Treatment Of Multiple Myeloma Cells Research Articles

In Vitro and In Vivo Selective Antitumor Activity of a Novel Orally Bioavailable Proteasome Inhibitor MLN9708 against Multiple Myeloma Cells

The success of bortezomib therapy for treatment of multiple myeloma (MM) led to the development of structurally and pharmacologically distinct novel proteasome inhibitors. In the present study, we evaluated the efficacy of one such novel orally bioactive proteasome inhibitor MLN9708/MLN2238 in MM using well-established in vitro and in vivo models. MM cell lines, primary patient cells, and the human MM xenograft animal model were used to study the antitumor activity of MN2238. Treatment of MM cells with MLN2238 predominantly inhibits chymotrypsin-like activity of the proteasome and induces accumulation of ubiquitinated proteins. MLN2238 inhibits growth and induces apoptosis in MM cells resistant to conventional and bortezomib therapies without affecting the viability of normal cells. In animal tumor model studies, MLN2238 is well tolerated and inhibits tumor growth with significantly reduced tumor recurrence. A head-to-head analysis of MLN2238 versus bortezomib showed a significantly longer survival time in mice treated with MLN2238 than mice receiving bortezomib. Immununostaining of MM tumors from MLN2238-treated mice showed growth inhibition, apoptosis, and a decrease in associated angiogenesis. Mechanistic studies showed that MLN2238-triggered apoptosis is associated with activation of caspase-3, caspase-8, and caspase-9; increase in p53, p21, NOXA, PUMA, and E2F; induction of endoplasmic reticulum (ER) stress response proteins Bip, phospho-eIF2-α, and CHOP; and inhibition of nuclear factor kappa B. Finally, combining MLN2238 with lenalidomide, histone deacetylase inhibitor suberoylanilide hydroxamic acid, or dexamethasone triggers synergistic anti-MM activity. Our preclinical study supports clinical evaluation of MLN9708, alone or in combination, as a potential MM therapy.

Small Molecule RITA Induces Apoptosis In Multiple Myeloma Cells through Activation of the p53 Pathway and Induction of ER-Stress response

AbstractAbstract 790TP53 mutations or deletions are relatively rare in multiple myeloma (MM). Thus, restoration of p53 tumor suppressor function in MM by blocking the inhibitory role of MDM2 is a promising and applicable therapeutic strategy. RITA (Reactivation of p53 and Induction of Tumor-cell Apoptosis), binds to p53, prevents the p53-MDM2 interaction and induces accumulation of p53 in solid tumors and leukemic cells. However, whether RITA exerts anti-myeloma activity is not known. Therefore, we examined the apoptotic effect of RITA and molecular mechanisms of RITA-induced apoptosis in MM cells. Treatment of MM cells with RITA caused a dose-dependent inhibition of survival and an increase in apoptosis of cells harboring wild type (MM.1S, H929) but not mutant (U266, LP1) or null p53 (8226R5) suggesting that RITA-induced apoptosis in MM cells is p53-dependent. MM cells were sensitive to low doses of RITA, killing about 80% of MM.1S or H929 cells at 2 or 6 μM, respectively. In addition, RITA elicited a dose-dependent inhibition of myeloma cell growth in 4 of the 5 primary MM samples tested. Importantly, RITA did not have a significant inhibitory effect on the survival of bone marrow or peripheral blood mononuclear cells obtained from 5 healthy donors at the concentrations that induced apoptosis of MM cells, indicating a preferential killing of myeloma cells by this drug. Furthermore, the combination of RITA with an MDM2 antagonist, nutlin displayed a strong synergism on the killing of H929 or MM.1S cells (CI=0.56-0.82). Western blot analysis showed that RITA induced an increase in the expression of p53 only in cells harboring wild type p53 but not mutant p53. The activation of p53 induced by RITA was mediated through modulation of multiple apoptotic regulatory proteins, including up-regulation of a pro-apoptotic protein Noxa, down-regulation of an anti-apoptotic protein Mcl-1 and activation of caspase-8 and -3. RITA-induced caspase-mediated apoptosis was followed by extrinsic pathway as shown by decrease in the percentage of annexin V-positive cells (p<0.05) as well as inhibition of cleavage of PARP and caspase-3 and/or caspase-8 activation in MM.1S or H929 cells when treated with either pan-caspase inhibitor, Z-VAD-FMK or caspase-8 specific inhibitor, Z-IETD-FMK. To further delineate the mechanisms of apoptosis, we performed gene expression profiling and identified up-regulation of transcription factors including JUN, DDIT3, ATF3 and ATF4, and down-regulation of survival factors, MYC and IGF1R in RITA-induced MM.1S cells, which were validated by qRT-pCR. Up-regulations of DDIT3, ATF3 and ATF4 are thought to be associated with the induction of endoplasmic reticulum (ER)-stress response. To examine whether apoptosis induction by RITA in MM cells is mediated through ER-stress response pathway, cells were treated with either an inhibitor of ER-stress-induced apoptosis, salubrinal, or a specific inhibitor of c-Jun N-terminal kinase (JNK), SP600125. Treatment of MM.1S or H929 cells with these inhibitors resulted in a significant inhibition of apoptosis induced by RITA as evidenced by inhibition of cleavage of caspase-3 and PARP, and decrease in the percentage of annexin V-positive cells (p<0.05). However, the above phenomenon was not observed in MM cells harboring mutant or null p53. Taken together, our results identify RITA as a promising agent in treatment of MM with two potential mechanisms of action: 1) activation of the p53 apoptotic pathway through up-regulation of Noxa and down-regulation of Mcl-1; and 2) p53-mediated induction of the ER-stress response pathways that may inhibit MM proliferation or survival. Our data provide the rationale for the clinical evaluation of RITA as a potential novel therapeutic intervention in MM. Disclosures:No relevant conflicts of interest to declare.

