cause of renal impairment in MM patients and light chain amyloid and FLC reduction is associated with reversal of tissue damage. Our data indicate that niclosamides anti-MM activity was mediated through the mitochondria with rapid loss of mitochondrial membrane potential, uncoupling of oxidative phosphorylation and production of mitochondrial superoxide. Niclosamide also modulated the nuclear factor-κB and STAT3 pathways in MM cells. In conclusion, our data indicate that MM cells can be selectively targeted using niclosamide while also reducing FLC secretion. Importantly, niclosamide is widely used at these concentrations with minimal toxicity.

Widespread Deregulation of Phosphorylation-Based Signaling Pathways in Multiple Myeloma Cells: Opportunities for Therapeutic Intervention

Multiple myeloma (MM) is a neoplasm of plasma cell origin that is largely confined to the bone marrow (BM). Chromosomal translocations and other genetic events are known to contribute to deregulation of signaling pathways that lead to transformation of plasma cells and progression to malignancy. However, the tumor stroma may also provide trophic support and enhance resistance to therapy. Phosphorylation of proteins on tyrosine, serine and threonine residues plays a pivotal role in cell growth and survival. Therefore, knowing the status of phosphorylation-based signaling pathways in cells may provide key insights into how cell growth and survival is promoted in tumor cells. To provide a more comprehensive molecular analysis of signaling disruptions in MM, we conducted a kinome profile comparison of normal plasma cells and MM plasma cells as well as their surrounding cells from normal BM and diseased BM. Integrated pathway analysis of the profiles obtained reveals deregulation of multiple signaling pathways in MM cells but also in surrounding bone marrow blood cells compared to their normal counterparts. The deregulated kinase activities identified herein, which include the mTOR (mammalian target of rapamycin)/p70S6K and ERK1/2 (extracellular signal-regulated kinases 1 and 2) pathways, are potential novel molecular targets in this lethal disease.

Isopentenyl Pyrophosphate (IPP), a Metabolite produced in Myeloma Cells Induces the Chemotaxis of γδT Cells

AbstractAbstract 2766γδT cells, which control the innate immune system, are classified into three subtypes on the basis of Vγ chain. Of these subtypes, Vγ2Vδ9 T cells display anti-tumor immunity. We have demonstrated that nitrogen-containing bisphosphonate (N-BP) treatment expands Vγ2Vδ9 T cells ex vivo and that these expanded cells can kill tumor cells in a major histocompatibility complex-unrestricted manner (Sato, Int J Cancer, 2005; Uchida, Biochem Biophys Res Commun, 2007; Sato, Cancer Immunol Immunother, 2008.). N-BP inhibits farnesyl pyrophosphate synthase in the mevalonate pathway, resulting in the accumulation of isopentenyl pyrophosphate (IPP), which is a stimulatory antigen for Vγ2Vδ9T cells. In the present study, we investigated the chemotactic factors for Vγ2Vδ9T cells by using a micro total analysis system-based microfluidic cellular analysis device (Kanai, Sens Actuators A, 2004; Munaka, Analyst, 2007.). This microchip possesses a minute-volume (240 nL) chamber integrated with a micro-sample injector that permits the injection of a small amount (several nL) of a solute (Figure 1). Because of the minute size of this chamber, a concentration gradient can be maintained free from the influence of fluid convection and stirring, and the solute can consequently spread in a diffusion-dependent manner. Therefore, administration of a humoral factor via the sample injector mimics its release from the cell surface. We first investigated whether the supernatant of RPMI8226 multiple myeloma (MM) cells treated with zoledronic acid (ZOL) induced chemotaxis of γδT cells. We treated RPMI8226 MM cells with ZOL (1 mM) overnight and collected the supernatant. Human γδT cells were obtained by the culture of peripheral blood mononuclear cells as previously reported (Uchida, Biochem Biophys Res Commun, 2007.), and these cells were cultured in the microchip. After the injection of supernatant, γδT cells migration was observed under a microscope and continuous time-lapse recording was performed for 30 min. γδT cells migrated toward the injector, indicating that the supernatant of ZOL-treated RPMI8226 cells includes a chemoattractant factor for γδT cells. We next applied soluble MICA (sMICA), sICAM-1, sVCAM-1, and IPP and examined the migration of γδT cells. Among them, sMICA and IPP were chemoattractive for γδT cells, and the velocity of γδT cell migration was increased by the injection of IPP compared to the solvent control (Figure 2). These observations indicate that IPP, a metabolite of the mevalonate pathway in MM cells, or sMICA is a chemotactic factor for γδT cells when the target MM ells are treated with ZOL. [Display omitted] [Display omitted] Disclosures:Munaka:Shimadzu Corporation: Employment. Kanai:Shimadzu Corporation: Employment. Abe:Shimadzu Corporation: Employment.

