Tryptophan depletion results in tryptophan-to-phenylalanine substitutants

Activated T cells secrete interferon-γ, which triggers intracellular tryptophan shortage by upregulating the indoleamine 2,3-dioxygenase 1 (IDO1) enzyme1–4. Here we show that despite tryptophan depletion, in-frame protein synthesis continues across tryptophan codons. We identified tryptophan-to-phenylalanine codon reassignment (W>F) as the major event facilitating this process, and pinpointed tryptophanyl-tRNA synthetase (WARS1) as its source. We call these W>F peptides ‘substitutants’ to distinguish them from genetically encoded mutants. Using large-scale proteomics analyses, we demonstrate W>F substitutants to be highly abundant in multiple cancer types. W>F substitutants were enriched in tumours relative to matching adjacent normal tissues, and were associated with increased IDO1 expression, oncogenic signalling and the tumour-immune microenvironment. Functionally, W>F substitutants can impair protein activity, but also expand the landscape of antigens presented at the cell surface to activate T cell responses. Thus, substitutants are generated by an alternative decoding mechanism with potential effects on gene function and tumour immunoreactivity

Mounting an attack on the glioblastoma triad : proliferation, invasion and resistance

Despite decades of research, glioblastoma (GBM) remains a formidable opponent and patients suffering from this primary brain tumor still have a dismal prognosis. Following extensive treatment involving surgery, ionizing radiotherapy and temozolomide chemotherapy, the median overall survival is still a mere 15 months. Large-scale efforts to characterize GBM have delivered major insights into its genomic landscape, epigenetic profile and micro-environmental context. Together, these studies have revealed that GBM is a collection of diseases. Today, we recognize up to 5 different subtypes of glioblastoma based on genomic, epigenomic and expression profiling: classical, proneural, G-CIMP, mesenchymal and neural GBM. Moreover, GBMs are very heterogeneous and typically consist of more than 1 subtype. Importantly, these subtypes appear to exhibit a high level of plasticity, as GBM cells can rapidly change their subtype as a result of selective pressures such as anticancer treatment. Although there is substantial heterogeneity, the aforementioned characterization efforts have also pinpointed various commonalities between GBMs that offer potential handles for therapeutic approaches. This thesis investigates several of these new approaches, but also studies ways to further improve the efficacy of classical treatment modalities such as chemotherapy and radiotherapy. The ultimate aim is to mount a full-blown attack on three main pillars of GBM biology: uncontrolled proliferation, an unparalleled level of invasion into surrounding healthy brain tissue, and tremendous resistance to therapy. Together, these properties form the glioblastoma triad. This thesis investigates all three pillars separately and identifies efficient ways to attack each individually. However, while modest antitumor efficacy can be achieved by individual targeting, an intelligently designed therapeutic approach that attacks the entire glioblastoma triad will be necessary to achieve durable clinical responses

Mounting an attack on the glioblastoma triad : proliferation, invasion and resistance

Despite decades of research, glioblastoma (GBM) remains a formidable opponent and patients suffering from this primary brain tumor still have a dismal prognosis. Following extensive treatment involving surgery, ionizing radiotherapy and temozolomide chemotherapy, the median overall survival is still a mere 15 months. Large-scale efforts to characterize GBM have delivered major insights into its genomic landscape, epigenetic profile and micro-environmental context. Together, these studies have revealed that GBM is a collection of diseases. Today, we recognize up to 5 different subtypes of glioblastoma based on genomic, epigenomic and expression profiling: classical, proneural, G-CIMP, mesenchymal and neural GBM. Moreover, GBMs are very heterogeneous and typically consist of more than 1 subtype. Importantly, these subtypes appear to exhibit a high level of plasticity, as GBM cells can rapidly change their subtype as a result of selective pressures such as anticancer treatment. Although there is substantial heterogeneity, the aforementioned characterization efforts have also pinpointed various commonalities between GBMs that offer potential handles for therapeutic approaches. This thesis investigates several of these new approaches, but also studies ways to further improve the efficacy of classical treatment modalities such as chemotherapy and radiotherapy. The ultimate aim is to mount a full-blown attack on three main pillars of GBM biology: uncontrolled proliferation, an unparalleled level of invasion into surrounding healthy brain tissue, and tremendous resistance to therapy. Together, these properties form the glioblastoma triad. This thesis investigates all three pillars separately and identifies efficient ways to attack each individually. However, while modest antitumor efficacy can be achieved by individual targeting, an intelligently designed therapeutic approach that attacks the entire glioblastoma triad will be necessary to achieve durable clinical responses