Prokineticin-1/endocrine gland-derived vascular endothelial growth factor is a survival factor for human multiple myeloma cells

Prokineticin-1 (PK1) has been identified as a mitogen-specific protein for the endothelium of steroidogenic glands. Here we report a novel function of PK1 in the regulation of multiple myeloma (MM) cells. PK1 activates multiple signals including mitogen-activated protein kinase (MAPK), PI3K-AKT, and Jak-STAT3, sphingosine kinase-1 (SPK1) in MM cells. Treatment of MM cells with PK1 causes a time- and dose-dependent phosphorylation of MAPK, AKT and STAT3 and upregulation of SPK1 expression and cellular activity. We also show that PK1 upregulates Mcl-1 expression in a time- and dose-dependent manner in human MM cell lines and in the cells of patients with MM. Pertussis toxin, a pan-PK1 receptor inhibitor, can block PK1-induced upregulation of Mcl-1, indicating it relates to a G-protein-coupled receptor. We also show that PK1 protects MM cells against apoptosis induced by starvation for fetal calf serum (FBS), or for FBS and IL-6. Taken together, PK1 activates multiple signaling pathways and, upregulates Mcl-1 expression, leading to proliferation and survival of MM cells.

Mapatumumab, A TRAIL Receptor 1 Agonist Antibody, Induces Apoptosis in Bortezomib Resistant Multiple Myeloma.

Abstract 2832Poster Board II-808Bortezomib, a novel proteasomal inhibitor, is an important treatment for patients with multiple myeloma (MM). Despite its clinical success, acquired resistance to bortezomib is an unresolved challenge experienced in the clinic. Limited therapeutic options are available for patients with bortezomib resistant disease and novel therapies are urgently required to target bortezomib resistant MM. In this report, we evaluated the efficacy of Mapatumumab, a fully human agonistic antibody that targets and activates the tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) receptor-1, on MM cell lines. Myeloma cell lines OPM2, KMS11 and U266 wild type (WT) and their respective bortezomib resistant (BR) derivatives were utilized to investigate the efficacy of mapatumumab to overcome bortezomib resistance. Biochemical analyses showed BR cells to have a higher proteasomal activity with cross-resistance to many of the conventional therapeutic agents. Treatment of both WT and BR cells with mapatumumab resulted in a significant (p<0.005) inhibition in their viability by 24h, compared to cells treated with the control antibody. Inhibition of tumor cell viability varied between 20-70%, depending on the cell line. The inhibitory response was higher in WT cells, as compared to the BR cells. Annexin-V staining of mapatumumab treated cells showed the decrease in viability was due to the induction of apoptosis. Immunoblot analyses revealed that mapatumumab treatment resulted in the activation of caspase-8 and caspase-3, suggesting activation of the extrinsic apoptosis pathway in the presence of the antibody. A pan caspase inhibitor, z-vad-fmk (at 25mM), blocked apoptosis induced by mapatumumab. Investigation into the effect of mapatumumab on intracellular signaling showed that treatment with the antibody inhibited the phosphorylation of AKT, and ERK1/2, as well as the expression of Bcl-2 in these cells as early as 4h post-treatment. This suggests mapatumumab treatment affects cellular proliferation and survival pathways. Furthermore, combined treatment of MM cells with mapatumumab and bortezomib resulted in enhanced cell killing compared to mapatumumab or bortezomib alone in both WT and BR cells, thus indicating that there a co-operation in apoptosis signaling induced by these agents. Results presented in this report suggest that TRAIL-R1 mediated apoptosis can be exploited as a therapeutic option in bortezomib resistant MM and warrant clinical evaluation of mapatumumab in bortezomib resistant MM. Disclosures:Chanan-Khan:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Immunogen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Targeting the Clonogenic Side Population of Multiple Myeloma by Immunomodulatory Drugs (IMiDs) in the Context of Stromal Cell Microenvironment: Pathophysiologic and Clinical Implications.

