Qianghu Wang

The landscape and therapeutic relevance of cancer-associated transcript fusions.

Transcript fusions as a result of chromosomal rearrangements have been a focus of attention in cancer as they provide attractive therapeutic targets. To identify novel fusion transcripts with the potential to be exploited therapeutically, we analyzed RNA sequencing, DNA copy number and gene mutation data from 4366 primary tumor samples. To avoid false positives, we implemented stringent quality criteria that included filtering of fusions detected in RNAseq data from 364 normal tissue samples. Our analysis identified 7887 high confidence fusion transcripts across 13 tumor types. Our fusion prediction was validated by evidence of a genomic rearrangement for 78 of 79 fusions in 48 glioma samples where whole-genome sequencing data were available. Cancers with higher levels of genomic instability showed a corresponding increase in fusion transcript frequency, whereas tumor samples harboring fusions contained statistically significantly fewer driver gene mutations, suggesting an important role for tumorigenesis. We identified at least one in-frame protein kinase fusion in 324 of 4366 samples (7.4%). Potentially druggable kinase fusions involving ALK, ROS, RET, NTRK and FGFR gene families were detected in bladder carcinoma (3.3%), glioblastoma (4.4%), head and neck cancer (1.0%), low-grade glioma (1.5%), lung adenocarcinoma (1.6%), lung squamous cell carcinoma (2.3%) and thyroid carcinoma (8.7%), suggesting a potential for application of kinase inhibitors across tumor types. In-frame fusion transcripts involving histone methyltransferase or histone demethylase genes were detected in 111 samples (2.5%) and may additionally be considered as therapeutic targets. In summary, we described the landscape of transcript fusions detected across a large number of tumor samples and revealed fusion events with clinical relevance that have not been previously recognized. Our results support the concept of basket clinical trials where patients are matched with experimental therapies based on their genomic profile rather than the tissue where the tumor originated.

PRADA: pipeline for RNA sequencing data analysis

SUMMARY Technological advances in high-throughput sequencing necessitate improved computational tools for processing and analyzing large-scale datasets in a systematic automated manner. For that purpose, we have developed PRADA (Pipeline for RNA-Sequencing Data Analysis), a flexible, modular and highly scalable software platform that provides many different types of information available by multifaceted analysis starting from raw paired-end RNA-seq data: gene expression levels, quality metrics, detection of unsupervised and supervised fusion transcripts, detection of intragenic fusion variants, homology scores and fusion frame classification. PRADA uses a dual-mapping strategy that increases sensitivity and refines the analytical endpoints. PRADA has been used extensively and successfully in the glioblastoma and renal clear cell projects of The Cancer Genome Atlas program. AVAILABILITY AND IMPLEMENTATION http://sourceforge.net/projects/prada/ CONTACT  gadgetz@broadinstitute.org or rverhaak@mdanderson.org SUPPLEMENTARY INFORMATION  Supplementary data are available at Bioinformatics online.

GlioVis data portal for visualization and analysis of brain tumor expression datasets.

© The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. doi:10.1093/neuonc/now199 Advance Access date 14 September 2016 the effects they observed could be influenced by inhibition of other types of Trk receptors or signaling molecules downstream of Trk. We have recently started a series of experiments aiming to verify whether specific TrkB inhibition reduces glioma cell proliferation using ANA-12, a small-molecule selective TrkB antagonist. Our first results showed that ANA-12 effectively and dose-dependently reduces the viability of a human glioblastoma cell line with almost complete disappearance of cultured cells 72 hours after treatment (Fig. 1). Therefore, selective TrkB inhibition might prove to be an effective experimental therapeutic strategy, possibly with fewer off-target toxicities compared with multitarget drugs in patients with astrocytomas harboring oncogenic TrkB.