ATP-binding cassette transporters limit the brain penetration of Wee1 inhibitors

Introduction Wee1 is an important kinase involved in the G2 cell cycle checkpoint and frequently upregulated in intracranial neoplasms such as glioblastoma (GBM) and diffuse intrinsic pontine glioma (DIPG). Two small molecules are available that target Wee1, AZD1775 and PD0166285, and clinical trials with AZD1775 have already been started. Since GBM and DIPG are highly invasive brain tumors, they are at least to some extent protected by the blood-brain barrier (BBB) and its ATP-binding cassette (ABC) efflux transporters. Methods We have here conducted a comprehensive set of in vitro and in vivo experiments to determine to what extent two dominant efflux transporters in the BBB, P-gp (ABCB1) and BCRP (ABCG2), exhibit affinity towards AZD1775 and PD0166285 and restrict their brain penetration. Results Using these studies, we demonstrate that AZD1775 is efficiently transported by both P-gp and BCRP, whereas PD0166285 is only a substrate of P-gp. Nonetheless, the brain penetration of both compounds was severely restricted in vivo, as indicated by a 5-fold (PD0166285) and 25-fold (AZD1775) lower brain-plasma ratio in wild type mice compared to Abcb1a/b;Abcg2-/-mice. Conclusion The brain penetration of these Wee1 inhibitors is severely limited by ABC transporters, which may compromise their clinical efficacy against intracranial neoplasms such as DIPG and GBM

An Experimenter's Guide to Glioblastoma Invasion Pathways

Glioblastoma is a highly aggressive brain tumor that is characterized by its unparalleled invasiveness. Invasive glioblastoma cells not only escape surgery and focal therapies but also are more resistant to current radio- and chemo-therapeutic approaches. Thus, any curative therapy for this deadly disease likely should include treatment strategies that interfere with glioblastoma invasiveness. Understanding glioblastoma invasion mechanisms is therefore critical. We discuss the strengths and weaknesses of various glioblastoma invasion models and conclude that robust experimental evidence has been obtained for a pro-invasive role of Ephrin receptors, Rho GTPases, and casein kinase 2 (CK2). Extensive interplay occurs between these proteins, suggesting the existence of a glioblastoma invasion signaling network that comprises several targets for therapy

ABCB1 and ABCG2 restrict the brain penetration of a panel of novel EZH2-Inhibitors

