Growth Inhibition of Human Multiple Myeloma Cells by a CD40 Ligand (CD40L, CD154) Transgene - Oncolytic Virus Construct.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3427-3427
Author(s):  
Margret S. Rodrigues ◽  
Erica M. Gomes ◽  
Reuben Hernandez ◽  
Jack Chang ◽  
Joe Kansopon ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Growth ◽  
Growth Inhibition ◽  
Cell Lines ◽  
Annexin V ◽  
Normal Fibroblast ◽  
Distribution Analysis ◽  
Untreated Culture ◽  
Human Multiple Myeloma ◽  
Rpmi 8226

Abstract Human multiple myeloma (MM) remains incurable despite recent advances in induction therapy. To explore biotherapeutic approaches that may potentially enhance the efficacy of conventional treatment, we examined the growth regulatory properties of CD40L (CD154), the natural ligand for the myeloma cell surface receptor, CD40. Based on our previous findings that the recombinant CD40L protein effectively inhibited human MM cell growth, a conditional-replicative adenoviral construct, AdEHCD40L was used for targeted delivery of the CD40L transgene. AdEHCD40L incorporates tumor/tissue specific promoters that limit viral and transgene expression to HIF (hypoxia inducing factor)-1α overexpressing cells, which are prevalent in the human bone marrow compartment. Conditional expression of the early adenoviral E1A gene and the CD154 transgene was validated in the IL-6 independent MM line RPMI 8226 (62% and 66%, respectively), and the IL-6 dependent cell line Kas-6/1 (32.68% and 30%, respectively). Further, treatment with AdEHCD40L at a multiplicity of infection (MOI) of 1 resulted in pronounced growth inhibition for both cell lines (95.5±2.1% and 80.5±9.8%, mean±SD, respectively). AdEHCD40L treatment was more effective than the parental construct without the CD154 transgene (AdEHNULL) in both cell lines (p=0.04). Both AdEHNULL and AdEHCD40L were minimally cytotoxic to normal peripheral blood mononuclear cells (0% at 48hrs) and normal fibroblast cells IMR-90 (2.8±0.3%). The in vivo antitumor activity of AdEHCD40L was examined with a subcutaneous RPMI 8226 heterotransplant model in SCID mice. Intratumoral injection of AdEHCD40L (5x107 pfu, x 5) reduced xenograft growth by 53% at day 29 (4.8±0.9 mm, vs. 10.5±1.2 mm in mock-treated animals; p=0.002), and was more effective than AdEHNULL (7.6±1.1 mm; p=0.03). Adenoviral hexon and CD40L expression was detectable at 29 days post-viral treatment, based on immunohistochemical analysis. Hence intratumoral treatment with AdEHCD40L likely involved oncolytic viral replication. To further characterize cellular events that accompany MM cell growth inhibition, apoptotic activity was measured by annexin V and propidium iodide incorporation. A marked elevation of the annexin V+ subset (21.3±6.5%, vs. 7.7±1.9% in untreated culture; p=0.007) was accompanied by decreased cell viability (52.8±1%, vs. 76.1±11% in untreated cultures; p = 0.04) following AdEHCD40L treatment. Cell cycle distribution analysis demonstrated a corresponding increase in the subG0/G1 compartment (AdEHCD40L, 23.9±3.6%; vs. untreated, 9.1±2.3%) that was consistent with elevated apoptosis. Further, AdEHCD40L increased S phase accumulation by 72 hrs (68.0±2.4%, vs. 54.0±5.5% in AdEHNULL and 48.9±4.6% in untreated culture; p<0.003). Currently, gene expression array analyses are underway to define molecular events that are pertubated by CD154 transgene activity and by viral oncolysis. These findings will further elucidate the mechanism of action of the CD154+oncolytic viral approach for experimental gene therapy of human MM.

