Targeting the ubiquitin ligase SKP2 in the myeloma stem-cell like side population.
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e13510 Background: While anti-myeloma therapies are effective in eradicating the bulk of tumor cells, intrinsically drug resistant, clonogenic tumor-initiating subpopulations contribute to disease relapse and poor outcomes in multiple myeloma (MM). Side population (SP) cells are an enriched source of therapy-resistant, tumor-initiating cells with stem cell-like features. Here, we investigated the molecular determinants responsible for chemoresistance in the myeloma SP to then identify actionable therapeutic targets. Methods: Side population (SP) cells were isolated from myeloma cell lines and patient samples using Hoechst 33342 dye-based flow cytometry. SP cells represented 1-2% of the tumor population and were used in cell-based assays to determine chemosensitivities relative to the non-SP cells. Results: Gene expression analysis indicated that components of the E3 ubiquitin ligase Skp1-Cul1-FboxSkp2 (SCFSkp2) complex were hyperexpressed in myeloma patients. SP cells were less sensitive to the anti-myeloma effects of the proteasome inhibitors (PIs) bortezomib, carfilzomib and ixazomib relative to non-SP cells. The relative percentage of SP cells was significantly greater in cultures of myeloma cells generated with acquired resistance to each PI. Genetic ablation of Skp2 or Cullin-1 synergistically enhanced the cytotoxic effect of bortezomib. A high-throughput assay was performed using large databases and chemical libraries to identify novel lead compounds that inhibited SCFSkp2-dependent ubiquitination activity. Bortezomib co-treatment with a lead compound identified here enhanced the anti-myeloma effect against SP cells isolated from cell lines and patient bone marrow samples. Conclusions: Taken together, we identify novel molecular determinants of chemoresistance in the myeloma stem cell-like SP. Functional assays demonstrated that SCFSkp2 is hyperexpressed in SP cells and represents an actionable drug target. Pharmacologics to selectively disrupt SCFSkp2 activity hold promise to selectively target chemoresistance with potential therapeutic benefit for myeloma patients.Keywords:
Side population
Clonogenic assay
Ixazomib
Vorinostat
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Mnohočetný myelom, maligní onemocnění plazmatických buněk, zůstává stále velmi obtížně léčitelným hematoonkologickým onemocněním, pro které je nutné hledat nové možnosti terapie ovlivňující jak plazmocyty samotné, tak i mikroprostředí kostní dřeně
Hematologic malignancy
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Abstract Proteasome inhibitors bortezomib and carfilzomib are the backbone of treatments of multiple myeloma, which remains uncurable despite many recent advances. With many patients relapsing despite high initial response rates to proteasome inhibitor-containing regimens, it is critical to understand the process of acquired resistance. In vitro generated resistant cell lines are important tools in this process. The majority of previously developed bortezomib-resistant cell lines bear mutations in the proteasome PSMB5 sites, the prime target of bortezomib and carfilzomib, which are rarely observed in patients. Here we present a novel bortezomib-resistant derivative of KMS-12-BM multiple myeloma cell line, KMS-12-BM-BPR. Unlike previously published bortezomib-resistant cell lines, this sub-line was created using a clinically relevant twice-weekly pulse treatments with bortezomib instead of continuous incubation. It does not contain mutations in proteasome active sites and retains its sensitivity to carfilzomib. Reduced load on proteasome due to decreased protein synthesis appears to be the main cause of resistance. In addition, KMS-12-BM-BPR cells express less Mcl-1 than wild type and are more sensitive to Bcl-2 inhibitor venetoclax. Overall, this study demonstrates the feasibility of creating a proteasome-resistant myeloma cell lines by using clinically relevant pulse treatments and provides a novel model of acquired resistance.
