Exocytosis of polyubiquitinated proteins in bortezomib-resistant leukemia cells: a role for MARCKS in acquired resistance to proteasome inhibitors

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.

Keywords: MARCKS; bortezomib; leukemia; proteasome; resistance.

Figure 1. Differential miRNA expression between BTZ-resistant CEM cells and CEM/WT

Red color represents upregulation, green color downregulation.

Figure 2. Top 50 upregulated and top 50 significantly down-regulated genes (p < 0.05) comparing CEM/WT_BTZ (CEM/WT cells incubated for 24 hrs with 7 nM BTZ) to CEM/BTZ7 (CEM cells resistant to 7 nM BTZ)

The figure depicts this selection of genes for all CEM variations as a ratio of the untreated CEM/WT cells. (Clustering performed using Cluster 3.0) An overview of top 50 upregulated and top 50 down-regulated genes is depicted. For genes in cluster 1 and cluster 2, gene annotation and function are provided.

Figure 3

(A) Top 20 up- and downregulated genes (p < 0.05) and (B) proteasomal genes comparing CEM/WT_BTZ with CEM/BTZ7 (p < 0.05). Results for all CEM variations are depicted as a ratio of the untreated CEM/WT. Red color represents upregulation, whereas green downregulation of expression.

Figure 4

(A) MARCKS protein expression of CEM and THP-1 cells and their BTZ resistant sublines CEM/BTZ200 and THP-1/BTZ200. In addition, the CEM/BTZ200 cultured in absence of BTZ for several months (CEM/BTZ-200) is also included. Western blot analysis of MARCKS (green) and β-actin (red) as a loading control, (B) Ratio of the quantification of MARCKS protein expression (β-actin normalized) of proteasome inhibitor (PR924 and Marizomib) resistant THP-1 and CEM cells relative to their parental cells. (C) Basal levels of phosphorylated MARCKS expression in CEM/WT and CEM/BTZ cells before and after incubation with PMA (50 nM for 1 hour,), and a combination of PMA and RNS (1 hour 100 nM) or MANS (1 hour 100 μM).

Figure 5. Fluorescence microscopy of vesicles staining in WT and BTZ-resistant CEM cells, primary patient samples, and HeLa cells

(A) MARCKS and ubiquitin protein expression in CEM/WT cells, CEM/BTZ7 cells treated with 30 nM BTZ for 24 hours and CEM/BTZ200 cells treated with 400 nM BTZ for 24 hours. Left panel: DAPI nuclear staining (Blue), ubiquitin (Green), and MARCKS (Red), right panel: level of co-localization of MARCKS with ubiquitin as calculated by the SlideBook microscope software as depicted in blue (normal) to magenta (strong co-localization). Single channel images are shown in Supplementary Figure S4. (B) Ubiquitin (green) and DAPI (blue) staining in HeLa cells after 24 hour incubation with the supernatants of cultures of CEM/WT, CEM/BTZ7 and CEM/BTZ200 cells untreated or treated with the indicated concentrations of BTZ for 1 hour. (C) PKH labeling and ubiquitin protein expression in CEM/WT cells, CEM/BTZ7 and CEM/BTZ200 cells treated with the abovementioned concentrations of BTZ for 24 hours. DAPI nuclear staining (Blue), PKH (Green), and ubiquitin (Red) and level of colocalization of MARCKS with ubiquitin as calculated by the SlideBook microscope software as depicted in blue to magenta (Right panel). (D) Ubiquitin (red) and PKH (green) staining in HeLa cells after 24 hour incubation with the supernatants of cultures of CEM/WT, CEM/BTZ7 and CEM/BTZ200 cells untreated or treated with the indicated concentrations of BTZ. Calculated co-localization is depicted in yellow. (E) Ubiquitin (green) and MARCKS (red) staining in a primary ALL patient samples, one sensitive to BTZ (LC50: BTZ 6 nM) and one resistant to BTZ (LC50 BTZ: 262 nM) (F) without and after incubation with low concentration (7 nM) or high concentration of BTZ (400 nM). In the merge figure, the co-localization of the two proteins is shown in yellow. The right top of the figures depict a quantification using a line scan through the corresponding area of interest (α and β).

Figure 6. Pre-treatment MARCKS protein expression (Western blot) in primary ALL samples stratified according to response to BTZ-containing treatment (Complete remission (CR) after induction chemotherapy vs no CR)

Data are presented after normalization to β-actin. For both groups, the mean and standard error of the mean (SEM) is depicted.

Figure 7. Summary model of the involvement of MARCKS and vesicular exocytosis of ubiquitinated proteins in BTZ-resistant leukemia cells

CEM/BTZ cells harboring PSMB5 mutations (indicated by *) have a diminished capacity of inhibition of proteasomal catalytic activity by BTZ [29, 31]. Upon exposure of CEM/BTZ7 and CEM/BTZ200 cells to BTZ concentrations that block PSMB5 activity (30 nM and 400 nM BTZ, respectively), these cells accumulate polyubiquitinated proteins. This coincides with the biogenesis of vesicle-like structures incorporating these ubiquitinated proteins. These vesicles then traffic along actin/cytoskeleton axis to the plasma membrane with MARCKS protein serving as partner protein. Here myristoylated-anchored MARCKS facilitates exocytosis of vesicles which subsequently can be taken up by recipient (HeLa) cells. Proficient proteasomal activity in recipient cells would allow degradation of ubiquitinated proteins from BTZ-resistant cells. Through a mechanism of exocytosis-mediated extrusion of vesicles containing ubiquitinated proteins, BTZ-resistant cells can overcome proteolytic stress over a broad range of BTZ concentrations.