Isoprenoid biosynthetic pathway inhibition disrupts monoclonal protein secretion and induces the unfolded protein response pathway in multiple myeloma cells

Myeloma is characterized by the overproduction and secretion of monoclonal protein. Inhibitors of the isoprenoid biosynthetic pathway (IBP) have pleiotropic effects in myeloma cells. To investigate whether IBP inhibition interferes with monoclonal protein secretion, human myeloma cells were treated with specific inhibitors of the IBP or prenyltransferases. These studies demonstrate that agents that inhibit Rab geranylgeranylation disrupt light chain trafficking, lead to accumulation of light chain in the endoplasmic reticulum, activate the unfolded protein response pathway and induce apoptosis. These studies provide a novel mechanism of action for IBP inhibitors and suggest that further exploration of Rab-targeted agents in myeloma is warranted.

Bcl6 as a Novel Therapeutic Target in Multiple Myeloma (MM).

Abstract 295BCL6 (B- cell lymphoma 6) is a proto-oncogene encoding a transcriptional repressor and regulates germinal center B cell differentiation. It is deregulated by chromosomal translocations or aberrant somatic hypermutation in a subset of diffuse large B cell lymphomas (Polo, JM. et al. PNAS, 2007). Importantly, small peptide inhibitor of BCL6 mediates cytotoxicity in primary human DLBCL cells both in vitro and in vivo, without effecting normal lymphoid tissue (Cerchietti, LC. et al. Blood, 2009). In this study, we examined modulation of Bcl6 expression and its sequelae in human MM cells in the context of bone marrow (BM) microenvironment.By Western blot analysis, constitutive expression of Bcl6 was either undetectable or very weakly expressed in all MM cell lines (MM.1S, MM.1R, RPMI8226, RPMI-LR5 H929, OPM1 and OPM2) except U266 cells. Importantly, however, Bcl6 expression was markedly upregulated by co-culture of MM cells with bone marrow stromal cells (BMSCs). Since U266 has high baseline phopho-STAT3 and BMSC-induced Bcl6 upregulation in other MM cell lines was associated with phosphorylation of STAT3, we hypothesized that JAK/STAT3 signaling might mediate Bcl6 expression in MM cells in the context of BM microenvironment. Indeed, anti-IL-6 neutralizing Ab significantly blocked BMSC co-culture-induced Bcl6 expression. To assess the clinical relevance of Bcl6 expression observed in MM cell lines, we examined its expression in patient MM cells by tissue microarray immunohistochemical analysis, confirming that Bcl6 was strongly positive within the nucleus in all cases. To obtain direct evidence that IL-6 triggered Bcl6 expression, we cultured MM cells with recombinant human IL-6; as expected, IL-6 strongly triggered Bcl6 expression in MM cell lines in a dose- and time-dependent fashion whereas other cytokines (ie, IGF-1, VEGF, IL-3) did not. Importantly, Bcl6 was also induced by IL-6 in primary tumor cells from MM patients. Real time RT-PCR confirmed that both BMSC co-culture and IL-6 treatment upregulated expression of Bcl6 mRNA. Furthermore, oncostatin-M, which also induces phospho-STAT3, similarly upregulated Bcl6. Conversely, pan-JAK inhibitor AG490 and STAT3 siRNA markedly downregulated Bcl6 expression in U266 cells, associated with downregulation of phospho-STAT3. Taken together, these results indicate that gp130 family member cytokines upregulate Bcl6 expression in MM cells via JAK/STAT3 signaling. We also found that TNFα also triggered Bcl6 in MM cell lines (MM.1S, RPMI8226, OPM1 and OPM2) and primary MM tumor cells; however, TNFα-induced upregulation of Bcl6 was independent on STAT3 activation. Importantly, IKKβ inhibitor MLN120B significantly reduced Bcl6 expression in MM cells triggered by TNFα. Taken together, these results suggested that TNFα-induced Bcl6 expression is mediated via the canonical NF-κB signaling pathway. Finally, to examine the biologic significance of Bcl6 inhibition in MM cells, we downregulated Bcl6 expression using lentiviral shRNA, in the presence or absence of IL-6. Importantly, downregulation of Bcl6 significantly decreased the number of MM cells, associated with decreased S and G2/M phase on cell cycle analysis. Our results therefore suggest that Bcl-6 expression is modulated via both JAk/STAT3 and NF-κB pathways in MM cells. Therefore Bcl6 represents a novel therapeutic target in MM.since targeting these cascades could downregulate Bcl-6 expression, and inhibit growth of MM cells in the bone marrow milieu. Disclosures:Raje:Millennium: Speakers Bureau. Munshi:: . Richardson:Keryx Biopharmaceuticals: Honoraria. Anderson:Millennium: Research Funding.

