Karianne Schuurman

Boronic acid-based inhibitor of autotaxin reveals rapid turnover of LPA in the circulation

Autotaxin (ATX) is a secreted nucleotide pyrophosphatase/phosphodiesterase that functions as a lysophospholipase D to produce the lipid mediator lysophosphatidic acid (LPA), a mitogen, chemoattractant, and survival factor for many cell types. The ATX-LPA signaling axis has been implicated in angiogenesis, chronic inflammation, fibrotic diseases and tumor progression, making this system an attractive target for therapy. However, potent and selective nonlipid inhibitors of ATX are currently not available. By screening a chemical library, we have identified thiazolidinediones that selectively inhibit ATX-mediated LPA production both in vitro and in vivo. Inhibitor potency was approximately 100-fold increased (IC50 ∼ 30 nM) after the incorporation of a boronic acid moiety, designed to target the active-site threonine (T210) in ATX. Intravenous injection of this inhibitor into mice resulted in a surprisingly rapid decrease in plasma LPA levels, indicating that turnover of LPA in the circulation is much more dynamic than previously appreciated. Thus, boronic acid-based small molecules hold promise as candidate drugs to target ATX.

Definition of Proteasomal Peptide Splicing Rules for High-Efficiency Spliced Peptide Presentation by MHC Class I Molecules

Peptide splicing, in which two distant parts of a protein are excised and then ligated to form a novel peptide, can generate unique MHC class I–restricted responses. Because these peptides are not genetically encoded and the rules behind proteasomal splicing are unknown, it is difficult to predict these spliced Ags. In the current study, small libraries of short peptides were used to identify amino acid sequences that affect the efficiency of this transpeptidation process. We observed that splicing does not occur at random, neither in terms of the amino acid sequences nor through random splicing of peptides from different sources. In contrast, splicing followed distinct rules that we deduced and validated both in vitro and in cells. Peptide ligation was quantified using a model peptide and demonstrated to occur with up to 30% ligation efficiency in vitro, provided that optimal structural requirements for ligation were met by both ligating partners. In addition, many splicing products could be formed from a single protein. Our splicing rules will facilitate prediction and detection of new spliced Ags to expand the peptidome presented by MHC class I Ags.

Probing the specificity and activity profiles of the proteasome inhibitors bortezomib and delanzomib.

The ubiquitin proteasome system is an attractive pharmacological target for the treatment of cancer. The proteasome inhibitor bortezomib has been approved for the treatment of multiple myeloma and mantle cell lymphoma but is associated with substantial adverse effects and the occurrence of resistance, underscoring the continued need for novel proteasome inhibitors. In this study, bortezomib and the novel proteasome inhibitor delanzomib were compared for their ability to inhibit proteasome activity using both fluorogenic substrates and a recently developed fluorescent proteasome activity probe. Bortezomib and delanzomib were equipotent in inhibiting distinct subunits of the proteasome in a panel of cell lines in vitro. In a preclinical multiple myeloma model, both inhibitors inhibited the proteasome in normal tissues to a similar extent. Tumor proteasome activity was inhibited to a significantly higher extent by delanzomib (60%) compared to bortezomib (32%). In addition, delanzomib was able to overcome bortezomib resistance in vitro. The present findings demonstrate that proteasome activity probes can accurately monitor the effects of proteasome inhibitors on both normal and tumor tissues in preclinical models and can be used as a diagnostic approach to predict resistance against treatment with proteasome inhibitors. Furthermore, the data presented here provide rationale for further clinical development of delanzomib.

Quantifying cross-tissue diversity in proteasome complexes by mass spectrometry

The composition of 20S mouse proteasome complexes isolated from mice heart, kidney, liver, lung, thymus and spleen was compared using quantitative mass spectrometry. Significant variety was observed in hybrid classes of immunoproteasomes which may have implications for the use of proteasome targeted inhibitors.