CD40 activation mediates p53-dependent cell cycle regulation in human multiple myeloma cell lines

Blood ◽  
2000 ◽  
Vol 95 (3) ◽  
pp. 1039-1046 ◽  
Author(s):  
G. Teoh ◽  
Y.-T. Tai ◽  
M. Urashima ◽  
S. Shirahama ◽  
M. Matsuzaki ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Growth Arrest ◽  
Myeloma Cell ◽  
Luciferase Reporter ◽  
Temperature Sensitive ◽  
Multiple Myeloma Cell ◽  
Cd40 Activation ◽  
Human Multiple Myeloma ◽  
Rpmi 8226

It has been reported that the activation of multiple myeloma (MM) cells by CD40 induces proliferation, growth arrest, and apoptosis. To determine whether the biologic sequelae of CD40 activation in MM cells depends on p53 function, we identified temperature-sensitive p53 mutations in the RPMI 8226 (tsp53E285K) and the HS Sultan (tsp53Y163H) MM cell lines. These cells were then used as a model system of inducible wtp53-like function because wild-type-like p53 is induced at permissive (30°C) but not at restrictive (37°C) temperatures. Using p21-luciferase reporter assays, we confirmed that CD40 induces p53 transactivation in RPMI 8226 and HS Sultan cells cultured under permissive, but not restrictive, conditions. Furthermore, CD40 activation of these MM cells under permissive, but not restrictive, temperatures increased the expression of p53 and p21 mRNA and protein. Importantly, CD40 activation induced the proliferation of RPMI 8226 and HS Sultan cells at restrictive temperatures and growth arrest and increased subG1 phase cells at permissive temperatures. These data confirmed that CD40 activation might have distinct biologic sequelae in MM cells, depending on their p53 status.

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

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

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

ABT-737, an Inhibitor of Bcl-2/Bcl-XL Proteins, Is Cytotoxic in Multiple Myeloma Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1589-1589
Author(s):  
Michael Kline ◽  
Terry Kimlinger ◽  
Michael Timm ◽  
Jessica Haug ◽  
John A. Lust ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Tumor Microenvironment ◽  
Cell Lines ◽  
Plasma Cells ◽  
Annexin V ◽  
Significant Activity ◽  
Rpmi 8226 ◽  
Dose Dependent

Abstract Background: Multiple myeloma (MM) is a plasma cell proliferative disorder that is incurable with the currently available therapeutics. New therapies based on better understanding of the disease biology are urgently needed. MM is characterized by accumulation of malignant plasma cells predominantly in the bone marrow. These plasma cells exhibit a relatively low proliferative rate as well as a low rate of apoptosis. Elevated expression of the anti-apoptotic Bcl-2 family members has been reported in MM cell lines as well as in primary patient samples and may be correlated with disease stage as well as resistance to therapy. ABT-737 (Abbott Laboratories, Abbott Park, IL) is a small-molecule inhibitor designed to specifically inhibit anti-apoptotic proteins of the Bcl-2 family and binds with high affinity to Bcl-XL, Bcl-2, and Bcl-w. ABT-737 exhibits toxicity in human tumor cell lines, malignant primary cells, and mouse tumor models. We have examined the in vitro activity of this compound in the context of MM to develop a rationale for future clinical evaluation. Methods: MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum supplemented with L-Glutamine, penicillin, and streptomycin. The KAS-6/1 cell line was also supplemented with 1 ng/ml IL-6. Cytotoxicity of ABT-737 was measured using the MTT viability assay. Apoptosis was measured using flow cytometry upon cell staining with Annexin V-FITC and propidium iodide (PI). Flow cytometry was also used to measure BAX: Bcl-2 ratios after ABT-737 treatment and cell permeabilization with FIX & PERM (Caltag Laboratories, Burlingame, CA) Results: ABT-737 exhibited cytotoxicity in several MM cell lines including RPMI 8226, KAS-6/1, OPM-1, OPM-2, and U266 with an LC50 of 5-10μM. The drug also had significant activity against MM cell lines resistant to conventional agents such as melphalan (LR5) and dexamethasone (MM1.R) with similar LC50 (5-10 μM), as well as against doxorubicin resistant cells (Dox40), albeit at higher doses. Furthermore, ABT-737 retained activity in culture conditions reflective of the permissive tumor microenvironment, namely in the presence of VEGF, IL-6, or in co-culture with marrow-derived stromal cells. ABT-737 was also cytotoxic to freshly isolated primary patient MM cells. Time and dose dependent induction of apoptosis was confirmed using Annexin V/PI staining of the MM cell line RPMI 8226. Flow cytometry analysis of cells treated with ABT-737 demonstrated a time and dose dependent increase in pro-apoptotic BAX protein expression without significant change in the Bcl-XL or Bcl-2 expression. Ongoing studies are examining the parameters and mechanisms of ABT-737 cytotoxicity to MM cells in more detail. Conclusion: ABT-737 has significant activity against MM cell lines and patient derived primary MM cells in vitro. It is able to overcome resistance to conventional anti-myeloma agents suggesting a different mechanism of toxicity that may replace or supplement these therapies. Additionally, it appears to be able to overcome resistance offered by elements of the tumor microenvironment. The results of these studies will form the framework for future clinical evaluation of this agent in the clinical setting.

