Dataset Information

Overcoming bortezomib resistance in human B cells by anti-CD20/rituximab-mediated complement-dependent cytotoxicity and epoxyketone-based irreversible proteasome inhibitors.

ABSTRACT:

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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 ?5 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 ?5 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.

SUBMITTER: Verbrugge SE

PROVIDER: S-EPMC3560160 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Publications

Overcoming bortezomib resistance in human B cells by anti-CD20/rituximab-mediated complement-dependent cytotoxicity and epoxyketone-based irreversible proteasome inhibitors.

Verbrugge Sue Ellen SE Al Marjon M Assaraf Yehuda G YG Niewerth Denise D van Meerloo Johan J Cloos Jacqueline J van der Veer Michael M Scheffer George L GL Peters Godefridus J GJ Chan Elena T ET Anderl Janet L JL Kirk Christopher J CJ Zweegman Sonja S Dijkmans Ben Ac BA Lems Willem F WF Scheper Rik J RJ de Gruijl Tanja D TD Jansen Gerrit G

Experimental hematology & oncology 20130110 1

<h4>Unlabelled</h4><h4>Background</h4>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.<h4>Methods</h4>Here we studied the molecular basis of acquired r ...[more]

PMID: 23305345

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Project description:Streptomyces sp. BRA-346 is an Actinobacteria isolated from the Brazilian endemic tunicate Euherdmania sp. We have reported that this strain produces epoxyketone peptides, as dihydroeponemycin (DHE) and structurally related analogs. This cocktail of epoxyketone peptides inhibits the proteasome chymotrypsin-like activity and shows high cytotoxicity to glioma cells. However, low yields and poor reproducibility of epoxyketone peptides production by BRA-346 under laboratory cultivation have limited the isolation of epoxyketone peptides for additional studies. Here, we evaluated several cultivation methods using different culture media and chemical elicitors to increase the repertoire of peptide epoxyketone production by this bacterium. Furthermore, BRA-346 genome was sequenced, revealing its broad genetic potential, which is mostly hidden under laboratory conditions. By using specific growth conditions, we were able to evidence different classes of secondary metabolites produced by BRA-346. In addition, by combining genome mining with untargeted metabolomics, we could link the metabolites produced by BRA-346 to its genetic capacity and potential regulators. A single biosynthetic gene cluster (BGC) was related to the production of the target epoxyketone peptides by BRA-346. The candidate BGC displays conserved biosynthetic enzymes with the reported eponemycin (EPN) and TMC-86A (TMC) BGCs. The core of the putative epoxyketone peptide BGC (ORFs A-L), in which ORF A is a LuxR-like transcription factor, was cloned into a heterologous host. The recombinant organism was capable to produce TMC and EPN natural products, along with the biosynthetic intermediates DH-TMC and DHE, and additional congeners. A phylogenetic analysis of the epn/tmc BGC revealed related BGCs in public databases. Most of them carry a proteasome beta-subunit, however, lacking an assigned specialized metabolite. The retrieved BGCs also display a diversity of regulatory genes and TTA codons, indicating tight regulation of this BGC at the transcription and translational levels. These results demonstrate the plasticity of the epn/tmc BGC of BRA-346 in producing epoxyketone peptides and the feasibility of their production in a heterologous host. This work also highlights the capacity of BRA-346 to tightly regulate its secondary metabolism and shed light on how to awake silent gene clusters of Streptomyces sp. BRA-346 to allow the production of pharmacologically important biosynthetic products.

Dataset Information

Overcoming bortezomib resistance in human B cells by anti-CD20/rituximab-mediated complement-dependent cytotoxicity and epoxyketone-based irreversible proteasome inhibitors.

Unlabelled

Background

Methods

Results

Conclusions

Publications

Overcoming bortezomib resistance in human B cells by anti-CD20/rituximab-mediated complement-dependent cytotoxicity and epoxyketone-based irreversible proteasome inhibitors.

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