Peptide Splicing in the Proteasome Creates a Novel Type of Antigen with an Isopeptide Linkage

The proteasome is able to create spliced Ags, in which two distant parts of a protein are excised and ligated together to form a novel peptide, for presentation by MHC class I molecules. These noncontiguous epitopes are generated via a transpeptidation reaction catalyzed by the proteasomal active sites. Transpeptidation reactions in the proteasome follow explicit rules and occur particularly efficiently when the C-terminal ligation partner contains a lysine or arginine residue at the site of ligation. Lysine contains two amino groups that theoretically may both participate in ligation reactions, implying that potentially not only peptide but also isopeptide linkages could be formed. Using nuclear magnetic resonance spectroscopy, we demonstrate in the present study that the proteasome can use the ε-amino group of an N-terminal lysine residue in transpeptidation reactions to create a novel type of posttranslationally modified epitopes. We show that the overall efficiency of ε ligation is only 10-fold lower as compared with α ligation, suggesting that the proteasome can produce sufficient isopeptide Ag to evoke a T cell response. Additionally, we show that isopeptides are more stable toward further proteasomal processing than are normal peptides, and we demonstrate that isopeptides can bind to HLA-A2.1 and HLA-A3 with high affinity. These properties likely increase the fraction of ε-ligated peptides presented on the cell surface for CD8+ T cell surveillance. Finally, we show that isopeptide Ags are immunogenic in vivo. We postulate that ε ligation is a genuine posttranslational modification, suggesting that the proteasome can create a novel type of Ag that is likely to play a role in immunity.

Fluorescence-Based Proteasome Activity Profiling

With the proteasome emerging as a therapeutic target for cancer treatment, accurate tools for monitoring proteasome (inhibitor) activity are in demand. In this chapter, we describe the synthesis and use of a fluorescent proteasome activity probe that allows for accurate profiling of proteasomal activity in cell lysates, intact cells, and murine and human patient-derived material, with high sensitivity using SDS-PAGE. The probe allows for direct scanning of the gel for fluorescent emission of the distinct proteasomal subunits and circumvents the use of Western blot analysis. Due to its suitable biochemical and biophysical properties, the fluorescent probe can also be used for confocal laser scanning microscopy and flow cytometry-based experiments.

Comparative Cistromics Reveals Genomic Cross-talk between FOXA1 and ERα in Tamoxifen-Associated Endometrial Carcinomas.

Tamoxifen, a small-molecule antagonist of the transcription factor estrogen receptor alpha (ERα) used to treat breast cancer, increases risks of endometrial cancer. However, no parallels of ERα transcriptional action in breast and endometrial tumors have been found that might explain this effect. In this study, we addressed this issue with a genome-wide assessment of ERα-chromatin interactions in surgical specimens obtained from patients with tamoxifen-associated endometrial cancer. ERα was found at active enhancers in endometrial cancer cells as marked by the presence of RNA polymerase II and the histone marker H3K27Ac. These ERα binding sites were highly conserved between breast and endometrial cancer and enriched in binding motifs for the transcription factor FOXA1, which displayed substantial overlap with ERα binding sites proximal to genes involved in classical ERα target genes. Multifactorial ChIP-seq data integration from the endometrial cancer cell line Ishikawa illustrated a functional genomic network involving ERα and FOXA1 together with the enhancer-enriched transcriptional regulators p300, FOXM1, TEAD4, FNFIC, CEBP8, and TCF12. Immunohistochemical analysis of 230 primary endometrial tumor specimens showed that lack of FOXA1 and ERα expression was associated with a longer interval between breast cancer and the emergence of endometrial cancer, exclusively in tamoxifen-treated patients. Our results define conserved sites for a genomic interplay between FOXA1 and ERα in breast cancer and tamoxifen-associated endometrial cancer. In addition, FOXA1 and ERα are associated with the interval time between breast cancer and endometrial cancer only in tamoxifen-treated breast cancer patients. Cancer Res; 76(13); 3773-84. ©2016 AACR.

