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2.
Blood ; 141(21): 2599-2614, 2023 05 25.
Article in English | MEDLINE | ID: mdl-36630605

ABSTRACT

PSMD4/Rpn10 is a subunit of the 19S proteasome unit that is involved with feeding target proteins into the catalytic machinery of the 26S proteasome. Because proteasome inhibition is a common therapeutic strategy in multiple myeloma (MM), we investigated Rpn10 and found that it is highly expressed in MM cells compared with normal plasma cells. Rpn10 levels inversely correlated with overall survival in patients with MM. Inducible knockout or knockdown of Rpn10 decreased MM cell viability both in vitro and in vivo by triggering the accumulation of polyubiquitinated proteins, cell cycle arrest, and apoptosis associated with the activation of caspases and unfolded protein response-related pathways. Proteomic analysis revealed that inhibiting Rpn10 increased autophagy, antigen presentation, and the activation of CD4+ T and natural killer cells. We developed an in vitro AlphaScreen binding assay for high-throughput screening and identified a novel Rpn10 inhibitor, SB699551 (SB). Treating MM cell lines, leukemic cell lines, and primary cells from patients with MM with SB decreased cell viability without affecting the viability of normal peripheral blood mononuclear cells. SB inhibited the proliferation of MM cells even in the presence of the tumor-promoting bone marrow milieu and overcame proteasome inhibitor (PI) resistance without blocking the 20S proteasome catalytic function or the 19S deubiquitinating activity. Rpn10 blockade by SB triggered MM cell death via similar pathways as the genetic strategy. In MM xenograft models, SB was well tolerated, inhibited tumor growth, and prolonged survival. Our data suggest that inhibiting Rpn10 will enhance cytotoxicity and overcome PI resistance in MM, providing the basis for further optimization studies of Rpn10 inhibitors for clinical application.


Subject(s)
Multiple Myeloma , Proteasome Endopeptidase Complex , Humans , Proteasome Endopeptidase Complex/metabolism , Ubiquitin/metabolism , Multiple Myeloma/drug therapy , Multiple Myeloma/genetics , Proteomics , Leukocytes, Mononuclear/metabolism , Carrier Proteins/genetics , Proteins/metabolism , RNA-Binding Proteins
3.
J Bone Jt Infect ; 7(6): 241-248, 2022.
Article in English | MEDLINE | ID: mdl-36532293

ABSTRACT

The aim of this study was to review the available literature concerning Madura foot ("mycetoma") caused by Madurella mycetomatis in immunocompromised patients. With a systematic literature search, we identified only three papers, describing a total of three immunocompromised patients. Hence, the clinical presentation and prognosis of the disease in this patient population have not yet been well described. In addition, we present a case from our institution, illustrating the complexity of the treatment of this rare disease. Although very rare in non-endemic countries, we emphasize that mycetoma should be included in the differential diagnoses of (immunocompromised) patients who have been residing in a geographical area where the disease is endemic and presenting with soft tissue inflammation of one of the extremities.

4.
Blood Cancer J ; 12(4): 50, 2022 04 01.
Article in English | MEDLINE | ID: mdl-35365613

ABSTRACT

Interaction of plasmacytoid dendritic cells (pDCs) with multiple myeloma (MM) cells, T- or NK-effector cells in the bone marrow (BM) microenvironment induces tumor cell growth, as well as inhibits innate and adaptive immune responses. Defining pDC-MM interaction-triggered immunosuppressive mechanism(s) will enable design of interventional therapies to augment anti-MM immunity. In the present study, we show that pDC-MM interactions induce metabolic enzyme Ecto-5' Nucleotidase/CD73 in both pDCs and MM cells. Gene expression database from MM patients showed that CD73 levels inversely correlate with overall survival. Using our pDC-MM coculture models, we found that blockade of CD73 with anti-CD73 Abs: decreases adenosine levels; activates MM patient pDCs; triggers cytotoxic T lymphocytes (CTL) activity against autologous patient MM cells. Combination of anti-CD73 Abs and an immune-stimulating agent TLR-7 agonist enhances autologous MM-specific CD8+ CTL activity. Taken together, our preclinical data suggest that the therapeutic targeting of CD73, alone or in combination with TLR-7 agonist, represents a promising novel strategy to restore host anti-MM immunity.