Abstract 954 Introduction:Multiple myeloma (MM), a malignancy of plasma cells, remains incurable even though newly established regimens incorporating conventional and novel agents are capable of achieving complete clinical and biochemical remissions in a sizeable subset of MM patients. This indicates the presence of a distinctive drug-resistant sub-population of MM cells, which might be responsible for tumor re-growth. Methods and Results:To evaluate this phenomenon, we performed flow cytometry-based Hoechst 33342 staining assay to evaluate the existence and characteristics in MM of a population known as side population (SP), which has been identified in diverse neoplasias and has been reported to exhibit “stem-like” features. We specifically performed flow cytometry or ImageStream analyses to quantify the SP population in a panel of 18 MM cell lines. These analyses revealed heterogeneity in the proportion of SP fractions in different cell lines and indicated the lack of correlation between CD138 expression and SP fraction. We observed that, after sorting of the SP and non-SP fractions, SP cells have the potential to generate colonies in CFU assays, and regenerate a population resembling the original, pre-sorted, one. Moreover, SP cells had a high proliferation index compared to non-SP cells. We also observed that most MM cells lines express significantly higher levels of ABCG2 transcripts in their sorted SP fraction compared to their non-SP cells. The mRNA profile results were further corroborated by functional assays, where SP cells showed more pronounced efflux of the known ABCG2 substrate mitoxantrone, consistent with the association of SP phenotype with ABCG2 expression. Interestingly, at concentrations and durations of drug exposure that did not substantially affect the viability of non-SP cells, the immunomodulatory thalidomide derivative (IMiDs) lenalidomide significantly decreased the percentage and clonogenicity of SP cells. This response was accompanied by a distinct pattern of changes in diverse signaling pathways in SP cells (including phosphorylation changes in Akt, GSK-3a/b, MEK1, c-Jun, p53, p38 MAPK, p70S6K and STAT3) and increase of CD138-/low+ phenotype. Because the BM microenvironment is considered a key regulator of MM cell biological behavior, we evaluated the impact of bone marrow stromal cells (BMSCs) on the behavior of SP cells and observed that BMSCs led to increased percentage, viability, as well as proliferation potential of SP cells. Interestingly, IMiDs abrogated this stimulatory effect of stromal cells by significantly decreasing SP cell percentages. In these co-culture models, we identified several cytokines, including IL-1b, IL-10, IL-17; chemokines (MIP-1a, MIP-1b and IP-10) and growth factors such as GM-CSF and VEGF, which were up-regulated in co-culture MM cells with stromal cells compared to stroma alone or MM cells alone. Significant modulation in the production of these cytokines and chemokines was detected after IMiD treatment of MM cells only, as well as in co-culture model. Conclusion:Identifying the clonogenic population of SP cells could facilitate the development of new strategies targeting subpopulations of MM cells with drug resistant properties. Our study raises the intriguing possibility that IMiDs could represent such a strategy that can target SP cells and counteract their resistance to different conventional, and perhaps some novel, therapies. In addition, our data suggest that further studies of this tumor cell population are warranted towards to the goal of developing new strategies to treat MM. Disclosures:Laubach:Novartis: Consultancy, Honoraria. Richardson:Millenium: (Speakers' bureau until 7/1/09), Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: (Speakers' bureau until 7/1/09), Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Anderson:Novartis: Consultancy, Honoraria, Research Funding; Millennium: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding. Mitsiades:Millennium: Consultancy, Honoraria; Novartis : Consultancy, Honoraria; Bristol-Myers Squibb : Consultancy, Honoraria; Merck &Co: Consultancy, Honoraria; Kosan Pharmaceuticals: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; PharmaMar: Patents & Royalties; Amgen: Research Funding; AVEO Pharma: Research Funding; EMD Serono : Research Funding; Sunesis Pharmaceuticals: Research Funding.

Lenalidomide Inhibits Multiple Myeloma Cell Proliferation in Vitro Via Its Effect On Expression of Oncogenes and Tumor Suppressor Genes.

Abstract 2855Poster Board II-831Lenalidomide is an oral anti-proliferative and immunomodulatory drug. In combination with dexamethasone, it is indicated for the treatment of patients with multiple myeloma (MM) who have received at least one prior therapy. In this study we investigated direct antimyeloma effects of lenalidomide in vitro using a panel of human MM cell lines with various cytogenetic features and bone marrow cells from patients with active MM. We also assessed the effect of lenalidomide on expression of tumor suppressor and enhancer genes such as p21cip1, SPARC, ING1/4, p57kip2, p53, cyclin D1/2, IRF4/MUM1 and IRF8/ICSBP. At attainable plasma levels in treated patients, lenalidomide directly inhibited human MM cell proliferation. Lenalidomide strongly increased expression of tumor suppressor genes such as p21waf1/cip1, SPARC, IRF8, ING4 and p57kip2. In the MM cell lines tested, lenalidomide had partial but consistent inhibitory effects on expression of IRF4, an important MM survival factor. However, lenalidomide had no marked effect on expression of tumor enhancer genes such as VEGF, cyclin D1/2 and MAF or tumor suppressor genes such as ING1 and p53 in most lines of cells. This suggests that the antiproliferative effects of lenalidomide on MM cells may be related to the upregulation of some tumor suppressor gene expression. Statistical analyses show that the antiproliferative effect of lenalidomide is significantly correlated with the drug induced upregulation of SPARC and IRF8 expression (p=0.0016; p=0.012, respectively), but not with the drug induced changes of p21 (p> 0.05) and IRF4 expression (p> 0.05). Furthermore, the antiproliferative effect of lenalidomide was significantly correlated with the constitutive expression levels of cyclin D1 (p=0.021) and IRF4 (p=0.027), and inversely correlated with the constitutive level of cyclin D2 (p=0.041) in these MM cell lines. Using bone marrow myeloma cells from patients, we confirmed that the sensitivity of cells to lenalidomide was associated with SPARC and IRF8 upregulation and baseline levels of cyclin D1/2 and IRF4 expression. Using MM cell lines adapted to prolonged exposure (5 months) to lenalidomide, we found that cells became resistant to the drug in association with decreased baseline levels of cyclin D1 and IRF4. In conclusion, lenalidomide demonstrates direct inhibitory effects on proliferation various MM cells. These antimyeloma activities may help explain the clinical efficacy seen in patients treated with lenalidomide. Lenalidomide treatment of MM cells increased SPARC and IRF8 mRNA expression, whereas pre-treatment cyclin D1/2 and IRF4 mRNA levels were associated with increased sensitivity and may have prognostic potential for MM therapy with lenalidomide. Disclosures:Zhang:Celgene Corporation: Employment. Adams:Celgene Corporation: Employment. Kosek:Celgene Corporation: Employment. Schafer:Celgene Corporation: Employment. Bartlett:Celgene Corporation: Employment.

