Therapeutic Agent For Multiple Myeloma Research Articles

Anti-DKK1 mAb (BHQ880) as a potential therapeutic agent for multiple myeloma

AbstractDecreased activity of osteoblasts (OBs) contributes to osteolytic lesions in multiple myeloma (MM). The production of the soluble Wnt inhibitor Dickkopf-1 (DKK1) by MM cells inhibits OB activity, and its serum level correlates with focal bone lesions in MM. Therefore, we have evaluated bone anabolic effects of a DKK1 neutralizing antibody (BHQ880) in MM. In vitro BHQ880 increased OB differentiation, neutralized the negative effect of MM cells on osteoblastogenesis, and reduced IL-6 secretion. In a severe combined immunodeficiency (SCID)–hu murine model of human MM, BHQ880 treatment led to a significant increase in OB number, serum human osteocalcin level, and trabecular bone. Although BHQ880 had no direct effect on MM cell growth, it significantly inhibited growth of MM cells in the presence of bone marrow stromal cells (BMSCs) in vitro. This effect was associated with inhibition of BMSC/MM cell adhesion and production of IL-6. In addition, BHQ880 up-regulated β-catenin level while down-regulating nuclear factor-κB (NF-κB) activity in BMSC. Interestingly, we also observed in vivo inhibition of MM cell growth by BHQ880 treatment in the SCID-hu murine model. These results confirm DKK1 as an important therapeutic target in myeloma and provide the rationale for clinical evaluation of BHQ880 to improve bone disease and to inhibit MM growth.

Human-like mouse models for testing the efficacy and safety of anti-beta2-microglobulin monoclonal antibodies to treat myeloma.

We showed recently that anti-beta2-microglobulin (beta2M) monoclonal antibodies (mAb) have remarkably strong apoptotic effects on myeloma cells in vitro and in SCID-hu mice. However, whether the mAbs will be therapeutic and safe in the treatment of myeloma patients, in whom every tissue expresses low densities of MHC class I molecules and elevated levels of soluble beta2M are present, remains to be determined. In this study, human-like myeloma mouse models (HLA-A2-transgenic NOD/SCID mice) were developed, which express mature and functional human MHC class I (HLA-A2 and human beta2M) on murine organs and present high levels of circulating human beta2M derived from human myeloma cells. Myeloma-bearing mice were treated intraperitoneally with anti-beta2M mAbs, and the distribution and effects of the mAbs on normal organs and established tumors were examined. Our results show that anti-beta2M mAbs were effective in suppressing myeloma growth in treated mice. The therapeutic efficacy of the mAbs in these mice are comparable with those observed in myeloma-bearing nontransgenic NOD/SCID mice in which no human MHC class I is expressed on murine organs. Furthermore, although the mAbs can be detected on different organs, no tissue damage or cell apoptosis was observed in the mice. Based on the antimyeloma efficacy and low toxicity in the mice, our study suggests that anti-beta2M mAbs may be safe and the tissue-expressing and soluble beta2M may not compromise their therapeutic effects in myeloma patients. This study provides further support for the future application of the mAbs as therapeutic agents for multiple myeloma.

B563 Anti-DKK1 mAb (BHQ880) as a Potential Therapeutic Agent for Multiple Myeloma

B563 Anti-DKK1 mAb (BHQ880) as a Potential Therapeutic Agent for Multiple Myeloma

Human-Like Mouse Models for Testing the Efficacy and Safety of Anti-β2-Microglobulin Monoclonal Antibodies to Treat Myeloma