Glioblastoma-infiltrated innate immune cells resemble M0 macrophage phenotype

Glioblastomas are highly infiltrated by diverse immune cells, including microglia, macrophages, and myeloid-derived suppressor cells (MDSCs). Understanding the mechanisms by which glioblastoma-associated myeloid cells (GAMs) undergo metamorphosis into tumor-supportive cells, characterizing the heterogeneity of immune cell phenotypes within glioblastoma subtypes, and discovering new targets can help the design of new efficient immunotherapies. In this study, we performed a comprehensive battery of immune phenotyping, whole-genome microarray analysis, and microRNA expression profiling of GAMs with matched blood monocytes, healthy donor monocytes, normal brain microglia, nonpolarized M0 macrophages, and polarized M1, M2a, M2c macrophages. Glioblastoma patients had an elevated number of monocytes relative to healthy donors. Among CD11b+ cells, microglia and MDSCs constituted a higher percentage of GAMs than did macrophages. GAM profiling using flow cytometry studies revealed a continuum between the M1- and M2-like phenotype. Contrary to current dogma, GAMs exhibited distinct immunological functions, with the former aligned close to nonpolarized M0 macrophages.

Systematic analysis of telomere length and somatic alterations in 31 cancer types

Cancer cells survive cellular crisis through telomere maintenance mechanisms. We report telomere lengths in 18,430 samples, including tumors and non-neoplastic samples, across 31 cancer types. Telomeres were shorter in tumors than in normal tissues and longer in sarcomas and gliomas than in other cancers. Among 6,835 cancers, 73% expressed telomerase reverse transcriptase (TERT), which was associated with TERT point mutations, rearrangements, DNA amplifications and transcript fusions and predictive of telomerase activity. TERT promoter methylation provided an additional deregulatory TERT expression mechanism. Five percent of cases, characterized by undetectable TERT expression and alterations in ATRX or DAXX, demonstrated elongated telomeres and increased telomeric repeat–containing RNA (TERRA). The remaining 22% of tumors neither expressed TERT nor harbored alterations in ATRX or DAXX. In this group, telomere length positively correlated with TP53 and RB1 mutations. Our analysis integrates TERT abnormalities, telomerase activity and genomic alterations with telomere length in cancer.

TERT Promoter Mutations and Risk of Recurrence in Meningioma

The World Health Organization (WHO) classification and grading system attempts to predict the clinical course of meningiomas based on morphological parameters. However, because of high interobserver variation of some criteria, more reliable prognostic markers are required. Here, we assessed the TERT promoter for mutations in the hotspot regions C228T and C250T in meningioma samples from 252 patients. Mutations were detected in 16 samples (6.4% across the cohort, 1.7%, 5.7%, and 20.0% of WHO grade I, II, and III cases, respectively). Data were analyzed by t test, Fishers exact test, log-rank test, and Cox proportional hazard model. All statistical tests were two-sided. Within a mean follow-up time in surviving patients of 68.1 months, TERT promoter mutations were statistically significantly associated with shorter time to progression (P < .001). Median time to progression among mutant cases was 10.1 months compared with 179.0 months among wild-type cases. Our results indicate that the inclusion of molecular data (ie, analysis of TERT promoter status) into a histologically and genetically integrated classification and grading system for meningiomas increases prognostic power. Consequently, we propose to incorporate the assessment of TERT promoter status in upcoming grading schemes for meningioma.

Mesenchymal Stem Cells Isolated From Human Gliomas Increase Proliferation and Maintain Stemness of Glioma Stem Cells Through the IL-6/gp130/STAT3 Pathway.

Although mesenchymal stem cells (MSCs) have been implicated as stromal components of several cancers, their ultimate contribution to tumorigenesis and their potential to drive cancer stem cells, particularly in the unique microenvironment of human brain tumors, remain largely undefined. Consequently, using established criteria, we isolated glioma‐associated‐human MSCs (GA‐hMSCs) from fresh human glioma surgical specimens for the first time. We show that these GA‐hMSCs are nontumorigenic stromal cells that are phenotypically similar to prototypical bone marrow‐MSCs. Low‐passage genomic sequencing analyses comparing GA‐hMSCs with matched tumor‐initiating glioma stem cells (GSCs) suggest that most GA‐hMSCs (60%) are normal cells recruited to the tumor (group 1 GA‐hMSCs), although, rarely (10%), GA‐hMSCs may differentiate directly from GSCs (group 2 GA‐hMSCs) or display genetic patterns intermediate between these groups (group 3 GA‐hMSCs). Importantly, GA‐hMSCs increase proliferation and self‐renewal of GSCs in vitro and enhance GSC tumorigenicity and mesenchymal features in vivo, confirming their functional significance within the GSC niche. These effects are mediated by GA‐hMSC‐secreted interleukin‐6, which activates STAT3 in GSCs. Our results establish GA‐hMSCs as a potentially new stromal component of gliomas that drives the aggressiveness of GSCs, and point to GA‐hMSCs as a novel therapeutic target within gliomas. Stem Cells 2015;33:2400–2415