Venetoclax
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Acquired proteasome-inhibitor (PI) resistance is a major obstacle in the treatment of multiple myeloma (MM). We investigated whether the clinical XPO1-inhibitor selinexor, when combined with bortezomib or carfilzomib, could overcome acquired resistance in MM. PI-resistant myeloma cell lines both in vitro and in vivo and refractory myeloma patient biopsies were treated with selinexor/bortezomib or carfilzomib and assayed for apoptosis. Mechanistic studies included NFκB pathway protein expression assays, immunofluorescence microscopy, ImageStream flow-cytometry, and proximity-ligation assays. IκBα knockdown and NFκB activity were measured in selinexor/bortezomib-treated MM cells. We found that selinexor restored sensitivity of PI-resistant MM to bortezomib and carfilzomib. Selinexor/bortezomib treatment inhibited PI-resistant MM tumor growth and increased survival in mice. Myeloma cells from PI-refractory MM patients were sensitized by selinexor to bortezomib and carfilzomib without affecting non-myeloma cells. Immunofluorescence microscopy, Western blot, and ImageStream analyses of MM cells showed increases in total and nuclear IκBα by selinexor/bortezomib. Proximity ligation found increased IκBα-NFκB complexes in treated MM cells. IκBα knockdown abrogated selinexor/bortezomib-induced cytotoxicity in MM cells. Selinexor/bortezomib treatment decreased NFκB transcriptional activity. Selinexor, when used with bortezomib or carfilzomib, has the potential to overcome PI drug resistance in MM. Sensitization may be due to inactivation of the NFκB pathway by IκBα.
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Proteasome inhibitor resistance is a challenge for myeloma therapy. Bortezomib targets the β5 and β1 activity, but not the β2 activity of the proteasome. Bortezomib-resistant myeloma cells down-regulate the activation status of the unfolded protein response, and up-regulate β2 proteasome activity. To improve proteasome inhibition in bortezomib-resistant myeloma and to achieve more efficient UPR activation, we have developed LU-102, a selective inhibitor of the β2 proteasome activity. LU-102 inhibited the β2 activity in intact myeloma cells at low micromolar concentrations without relevant co-inhibition of β1 and β5 proteasome subunits. In proteasome inhibitor-resistant myeloma cells, significantly more potent proteasome inhibition was achieved by bortezomib or carfilzomib in combination with LU-102, compared to bortezomib/carfilzomib alone, resulting in highly synergistic cytotoxic activity of the drug combination via endoplasmatic reticulum stress-induced apoptosis. Combining bortezomib/carfilzomib with LU-102 significantly prolonged proteasome inhibition and increased activation of the unfolded protein response and IRE1-a activity. IRE1-α has recently been shown to control myeloma cell differentiation and bortezomib sensitivity (Leung-Hagesteijn, Cancer Cell 24:3, 289-304). Thus, β2-selective proteasome inhibition by LU-102 in combination with bortezomib or carfilzomib results in synergistic proteasome inhibition, activation of the unfolded protein response, and cytotoxicity, and overcomes bortezomib/carfilzomib resistance in myeloma cells in vitro
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Ixazomib
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Propidium iodide
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Ixazomib
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Proteasome inhibitors bortezomib and carfilzomib are the backbones of treatments of multiple myeloma, which remains incurable despite many recent advances. With many patients relapsing despite high initial response rates to proteasome inhibitor-containing regimens, it is critical to understand the process of acquired resistance. In vitro generated resistant cell lines are important tools in this process. The majority of previously developed bortezomib-resistant cell lines bear mutations in the proteasome PSMB5 sites, the prime target of bortezomib and carfilzomib, which are rarely observed in patients. Here we present a novel bortezomib-resistant derivative of the KMS-12-BM multiple myeloma cell line, KMS-12-BM-BPR. Unlike previously published bortezomib-resistant cell lines, it was created using clinically relevant twice-weekly pulse treatments with bortezomib instead of continuous incubation. It does not contain mutations in the PSMB5 site and retains its sensitivity to carfilzomib. Reduced load on proteasome due to decreased protein synthesis appears to be the main cause of resistance. In addition, KMS-12-BM-BPR cells are more sensitive to Bcl-2 inhibitor venetoclax. Overall, this study demonstrates the feasibility of creating a proteasome inhibitor resistant myeloma cell lines by using clinically relevant pulse treatments and provides a novel model of acquired resistance.
Venetoclax
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