Enhancer of Zeste Homolog 2 (EZH2) has emerged as a promising therapeutic target for treatment of a broad spectrum of tumors including gliomas. We explored the interactions of five novel, structurally similar EZH2 inhibitors (EPZ005687, EPZ-6438, UNC1999, GSK343 and GSK126) with P-glycoprotein (P-gp/ABCB1) and breast cancer resistance protein (BCRP/ABCG2). The compounds were screened by in vitro transwell assays and EPZ005687, EPZ-6438 and GSK126 were further tested in vivo using wild-type (WT), Abcb1 and/or Abcg2 knockout mice. All EZH2 inhibitors are transported by P-gp and BCRP, although in vitro the transporter affinity of GSK126 was obscured by very low membrane permeability. Both P-gp and Bcrp1 restrict the brain penetration of EPZ005687 and GSK126, whereas the brain accumulation of EPZ-6438 is limited by P-gp only and efflux of EPZ-6438 was completely abrogated by elacridar. Intriguingly, an unknown factor present in all knockout mouse strains causes EPZ005687 and EPZ-6438 retention in plasma relative to WT mice, a phenomenon not seen with GSK126. In WT mice, the GSK126 tissue-to-plasma ratio for all tissues is lower than for EPZ005687 or EPZ-6438. Moreover, the oral bioavailability of GSK126 is only 0.2% in WT mice, which increases to 14.4% in Abcb1;Abcg2 knockout mice. These results are likely due to poor membrane permeability and question the clinical usefulness of GSK126. Although all tested EZH2 inhibitors are substrates of P-gp and BCRP, restricting the brain penetration and potential utility for treatment of glioma, EPZ-6438 would be the most suitable candidate of this series. What's New? Enhancer of zeste homolog 2 (EZH2) maintains the stemness of tumor cells and may have a critical role in glioma progression. As a promising therapeutic target in various cancers, there are now several candidate EZH2 inhibitors in the pipeline. The interactions of five of them were explored in this study. Experiments conducted in vitro and in vivo show that EZH2 inhibitors are substrates of ABCB1 and ABCG2, efflux transporters that severely restrict the distribution of drug into the brain. The findings suggest that EZH2 inhibitors are unlikely to play a meaningful role in the treatment of intracranial tumors

Targeting core (mutated) pathways of high-grade gliomas: challenges of intrinsic resistance and drug efflux

High-grade gliomas are the most common type of primary brain tumor and are among the most lethal types of human cancer. Most patients with a high-grade glioma have glioblastoma multiforme (GBM), the most malignant glioma subtype that is associated with a very aggressive disease course and short overall survival. Standard treatment of newly diagnosed GBM involves surgery followed by chemoradiation with temozolomide. However, despite this extensive treatment the mean overall survival is still only 14.6 months and more effective treatments are urgently needed. Although different types of GBMs are indistinguishable by histopathology, novel molecular pathological techniques allow discrimination between the four main GBM subtypes. Targeting the aberrations in the molecular pathways underlying these subtypes is a promising strategy to improve therapy. In this article, we will discuss the potential avenues and pitfalls of molecularly targeted therapies for the treatment of GBM

The impact of P-glycoprotein and breast cancer resistance protein on the brain pharmacokinetics and pharmacodynamics of a panel of MEK inhibitors

Mitogen/extracellular signal-regulated kinase (MEK) inhibitors have been tested in clinical trials for treatment of intracranial neoplasms, including glioblastoma (GBM), but efficacy of these drugs has not yet been demonstrated. The blood-brain barrier (BBB) is a major impediment to adequate delivery of drugs into the brain and may thereby also limit the successful implementation of MEK inhibitors against intracranial malignancies. The BBB is equipped with a range of ATP-dependent efflux transport proteins, of which P-gp (ABCB1) and BCRP (ABCG2) are the two most dominant for drug efflux from the brain. We investigated their impact on the pharmacokinetics and target engagement of a panel of clinically applied MEK inhibitors, in order to select the most promising candidate for brain cancers in the context of clinical pharmacokinetics and inhibitor characteristics. To this end, we used in vitro drug transport assays and conducted pharmacokinetic and pharmacodynamic studies in wildtype and ABC-transporter knockout mice. PD0325901 displayed more promising characteristics than trametinib (GSK1120212), binimetinib (MEK162), selumetinib (AZD6244), and pimasertib (AS703026): PD0325901 was the weakest substrate of P-gp and BCRP in vitro, its brain penetration was only marginally higher in Abcb1a/b;Abcg2-/-mice, and efficient target inhibition in the brain could be achieved at clinically relevant plasma levels. Notably, target inhibition could also be demonstrated for selumetinib, but only at plasma levels far above levels in patients receiving the maximum tolerated dose. In summary, our study recommends further development of PD0325901 for the treatment of intracranial neoplasms