Celia R. Berkers

Serine starvation induces stress and p53-dependent metabolic remodelling in cancer cells

Cancer cells acquire distinct metabolic adaptations to survive stress associated with tumour growth and to satisfy the anabolic demands of proliferation. The tumour suppressor protein p53 (also known as TP53) influences a range of cellular metabolic processes, including glycolysis, oxidative phosphorylation, glutaminolysis and anti-oxidant response. In contrast to its role in promoting apoptosis during DNA-damaging stress, p53 can promote cell survival during metabolic stress, a function that may contribute not only to tumour suppression but also to non-cancer-associated functions of p53. Here we show that human cancer cells rapidly use exogenous serine and that serine deprivation triggered activation of the serine synthesis pathway and rapidly suppressed aerobic glycolysis, resulting in an increased flux to the tricarboxylic acid cycle. Transient p53-p21 (also known as CDKN1A) activation and cell-cycle arrest promoted cell survival by efficiently channelling depleted serine stores to glutathione synthesis, thus preserving cellular anti-oxidant capacity. Cells lacking p53 failed to complete the response to serine depletion, resulting in oxidative stress, reduced viability and severely impaired proliferation. The role of p53 in supporting cancer cell proliferation under serine starvation was translated to an in vivo model, indicating that serine depletion has a potential role in the treatment of p53-deficient tumours.

Molecular basis of bortezomib resistance: proteasome subunit β5 (PSMB5) gene mutation and overexpression of PSMB5 protein

The proteasome inhibitor bortezomib is a novel anticancer drug that has shown promise in the treatment of refractory multiple myeloma. However, its clinical efficacy has been hampered by the emergence of drug-resistance phenomena, the molecular basis of which remains elusive. Toward this end, we here developed high levels (45- to 129-fold) of acquired resistance to bortezomib in human myelomonocytic THP1 cells by exposure to stepwise increasing (2.5-200 nM) concentrations of bortezomib. Study of the molecular mechanism of bortezomib resistance in these cells revealed (1) an Ala49Thr mutation residing in a highly conserved bortezomib-binding pocket in the proteasome beta5-subunit (PSMB5) protein, (2) a dramatic overexpression (up to 60-fold) of PSMB5 protein but not of other proteasome subunits including PSMB6, PSMB7, and PSMA7, (3) high levels of cross-resistance to beta5 subunit-targeted cytotoxic peptides 4A6, MG132, MG262, and ALLN, but not to a broad spectrum of chemotherapeutic drugs, (4) no marked changes in chymotrypsin-like proteasome activity, and (5) restoration of bortezomib sensitivity in bortezomib-resistant cells by siRNA-mediated silencing of PSMB5 gene expression. Collectively, these findings establish a novel mechanism of bortezomib resistance associated with the selective overexpression of a mutant PSMB5 protein.

Metabolic Regulation by p53 Family Members

The function of p53 is best understood in response to genotoxic stress, but increasing evidence suggests that p53 also plays a key role in the regulation of metabolic homeostasis. p53 and its family members directly influence various metabolic pathways, enabling cells to respond to metabolic stress. These functions are likely to be important for restraining the development of cancer but could also have a profound effect on the development of metabolic diseases, including diabetes. A better understanding of the metabolic functions of p53 family members may aid in the identification of therapeutic targets and reveal novel uses for p53-modulating drugs.

Activity probe for in vivo profiling of the specificity of proteasome inhibitor bortezomib

Proteasome inhibitors, such as the dipeptide boronic acid bortezomib, are emerging as important tools in the treatment of the fatal hematologic malignancy multiple myeloma. Despite the recent US Food and Drug Administration approval of bortezomib (PS341, Velcade) for the treatment of refractory multiple myeloma, many of the basic pharmacologic parameters of bortezomib and its mode of action on myeloma cells remain to be determined. We describe the synthesis and use of a cell-permeant active site–directed probe, which allows profiling of proteasomal activities in living cells. When we compared proteasome activity patterns in cultured cells and crude cell extracts with this probe, we observed substantial differences, stressing the importance for bioassays compatible with live cells to ensure accuracy of such measurements. Using this probe, we investigated the in vivo subunit specificities of bortezomib and another inhibitor, MG132.

