pr-957 and carfilzomib

Proteasome inhibitors bortezomib, carfilzomib and ixazomib (approved by the US Food and Drug Administration [FDA]) induce remissions in patients with multiple myeloma (MM), but most patients eventually become resistant. MM and other hematologic malignancies express ubiquitous constitutive proteasomes and lymphoid tissue-specific immunoproteasomes; immunoproteasome expression is increased in resistant patients. Immunoproteasomes contain 3 distinct pairs of active sites, β5i, β1i, and β2i, which are different from their constitutive β5c, β1c, and β2c counterparts. Bortezomib and carfilzomib block β5c and β5i sites. We report here that pharmacologically relevant concentrations of β5i-specific inhibitor ONX-0914 show cytotoxicity in MM cell lines similar to that of carfilzomib and bortezomib. In addition, increasing immunoproteasome expression by interferon-γ increases sensitivity to ONX-0914 but not to carfilzomib. LU-102, an inhibitor of β2 sites, dramatically sensitizes MM cell lines and primary cells to ONX-0914. ONX-0914 synergizes with all FDA-approved proteasome inhibitors in MM in vitro and in vivo. Thus, immunoproteasome inhibitors, currently in clinical trials for the treatment of autoimmune diseases, should also be considered for the treatment of MM.

Topics: Animals; Antineoplastic Agents; Biomarkers; Bortezomib; Cell Survival; Dose-Response Relationship, Drug; Drug Synergism; Humans; Male; Mice; Multiple Myeloma; Oligopeptides; Proteasome Endopeptidase Complex; Proteasome Inhibitors

Inhibition of the 20S proteasome by bortezomib (Velcade) constitutes a successfully applied therapy for blood cancer. However, emerging resistance restricts its medicinal use. For example, mutations in the proteolytically active β5-subunit of the proteasome, the main target of inhibitors, were reported to impair drug binding and thus to reduce therapeutic efficacy. Using yeast as a model system, we describe here a systematic evaluation of these mutations by cell growth analysis, proteasome inhibition assays, and X-ray crystallography. The 11 mutants examined display decreased proliferation rates, impaired proteolytic activity, and marked resistance to bortezomib as well as the α',β'-epoxyketone inhibitors carfilzomib (Kyprolis) and ONX 0914, while the second-generation compound carfilzomib was the least affected. In total, 49 proteasome X-ray structures, including structural data on proteasome-carfilzomib complexes, reveal three distinct molecular mechanisms that hamper both drug binding and natural substrate turnover to an extent that is still compatible with cell survival.

Topics: Boronic Acids; Bortezomib; Catalytic Domain; Crystallography, X-Ray; Drug Resistance, Neoplasm; Models, Molecular; Molecular Structure; Mutagenesis; Mutation; Oligopeptides; Proteasome Endopeptidase Complex; Protein Conformation; Pyrazines; X-Ray Diffraction; Yeasts

Aside from antimalarials, there is currently no treatment for cerebral malaria, a fulminant neurological complication of P. falciparum infection that is a leading cause of death in African children. In the mouse model of cerebral malaria, cross-presentation of parasite antigens by brain endothelial cells is thought to be a crucial late step in pathogenesis. We have investigated three proteasome inhibitors as potential adjunct therapies: bortezomib, carfilzomib and ONX-0914. Only carfilzomib, an irreversible inhibitor of both constitutive proteasomes and immunoproteasomes, was able to inhibit cross-presentation of malaria antigen by murine brain endothelial cells in vitro. To mimic the clinical setting, carfilzomib was co-administered with artesunate only when infected mice exhibited neurological defects. However, there was no improvement in survival compared to artesunate monotherapy. The treatment failure was explained by the inability of daily or twice daily bolus doses of carfilzomib to inhibit cross-presentation by brain endothelial cells in vivo. We also report here that bortezomib, which has been associated with neurological adverse events, accelerated death in ECM-infected mice. Future investigations of proteasome inhibitors for modulating cross-presentation during malaria infection should focus on sustained and targeted delivery to brain endothelial cells.

