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.

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.

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.

ABCB1 Attenuates the Brain Penetration of the PARP Inhibitor AZD2461.

Poly (ADP-ribose) polymerase (PARP) inhibitors are a relatively new class of anticancer agents that have attracted attention for treatment of glioblastoma because of their ability to potentiate temozolomide chemotherapy. Previous studies have demonstrated that sufficient brain penetration is a prerequisite for efficacy of PARP inhibitors in glioma mouse models. Unfortunately, however, most of the PARP inhibitors developed to date have a limited brain penetration due to the presence of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) at the blood-brain barrier. AZD2461 is a novel PARP inhibitor that is unaffected by P-gp mediated resistance in breast cancer models and thus appears to have promising characteristics for brain penetration. We here use a comprehensive set of in vitro and in vivo models to study the brain penetration and oral bioavailability of AZD2461. We report that AZD2461 has a good membrane permeability. However, it is a substrate of P-gp and BCRP, and P-gp in particular limits its brain penetration in vivo. We show that AZD2461 has a low oral bioavailability, although it is not affected by P-gp and BCRP. Together, these findings are not in favor of further development of AZD2461 for treatment of glioblastoma.

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.

P-glycoprotein and breast cancer resistance protein restrict the brain penetration of the CDK4/6 inhibitor palbociclib.

INTRODUCTION: Palbociclib is a cyclin dependent kinase (CDK) 4/6 inhibitor with nanomolar potency and was recently approved for treatment of breast cancer. The drug may also be useful in glioblastoma (GBM) and diffuse intrinsic pontine gliomas (DIPG), which often have an activated CDK4/6-retinoblastoma signaling pathway. However, GBM and DIPG spread widely into the surrounding brain, which calls for a CDK4/6 inhibitor with sufficient blood-brain barrier penetration. METHODS: We first performed in vitro transwell assays and demonstrate that palbociclib is a substrate of both P-gp and BCRP. Next, we conducted pharmacokinetic studies using wildtype, Abcg2(-/-), Abcb1a/b(-/-) and Abcg2; Abcb1a/b(-/-) mice. RESULTS: The plasma levels were about 3000 and 500 nM and similar in all genotypes at 1 and 4 h after i.v. administration of 10 mg/kg. At 4 h the brain-to-plasma ratios were 0.3 in WT and Abcg2(-/-) mice versus 5.5 and 15 in Abcb1a/b(-/-) and Abcg2; Abcb1a/b(-/-) mice, respectively. The oral bioavailability of palbociclib was high (63 %) in WT mice and increased only modestly and non-significantly in Abcg2; Abcb1a/b(-/-) mice. The plasma level after oral dosing of 150 mg/kg was already much higher than observed in patients (200-400 nM) and exceeded 2500 nM for up to 24 h. This latter dose is commonly used in preclinical studies, which calls into question their predictive value as they were conducted at dose levels causing a clinically non-relevant systemic drug exposure. CONCLUSION: Thus, the brain penetration of palbociclib is restricted by P-gp and BCRP, which may restrict the efficacy against GBM and DIPG. Moreover, preclinical studies with this agent should be conducted at a more clinically relevant dose level.

The impact of ATP-binding cassette transporters in the diseased brain: Context matters.

ATP-binding cassette (ABC) transporters facilitate the movement of diverse molecules across cellular membranes, including those within the CNS. While most extensively studied in microvascular endothelial cells forming the blood-brain barrier (BBB), other CNS cell types also express these transporters. Importantly, disruptions in the CNS microenvironment during disease can alter transporter expression and function. Through this comprehensive review, we explore the modulation of ABC transporters in various brain pathologies and the context-dependent consequences of these changes. For instance, downregulation of ABCB1 may exacerbate amyloid beta plaque deposition in Alzheimer's disease and facilitate neurotoxic compound entry in Parkinson's disease. Upregulation may worsen neuroinflammation by aiding chemokine-mediated CD8 T cell influx into multiple sclerosis lesions. Overall, ABC transporters at the BBB hinder drug entry, presenting challenges for effective pharmacotherapy. Understanding the context-dependent changes in ABC transporter expression and function is crucial for elucidating the etiology and developing treatments for brain diseases.

Directed molecular evolution reveals Gaussia luciferase variants with enhanced light output stability.