Characterizing steroid hormone receptor chromatin binding landscapes in male and female breast cancer

Male breast cancer (MBC) is rare and poorly characterized. Like the female counterpart, most MBCs are hormonally driven, but relapse after hormonal treatment is also noted. The pan-hormonal action of steroid hormonal receptors, including estrogen receptor alpha (ERα), androgen receptor (AR), progesterone receptor (PR), and glucocorticoid receptor (GR) in this understudied tumor type remains wholly unexamined. This study reveals genomic cross-talk of steroid hormone receptor action and interplay in human tumors, here in the context of MBC, in relation to the female disease and patient outcome. Here we report the characterization of human breast tumors of both genders for cistromic make-up of hormonal regulation in human tumors, revealing genome-wide chromatin binding landscapes of ERα, AR, PR, GR, FOXA1, and GATA3 and enhancer-enriched histone mark H3K4me1. We integrate these data with transcriptomics to reveal gender-selective and genomic location-specific hormone receptor actions, which associate with survival in MBC patients.Male breast cancer (MBC) is rare and largely hormonally driven. Here, the authors examine the action of steroid hormone receptors in male and female breast cancers and find gender selective hormone receptor action that associates with the survival of MBC patients.

Loss of steroid hormone receptors is common in malignant pleural and peritoneal effusions of breast cancer patients treated with endocrine therapy

Discordance in estrogen receptor alpha (ERα), progesterone receptor (PR), androgen receptor (AR) and human epidermal growth factor receptor 2 (HER2) status between primary breast cancers and solid distant metastases (“conversion”) has been reported previously. Even though metastatic spread to the peritoneal and pleural cavities occurs frequently and is associated with high mortality, the rate of receptor conversion and the prognostic implications thereof remain elusive. We therefore determined receptor conversion in 91 effusion metastases (78 pleural, 13 peritoneal effusions) of 69 patients by immunohistochemistry (IHC) and in situ hybridization. Data were coupled to clinical variables and treatment history. ERα, PR and AR receptor status converted from positive in the primary tumor to negative in the effusion metastases or vice versa in 25-30%, 30-35% and 46-51% of cases for the 1% and 10% thresholds for positivity, respectively. 19-25% of patients converted clinically relevant from “ERα+ or PR+” to ERα-/PR- and 3-4% from ERα-/PR- to “ERα+ or PR+”. For HER2, conversion was observed in 6% of cases. Importantly, receptor conversion for ERα (p = 0.058) and AR (p < 0.001) was more often seen in patients adjuvantly treated with endocrine therapy. Analogous to this observation, HER2-loss was more frequent in patients adjuvantly treated with trastuzumab (p < 0.001). Alike solid distant metastases, receptor conversion for ERα, PR, AR and HER2 is a frequent phenomenon in peritoneal and pleural effusion metastases. Adjuvant endocrine and trastuzumab therapy imposes an evolutionary selection pressure on the tumor, leading to receptor loss in effusion metastases. Determination of receptor status in malignant effusion specimens will facilitate endocrine treatment decision-making at this lethal state of the disease, and is hence recommended whenever possible.

FOXA1 levels are decreased in pleural breast cancer metastases after adjuvant endocrine therapy, and this is associated with poor outcome

Estrogen receptor‐alpha (ERα)‐positive breast cancer is often treated with antihormonal regimens. However, resistance to treatment is common, leading to metastatic disease. ERα activity requires the functional involvement of pioneer factors FOXA1 and GATA3, which enable ERα–chromatin binding and are crucial for ERα‐driven cell proliferation. FOXA1 was found increased in metastatic breast cancers in relation to the primary tumor, but a comprehensive clinical assessment thereof, in relation to different metastatic sites and endocrine therapy usage, is currently lacking. Prior cell line‐based reports, however, have revealed that FOXA1 is required for tamoxifen‐resistant tumor cell proliferation. We studied expression levels of ERα, GATA3, and FOXA1 by immunohistochemistry in samples from both primary tumors and various metastatic sites. For all factors, expression levels varied between the metastatic sites. For pleural metastases, strong variation was found in FOXA1 and GATA3 levels. Although GATA3 levels remained unaltered between primary breast cancer and pleural metastases, FOXA1 levels were reduced exclusively in metastases of patients who received endocrine therapies in the adjuvant setting, even though ERα was still expressed. Importantly, decreased FOXA1 levels in pleural metastases correlated with hormone irresponsiveness in the palliative setting, while no such correlation was found for GATA3. With this, we show divergent clinical correlations of the two ERα pioneer factors FOXA1 and GATA3 in metastatic breast cancer, where endocrine therapy resistance was associated with decreased FOXA1 levels in pleural metastases.