ATM-ATR–dependent up-regulation of DNAM-1 and NKG2D ligands on multiple myeloma cells by therapeutic agents results in enhanced NK-cell susceptibility and is associated with a senescent phenotype

AbstractThere is much evidence to support a role for natural killer (NK) cells in controlling the progression of multiple myeloma (MM), a malignancy characterized by an abnormal plasma cell proliferation in the bone marrow (BM). Induction of DNA damage response has been recently shown capable of enhancing NKG2D ligand (NKG2DL) expression, but nothing is known about DNAM-1 ligand (DNAM-1L) regulation. In this study, we show that myeloma cells treated with low doses of therapeutic agents commonly used in the management of patients with MM, such as doxorubicin, melphalan, and bortezomib, up-regulate DNAM-1 and NKG2D ligands. Accordingly, therapeutic drug treatment of MM cells increases NK-cell degranulation, the NKG2D and DNAM-1 receptors being the major triggering molecules. Similar data were also obtained using ex vivo primary plasma cells derived from MM patients. Drug-induced DNAM-1 and NKG2D ligand expression was abolished after treatment with the ATM (ataxia telangiectasia mutated) and ATR (ATM- and RAD3-related) pharmacologic inhibitors caffeine and KU-55933, and was preferentially associated with senescent cells arrested in the G2 phase of the cell cycle. Altogether, our findings have identified a common pathway that can trigger the up-regulation of different NK cell–activating ligands and suggest that NK cells represent an immunosurveillance mechanism toward cells undergoing stress-induced senescent programs.

Effects of CK2 Inhibition on Multiple Signaling Pathways in Myeloma Cells

CK2 serine-threonine kinase plays a key role in promoting cell survival of multiple myeloma (MM) cells through modulation of extrinsic and intrinsic apoptotis and by activating pro-survival signaling such as the NF-κB and STAT3 pathways (Piazza FA et al. 2006, Blood 105:1698–1707). Here we deepened our previous results and placed the CK2 action inside critical pathways for MM cell growth, like the MAPK, STAT3 and Wnt regulated cascades.We employed MM cell lines, primary cells from MM patients and human cervical carcinoma cell line HeLa. Inhibition of CK2 was obtained with well characterized chemical compounds (TBB, K27 and K17) or by RNA interference.CK2 inhibition led to an increased p38 MAPK activation and to a decreased basal and growth factor stimulated-ERK1,2 phosphorylation. These changes were associated to increased activation of apoptotis and cell death. Inhibition of PP1 and PP2A phosphatases led to a decreased p38 MAPK phosphorylation upon CK2 inhibition, suggesting that CK2 may control p38 activity through the modulation of these molecules. Activation of upstream JAK tyrosine kinase upon cytokine or growth factor stimulation are a prerequisite for STAT3 activation. Following inhibition of CK2 activity, Tyk2 phosphorylation and JAK2 activation were found impaired and these effects were associated to a decreased STAT3 phosphorylation in tyr705 and ser727. In turn, activated-STAT3 nuclear migration and STAT3-dependent transcription of target genes, like the pro-survival bcl-2 family member mcl-1 and the cell-cycle promoter cyclin-D1, were significantly reduced.Finally, Wnt activation was also affected by CK2 manipulation. Indeed, β-catenin activation and transcriptional activity were impaired by the treatment of MM cells with CK2 inhibitors; these effects were associated with reduced cell proliferation and cell death.Altogether, these data demonstrate that CK2 is a key player placed at the cross-road of important signaling pathways for MM cell growth and reinforce the possibility that it may represent an ideal target molecule for therapeutic purposes.

Inhibition of fatty acid synthase induces anti-tumor effect in multiple myeloma

19522 Background: Fatty acid synthase (FAS) is a key metabolic enzyme that catalyzes the terminal steps in the synthesis of long-chain fatty acids. Several reports have shown that FAS is expressed at significantly higher levels in a variety of human cancers, and FAS inhibitors inhibit tumor cell growth and induce apoptosis in various human cancers both in vitro and in vivo. In this study, we investigate the biological significance of inhibition of FAS in multiple myeloma (MM) cells using the small molecule inhibitor Cerulenin. Methods: FAS expression were analyzed by western blotting (WB) and immunohistochemical analysis. Cell growth was assesed by MTT assay or 3H uptake analysis. Apoptosis was evaluated by Flow cytometric analysis using Apo2.7 staining and WB. ER stress response was analyzed by WB. Assesement of combination effects with Cerulenin and other agents was performed by MTT assay and isobologram analysis. Results: FAS protein is more highly expressed in MM than normal cells. Cerulenin triggers growth inhibition in both MM cell lines and MM patient cells, as well as overcomes the survival and growth advantages conferred by IL-6, IGF-1 and bone marrow stromal cells (BMSC). It induces apoptosis in MM cell lines with only modest activation of caspase-8,-9,-3 and PARP. Moreover, the pan-caspase inhibitor Z- VAD-FMK does not significantly inhibit Cerulenin-induced apoptosis and cell death. In addition, treatment of MM cells with Cerulenin primarily up- regulates both AIF and Endo G, mediators of caspase-independent apoptosis. Importantly, Cerulenin also induces ER stress response via up- regulation of Grp78/IRE1α/JNK pathway. Although C-Jun-NH2-terminal kinase (JNK) inhibitor SP600215 blocks Cerulenin- induced cytotoxicity, it does not inhibit apoptosis and caspase cleavage triggered by Cerulenin. Furthermore, Cerulenin triggers synergistic cytotoxicity with Bortezomib, Melphalan and Doxorubicin, which induce apoptosis via activation of caspase. Conclusions: Our results indicate that inhibition of FAS by Cerulenin triggers primarily non-caspase-dependent apoptosis and JNK-dependent cell death in MM. This is the first report to demonstrate that inhibition of FAS has anti-tumor activity against MM cells, suggesting that it represents a novel therapeutic target in MM. No significant financial relationships to disclose.

Sphingosine 1-phosphate induces Mcl-1 upregulation and protects multiple myeloma cells against apoptosis

Sphingosine 1-phosphate (S1P) is a bioactive lysophospholipid which is known to induce diverse cellular responses through at least five G-protein-coupled receptors on various cell types. However, neither the distribution of S1P receptors nor the effects of S1P on multiple myeloma (MM) cells are fully understood. Here, we show that MM cells express the S1P receptors, S1P1, S1P2, and S1P3. Furthermore, S1P protects MM cells against Dex-induced apoptosis. Importantly, S1P upregulates Mcl-1 expression in a time- and concentration-dependent manner in human MM cell lines. Treatment of MM cells with pertussis toxin (PTX), a pan-S1P receptor inhibitor, results in blockage of S1P-induced upregulation of Mcl-1. These data demonstrate that S1P upregulates the expression of Mcl-1 and protects MM cells from Dex-induced apoptosis, providing the preclinical framework for novel therapeutics targeting at both Mcl-1 and/or S1P to improve the patient outcome in MM.