Multiple myeloma (MM) remains fatal in the majority of patients with the disease, even though the application of new therapeutic agents has greatly advanced the treatment of the disease. There is an urgent need to develop novel therapeutic agents. We recently demonstrated that anti-β2-microglobulin monoclonal antibodies (anti-β2M mAbs) have remarkably strong apoptotic effects on myeloma in vitro and in SCID mice. However, whether the mAbs will be therapeutic and safe in treating myeloma patients, in whom every tissue expresses low densities of MHC class I molecules and elevated levels of soluble β2M are present, remains to be determined. In this study, we examined the antimyeloma activity and potential toxicity of the mAbs in a human-like situation. We developed and used a myeloma-HLA-A2-transgenic NOD/SCID mouse model. After myeloma establishment in NOD/SCID mice transgenic for HLA-A2 α-chain, we found that all tissues express human HLA-A2 and β2M, and detected high levels (2 mg/mL) of circulating human β2M. After one intraperitoneal (i.p.) injection of the mAbs (1 mg per mouse), the mAbs could be detected on the surface of tumors and murine organs. Furthermore, murine splenocytes were incubated with immobilized mAb W6/32 to crosslink MHC class I. Western blotting analysis showed that cross-linking human MHC class I triggers phosphorylation of Lyn, Syk, and PLC-γ2 in the treated cells, indicating that indeed human MHC class I are functional and able to transduce signals in murine cells. Thus, these findings demonstrate that the mouse model is suitable for our studies because myeloma-derived human β2M was highly elevated in the serum, and bound with the α-chain to form mature and functional human MHC class I on all tissues. By using the model, we first examined the therapeutic efficacy of the mAbs on established myeloma. Myeloma-bearing mice were i.p. injected with the mAbs (1 mg per mouse) twice a week for a total of 4 injections. The results showed that the mAbs effectively suppress myeloma growth, and activated caspase-9 and -3 and induced myeloma cell apoptosis in vivo. We also examined whether the mAbs damage human MHC class I-expressing normal cells and tissues. After treatment with the mAbs, murine organs were removed for histological examination. We found that although the mAbs were detected on different organs, no tissue damage or cell apoptosis was observed in the mice. Furthermore, we elucidated the mechanisms underlying the selectivity of the mAbs in killing myeloma cells without damaging normal cells. By using the QuantiBRITE beads, we quantified the numbers of surface β2M and found that myeloma cells have approximately 265,918 surface β2M molecules whereas normal blood lymphocytes have approximately 79,521, indicating that myeloma cells express 3-fold more surface β2M than lymphocytes. Myeloma cells treated with specific siRNAs for human β2M expressed a similarly low level of β2M (91,723) and became resistant to mAb-induced apoptosis. Thus, our findings indicate that the mAbs may be safe and that the tissue-expressed and soluble β2M may not compromise their therapeutic effects in myeloma patients. This study provides further support for the future application of the mAbs as therapeutic agents for MM.

New Tubulin Polymerization Inhibitor Derived from Thalidomide: Implications for Anti-Myeloma Therapy

Despite the conventional and high-dose chemotherapy with hematopoietic stem cell transplantation, multiple myeloma eventually relapses, resulting in an incurable hematological malignancy. Therefore, novel therapeutic approaches in clinical settings are desired. Recently, thalidomide was introduced for the treatment of myeloma, and many clinical trials have since confirmed its efficacy in patients with relapsed/refractory or newly diagnosed multiple myeloma. Multiple mechanisms have been proposed to explain thalidomide's anti-myeloma activity. However, the precise mechanism underlying this activity remains unclear, because thalidomide rapidly undergoes spontaneous, nonenzymatic, hydrolytic cleavage to numerous metabolites in vivo. To elucidate the exact anti-myeloma mechanism of thalidomide in vivo, we have performed structural development studies of thalidomide, and obtained various analogs with specific molecular properties. Among these derivatives, we found that a new thalidomide analog, 2-(2,6-diisopropylphenyl)-5-hydroxy-1H-isoindole-1,3-dione (5HPP-33), has the most potent anti-myeloma effect with tubulin polymerization inhibiting activity. 5HPP-33 directly inhibited the growth and survival of various myeloma cells in a dose-dependent manner with IC(50) of 1-10 microM. In contrast, thalidomide itself did not inhibit RPMI8226 cell growth. A tubulin polymerization assay using microtubule protein from porcine brain revealed that 5HPP-33 had potent tubulin polymerization inhibiting activity with IC(50) of 8.1 microM, comparable to that of rhizoxin, a known tubulin polymerization inhibitor. Moreover, its activity was more potent than that of a known thalidomide metabolite, 5-hydroxythalidomide. Our data suggest that 5HPP-33 is a promising candidate as a therapeutic agent for multiple myeloma. In addition, the results suggest that thalidomide's tubulin polymerization inhibiting activity might be the mechanism underlying the induction of apoptosis in myeloma cells.

Emerging drugs in multiple myeloma

The treatment of multiple myeloma has seen significant changes from the time of the initial use of cytotoxic agents such as melphalan, to the introduction of high-dose chemotherapy and stem cell transplantation, and most recently the era of novel targeted agents. These new drugs have rapidly become the mainstay of therapy of this disease and transformed the treatment paradigm, leading to improvements in survival and quality of life. Existing therapeutic options include agents such as thalidomide, bortezomib and lenalidomide, either used alone or in combination with standard agents, including glucocorticoids, and in conjunction with high-dose chemotherapy supported with stem cell transplantation. Several other targeted agents have demonstrated exciting preclinical activity, and are presently being tested in early Phase I and II clinical trials. This review summarizes the role of novel therapeutic agents in multiple myeloma, and the promising effect of multiple new agents in development.

