Bortezomib Treatment Modulates Autophagy in Multiple Myeloma

Abstract

Although the introduction of bortezomib as a therapeutic strategy has improved the overall survival of multiple myeloma (MM) patients, 15-20% of high-risk patients do not respond to bortezomib over time or become resistant to treatment. Therefore, the development of new therapeutic strategies, such as combination therapies, is urgently needed.

Methods: Given that bortezomib resistance may be mediated by activation of the autophagy pathway as an alternative mechanism of protein degradation, and that an enormous amounts of misfolded protein is generated in myeloma plasma cells (PCs), we investigated the effect of the simultaneous inhibition of proteasome by bortezomib and autophagy by hydroxychloroquine (HCQ) treatment on PCs and endothelial cells (ECs) isolated from patients with monoclonal gammopathy of undetermined significance (MGUS) and MM.

Results: We found that bortezomib combined with HCQ induces synergistic cytotoxicity in myeloma PCs whereas this effect is lost on ECs. Levels of microtubule-associated protein light chain beta (LC3B) and p62 are differentially modulated in PCs and ECs, with effects on cell viability and proliferation.

Conclusions: Our results suggest that treatment with bortezomib and HCQ should be associated with an anti-angiogenic drug to prevent the pro-angiogenic effect of bortezomib, the proliferation of a small residual tumor PC clone, and thus the relapse.

Keywords: angiogenesis; autophagy; bortezomib; drug resistance; endothelial cells; multiple myeloma; plasma cells.

Figure 1

Schematic overview of the experimental design. Cytoplasmic cargo material including mitochondria, membrane, and unfolded proteins is engulfed by a double-membrane vesicle to form and elongate into an autophagosome. Autophagy related (ATG) proteins create two “ubiquitin-like conjugation systems” that catalyze the conversion of LC3B-I to its phosphatidylethanolamine (PE)-conjugated LC3B-II form. LC3B-II protein is recruited to the autophagosome and P62 along with the attached ubiquitinated proteins bind to LC3B-II protein. The complex LC3B-II-p62 is then incorporated into the autophagosome membrane, where it serves as a docking site of adaptor proteins and bound cargo. The mature autophagosome then fuses with the lysosome and forms an autolysosome, leading to cargo degradation and recycling of nutrients and metabolites. Hydroxychloroquine inhibits autophagosome–lysosome fusion and degradation. Thus, HCQ treatment promotes intracellular autophagosome accumulation that correlates with LC3B-II and p62 levels.

Figure 2

Bortezomib and hydroxychloroquine combination decreases autophagosome formation acting as a downregulator of autophagic flux in plasma cells (PCs). RPMI 8226 (A) and JJN-3 (D) cells were treated with or without bortezomib (10 nM), hydroxychloroquine (HCQ, 100 uM), or with both drugs for 24 h, followed by immunoblotting analysis to determine LC3B-II and p62 expression levels under each condition. Densitometric analysis of RPMI 8226 (B,C) and JJN-3 (E,F) lysates for LC3B-II (B,E) and p62 (C,F) expression. The results are expressed as fold-change normalized to the β-actin level and relative to the control. Mann–Whitney U test.

Figure 3

Bortezomib and hydroxychloroquine combination downregulates the autophagic flux in plasma cells (PCs). Changes in LC3B-II and p62 levels upon treatment with hydroxychloroquine (HCQ, 100 uM) alone or both bortezomib (10 nM) and HCQ for 24 h, determined by flow cytometry. Mean Fluorescence intensity (A,C) and representative plots (B,D) of primary plasma cells isolated from MM patients (n = 6). Mann–Whitney U test.

Figure 4

Bortezomib and hydroxychloroquine combination upregulates autophagosome formation in endothelial cells (ECs). (A) Human umbilical vein endothelial cells (HUVECs), (D) ECs from monoclonal gammopathy of undetermined significance (MGECs, n = 9) and ECs from multiple myeloma (MMECs, n = 11) were treated with or without bortezomib (10 nM), HCQ (100 uM), or with both drugs for 24 h, followed by immunoblotting to determine LC3B-II and p62 levels under each condition. (B–F) Densitometric analysis of HUVEC (B,C), MGEC (black bars), and MMEC (gray bars) (E,F) lysates for LC3B-II (B,E) and p62 (C,F) expression. The results are expressed as fold-change normalized to the β-actin level and relative to the control. Mann–Whitney U test.

Figure 5

Bortezomib and hydroxychloroquine combination increases autophagosome number in ECs. (A) HUVECs, MGECs (n = 4), and MMECs (n = 4) were treated with HCQ (100 uM), bortezomib (10 nM), or both drugs. Representative photomicrographs (on the left) and flow cytometry plots (on the right) of four independent experiments are shown. Black arrows indicate autophagosome vacuoles (magnification 20×).

Figure 6

Bortezomib and HCQ differentially modulate MM cell proliferation. RPMI 8226 cells (A), MGECs (n = 6) (B), and MMECs (n = 6) (C) were treated with or without bortezomib (10 nM), HCQ (100 uM) or with both drugs for 24 h, after which the inhibition of proliferation was evaluated by measuring cell viability. Mann–Whitney U test. ns = not significant.

Figure 7

Effect of combination treatment with bortezomib and HCQ on cell cytotoxicity. Representative flow cytometry plots show changes in cytotoxicity for (A) RPMI 8226 cells, (B) MGECs (n = 9), and (C) MMECs (n = 11) treated with or without bortezomib (10 nM), HCQ (100 uM), or with both drugs for 24 h.