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.

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

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2635-2635
Author(s):  
Sarah A. Holstein ◽  
Huaxiang Tong ◽  
Raymond J. Hohl
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Annexin V ◽  
Differential Sensitivity ◽  
Parp Cleavage ◽  
Myeloma Cells ◽  
Protein Isoprenylation ◽  
Geranylgeranyl Transferase ◽  
Synthase Inhibitor ◽  
Rpmi 8226

Abstract 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. Figure Figure

Dual Antitumoral and Bone Antiresorptive Effect Of The Pan-Pim Kinase Inhibitor, LGH447, In Multiple Myeloma

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4435-4435
Author(s):  
Teresa Paíno ◽  
Antonio Garcia-Gomez ◽  
Lorena González-Méndez ◽  
Laura San-Segundo ◽  
Montserrat Martín-Sánchez ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Annexin V ◽  
Myeloma Cells ◽  
Pim Kinases ◽  
Castilla Y Leon ◽  
Ic50 Values ◽  
Rpmi 8226 ◽  
Resorptive Activity

Introduction Multiple myeloma (MM) is characterized by the accumulation of malignant plasma cells in the bone marrow (BM) and is closely associated with osteolytic lesions, in part due to an increase in the bone-resorptive activity and number of osteoclasts (OCs). The activation of survival pathways in myeloma cells could be the cause of treatment failure rendering the disease incurable. Pim kinases are a family of survival serine/threonine kinases composed of three members (Pim1, Pim2 and Pim3) that are overexpressed in MM cells and may have a role in MM pathogenesis. However, little is known about the role of Pim kinases in OCs and its involvement in myeloma bone disease. Here, we have evaluated the preclinical activity of a new pan-Pim kinase inhibitor, LGH447, on MM cells and OCs. Cell lines, primary samples, material and methods LGH447 was provided by Novartis Pharmaceuticals. The human MM cell lines MM1S, MM1R, RPMI-8226 (or RPMI-8226-luc), RPMI-LR5, MM144, NCI-H929, OPM-2, U266, U266-Dox4 and U266-LR7 were employed. PBMCs from healthy volunteers were used to generate OCs, whereas primary mesenchymal stromal cells (MSCs) were obtained from bone marrow aspirates of MM patients. Cell viability was studied using MTT colorimetric assay or bioluminescence. Apoptosis was measured by annexin-V staining. For cell cycle analysis, propidium iodide staining was used. OC formation was assessed by enumeration of multinucleated (≥3) TRAP-positive cells and OC resorption was assessed on calcium-coated slides. Immunoblotting, quantitative PCR and immunofluorescence were used to further investigate the mechanism of action of LGH447. Results All MM cell lines expressed the three isoforms of Pim kinases with higher levels of Pim2. The dose-response curves to LGH447 after a 48 hour treatment revealed two groups of MM cell lines with regard to sensitivity to this drug: high sensitive, with IC50 values ranging from 0.2 to 3.3 µM (MM1S, MM1R, RPMI-8226, MM144, U266 and NCI-H929); and low sensitive, with IC50 values >7 µM (OPM-2, RPMI-LR5, U266-Dox4 and U266-LR7). Our results indicated that LGH447 promoted apoptosis in myeloma cells as shown by the increase in annexin-V positive cells and by the cleavage of initiator (caspases 8 and 9) and effector caspases (caspases 3 and 7) and of PARP. LGH447 also blocked the cell cycle in MM cells as demonstrated by the increase in G0-G1 and the decrease in S-G2-M phases. Importantly, LGH447 was also able to overcome the growth advantage conferred to RPMI-8226-luc cells by co-culture with MSCs or OCs. Regarding the mechanisms involved in these effects, LGH447 inhibited the mTOR pathway, demonstrated by a decreased phosphorylation of the downstream mTOR effectors, 4EBP1 and S6 in residues Thr37/46 and Ser235/236, respectively. Interestingly, LGH447 also inhibited OC formation and resorption activity. LGH447 treatment of human pre-OCs diminished the expression of key molecules involved in OC differentiation (p-Erk1/2 and NFATc1) and function [CAII (carbonic anhidrase II), CLCN7 (chloride channel 7), ATP6V1A (vacuolar-H+-ATPase catalytic subunit A1) and MMP9 (matrix metalloproteinase 9)] and also disrupted the F-actin ring necessary for OC effective resorption. Conclusion Overall, our results demonstrate that both MM cells and OCs are targets of the pan-Pim kinase inhibitor, LGH447. Therefore, the inhibition of Pim kinases could potentially provide a dual benefit in myeloma patients as a consequence of cytotoxic effects exerted on MM cells and an anti-resorptive activity on bone. This work was supported by funding from the Fundación Española de Hematología y Hemoterapia (AG-G), Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León, the RTICC-Hematology Group (RD12/0036/0058), Spanish FIS (PI12/02591) and the Junta de Castilla y León, Gerencia Regional de Salud (GRS 862/A/13). Disclosures: Off Label Use: LGH447 is a pan-Pim kinase inhibitor (Novartis Pharmaceuticals). It has been used for pre-clinical studies in multiple myeloma.

