nitrophenols and Multiple-Myeloma

Topics: Antibodies, Bacterial; Antigens; Blood Proteins; Electrolytes; Erythrocytes; Humans; Immunoglobulins; Ligands; Lipoproteins; Multiple Myeloma; Nitrophenols; Paraproteinemias; Peripheral Nerves; Phospholipids; Polysaccharides; Rheumatoid Factor; Waldenstrom Macroglobulinemia

Waldenström macroglobulinemia (WM) is a proliferative disorder of IgM-secreting, lymphoplasmacytoid cells that inhabit the lymph nodes and bone marrow. The disease carries a high prevalence of activating mutations in MyD88 (91%) and CXCR4 (28%). Because signaling through these pathways leads to Bcl-xL induction, we examined Bcl-2 family expression in WM patients and cell lines. Unlike other B-lymphocyte-derived malignancies, which become dependent on expression of anti-apoptotic proteins to counter expression of pro-apoptotic proteins, WM samples expressed both pro- and anti-apoptotic Bcl-2 proteins at low levels similar to their normal B-cell and plasma cell counterparts. Three WM cell lines expressed pro-apoptotic Bcl-2 family members Bim or Bax and Bak at low levels, which determined their sensitivity to inducers of intrinsic apoptosis. In two cell lines, miR-155 upregulation, which is common in WM, was responsible for the inhibition of FOXO3a and Bim expression. Both antagonizing miR-155 to induce Bim and proteasome inhibition increased the sensitivity to ABT-737 in these lines indicating a lowering of the apoptotic threshold. In this manner, treatments that increase pro-apoptotic protein expression increase the efficacy of agents treated in combination in addition to direct killing.

Topics: Apoptosis; Apoptosis Regulatory Proteins; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Bcl-2-Like Protein 11; Biphenyl Compounds; Bortezomib; Cell Line, Tumor; Forkhead Box Protein O3; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Humans; Membrane Proteins; MicroRNAs; Multiple Myeloma; Nitrophenols; Piperazines; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Sulfonamides; Waldenstrom Macroglobulinemia

The ABT-analogous 737, 263 and 199 are BH3 mimetics showing potent anti-myeloma (MM) activity, but only on defined molecular subgroups of MM patients presenting a Bcl-2high/Mcl-1low profile. IGF-1 is a major survival factor in MM regulating the expression of Bcl-2 proteins and might therefore be a resistance factor to these ABT-analogous. We first show that IGF-1 protected human MM cell lines (HMCLs) against ABT-737. Concurrently, the IGF-1 receptor inhibitor picropodophyllin (PPP) synergistically sensitized HMCL, primary human MM and murine 5T33MM cells to ABT-737 and ABT-199 by further decreasing cell viability and enhancing apoptosis. Knockdown of Bcl-2 by shRNA protected MM cells to ABT-737, while Mcl-1 shRNA sensitized the cells. PPP overcame the Bcl-2 dependency of ABT-737, but failed to completely overcome the protective effect of Mcl-1. In vivo, co-treatment of 5T33MM bearing mice significantly decreased tumor burden and prolonged overall survival both in a prophylactic and therapeutic setting. Interestingly, proteasome inhibitor resistant CD138- 5T33MM cells were more sensitive to ABT-737, whereas PPP alone targeted the CD138+ cells more effectively. After co-treatment, both subpopulations were targeted equally. Together, the combination of an IGF-1R inhibitor and an ABT-analogue displays synergistic anti-myeloma activity providing the rational for further (pre)clinical testing.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Biomimetic Materials; Biphenyl Compounds; Cell Line, Tumor; Drug Synergism; Humans; Mice; Mice, Inbred C57BL; Multiple Myeloma; Nitrophenols; Piperazines; Podophyllotoxin; Receptor, IGF Type 1; Sulfonamides

Intracellular proteolytic pathways have been validated as rational targets in multiple myeloma with the approval of two proteasome inhibitors in this disease, and with the finding that immunomodulatory agents work through an E3 ubiquitin ligase containing Cereblon. Another E3 ligase that could be a rational target is the murine double minute (MDM) 2 protein, which plays a role in p53 turnover. A novel inhibitor of this complex, MI-63, was found to induce apoptosis in p53 wild-type myeloma models in association with activation of a p53-mediated cell death program. MI-63 overcame adhesion-mediated drug resistance, showed anti-tumor activity in vivo, enhanced the activity of bortezomib and lenalidomide, and also overcame lenalidomide resistance. In mutant p53 models, inhibition of MDM2 with MI-63 also activated apoptosis, albeit at higher concentrations, and this was associated with activation of autophagy. When MI-63 was combined with the BH3 mimetic ABT-737, enhanced activity was seen in both wild-type and mutant p53 models. Finally, this regimen showed efficacy against primary plasma cells from patients with newly diagnosed and relapsed/refractory myeloma. These findings support the translation of novel MDM2 inhibitors both alone, and in combination with other novel agents, to the clinic for patients with multiple myeloma.