Subject(s)
Multiple Myeloma , 5'-Nucleotidase/metabolism , 5'-Nucleotidase/therapeutic use , Dendritic Cells/metabolism , Humans , Immunotherapy , Killer Cells, Natural , Multiple Myeloma/drug therapy , Tumor Microenvironment
8.
Leukemia ; 35(2): 550-561, 2021 02.
Article in English | MEDLINE | ID: mdl-32424294

ABSTRACT

Our prior study showed that inhibition of 19S proteasome-associated ubiquitin receptor Rpn13 can overcome bortezomib resistance in MM cells. Here, we performed proteomic analysis of Rpn13 inhibitor (RA190)-treated MM cells and identified an antioxidant enzyme superoxide dismutase (SOD1) as a mediator of Rpn13 signaling. SOD1 levels are higher in MM patient cells versus normal PBMCs; and importantly, SOD1 expression correlates with the progression of disease and shorter survival. Functional validation studies show that RA190-induced cytotoxicity in bortezomib-sensitive and -resistant MM cells is associated with decrease in SOD1 levels; conversely, forced expression of SOD1 inhibits RA190-induced cell death. Genetic knockdown and biochemical blockade of SOD1 with LCS-1 sensitizes bortezomib-resistant MM cells to bortezomib. SOD1 inhibitor LCS-1 decreases viability in MM cell lines and patient cells. LCS-1-induced cell death is associated with: (1) increase in superoxide and ROS levels; (2) activation of caspases, and p53/p21 signaling; (3) decrease in MCL-1, BCLxL, CDC2, cyclin-B1, and c-Myc; (4) ER stress response; and (5) inhibition of proteasome function. In animal model studies, LCS-1 inhibits xenografted bortezomib-resistant human MM cell growth and prolongs host survival. Our studies therefore show that targeting Rpn13 overcomes bortezomib resistance by decreasing cellular SOD1 levels, and provide the rationale for novel therapeutics targeting SOD1 to improve patient outcome in MM.


Subject(s)
Biomarkers, Tumor/metabolism , Bortezomib/pharmacology , Drug Resistance, Neoplasm , Gene Expression Regulation, Neoplastic/drug effects , Intracellular Signaling Peptides and Proteins/metabolism , Multiple Myeloma/drug therapy , Proteome/analysis , Animals , Antineoplastic Agents/pharmacology , Apoptosis , Caspases/metabolism , Cell Proliferation , Female , Humans , Intracellular Signaling Peptides and Proteins/genetics , Mice , Mice, SCID , Multiple Myeloma/metabolism , Multiple Myeloma/pathology , Prognosis , Proteasome Endopeptidase Complex/drug effects , Survival Rate , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
9.
Teach Learn Med ; 33(1): 78-88, 2021.
Article in English | MEDLINE | ID: mdl-32917107

ABSTRACT

Problem: Effective clinical workplace learning depends on interprofessional and multidisciplinary learning. However, traditional patient wards are centered around patient care and not so much around education. Other barriers such as time constraints also contribute to suboptimal interprofessional and multidisciplinary learning. Intervention: Six formal and informal learning activities that aimed at stimulation of interprofessional and multidisciplinary learning were designed and introduced in our patient ward to enable optimal integration of clinical practice and learning. Context: The study took place in an internal medicine inpatient ward where daily patient care is performed by specialized teams consisting of different healthcare professionals from the departments of Endocrinology, Nephrology, and Infectious Diseases. In the traditional ward setting, interprofessional and multidisciplinary learning mostly takes place during shared clinical activities. In this article, we describe the development and implementation of a Clinical Teaching Unit to support learning between different healthcare professionals. Impact: The intervention was evaluated with an online questionnaire among 108 nurses, student nurses, clerks, residents, supervising clinicians, and managers. Open-ended questions (response rate 65%) were used to determine the changes in the workplace experienced by the participants since the introduction of the Clinical Teaching Unit and what influenced their learning process and motivation to learn. Closed questions (response rate 46%) aimed to measure the effect of our intervention on collaboration, learning, and the quality of care and education. The results of the open-ended questions showed that participants experienced more interprofessional collaboration and learning. This took place in a less hierarchical, safer work climate which also resulted in perceptions of a better quality of patient care and education. The closed-ended questions showed that the intervention resulted in perceptions of improved collaboration, work culture, quality of care, education, and learning conditions. Lessons Learned: The findings imply that implementation of a Clinical Teaching Unit not only facilitates the integration of patient care and education but also the integration of different professions working together. From the intervention, we also learned that a successful Clinical Teaching Unit requires investment of time and staff, clear communication between healthcare professionals, and dedication of teachers within all professions.