Marizomib, a Proteasome Inhibitor for All Seasons: Preclinical Profile and a Framework for Clinical Trials

The proteasome has emerged as an important clinically relevant target for the treatment of hematologic malignancies. Since the Food and Drug Administration approved the first-in-class proteasome inhibitor bortezomib (Velcade) for the treatment of relapsed/refractory multiple myeloma (MM) and mantle cell lymphoma, it has become clear that new inhibitors are needed that have a better therapeutic ratio, can overcome inherent and acquired bortezomib resistance and exhibit broader anti-cancer activities. Marizomib (NPI-0052; salinosporamide A) is a structurally and pharmacologically unique β-lactone-γ-lactam proteasome inhibitor that may fulfill these unmet needs. The potent and sustained inhibition of all three proteolytic activities of the proteasome by marizomib has inspired extensive preclinical evaluation in a variety of hematologic and solid tumor models, where it is efficacious as a single agent and in combination with biologics, chemotherapeutics and targeted therapeutic agents. Specifically, marizomib has been evaluated in models for multiple myeloma, mantle cell lymphoma, Waldenstroms macroglobulinemia, chronic and acute lymphocytic leukemia, as well as glioma, colorectal and pancreatic cancer models, and has exhibited synergistic activities in tumor models in combination with bortezomib, the immunomodulatory agent lenalidomide (Revlimid), and various histone deacetylase inhibitors. These and other studies provided the framework for ongoing clinical trials in patients with MM, lymphomas, leukemias and solid tumors, including those who have failed bortezomib treatment, as well as in patients with diagnoses where other proteasome inhibitors have not demonstrated significant efficacy. This review captures the remarkable translational studies and contributions from many collaborators that have advanced marizomib from seabed to bench to bedside.

Endoplasmic reticulum stress activates autophagy but not the proteasome in neuronal cells: implications for Alzheimer's disease

Protein folding stress in the endoplasmic reticulum (ER) may lead to activation of the unfolded protein response (UPR), aimed to restore cellular homeostasis via transcriptional and post-transcriptional mechanisms. ER stress is also reported to activate the ER overload response (EOR), which activates transcription via NF-κB. We previously demonstrated that UPR activation is an early event in pre-tangle neurons in Alzheimers disease (AD) brain. Misfolded and unfolded proteins are degraded via the ubiquitin proteasome system (UPS) or autophagy. UPR activation is found in AD neurons displaying both early UPS pathology and autophagic pathology. Here we investigate whether activation of the UPR and/or EOR is employed to enhance the proteolytic capacity of neuronal cells. Expression of the immunoproteasome subunits β2i and β5i is increased in AD brain. However, expression of the proteasome subunits is not increased by the UPR or EOR. UPR activation does not relocalize the proteasome or increase overall proteasome activity. Therefore proteasomal degradation is not increased by ER stress. In contrast, UPR activation enhances autophagy and LC3 levels are increased in neurons displaying UPR activation in AD brain. Our data suggest that autophagy is the major degradational pathway following UPR activation in neuronal cells and indicate a connection between UPR activation and autophagic pathology in AD brain.

Activity patterns of proteasome subunits reflect bortezomib sensitivity of hematologic malignancies and are variable in primary human leukemia cells.