DNA Damage Induced Sumoylation of p53 in Multiple Myeloma: Implications for Cell Proliferation and Apoptosis.

The tumor suppressor protein p53 has a critical role in malignant transformation through its critical functions in the regulation of cell proliferation, DNA repair, and apoptosis. The level and activity of the p53 tumor suppressor protein is regulated by post-translational modifications such as phosphorylation and ubiquitination. To address the role of p53 in the pathogenesis of Multiple Myeloma (MM), a number of patient-derived MM cell lines were probed using a p53-specific antibody and immunoblotting indicated a significantly elevated level of the p53 protein in the lysate of a vast majority of cell lines relative to that of normal CD138+ plasma cells. Since p53 is regulated by a number of post-translational modifications, the MM cell line RPMI-8226 was then treated with g-radiation (5 Gy), total lysate prepared and probed with a p53 monoclonal antibody As early as 30 minutes following treatment with radiation, a dramatic induction in the steady-state levels of the p53 protein and, in addition, a new higher molecular weight (∼68kDa) immunoreactive form of p53 was observed. The 68 kDa form of p53 was immunoprecipitable from MM total cell lysates with an antibody to the small-ubiquitin-like modifier (Sumo-1) but importantly, was not immunoprecipitated by an antibody generated to ubiquitin. Sumo-1 is covalently conjugated to target proteins through a conjugating enzyme, Ubc9, and a substrate ligase, PIAS1. Both Ubc9 and PIAS1 were rapidly induced at the protein level upon treatment of MM cells with g-radiation. Sumoylation of p53 was detected following treatment of MM cells with a number of genotoxic stressors in addition to g-irradiation, such as etoposide, doxorubicin, methylethylsulfonate and Ni++ and was detected using cell lysate from a number of MM cell lines. MM cells were transformed with a plasmid that expressed a dominant negative mutant form of UBC9 that abolished sumoylation of p53. Transfectants displayed increased sensitivity to g-radiation. A second plasmid that over expressed a mutant form of p53 that had mutated attachment site for covalent linkage of Sumo-1 (p53-K386R) was transfected into MM cells. These cells displayed a greater proliferative capacity relative to mock or wild-type p53 transfected cells. We then examined CD138+ plasma cells that had been immunoaffinity purified from MM patient bone marrow samples. Similar to the MM cell lines, the steady-state level of p53 in MM patient samples was significantly elevated relative to that of normal CD138+ cells. Importantly, the sumoylated form of p53 was also detected in the cell lysate prepared from MM patient samples (but not MGUS samples). Our results indicate that p53 is rapidly sumoylated upon exposure of MM cells to genotoxic stressors. Importantly, the sumoylated form of p53 was readily detected in the purified plasma cells of MM patients and led to increased cell proliferation. The results indicate a critical role for the sumoylation pathway in the DNA damage response, the proliferative and apoptotic functions of p53 and the pathogenesis of MM.

Activity of CDK1/2 Inhibitor LCQ195 Against Multiple Myeloma Cells.

Context: Conceptually, targeting proteins involved in cell cycle regulation is an appealing therapeutic strategy for multiple myeloma (MM), given the increased proliferative rate of biologically aggressive multi-drug resistant MM cells compared to not only their normal plasma cell counterparts, but also MM cells from earlier stages of the disease. Within the intricate network of signaling cascades and regulatory checkpoints involved in cell cycle progression and survival of neoplastic cells, cyclin-dependent kinases (CDKs) have emerged as intriguing therapeutic targets. We report the results of preclinical studies on the anti-MM activity of the selective CDK1/2 small molecule inhibitor NVP-LCQ195/AT9311 (Novartis Pharma/Astex Therapeutics).Methods/Results: In MTT colorimetric survival assays against a panel of 28 human MM cell lines, NVP-LCQ195 exhibited dose-dependent anti-MM activity, with IC50 values at or below ~0.5 μM (a pharmacologically relevant concentration) for 12 of 28 cell lines many of which are resistant to conventional or novel anti-MM therapies (e.g. dexamethasone, melphalan, thalidomide derivatives). NVP-LCQ195 was also active against primary MM cells isolated from heavily-pretreated/drug-resistant cases of MM. In marked contrast, the IC50 values of NVP-LCQ195 against non-malignant cells such as bone marrow stromal cells (BMSCs) and immortalized hepatocyte cells was >4 μM. In addition, both PHA-stimulated and unstimulated normal donor PBMCs were less sensitive than the majority of MM cell lines of our panel. Treatment with NVP-LCQ195 was able to overcome the protective effects conferred on MM cells by exogenous IL-6 (10ng/mL) or IGF-1 (50ng/mL), as well as by co-culture of MM cells with bone marrow stromal cells (BMSCs). NVP-LCQ195 was observed to trigger a distinct pattern of S-phase arrest, followed by eventual induction of apoptotic cell death. Mechanistic studies revealed that hsp90, B-raf, cyclin D1, and cyclin E2 levels decrease in response to NVP-LCQ195 treatment, while caspases- 3, -8 and PARP are cleaved within 16 and 24 hrs of drug treatment. We evaluated a series of combinations of this agent with conventional (e.g. dexamethasone, doxorubicin) and novel (e.g. bortezomib) anti-MM agents. No evidence of antagonism with any of these anti-MM agents was observed, indicating that combinations of NVP-LCQ195 with the aforementioned anti-MM agents can be feasible in clinical settings.Conclusion: Functional inhibition of proteins involved in cell cycle regulation remains an attractive approach for the treatment of MM. Treatment of MM cells with the CDK1/2 inhibitor NVP-LCQ195 induces, in a subset of MM cell lines, a distinct pattern of initial cell cycle arrest, anti-proliferative/pro-apoptotic molecular sequleae and eventual induction of cell death. Our results determine intriguing potential combination regimens for MM treatment. Further in vitro and in vivo studies are attempting to delineate molecular markers of MM cell responsiveness vs. resistance to NVP-LCQ195 and provide a framework for individualized treatment of select MM patients with this interesting class of compounds.

