Celia R. Berkers(1), Esther A. Zaal(1), Pieter Langerhorst(1), Gerrit Jansen(2), Jacqueline Cloos(3)
(1)Biomolecular Mass Spectrometry and Proteomics, Utrecht University, (2)Department of Rheumatology, VU University Medical Center, (3)Department of Hematology, VU University Medical Center
Proteasome inhibition has emerged as an important strategy for the treatment of cancer. However, treatment with proteasome inhibitors is often hampered by the occurrence of both primary and acquired resistance. We embarked on unravelling the metabolic mode of resistance to the proteasome inhibitor bortezomib. To this end, we profiled bortezomib-sensitive and -resistant cell lines, combining steady-state metabolomics screens with metabolic flux studies using stable isotope labelling approaches. Our studies revealed that the metabolic profiles of bortezomib-sensitive and resistant cells differed significantly. In particular, resistant cells were more dependent on the uptake of specific nutrients from their environment. Together, our data indicate a potential role for nutrient starvation in the treatment of bortezomib-resistant tumours.
The 26S proteasome, a 2.5 MDa protease complex, is responsible for the controlled turnover of misfolded and damaged proteins and for the degradation of many short-lived proteins. In the latter capability, the proteasome controls a wide range of cellular processes including cell cycle progression, cell differentiation, apoptosis, stress response and DNA repair, and is thereby vital for maintaining cellular homeostasis. Proteasome inhibition is more cytotoxic to malignant cells than to healthy cells, establishing the proteasome as an attractive pharmacological target for the treatment of cancer. Bortezomib (velcade®), the first-in-class proteasome inhibitor, has been approved for the treatment of of multiple myeloma and mantle cell lymphoma and several second-generation proteasome inhibitors are in clinical trials. Results of bortezomib treatment in solid tumours, however, have been disappointing, and although many hematologic malignancies initially seem to respond well to proteasome inhibitor therapy, treatment is often hampered by the rapid development of resistance. Mechanisms contributing to primary and/or acquired resistance have been reported, but the factors determining the occurrence of resistance remain largely elusive. We hypothesise that bortezomib resistance is associated with changes in the metabolism of tumour cells, also called metabolic remodelling. Interestingly, cells may become reliant on such drug-induced metabolic reprogramming, a vulnerability that may be exploited for therapy. In this study, we aim to elucidate the metabolic pathways involved in bortezomib resistance using a mass spectrometry-based metabolomics approach.
To study which metabolic pathways are involved in bortezomib resistance, bortezomib-sensitive and -resistant RPMI8226 multiple myeloma cell lines were used as a model system. These cells have acquired resistance by exposure to increasing concentrations of bortezomib. To investigate up- or down-regulation of metabolic pathway activities in bortezomib-sensitive versus bortezomib-resistant cells, metabolomics studies were performed, consisting of two complementary types of analyses. First, comprehensive steady-state metabolomic screens were performed to obtain unbiased insight in changes in metabolite concentrations in major metabolic pathways in the cell. Identified pathways of interest were subsequently studied in further detail in metabolic flux studies using stable isotope labelling approaches. To this end, we established LC/MS methods to study a wide variety of pathways involved in energy, amino acid, fatty acid and nucleotide metabolism and to monitor the oxidative stress response, using different stable isotope-labelled nutrients in combination with various extraction and separation methods, including HILIC and reversed-phase chromatography. As many pathways result in the creation of the same metabolites, such flux studies are indispensable to resolve intersecting pathways.
Metabolic screens of bortezomib sensitive and -resistant cell lines showed substantial differences in the metabolic profiles of these cells, suggesting that metabolic changes contribute to bortezomib resistance. Metabolic flux studies demonstrated that bortezomib-resistant cells are forced to use a large part of their glucose uptake for anti-oxidant purposes. Because glucose supply is limited, this resulted in metabolic stress on other pathways that also depend on glucose. Importantly, this metabolic stress could be exploited. Bortezomib-resistant cell showed increased uptake of specific nutrients from their environment and cell viability assays showed that bortezomib-resistant cells were less viable when starved for these nutrients compared to bortezomib-sensitive cells. In addition, nutrient starvation resensitised bortezomib-resistant cells to the drug.
Taken together, our data identify metabolic remodelling of specific pathways as a novel mechanism that contributes to bortezomib-resistance in multiple myeloma cells. In addition, bortezomib-resistant cells appear to become reliant on their rewired metabolism, and in particular on the uptake of specific nutrients from their environment. These findings indicate a potential role for nutrient starvation in the treatment of bortezomib-resistant tumours.