ALL-tRNAseq enables robust tRNA profiling in tissue samples.

Transfer RNAs (tRNAs) are small adaptor RNAs essential for mRNA translation. Alterations in the cellular tRNA population can directly affect mRNA decoding rates and translational efficiency during cancer development and progression. To evaluate changes in the composition of the tRNA pool, multiple sequencing approaches have been developed to overcome reverse transcription blocks caused by the stable structures of these molecules and their numerous base modifications. However, it remains unclear whether current sequencing protocols faithfully capture tRNAs existing in cells or tissues. This is specifically challenging for clinical tissue samples that often present variable RNA qualities. For this reason, we developed ALL-tRNAseq, which combines the highly processive MarathonRT and RNA demethylation for the robust assessment of tRNA expression, together with a randomized adapter ligation strategy prior to reverse transcription to assess tRNA fragmentation levels in both cell lines and tissues. Incorporation of tRNA fragments not only informed on sample integrity but also significantly improved tRNA profiling of tissue samples. Our data showed that our profiling strategy effectively improves classification of oncogenic signatures in glioblastoma and diffuse large B-cell lymphoma tissues, particularly for samples presenting higher levels of RNA fragmentation, further highlighting the utility of ALL-tRNAseq for translational research.

Culture methods of diffuse intrinsic pontine glioma cells determine response to targeted therapies.

Diffuse intrinsic pontine glioma (DIPG) is an aggressive type of brainstem cancer occurring mainly in children, for which there currently is no effective therapy. Current efforts to develop novel therapeutics for this tumor make use of primary cultures of DIPG cells, maintained either as adherent monolayer in serum containing medium, or as neurospheres in serum-free medium. In this manuscript, we demonstrate that the response of DIPG cells to targeted therapies in vitro is mainly determined by the culture conditions. We show that particular culture conditions induce the activation of different receptor tyrosine kinases and signal transduction pathways, as well as major changes in gene expression profiles of DIPG cells in culture. These differences correlate strongly with the observed discrepancies in response to targeted therapies of DIPG cells cultured as either adherent monolayers or neurospheres. With this research, we provide an argument for the concurrent use of both culture conditions to avoid false positive and false negative results due to the chosen method.

Evaluation of potential circulating biomarkers for prediction of response to chemoradiation in patients with glioblastoma.

Surgery followed by chemoradiation and adjuvant chemotherapy is standard of care for patients with a glioblastoma (GBM). Due to its limited benefit, an upfront method to predict dismal outcome would prevent unnecessary toxic treatment. We searched for a predictive blood derived biomarker in a cohort of 55 patients with GBM. Increasing age (HR 1.03, 95 % CI 1.01-1.06), and postoperative tumor residue (HR 1.07, 95 % CI 1.02-1.15) were independently associated with unfavourable progression free survival (PFS) in these patients. Corticosteroid use before start of chemoradiaton was strongly predictive for outcome (HR 3.26, 95 % CI 1.67-6.39) with a mean PFS and OS in patients using corticosteroids of 7.3 and 14.6 months, versus 16.1 and 21.6 months in patients not using corticosteroids (p = 0.0005, p < 0.0067 respectively). Despite earlier reports, blood concentrations of YKL-40, Fetuin-a and haptoglobin were not predictive for response. In addition, serum peptide profiles, determined by MALDI-TOF mass spectroscopy, were not predictive as well. In conclusion, further biomarker discovery studies are needed to predict treatment outcome for patients with GBM in the near future.

Tumor-educated platelet blood tests for Non-Small Cell Lung Cancer detection and management.

Liquid biopsy approaches offer a promising technology for early and minimally invasive cancer detection. Tumor-educated platelets (TEPs) have emerged as a promising liquid biopsy biosource for the detection of various cancer types. In this study, we processed and analyzed the TEPs collected from 466 Non-small Cell Lung Carcinoma (NSCLC) patients and 410 asymptomatic individuals (controls) using the previously established thromboSeq protocol. We developed a novel particle-swarm optimization machine learning algorithm which enabled the selection of an 881 RNA biomarker panel (AUC 0.88). Herein we propose and validate in an independent cohort of samples (n = 558) two approaches for blood samples testing: one with high sensitivity (95% NSCLC detected) and another with high specificity (94% controls detected). Our data explain how TEP-derived spliced RNAs may serve as a biomarker for minimally-invasive clinical blood tests, complement existing imaging tests, and assist the detection and management of lung cancer patients.

Detection and localization of early- and late-stage cancers using platelet RNA.

Cancer patients benefit from early tumor detection since treatment outcomes are more favorable for less advanced cancers. Platelets are involved in cancer progression and are considered a promising biosource for cancer detection, as they alter their RNA content upon local and systemic cues. We show that tumor-educated platelet (TEP) RNA-based blood tests enable the detection of 18 cancer types. With 99% specificity in asymptomatic controls, thromboSeq correctly detected the presence of cancer in two-thirds of 1,096 blood samples from stage I-IV cancer patients and in half of 352 stage I-III tumors. Symptomatic controls, including inflammatory and cardiovascular diseases, and benign tumors had increased false-positive test results with an average specificity of 78%. Moreover, thromboSeq determined the tumor site of origin in five different tumor types correctly in over 80% of the cancer patients. These results highlight the potential properties of TEP-derived RNA panels to supplement current approaches for blood-based cancer screening.