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

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

Modulation of ER Stress/Unfolded Protein Response (UPR) Pathways in Multiple Myeloma Cells by Inhibition of Hsp90 and Serine-Threonine Kinase CK2.

Abstract 3840Poster Board III-776Hsp90 is an essential chaperone molecule that helps in the maturation and folding of a number of cellular client proteins. Hsp90 function is essential for malignant plasma cell survival, since its inhibition in multiple myeloma (MM) cells results in cell death and activation of apoptosis. Clinical trials using Hsp90 inhibitors are currently ongoing in MM patients.Hsp90 inactivation in MM cells causes perturbation of the endoplasmic reticulum (ER) stress/unfolded protein response (UPR), eventually triggering the apoptotic cascades. Protein kinase CK2 critically regulates the activity of the chaperone complex formed by the Cdc37 and Hsp90 proteins. We already described that CK2 is over-expressed in a fraction of MM patients and is an essential MM pro-survival molecule. We have here investigated its role in the ER stress/UPR pathways and in Hsp90 inhibition-induced apoptosis in MM cells.Down-regulation of the catalytic CK2 alpha subunit with selective chemical inhibitors or RNA interference resulted in significant modifications of the main UPR regulating signaling cascades: 1) a marked reduction of IRE1alpha protein levels; 2) a reduction of BiP/GRP78 and Hsp70 chaperone protein levels; 3) an increase of PERK activity and phospho eIF2alpha levels. When UPR was triggered by thapsigargin in CK2-inactivated cells, we observed that the IRE1alpha-dependent axis of the UPR was greatly impaired, as XBP-1short isoform generation and the levels of some induced chaperones were reduced. Interestingly, thapsigargin was able to induce CK2 kinase activity. Remarkably, treatment of CK2-silenced MM cells with Hsp90 inhibitors geldanamycin or its derivative 17-AAG (17-(demethoxy)-17-allylamino geldanamycin) resulted in 1) an even more pronounced reduction of IRE1 alpha protein levels; 2) a marked inhibition of GA or 17-AAG-triggered BiP/GRP78 protein level raise; 3) a more evident increase of eIF2 alpha phosphorylation. Of note, CK2 plus Hsp90 inhibition was followed by apoptotic cell death to a much greater extent than that obtained with the single inhibition of the two molecules. Noteworthy, these effects were also reproduced upon modelling the MM bone marrow (BM) microenvironment by co-culturing MM cells with BM stromal cells.These data suggest that CK2-mediated signaling regulates the ER stress/UPR pathways and modulates the threshold to apoptosis of ER stressed MM cells. CK2 interacts with Hsp90, since its inhibition synergizes with GA or 17-AAG treatments in terms of induction of apoptosis and shift of the ER stress/UPR pathways towards the terminal phase. These results might be useful to set the groundwork in designing novel combination treatments for MM patients. Disclosures:No relevant conflicts of interest to declare.