HDAC3 Maintains DNMT1 through Dual Mechanisms in Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4472-4472 ◽  
Author(s):  
Takeshi Harada ◽  
Hiroto Ohguchi ◽  
Yohann Grondin ◽  
Shohei Kikuchi ◽  
Morihiko Sagawa ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Growth ◽  
Growth Inhibition ◽  
Board Of Directors ◽  
Combination Treatment ◽  
Hdac Inhibitors ◽  
Advisory Committees ◽  
Equity Ownership ◽  
Rpmi 8226 ◽  
Dnmt1 Expression

Abstract Histone deacetylases (HDACs) represent novel therapeutic targets for the treatment of multiple myeloma (MM). Although non-selective HDAC inhibitors demonstrate remarkable anti-MM activity, they also are associated with side effects. To avoid these adverse events without reducing anti-MM efficacy, we have been developing isoform- or class-selective HDAC inhibitors. Specifically, we showed that HDAC3 plays an important role in MM cell proliferation (Minami J, et al, Leukemia. 2014), and here delineate the mechanism whereby HDAC3 inhibition abrogates MM cell growth. We first carried out gene expression profiling before and after knocking down of HDAC3 in MM.1S cells. Among significantly downregulated genes (adjusted P values < 0.001, log fold change > 1.0), we selected DNA methyltranseferase 1 (DNMT1) for further studies. Downregulation of DNMT1 by HDAC3 knockdown was first confirmed by quantitative real time PCR (Q-PCR) and immunoblotting in both MM.1S and RPMI 8226 cells. HDAC3 selective inhibitor BG45 also downregulated DNMT1 expression. Importantly, knockdown of DNMT1triggers apoptosis in MM cells, suggesting that DNMT1 downregulation plays, at least in part, a role in HDAC3 inhibitor-induced MM cell growth inhibition. Previous studies show that HDAC inhibitors downregulate c-Myc expression (Hideshima T, et al. Blood Cancer J. 2015), and we confirmed that c-Myc was downregulated by genetic downregulation and pharmacological inhibition of HDAC3 by HDAC3 shRNA and BG45, respectively. Moreover, treatment of MM.1S cells with BG45 markedly increased c-Myc acetylation. Importantly, c-Myc was significantly degraded after treatment of MM.1S with HDAC3 inhibitor BG45 in the presence of cycloheximide (CHX), indicating that downregulation of c-Myc by HDAC3 inhibition is due to loss of protein stability. To determine whether DNMT1 expression is regulated by c-Myc, we next analyzed ChIP-Seq data in MM.1S cells (GSE36354) and found that c-Myc binds to DNMT1 promoter region. We confirmed downregulation of DNMT1 after knockdown of MYC in MM.1S and RPMI 8226 cells by Q-PCR and immunoblotting. These results suggest that HDAC3 inhibition downregulates DNMT1 through downregulation of c-Myc. A recent study reported that acetylation of DNMT1 leads to its ubiquitination, resulting in degradation of DNMT1 (Cheng J, et al. Nat Commun. 