Proteasomal proteolysis relies on the activity of six catalytically active proteasomal subunits (β1, β2, β5, β1i, β2i and β5i). Applying a functional proteomics approach, we used a recently developed activity-based, cell-permeable proteasome-specific probe that for the first time allows differential visualization of individual active proteasomal subunits in intact primary cells. In primary leukemia samples, we observed remarkable variability in the amounts of active β1/1i-, β2/2i- and β5/5i-type of subunits, contrasting with their constant protein expression. Bortezomib inhibited β5- and β1-type, but to a lesser extend β2-type of subunits in live primary cells in vitro and in vivo. When we adapted the bortezomib-sensitive human acute myeloid leukemia cell line HL-60 to bortezomib 40 nM (HL-60a), proteasomal activity profiling revealed an upregulation of active subunits, and residual β1/β5-type of activity could be visualized in the presence of bortezomib 20 nM, in contrast to control cells. In a panel of cell lines from hematologic malignancies, the ratio between β2-type and (β1+β5)-type of active proteasomal polypeptides mirrored different degrees of bortezomib sensitivity. We thus conclude that the proteasomal activity profile varies in primary leukemia cells, and that the pattern of proteasomal subunit activity influences the sensitivity of hematologic malignancies toward bortezomib.

Conditional MHC class I ligands and peptide exchange technology for the human MHC gene products HLA-A1, -A3, -A11, and -B7

Major histocompatibility complex (MHC) class I multimer technology has become an indispensable immunological assay system to dissect antigen-specific cytotoxic CD8+ T cell responses by flow cytometry. However, the development of high-throughput assay systems, in which T cell responses against a multitude of epitopes are analyzed, has been precluded by the fact that for each T cell epitope, a separate in vitro MHC refolding reaction is required. We have recently demonstrated that conditional ligands that disintegrate upon exposure to long-wavelength UV light can be designed for the human MHC molecule HLA-A2. To determine whether this peptide-exchange technology can be developed into a generally applicable approach for high throughput MHC based applications we set out to design conditional ligands for the human MHC gene products HLA-A1, -A3, -A11, and -B7. Here, we describe the development and characterization of conditional ligands for this set of human MHC molecules and apply the peptide-exchange technology to identify melanoma-associated peptides that bind to HLA-A3 with high affinity. The conditional ligand technology developed here will allow high-throughput MHC-based analysis of cytotoxic T cell immunity in the vast majority of Western European individuals.

Automated Online Sequential Isotope Labeling for Protein Quantitation Applied to Proteasome Tissue-specific Diversity

Quantitation of protein abundance is a vital component in the proteomic analysis of biological systems, which can be achieved by differential stable isotopic labeling. To analyze tissue-derived samples, the isotopic labeling can be performed using chemical labeling of the peptides post-digestion. Standard chemical labeling procedures often require many manual sample handling steps, reducing the accuracy of measurements. Here, we describe a fully automated, online (in nanoLC columns), labeling procedure, which allows protein quantitation using differential isotopic dimethyl labeling of peptide N termini and lysine residues. We show that the method allows reliable quantitation over a wide dynamic range and can be used to quantify differential protein abundances in lysates and, more targeted, differences in composition between purified protein complexes. We apply the method to determine the differences in composition between bovine liver and spleen 20 S core proteasome complexes. We find that although all catalytically active immunoproteasome subunits were up-regulated in spleen (compared with liver), only one of the normal catalytic subunits was down-regulated, suggesting that the tissue-specific immunoproteasome assembly is more diverse than previously assumed.

Early adipogenesis is regulated through USP7-mediated deubiquitination of the histone acetyltransferase TIP60

Transcriptional coregulators, including the acetyltransferase Tip60, have a key role in complex cellular processes such as differentiation. Whereas post-translational modifications have emerged as an important mechanism to regulate transcriptional coregulator activity, the identification of the corresponding demodifying enzymes has remained elusive. Here we show that the expression of the Tip60 protein, which is essential for adipocyte differentiation, is regulated through polyubiquitination on multiple residues. USP7, a dominant deubiquitinating enzyme in 3T3-L1 adipocytes and mouse adipose tissue, deubiquitinates Tip60 both in intact cells and in vitro and increases Tip60 protein levels. Furthermore, inhibition of USP7 expression and activity decreases adipogenesis. Transcriptome analysis reveals several cell cycle genes to be co-regulated by both Tip60 and USP7. Knockdown of either factor results in impaired mitotic clonal expansion, an early step in adipogenesis. These results reveal deubiquitination of a transcriptional coregulator to be a key mechanism in the regulation of early adipogenesis.