Sp1 Transcription Factor as a Novel Therapeutic Target in Multiple Myeloma (MM)

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3664-3664
Author(s):  
Mariateresa Fulciniti ◽  
Pierfrancesco Tassone ◽  
Teru Hideshima ◽  
Puru Nanjappa ◽  
Klaus Podar ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Transcription Factor ◽  
Cell Growth ◽  
Expression Profiling ◽  
Dependent Manner ◽  
Sp1 Transcription Factor ◽  
Dose Dependent ◽  
Novel Therapeutic

Abstract Sp1 transcription factor has an important role in regulating expression of cell differentiation, cell cycle and apoptosis related genes. Increased Sp1 activity has been shown to augment growth and metastatic potential of tumor cells. In multiple myeloma (MM), modulation of autocrine IL-6 secretion by Sp1 has been described. Using genome expression profiling analysis we observed high expression of Sp1 in primary myeloma cells obtained from bone marrow aspirates of MM patients. This observation was confirmed by western blot analysis. Therefore, we evaluated Sp1 as a novel therapeutic target in MM, analyzing both in vitro and in vivo anti-MM activity of a Sp1 inhibitor (Terameprocol, EM-1421). Terameprocol significantly (≥80% inhibition, P≤0.001) inhibited DNA synthesis in 12 MM cells lines tested in a dose-dependent fashion at 24 hour with an IC50 of 1–10 uM. Longer exposure of MM cells to Terameprocol resulted also in cytotoxity, as assessed by MTT assay, in all cell lines tested (IC50 of 20–40 uM at 72 hours). Terameprocol induced apoptosis in a time- and dose-dependent manner in all MM cell lines examined, as assessed by Annexin V+/PI staining, and triggered caspase-9/3 and PARP cleavage. Since Sp1 modulates transcription of survivin and survivin is an anti-apoptotic protein overexpressed in most human cancers that inhibits caspases-3 and -9 activity, we next examined effect of Terameprocol on survivin protein expression. Consistent with induction of caspase-3 and apoptosis, Terameprocol significantly reduced survivin protein in a time-dependent manner. To identify additional potential downstream targets of Sp1 inhibition in MM, we evaluated change in expression profiling in MM cells following Terameprocol treatment (10 uM for 12 hours). We observed alteration (>2-fold changes) in 68 genes. In particular, we recognized significant down-regulation of c-Jun and up-regulation of p53 and p21 following exposure to Terameprocol. Changes in protein expression of these genes were also confirmed by western blot analysis. Since bone marrow stromal cells (BMSCs) trigger growth, survival, and drug resistance of MM cells we next confirmed the ability of Terameprocol to overcome protective effects of BMSCs on MM cell growth and survival. Finally, we have confirmed in vivo activity of Terameprocol on MM cell growth in a xenograft murine model of human MM. In conclusion, our results demonstrate that Sp1 may be an attractive target for MM therapy and provide the rationale for clinical evaluation of Terameprocol in MM.

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

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1844-1844 ◽  
Author(s):  
Aldo M Roccaro ◽  
Antonio Sacco ◽  
Michelle Kuhne ◽  
AbdelKareem Azab ◽  
Patricia Maiso ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Tumor Cells ◽  
Mouse Models ◽  
Research Funding ◽  
Therapeutic Agents ◽  
Dependent Manner ◽  
Apoptotic Pathways ◽  
Dose Dependent

Abstract 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:.

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

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4009-4009
Author(s):  
Aldo M Roccaro ◽  
Antonio Sacco ◽  
Michelle R. Kuhne ◽  
Abdel Kareem Azab ◽  
Patricia Maiso ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Advisory Board ◽  
Dependent Manner ◽  
In Vivo Confocal Microscopy ◽  
Transfected Cells ◽  
Cell Dissemination ◽  
Dose Dependent ◽  
Bone Marrow Niches

Abstract Abstract 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.

scholarly journals Anti-tumor activity of a novel proteasome inhibitor D395 against multiple myeloma and its lower cardiotoxicity compared with carfilzomib

2021 ◽  
Vol 12 (5) ◽  
Author(s):  
Xuxing Shen ◽  
Chao Wu ◽  
Meng Lei ◽  
Qing Yan ◽  
Haoyang Zhang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Proteasome Inhibitor ◽  
Osteoclast Differentiation ◽  
Dependent Manner ◽  
Serum Enzyme ◽  
Herg Channels ◽  
Anti Tumor Activity ◽  
Dose Dependent