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.

Tumor-Educated Platelet RNA for the Detection and (Pseudo)progression Monitoring of Glioblastoma.

Tumor-educated platelets (TEPs) are potential biomarkers for cancer diagnostics. We employ TEP-derived RNA panels, determined by swarm intelligence, to detect and monitor glioblastoma. We assessed specificity by comparing the spliced RNA profile of TEPs from glioblastoma patients with multiple sclerosis and brain metastasis patients (validation series, n = 157; accuracy, 80%; AUC, 0.81 [95% CI, 0.74-0.89; p < 0.001]). Second, analysis of patients with glioblastoma versus asymptomatic healthy controls in an independent validation series (n = 347) provided a detection accuracy of 95% and AUC of 0.97 (95% CI, 0.95-0.99; p < 0.001). Finally, we developed the digitalSWARM algorithm to improve monitoring of glioblastoma progression and demonstrate that the TEP tumor scores of individual glioblastoma patients represent tumor behavior and could be used to distinguish false positive progression from true progression (validation series, n = 20; accuracy, 85%; AUC, 0.86 [95% CI, 0.70-1.00; p < 0.012]). In conclusion, TEPs have potential as a minimally invasive biosource for blood-based diagnostics and monitoring of glioblastoma patients.

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.

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.

Radiosensitization of human glioma cells by cyclooxygenase-2 (COX-2) inhibition: independent on COX-2 expression and dependent on the COX-2 inhibitor and sequence of administration.

PURPOSE: Patients with a malignant glioma have a very poor prognosis. Cyclooxygenase-2 (COX-2) protein is regularly upregulated in gliomas and might be a potential therapeutic target. The effects of three selective COX-2 inhibitors were studied on three human glioma cell lines. MATERIALS AND METHODS: The selective COX-2 inhibitors NS-398, Celecoxib and Meloxicam and three human glioma cell lines (D384, U251 and U87) were used. Cell growth was assessed by a proliferation assay, the interaction with radiation (0 - 6 Gy) was studied using the clonogenic assay and cell cycle distribution was determined by FACS (fluorescence-activated cell sorting) analysis. RESULTS: All COX-2 inhibitors reduced proliferation of the glioma cell lines irrespective of their COX-2 expression level. Incubation with 200 microM NS-398 24 h before radiation enhanced radiation-induced cell death of D384 cells and 750 microM Meloxicam resulted in radiosensitization of D384 and U87 cells. No radiosensitization was observed with COX-2 inhibitor administration after radiotherapy. Treatment of D384 with NS-398 (200 microM) or Celecoxib (50 microM) and U87 with NS-398 (200 microM) after radiation resulted even in radioprotection. CONCLUSIONS: Effectiveness of COX-2 inhibitors on cell proliferation and radio-enhancement was independent of COX-2 protein expression. The sequence of COX-2 inhibitor addition and irradiation is very important.

Swarm Intelligence-Enhanced Detection of Non-Small-Cell Lung Cancer Using Tumor-Educated Platelets.

Blood-based liquid biopsies, including tumor-educated blood platelets (TEPs), have emerged as promising biomarker sources for non-invasive detection of cancer. Here we demonstrate that particle-swarm optimization (PSO)-enhanced algorithms enable efficient selection of RNA biomarker panels from platelet RNA-sequencing libraries (n = 779). This resulted in accurate TEP-based detection of early- and late-stage non-small-cell lung cancer (n = 518 late-stage validation cohort, accuracy, 88%; AUC, 0.94; 95% CI, 0.92-0.96; p < 0.001; n = 106 early-stage validation cohort, accuracy, 81%; AUC, 0.89; 95% CI, 0.83-0.95; p < 0.001), independent of age of the individuals, smoking habits, whole-blood storage time, and various inflammatory conditions. PSO enabled selection of gene panels to diagnose cancer from TEPs, suggesting that swarm intelligence may also benefit the optimization of diagnostics readout of other liquid biopsy biosources.

Pre-B-cell leukemia homeobox interacting protein 1 is overexpressed in astrocytoma and promotes tumor cell growth and migration.