Arsenic Trioxide Induces Up-Regulation of BMF and NOXA and Overcomes an Anti-Oxidative Response To Induce Apoptosis in Myeloma Cells.

Arsenic Trioxide (ATO) has been successfully used for the treatment of acute promyelocytic leukemia (APL) and has shown promise as a therapeutic agent in multiple myeloma (MM). We have been characterizing the response of MM cell lines to ATO and have demonstrated that four human MM cell lines, including U266, MM.1s, 8226/S and KMS11 were sensitive to ATO. Affymetrix Hu133 2.0 Plus arrays were used for expression profiling at 0, 6, 24 and 48 h after ATO treatment. The pattern observed in all four cell lines was consistent with anti-oxidative responses and an imbalance of Bcl-2 family members favoring apoptosis. Most notably expression changes were associated with a response driven by the Nrf2-Keap1 pathway. Nrf2 is expressed at the mRNA level however the protein is not observed in untreated cells. Consistent with inactivation of Keap1, Nrf2 protein is stabilized within 6 h of ATO treatment. Importantly, this up-regulation is associated with Nrf2 accumulation in the nucleus where it functions. The activation of the Nrf2 is associated with the changes observed during ATO treatment, including up-regulation of the following genes: metallothionein-1 (MT-1), phase II detoxification enzymes (NQO-1), anti-oxidant enzymes [heme oxygenase-1 (HO-1), thioredoxin reductase, glutathione reductase and γ-glutamate cysteine ligase], NADPH-generating enzymes (malic enzyme), chaperone proteins (Hsp70, Hsp40, Hsp27KDa and Hsp105KDa) and the transcriptional repression of genes involved in the cholesterol/lipid biosynthesis pathway. HO-1 and MT-1 expression has been previously suggested to be associated with ATO-resistance in myeloma. However, the up-regulation of this gene (greater than 50 fold in MM.1s and KMS11 as early as 6 hrs) could not protect cells from ATO-induced apoptosis. Similarly up-regulation of MT-1 did not correlate with cell survival. Moreover, up-regulation of HO-1 or MT-1 prior to ATO treatment had no effect on ATO-induced apoptosis in U266 or MM.1s cells, consistent with each of these being components of a larger anti-oxidative response. Bcl-x, was down-regulated in all the four cell lines during ATO-induced apoptosis while one of its ligands, Bmf, a BH3-only pro-apoptotic Bcl-2 family member was up-regulated 1.5–3.0 fold at the mRNA level and correlated with response. An additional BH3-only protein Noxa was up-regulated in three out four cell lines at the mRNA and protein level. In U266, Noxa is expressed constitutively and not affected by ATO. Interestingly however, ATO induced a down-regulation of Mcl-1, the Bcl-2 anti-apoptotic partner of Noxa in U266 cells. Taken together the Bmf/Bcl-x and Noxa/Mcl-1 ratios favored an apoptotic response following addition of ATO. Additionally Noxa up-regulation was enhanced when glutathione was depleted with BSO and blocked by addition of N-acetylcysteine. Taken together these data indicate that while the cells initially respond to ATO treatment in a manner consistent with a strong anti-oxidative response, these protective mechanisms are not sufficient to block cell death induced by the up-regulation of two BH3-only proteins (Bmf and Noxa) and the down-regulation of Bcl-x and Mcl-1 resulting in the activation of the intrinsic apoptotic pathway as part of the ATO-induced apoptotic mechanism(s). New arsenical compounds or combined therapies, could avoid this protective anti-oxidative pathway activated by ATO increasing its activity.

Inhibitors of the mevalonate pathway as potential therapeutic agents in multiple myeloma

Clinical studies have suggested that bisphosphonates may prolong the survival of sub-sets of myeloma patients. Newer nitrogen containing bisphosphonates such as zoledronate act, at least in part, by inhibiting farnesyl diphosphate synthase and subsequent protein prenylation, furthermore, limited data suggests that zoledronate exerts a direct anti-tumour effect against human myeloma cell lines. We therefore investigated the anti-myeloma potential of zoledronate in comparison to, and in combination with, two other inhibitors of the mevalonate pathway: the HMGCoA reductase inhibitor fluvastatin and the farnesyl transferase inhibitor SCH66336. We found that fluvastatin was able to inhibit the proliferation of myeloma cells more effectively than zoledronate or SCH66336 and that combinations of zoledronate and fluvastatin, but not zoledronate and SCH66336 acted synergistically. Our data indicated that the anti-proliferative effect of mevalonate pathway inhibitors is mediated principally via prevention of geranylgeranylation and is the result of both cell cycle arrest and apoptosis induction. Microarray and quantitative real-time PCR analyses further demonstrated that genes related to apoptosis, cell cycle control, and the mevalonate pathway were particularly affected by zoledronate and fluvastatin, and that some of these genetic effects were synergistic. We conclude that the mechanisms of geranylgeranylation inhibition mediated anti-myeloma effects warrant further evaluation and may provide novel targets for future therapeutic development.