AbstractCarfilzomib, a second-generation proteasome inhibitor, has significantly improved the survival rate of multiple myeloma (MM) patients, but its clinical application is still restricted by drug resistance and cardiotoxicity. Here, we identified a novel proteasome inhibitor, D395, and assessed its efficacy in treating MM as well as its cardiotoxicity at the preclinical level. The activities of purified and intracellular proteasomes were measured to determine the effect of D395 on the proteasome. CCK-8 and flow cytometry experiments were designed to evaluate the effects of D395 on cell growth and apoptosis. The effects of D395 and carfilzomib on serum enzyme activity, echocardiography features, cardiomyocyte morphology, and hERG channels were also compared. In our study, D395 was highly cytotoxic to MM cell lines and primary MM cells but not normal cells, and it was well tolerated in vivo. Similar to carfilzomib, D395 inhibited osteoclast differentiation in a dose-dependent manner. In particular, D395 exhibited lower cardiotoxicity than carfilzomib in all experiments. In conclusion, D395 is a novel irreversible proteasome inhibitor that has remarkable anti-MM activity and mild cardiotoxicity in vitro and in vivo.

scholarly journals TB-4 Antitumor effects of a novel curcumin derivative curcumin monoglucuronide on glioblastoma cells in vitro and in vivo

2021 ◽  
Vol 3 (Supplement_6) ◽  
pp. vi6-vi6
Author(s):  
Takashi Fujii ◽  
Shun Yamamuro ◽  
Masamichi Takahashi ◽  
Akihide Kondo ◽  
Yoshitaka Narita ◽  
...  
Keyword(s):  
Cell Death ◽  
Water Soluble ◽  
Dependent Manner ◽  
Antitumor Effects ◽  
Multiple Cell ◽  
Human Gbm ◽  
Dose Dependent ◽  
Novel Therapeutic

Abstract The therapeutic outcome of glioblastomas (GBMs) is still very poor. Therefore, invention of novel therapeutic methods against GBM cases is considered urgent. The antitumor effects of naturally-derived compounds are attracting attention recently, and therapeutic efficacy of curcumin, a plant-derived compound previously used for multiple purpose, has been indicated in many cancer systems; however, clinical application of curcumin is considered difficult because of its poor bioavailability (under 1 %). Curcumin monoglucuronide (CMG), a water-soluble prodrug of curcumin recently developed for overcoming this weakness, has been demonstrated excellent antitumor effects for several malignancies in vitro and in vivo; therefore, we investigated the effects of CMG against GBM cells. CMG induced cell death of human GBM cells lines (T98G, U251MG, and U87MG) by dose dependent manner by triggering multiple forms of cell death such as apoptosis and perthanatos. Immunoblotting of CMG-treated GBM cell lysates demonstrated activation of multiple cell death signaling. Furthermore, immunodeficiency mice harboring intracerebral U87MG cell xenografts systemically treated by CMG showed significantly prolonged survival compared with control mice. These results suggest CMG would be a novel therapeutic agent against GBM cases.

Novel Murine Model To Study Modulation of Genes and Molecular Pathways Induced Following In Vivo Interaction between Multiple Myeloma Cells and Human BM Milieu.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3409-3409
Author(s):  
Paola Neri ◽  
Pierfrancesco Tassone ◽  
Masood Shammas ◽  
Mariateresa Fulciniti ◽  
Yu-Tzu Tai ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Growth ◽  
Gene Expression Profile ◽  
Expression Profile ◽  
Murine Model ◽  
Cell Signalling

