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1.  Inhibition of the immunoproteasome ameliorates experimental autoimmune encephalomyelitis 
EMBO Molecular Medicine  2014;6(2):226-238.
Multiple sclerosis (MS) is a chronic demyelinating immune mediated disease of the central nervous system. The immunoproteasome is a distinct class of proteasomes found predominantly in monocytes and lymphocytes. Recently, we demonstrated a novel function of immunoproteasomes in cytokine production and T cell differentiation. In this study, we investigated the therapeutic efficacy of an inhibitor of the immunoproteasome (ONX 0914) in two different mouse models of MS. ONX 0914 attenuated disease progression after active and passive induction of experimental autoimmune encephalomyelitis (EAE), both in MOG35–55 and PLP139–151-induced EAE. Isolation of lymphocytes from the brain or spinal cord revealed a strong reduction of cytokine-producing CD4+ cells in ONX 0914 treated mice. Additionally, ONX 0914 treatment prevented disease exacerbation in a relapsing-remitting model. An analysis of draining lymph nodes after induction of EAE revealed that the differentiation to Th17 or Th1 cells was strongly impaired in ONX 0914 treated mice. These results implicate the immunoproteasome in the development of EAE and suggest that immunoproteasome inhibitors are promising drugs for the treatment of MS.
doi:10.1002/emmm.201303543
PMCID: PMC3927957  PMID: 24399752
experimental autoimmune encephalomyelitis; immunoproteasome; multiple sclerosis; proteasome
2.  Interferon-γ-induced upregulation of immunoproteasome subunit assembly overcomes bortezomib resistance in human hematological cell lines 
Background
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.
Methods
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.
Results
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.
Conclusion
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.
doi:10.1186/1756-8722-7-7
PMCID: PMC3896789  PMID: 24418325
Leukemia; Proteasome; Immunoproteasome; Proteasome inhibitor; Bortezomib; Interferon-gamma
3.  Validation of the proteasome as a therapeutic target in Plasmodium using an epoxyketone inhibitor with parasite-specific toxicity 
Chemistry & biology  2012;19(12):1535-1545.
Summary
The Plasmodium proteasome has been suggested to be a potential anti-malarial drug target, however toxicity of inhibitors has prevented validation of this enzyme in vivo. We report here a screen of a library of 670 analogs of the recently FDA approved inhibitor, carfilzomib, to identify compounds that selectively kill parasites. We identified one compound, PR3, that has significant parasite killing activity in vitro but dramatically reduced toxicity in host cells. We found that this parasite-specific toxicity is not due to selective targeting of the Plasmodium proteasome over the host proteasome, but instead is due to a lack of activity against one of the human proteasome subunits. Subsequently, we used PR3 to significantly reduce parasite load in P. berghei infected mice without host toxicity, thus validating the proteasome as a viable anti-malarial drug target.
doi:10.1016/j.chembiol.2012.09.019
PMCID: PMC3529830  PMID: 23142757
4.  The next generation proteasome inhibitors carfilzomib and oprozomib activate prosurvival autophagy via induction of the unfolded protein response and ATF4 
Autophagy  2012;8(12):1873-1874.
The proteasome inhibitor bortezomib has shown remarkable clinical success in the treatment of multiple myeloma. However, the efficacy and mechanism of action of bortezomib in solid tumor malignancies is less well understood. In addition, the use of this first-in-class proteasome inhibitor is limited by several factors, including off-target effects that lead to adverse toxicities. We recently reported the impact and mechanisms of carfilzomib and oprozomib, second-in-class proteasome inhibitors with higher specificities and reduced toxicities, against head and neck squamous cell carcinoma (HNSCC). Carfilzomib and oprozomib potently inhibit HNSCC cell survival and the growth of HNSCC tumors. Both compounds promote upregulation of proapoptotic BIK and antiapoptotic MCL1, which serves to mediate and attenuate, respectively, the killing activities of these proteasome inhibitors. Both compounds also induce complete autophagic flux that is partially dependent on activation of the unfolded protein response (UPR) and upregulation of ATF4. Carfilzomib- and oprozomib-induced autophagy acts to promote HNSCC cell survival. Our study indicates that the therapeutic benefit of these promising proteasome inhibitors may be improved by inhibiting MCL1 expression or autophagy.