Targeting Histone Deacetylase 6 and the Aggresome Pathway in Acute Lymphoblastic Leukemia Cells.

Acute lymphoblastic leukemia (ALL) is the most common form of childhood cancer. Despite effective chemotherapy, 20% of patients will relapse. Therefore, it is critical that we identify novel therapies to treat ALL. We are studying a new small molecule compound known as tubacin (tubulin acetylation inducer) that selectively inhibits histone deacetylase 6 (HDAC6). HDAC6 binds to polyubiquitinated misfolded proteins and to dynein motor proteins, including alpha-tubulin, thereby recruiting misfolded or unwanted proteins to aggresomes and subsequent degradation by the lysosome. Tubacin was discovered through a chemical genetic screen of a 7,392 small molecule library and was found to induce acetylation of alpha-tubulin by inhibiting one of the two catalytic domains of HDAC6. This inhibition disrupts the interaction of HDAC6 with dynein resulting in marked accumulation of ubiquitinated proteins. Previous work demonstrated that treatment of multiple myeloma cells with tubacin inhibited growth at low micromolar concentrations. Tubacin does not appear to affect global histone acetylation, gene expression, or cell cycle regulation. To determine the effects of tubacin in human ALL cells, we treated both B- and T-cell ALL cell lines with varying concentrations of the drug and performed MTT assays. In T-ALL cells (Jurkat, Loucy), the IC50 of tubacin was found to be 1 to 3 uM, while in B-cell ALL cells (REH, Nalm-6), the IC50 was 2 to 5uM. Tubacin induced apoptosis of Jurkat and Loucy cells stained with Annexin V and propidium iodide. Within 12 hours, we observed increased protein polyubiquitination and PARP cleavage, but no difference in Rb phosphorylation in cells treated with tubacin. Furthermore, tubacin treatment increased acetylation of alpha-tubulin in Loucy and Jurkat cells within 3 hours. To study whether tubacin was toxic to normal hematopoietic cells, we treated human bone marrow cells cultured in methylcellulose containing IL-3, IL-6, and Stem Cell Factor with varying concentrations of tubacin. The IC50 of 20uM was determined from numbers of colonies plated in triplicate. Similarly, treatment of normal human lymphocytes cultured in IL-2, demonstrated an IC50 of 16uM. Finally, we examined the effects of tubacin on growth of primary ALL cells. Bone marrow cells from three patients with B-cell ALL at diagnosis were cultured in tubacin at varying concentrations and MTT assays were performed. In two of the three ALL samples, the IC50 was less than 5uM. Experiments to study the effects of tubacin in mouse models of leukemia are in progress. Our results suggest that inhibition of HDAC6 and the aggresome pathway provides a novel approach to treat ALL.

Zoledronate has an antitumor effect and induces actin rearrangement in dexamethasone‐resistant myeloma cells

New strategies are needed to overcome the resistance of multiple myeloma (MM) to dexamethasone (Dex). Several recent in vitro studies demonstrated the antitumor effect of nitrogen-containing amino-bisphosphonates (N-BPs) in various tumor cell lines. Inhibition of the prenylation of small G proteins is assumed to be one of the principal mechanisms by which N-BPs exert their effects. There have been few reports on N-BP treatment of MM cells that are resistant to Dex. Additionally, it is not known how small G proteins are altered in N-BP-treated MM cells. In this study, we evaluated the effect of the most potent N-BP, zoledronate (ZOL), on a Dex-resistant human MM cell subline (Dex-R) that we established from the well-documented RPMI8226 cell line. ZOL reduced the viability and induced apoptosis of Dex-R cells. Some of the ZOL-treated RPMI8226 cells and ZOL-treated Dex-R cells were elongated; however, elongated cells were not seen among the Dex-treated RPMI8226 cells. Furthermore, we found that portions of the small G proteins, Rho and Rap1A, were unprenylated in the ZOL-treated MM cells. Geranylgeraniol reduced the above-mentioned ZOL-induced effects. These findings suggest that ZOL may be beneficial for the treatment of Dex-resistant MM by suppressing the processing of RhoA and Rap1A.

Activation of sphingosine kinase mediates suppressive effect of interleukin‐6 on human multiple myeloma cell apoptosis

Interleukin 6 (IL-6) influences the growth and survival of multiple myeloma (MM) cells via the activation of multiple signalling cascades. Although sphingosine kinase (SPHK) signalling is known to play important roles in the regulation of cell proliferation and apoptosis, the role of SPHK activation in IL-6 signalling and in the pathology of MM remains unclear. This study found that IL-6 activated SPHK in MM cells, which mediates the suppressive effects of IL-6 on MM cell apoptosis. Both MM cell lines and primary MM cells constitutively expressed SPHK, and treatment of MM cells with IL-6 resulted in activation of SPHK in a concentration-dependent manner. Specific inhibitors of the phosphatidylinositol-3 kinase and extracellular signal-regulated kinase/mitogen-activated protein kinase pathways blocked the IL-6-induced activation of SPHK. It was further demonstrated that IL-6-induced activation of SPHK inhibited dexamethasone-induced apoptosis of MM cells. IL-6 stimulation or retroviral-mediated overexpression of SPHK1 in MM cells resulted in increased intracellular SPHK activity and upregulation of myeloid cell leukaemia-1 (Mcl-1), leading to increased cell proliferation and survival. Conversely, inhibition of SPHK1 by small interfering RNA reduced IL-6-induced upregulation of Mcl-1 and blocked the suppressive effect of IL-6 on MM cell apoptosis. Taken together, these results delineate a key role for SPHK activation in IL-6-induced proliferation and survival of MM cells, and suggest that SPHK may be a potential new therapeutic target in MM.