Clinicopathological and prognostic characteristics of CD33‐positive multiple myeloma

There have been few reports about the CD33 expression on multiple myeloma (MM) cells so far, showing that only a few patients expressed CD33 homogenously on their MM cells. However, in these reports, neither detailed clinical information nor its prognostic significance was described. Therefore, we analyzed the CD33 expression on MM cells from 63 newly diagnosed patients by flow cytometry and the correlation with other clinical parameters to determine the clinicopathological significance of this molecule. Fourteen (22%) patients were positive for CD33. Of the 14 patients with CD33+ MM, >80% of MM cells were positive in six (9.5%). The CD33+ patients had higher beta 2 microglobulin and lactate dehydrogenase levels and higher incidence of anemia and thrombocytopenia than did CD33- patients. The estimated 3-yr overall survival in CD33+ patients was significantly lower than in the CD33- ones (31% and 50%, respectively, P = 0.042). Especially, mortality within a year from diagnosis in the CD33+patients was higher than that in CD33- patients (43% and 10%, respectively, P = 0.005). Serial evaluation of CD33 expression showed that the amount of CD33 significantly increased after a variety of treatment including melphalan and steroid in individual patients. These results suggest that the CD33 expression might be associated with drug resistance to these conventional agents, and CD33 might be a useful target for the development of new therapeutic agents in MM.

The Role of the AKT Inhibitor Perifosine in Migration and Adhesion in Multiple Myeloma (MM).

Homing of plasma cells to the bone marrow microenvironment supports the cells' survival and growth. We have previously demonstrated that MM cells migrate in response to the chemokine SDF-1 and that the PI3K/AKT pathway is an important regulator of migration in MM. The AKT inhibitor, perifosine (KRX-0401, Keryx, NY, NY, provided by the NCI) is an alkylphosphocholine, a member of a new class of potent AKT inhibitors that is being tested preclinically as a therapeutic agent in MM. The objective of this study was to determine the effect of this novel inhibitor on the migration and adhesion of MM cells and to determine differences in response between MM cells with high AKT activity (OPM2 cells) and lower AKT activity (MM.1S). MM cell lines were treated for 16 hrs with serial concentrations of perifosine 2–50uM or vehicle (sterile water). Apoptosis was determined using Annexin V/PI flow cytometry analysis (BD Biosciences, CA). Migration was determined using a transwell migration assay (Costar, Corning, NY). Treated cells were washed, placed in serum free medium for two hours, then placed in the upper chambers of the migration assay with 1% FCS medium in the presence of the chemoattractant SDF-1 in the lower chambers (SDF-1 20nM, a concentration previously determined to induce maximum migration in MM cells). After 4 hours of incubation, cells that migrated to the lower chambers were counted. Similarly, adhesion was determined using an adhesion assay (EMD Biosciences, San Diego, CA) with 96 well plated coated with fibronectin in the presence or absence of serial concentrations of SDF-1 (10–30nM). A two-tailed student t-test was used for statistical analysis. Perifosine induced a dose dependent inhibition of migration in both MM.1S and OPM2. In MM.1S, 2uM perifosine inhibited migration to 82% while 5uM inhibited migration to 44% of control. There was no further significant reduction of migration at 20nM of perifosine (40% migration). In OPM2, 2uM perifosine inhibited migration to a greater extent than MM.1S at 59% of control (p=0.03), and 5uM induced 41% migration similar to the effect observed in MM.1S (p=0.7). There was no induction of apoptosis up to 48hrs of treatment with the low concentrations of perifosine (2–5uM) indicating that the effect of low dose perifosine on MM cells is specific to migration and not due to apoptosis of cells. We have previously demonstrated that SDF-1 increases adhesion of MM cells to fibronectin indicating activation of alpha4beta integrin receptor, with 30nM SDF-1 inducing 296% increase in adhesion. Perifosine alone (2–20uM) inhibited adhesion of MM cells (38% with 2uM, and 12% with 5–20uM as compared to control). Perifosine 2–20uM inhibited adhesion even in the presence of 30nM SDF-1 (55% to 12% respectively). There was no difference in the adhesion response of MM.1S and OPM2 to perifosine. We demonstrate that low doses of the AKT inhibitor perifosine inhibit migration and adhesion of MM cells in response to SDF-1. These doses do not induce apoptosis, and therefore are specific to inhibiting migration. Future studies to test the AKT inhibitor perifosine as a modulator of MM homing, migration and adhesion in vivo are warranted. Supported in part by an ASH scholar award and an MMRF grant.