CS1 promotes multiple myeloma cell adhesion, clonogenic growth, and tumorigenicity via c-maf–mediated interactions with bone marrow stromal cells

AbstractCS1 is highly expressed on tumor cells from the majority of multiple myeloma (MM) patients regardless of cytogenetic abnormalities or response to current treatments. Furthermore, CS1 is detected in MM patient sera and correlates with active disease. However, its contribution to MM pathophysiology is undefined. We here show that CS1 knockdown using lentiviral short-interfering RNA decreased phosphorylation of ERK1/2, AKT, and STAT3, suggesting that CS1 induces central growth and survival signaling pathways in MM cells. Serum deprivation markedly blocked survival at earlier time points in CS1 knockdown compared with control MM cells, associated with earlier activation of caspases, poly(ADP-ribose) polymerase, and proapoptotic proteins BNIP3 and BIK. CS1 knockdown further delayed development of MM tumor and prolonged survival in mice. Conversely, CS1 overexpression promoted myeloma cell growth and survival by significantly increasing myeloma adhesion to bone marrow stromal cells (BMSCs) and enhancing myeloma colony formation in semisolid culture. Moreover, CS1 increased c-maf–targeted cyclin D2-dependent proliferation, -integrin β7/αE-mediated myeloma adhesion to BMSCs, and -vascular endothelial growth factor-induced bone marrow angiogenesis in vivo. These studies provide direct evidence of the role of CS1 in myeloma pathogenesis, define molecular mechanisms regulating its effects, and further support novel therapies targeting CS1 in MM.

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

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. [Display omitted]

MUC1 oncoprotein promotes growth and survival of human multiple myeloma cells

The MUC1 oncoprotein is aberrantly expressed in human multiple myeloma cells by mechanisms that are not understood. Moreover, the functional role of MUC1 in multiple myeloma is not known. The present studies demonstrate that the MUC1 gene locus is amplified in multiple myeloma cell lines and in primary cells from patients. The KMS28PE multiple myeloma cell line, which was found to have MUC1 gene amplification, was stably silenced for MUC1 using different siRNAs. Silencing MUC1 was associated with a decrease in nuclear beta-catenin levels, consistent with the function of MUC1 in stabilizing beta-catenin. MUC1 is also known to activate the IKKbeta-->NF-kappaB pathway and KMS28PE cells silenced for MUC1 were found to have downregulation of IKKbeta and IkappaBalpha phosphorylation, and decreased nuclear targeting of NF-kappaB p65. The results also demonstrate that MUC1: i) contributes to KMS28PE cell proliferation, and ii) protects against apoptosis and loss of self-renewal in the response to melphalan and dexamethasone. These findings indicate that MUC1 activates the beta-catenin and NF-kappaB pathways in multiple myeloma cells and contributes to their growth and survival.

Activation of B-Cell Maturation Antigen (BCMA) on Human Multiple Myeloma Cells by a Proliferation-Inducing Ligand (APRIL) Promotes Myeloma Cell Function in the Bone Marrow Microenvironment.