A New Target in Multiple Myeloma: Inhibition of Notch Pathway Induces Apoptosis and Enhances Drug Sensitivity of Myeloma Cells In Vitro and In Vivo.

Bone marrow (BM) microenvironment and particularly BM stromal cells play a critical role in de novo drug resistance of multiple myeloma (MM) cells. BM stromal cells express Notch ligands and activate Notch signaling in MM cells. We have previously demonstrated that Notch signaling is one of the major mechanisms of BM stroma mediated MM cell protection from chemotherapeutic drugs. Here we investigated whether the pharmacological inhibition of Notch signaling with γ-secretase inhibitor (GSI) could affect viability of MM cells and overcome BM stroma mediated resistance of MM cells to chemotherapy. GSI (6–8 μM) induced apoptosis of MM cells cultured in suspension or on monolayer of BM stroma via specific inhibition of Notch signaling. The effect of GSI was evaluated in four different MM cell lines and primary MM cells isolated from BM of four patients with MM. Treatment with GSI alone significantly reduced the viability of MM cells with IC50 almost 5-fold lower than that for peripheral blood mononuclear cells or BM cells from healthy donors. In addition, treatment of MM cells with GSI reversed BM stroma mediated protection of MM cells from drug-induced apoptosis. This effect of GSI was associated by dramatic up-regulation of pro-apoptotic protein NOXA and down-regulation of anti-apoptotic proteins bcl-xL, bcl-2, and Akt. Inhibition of NOXA with siRNA canceled the cytotoxic effect of GSI on MM cells indicating that NOXA could mediate the direct effect of GSI on MM cells. To test the effect of GSI on MM cells in vivo we used SCID/NOD mouse model. Mice were injected s.c. with human MM RPMI-8226 cells. These cells express both Notch ligands and Notch receptors so Notch signaling is activated by interaction between MM cells themselves. Tumor became visible in ~3 weeks after inoculation and grew as a solid tumor which allowed easily monitoring tumor size. Mice were treated with doxorubicin (1.5 mg/kg, i.p., once a 4 days, 3 times), GSI (5mg/kg/day i.p. for 14 days), combinations of doxorubicin and GSI, or vehicle control (PBS). Tumor size was constantly monitored during treatment and 3 weeks after the treatment. Treatment with doxorubicin and GSI alone resulted in moderate decreased in tumor burden as compared with control group. In contrast, combination of GSI and doxorubicin induced dramatic antitumor effect. Thus, our study, for the first time, demonstrates that inhibition of Notch signaling with GSI can be a new promising approach for therapeutic intervention in MM.

Antiproliferative and Proapoptotic Effect of Vitamin D in Bortezomib (B) Resistant Human Myeloma Cell Line (HMCL).

-Introduction: B is a proteasome inhibitor approved for treatment of multiple myeloma (MM) patients. Despite initial clinical efficacy all patients eventually become resistant to bortezomib and die of progressive disease. Development of newer agents with novel antitumor mechanisms is thus warranted. In vitro antiproliferative and proapoptotic effects of vitamin D are reported in various tumor models including MM. This is mediated through vitamin D receptor (VDR) which is also expressed on MM cells. Effect of calcitriol (D3) or its analog paricalcitol (D2) in HMCL resistant to B have not been previously examined. We investigated the antitumor potential of D3 and D2 in HMCL sensitive or resistant to B.Method & Results: HMCL sensitive (OPM-2) or resistant to B (OPM-2/BR) were treated with various concentration of D2 or D3. Significant growth inhibition and induction of apoptosis was noted with D2 and D3 that was maximal at 3 days (p<0.01). This antitumor response was noted despite resistance to B. Treatment of MM cells with vitamin D (D2 or D3) was associated with a loss of full-length PARP, decrease in p21 and induction of p27. Levels of the pro-survival molecules P-Akt and P-Erk were also reduced while total Akt and Erk remain unchanged.Conclusion: We demonstrate that the antiproliferative and proapoptotitc effect of vitamin D in HMCL are mediated through interruption of the Akt pathway and were independent of resistance to B. We have initiated a phase I clinical trial to investigate the clinical utility of paricalcitol in relapsed or refractory MM patients.

Inhibition of p38α MAPK enhances proteasome inhibitor-induced apoptosis of myeloma cells by modulating Hsp27, Bcl-XL, Mcl-1 and p53 levels in vitro and inhibits tumor growth in vivo

Inhibition of p38 kinase blocks the production of tumor-promoting factors in the multiple myeloma (MM) bone marrow microenvironment. Proteasome inhibitors MG132 and bortezomib have been shown to have direct cytotoxic effects on MM cells. We show that a selective inhibitor of p38alpha, SCIO-469, enhances the ability of MG132 and bortezomib to induce the apoptosis of MM cells. Previously, we showed that p38 inhibition with SCIO-469 enhances MM cytotoxicity of bortezomib by inhibiting the transient expression and phosphorylation of Hsp27, a downstream target of p38. Here we show that continued treatment of MM cells with bortezomib leads to a SCIO-469-enhanced downregulation of Hsp27 and to increased MM apoptosis. Furthermore, we show that p38 inhibition enhances the bortezomib-induced MM apoptosis by upregulation of p53 and downregulation of Bcl-X(L) and Mcl-1. In a mouse xenograft plasmacytoma model of MM, we found that inhibiting p38 augments the effects of bortezomib in decreasing MM tumor growth in vivo. Thus, in addition to its role in suppressing an activated MM microenvironment, co-treatment with a p38 inhibitor, such as SCIO-469, may enhance the cytotoxicity of bortezomib by modulating pro-apoptotic and anti-apoptotic factors in MM cells, suggesting great potential for co-therapy.