Azacytidine Suppressors Autocrine IL-6 Secretion and Demonstrates In Vitro and In Vivo Activity Against Multiple Myeloma.

Azacytidine (AZA), a DNA methyltransferase inhibitor, has been shown to inhibit cell growth and induce apoptosis in some cancer cells. We determined the impact of AZA on a panel of human myeloma cell lines (HMCL); KMS 12PE, KMS 18, LP-1, NCI-H929, OPM-2, RPMI-8226 and U266 and in an in vivo murine model of multiple myeloma (5T33 model). Dose responsiveness to AZA was determined via MTS assays with a range of AZA doses (1–10mM) for 72 hours. FACS and cell cycle analysis were used to evaluate the profile of the cells after exposure to AZA for 72 hours. MTS assays demonstrated a dose and time dependent AZA-induced inhibition of HMCL viability with effective concentrations of AZA ranging from 1–10 mM. This was associated with accumulation of cells in the Go/G1 phase with decreasing number of cells in the S and G2/M phases. Western Blot analysis using antibodies against caspases 3,8,10, PARP, phospho-ERK, ERK, Stat3 and phospho -Stat3 were performed to help characterize the mechanism(s) of cell killing. Cleavage of caspases 3,8,10 and PARP within 24 hours of AZA treatment confirmed early AZA-induced HMCL apoptosis. phospho-ERK which was absent in untreated U266 appeared after 48 hours exposure to 5mM AZA. Similarly inhibitors of caspases 3,8 and 9 were used to determine which apoptotic pathway was being preferentially activated by AZA. Inhibitors of both caspase 3 and 9 effectively abrogated AZA-induced apoptosis in U266 and NCI-H929. In contrast caspase 8 inhibitor was less effective which is consistent with AZA acting via the mitochondrial apoptotic pathway. Reactivation of p16 gene by AZA-induced hypomethylation was assessed with methylation specific PCR. MSP-PCR of the p16 gene indicated a loss of methylation and up-regulated transcription after 48 hours treatment with 5 mM AZA. The level of IL-6 in conditioned media from U266 cells treated with AZA was determined by ELISA assay and demonstrated a rapid fall in autocrine IL-6 production. RT-PCR demonstrated rapid AZA-induced cessation of IL-6 transcription temporarily associated with the disappearance of upstream phospho -Stat3. Addition of exogenous IL-6 did not rescue U266 from AZA-induced apoptosis. AZA was also administered to a 5T33 murine model of multiple myeloma at increasing concentrations (1, 3, 10 mg/kg). At 10 mg/kg the median survival of vehicle versus AZA treated mice was 28 days versus 30+ days (p=0.003). These findings justify further evaluation of AZA as a potential therapeutic agent for multiple myeloma.

Flavopiridol Decreases the Level of p21 and Induces Apoptosis in Human Myeloma Cell Lines.