Abstract Interaction between multiple myeloma (MM) cells and the bone marrow (BM) microenvironment plays a critical role in promoting MM cell growth, survival, migration and development of drug resistance. This interaction within the bone marrow milieu is unique and its understanding is important in evaluating effects of novel agents in vitro and in vivo. We here describe a novel murine model that allows us to study the expression changes in vivo in MM cells within the human BM milieu. In this model, the green fluorescent protein (INA-6 GFP+) transduced IL-6-dependent human MM cell line, INA-6, was injected in human bone chip implanted into SCID mice. At different time points the bone chip was retrieved, cells flushed out and GFP+ MM cells were purified by CD138 MACS microbeads. Similar isolation process was used on INA-6 GFP+ cells cultured in vitro and used as control. Total RNA was isolated from these cells and gene expression profile analyzed using the HG-U133 array chip (Affymetrix) and DChip analyzer program. We have identified significant changes in expression of several genes following in vivo interaction between INA-6 and the BM microenvironment. Specifically, we observed up-regulation of genes associated with cytokines (IL-4, IL-8, IGFB 2–5) and chemokines (CCL2, 5, 6, 18, 24, CCR1, 2, 4), implicated in cell-cell signalling. Moreover genes implicated in DNA transcription (V-Fos, V-Jun, V-kit), adhesion (Integrin alpha 2b, 7, cadherin 1 and 11) and cell growth (CDC14, Cyclin G2, ADRA1A) were also up-regulated and genes involved in apoptosis and cell death (p-57, BCL2, TNF1a) were down-regulated. Using the Ingenuity Pathway Analysis the most relevant pathways modulated by the in vivo interaction between MM cells and BMSCs were IL-6, IGF1, TGF-beta and ERK/MAPK-mediated pathways as well as cell-cycle regulation and chemokine signalling. These results are consistent with previously observed in vitro cell signalling studies. Taken together these results highlight the ability of BM microenvironment to modulate the gene expression profile of the MM cells and our ability to in vivo monitor the changes. This model thus provides us with an ability to study in vivo effects of novel agents on expression profile of MM cells in BM milieu, to pre-clinically characterize their activity.

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

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

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

KRN5500, a Spicamycin Derivative, Exerts Anti-Myeloma Effects through Impairing Both Myeloma Cells and Osteoclasts.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1669-1669
Author(s):  
Hirokazu Miki ◽  
Shuji Ozaki ◽  
Osamu Tanaka ◽  
Shingen Nakamura ◽  
Ayako Nakano ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Drug Resistance ◽  
Cell Death ◽  
Cell Growth ◽  
Cell Lines ◽  
G1 Arrest ◽  
Dependent Manner ◽  
Osteolytic Bone Disease ◽  
Rpmi 8226

Abstract Multiple myeloma (MM) is a plasma cell malignancy characterized by devastating bone destruction due to enhanced bone resorption and suppressed bone formation. Although high-dose chemotherapy and new agents such as thalidomide, lenalidomide, and bortezomib have shown marked anti-MM activity in clinical settings, MM remains incurable due to drug resistance mediated by interactions with osteoclasts or stroma cells. Moreover, osteolytic bone disease continues to be a major problem for many patients. Therefore, alternative approaches are necessary to overcome drug resistance and inhibit osteoclasts activity in MM. KRN5500 is a new derivative of spicamycin produced by Streptomyces alanosinicus (Kirin Pharma, Tokyo, Japan), which potently inhibits protein synthesis and induces cell death in human tumor cell lines. Phase I studies of KRN5500 in patients with solid tumors such as colon cancer and gastric cancer showed acceptable toxicity with Cmax values of 1000––3000 nM. In this study, we investigated the effects of KRN5500 against MM cells and osteoclasts in vitro and in vivo. MM cell lines such as RPMI 8226, MM.1S, INA-6, KMS12-BM, UTMC-2, TSPC-1, and OPC were incubated with various concentrations of KRN5500 for 3 days. Cell proliferation assay showed marked inhibition of cell growth with G1 arrest in these MM cells (IC50: 4–100 nM). KRN5500 (100 nM) also induced 30–90% of cell death in primary MM cells (n=7). Annexin V/propidium iodide staining showed that KRN5500 induced apoptosis of MM cells in a dose- and time-dependent manner. Western blot analysis confirmed activation of caspase-8, -9, and −3, cleavage of poly (ADP-ribose) polymerase (PARP), and down-regulation of Mcl-1. We next examined the effect of KRN5500 against MM cell lines and primary MM cells in the presence of bone marrow stroma cells and osteoclasts. Co-culture of these cells enhanced viability of MM cells; however, KRN5500 still induced strong cytotoxicity to MM cells. Of interest, KRN5500 specifically mediated apoptosis in osteoclasts but not stroma cells as assessed by TUNEL staining. More than 90% of osteoclasts were killed even at a low concentration of KRN5500 (20 nM). Finally, we evaluated the effect of KRN5500 against MM cells and osteoclasts in vivo. Two xenograft models were established in SCID mice by either subcutaneous injection of RPMI 8226 cells or intra-bone injection of INA-6 cells into subcutaneously implanted rabbit bones (SCID-rab model). These mice were treated with intraperitoneal injection of KRN5500 (5 mg/kg/dose) or saline thrice a week for 3 weeks after tumor development. In a subcutaneous tumor model, KRN5500 inhibited the tumor growth compared with control mice (increased tumor size, 232 ± 54% vs 950 ± 422%, p<0.001, n=6 per group). In a SCID-rab model, KRN5500 also inhibited MM cell growth in the bone marrow (increase of serum human sIL6-R derived from INA-6, 134 ± 19% vs 1112 ± 101%, p<0.001, n=5 per group). Notably, the destruction of the rabbit bones was also prevented in the KRN5500-treated mice as evaluated by radiography. Therefore, these results suggest that KRN5500 exerts anti-MM effects through impairing both MM cells and osteoclasts and that this unique mechanism of action provides a valuable therapeutic option to improve the prognosis in patients with MM.