Expression and Modulation of Carbohydrate-Binding Protein Galectin-3 in Multiple Myeloma Cells by Combined Treatment with GCS-100 and Dexamethasone.

Galectins belong to the family of mammalian lectins sharing an evolutionary conserved sequence in their carbohydrate recognition binding (CRD) domains and exhibit a characteristic affinity for beta-galactoside-sugars. Based on their structure and CRD, galectins have been categorized into three different categories: 1) Prototype (Gal-1, 2, 5, 7, 10, 11, 13, and 14), 2) Chimera type (Gal-3), and 3) Tandem repeat type (Gal-6, 8, 9, and 12). In particular, Galcetin-3 (Gal-3) is associated with growth, differentiation, adhesion, RNA processing, and apoptosis ( Apoptosis , 2005, 10: 267–75). Previous studies show that Gal-3 is expressed on tumor cell surface and mediate the transformation and metastasis of tumor cells in vivo ( Apoptosis , 2005, 10: 267–75). Here, we show that Gal-3 is differentially expressed in multiple myeloma (MM) cell lines and purified patient cells, as determined by both western blotting and polymerase chain reaction (PCR). Importantly, Gal-3 shares structural similarities with the anti-apoptotic protein Bcl-2: both proteins are rich in proline, glycine, and alanine amino acid residues in their NH-2-terminal domains and contain anti-death motif Asp-Trp-Gly-Arg (NWGR) in their C-terminal domains ( Proc Natl Acad Sci USA , 1996, 93: 6737–42). Gal-3, like Bcl-2, is therefore a potential therapeutic target. In this context, modified citrus pectins (MCP) have been reported to bind and inhibit Gal-3-induced human umbilical endothelial cell migration and micro vessel tube formation ( Am J Pathol. 2000, 156:899–909) as well as block tumor growth and metastasis (J Natl Cancer Inst. 2002, 94:1854–62). GCS-100 is a MCP currently under clinical development. Here we show that GCS-100 binds to human recombinant Gal-3. In MM cells, GCS-100 triggers apoptosis and enhances anti-tumor activity of the conventional agent Dexamethasone. We examined whether GCS-100-, Dex- or GCS-100 + Dex-induced MM cell death modulates Gal-3 or Bcl-2 expression. Treatment of MM.1S cells with either GCS-100 or Dex alone does not alter Gal-3 or Bcl-2 expression. However, combined treatment of MM cells with sub-toxic concentrations of GCS-100 and Dex markedly attenuates Gal-3 expression, without any changes in Bcl-2 levels, suggesting that alterations in Gal-3 are specific. Previous studies showed that Gal-3 inhibits mitochondrial pro-apoptotic protein cytochrome-c (cyto-c), and our results show that GCS-100 + Dex triggers the release of cyto-c from mitochondria to cytosol. Together, these data suggest that the combination of GCS-100 + Dex overcomes the inhibitory effect of Gal-3 on cyto-c, thereby inducing apoptosis. Since Gal-3 is widely expressed in MM cells, its inhibition may augment the anti-tumor activity of therapeutic regimens.

Human Anti-CD40 Antagonist Antibody Triggers Significant Antitumor Activity against Human Multiple Myeloma

Monoclonal antibodies (mAb) directed against lineage-specific B-cell antigens have provided clinical benefit for patients with hematologic malignancies, but to date no antibody-mediated immunotherapy is available for multiple myeloma. In the present study, we assessed the efficacy of a fully human anti-CD40 mAb CHIR-12.12 against human multiple myeloma cells. CHIR-12.12, generated in XenoMouse mice, binds to CD138-expressing multiple myeloma lines and freshly purified CD138-expressing cells from >80% multiple myeloma patients, as assessed by flow cytometry. Importantly, CHIR-12.12 abrogates CD40L-induced growth and survival of CD40-expressing patient multiple myeloma cells in the presence or absence of bone marrow stromal cells (BMSC), without altering constitutive multiple myeloma cell proliferation. Immunoblotting analysis specifically showed that PI3-K/AKT, nuclear factor-kappaB (NF-kappaB), and extracellular signal-regulated kinase activation induced by CD40L (5 mug/mL) was inhibited by CHIR-12.12 (5 mug/mL). Because CD40 activation induces multiple myeloma cell adhesion to both fibronectin and BMSCs, we next determined whether CHIR-12.12 inhibits this process. CHIR-12.12 decreased CD40L-induced multiple myeloma cell adhesion to fibronectin and BMSCs, whereas control human IgG1 did not. Adhesion of multiple myeloma cells to BMSCs induces interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) secretion, and treatment of multiple myeloma cells with CD40L further enhanced adhesion-induced cytokine secretion; conversely, CHIR-12.12 blocks CD40L-enhanced IL-6 and VEGF secretion in cocultures of multiple myeloma cells with BMSCs. Finally, CHIR-12.12 triggered lysis of multiple myeloma cells via antibody-dependent cellular cytotoxicity (ADCC) but did not induce ADCC against CD40-negative multiple myeloma cells, confirming specificity against CD40-expressing multiple myeloma cells. These results provide the preclinical rationale for clinical trials of CHIR-12.12 to improve patient outcome in multiple myeloma.