Integrated Chromosome 19 Transcriptomic and Proteomic Data Sets Derived from Glioma Cancer Stem-Cell Lines

One subproject within the global Chromosome 19 Consortium is to define chromosome 19 gene and protein expression in glioma-derived cancer stem cells (GSCs). Chromosome 19 is notoriously linked to glioma by 1p/19q codeletions, and clinical tests are established to detect that specific aberration. GSCs are tumor-initiating cells and are hypothesized to provide a repository of cells in tumors that can self-replicate and be refractory to radiation and chemotherapeutic agents developed for the treatment of tumors. In this pilot study, we performed RNA-Seq, label-free quantitative protein measurements in six GSC lines, and targeted transcriptomic analysis using a chromosome 19-specific microarray in an additional six GSC lines. The data have been deposited to the ProteomeXchange with identifier PXD000563. Here we present insights into differences in GSC gene and protein expression, including the identification of proteins listed as having no or low evidence at the protein level in the Human Protein Atlas, as correlated to chromosome 19 and GSC subtype. Furthermore, the upregulation of proteins downstream of adenovirus-associated viral integration site 1 (AAVS1) in GSC11 in response to oncolytic adenovirus treatment was demonstrated. Taken together, our results may indicate new roles for chromosome 19, beyond the 1p/19q codeletion, in the future of personalized medicine for glioma patients.

Systematic Identification of Single Amino Acid Variants in Glioma Stem-Cell-Derived Chromosome 19 Proteins

Novel proteoforms with single amino acid variations represent proteins that often have altered biological functions but are less explored in the human proteome. We have developed an approach, searching high quality shotgun proteomic data against an extended protein database, to identify expressed mutant proteoforms in glioma stem cell (GSC) lines. The systematic search of MS/MS spectra using PEAKS 7.0 as the search engine has recognized 17 chromosome 19 proteins in GSCs with altered amino acid sequences. The results were further verified by manual spectral examination, validating 19 proteoforms. One of the novel findings, a mutant form of branched-chain aminotransferase 2 (p.Thr186Arg), was verified at the transcript level and by targeted proteomics in several glioma stem cell lines. The structure of this proteoform was examined by molecular modeling in order to estimate conformational changes due to mutation that might lead to functional modifications potentially linked to glioma. Based on our initial findings, we believe that our approach presented could contribute to construct a more complete map of the human functional proteome.

Qki deficiency maintains stemness of glioma stem cells in suboptimal environment by downregulating endolysosomal degradation

Stem cells, including cancer stem cells (CSCs), require niches to maintain stemness, yet it is unclear how CSCs maintain stemness in the suboptimal environment outside their niches during invasion. Postnatal co-deletion of Pten and Trp53 in mouse neural stem cells (NSCs) leads to the expansion of these cells in their subventricular zone (SVZ) niches but fails to maintain stemness outside the SVZ. We discovered that Qki is a major regulator of NSC stemness. Qk deletion on a Pten−/−; Trp53−/− background helps NSCs maintain their stemness outside the SVZ in Nes-CreERT2; QkL/L; PtenL/L; Trp53L/L mice, which develop glioblastoma with a penetrance of 92% and a median survival time of 105 d. Mechanistically, Qk deletion decreases endolysosome-mediated degradation and enriches receptors essential for maintaining self-renewal on the cytoplasmic membrane to cope with low ligand levels outside niches. Thus, downregulation of endolysosome levels by Qki loss helps glioma stem cells (GSCs) maintain their stemness in suboptimal environments outside their niches.