Inhibition Of PI3K Classia Kinases Using GDC0941 Overcomes Protection Of Multiple Myeloma Cells In The Bone Marrow Microenvironment

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3169-3169
Author(s):  
Hugh Kikuchi ◽  
Amofa Eunice ◽  
Maeve McEnery ◽  
Farzin Farzaneh ◽  
Stephen A Schey ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Proliferation ◽  
Bone Resorption ◽  
Cell Lines ◽  
Stromal Cells ◽  
Conflicts Of Interest ◽  
Pi3k Pathway ◽  
Dependent Manner ◽  
Dose Dependent

Abstract Despite of newly developed and more efficacious therapies, multiple myeloma (MM) remains incurable as most patient will eventually relapse and become refractory. The bone marrow (BM) microenvironment provides niches that are advantageous for drug resistance. Effective therapies against MM should ideally target the various protective BM niches that promote MM cell survival and relapse. In addition to stromal mesenchymal/myofibroblastic cells, osteoclasts play a key supportive role in MM cell viability. Additionally, 80% of patients develop osteolytic lesions, which is a major cause of morbidity. Increased osteoclast activity is characteristic in these patients and targeting osteoclast function is desirable to improve therapies against MM. Osteoclasts need to form an F-actin containing ring along the cell margin that defines a resorbing compartment where protons and degradative enzymes are secreted for dissolution of bone mineral. Remodelling of F-actin and vesicle secretion are regulated by the class IA PI3K pathway during osteoclastic bone resorption. Additionally, it has recently been shown that inhibition of the class IA PI3K pathway in MM cells with GDC0941 induces apoptosis-mediated killing. We hypothesised that GDC0941 could be used as a therapeutic agent to overcome MM-induced osteoclast activation. GDC0941 inhibited maturation of osteoclasts derived from BM aspirates from MM patients in a dose dependent manner. This correlated with decreased bone resorption of osteoclasts cultured on dentine discs. Exposure of mature osteoclasts to GC0941 resulted in abnormal organisation of larger F-actin rings, suggesting a negative effect on the dynamics of the actin cytoskeleton required for bone resorption. We also found that GDC-0941 can prevent protection of the MM cell lines MM1.S and MM1.R by osteoclasts against killing. GDC-0941 alone blocked MM cell proliferation independently of the presence of BM stromal cells and synergised with other therapeutic agents including Lenalidomide, Pomalidomide, Bortezomid and Dexamethasone. We also found that in the presence of MM cells, Dexamethasone (a drug commonly used alone or in combination with new drugs against MM) induced the proliferation of BM stromal cells and adhesion of MM cells on this protective stroma in a dose dependent manner. Dexamethasone is highly effective at MM cell killing when cells are cultured alone. However, we found that at low doses (below 1 uM) and in the presence of BM stromal cells, Dexamethasone could induce MM cell proliferation. GDC0941 enhanced Dexamethasone killing even in the presence of BM stromal cells by blocking Dexamethasone-induced stromal cell proliferation and adhesion of MM cells on the stroma. Targeting individual the PI3K Class IA isoforms alpha, beta, delta or gamma proved to be a less efficient strategy to enhance Dexamethasone killing. Previous work has shown that efficacy of targeting individual PI3K Class I A isoforms would be low for activation of caspases in MM cells as it would be dependent on relative amounts of isoforms expressed by the MM patient. GDC-0941 also inhibited the proliferation of MM1.R and RPMI8266 MM cell lines, which are less sensitive to treatment to Dexamethasone. Co-culture of MM cells with BM stromal cells induced the secretion of IL-10, IL-6, IL-8, MCP-1 and MIP1-alpha. The dose-dependant increased proliferation of Dexamethasone-treated MM cells in the presence of the BM stroma correlated with the pattern of secretion of IL-10 (a cytokine that can induce B-cell proliferation) and this was blocked by the combination of Dexamethasone with GDC0941. GDC-0941 alone or in combination with Dexamethasone was more efficacious at inducing MM cell apoptosis in the presence of the BM stroma cells vs treatment of MM cells alone. These are very encouraging results as they suggest that GDC-0941 in combination with Dexamethasone would be potentially highly efficacious for targeting MM cells in the BM microenvironment. We are currently performing in vivo data using C57BL/KaLwRij mice injected with 5T33-eGFP MM cells that will be discussed at the meeting. We propose that MM patients with active bony disease may benefit from treatment with GDC0941 alone or in combination with currently used therapeutic drugs against MM. Disclosures: No relevant conflicts of interest to declare.

