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// R. Jonas A. Nilsson 1,2,3,* , Niki Karachaliou 4,* , Jordi Berenguer 1 , Ana Gimenez-Capitan 5 , Pepijn Schellen 1,3 , Cristina Teixido 5 , Jihane Tannous 6 , Justine L. Kuiper 7 , Esther Drees 1 , Magda Grabowska 1 , Marte van Keulen 6 , Danielle A. M. Heideman 8 , Erik Thunnissen 8 , Anne-Marie C. Dingemans 9 , Santiago Viteri 4 , Bakhos A. Tannous 6 , Ana Drozdowskyj 10 , Rafael Rosell 4,5,11,12,** , Egbert F. Smit 7,** and Thomas Wurdinger 1,3,6,** 1 Cancer Center Amsterdam, Department of Neurosurgery, VU University Medical Center, Amsterdam, The Netherlands 2 Department of Radiation Sciences, Oncology, Umea University, Umea, Sweden 3 ThromboDx B.V., Amsterdam, The Netherlands 4 Translational Research Unit, Dr, Rosell Oncology Institute, Quiron Dexeus University Hospital, Barcelona, Spain 5 Pangaea Biotech SL, Barcelona, Spain 6 Department of Neurology, Massachusetts General Hospital and Neuroscience Program, Harvard Medical School, Boston, MA, USA 7 Cancer Center Amsterdam, Department of Pulmonary Diseases, VU University Medical Center, Amsterdam, The Netherlands 8 Cancer Center Amsterdam, Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands 9 Department of Pulmonary Diseases, Maastricht University Medical Center, Maastricht, The Netherlands 10 Pivotal, Madrid, Spain 11 Catalan Institute of Oncology, Hospital Germans Trias i Pujol, Barcelona, Spain 12 Molecular Oncology Research (MORe) Foundation, Barcelona, Spain * These two authors are co-first authors of the manuscript ** These three authors are co-senior authors of the manuscript Correspondence to: Thomas Wurdinger, email: // Keywords : diagnostics, NSCLC, liquid biopsies, platelets, EML4-ALK Received : August 23, 2015 Accepted : October 06, 2015 Published : November 02, 2015 Abstract Purpose: Non-small-cell lung cancers harboring EML4-ALK rearrangements are sensitive to crizotinib. However, despite initial response, most patients will eventually relapse, and monitoring EML4-ALK rearrangements over the course of treatment may help identify these patients. However, challenges associated with serial tumor biopsies have highlighted the need for blood-based assays for the monitoring of biomarkers. Platelets can sequester RNA released by tumor cells and are thus an attractive source for the non-invasive assessment of biomarkers. Methods: EML4-ALK rearrangements were analyzed by RT-PCR in platelets and plasma isolated from blood obtained from 77 patients with non-small-cell lung cancer, 38 of whom had EML4-ALK-rearranged tumors. In a subset of 29 patients with EML4-ALK-rearranged tumors who were treated with crizotinib, EML4-ALK rearrangements in platelets were correlated with progression-free and overall survival. Results: RT-PCR demonstrated 65% sensitivity and 100% specificity for the detection of EML4-ALK rearrangements in platelets. In the subset of 29 patients treated with crizotinib, progression-free survival was 3.7 months for patients with EML4-ALK+ platelets and 16 months for those with EML4-ALK− platelets (hazard ratio, 3.5; P = 0.02). Monitoring of EML4-ALK rearrangements in the platelets of one patient over a period of 30 months revealed crizotinib resistance two months prior to radiographic disease progression. Conclusions: Platelets are a valuable source for the non-invasive detection of EML4-ALK rearrangements and may prove useful for predicting and monitoring outcome to crizotinib, thereby improving clinical decisions based on radiographic imaging alone.

