leupeptins and Multiple-Myeloma

The proteasome, which plays a pivotal role in the control of many cell cycle-regulatory processes, has become the focus of new approaches to the treatment of cancer, including B-cell malignancies, and the first proteasome inhibitor, bortezomib (VELCADE; formerly PS-341), has entered clinical trials. The proteasome controls the stability of numerous proteins that regulate progression through the cell cycle and apoptosis, such as cyclins, cyclin-dependent kinases, tumor suppressors, and the nuclear factor-kB. By altering the stability or activity of these proteins, proteasome inhibitors sensitize malignant cells to apoptosis. Bortezomib is a dipeptidyl boronic acid proteasome inhibitor that effectively and specifically inhibits proteasome activity. In preclinical studies, bortezomib and other proteasome inhibitors have shown activity against a variety of B-cell malignancies, including multiple myeloma, diffuse large B-cell lymphoma, mantle cell lymphoma, and Hodgkin's lymphoma. These agents can induce apoptosis and sensitize tumor cells to radiation or chemotherapy. Based on these findings, phase I clinical trials were conducted with bortezomib in various solid and hematologic malignancies. In these studies, bortezomib was generally well tolerated with manageable toxicities. Phase II trials have been initiated for relapsed and refractory multiple myeloma, refractory chronic lymphocytic leukemia, and non-Hodgkin's lymphoma. Preliminary data from the multiple myeloma phase II study indicate that a significant number of patients responded to therapy or exhibited stable disease and that the drug had manageable toxicities. These findings, along with extensive preclinical data, suggest that bortezomib and other proteasome inhibitors may have far-reaching potential in the treatment of various cancers, including B-cell malignancies.

Topics: Acetylcysteine; Animals; Boronic Acids; Bortezomib; Cell Cycle Proteins; Clinical Trials as Topic; Drug Screening Assays, Antitumor; Enzymes; Gene Expression Regulation; Hodgkin Disease; Humans; Leukemia, B-Cell; Leupeptins; Lymphoma, B-Cell; Lymphoma, Large B-Cell, Diffuse; Lymphoma, Mantle-Cell; Mice; Multiple Myeloma; Neoplasm Proteins; NF-kappa B; Oncogene Proteins; Peptide Hydrolases; Protease Inhibitors; Proteasome Endopeptidase Complex; Pyrazines; Substrate Specificity; Transcription Factors; Treatment Outcome

Proteasome inhibitors are an important class of chemotherapeutic drugs. In this study, we performed a large-scale ubiquitylome analysis of the three proteasome inhibitors MG132, bortezomib and carfilzomib. Although carfilzomib is currently being used for the treatment of multiple myeloma, it has not yet been subjected to a global ubiquitylome analysis. In this study, we identified more than 14,000 unique sites of ubiquitylation in more than 4400 protein groups. We introduced stringent criteria to determine the correct ubiquitylation site ratios and used five biological replicates to achieve increased statistical power. With the vast amount of data acquired, we made proteome-wide comparisons between the proteasome inhibitors and indicate candidate proteins that will benefit from further study. We find that in addition to the expected increase in ubiquitylation in the majority of proteins, unexpectedly a select few are specifically and significantly decreased in ubiquitylation at specific sites after treatment with proteasome inhibitors. We chose to follow-up on Mortality factor 4-like 1 (MORF4L1), which was significantly decreased in ubiquitylation at lysine 187 and lysine 104 upon proteasome inhibition, but increased in protein abundance by approximately two-fold. We demonstrate that the endogenous protein level of MORF4L1 is highly regulated by the ubiquitin proteasome system. SIGNIFICANCE: This study provides a highly curated dataset of more than 14,000 unique sites of ubiquitylation in more than 4400 protein groups. For the proper quantification of ubiquitylation sites, we introduced a higher standard by quantifying only those ubiquitylation sites that are not flanked by neighboring ubiquitylation, thereby avoiding the report of incorrect ratios. The sites identified will serve to identify important targets of the ubiquitin proteasome system and aid to better understand the repertoire of proteins that are affected by inhibiting the proteasome with MG132, bortezomib, and carfilzomib. In addition, we investigated the unusual observation that ubiquitylation of the tumor suppressor Mortality factor 4-like (MORF4L1) protein decreases rather than increases upon proteasome inhibition, which may contribute to an additional anti-tumor effect of bortezomib and carfilzomib.