doi:10.4161/auto.22185
PMCID: PMC3541310  PMID: 22995770
ATF4; ONX 0912; apoptosis; autophagy; carfilzomib; oprozomib; proteasome inhibitor; unfolded protein response
5.  Carfilzomib and ONX 0912 Inhibit Cell Survival and Tumor Growth of Head and Neck Cancer and Their Activities are Enhanced by Suppression of Mcl-1 or Autophagy 
Purpose
Carfilzomib is a selective, irreversible inhibitor of the chymotrypsin-like activity of the proteasome and is undergoing clinical evaluation in myeloma. ONX 0912 (oprozomib) is an orally bioavailable derivative. The activities of carfilzomib and ONX 0912 against solid tumor malignancies are less well understood. We investigated the impact and mechanisms of action of carfilzomib and ONX 0912 in preclinical models of head and neck squamous cell carcinoma (HNSCC).
Experimental Design
The effects of carfilzomib and ONX 0912 on HNSCC cell survival and xenograft tumor growth were evaluated. The impact and mechanisms of both agents on apoptosis and autophagy induction were also investigated. The contribution of the unfolded protein response (UPR) to autophagy induction and the role of autophagy in attenuating HNSCC cell death was determined.
Results
Carfilzomib and ONX 0912 potently induced apoptosis in HNSCC cell lines via upregulation of proapoptotic Bik. Upregulation of Mcl-1 by these agents served to dampen their efficacies. Carfilzomib and ONX 0912 also induced autophagy, mediated, in part, by activation of the UPR pathway involving upregulation of ATF4 transcription factor. Autophagy induction served a prosurvival role. Oral administration of ONX 0912 inhibited the growth of HNSCC xenograft tumors in a dose-dependent manner.
Conclusions
These results show that carfilzomib and ONX 0912 are potently active against HNSCC cells, and the activities of these agents can be enhanced via suppresion of Mcl-1 or inhibition of autophagy. Oral ONX 0912 exhibits in vivo activity against HNSCC tumors, and may represent a useful therapeutic agent for this malignancy.
doi:10.1158/1078-0432.CCR-12-1213
PMCID: PMC3473099  PMID: 22929803
6.  Overcoming bortezomib resistance in human B cells by anti-CD20/rituximab-mediated complement-dependent cytotoxicity and epoxyketone-based irreversible proteasome inhibitors 
Background
In clinical and experimental settings, antibody-based anti-CD20/rituximab and small molecule proteasome inhibitor (PI) bortezomib (BTZ) treatment proved effective modalities for B cell depletion in lymphoproliferative disorders as well as autoimmune diseases. However, the chronic nature of these diseases requires either prolonged or re-treatment, often with acquired resistance as a consequence.
Methods
Here we studied the molecular basis of acquired resistance to BTZ in JY human B lymphoblastic cells following prolonged exposure to this drug and examined possibilities to overcome resistance by next generation PIs and anti-CD20/rituximab-mediated complement-dependent cytotoxicity (CDC).
Results
Characterization of BTZ-resistant JY/BTZ cells compared to parental JY/WT cells revealed the following features: (a) 10–12 fold resistance to BTZ associated with the acquisition of a mutation in the PSMB5 gene (encoding the constitutive β5 proteasome subunit) introducing an amino acid substitution (Met45Ile) in the BTZ-binding pocket, (b) a significant 2–4 fold increase in the mRNA and protein levels of the constitutive β5 proteasome subunit along with unaltered immunoproteasome expression, (c) full sensitivity to the irreversible epoxyketone-based PIs carfilzomib and (to a lesser extent) the immunoproteasome inhibitor ONX 0914. Finally, in association with impaired ubiquitination and attenuated breakdown of CD20, JY/BTZ cells harbored a net 3-fold increase in CD20 cell surface expression, which was functionally implicated in conferring a significantly increased anti-CD20/rituximab-mediated CDC.
Conclusions
These results demonstrate that acquired resistance to BTZ in B cells can be overcome by next generation PIs and by anti-CD20/rituximab-induced CDC, thereby paving the way for salvage therapy in BTZ-resistant disease.
doi:10.1186/2162-3619-2-2
PMCID: PMC3560160  PMID: 23305345
Proteasome inhibitors; Anti-CD20/rituximab therapy; B cells; Autoimmune disorders; Resistance
7.  PR-924, a Selective Inhibitor of the Immunoproteasome Subunit LMP-7, Blocks Multiple Myeloma Cell Growth both in Vitro and in Vivo 
British journal of haematology  2010;152(2):155-163.