A proliferation-inducing ligand (APRIL), a close member of B-cell-activating factor (BAFF) belonging to the TNF superfamily that stimulates growth and survival of multiple myeloma (MM) cells, is mainly produced by MM patient bone marrow stromal cells (BMSCs) and osteoclasts in the BM microenvironment. Like BAFF, APRIL binds two receptors, B-cell maturation antigen (BCMA) and transmembrane activator and calcium modulator (TACI). Since BCMA has significantly higher affinity for APRIL than for BAFF (nM vs. mM range), APRIL/BCMA signaling may be more important than BAFF/BCMA in MM. Specifically, BCMA but not TACI is unequivocally expressed at mRNA level in the majority of CD138-expressing MM cell lines and patient MM cells. To date, it is unclear how APRIL signaling through BCMA contributes to BM microenvironment in support of MM cells. We here studied the functional sequelae of APRIL/BCMA pathway in MM cells, and further identified molecular mechanisms regulating these processes. Using flow cytometric analysis, cell surface BCMA expression was confirmed in the majority of MM cell lines. Microarray gene expression analysis showed significant expression of BCMA mRNA in CD138+ patient MM cells (n=105). Higher BCMA mRNA levels were observed in patient MM cells than in normal plasma cells (n=27) (p< 0.03). Importantly, CD138+ patient MM cells express BCMA protein on the cell surface (n=15), as confirmed by CD38+BCMA+ dual immunofluorescence staining followed by flow cytometric analysis and immunohistochemical (IHC) study. Next, H929 MM cell line that expresses only BCMA, but not TACI, and Daudi B cells transduced with huBCMA (huBCMA-Daudi) were stimulated with APRIL. These cells were subjected to mRNA extraction at different time points followed by microarray analysis to identify downstream transcriptional targets. Stimulation of these two cell lines by APRIL activated the NF-kB signaling in a dose-dependent manner. More than 6-fold increase in NF-kB activity was induced by APRIL (100 ng/ml) using a p65 enzyme-linked DNA/protein interaction assay. IL-6 and PI3K/AKT signaling cascades were concurrently induced. Additionally, APRIL upregulated chemotactic/osteoclast activating factors, i.e., MIP-1a and MIP1b, in a dose-dependent manner, which was further validated by specific ELISA. Multi-Analyte Profiles also confirmed that angiogenesis and adhesion/chemoattractant factors, i.e., IL-8, CXCL10, RANTES, MDC (ccl22), were significantly induced upon APRIL stimulation. Conversely, a soluble BCMA-Fc protein inhibited APRIL binding to BCMA and blocked secretion of APRIL-induced chemokines, indicating specific functional interaction of BCMA with APRIL. Importantly, APRIL similarly induced these signaling cascades in CD138+ patient MM cells. Taken together, our studies are the first to demonstrate cell surface BCMA on the majority of CD138+ patient MM cells. Furthermore, APRIL induced osteoclasts-, migration-, and angiogenesis-associated factors in MM cells. These studies therefore establish a role of BCMA activation in the BM microenvironment that provides a niche for MM disease progression, supporting novel therapeutics specifically targeting APRIL/BCMA pathway in MM.

Role of GSK3 in Multiple Myeloma Cell Growth and Survival.

The serine-threonine GSK3 displays a crucial role in different cancer-pathogenetic pathways, including the PI3K/AKT, Wnt β-catenin and NF-κB signaling cascades, either promoting or counteracting cell survival. The aim of this study was to investigate the role of GSK3 in multiple myeloma (MM) cell growth. GSK3α and β total and phosphorylated protein levels were found differentially expressed in malignant plasma cells as compared to normal resting B-lymphocytes and to normal in vitro generated plasmablasts. Intriguingly, in freshly isolated malignant plasmacells from patients, most of GSK3 was found colocalized with Wnt receptor LRP6 and casein kinase I next to the cell membrane as compared to normal plasmacells or B-cells from other malignancies, wher it was distributed in the cytosol and in the nucleus, thus suggesting a peculiar role of this kinase in these cells. Upon stimulation of MM cells with IL-6 and IGF-I, GSK3 enzymatic activity was hampered, while stimulation with TNFα did not affect GSK3 function nor the early events in NF-κB activation. Basal and IL-6 and IGF-I driven proliferation of MM cells was slightly impaired by GSK3 blockade. Interestingly, GSK3β−/− mouse embryo fibroblasts (MEFs) proliferated slightly slower as compared to GSK3β+/+ cells; however, GSK3α inhibition and IL-6 and IGF-I stimulation, resulted in much higher proliferation of GSK3β −/− cells. Intriguingly, GSK3 inhibition with specific compounds (SB216763 and SB415286) caused a significant rescue from cell death of growth factor-deprived MM cells while resulted in reduced cell proliferation and apoptosis of MM cells grown with serum or growth factors. When GSK3 inhibitors were added to MM cell cultured with bone marrow stromal cells (BMSC), MM cells survival increased and NF-κB and β-catenin-mediated gene transcription (of IAPs and cyclinD1, respectively) was deregulated. GSK3 activity inhibition did not modify the rate of proteasome inhibitor-induced cell death in co-colture experiments with BMSC, suggesting that the sensitivity to bortezomib-induced MM cell apoptosis is independent on GSK3. Altogether, our data indicate thati.GSK3 localizes on the cell membrane in primary MM cells;ii.GSK3 is differently regulated downstream from growth factors or TNFα-induced signaling pathways in MM cells;iii.a peculiar role of GSK3 in malignant MM cells as compared to normal MEFs with regard to cell proliferation; andiv.a critical role of this kinase in regulating the MM microenvironment.