Subject(s)
Cooperative Behavior , Curriculum/standards , Interdisciplinary Communication , Internal Medicine/education , Interprofessional Relations , Professional Competence/standards , Communication , Humans , Interdisciplinary Studies , Students, Medical/statistics & numerical data
10.
Sci Rep ; 10(1): 5324, 2020 03 24.
Article in English | MEDLINE | ID: mdl-32210275

ABSTRACT

Ubiquitin specific peptidase 7 (USP7) is a deubiquitinating enzyme (DUB) that removes ubiquitin tags from specific protein substrates in order to alter their degradation rate and sub-cellular localization. USP7 has been proposed as a therapeutic target in several cancers because it has many reported substrates with a role in cancer progression, including FOXO4, MDM2, N-Myc, and PTEN. The multi-substrate nature of USP7, combined with the modest potency and selectivity of early generation USP7 inhibitors, has presented a challenge in defining predictors of response to USP7 and potential patient populations that would benefit most from USP7-targeted drugs. Here, we describe the structure-guided development of XL177A, which irreversibly inhibits USP7 with sub-nM potency and selectivity across the human proteome. Evaluation of the cellular effects of XL177A reveals that selective USP7 inhibition suppresses cancer cell growth predominantly through a p53-dependent mechanism: XL177A specifically upregulates p53 transcriptional targets transcriptome-wide, hotspot mutations in TP53 but not any other genes predict response to XL177A across a panel of ~500 cancer cell lines, and TP53 knockout rescues XL177A-mediated growth suppression of TP53 wild-type (WT) cells. Together, these findings suggest TP53 mutational status as a biomarker for response to USP7 inhibition. We find that Ewing sarcoma and malignant rhabdoid tumor (MRT), two pediatric cancers that are sensitive to other p53-dependent cytotoxic drugs, also display increased sensitivity to XL177A.


Subject(s)
Protease Inhibitors/pharmacology , Tumor Suppressor Protein p53/metabolism , Ubiquitin-Specific Peptidase 7/antagonists & inhibitors , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Humans , MCF-7 Cells , Protease Inhibitors/chemistry , Ubiquitin-Specific Peptidase 7/chemistry , Ubiquitin-Specific Peptidase 7/metabolism , Ubiquitination/drug effects
11.
Oncogene ; 39(13): 2786-2796, 2020 03.
Article in English | MEDLINE | ID: mdl-32024967