scholarly journals MicroRNAs 15a and 16 regulate tumor proliferation in multiple myeloma

Blood ◽  
2009 ◽  
Vol 113 (26) ◽  
pp. 6669-6680 ◽  
Author(s):  
Aldo M. Roccaro ◽  
Antonio Sacco ◽  
Brian Thompson ◽  
Xavier Leleu ◽  
Abdel Kareem Azab ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Expression Profiling ◽  
Map Kinases ◽  
Qrt Pcr ◽  
Ribosomal Protein S6 ◽  
Genomic Studies

Abstract Detailed genomic studies have shown that cytogenetic abnormalities contribute to multiple myeloma (MM) pathogenesis and disease progression. Nevertheless, little is known about the characteristics of MM at the epigenetic level and specifically how microRNAs regulate MM progression in the context of the bone marrow milieu. Therefore, we performed microRNA expression profiling of bone marrow derived CD138+ MM cells versus their normal cellular counterparts and validated data by qRT-PCR. We identified a MM-specific microRNA signature characterized by down-expression of microRNA-15a/-16 and overexpression of microRNA-222/-221/-382/-181a/-181b (P < .01). We investigated the functional role of microRNA-15a and -16 and showed that they regulate proliferation and growth of MM cells in vitro and in vivo by inhibiting AKT serine/threonine-protein-kinase (AKT3), ribosomal-protein-S6, MAP-kinases, and NF-κB-activator MAP3KIP3. Moreover, miRNA-15a and -16 exerted their anti-MM activity even in the context of the bone marrow milieu in vitro and in vivo. These data indicate that microRNAs play a pivotal role in the biology of MM and represent important targets for novel therapies in MM.

NVP-LAQ824 is a potent novel histone deacetylase inhibitor with significant activity against multiple myeloma

Blood ◽  
2003 ◽  
Vol 102 (7) ◽  
pp. 2615-2622 ◽  
Author(s):  
Laurence Catley ◽  
Ellen Weisberg ◽  
Yu-Tzu Tai ◽  
Peter Atadja ◽  
Stacy Remiszewski ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Histone Deacetylase ◽  
Hdac Inhibitors ◽  
Myeloma Cell ◽  
Parp Cleavage ◽  
Significant Activity ◽  
Dependent Manner

Abstract Histone deacetylase (HDAC) inhibitors are emerging as a promising new treatment strategy in hematologic malignancies. Here we show that NVP-LAQ824, a novel hydroxamic acid derivative, induces apoptosis at physiologically achievable concentrations (median inhibitory concentration [IC50] of 100 nM at 24 hours) in multiple myeloma (MM) cell lines resistant to conventional therapies. MM.1S myeloma cell proliferation was also inhibited when cocultured with bone marrow stromal cells, demonstrating ability to overcome the stimulatory effects of the bone marrow microenvironment. Importantly, NVP-LAQ824 also inhibited patient MM cell growth in a dose- and time-dependent manner. NVP-LAQ824-induced apoptotic signaling includes up-regulation of p21, caspase cascade activation, and poly (adenosine diphosphate [ADP]) ribose (PARP) cleavage. Apoptosis was confirmed with cell cycle analysis and annexin-propidium iodide staining. Interestingly, treatment of MM cells with NVPLAQ824 also led to proteasome inhibition, as determined by reduced proteasome chymotrypsin-like activity and increased levels of cellular polyubiquitin conjugates. Finally, a study using NVP-LAQ824 in a preclinical murine myeloma model provides in vivo relevance to our in vitro studies. Taken together, these findings provide the framework for NVP-LAQ824 as a novel therapeutic in MM. (Blood. 2003;102:2615-2622)

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