Topics: Animals; Antigen Presentation; Antimalarials; Bortezomib; Brain; Cells, Cultured; Disease Models, Animal; Endothelial Cells; In Vitro Techniques; Malaria, Cerebral; Malaria, Falciparum; Mice; Mice, Inbred C57BL; Oligopeptides; Plasmodium berghei; Proteasome Inhibitors

Despite encouraging results with the proteasome inhibitor bortezomib in the treatment of hematologic malignancies, emergence of resistance can limit its efficacy, hence calling for novel strategies to overcome bortezomib-resistance. We previously showed that bortezomib-resistant human leukemia cell lines expressed significantly lower levels of immunoproteasome at the expense of constitutive proteasomes, which harbored point mutations in exon 2 of the PSMB5 gene encoding the β5 subunit. Here we investigated whether up-regulation of immunoproteasomes by exposure to interferon-γ restores sensitivity to bortezomib in myeloma and leukemia cell lines with acquired resistance to bortezomib.. RPMI-8226 myeloma, THP1 monocytic/macrophage and CCRF-CEM (T) parental cells and sub lines with acquired resistance to bortezomib were exposed to Interferon-γ for 24-48 h where after the effects on proteasome subunit expression and activity were measured, next to sensitivity measurements to proteasome inhibitors bortezomib, carfilzomib, and the immunoproteasome selective inhibitor ONX 0914. At last, siRNA knockdown experiments of β5i and β1i were performed to identify the contribution of these subunits to sensitivity to proteasome inhibition. Statistical significance of the differences were determined using the Mann-Whitney U test.. Interferon-γ exposure markedly increased immunoproteasome subunit mRNA to a significantly higher level in bortezomib-resistant cells (up to 30-fold, 10-fold, and 6-fold, in β1i, β5i, and β2i, respectively) than in parental cells. These increases were paralleled by elevated immunoproteasome protein levels and catalytic activity, as well as HLA class-I. Moreover, interferon-γ exposure reinforced sensitization of bortezomib-resistant tumor cells to bortezomib and carfilzomib, but most prominently to ONX 0914, as confirmed by cell growth inhibition studies, proteasome inhibitor-induced apoptosis, activation of PARP cleavage and accumulation of polyubiquitinated proteins. This sensitization was abrogated by siRNA silencing of β5i but not by β1i silencing, prior to pulse exposure to interferon-γ.. Downregulation of β5i subunit expression is a major determinant in acquisition of bortezomib-resistance and enhancement of its proteasomal assembly after induction by interferon-γ facilitates restoration of sensitivity in bortezomib-resistant leukemia cells towards bortezomib and next generation (immuno) proteasome inhibitors.

Topics: Apoptosis; Blotting, Western; Boronic Acids; Bortezomib; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Hematologic Neoplasms; Histocompatibility Antigens Class I; HLA Antigens; Humans; Interferon-gamma; Oligopeptides; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Subunits; Pyrazines; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Up-Regulation

Proteasome inhibitors, although initially developed for the treatment of malignancies, have been found to affect normal plasma cells by efficaciously inducing apoptosis. One proteasome inhibitor, bortezomib, has been used in transplantation settings to deplete human leukocyte antigen antibody-producing plasma cells to reverse humoral allograft rejection.. To establish whether proteasome inhibitors are active on B cells, being plasma cell precursors, we examined a set of four proteasome inhibitors, including bortezomib, carfilzomib, ONX 0912, and ONX 0914, for their potential to impact the functionalities of activated B cells in vitro.. All proteasome inhibitors dose-dependently abrogated IgM and IgG production by activated B cells, as well as their proliferation, with varying efficiencies. The bortezomib-induced decline in immunoglobulin production was mainly due to a decrease in the number of B cells capable of immunoglobulin secretion, caused by apoptosis.. The action of proteasome inhibitors is not confined to plasma cells but also has impact on activated naïve and memory B cells.

Topics: Apoptosis; B-Lymphocytes; Boronic Acids; Bortezomib; Cell Proliferation; Cells, Cultured; Humans; Immunoglobulin G; Immunoglobulin M; In Vitro Techniques; Oligopeptides; Plasma Cells; Proteasome Endopeptidase Complex; Pyrazines