Gaussia Luciferase (Gluc) has proven to be a powerful mammalian cell reporter for monitoring numerous biological processes in immunology, virology, oncology, and neuroscience. Current limitations of Gluc as a reporter include its emission of blue light, which is absorbed by mammalian tissues, limiting its use in vivo, and a flash-type bioluminescence reaction, making it unsuited for high-throughput applications. To overcome these limitations, a library of Gluc variants was generated using directed molecular evolution and screened for relative light output, a shift in emission spectrum, and glow-type light emission kinetics. Several variants with a 10-15 nm shift in their light emission peak were found. Further, a Gluc variant that catalyzes a glow-type bioluminescence reaction, suited for high-throughput applications, was also identified. These results indicate that molecular evolution could be used to modulate Gluc bioluminescence reaction characteristics.

BCRP drives intrinsic chemoresistance in chemotherapy-naïve breast cancer brain metastasis.

Although initially successful, treatments with chemotherapy often fail because of the recurrence of chemoresistant metastases. Since these tumors develop after treatment, resistance is generally thought to occur in response to chemotherapy. However, alternative mechanisms of intrinsic chemoresistance in the chemotherapy-naïve setting may exist but remain poorly understood. Here, we study drug-naïve murine breast cancer brain metastases (BCBMs) to identify how cancer cells growing in a secondary site can acquire intrinsic chemoresistance without cytotoxic agent exposure. We demonstrate that drug-naïve murine breast cancer cells that form cancer lesions in the brain undergo vascular mimicry and concomitantly express the adenosine 5'-triphosphate-binding cassette transporter breast cancer resistance protein (BCRP), a common marker of brain endothelial cells. We reveal that expression of BCRP by the BCBM tumor cells protects them against doxorubicin and topotecan. We conclude that BCRP overexpression can cause intrinsic chemoresistance in cancer cells growing in metastatic sites without prior chemotherapy exposure.

Protocol for live-cell imaging during Tumor Treating Fields treatment with Inovitro Live.

Tumor Treating Fields (TTFields) are an FDA-approved anticancer treatment using alternating electric fields. Here, we present a protocol to perform live-cell imaging (LCI) of cells during TTFields treatment with the Inovitro LiveTM system. The setup we describe dissipates TTFields-related heat production and can be used in conjunction with any LCI-compatible microscope setup. This approach will enable further elucidation of TTFields' mechanism of action at the molecular level and facilitate the development of promising combination strategies.

ATP-binding cassette transporters restrict drug delivery and efficacy against brain tumors even when blood-brain barrier integrity is lost.

The impact of a compromised blood-brain barrier (BBB) on the drug treatment of intracranial tumors remains controversial. We characterize the BBB integrity in several intracranial tumor models using magnetic resonance imaging, fluorescent dyes, and autoradiography and determine the distribution and efficacy of docetaxel in brain tumors grafted in Abcb1-proficient and Abcb1-deficient mice. Leakiness of the tumor vasculature varies from extensive to absent. Regardless of the extent of leakiness, tumor blood vessels express ATP-binding cassette transporters (Abcb1 and Abcg2). A leaky vasculature results in higher docetaxel tumor levels compared to normal brain. Nevertheless, Abcb1 can reduce drug distribution and efficacy even in leaky models. Thus, BBB leakiness does not ensure the unimpeded access of ATP-binding cassette transporter substrate drugs. Therapeutic responses may be observed, but the full potential of such therapeutics may still be attenuated. Consequently, BBB-penetrable drugs with little to no affinity for efflux transporters are preferred for the treatment of intracranial tumors.

MEK/MELK inhibition and blood-brain barrier deficiencies in atypical teratoid/rhabdoid tumors.

BACKGROUND: Atypical teratoid/rhabdoid tumors (AT/RT) are rare, but highly aggressive. These entities are of embryonal origin occurring in the central nervous system (CNS) of young children. Molecularly these tumors are driven by a single hallmark mutation, resulting in inactivation of SMARCB1 or SMARCA4. Additionally, activation of the MAPK signaling axis and preclinical antitumor efficacy of its inhibition have been described in AT/RT. METHODS: We established and validated a patient-derived neurosphere culture and xenograft model of sonic hedgehog (SHH) subtype AT/RT, at diagnosis and relapse from the same patient. We set out to study the vascular phenotype of these tumors to evaluate the integrity of the blood-brain barrier (BBB) in AT/RT. We also used the model to study combined mitogen-activated protein kinase kinase (MEK) and maternal embryonic leucine zipper kinase (MELK) inhibition as a therapeutic strategy for AT/RT. RESULTS: We found MELK to be highly overexpressed in both patient samples of AT/RT and our primary cultures and xenografts. We identified a potent antitumor efficacy of the MELK inhibitor OTSSP167, as well as strong synergy with the MEK inhibitor trametinib, against primary AT/RT neurospheres. Additionally, vascular phenotyping of AT/RT patient material and xenografts revealed significant BBB aberrancies in these tumors. Finally, we show in vivo efficacy of the non-BBB penetrable drugs OTSSP167 and trametinib in AT/RT xenografts, demonstrating the therapeutic implications of the observed BBB deficiencies and validating MEK/MELK inhibition as a potential treatment. CONCLUSION: Altogether, we developed a combination treatment strategy for AT/RT based on MEK/MELK inhibition and identify therapeutically exploitable BBB deficiencies in these tumors.