Topics: Animals; Apoptosis; Autophagy; Biphenyl Compounds; Cell Line, Tumor; Drug Screening Assays, Antitumor; Drug Synergism; Indoles; Mice, Inbred NOD; Mice, SCID; Multiple Myeloma; Mutation; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-mdm2; Spiro Compounds; Sulfonamides; Tumor Suppressor Protein p53

Multiple myeloma (MM) is an incurable malignancy with an unmet need for innovative treatment options. Histone deacetylase inhibitors (HDACi) are a new class of anticancer agent that have demonstrated activity in hematological malignancies. Here, we investigated the efficacy and safety of HDACi (vorinostat, panobinostat, romidepsin) and novel combination therapies using in vitro human MM cell lines and in vivo preclinical screening utilizing syngeneic transplanted Vk*MYC MM. HDACi were combined with ABT-737, which targets the intrinsic apoptosis pathway, recombinant human tumour necrosis factor-related apoptosis-inducing ligand (rhTRAIL/MD5-1), that activates the extrinsic apoptosis pathway or the DNA methyl transferase inhibitor 5-azacytidine. We demonstrate that in vitro cell line-based studies provide some insight into drug activity and combination therapies that synergistically kill MM cells; however, they do not always predict in vivo preclinical efficacy or toxicity. Importantly, utilizing transplanted Vk*MYC MM, we report that panobinostat and 5-azacytidine synergize to prolong the survival of tumor-bearing mice. In contrast, combined HDACi/rhTRAIL-based strategies, while efficacious, demonstrated on-target dose-limiting toxicities that precluded prolonged treatment. Taken together, our studies provide evidence that the transplanted Vk*MYC model of MM is a useful screening tool for anti-MM drugs and should aid in the prioritization of novel drug testing in the clinic.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Azacitidine; Biphenyl Compounds; Drug Screening Assays, Antitumor; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Indoles; Mice; Mice, Inbred C57BL; Mice, Transgenic; Multiple Myeloma; Nitrophenols; Panobinostat; Piperazines; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; Receptors, Death Domain; Recombinant Proteins; Sulfonamides; TNF-Related Apoptosis-Inducing Ligand

Dependence on Bcl-2 proteins is a common feature of cancer cells and provides a therapeutic opportunity. ABT-737 is an antagonist of antiapoptotic Bcl-2 proteins and therefore is a good predictor of Bcl-x(L)/Bcl-2 dependence. Surprisingly, analysis of Mcl-1-dependent multiple myeloma cell lines revealed codependence on Bcl-2/Bcl-x(L) in half the cells tested. Codependence is not predicted by the expression level of antiapoptotic proteins, rather through interactions with Bim. Consistent with these findings, acquired resistance to ABT-737 results in loss of codependence through redistribution of Bim to Mcl-1. Overall, these results suggest that complex interactions, and not simply expression patterns of Bcl-2 proteins, need to be investigated to understand Bcl-2 dependence and how to better use agents, such as ABT-737.

Topics: Antineoplastic Agents; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; bcl-X Protein; Biphenyl Compounds; Cell Line, Tumor; Cell Survival; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; Membrane Proteins; Multiple Myeloma; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Piperazines; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; RNA, Small Interfering; Sulfonamides; Tissue Distribution