Topics: Antineoplastic Agents; Bortezomib; Humans; Leupeptins; Multiple Myeloma; Oligopeptides; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proteomics; Ubiquitination

The importance of telomerase, the enzyme that maintains telomere length, has been reported in many malignancies in general and in multiple myeloma (MM) in particular. Proteasome inhibitors are clinically used to combat effectively MM. Since the mechanism of action of proteasome inhibitors has not been fully described we sought to clarify its potential effect on telomerase activity (TA) in MM cells. Previously we showed that the first generation proteasome inhibitor bortezomib (Brt) inhibits TA in MM cells by both transcriptional and post-translational mechanisms and has a potential clinical significance. In the current study we focused around the anti- telomerase activity of the new generation of proteasome inhibitors, epoxomicin (EP) and MG-132 in order to clarify whether telomerase inhibition represents a class effect. We have exposed MM cell lines, ARP-1, CAG, RPMI 8226 and U266 to EP or MG and the following parameters were assessed: viability; TA, hTERT expression, the binding of hTERT (human telomerase reverse transcriptase) transcription factors and post-translational modifications. Epoxomicin and MG-132 differentially downregulated the proliferation and TA in all MM cell lines. The downregulation of TA and the expression of hTERT were faster in CAG than in ARP-1 cells. Epoxomicin was more potent than MG-132 and therefore further mechanistic studies were performed using this compound. The inhibition of TA was mainly transcriptionally regulated. The binding of three positive regulator transcription factors: SP1, c-Myc and NF-κB to the hTERT promoter was decreased by EP in CAG cells as well as their total cellular expression. In ARP-1 cells the SP1 and c-MYC binding and protein levels were similarly affected by EP while NF-κB was not affected. Interestingly, the transcription factor WT-1 (Wilms' tumor-1) exhibited an increased binding to the hTERT promoter while its total cellular amount remained unchanged. Our results combined with our previous study of bortezomib define telomerase as a general target for proteasome inhibitors. The inhibitory effect of TA is exerted by several regulatory levels, transcriptional and post translational. SP1, C-Myc and NF-κB were involved in mediating these effects. A novel finding of this study is the role of WT-1 in the regulation of telomerase which appears as a negative regulator of hTERT expression. The results of this study may contribute to future development of telomerase inhibition as a therapeutic modality in

Topics: Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; Leupeptins; Multiple Myeloma; NF-kappa B; Oligopeptides; Promoter Regions, Genetic; Proteasome Inhibitors; Sp1 Transcription Factor; Telomerase; Transcription, Genetic

The immunomodulatory drugs (IMiDs) thalidomide and its analogs, lenalidomide and pomalidomide, all FDA approved drugs for the treatment of multiple myeloma, induce ubiquitination and degradation of the lymphoid transcription factors Ikaros (IKZF1) and Aiolos (IKZF3) via the cereblon (CRBN) E3 ubiquitin ligase for proteasomal degradation. IMiDs have recently been utilized for the generation of bifunctional proteolysis targeting chimeras (PROTACs) to target other proteins for ubiquitination and proteasomal degradation by the CRBN E3 ligase. We designed and synthesized pomalidomide-based homobifunctional PROTACs and analyzed their ability to induce self-directed ubiquitination and degradation of CRBN. Here, CRBN serves as both, the E3 ubiquitin ligase and the target at the same time. The homo-PROTAC compound 8 degrades CRBN with a high potency with only minimal remaining effects on IKZF1 and IKZF3. CRBN inactivation by compound 8 had no effect on cell viability and proliferation of different multiple myeloma cell lines. This homo-PROTAC abrogates the effects of IMiDs in multiple myeloma cells. Therefore, our homodimeric pomalidomide-based compounds may help to identify CRBN's endogenous substrates and physiological functions and investigate the molecular mechanism of IMiDs.

Topics: Adaptor Proteins, Signal Transducing; Carbon-13 Magnetic Resonance Spectroscopy; Cell Line, Tumor; Cell Survival; Humans; Leupeptins; Multiple Myeloma; Protein Multimerization; Proteolysis; Proton Magnetic Resonance Spectroscopy; Thalidomide; Ubiquitin-Protein Ligases