Summary Tumor-educated blood platelets (TEPs) are implicated as central players in the systemic and local responses to tumor growth, thereby altering their RNA profile. We determined the diagnostic potential of TEPs by mRNA sequencing of 283 platelet samples. We distinguished 228 patients with localized and metastasized tumors from 55 healthy individuals with 96% accuracy. Across six different tumor types, the location of the primary tumor was correctly identified with 71% accuracy. Also, MET or HER2-positive, and mutant KRAS, EGFR, or PIK3CA tumors were accurately distinguished using surrogate TEP mRNA profiles. Our results indicate that blood platelets provide a valuable platform for pan-cancer, multiclass cancer, and companion diagnostics, possibly enabling clinical advances in blood-based “liquid biopsies”., Graphical Abstract, Highlights • Tumors “educate” platelets (TEPs) by altering the platelet RNA profile • TEPs provide a RNA biosource for pan-cancer, multiclass, and companion diagnostics • TEP-based liquid biopsies may guide clinical diagnostics and therapy selection • A total of 100–500 pg of total platelet RNA is sufficient for TEP-based diagnostics, Best et al. show that mRNA sequencing of tumor-educated blood platelets distinguishes cancer patients from healthy individuals with 96% accuracy, differentiates between six primary tumor types of patients with 71% accuracy, and identifies several genetic alterations found in tumors.

Summary 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, Graphical Abstract, Highlights • Tumor-educated platelet (TEP) RNA profiles allow for blood-based cancer diagnostics • Inflammatory conditions only minimally confound TEP-based cancer detection • Swarm intelligence algorithms enable efficient selection of biomarker gene panels • TEP gene panels enable support vector machine-based classification of lung cancer, Best et al. use particle-swarm optimization algorithms and RNA-seq of tumor-educated platelets from patients to generate RNA sets capable of identifying patients with non-small-cell lung cancer, including those having early stage, from individuals without cancer, including those having inflammatory conditions.

BACKGROUND: Glioblastomas exhibit a high level of chemotherapeutic resistance, including to the antimitotic agents vincristine and taxol. During the mitotic agent-induced arrest, glioblastoma cells are able to perform damage-control and self-repair to continue proliferation. Monopolar spindle 1 (MPS1/TTK) is a checkpoint kinase and a gatekeeper of the mitotic arrest.METHODS: We used glioblastoma cells to determine the expression of MPS1 and to determine the effects of MPS1 inhibition on mitotic errors and cell viability in combination with vincristine and taxol. The effect of MPS1 inhibition was assessed in different orthotopic glioblastoma mouse models (n = 3-7 mice/group). MPS1 expression levels were examined in relation to patient survival.RESULTS: Using publicly available gene expression data, we determined that MPS1 overexpression corresponds positively with tumor grade and negatively with patient survival (two-sided t test, P < .001). Patients with high MPS1 expression (n = 203) had a median and mean survival of 487 and 913 days (95% confidence intervals [CI] = 751 to 1075), respectively, and a 2-year survival rate of 35%, whereas patients with intermediate MPS1 expression (n = 140) had a median and mean survival of 858 and 1183 days (95% CI = 1177 to 1189), respectively, and a 2-year survival rate of 56%. We demonstrate that MPS1 inhibition by RNAi results in sensitization to antimitotic agents. We developed a selective small-molecule inhibitor of MPS1, MPS1-IN-3, which caused mitotic aberrancies in glioblastoma cells and, in combination with vincristine, induced mitotic checkpoint override, increased aneuploidy, and augmented cell death. MPS1-IN-3 sensitizes glioblastoma cells to vincristine in orthotopic mouse models (two-sided log-rank test, P < .01), resulting in prolonged survival without toxicity.CONCLUSIONS: Our results collectively demonstrate that MPS1, a putative therapeutic target in glioblastoma, can be selectively inhibited by MPS1-IN-3 sensitizing glioblastoma cells to antimitotic drugs.

Diagnostic platforms providing biomarkers that are highly predictive for diagnosing, monitoring, and stratifying cancer patients are key instruments in the development of personalized medicine. We demonstrate that tumor cells transfer (mutant) RNA into blood platelets in vitro and in vivo, and show that blood platelets isolated from glioma and prostate cancer patients contain the cancer-associated RNA biomarkers EGFRvIII and PCA3, respectively. In addition, gene-expression profiling revealed a distinct RNA signature in platelets from glioma patients compared with normal control subjects. Because platelets are easily accessible and isolated, they may form an attractive platform for the companion diagnostics of cancer.