Flavopiridol is a cyclin dependent kinase (CDK) inhibitor with a broad spectrum of activity both in vitro and in vivo and has been demonstrated to inhibit cell growth and induce apoptosis in cancer cells. We have evaluated the effect of Flavopiridol on a panel of human myeloma cell lines (HMCL)( NCI H929, LP-1, RPMI 8226, OPM-2 and U266) and in an in vivo murine model of multiple myeloma (5T33). Dose response was determined by MTS assays with a range of Flavopiridol doses (0–5mM) for 72 hours. The profiles of the cells after treatment with Flavopiridol were evaluated via FACS and cell cycle analysis. MTS assays showed a dose and time dependent Flavopiridol-induced cell killing in HMCL with effective concentrations of Flavopiridol ranging from 10nM - 5mM. This observation was associated with increasing number of cells in the late apoptotic stage and accumulation of cells in the SubG0 and G0/G1 phases. Western analysis using antibodies against p21, cyclin A, p27, Mcl-1, Bcl-XL, Bcl-2a, pRB, and PARP were performed to help characterize the mechanism(s) of cell killing. Cleavage of PARP in NCI H929 was noted within 24 hours of Flavopiridol treatment indicating early Flavopiridol-induced HMCL apoptosis. Expression of p21 protein, a member of the Cip/Kip family of CDK that can bind and obstruct a broad range of cyclin/Cdk complexes, was found to decrease following 24 hours of exposure to Flavopiridol. A reduction of another CDK member, Cyclin A, was apparent following Flavopiridol treatment whilst p27 expression was unchanged even after 72 hours. Three anti apoptotic proteins, Mcl-1, Bcl-2a and Bcl-XL protein expression were evaluated. Mcl-1 protein expression increased with time in all three cell lines tested while Bcl-2a was unaltered and Bcl-XL decreased following treatment. Retinoblastoma protein (RB), a tumour suppressor which acts as a cell cycle checkpoint between G1 and S phases was evaluated. A decrease of hyperphosphorylated RB (ppRB) was demonstrated in all 3 cell lines indicating a decrease in proliferating cells. Furthermore, silencing of p21 by siRNA enhanced Flavopiridol induced apoptosis indicating cell death occurred via a p21 dependent mechanism. The impact of combination and scheduling with bortezomib was evaluated in NCI-H929 and primary myeloma cells and it was found that addition of bortezomib subsequent to Flavopiridol improved cell killing compared to single-agent treatment. Reversal of drug treatment was found to be not as efficient. 2.5 mg/ml Flavopiridol and 1% DMSO/saline (vehicle) were administered to a 5T33 murine model of multiple myeloma for 5 days intraperitoneally. Hind limp paralysis was observed later in the Flavopiridol treated mice compared to vehicle treated mice with the median survival for vehicle treated mice of 23 days compared to 24.5 days in the AZA treatment (p= 0.018). These data provide a rationale for further evaluation of Flavopiridol as a therapeutic agent in multiple myeloma. Furthermore, we propose that Flavopiridol induced killing of HMCL may be mediated by a novel mode of action involving suppression of p21.

Thalidomide-induced antiangiogenic action is mediated by ceramide through depletion of VEGF receptors, and is antagonized by sphingosine-1-phosphate

Thalidomide, which is clinically recognized as an efficient therapeutic agent for multiple myeloma, has been thought to exert antiangiogenic action through an unknown mechanism. We here show a novel mechanism of thalidomide-induced antiangiogenesis in zebrafish embryos. Thalidomide induces the defect of major blood vessels, which is demonstrated by their morphologic loss and confirmed by the depletion of vascular endothelial growth factor (VEGF) receptors such as neuropilin-1 and Flk-1. Transient increase of ceramide content through activation of neutral sphingomyelinase (nSMase) precedes thalidomide-induced vascular defect in the embryos. Synthetic cell permeable ceramide, N-acetylsphingosine (C2-ceramide) inhibits embryonic angiogenesis as well as thalidomide. The blockade of ceramide generation by antisense morpholino oligonucleotides for nSMase prevents thalidomide-induced ceramide generation and vascular defect. In contrast to ceramide, sphingosine-1-phosphate (S1P) inhibits nSMase-dependent ceramide generation and restores thalidomide-induced embryonic vascular defect with an increase of expression of VEGF receptors. In human umbilical vein endothelial cells (HUVECs), thalidomide-induced inhibition of cell growth, generation of ceramide through nSMase, and depletion of VEGF receptors are restored to the control levels by pretreatment with S1P. These results suggest that thalidomide-induced antiangiogenic action is regulated by the balance between ceramide and S1P signal.

Drug Insight: thalidomide as a treatment for multiple myeloma

Multiple myeloma (MM)--a malignancy of the bone marrow--remains incurable by current therapies, and there is an urgent need for new drugs based on a better understanding of the underlying disease biology. MM is characterized by monoclonal plasma cells that accumulate in the bone marrow, which provides a microenvironment that promotes tumor cell growth and survival and protection against various therapeutic agents. The MM cell interacts with bone marrow stromal cells and endothelial cells, as well as osteoblasts and osteoclasts. Our understanding of the tumor microenvironment has already prompted the development of new agents that are aimed at disrupting the multiple facets of these interactions. It has also enabled the development of a comprehensive and rational approach to preclinical evaluation of new agents, facilitating the translation of in vitro studies to in vivo tumor models and, subsequently, to clinical trials. In this review, we describe the preclinical studies that led to the development of clinical trials of thalidomide and its immunomodulatory derivatives as therapeutic agents for MM. These drugs, alone or in combination, have shown impressive activity at all stages of the disease, and these demonstrations of clinical benefit have in turn validated our model systems for drug discovery in MM. Integration of data from clinical trials and laboratory studies will allow the design of future clinical trials that combine thalidomide and its derivatives with other drugs, ultimately leading to more effective therapies and better outcomes in patients with MM.