PR-924 is an LMP-7-selective tripeptide epoxyketone proteasome inhibitor that covalently modifies proteasomal N-terminal threonine active sites. In the present study, we show that PR-924 inhibits growth and triggers apoptosis in multiple myeloma (MM) cell lines and primary patient MM cells, without significantly affecting normal peripheral blood mononuclear cells. PR-924-induced apoptosis in MM cells is associated with activation of caspase-3, caspase8, caspase-9, BID, and PARP, translocation of cleaved-BID to mitochondria, and cytochrome-c release. In vivo administration of PR-924 inhibits tumor growth in human plasmacytoma xenografts. Results from SCID-hu model show a significant reduction in the shIL-6R levels in mice treated with PR-924 versus vehicle-control. PR-924 treatment was well tolerated as evidenced by the lack of weight loss. Importantly, treatment of tumor-bearing mice with PR-924, but not vehicle alone, prolonged survival. Our preclinical findings therefore validate immunoproteasome LMP-7 subunit as a novel therapeutic target in MM.
doi:10.1111/j.1365-2141.2010.08491.x
PMCID: PMC3138210  PMID: 21114484
8.  Molecular Mechanisms of Bortezomib Resistant Adenocarcinoma Cells 
PLoS ONE  2011;6(12):e27996.
Bortezomib (Velcade™) is a reversible proteasome inhibitor that is approved for the treatment of multiple myeloma (MM). Despite its demonstrated clinical success, some patients are deprived of treatment due to primary refractoriness or development of resistance during therapy. To investigate the role of the duration of proteasome inhibition in the anti-tumor response of bortezomib, we established clonal isolates of HT-29 adenocarcinoma cells adapted to continuous exposure of bortezomib. These cells were ∼30-fold resistant to bortezomib. Two novel and distinct mutations in the β5 subunit, Cys63Phe, located distal to the binding site in a helix critical for drug binding, and Arg24Cys, found in the propeptide region were found in all resistant clones. The latter mutation is a natural variant found to be elevated in frequency in patients with MM. Proteasome activity and levels of both the constitutive and immunoproteasome were increased in resistant cells, which correlated to an increase in subunit gene expression. These changes correlated with a more rapid recovery of proteasome activity following brief exposure to bortezomib. Increased recovery rate was not due to increased proteasome turnover as similar findings were seen in cells co-treated with cycloheximide. When we exposed resistant cells to the irreversible proteasome inhibitor carfilzomib we noted a slower rate of recovery of proteasome activity as compared to bortezomib in both parental and resistant cells. Importantly, carfilzomib maintained its cytotoxic potential in the bortezomib resistant cell lines. Therefore, resistance to bortezomib, can be overcome with irreversible inhibitors, suggesting prolonged proteasome inhibition induces a more potent anti-tumor response.
doi:10.1371/journal.pone.0027996
PMCID: PMC3245226  PMID: 22216088
9.  The trans-Acting Protein Interacting with the DNA Motif Proximal to the Transcriptional Start Site of Plant l-Asparaginase Is Bacterial Sarcosine Oxidase 
Journal of Bacteriology  2004;186(3):811-817.
A trans-acting protein interacting with a specific sequence motif proximal to the transcriptional start site of the l-asparaginase promoter has been observed previously (E. Vincze, J. M. Reeves, E. Lamping, K. J. F. Farnden, and P. H. S. Reynolds, Plant Mol. Biol. 26:303-311, 1994). Gel retardation experiments in which protein extracts of Mesorhizobium loti and developing nodules were used suggested a bacterial origin for the repressor binding protein (rep2037). Nodulation tests were performed by using different Fix− Tn5 mutants of M. loti. Analyses of these mutants revealed a correlation between the presence of Mesorhizobium in the nodule-like structures and the ability of nodule protein extracts to bind the repressor binding domain (RBD). Through the use of mutated RBD sequences, the RBD sequence was identified as CTAAAAT. The repressor protein was isolated from M. loti NZP2037 by multiple chromatographic procedures and affinity separation by using concatemers of RBD attached to magnetic beads. Sequencing of the recovered protein resulted in identification of the repressor protein as the sarcosine oxidase α subunit. This was confirmed by expression of the gene encoding the M. loti α subunit of sarcosine oxidase in Escherichia coli. When the expressed peptide was bound to RBD, the gel retardation result was identical to the result obtained with rep2037 from M. loti strain NZP2037.
doi:10.1128/JB.186.3.811-817.2004
PMCID: PMC321473  PMID: 14729708

Results 1-9 (9)