Combined Therapy of AXL and HDAC Inhibition Reverses Mesenchymal Transition in Diffuse Intrinsic Pontine Glioma.

PURPOSE: Diffuse intrinsic pontine glioma (DIPG) is an incurable type of pediatric brain cancer, which in the majority of cases is driven by mutations in genes encoding histone 3 (H3K27M). We here determined the preclinical therapeutic potential of combined AXL and HDAC inhibition in these tumors to reverse their mesenchymal, therapy-resistant, phenotype. EXPERIMENTAL DESIGN: We used public databases and patient-derived DIPG cells to identify putative drivers of the mesenchymal transition in these tumors. Patient-derived neurospheres, xenografts, and allografts were used to determine the therapeutic potential of combined AXL/HDAC inhibition for the treatment of DIPG. RESULTS: We identified AXL as a therapeutic target and regulator of the mesenchymal transition in DIPG. Combined AXL and HDAC inhibition had a synergistic and selective antitumor effect on H3K27M DIPG cells. Treatment of DIPG cells with the AXL inhibitor BGB324 and the HDAC inhibitor panobinostat resulted in a decreased expression of mesenchymal and stem cell genes. Moreover, this combination treatment decreased expression of DNA damage repair genes in DIPG cells, strongly sensitizing them to radiation. Pharmacokinetic studies showed that BGB324, like panobinostat, crosses the blood-brain barrier. Consequently, treatment of patient-derived DIPG xenograft and murine DIPG allograft-bearing mice with BGB324 and panobinostat resulted in a synergistic antitumor effect and prolonged survival. CONCLUSIONS: Combined inhibition of AXL and HDACs in DIPG cells results in a synergistic antitumor effect by reversing their mesenchymal, stem cell-like, therapy-resistant phenotype. As such, this treatment combination may serve as part of a future multimodal therapeutic strategy for DIPG.

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.

Improved Brain Penetration and Antitumor Efficacy of Temozolomide by Inhibition of ABCB1 and ABCG2.

The anticancer drug temozolomide is the only drug with proven activity against high-grade gliomas and has therefore become a part of the standard treatment of these tumors. P-glycoprotein (P-gp; ABCB1) and breast cancer resistance protein (BCRP; ABCG2) are transport proteins, which are present at the blood-brain barrier and limit the brain uptake of substrate drugs. We have studied the effect of P-gp and BCRP on the pharmacokinetics and pharmacodynamics of temozolomide, making use of a comprehensive set of in vitro transport experiments and in vivo pharmacokinetic and antitumor efficacy experiments using wild-type, Abcg2-/-, Abcb1a/b-/-, and Abcb1a/b;Abcg2-/- mice. We here show that the combined deletion of Abcb1a/b and Abcg2 increases the brain penetration of temozolomide by 1.5-fold compared to wild-type controls (P < .001) without changing the systemic drug exposure. Moreover, the same increase was achieved when temozolomide was given to wild-type mice in combination with the dual P-gp/BCRP inhibitor elacridar (GF120918). The antitumor efficacy of temozolomide against three different intracranial tumor models was significantly enhanced when Abcb1a/b and Abcg2 were genetically deficient or pharmacologically inhibited in recipient mice. These findings call for further clinical testing of temozolomide in combination with elacridar for the treatment of gliomas, as this offers the perspective of further improving the antitumor efficacy of this already active agent.

Buparlisib is a brain penetrable pan-PI3K inhibitor.

Characterization of the genomic landscapes of intracranial tumours has revealed a clear role for the PI3K-AKT-mTOR pathway in tumorigenesis and tumour maintenance of these malignancies, making phosphatidylinositol 3-kinase (PI3K) inhibition a promising therapeutic strategy for these tumours. Buparlisib is a novel pan-PI3K inhibitor that is currently in clinical development for various cancers, including primary and secondary brain tumours. Importantly however, earlier studies have revealed that sufficient brain penetration is a prerequisite for antitumor efficacy against intracranial tumours. We therefore investigated the brain penetration of buparlisib using a comprehensive set of in vitro and in vivo mouse models. We demonstrate that buparlisib has an excellent brain penetration that is unaffected by efflux transporters at the blood-brain barrier, complete oral bioavailability and efficient intracranial target inhibition at clinically achievable plasma concentrations. Together, these characteristics make buparlisib the ideal candidate for intracranially-targeted therapeutic strategies that involve PI3K inhibition.