Proteomic Analysis of Targeted Therapeutic Agents in Multiple Myeloma (MM).

The objective of this study was to analyze protein changes that occur a the molecular level with novel therapeutic agents used in MM in order to identify potential combinations for clinical trials. We employed antibody protein microarrays (BD Clonetech, CA) to measure changes in the patterns of protein expression in Kas 6/1 MM cell line after treatment with bortezomib, dexamethasone, CC-5013, and 17-AAG. The antibody array is a new technique that assays protein differences directly by hybridizing fluorescently labeled protein mixtures from cell extracts onto glass slides spotted with 512 different monoclonal antibodies specific for human proteins. Induction of apoptosis was assessed by Annexin/Propidium iodide FACS analysis at 24 and 48 hours using different concentrations of the inhibitors. Protein arrays were performed at concentrations and treatment times that did not induce more than 25% apoptosis to ensure adequate analysis of early changes in signaling pathways. Bortezomib 5nM at 10 hours, dexamethasone 100mM, CC-5013 250nM, and 17-AAG 1mM at 24 hours were used. Control cells were treated with vehicles ETOH or DMSO at the same concentrations as the inhibitors. To assess differential protein expression, the mean of the Cy5/Cy3 ratios for each sample and its control were analyzed using the Clontech software and an internally normalized ratio (INR) was calculated. INR values >1.3 or <0.77 were considered as valid changes in protein abundance for this study. All inhibitors induced the upregulation of apoptosis proteins including BAK, BIK, SMAC/DIABLO, and caspase 14. Several proteins were inhibited by all 4 inhibitors indicating common pathways affected by these agents. These included members of the PI3K pathway (PI3K-p85a, p70S6kinase, and cyclin dependent kinases), MAPKinases (ERK1 and 2), antiapoptotic protein Bclx, heat shock proteins Hsp70 and HSP60, and ubiqutin/NFkB pathway (Ubch6, IKKalpha, and IKBe). These results indicate that inhibitors of the PI3K pathway such as mTOR inhibitors, IKK inhibitors or MAPkinase inhibitors may be used in the future in combination with bortezomib, dexamethasone, CC-5013 or 17-AAG. Fatty acid synthetase was downregulated by bortezomib and dexamethasone indicating potential use of fatty acid synthase inhibitors in combination with bortezomib or dexamethasone in future clinical trials. These results suggest that antibody protein arrays may be used in the rational design of combinations of targeted therapies in MM.

8-Amino-adenosine is a potential therapeutic agent for multiple myeloma

Multiple myeloma (MM) is a malignancy of clonal B-cells that accounts for 10% of all hematologic malignancies. We have shown previously that a novel purine analogue, 8-chloro-adenosine, has significant activity for MM in preclinical studies. Using MM cell lines, we investigated the molecular mechanism of related congener of adenosine, 8-amino-adenosine (8-NH2-Ado). We employed biological and biochemical assays in MM cell lines to evaluate the clinical potential of 8-NH2-Ado. In MM cell lines both sensitive and resistant to conventional chemotherapies, 8-NH2-Ado is cytotoxic, with IC50 ranging from 300 nmol/L to 3 micromol/L. A mouse leukemic cell line lacking adenosine kinase activity was resistant to 8-NH2-Ado, indicating that phosphorylation of 8-NH2-Ado to its triphosphate form is required for cytotoxicity. A 4-hour incubation of MM cells with 10 micromol/L analogue resulted in an accumulation of >7 mmol/L 8-NH2-ATP with a parallel decline in the endogenous ATP levels. Accumulation of 8-NH2-ATP was dependent on both exogenous concentration of 8-NH2-Ado and incubation time. The accumulation of 8-NH2-ATP was accompanied by a decrease in both RNA and DNA synthesis. The mechanism of 8-NH2-Ado-mediated cytotoxicity was due to apoptosis as measured by an increase in Annexin V binding, a decrease in mitochondrial membrane potential, an increase in caspase activity, cleavage of caspase substrates, and an increase in cells with a sub-G1 DNA content. Based on these results, we conclude that 8-NH2-Ado may hold great potential as a therapeutic agent for the treatment of MM.