The 3-Deazaneplanocin A (DZNep), one of S-adenosylhomocysteine (AdoHcy) hydrolase inhibitors, has shown antitumor activities in a broad range of solid tumors and acute myeloid leukemia. Here, we examined its effects on multiple myeloma (MM) cells and found that, at 500 nM, it potently inhibited growth and induced apoptosis in 2 of 8 MM cell lines. RNA from un-treated and DZNep treated cells was profiled by Affymetrix HG-U133 Plus 2.0 microarray and genes with a significant change in gene expression were determined by significance analysis of microarray (SAM) testing. ALOX5 was the most down-regulated gene (5.8-fold) in sensitive cells and was expressed at low level in resistant cells. The results were corroborated by quantitative RT-PCR. Western-blot analysis indicated ALOX5 was highly expressed only in sensitive cell line H929 and greatly decreased upon DZNep treatment. Ectopic expression of ALOX5 reduced sensitivity to DZNep in H929 cells. Furthermore, down-regulation of ALOX5 by RNA interference could also induce apoptosis in H929. Gene expression analysis on MM patient dataset indicated ALOX5 expression was significantly higher in MM patients compared to normal plasma cells. We also found that Bcl-2 was overexpressed in DZNep insensitive cells, and cotreatment with DZNep and ABT-737, a Bcl-2 family inhibitor, synergistically inhibited growth and induced apoptosis of DZNep insensitive MM cells. Taken together, this study shows one of mechanisms of the DZNep efficacy on MM correlates with its ability to down-regulate the ALOX5 levels. In addition, DZNep insensitivity might be associated with overexpression of Bcl-2, and the combination of ABT-737 and DZNep could synergistically induced apoptosis. These results suggest that DZNep may be exploited therapeutically for a subset of MM.

Topics: Adenosine; Apoptosis; Arachidonate 5-Lipoxygenase; Biphenyl Compounds; Caspases; Cell Line, Tumor; Cell Proliferation; DNA-Binding Proteins; Drug Screening Assays, Antitumor; Drug Synergism; Enhancer of Zeste Homolog 2 Protein; Gene Expression Regulation, Neoplastic; Humans; Multiple Myeloma; Nitrophenols; Piperazines; Polycomb Repressive Complex 2; Sulfonamides; Transcription Factors

Multiple myeloma is a plasma cell malignancy that is heterogeneous with respect to its causative molecular abnormalities and the treatment response of patients. The Bcl-2 protein family is critical for myeloma cell survival. ABT-737 is a cell-permeant compound that binds to Bcl-2 and Bcl-x(L) but not to Mcl-1. Using a myeloma cell line collection (n = 25) representative of different molecular translocations, we showed that ABT-737 effectively kills a subset of cell lines (n = 6), with a median lethal dose ranging from 7 ± 0.4 nM to 150 ± 7.5 nM. Of interest, all sensitive cell lines harbored a t(11;14). We demonstrated that ABT-737-sensitive and ABT-737-resistant cell lines could be differentiated by the BCL2/MCL1 expression ratio. A screen of a public expression database of myeloma patients indicates that the BCL2/MCL1 ratio of t(11;14) and hyperdiploid patients was significantly higher than in all other groups (P < .001). ABT-737 first induced the disruption of Bcl-2/Bax, Bcl-2/Bik, or Bcl-2/Puma complexes, followed by the disruption of Bcl-2 heterodimers with Bak and Bim. Altogether, the identification of a subset of cell lines and primary cells effectively killed by ABT-737 alone supported the evaluation of ABT-263, an orally active counterpart to ABT-737, for the treatment of t(11;14) and hyperdiploid groups of myeloma harboring a Bcl-2(high)/Mcl-1(low) profile.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Biphenyl Compounds; Cell Line, Tumor; Cyclin D1; Gene Expression Regulation, Neoplastic; Humans; Multiple Myeloma; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Sulfonamides; Tumor Cells, Cultured

Signaling through the receptor/transcriptional regulator Notch plays an important role in tumor cell survival. Recent studies have demonstrated that pharmacological inhibition of the Notch pathway with γ-secretase inhibitor (GSI) induces apoptosis of multiple myeloma (MM) cells via upregulation of the proapoptotic protein Noxa. ABT-737, a novel BH3 mimetic, was shown to block Bcl-2 and Bcl-xL and induce MM cell apoptosis. Here, we investigated whether the inhibition of Notch signaling could enhance the proapoptotic effect of ABT-737. The antimyeloma effect of ABT-737 on MM cell lines or primary cells was substantially increased by the addition of Notch inhibitor. The synergistic effect of the GSI+ABT-737 combination was mediated by activation of Bak and Bax and release of cytochrome c. While toxic for MM cells, the combination of GSI and ABT-737 did not affect survival of peripheral blood mononuclear cells isolated from healthy donors. In vivo experiments using xenograft and SCID-hu models of MM demonstrated a significant antitumor effect of the GSI/ABT-737 combination as compared to the effect of Notch or Bcl-2/Bcl-xL inhibitors alone. Thus, this drug combination may be therapeutically beneficial for patients with MM.

