Abstract
PSMB5 mutations and upregulation of the β5 subunit of the proteasome represent key determinants of acquired resistance to the proteasome inhibitor bortezomib (BTZ) in leukemic cells in vitro. We here undertook a multi-modality (DNA, mRNA, miRNA) array-based analysis of human CCRF-CEM leukemia cells and BTZ-resistant subclones to determine whether or not complementary mechanisms contribute to BTZ resistance. These studies revealed signatures of markedly reduced expression of proteolytic stress related genes in drug resistant cells over a broad range of BTZ concentrations along with a high upregulation of myristoylated alanine-rich C-kinase substrate (MARCKS) gene expression. MARCKS upregulation was confirmed on protein level and also observed in other BTZ-resistant tumor cell lines as well as in leukemia cells with acquired resistance to other proteasome inhibitors. Moreover, when MARCKS protein expression was demonstrated in specimens derived from therapy-refractory pediatric leukemia patients (n = 44), higher MARCKS protein expression trended (p = 0.073) towards a dismal response to BTZ-containing chemotherapy. Mechanistically, we show a BTZ concentration-dependent association of MARCKS protein levels with the emergence of ubiquitin-containing vesicles in BTZ-resistant CEM cells. These vesicles were found to be extruded and taken up in co-cultures with proteasome-proficient acceptor cells. Consistent with these observations, MARCKS protein associated with ubiquitin-containing vesicles was also more prominent in clinical leukemic specimen with ex vivo BTZ resistance compared to BTZ-sensitive leukemia cells. Collectively, we propose a role for MARCKS in a novel mechanism of BTZ resistance via exocytosis of ubiquitinated proteins in BTZ-resistant cells leading to quenching of proteolytic stress.
Highlights
The proteasome inhibitor Bortezomib (BTZ, Velcade®) is registered for the treatment of multiple myeloma (MM) and mantle cell lymphoma [1, 2] and is currently undergoing clinical evaluation in other hematological malignancies, such as pediatric acute leukemia [3]
Given the overexpression of myristoylated alanine-rich C-kinase substrate (MARCKS) in BTZresistant leukemia cells, we explored whether or not MARCKS overexpression may serve as predictive marker of BTZ unresponsiveness in clinical samples of acute leukemia patients
The current study reports a novel mechanism of exocytosis-mediated extrusion of vesicle-like structures containing ubiquitinated proteins in BTZ-resistant CEM leukemia cells as a novel modality to overcome proteolytic stress over a broad range of cytotoxic BTZ concentrations
Summary
The proteasome inhibitor Bortezomib (BTZ, Velcade®) is registered for the treatment of multiple myeloma (MM) and mantle cell lymphoma [1, 2] and is currently undergoing clinical evaluation in other hematological malignancies, such as pediatric acute leukemia [3]. Examining the ability of BTZ to inhibit the catalytic activity of the mutated β5 subunit revealed a 2-fold lower potency as compared to non-mutated β5 subunits, whereas the cell growth inhibitory capacity was repressed by a factor of > 100 fold [29, 41] These findings suggest that BTZ resistant cells acquired additional compensatory mechanism(s) to cope with the proteolytic stress. To gain further insight into these underlying molecular mechanisms, we undertook a multi-modality (DNA, mRNA, miRNA) array-based analysis of human CCRF-CEM leukemia cells and two subclones harboring PSMB5 mutations, one with a moderate and one with a high level BTZ resistance These studies revealed a highly upregulated myristoylated alanine-rich C-kinase substrate (MARCKS) gene expression which correlated with protein expression. The relevance of this novel function of MARCKs in BTZ resistance was further corroborated in BTZ and second generation proteasome inhibitor resistant hematological cell lines, ex vivo BTZresistant pediatric ALL cells, and clinical specimens of ALL children receiving BTZ-containing chemotherapy
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