ABSTRACT

Bone marrow plasmacytoid dendritic cells (pDCs) in patients with multiple myeloma (MM) promote tumor growth, survival, drug resistance, and immune suppression. Understanding the molecular signaling crosstalk among the tumor cells, pDCs and immune cells will identify novel therapeutic approaches to enhance anti-MM immunity. Using oligonucleotide arrays, we found that pDC-MM interactions induce metabolic enzyme Alpha-Enolase (ENO1) in both pDCs and MM cells. Analysis of MM patient gene expression profiling database showed that ENO1 expression inversely correlates with overall survival. Protein expression analysis showed that ENO1 is expressed in pDC and MM cells; and importantly, that pDC-MM coculture further increases ENO1 expression in both MM cells and pDCs. Using our coculture models of patient autologous pDC-T-NK-MM cells, we examined whether targeting ENO1 can enhance anti-MM immunity. Biochemical inhibition of ENO1 with ENO1 inhibitor (ENO1i) activates pDCs, as well as increases pDC-induced MM-specific CD8+ CTL and NK cell activity against autologous tumor cells. Combination of ENO1i and anti-PD-L1 Ab or HDAC6i ACY-241 enhances autologous MM-specific CD8+ CTL activity. Our preclinical data therefore provide the basis for novel immune-based therapeutic approaches targeting ENO1, alone or in combination with anti-PD-L1 Ab or ACY241, to restore anti-MM immunity, enhance MM cytotoxicity, and improve patient outcome.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Biomarkers, Tumor/antagonists & inhibitors , Cell Communication/drug effects , DNA-Binding Proteins/antagonists & inhibitors , Dendritic Cells/drug effects , Multiple Myeloma/drug therapy , Phosphopyruvate Hydratase/antagonists & inhibitors , Tumor Suppressor Proteins/antagonists & inhibitors , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , B7-H1 Antigen/antagonists & inhibitors , B7-H1 Antigen/immunology , Biomarkers, Tumor/metabolism , Cell Communication/immunology , Cell Culture Techniques , Coculture Techniques , DNA-Binding Proteins/metabolism , Dendritic Cells/immunology , Dendritic Cells/metabolism , Gene Expression Profiling , Glycolysis/drug effects , Glycolysis/immunology , Histone Deacetylase 6/antagonists & inhibitors , Histone Deacetylase 6/metabolism , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylase Inhibitors/therapeutic use , Humans , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Lymphocyte Activation/drug effects , Multiple Myeloma/immunology , Multiple Myeloma/metabolism , Multiple Myeloma/mortality , Phosphopyruvate Hydratase/metabolism , Primary Cell Culture , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/metabolism , Tumor Cells, Cultured , Tumor Suppressor Proteins/metabolism
12.
Leukemia ; 34(2): 567-577, 2020 02.
Article in English | MEDLINE | ID: mdl-31462737

ABSTRACT

Our prior studies showed that dysfunctional plasmacytoid dendritic cells (pDCs) contribute to multiple myeloma (MM) pathogenesis. Specifically, pDC interactions with tumor and T/NK effector cells in the bone marrow (BM) milieu induce immune suppression and MM cell proliferation. Delineation of the mechanism(s) mediating pDC-MM-T-NK cell interactions will identify novel therapeutic targets to both enhance cytotoxicity and anti-MM immunity. Here, we utilized gene expression profiling (GEP) to show that pDC-MM interactions trigger upregulation of immunosuppressive tryptophan catabolic kynurenine (Kyn) pathway. In particular, we show that Kyn pathway enzyme kynurenine-3-monooxygenase (KMO) is upregulated during pDC-MM interactions. Using our coculture models of patient autologous pDC-T-NK-MM cells, we show that pharmacological blockade of KMO activates pDCs and triggers both MM-specific cytotoxic T-cell lymphocytes (CTL) and NK cells cytolytic activity against tumor cells. Furthermore, we show that simultaneous inhibition of Kyn pathway and immune checkpoint PD-L1 enhances antitumor immunity and cytotoxicity in MM. Our preclinical data therefore provide the basis for novel immune-based therapeutic approaches targeting Kyn metabolic pathway enzyme KMO, alone or in combination with anti-PD-L1 Ab, to restore anti-MM immune responses in MM.


Subject(s)
Kynurenine/metabolism , Multiple Myeloma/metabolism , Tryptophan/metabolism , B7-H1 Antigen/metabolism , Bone Marrow/metabolism , Cell Line, Tumor , Dendritic Cells/metabolism , Humans , Killer Cells, Natural/metabolism , Multiple Myeloma/immunology , Multiple Myeloma/therapy , T-Lymphocytes/metabolism
14.
Leukemia ; 33(11): 2685-2694, 2019 11.
Article in English | MEDLINE | ID: mdl-30962579