Topics: Amyloid Precursor Protein Secretases; Animals; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-X Protein; Biphenyl Compounds; Cell Line, Tumor; Cell Survival; Drug Synergism; Enzyme Inhibitors; Humans; Mice; Multiple Myeloma; Nitrophenols; Oligopeptides; Piperazines; Receptors, Notch; Signal Transduction; Sulfonamides; Xenograft Model Antitumor Assays

The Bcl-2 antagonist ABT-737 kills transformed cells in association with displacement of Bim from Bcl-2. The histone deactetylase (HDAC) inhibitor suberoyl bis-hydroxamic acid (SBHA) was employed to determine whether and by what mechanism ABT-737 might interact with agents that upregulate Bim. Expression profiling of BH3-only proteins indicated that SBHA increased Bim, Puma, and Noxa expression, while SBHA concentrations that upregulated Bim significantly potentiated ABT-737 lethality. Concordance between SBHA-mediated Bim upregulation and interactions with ABT-737 was observed in various human leukemia and myeloma cells. SBHA-induced Bim was largely sequestered by Bcl-2 and Bcl-x(L), rather than Mcl-1; ABT-737 attenuated these interactions, thereby triggering Bak/Bax activation and mitochondrial outer membrane permeabilization. Knockdown of Bim (but not Puma or Noxa) by shRNA or ectopic overexpression of Bcl-2, Bcl-x(L), or Mcl-1 diminished Bax/Bak activation and apoptosis. Notably, ectopic expression of these antiapoptotic proteins disabled death signaling by sequestering different proapoptotic proteins, i.e., Bim by Bcl-2, both Bim and Bak by Bcl-x(L), and Bak by Mcl-1. Together, these findings indicate that HDAC inhibitor-inducible Bim is primarily neutralized by Bcl-2 and Bcl-x(L), thus providing a mechanistic framework by which Bcl-2 antagonists potentiate the lethality of agents, such as HDAC inhibitors, which upregulate Bim.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; bcl-X Protein; Biphenyl Compounds; Cell Line, Tumor; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Leukemia; Membrane Proteins; Multiple Myeloma; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Piperazines; Protein Binding; Protein Transport; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; RNA Interference; Sulfonamides; Up-Regulation

Bcl-2 or Bcl-X(L) confers resistance to chemotherapy in multiple myeloma (MM). Here we characterized the effects of ABT-737, a potent small-molecule inhibitor of antiapoptotic proteins Bcl-2, Bcl-X(L) and Bcl-w with markedly higher affinity than previously reported compounds, on human MM cells. ABT-737 induces apoptosis in MM cells, including those resistant to conventional therapy. Examination of purified patient MM cells demonstrated similar results, without significant toxicity against normal peripheral blood mononuclear cells and MM bone marrow stromal cells. Importantly, ABT-737 decreases the viability of bortezomib-, dexamethasone-(Dex) and thalidomide-refractory patient MM cells. Additionally, ABT-737 abrogates MM cell growth triggered by interleukin-6 or insulin-like growth factor-1. Mechanistic studies show that ABT-737-induced apoptosis is associated with activation of caspase-8, caspase-9 and caspase-3, followed by poly(ADP-ribose) polymerase cleavage. Combining ABT-737 with proteasome inhibitor bortezomib, melphalan or dexamethasone induces additive anti-MM activity. Taken together, our study provides the rationale for clinical protocols evaluating ABT-737, alone and together with botezomib, mephalan or dexamethasone, to enhance MM cell killing, overcome drug resistance conferred by Bcl-2 and improve patient outcome in MM.

Topics: Antineoplastic Agents; Apoptosis Regulatory Proteins; bcl-X Protein; Biphenyl Compounds; Bone Marrow Cells; Boronic Acids; Bortezomib; Cell Division; Cell Survival; Humans; Multiple Myeloma; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Pyrazines; Sulfonamides; Tumor Cells, Cultured