Biologic Sequelae of CDC2 Inhibition in MM.

Multiple Myeloma (MM) is a malignant proliferation of plasma cells characterized by disruption of cell cycle checkpoint controls which maintain G2M transition and/or mitosis. CDC2 is the cyclin-dependent kinase that normally drives cells into mitosis and is universally expressed in MM. To examine the biologic role of CDC2 in MM, we evaluated cellular and molecular effects of Terameprocol (M4N, tetra-O-methyl nordihydroguaiaretic acid) that has been shown to inhibit cell cycle progression at the G2/M phase by inhibiting the transcription of sp-1 dependent expression of CDC2. We observed that Terameprocol downregulated the expression of cdc2 in a time-dependent manner, with a maximal effect at 24h. This was associated with induction of G2/M growth arrest in a panel of MM cell lines (INA6, OPM1, OPM2, MM1S, RPMI-8226, U266), as determined by PI staining. Interestingly, Terameprocol treatment led to increase in p21waf1 protein levels. Importantly, we observed inhibition of DNA synthesis by Terameprocol in a dose- and time-dependent manner, with IC50 range from 1–20 uM for a 24 hours period of treatment, as assessed by 3H-thymidine uptake. Longer exposure of MM cells to Terameprocol resulted in cytoxicity, as assessed by MTT assay, via induction of apoptosis, evidenced by Annexin V+ /PI staining, in all the MM cell lines tested. Terameprocol -induced apoptosis is predominantly associated with caspase-9 and caspase-3, but not caspase-8 activation, suggesting that Terameprocol triggers intrinsic apoptotic pathway in MM cells. Our results show that genes that control entry and progression of G2/M phase, especially cdc2, may be an attractive target for MM therapy and Terameprocol represents a prototypic agent for the control of unregulated cellular proliferation in MM.

The MEK1/2 Inhibitor AZD6244 (ARRY-142886) Downregulates Constitutive and Adhesion-Induced c-MAF Oncogene Expression and Its Downstream Targets in Human Multiple Myeloma.

Human multiple myeloma (MM), an incurable plasma cell tumor, is highly dependent on its bone marrow (BM) microenvironment which secretes cytokines and growth factors (i.e., IL-6, IGF-1, BAFF) for myeloma cell growth, survival, and resistance to chemotherapeutic drugs. Studies of interactions of MM and BMSCs have demonstrated that activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) mediates these processes. Recently, gene expression profiling studies have revealed that the c-MAF oncogene, which is overexpressed in approximately 50% of myeloma cell lines and patient MM cells, not only stimulates cell cycle progression but also promotes pathological interactions between tumor and BMSCs. In this study, we examined effects of the MEK1/2 inhibitor AZD6244 (ARRY-142886) on the c-MAF signaling and adhesion-induced c-MAF expression in MM cells. We first demonstrated that AZD6244 inhibits growth of INA6, MM1S, MM1R and MCCAR MM cells in a dose-dependent manner (see also Abstract #553572, ASH 2006). We next found that AZD6244 significantly downregulates c-MAF mRNA and c-MAF protein expression in these MM lines, as early as 2h and this was sustained until 24h after drug treatment. Hypoxia-induced factor 2α (HIF-2α), a pro-angiogenic mediator, which regulates c-MAF gene expression and is regulated by ERK1/2, was significantly inhibited in AZD6244-treated MM cell lines. Since three c-MAF target genes, integrin β7, the C-C chemokines receptor-1 (CCR1), and cyclin D2, were recently identified in MM, we next asked whether the expression of these genes was concomitantly inhibited by AZD6244 in INA6 MM cells that lacks c-MAF translocation. Integrin β7 and CCR1 were inhibited by AZD6244 in a time-dependent manner, whereas cyclin D2 was not expressed in INA6 MM cells. Importantly, downregulation of c-MAF and integrin β7 by AZD6244 is associated with decreased MM cell adhesion to BMSCs and reduced VEGF secretion. Although the role of CCR1 in MM pathophysiology remains elusive, AZD6244 significantly decreased the expression of CCR1 as well as its cognate ligand MIP-1α in INA6 MM cells, which might inhibit a potential autocrine loop in MM cells. We next determined whether MM adhesion to BMSCs could upregulate the c-MAF pathway in MM cells, in the presence or absence of AZD6244. Adhesion of INA6 MM cells to BMSCs strongly induced ERK1/2 and STAT3 activation and augmented expression of c-MAF and its downstream targets (cyclin D2 and integrin β7). The induction of c-MAF and its downstream targets was also seen, although with less extent, in IL-6-induced INA6 MM cells. Conversely, AZD6244 inhibited adhesion-induced c-MAF and its target protein expression (cyclin D2, integrin β7) in INA6 MM cells. AZD6244 further blocked autocrine and paracrine VEGF secretion in a dose-dependent manner. In conclusion, these results confirm a role for c-MAF in the pathological interactions between MM cells and BMSCs and define an important role of the MEK/ERK activation in the ~40% of c-MAF-overexpressing myeloma cells that lack c-MAF translocations. Moreover, this study strongly supports AZD6244 (ARRY-142886) as a promising targeted therapeutic intervention in MM.