ABSTRACT

Proteasome inhibition is an effective treatment for multiple myeloma (MM); however, targeting different components of the ubiquitin-proteasome system (UPS) remains elusive. Our RNA-interference studies identified proteasome-associated ubiquitin-receptor Rpn13 as a mediator of MM cell growth and survival. Here, we developed the first degrader of Rpn13, WL40, using a small-molecule-induced targeted protein degradation strategy to selectively degrade this component of the UPS. WL40 was synthesized by linking the Rpn13 covalent inhibitor RA190 with the cereblon (CRBN) binding ligand thalidomide. We show that WL40 binds to both Rpn13 and CRBN and triggers degradation of cellular Rpn13, and is therefore first-in-class in exploiting a covalent inhibitor for the development of degraders. Biochemical and cellular studies show that WL40-induced Rpn13 degradation is both CRBN E3 ligase- and Rpn13-dependent. Importantly, WL40 decreases viability in MM cell lines and patient MM cells, even those resistant to bortezomib. Mechanistically, WL40 interrupts Rpn13 function and activates caspase apoptotic cascade, ER stress response and p53/p21 signaling. In animal model studies, WL40 inhibits xenografted human MM cell growth and prolongs survival. Overall, our data show the development of the first UbR Rpn13 degrader with potent anti-MM activity, and provide proof of principle for the development of degraders targeting components of the UPS for therapeutic application.


Subject(s)
Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Intracellular Signaling Peptides and Proteins/metabolism , Multiple Myeloma/metabolism , Proteasome Inhibitors/pharmacology , Animals , Antineoplastic Agents/pharmacology , Apoptosis , Bortezomib/pharmacology , CRISPR-Cas Systems , Caspases/metabolism , Cell Line, Tumor , Cell Proliferation , Cell Survival , Dendritic Cells/cytology , Drug Evaluation, Preclinical , HCT116 Cells , Humans , Lenalidomide/pharmacology , Mice , Mice, SCID , Multiple Myeloma/therapy , Neoplasm Transplantation , Proteasome Endopeptidase Complex/metabolism , RNA Interference , Ubiquitin/chemistry
15.
Clin Cancer Res ; 23(15): 4280-4289, 2017 Aug 01.
Article in English | MEDLINE | ID: mdl-28270494

ABSTRACT

Purpose: The ubiquitin proteasome pathway is a validated therapeutic target in multiple myeloma. Deubiquitylating enzyme USP1 participates in DNA damage response and cellular differentiation pathways. To date, the role of USP1 in multiple myeloma biology is not defined. In the present study, we investigated the functional significance of USP1 in multiple myeloma using genetic and biochemical approaches.Experimental Design: To investigate the role of USP1 in myeloma, we utilized USP1 inhibitor SJB3-019A (SJB) for studies in myeloma cell lines and patient multiple myeloma cells.Results: USP1-siRNA knockdown decreases multiple myeloma cell viability. USP1 inhibitor SJB selectively blocks USP1 enzymatic activity without blocking other DUBs. SJB also decreases the viability of multiple myeloma cell lines and patient tumor cells, inhibits bone marrow plasmacytoid dendritic cell-induced multiple myeloma cell growth, and overcomes bortezomib resistance. SJB triggers apoptosis in multiple myeloma cells via activation of caspase-3, caspase-8, and caspase-9. Moreover, SJB degrades USP1 and downstream inhibitor of DNA-binding proteins as well as inhibits DNA repair via blockade of Fanconi anemia pathway and homologous recombination. SJB also downregulates multiple myeloma stem cell renewal/survival-associated proteins Notch-1, Notch-2, SOX-4, and SOX-2. Moreover, SJB induced generation of more mature and differentiated plasma cells. Combination of SJB and HDACi ACY-1215, bortezomib, lenalidomide, or pomalidomide triggers synergistic cytotoxicity.Conclusions: Our preclinical studies provide the framework for clinical evaluation of USP1 inhibitors, alone or in combination, as a potential novel multiple myeloma therapy. Clin Cancer Res; 23(15); 4280-9. ©2017 AACR.


Subject(s)
DNA-Binding Proteins/genetics , Drug Synergism , Multiple Myeloma/drug therapy , Neoplasm Proteins/genetics , Ubiquitin-Specific Proteases/genetics , Apoptosis/drug effects , Bortezomib/administration & dosage , Cell Line, Tumor , Cell Survival/drug effects , DNA Damage/drug effects , DNA Repair/drug effects , DNA-Binding Proteins/drug effects , Drug Resistance, Neoplasm/genetics , Gene Expression Regulation, Neoplastic/drug effects , Humans , Lenalidomide , Multiple Myeloma/genetics , Multiple Myeloma/pathology , Neoplasm Proteins/drug effects , Thalidomide/administration & dosage , Thalidomide/analogs & derivatives , Ubiquitin-Specific Proteases/antagonists & inhibitors , Xenograft Model Antitumor Assays
16.
Br J Haematol ; 174(3): 397-409, 2016 08.
Article in English | MEDLINE | ID: mdl-27098276