Phosphoinositide‑dependent kinase 1 (PDK1) is generally active in multiple myeloma (MM) and higher expression than other hematopoietic cells, which is associated with the drug resistance and the disease progression. Previous studies have demonstrated that PDK1 can be targeted therapeutically in MM. In the present study, we examined the combination effect of GSK2334470 (GSK‑470), a novel and highly specific inhibitor of PDK1, with proteasome inhibitor MG‑132 in MM cell lines. GSK‑470 monotherapy significantly inhibited growth of MM cell lines and induced apoptosis that was associated with the activation of both the intrinsic mitochondrial pathway and the extrinsic death receptor pathway. Moreover, GSK‑470 demonstrated synergistic growth inhibitory effects with MG‑132. Notably, treatment with these inhibitors resulted in an almost complete inhibition of phosphorylation of mammalian target of rapamycin on Ser2448 and Ser2481 and full activation of AKT. The combination therapy also caused an upregulation of PTEN and an increased nuclear accumulation of PTEN protein. Collectively, our results provide the rationale for novel combination treatment with PDK1 inhibitor and proteasome inhibitors to improve outcomes in patients with MM.

Topics: Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Cell Survival; Drug Synergism; Gene Expression Regulation, Neoplastic; Humans; Indazoles; Leupeptins; Multiple Myeloma; Phosphorylation; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Pyrimidines; TOR Serine-Threonine Kinases

In hematology there has recently been increasing interest in inorganic polyphosphate. This polymer accumulates in platelet granules and its functions include modulating various stages of blood coagulation, inducing angiogenesis, and provoking apoptosis of plasma cells. In this study we evaluated the characteristics of intracellular polyphosphate in myeloma cell lines, in primary myeloma cells from patients, and in other human B-cell populations from healthy donors.. We have developed a novel flow cytometric method for detecting levels of polyphosphate in cell populations. We also used confocal microscopy and enzymatic analysis to study polyphosphate localization and characteristics.. We found that myeloma plasma cells contain higher levels of intracellular polyphosphate than normal plasma cells and other B-cell populations. Localization experiments indicated that high levels of polyphosphate accumulate in the nucleolus of myeloma cells. As the principal function of the nucleolus involves transcription of ribosomal DNA genes, we found changes in the cellular distribution of polyphosphate after the inhibition of nucleolar transcription. In addition, we found that RNA polymerase I activity, responsible for transcription in the nucleolus, is also modulated by polyphosphate, in a dose-dependent manner.. Our results show an unusually high accumulation of polyphosphate in the nucleoli of myeloma cells and a functional relationship of this polymer with nucleolar transcription.

Topics: Antineoplastic Agents; B-Lymphocytes; Biological Transport; Cell Line, Tumor; Cell Nucleolus; Humans; Leupeptins; Multiple Myeloma; Plasma Cells; Polyphosphates; RNA Polymerase I; Transcription, Genetic

Multiple myeloma (MM) is an incurable B lymphocyte cancer. To date, a comparative analysis of global protein metabolism for the MM cell line CCL-155 (RPMI-8226) and the non-cancerous B lymphocyte cell line CCL-156 (RPMI‑1788) has not been published. Here, we report that both global protein synthesis and degradation occur at a higher rate in MM cells and demonstrate that alkylating agents can reduce global protein degradation in both cell lines, but the effect is greater in CCL-156 cells. Treatment with melphalan plus the proteasome inhibitor MG132 reduced global protein degradation for MM cells to roughly 60% of that seen without drugs, but the reduction was approximately three times greater for CCL-156 cells. This drug combination was growth inhibitory for both cell lines, but CCL-156 inhibition was 2-fold greater than that of the MM cell line. Additionally, treatment with melphalan plus the lysosomal inhibitor chloroquine did not affect growth of MM cells more than melphalan alone, whereas this combination drastically inhibited growth of CCL-156 cells despite protein degradation being maintained at 60% level for both cell lines. This suggests that a lysosomal function other than protein degradation is required for recovery from alkylation damage in CCL-156 cells. In general, CCL-156 cells were affected to a greater extent for both protein degradation and growth inhibition with most drug combinations tested. Statistical analysis of our data (p=0.066) provides evidence that aberrant proteasome-mediated protein degradation correlates with chemoresistance in MM cells, but that lysosome-mediated protein degradation does not.