Absence of JAK-2 (Val617Phe) Point Mutations in Human Plasma Cell Myeloma.

Multiple myeloma (MM) is a terminal differentiated B cell chronic lymphoproliferative disorder characterized by the latent accumulation of plasma cells with a low proliferative index and an extended life span in bone marrow or extramedullary tissues. While studies on neoplastic cells isolated directly from MM patients or on MM-derived cell lines demonstrated that IL-6 is a major growth and survival factor for initiation of signaling in MM cells in an autocrine or paracrine manner, some MM cells independent on extracellular signaling such as IL-6 have been reported, suggesting other mechanisms may be involved in signal transduction pathways in MM cells. Accumulating evidence showed that IL-6 induces intracellular signaling through members of the signal transducers and activators of transcription (STAT) family of proteins by activating the janus kinase(JAK) family of protein tyrosine kinases, which subsequently phosphorylate and activate cytoplasmic Stat proteins. Activated Stat proteins dimerize and translocate to the nucleus, where they bind to specific DNA response elements and induce expression of Stat-regulated genes. Our previous studies demonstrated that one of the Stat family proteins, Stat3, was constitutively activated (phosphorylated) in the majority of MM patients, suggesting constitutive activation of JAK-2 kinase activities. However, other mechanisms might be also responsible JAK2 constitutive activation in MM cells, in particular in IL-6 independent MM cells. A specific point mutation of JAK-2 kinase gene has been identified recently showing its presence in higher frequency of certain types of chronic myeloproliferative disorders. The point mutation yields constitutive activation of JAK-2 kinase activity in the absence of extracellular signaling and plays an important role in the pathogenesis of those disorders. In this study, we try to determine whether JAK-2 (Val617Phe) point mutation was present in MM patients and if so, what kind of role it may play in the pathogenesis of MM. JAK-2 (Val617Phe) point mutation was analyzed by using an allele-specific polymerase chain reaction followed by separation and detection with capillary electrophoresis. The test has a detection sensitivity of up to 200 picogram mutated DNA. Bone marrow aspirates with various degrees of involvement by MM (ranged10–80%) were selected and all the cases have been verified by morphologic examination and demonstrated to have positive immunoglobulin heavy or kappa light gene rearrangements. Bone marrow specimens from total 59 MM patients were tested for JAK2 (Val617Phe) mutation and none of any MM cases with the specific point mutation were identified (0/59). The results indicated that JAK2 (Val617Phe) mutation was very infrequency or absence in MM patients, despite the fact that constitutional activation of JAK2 kinase as well as Stat3 activation was commonly seen in MM patients. The study suggests that other yet unknown mechanisms may also involve JAK2 activation, particularly in IL-6 independent MM cells.