2015). We showed that treatment of MM.1S cells with BG45 in the presence of CHX triggered hyperacetylation of DNMT1, followed by its degradation. We further confirmed this association of acetylation and ubiquitination of DNMT1 protein using a dequbiquitination assay in 293T cells. As expected, HDAC3 blocked DNMT1 ubiquitination. Taken together, these results suggest that HDAC3 inhibition modulates DNMT1 via both c-Myc and by acetylation and thereby altering protein stability. Finally, Azacytidine (AZA) is used as a DNMT1 inhibitor in the treatment of acute myeloid leukemia and myelodysplastic syndrome. We therefore examined combination treatment of MM cells with BG45 combined with AZA. Importantly, this combination triggered synergistic downregulation of DNMT1 and growth inhibition through apoptosis in both MM cell lines and patient MM cells. Efficacy of combination treatment was confirmed in a murine xenograft MM model, evidenced by both tumor growth inhibition and prolonged overall host survival. Our results therefore provide the rationale for combination treatment with HDAC3 inhibitor and DNMT1 inhibitor to improve patient outcome in MM. Disclosures Mazitschek: Acetylon: Equity Ownership. Hideshima:Acetylon: Consultancy; C4 Therapeutics: Equity Ownership. Anderson:Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Millennuim: Membership on an entity's Board of Directors or advisory committees; Oncoprep: Equity Ownership; Gilead: Membership on an entity's Board of Directors or advisory committees; Acetylon: Equity Ownership; Oncoprep: Equity Ownership; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acetylon: Equity Ownership; Millennuim: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Equity Ownership; C4 Therapeutics: Equity Ownership; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Elimination of clonogenic stem cells from human multiple myeloma cell lines by a plasma cell-reactive monoclonal antibody and complement

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1482-1489 ◽  
Author(s):  
AW Tong ◽  
JC Lee ◽  
JW Fay ◽  
MJ Stone
Keyword(s):  
Stem Cells ◽  
Multiple Myeloma ◽  
Monoclonal Antibody ◽  
Bone Marrow ◽  
Cell Lines ◽  
Plasma Cell ◽  
Colony Formation ◽  
Human Multiple Myeloma ◽  
Rpmi 8226 ◽  
Marrow Cells

Abstract The monoclonal antibody (MoAb) MM4 reacts with human multiple myeloma (MM) cell lines and bone marrow from patients with plasma cell dyscrasias but not with normal peripheral blood or bone marrow cells. Treatment with MM4 and rabbit complement (C') was cytotoxic to the plasma cell-derived cell lines GM 1312, RPMI 8226, and ARH-77, as demonstrated by chromium release microcytotoxicity and trypan blue exclusion assays. The same treatment eliminated greater than 99% of clonogenic myeloma stem cell colony formation of these cell lines, with less than 20% inhibition of normal human bone marrow pleuripotent progenitor colony formation in vitro. As an experimental model to explore the efficacy of MM4 + C' in purging MM-involved bone marrow, normal marrow cells were mixed with RPMI 8226 or GM 1312 cells in the ratio of 90:10 or 50:50 (marrow:myeloma cells). Colony growth assays indicated that MM4 + C' eliminated at least 2 logs of clonogenic myeloma stem cells in both 90:10 and 50:50 preparations, while sparing the majority of normal marrow progenitors (inhibition of CFU-C:10% to 13%; BFU-E:0%). The selectivity of MM4-mediated cytotoxicity may be useful for eliminating myeloma clonogenic stem cells from bone marrow of patients with multiple myeloma.