ABSTRACT

Our prior study utilized both in vitro and in vivo multiple myeloma (MM) xenograft models to show that a novel alkylator melphalan-flufenamide (Melflufen) is a more potent anti-MM agent than melphalan and overcomes conventional drug resistance. Here we examined whether this potent anti-MM activity of melflufen versus melphalan is due to their differential effect on DNA damage and repair signalling pathways via γ-H2AX/ATR/CHK1/Ku80. Melflufen-induced apoptosis was associated with dose- and time-dependent rapid phosphorylation of γ-H2AX. Melflufen induces γ-H2AX, ATR, and CHK1 as early as after 2 h exposure in both melphalan-sensitive and -resistant cells. However, melphalan induces γ-H2AX in melphalan-sensitive cells at 6 h and 24 h; no γ-H2AX induction was observed in melphalan-resistant cells even after 24 h exposure. Similar kinetics was observed for ATR and CHK1 in meflufen- versus melphalan-treated cells. DNA repair is linked to melphalan-resistance; and importantly, we found that melphalan, but not melflufen, upregulates Ku80 that repairs DNA double-strand breaks. Washout experiments showed that a brief (2 h) exposure of MM cells to melflufen is sufficient to initiate an irreversible DNA damage and cytotoxicity. Our data therefore suggest that melflufen triggers a rapid, robust, and an irreversible DNA damage which may account for its ability to overcome melphalan-resistance in MM cells.


Subject(s)
Apoptosis/drug effects , DNA Damage , Melphalan/analogs & derivatives , Multiple Myeloma/pathology , Phenylalanine/analogs & derivatives , Antineoplastic Agents, Alkylating/pharmacology , DNA Repair/drug effects , Drug Resistance, Neoplasm/drug effects , Histones/metabolism , Humans , Kinetics , Melphalan/pharmacology , Multiple Myeloma/drug therapy , Phenylalanine/pharmacology , Phosphorylation/drug effects , Signal Transduction/drug effects , Tumor Cells, Cultured
17.
Br J Haematol ; 171(5): 798-812, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26456076

ABSTRACT

The proteasome inhibitor bortezomib is an effective therapy for the treatment of relapsed and refractory multiple myeloma (RRMM); however, prolonged treatment can be associated with toxicity, peripheral neuropathy and drug resistance. Our earlier studies showed that the novel proteasome inhibitor marizomib is distinct from bortezomib in its chemical structure, mechanisms of action and effects on proteasomal activities, and that it can overcome bortezomib resistance. Pomalidomide, like lenalidomide, has potent immunomodulatory activity and has been approved by the US Food and Drug Administration for the treatment of RRMM. Here, we demonstrate that combining low concentrations of marizomib with pomalidomide induces synergistic anti-MM activity. Marizomib plus pomalidomide-induced apoptosis is associated with: (i) activation of caspase-8, caspase-9, caspase-3 and PARP cleavage, (ii) downregulation of cereblon (CRBN), IRF4, MYC and MCL1, and (iii) suppression of chymotrypsin-like, caspase-like, and trypsin-like proteasome activities. CRBN-siRNA attenuates marizomib plus pomalidomide-induced MM cells death. Furthermore, marizomib plus pomalidomide inhibits the migration of MM cells and tumour-associated angiogenesis, as well as overcomes cytoprotective effects of bone marrow microenvironment. In human MM xenograft model studies, the combination of marizomib and pomalidomide is well tolerated, inhibits tumour growth and prolongs survival. These preclinical studies provide the rationale for on-going clinical trials of combined marizomib and pomalidomide to improve outcome in patients with RRMM.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Multiple Myeloma/drug therapy , Adaptor Proteins, Signal Transducing , Angiogenesis Inhibitors/pharmacology , Animals , Apoptosis/drug effects , Caspases/metabolism , Cell Line, Tumor , Down-Regulation , Drug Resistance, Neoplasm/drug effects , Drug Synergism , Humans , Lactones/administration & dosage , Lactones/pharmacology , Mice, SCID , Peptide Hydrolases/metabolism , Poly(ADP-ribose) Polymerases/metabolism , Proteasome Inhibitors/pharmacology , Pyrroles/administration & dosage , Pyrroles/pharmacology , RNA, Small Interfering/metabolism , Thalidomide/administration & dosage , Thalidomide/analogs & derivatives , Thalidomide/pharmacology , Transplantation, Heterologous , Ubiquitin-Protein Ligases
18.
Blood ; 123(5): 706-16, 2014 Jan 30.
Article in English | MEDLINE | ID: mdl-24319254