Recycling drug screen repurposes hydroxyurea as a sensitizer of glioblastomas to temozolomide targeting de novo DNA synthesis, irrespective of molecular subtype.

Background: Glioblastoma (GBM) is the most common and most aggressive primary malignant brain tumor. Standard-of-care treatment involves maximal surgical resection of the tumor followed by radiation and chemotherapy (temozolomide [TMZ]). The 5-year survival rate of patients with GBM is <10%, a colossal failure that has been partially attributed to intrinsic and/or acquired resistance to TMZ through O6-methylguanine DNA methyltransferase (MGMT) promoter methylation status in the tumor. Methods: A drug screening aimed at evaluating the potential recycling and repurposing of known drugs was conducted in TMZ-resistant GBM cell lines and primary cultures of newly diagnosed GBM with different MGMT promoter methylation status, phenotypic/genotypic background and subtype, and validated with sphere formation, cell migration assays, and quantitative invasive orthotopic in vivo models. Results: We identified hydroxyurea (HU) to synergize with TMZ in GBM cells in culture and in vivo, irrespective of MGMT promoter methylation status, subtype, and/or stemness. HU acts specifically on the S-phase of the cell cycle by inhibiting the M2 unit of enzyme ribonucleotide reductase. Knockdown of this enzyme using RNA interference and other known chemical inhibitors exerted a similar effect to HU in combination with TMZ both in culture and in vivo. Conclusions: We demonstrate preclinical efficacy of repurposing hydroxyurea in combination with TMZ for adjuvant GBM therapy. This combination benefit is of direct clinical interest given the extensive use of TMZ and the associated problems with TMZ-related resistance and treatment failure.

MELK Inhibition in Diffuse Intrinsic Pontine Glioma.

Purpose: Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive pediatric brain tumor, for which no effective therapeutic options currently exist. We here determined the potential of inhibition of the maternal embryonic leucine zipper kinase (MELK) for the treatment of DIPG.Experimental Design: We evaluated the antitumor efficacy of the small-molecule MELK inhibitor OTSSP167 in vitro in patient-derived DIPG cultures, and identified the mechanism of action of MELK inhibition in DIPG by RNA sequencing of treated cells. In addition, we determined the blood-brain barrier (BBB) penetration of OTSSP167 and evaluated its translational potential by treating mice bearing patient-derived DIPG xenografts.Results: This study shows that MELK is highly expressed in DIPG cells, both in patient samples and in relevant in vitro and in vivo models, and that treatment with OTSSP167 strongly decreases proliferation of patient-derived DIPG cultures. Inhibition of MELK in DIPG cells functions through reducing inhibitory phosphorylation of PPARγ, resulting in an increase in nuclear translocation and consequent transcriptional activity. Brain pharmacokinetic analyses show that OTSSP167 is a strong substrate for both MDR1 and BCRP, limiting its BBB penetration. Nonetheless, treatment of Mdr1a/b;Bcrp1 knockout mice carrying patient-derived DIPG xenografts with OTSSP167 decreased tumor growth, induced remissions, and resulted in improved survival.Conclusions: We show a strong preclinical effect of the kinase inhibitor OTSSP167 in the treatment of DIPG and identify the MELK-PPARγ signaling axis as a putative therapeutic target in this disease. Clin Cancer Res; 24(22); 5645-57. ©2018 AACR.

Glycosylated extracellular vesicles released by glioblastoma cells are decorated by CCL18 allowing for cellular uptake via chemokine receptor CCR8.

Cancer cells release extracellular vesicles (EVs) that contain functional biomolecules such as RNA and proteins. EVs are transferred to recipient cancer cells and can promote tumour progression and therapy resistance. Through RNAi screening, we identified a novel EV uptake mechanism involving a triple interaction between the chemokine receptor CCR8 on the cells, glycans exposed on EVs and the soluble ligand CCL18. This ligand acts as bridging molecule, connecting EVs to cancer cells. We show that glioblastoma EVs promote cell proliferation and resistance to the alkylating agent temozolomide (TMZ). Using in vitro and in vivo stem-like glioblastoma models, we demonstrate that EV-induced phenotypes are neutralised by a small molecule CCR8 inhibitor, R243. Interference with chemokine receptors may offer therapeutic opportunities against EV-mediated cross-talk in glioblastoma.