Tetraspanin Overexpression Induces Multiple Myeloma Cell Death.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5067-5067
Author(s):  
Tali Tohami ◽  
Liat Drucker ◽  
Judith Radnay ◽  
Hava Shapiro ◽  
Michael Lishner
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Lines ◽  
Caspase 3 ◽  
Annexin V ◽  
Myeloma Cell ◽  
Transfected Cells ◽  
Over Expression ◽  
Rpmi 8226

Abstract Background: Medullary and extra-medullary dissemination of multiple myeloma (MM) cells involves cell-cell and cell-extracellular matrix (ECM) interactions. Proteins coordinating these intricate networks regulate the signaling cascades in a spatial and time dependent manner. Tetraspanins facilitate multiprotein complexing in defined membranal microdomains and select family members have been identified as metastasis suppressors. In preliminary studies, we observed that tetraspanins CD82, frequently down regulated or lost at the advanced clinical stages of various cancers, was absent in MM (8 BM samples, 5 cell lines) and CD81, characteristically expressed in leukocytes plasma membranes, was under-expressed (4/8 BM samples, 4/5 cell lines). We aimed to investigate the consequences of CD81 and CD82 over-expression in myeloma cell lines. Methods: CAG and RPMI 8226 were transfected with pEGFP-N1/C1 fusion vectors of CD81 and CD82. Transfected cells were assessed for - cell morphology (light and fluorescent microscope); cell survival (eGFP+/PI- cells); cell death (Annexin V/7AAD, pre-G1, activated caspase-3 (IC), caspase dependence with pan caspase inhibitor z-VAD-fmk); cell cycle (PI staining). Results: CD82 induced cell death was determined by morphologic characteristics in stably transfected CAG cells (50%) compared to their mock-transfected counterparts (8%) (p&lt;0.05). Activated caspase-3 was also detected (40% of the CD82 transfected cells) (p&lt;0.05). In CD82 transiently transfected MM cell lines a reduced fraction of surviving cells was observed compared to mocks (~60%) (p&lt;0.05) yet, no increases in pre-G1 or Annexin V+/7AAD- subgroups were observed. Moreover, CD82 induced cell death could not be inhibited by the use of z-VAD-fmk. CD82 transfection did not affect the cell cycle of CAG and RPMI 8226 lines. CD81 stably transfected cell lines (CAG and RPMI 8226) could not be established. Indeed, in transiently transfected cells we determined a massive rate of CD81 induced cell death. This is demonstrated in a surviving fraction of only 10% CAG cells and 30% RPMI 8226 (compared to mock) (p&lt;0.05). The CD81 transfected cells were negative for PS exposure, pre-G1 sub-population, or inhibition of death with z-VAD-fmk. The death inducing effect of both tetraspanins in the two cell lines was evident with the pEGFP-N1 orientation vector only. Conclusions: CD81 and CD82 over-expression in MM cell lines causes cell death. Based on the restriction of the killing effect to the pEGFP-N1 clone it may be speculated that its implementation is either dependent on the interactions of the N1 tetraspanin terminus or the proteins’ conformation. It is of interest that CD81 though normally expressed in RPMI 8226 still induced cell death when over-expressed, possibly indicative of ’negative signaling’. Tetraspanins’ suppressive effects on adhesion, motility, and metastasis in solid tumors combined with its capacity to induce myeloma cell death underscore the significance of its absence in MM cell lines and patients. We suspect that a better understanding of CD81/82 mediated signaling pathways will promote future treatment of myeloma cell in their microenvironment. Current studies designed to assess the involvement of oxidative stress in CD81/CD82 induced death are underway.

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