ABSTRACT

Proteasome inhibitors have demonstrated that targeting protein degradation is effective therapy in multiple myeloma (MM). Here we show that deubiquitylating enzymes (DUBs) USP14 and UCHL5 are more highly expressed in MM cells than in normal plasma cells. USP14 and UCHL5 short interfering RNA knockdown decreases MM cell viability. A novel 19S regulatory particle inhibitor b-AP15 selectively blocks deubiquitylating activity of USP14 and UCHL5 without inhibiting proteasome activity. b-AP15 decreases viability in MM cell lines and patient MM cells, inhibits proliferation of MM cells even in the presence of bone marrow stroma cells, and overcomes bortezomib resistance. Anti-MM activity of b-AP15 is associated with growth arrest via downregulation of CDC25C, CDC2, and cyclin B1 as well as induction of caspase-dependent apoptosis and activation of unfolded protein response. In vivo studies using distinct human MM xenograft models show that b-AP15 is well tolerated, inhibits tumor growth, and prolongs survival. Combining b-AP15 with suberoylanilide hydroxamic acid, lenalidomide, or dexamethasone induces synergistic anti-MM activity. Our preclinical data showing efficacy of b-AP15 in MM disease models validates targeting DUBs in the ubiquitin proteasomal cascade to overcome proteasome inhibitor resistance and provides the framework for clinical evaluation of USP14/UCHL5 inhibitors to improve patient outcome in MM.


Subject(s)
Antineoplastic Agents/pharmacology , Boronic Acids/pharmacology , Multiple Myeloma/drug therapy , Piperidones/pharmacology , Protease Inhibitors/pharmacology , Pyrazines/pharmacology , Ubiquitin Thiolesterase/antagonists & inhibitors , Animals , Apoptosis/drug effects , Bortezomib , Cell Line, Tumor , Drug Resistance, Neoplasm/drug effects , Endoplasmic Reticulum Stress/drug effects , Female , Gene Expression Regulation, Neoplastic , Humans , Mice , Multiple Myeloma/genetics , Multiple Myeloma/pathology , Ubiquitin Thiolesterase/genetics , Up-Regulation
19.
Clin Cancer Res ; 19(11): 3019-31, 2013 Jun 01.
Article in English | MEDLINE | ID: mdl-23584492

ABSTRACT

PURPOSE: The alkylating agent melphalan prolongs survival in patients with multiple myeloma; however, it is associated with toxicities and development of drug-resistance. Here, we evaluated the efficacy of melphalan-flufenamide (mel-flufen), a novel dipeptide prodrug of melphalan in multiple myeloma. EXPERIMENTAL DESIGN: Multiple myeloma cell lines, primary patient cells, and the human multiple myeloma xenograft animal model were used to study the antitumor activity of mel-flufen. RESULTS: Low doses of mel-flufen trigger more rapid and higher intracellular concentrations of melphalan in multiple myeloma cells than are achievable by free melphalan. Cytotoxicity analysis showed significantly lower IC50 of mel-flufen than melphalan in multiple myeloma cells. Importantly, mel-flufen induces apoptosis even in melphalan- and bortezomib-resistant multiple myeloma cells. Mechanistic studies show that siRNA knockdown of aminopeptidase N, a key enzyme mediating intracellular conversion of mel-flufen to melphalan, attenuates anti-multiple myeloma activity of mel-flufen. Furthermore, mel-flufen-induced apoptosis was associated with: (i) activation of caspases and PARP cleavage; (ii) reactive oxygen species generation; (iii) mitochondrial dysfunction and release of cytochrome c; and (iv) induction of DNA damage. Moreover, mel-flufen inhibits multiple myeloma cell migration and tumor-associated angiogenesis. Human multiple myeloma xenograft studies showed a more potent inhibition of tumor growth in mice treated with mel-flufen than mice receiving equimolar doses of melphalan. Finally, combining mel-flufen with lenalidomide, bortezomib, or dexamethasone triggers synergistic anti-multiple myeloma activity. CONCLUSION: Our preclinical study supports clinical evaluation of mel-flufen to enhance therapeutic potential of melphalan, overcome drug-resistance, and improve multiple myeloma patient outcome.