Topics: Antineoplastic Agents; B-Lymphocytes; Cell Line, Tumor; Cell Proliferation; Chloroquine; Drug Resistance, Neoplasm; Humans; Leupeptins; Lysosomes; Male; Melphalan; Multiple Myeloma; Neoplasm Proteins; Proteasome Endopeptidase Complex; Proteins; Proteolysis

Proteasome inhibitors (PIs) are effective against multiple myeloma (MM), but the mechanisms of action and bases of individual susceptibility remain unclear. Recent work linked PI sensitivity to protein synthesis and proteasome activity, raising the question whether different levels of proteasome expression and workload underlie PI sensitivity in MM cells (MMCs). Exploiting human MM lines characterized by differential PI sensitivity, we report that highly sensitive MMCs express lower proteasome levels and higher proteasomal workload than relatively PI-resistant MMCs, resulting in the accumulation of polyubiquitinated proteins at the expense of free ubiquitin (proteasome stress). Manipulating proteasome expression or workload alters apoptotic sensitivity to PI, demonstrating a cause-effect relationship between proteasome stress and apoptotic responses in MMCs. Intracellular immunostaining in primary, patient-derived MMCs reveals that polyubiquitinated proteins hallmark neoplastic plasma cells, in positive correlation with immunoglobulin (Ig) content, both intra- and interpatient. Moreover, overall proteasome activity of primary MMCs inversely correlates with apoptotic sensitivity to PI. Altogether, our data indicate that the balance between proteasome workload and degradative capacity represents a critical determinant of apoptotic sensitivity of MMCs to PI, potentially providing a framework for identifying indicators of responsiveness and designing novel combination therapies.

Topics: Animals; Apoptosis; B-Lymphocytes; Cell Differentiation; Cysteine Proteinase Inhibitors; Drug Resistance, Neoplasm; Endoplasmic Reticulum; Enzyme Activation; HeLa Cells; Humans; Leupeptins; Mice; Multiple Myeloma; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Stress, Physiological; Tumor Cells, Cultured

The proteasome inhibitor bortezomib (Velcade) effectively eradicates multiple myeloma (MM) cells, partly by activating endoplasmic reticulum (ER) stress apoptotic signaling. However, MM recurrences in bortezomib-treated patients are invariable. We have shown that ER stress signaling can also induce growth arrest and survival in cancer cells. Thus, we hypothesized that bortezomib therapy could induce quiescence and survival of residual MM cells, contributing to disease recurrence. Here, we report that in MM cells, proteasome inhibition with MG-132 or bortezomib results in a surviving cell fraction that enters a prolonged quiescent state (G(0)-G(1) arrest). Mechanism analysis revealed that bortezomib-surviving quiescent cells attenuate eIF2alpha phosphorylation and induction of the ER stress proapoptotic gene GADD153. This occurs independently of the eIF2alpha upstream kinases PERK, GCN2, and PKR. In contrast, the prosurvival ER-chaperone BiP/Grp78 was persistently induced. The bortezomib-surviving quiescent fraction could be eradicated by a simultaneous or sequential combination therapy with salubrinal, an inhibitor of GADD34-PP1C phosphatase complex, and, in consequence, eIF2alpha dephosphorylation. This effect was mimicked by expression of a phosphorylated mimetic eIF2alpha-S51D mutant. Our data indicate that bortezomib can induce growth arrest in therapy-surviving MM cells and that attenuation of eIF2alpha phosphorylation contributes to this survival. Most importantly, this survival mechanism can be blocked by inhibiting eIF2alpha dephosphorylation. Thus, strategies that maintain eIF2alpha in a hyperphosphorylated state may be a novel therapeutic approach to maximize bortezomib-induced apoptosis and reduce residual disease and recurrences in this type of cancer.

Topics: Antineoplastic Agents; Boronic Acids; Bortezomib; Cell Cycle; Cell Death; Cell Survival; Endoplasmic Reticulum Chaperone BiP; Eukaryotic Initiation Factor-2; G1 Phase; Humans; Leupeptins; Multiple Myeloma; Phosphorylation; Pyrazines; Resting Phase, Cell Cycle

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

Topics: Administration, Oral; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; bcl-X Protein; Boronic Acids; Bortezomib; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Down-Regulation; Enzyme Activation; Heat-Shock Proteins; HSP27 Heat-Shock Proteins; Humans; In Vitro Techniques; Indoles; Injections, Intravenous; Leupeptins; Mice; Mice, Nude; Mitogen-Activated Protein Kinase 14; Molecular Chaperones; Multiple Myeloma; Neoplasm Proteins; Protease Inhibitors; Pyrazines; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

Proteasome inhibitors, such as the dipeptide boronic acid bortezomib, are emerging as important tools in the treatment of the fatal hematologic malignancy multiple myeloma. Despite the recent US Food and Drug Administration approval of bortezomib (PS341, Velcade) for the treatment of refractory multiple myeloma, many of the basic pharmacologic parameters of bortezomib and its mode of action on myeloma cells remain to be determined. We describe the synthesis and use of a cell-permeant active site-directed probe, which allows profiling of proteasomal activities in living cells. When we compared proteasome activity patterns in cultured cells and crude cell extracts with this probe, we observed substantial differences, stressing the importance for bioassays compatible with live cells to ensure accuracy of such measurements. Using this probe, we investigated the in vivo subunit specificities of bortezomib and another inhibitor, MG132.