Angiogenesis is a balanced process controlled by pro- and anti-angiogenic molecules of which the regulation is not fully understood. Besides classical gene regulation, miRNAs have emerged as post-transcriptional regulators of angiogenesis. Furthermore, epigenetic changes caused by histone-modifying enzymes were shown to modulate angiogenesis as well. However, a possible interplay between miRNAs and histone-modulating enzymes during angiogenesis has not been described. Here we show that VEGF-mediated down-regulation of miR-101 caused pro-angiogenic effects. We found that the pro-angiogenic effects are partly mediated through reduced repression by miR-101 of the histone-methyltransferase EZH2, a member of the Polycomb group family, thereby increasing methylation of histone H3 at lysine 27 and transcriptome alterations. In vitro, the sprouting and migratory properties of primary endothelial cell cultures were reduced by inhibiting EZH2 through up-regulation of miR-101, siRNA-mediated knockdown of EZH2, or treatment with 3-Deazaneplanocin-A (DZNep), a small molecule inhibitor of EZH2 methyltransferase activity. In addition, we found that systemic DZNep administration reduced the number of blood vessels in a subcutaneous glioblastoma mouse model, without showing adverse toxicities. Altogether, by identifying a pro-angiogenic VEGF/miR-101/EZH2 axis in endothelial cells we provide evidence for a functional link between growth factor-mediated signaling, post-transcriptional silencing, and histone-methylation in the angiogenesis process. Inhibition of EZH2 may prove therapeutic in diseases in which aberrant vascularization plays a role.

Susceptibility to cell death is a prerequisite for the elimination of tumour cells by cytotoxic immune cells, chemotherapy or irradiation. Activation of the death receptor Fas is critical for the regulation of immune cell homeostasis and efficient killing of tumour cells by apoptosis. To define the molecular changes that occur during selection for insensitivity to Fas-induced apoptosis, a resistant variant of the U937 cell line was established. Individual resistant clones were isolated and characterized. The most frequently observed defect in the resistant cells was reduced Fas expression, which correlated with decreased FAS transcription. Clones with such reduced Fas expression also displayed partial cross-resistance to tumour necrosis factor-alpha stimulation, but the mRNA expression of tumour necrosis factor receptors was not decreased. Reintroduction of Fas conferred susceptibility to Fas but not to tumour necrosis factor-alpha stimulation, suggesting that several alterations could be present in the clones. The reduced Fas expression could not be explained by mutations in the FAS coding sequence or promoter region, or by silencing through methylations. Protein kinase B and extracellular signal-regulated kinase, components of signalling pathways downstream of Ras, were shown to be activated in some of the resistant clones, but none of the three RAS genes was mutated, and experiments using chemical inhibitors could not establish that the activation of these proteins was the cause of Fas resistance as described in other systems. Taken together, the data illustrate that Fas resistance can be caused by reduced Fas expression, which is a result of an unidentified mode of regulation.

8082 Background: Non-small-cell lung cancer (NSCLC) with EML4-ALK rearrangements is sensitive to crizotinib. However, despite initial response most patients (p) will eventually relapse and monitori...

Recent findings indicate that we should rethink how Myc oncoproteins transactivate their target genes. It appears that Mnt, a transcription factor that mediates transrepression at Myc's E-boxes, plays a crucial role in keeping the cell cycle in check. By removing Mnt, either via conventional knockout techniques or via RNA interference, cells become hyper-proliferative, susceptible to apoptosis and can be transformed by Ras--all hallmarks of Myc overexpression. These findings indicate that Myc's ability to function as an oncogene may rely, at least in part, on its ability to effectively antagonize Mnt's transrepression and tumor suppressor functions.

Over the last few years much attention was paid to 3D-QSAR studies1,2 owing to the incorporation of information derived from molecular modelling techniques such as GRID3, CoMFA4 and many others into the chemometrics procedures aimed at deriving a statistical model. Our research group has contributed to the field by suggesting the GOLPE5 approach, which is based on a severe validation criterion and on a region selection procedure6 in order to produce easily interpretable results and reliable predictions of the activity. Such studies are generally carried out on small molecules acting as ligands or enzyme inhibitors. On the contrary, relatively less attention was devoted to a quantitative description of the receptors.