Subject(s)
Antineoplastic Agents, Alkylating/pharmacology , Melphalan/analogs & derivatives , Multiple Myeloma/pathology , Phenylalanine/analogs & derivatives , Angiogenesis Inhibitors/administration & dosage , Angiogenesis Inhibitors/pharmacology , Animals , Antineoplastic Agents, Alkylating/administration & dosage , Cell Line, Tumor , Cell Movement/drug effects , Cell Survival/drug effects , Disease Models, Animal , Drug Synergism , Female , Humans , Melphalan/administration & dosage , Melphalan/pharmacology , Mice , Multiple Myeloma/drug therapy , Multiple Myeloma/mortality , Neovascularization, Pathologic/drug therapy , Phenylalanine/administration & dosage , Phenylalanine/pharmacology , Tumor Burden/drug effects , Xenograft Model Antitumor Assays
20.
PLoS One ; 6(9): e25351, 2011.
Article in English | MEDLINE | ID: mdl-21980431

ABSTRACT

OBJECTIVES: Clinical responses achieved with FLT3 kinase inhibitors in acute myeloid leukemia (AML) are typically transient and partial. Thus, there is a need for identification of molecular mechanisms of clinical resistance to these drugs. In response, we characterized MOLM13 AML cell lines made resistant to two structurally-independent FLT3 inhibitors. METHODS: MOLM13 cells were made drug resistant via prolonged exposure to midostaurin and HG-7-85-01, respectively. Cell proliferation was determined by Trypan blue exclusion. Protein expression was assessed by immunoblotting, immunoprecipitation, and flow cytometry. Cycloheximide was used to determine protein half-life. RT-PCR was performed to determine FLT3 mRNA levels, and FISH analysis was performed to determine FLT3 gene expression. RESULTS AND CONCLUSIONS: We found that MOLM13 cells readily developed cross-resistance when exposed to either midostaurin or HG-7-85-01. Resistance in both lines was associated with dramatically elevated levels of cell surface FLT3 and elevated levels of phosphor-MAPK, but not phospho-STAT5. The increase in FLT3-ITD expression was at least in part due to reduced turnover of the receptor, with prolonged half-life. Importantly, the drug-resistant phenotype could be rapidly reversed upon withdrawal of either inhibitor. Consistent with this phenotype, no significant evidence of FLT3 gene amplification, kinase domain mutations, or elevated levels of mRNA was observed, suggesting that protein turnover may be part of an auto-regulatory pathway initiated by FLT3 kinase activity. Interestingly, FLT3 inhibitor resistance also correlated with resistance to cytosine arabinoside. Over-expression of FLT3 protein in response to kinase inhibitors may be part of a novel mechanism that could contribute to clinical resistance.


Subject(s)
Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Gene Expression Regulation, Neoplastic/drug effects , Mutation , Protein Kinase Inhibitors/pharmacology , fms-Like Tyrosine Kinase 3/antagonists & inhibitors , fms-Like Tyrosine Kinase 3/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Enzyme Stability/drug effects , Enzyme Stability/genetics , Gene Expression Regulation, Neoplastic/genetics , Half-Life , Humans , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Phosphorylation/drug effects , Phosphorylation/genetics , Piperazines/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Signal Transduction/drug effects , Signal Transduction/genetics , Staurosporine/analogs & derivatives , Staurosporine/pharmacology , Thiazoles/pharmacology , Tyrosine/metabolism , Up-Regulation/drug effects , fms-Like Tyrosine Kinase 3/chemistry , fms-Like Tyrosine Kinase 3/metabolism
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