Topics: Animals; Antineoplastic Agents; Boronic Acids; Bortezomib; Cell Line, Tumor; Humans; Leupeptins; Mice; Multiple Myeloma; Protease Inhibitors; Proteasome Endopeptidase Complex; Pyrazines

In this study, we have evaluated the cytotoxic effect of combining two HDAC inhibitors, SAHA and TSA, with TRAIL in human multiple myeloma cell lines. Low doses of SAHA or TSA enhanced the cytotoxic and apoptotic effects of TRAIL and upregulated the surface expression of TRAIL death receptors (DR4 and/or DR5). SAHA and TSA induced G1 phase cell cycle growth arrest by upregulating p21(WAF1) and p27(Kip1) expression and by inhibiting E2F transcriptional activity. The enhanced TRAIL effect after pretreatment with HDAC inhibitors was consistent with the upregulation of the proapoptotic Bcl-2 family members (Bim, Bak, Bax, Noxa, and PUMA), the downregulation of the anti-apoptotic members of the Bcl-2 family (Bcl-2 and Bcl-X(L)), and IAPs. SAHA and TSA dissipated the mitochondrial membrane potential and enhanced the release of Omi/HtrA2 and AIF from the mitochondria to the cytosol. The cytotoxic effect of both SAHA and TSA was caspase- and calpain-independent. Inhibition of NF(kappa)B activation by the proteasome inhibitor, MG132, enhanced the apoptotic effect of TSA. Our study demonstrated the enhancing effects of HDAC inhibitors on apoptosis when combined with TRAIL and, for the first time, emphasized the role of AIF in mediating the cytotoxic effects of HDAC inhibitors.

Topics: Amino Acid Chloromethyl Ketones; Annexin A5; Apoptosis; Apoptosis Regulatory Proteins; Calpain; Caspases; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p27; DNA-Binding Proteins; Dose-Response Relationship, Drug; Down-Regulation; E2F Transcription Factors; Enzyme Inhibitors; Flow Cytometry; G1 Phase; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histones; Humans; Immunoblotting; Inhibitory Concentration 50; Leupeptins; Luciferases; Membrane Glycoproteins; Membrane Potentials; Microscopy, Fluorescence; Mitochondria; Multiple Myeloma; NF-kappa B; Phosphorylation; Propidium; Protein Binding; Ribonucleases; Subcellular Fractions; Time Factors; TNF-Related Apoptosis-Inducing Ligand; Transcription Factors; Tumor Necrosis Factor-alpha; Tumor Suppressor Proteins; Up-Regulation

Novel agents that target the proteasome, a proteolytic complex responsible for the degradation of ubiquitinated proteins, have demonstrated remarkable therapeutic efficacy in multiple myeloma, a plasma cell malignancy. However, the mechanism by which these compounds act remains unknown. A signaling pathway called the unfolded protein response (UPR) allows cells to handle the proper folding of proteins. The transcription factor XBP-1, a regulator of the UPR, is also required for plasma cell differentiation, suggesting a link between the UPR and plasma cell differentiation. Here we show that proteasome inhibitors target XBP-1 and the UPR in myeloma cells. Proteasome inhibitors suppress the activity of the translumenal endoplasmic reticulum endoribonuclease/kinase, IRE1 alpha, to impair the generation of the active, spliced XBP-1 species and simultaneously stabilize the unspliced species that acts as a dominant negative. Myeloma cells rendered functionally deficient in XBP-1 undergo increased apoptosis in response to endoplasmic reticulum stress. Identification of compounds that target the activity of IRE1 alpha/XBP-1 may yield novel therapies for the treatment of multiple myeloma and other malignancies that rely on an intact UPR.

Topics: 3T3 Cells; Animals; Apoptosis; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; Endoplasmic Reticulum; Endoribonucleases; Humans; Leupeptins; Membrane Proteins; Mice; Multienzyme Complexes; Multiple Myeloma; Neoplasm Proteins; Proteasome Endopeptidase Complex; Protein Folding; Protein Serine-Threonine Kinases; Regulatory Factor X Transcription Factors; RNA Splicing; Transcription Factors; Tumor Cells, Cultured; Tunicamycin; X-Box Binding Protein 1