BACKGROUND: Glial brain tumors cause considerable mortality and morbidity in children and adults. Innovative targets for therapy are needed to improve survival and reduce long-term sequelae. The aim of this study was to find a candidate tumor-promoting protein, abundantly expressed in tumor cells but not in normal brain tissues, as a potential target for therapy. METHODS: In silico proteomics and genomics, immunohistochemistry, and immunofluorescence microscopy validation were performed. RNA interference was used to ascertain the functional role of the overexpressed candidate target protein. RESULTS: In silico proteomics and genomics revealed pre-B-cell leukemia homeobox (PBX) interacting protein 1 (PBXIP1) overexpression in adult and childhood high-grade glioma and ependymoma compared with normal brain. PBXIP1 is a PBX-family interacting microtubule-binding protein with a putative role in migration and proliferation of cancer cells. Immunohistochemical studies in glial tumors validated PBXIP1 expression in astrocytoma and ependymoma but not in oligodendroglioma. RNAi-mediated PBXIP1-knockdown in glioblastoma cell lines strongly reduced proliferation and migration and induced morphological changes, indicating that PBXIP1 knockdown decreases glioma cell viability and motility through rearrangements of the actin cytoskeleton. Furthermore, expression of PBXIP1 was observed in radial glia and astrocytic progenitor cells in human fetal tissues, suggesting that PBXIP1 is an astroglial progenitor cell marker during human embryonic development. CONCLUSION: PBXIP1 is a novel protein overexpressed in astrocytoma and ependymoma, involved in tumor cell proliferation and migration, that warrants further exploration as a novel therapeutic target in these tumors.

In vitro drug response and efflux transporters associated with drug resistance in pediatric high grade glioma and diffuse intrinsic pontine glioma.

Pediatric high-grade gliomas (pHGG), including diffuse intrinsic pontine gliomas (DIPG), are the leading cause of cancer-related death in children. While it is clear that surgery (if possible), and radiotherapy are beneficial for treatment, the role of chemotherapy for these tumors is still unclear. Therefore, we performed an in vitro drug screen on primary glioma cells, including three DIPG cultures, to determine drug sensitivity of these tumours, without the possible confounding effect of insufficient drug delivery. This screen revealed a high in vitro cytotoxicity for melphalan, doxorubicine, mitoxantrone, and BCNU, and for the novel, targeted agents vandetanib and bortezomib in pHGG and DIPG cells. We subsequently determined the expression of the drug efflux transporters P-gp, BCRP1, and MRP1 in glioma cultures and their corresponding tumor tissues. Results indicate the presence of P-gp, MRP1 and BCRP1 in the tumor vasculature, and expression of MRP1 in the glioma cells themselves. Our results show that pediatric glioma and DIPG tumors per se are not resistant to chemotherapy. Treatment failure observed in clinical trials, may rather be contributed to the presence of drug efflux transporters that constitute a first line of drug resistance located at the blood-brain barrier or other resistance mechanism. As such, we suggest that alternative ways of drug delivery may offer new possibilities for the treatment of pediatric high-grade glioma patients, and DIPG in particular.

PARP inhibition sensitizes childhood high grade glioma, medulloblastoma and ependymoma to radiation.

Poly ADP-ribose polymerase (PARP) is a protein involved in single strand break repair. Recently, PARP inhibitors have shown considerable promise in the treatment of several cancers, both in monotherapy and in combination with cytotoxic agents. Synthetic lethal action of PARP inhibitors has been observed in tumors with mutations in double strand break repair pathways. In addition, PARP inhibition potentially enhances sensitivity of tumor cells to DNA damaging agents, including radiotherapy. Aim of this study is to determine the radiosensitizing properties of the PARP inhibitor Olaparib in childhood medulloblastoma, ependymoma and high grade glioma (HGG). Increased PARP1 expression was observed in medulloblastoma, ependymoma and HGG, as compared to non-neoplastic brain tissue. Pediatric high grade glioma, medulloblastoma and ependymoma gene expression profiling revealed that high PARP1 expression is associated with poor prognosis. Cell growth inhibition assays with Olaparib resulted in differential sensitivity, with IC50 values ranging from 1.4 to 8.4 µM, irrespective of tumor type and PARP1 protein expression. Sensitization to radiation was observed in medulloblastoma, ependymoma and HGG cell lines with subcytotoxic concentrations of Olaparib, which coincided with persistence of double strand breaks. Combining PARP inhibitors with radiotherapy in clinical studies in childhood high grade brain tumors may improve therapeutic outcome.

Radiosensitizing potential of the selective cyclooygenase-2 (COX-2) inhibitor meloxicam on human glioma cells.

The COX-2 protein is frequently overexpressed in human malignant gliomas. This expression has been associated with their aggressive growth characteristics and poor prognosis for patients. Targeting the COX-2 pathway might improve glioma therapy. In this study, the effects of the selective COX-2 inhibitor meloxicam alone and in combination with irradiation were investigated on human glioma cells in vitro. A panel of three glioma cell lines (D384, U87 and U251) was used in the experiments from which U87 cells expressed constitutive COX-2. The response to meloxicam and irradiation (dose-range of 0-6 Gy) was determined by the clonogenic assay, cell proliferation was evaluated by growth analysis and cell cycle distribution by FACS. 24-72 h exposure to 250-750 microM meloxicam resulted in a time and dose dependent growth inhibition with an almost complete inhibition after 24 h for all cell lines. Exposure to 750 microM meloxicam for 24 h increased the fraction of cells in the radiosensitive G(2)/M cell cycle phase in D384 (18-27%) and U251 (17-41%) cells. 750 microM meloxicam resulted in radiosensitization of D384 (DMF:2.19) and U87 (DMF:1.25) cells, but not U251 cells (DMF:1.08). The selective COX-2 inhibitor meloxicam exerted COX-2 independent growth inhibition and radiosensitization of human glioma cells.