Telomerase inhibition and cell growth arrest after telomestatin treatment in multiple myeloma.

The aim of this study was to test the efficacy of telomestatin, an intramolecular G-quadruplex intercalating drug with specificity for telomeric sequences, as a potential therapeutic agent for multiple myeloma. We treated ARD, ARP, and MM1S myeloma cells with various concentrations of telomestatin for 7 days and evaluated for telomerase activity. Myeloma cells were treated with the minimal effective telomestatin concentration for 3-5 weeks. Every 7(th) day the fraction of live cells was determined by trypan blue exclusion, aliquots of cells were removed for various molecular assays, and the remaining cells were replated at the same cell number and at the same concentration of telomestatin. Telomere length, apoptosis, and gene expression changes were monitored as described in detail in "Materials and Methods." Telomestatin treatment led to inhibition of telomerase activity, reduction in telomere length, and apoptotic cell death in ARD, MM1S, and ARP myeloma cells. Gene expression profile after 1 and 7 days of telomestatin treatment revealed >/==" BORDER="0">2-fold change in only 6 (0.027%) and 51 (0.23%) of 33,000 genes surveyed, respectively. No changes were seen in expression of genes involved in cell cycle, apoptosis, DNA repair, or recombination. These results demonstrate that telomestatin exerts its antiproliferative and proapoptotic effects in myeloma cells via inhibition of telomerase and subsequent reduction in telomere length. We conclude that telomerase is an important potential therapeutic target for multiple myeloma therapy, and G-quadruplex interacting agents with specificity for binding to telomeric sequences can be important agents for additional evaluation.

サリドマイドの新しい薬理学：多発性骨髄腫の治療経験から

Thalidomide was developed in the 1950s as a sedative having only a low toxicity. However, McBride and Lenz reported in 1961 a close correlation between oral administration of thalidomide by pregnant women and a particular deformity (phocomelia) of their babies. In the 1990s, the biological activities of thalidomide were determined to include the control of tumor necrosis factor-alpha production and inhibition of angiogenesis. In 1994, Folkman et al. reported that thalidomide exhibited a strong inhibition of angiogenesis in their experiments with rabbits and that this effect had a significant relationship to phocomelia. They suggested a utility of thalidomide as a therapeutic agent for diseases that involve angiogenesis, particularly tumorous diseases. Furthermore, in 1994, Vacca et al. reported that the bone marrow of multiple myeloma (MM) patients was rich in blood vessels and that there is a causal relationship between the activity of MM and marrow angiogenesis. According to these data, thalidomide was tested in many countries as a new therapeutic agent for MM. In this review, new pharmacological availability of thalidomide is described on the basis of our experiences.

Understanding novel therapeutic agents for multiple myeloma.

Multiple myeloma, a B cell malignancy of the plasma cells, remains incurable. Advances in high-dose chemotherapy and stem cell transplantation have improved overall survival and event-free disease periods, but relapses are inevitable. New therapeutic agents have shown promising clinical use in patients with relapsed or refractory multiple myeloma. This article discusses the therapeutic applications of these novel agents with a focus on immunomodulatory drugs, proteasome inhibitors, and arsenic compounds.

Bone morphogenetic protein-4 inhibits proliferation and induces apoptosis of multiple myeloma cells: Presented in part at the 41st annual meeting of the American Society of Hematology, New Orleans, December 1999.

AbstractBone morphogenetic proteins (BMPs) can be isolated from organic bone matrix and are able to initiate de novo cartilage and bone formation. Here it is shown that BMP-4 inhibited DNA synthesis in a dose-dependent manner in 3 IL-6–dependent multiple myeloma (MM) cell lines (OH-2, IH-1, and ANBL-6). In contrast, no effect on DNA synthesis was observed in 3 IL-6–independent MM cell lines (JJN-3, U266, and RPMI 8226). BMP-4 induced cell cycle growth arrest in the G0/G1 phase in OH-2 and ANBL-6 cells but not in IH-1 cells. BMP-4 induced apoptosis in OH-2 and IH-1 cells, but not significantly in ANBL-6 cells. Furthermore, BMP-4 induced apoptosis in freshly isolated MM cells from 4 of 13 patients. In the OH-2 and ANBL-6 cell lines and in a patient sample, immunoblotting showed that BMP-4 down-regulated IL-6–induced tyrosine phosphorylation of Stat3, suggesting a mechanism for the apparent antagonism between IL-6 and BMP-4. BMP-4 or analogues may be attractive therapeutic agents in MM because of possible beneficial effects on both tumor burden and bone disease.