The aim of this study is to investigate the antimyeloma activity of a novel Bcl-2 family inhibitor, ABT-737, in preclinical treatment of multiple myeloma.. The antimyeloma activity of ABT-737 was evaluated in cultured myeloma cell lines and patient myeloma samples, and in a xenograft mouse myeloma model. Drug combination therapy using ABT-737 with other commonly used myeloma drugs was also investigated.. MY5 and JJN3 cell lines exhibited the most sensitivity to ABT-737 with an EC(50) of 0.2 and 0.5 micromol/L, respectively, with increased cell apoptosis and elevated activated caspase-3. We identified two distinct groups of myeloma patient samples that were either sensitive or resistant to the drug. Four of 15 patient bone marrow samples (27%) were highly sensitive to ABT-737 at doses of 0.25 and 0.5 micromol/L, which eliminated 80% to 90% of myeloma cells as a result of cellular apoptosis 3 days after drug treatment. ABT-737 showed a synergistic effect when combined with dexamethasone or melphalan in inducing myeloma cell death. Furthermore, the dexamethasone-resistant MM1(Dex)R myeloma cell line was highly sensitive to 0.2 micromol/L ABT-737. As determined by colony assay, little or no detectable toxicity to patient hematologic progenitor cells was observed at 1 micromol/L ABT-737. ABT-737 dose dependently suppressed tumor growth in a xenograft MY5 mouse model.. These studies show substantial antimyeloma activity of ABT-737 as a single agent or in combination with dexamethasone or melphalan and suggest a rationale for future clinical trials.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Biphenyl Compounds; Cell Line, Tumor; Cell Survival; Dexamethasone; Drug Synergism; Humans; Interleukin-6; Melphalan; Mice; Multiple Myeloma; Neoplasm Transplantation; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Stromal Cells; Sulfonamides; Tumor Cells, Cultured

Disruption of pathways leading to programmed cell death plays a major role in most malignancies, including multiple myeloma (MM). ABT-737 is a BH3 mimetic small-molecule inhibitor that binds with high affinity to Bcl-2 and Bcl-xL, preventing the sequestration of proapoptotic molecules and shifting the cell survival/apoptosis balance toward apoptosis induction. In this study, we show that ABT-737 is cytotoxic to MM cell lines, including those resistant to conventional therapies, and primary tumor cells. Flow cytometric analysis of intracellular levels of Bcl-2 family proteins demonstrates a clear inversion of the Bax/Bcl-2 ratio leading to induction of apoptosis. Activation of the mitochondrial apoptosis pathway was indicated by mitochondrial membrane depolarization and caspase cleavage. Additionally, several signaling pathways known to be important for MM cell survival are disrupted following treatment with ABT-737. The impact of ABT-737 on survival could not be overcome by the addition of interleukin-6, vascular endothelial growth factor or insulin-like growth factor, suggesting that ABT-737 may be effective in preventing the growth and survival signals provided by the microenvironment. These data indicate that therapies targeting apoptotic pathways may be effective in MM treatment and warrant clinical evaluation of ABT-737 and similar drugs alone or in combination with other agents in the setting of MM.

Topics: Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Biphenyl Compounds; Caspases; Cell Line; Cells, Cultured; Flow Cytometry; Humans; Intercellular Signaling Peptides and Proteins; Multiple Myeloma; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Sulfonamides

Topics: Alkaline Phosphatase; Cysteamine; Hodgkin Disease; Humans; Infectious Mononucleosis; Intestines; Kinetics; Leukemia; Lymphoma; Lymphoproliferative Disorders; Multiple Myeloma; Nitrophenols; Organophosphorus Compounds; Placenta

Topics: Animals; Antibody Formation; Antibody Specificity; Cell Fusion; Cell Line; Genes; Haptens; Hybrid Cells; Immunoglobulin gamma-Chains; Immunoglobulin lambda-Chains; Immunoglobulin mu-Chains; Immunoglobulin Variable Region; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Multiple Myeloma; Nitrophenols

Topics: Antibody Specificity; Binding Sites, Antibody; Erythrocytes; Haptens; Hemagglutination Tests; Heparin; Humans; Hypergammaglobulinemia; Immunodiffusion; Immunoglobulin G; Immunoglobulin kappa-Chains; Immunoglobulin M; Immunoglobulins; Multiple Myeloma; Nitrophenols; Paraproteinemias; Phospholipids; Polysaccharides; Waldenstrom Macroglobulinemia

Topics: Animals; Antibody Formation; Cattle; Erythrocytes; Freund's Adjuvant; gamma-Globulins; Goats; Haptens; Hemolytic Plaque Technique; Immune Sera; Immunoglobulin A; Immunoglobulin Fragments; Immunoglobulin M; Ligands; Mice; Mice, Inbred BALB C; Multiple Myeloma; Myeloma Proteins; Neoplasms, Experimental; Nitrophenols; Rabbits; Sheep; Spleen; Trypsin