STEM-17. Characterization of the Cell Surface Proteome in Recurrent Glioblastoma Initiating Cells (original) (raw)
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AAV-mediated direct in vivo CRISPR screen identifies functional suppressors in glioblastoma
Nature neuroscience, 2017
A causative understanding of genetic factors that regulate glioblastoma pathogenesis is of central importance. Here we developed an adeno-associated virus-mediated, autochthonous genetic CRISPR screen in glioblastoma. Stereotaxic delivery of a virus library targeting genes commonly mutated in human cancers into the brains of conditional-Cas9 mice resulted in tumors that recapitulate human glioblastoma. Capture sequencing revealed diverse mutational profiles across tumors. The mutation frequencies in mice correlated with those in two independent patient cohorts. Co-mutation analysis identified co-occurring driver combinations such as B2m-Nf1, Mll3-Nf1 and Zc3h13-Rb1, which were subsequently validated using AAV minipools. Distinct from Nf1-mutant tumors, Rb1-mutant tumors are undifferentiated and aberrantly express homeobox gene clusters. The addition of Zc3h13 or Pten mutations altered the gene expression profiles of Rb1 mutants, rendering them more resistant to temozolomide. Our stu...
Directin vivomapping of functional suppressors in glioblastoma genome
2017
Glioblastoma (GBM) is one of the deadliest cancers, with limited effective treatments and single-digit five-year survival1-7. A causative understanding of genetic factors that regulate GBM formation is of central importance8-19. However, a global, quantitative and functional understanding of gliomagenesis in the native brain environment has been lacking due to multiple challenges. Here, we developed an adeno-associated virus (AAV) mediated autochthonous CRISPR screen and directly mapped functional suppressors in the GBM genome. Stereotaxic delivery of an AAV library targeting significantly mutated genes into fully immunocompetent conditional Cas9 mice robustly led to gliomagenesis, resulting in tumors that recapitulate features of human GBM. Targeted capture sequencing revealed deep mutational profiles with diverse patterns across mice, uncoveringin vivoroles of previously uncharacterized factors in GBM such as immune regulatorB2m,zinc finger proteinZc3h13,transcription repressorCic...
Delineating the cytogenomic and epigenomic landscapes of glioma stem cell lines
PloS one, 2013
Glioblastoma multiforme (GBM), the most common and malignant type of glioma, is characterized by a poor prognosis and the lack of an effective treatment, which are due to a small sub-population of cells with stem-like properties, termed glioma stem cells (GSCs). The term ''multiforme'' describes the histological features of this tumor, that is, the cellular and morphological heterogeneity. At the molecular level multiple layers of alterations may reflect this heterogeneity providing together the driving force for tumor initiation and development. In order to decipher the common ''signature'' of the ancestral GSC population, we examined six already characterized GSC lines evaluating their cytogenomic and epigenomic profiles through a multilevel approach (conventional cytogenetic, FISH, aCGH, MeDIP-Chip and functional bioinformatic analysis). We found several canonical cytogenetic alterations associated with GBM and a common minimal deleted region (MDR) at 1p36.31, including CAMTA1 gene, a putative tumor suppressor gene, specific for the GSC population. Therefore, on one hand our data confirm a role of driver mutations for copy number alterations (CNAs) included in the GBM genomicsignature (gain of chromosome 7-EGFR gene, loss of chromosome 13-RB1 gene, loss of chromosome 10-PTEN gene); on the other, it is not obvious that the new identified CNAs are passenger mutations, as they may be necessary for tumor progression specific for the individual patient. Through our approach, we were able to demonstrate that not only individual genes into a pathway can be perturbed through multiple mechanisms and at different levels, but also that different combinations of perturbed genes can incapacitate functional modules within a cellular networks. Therefore, beyond the differences that can create apparent heterogeneity of alterations among GSC lines, there's a sort of selective force acting on them in order to converge towards the impairment of cell development and differentiation processes. This new overview could have a huge importance in therapy.
Cell Reports, 2015
To identify therapeutic targets for Glioblastoma (GBM), we performed genome-wide CRISPR-Cas9 "knockout" (KO) screens in patient-derived GBM stem-like cells (GSCs) and human neural stem/progenitors (NSCs), non-neoplastic stem cell controls, for genes required for their in vitro growth. Surprisingly, the vast majority GSC-lethal hits were found outside of molecular networks commonly altered in GBM and GSCs (e.g., oncogenic drivers). In vitro and in vivo validation of GSC-specific targets revealed several strong hits, including the wee1-like kinase, PKMYT1/Myt1. Mechanistic studies demonstrated that PKMYT1 acts redundantly with WEE1 to inhibit Cyclin B-CDK1 activity via CDK1-Y15 phosphorylation and to promote timely completion of mitosis in NSCs. However, in GSCs, this redundancy is lost, likely as a result of oncogenic signaling, causing GBM-specific lethality.
Functional discovery of targetable dependencies in recurrent glioblastoma
Research Square (Research Square), 2022
Resistance to genotoxic therapies and tumor recurrence are hallmarks of glioblastoma (GBM), an aggressive brain tumor. Here, we explore the functional drivers of post-treatment recurrent GBM. By conducting genome-wide CRISPR-Cas9 screens in patient-derived GBM models, we uncover distinct genetic dependencies in recurrent tumor cells that were absent in their patient-matched primary predecessors, accompanied by increased mutational burden and differential transcript and protein expression. These analyses map a multilayered genetic response to drive tumor recurrence, identifying protein tyrosine phosphatase 4A2 (PTP4A2) as a novel modulator of self-renewal, proliferation and tumorigenicity at GBM recurrence. Mechanistically, genetic perturbation or small molecule inhibition of PTP4A2 represses axon guidance activity through a dephosphorylation axis with roundabout guidance receptor 1 (ROBO1), exploiting a functional dependency on ROBO signaling. Importantly, engineered anti-ROBO1 single-domain antibodies also mimic the effects of PTP4A2 inhibition. We conclude that functional reprogramming drives tumorigenicity and dependence on a multi-targetable PTP4A2-ROBO1 signaling axis at GBM recurrence. Full Text For decades, clinicians have administered radiation therapy and chemotherapy to treat cancer patients 1. In parallel, resistance to these genotoxic treatments and tumor recurrence have become an inevitable reality for aggressive tumors. However, despite the clinical relevance and applications, functional drivers of disease recurrence remain poorly understood. Glioblastoma (GBM) remains the most aggressive and prevalent malignant primary brain tumor in adults 2. Unchanged since 2005, standard of care (SoC) consists of surgical resection, followed by radiation therapy (RT) plus concurrent and adjuvant chemotherapy with temozolomide (TMZ) 3,4. Despite these therapeutic efforts, patients inevitably succumb to recurrent disease with a median overall survival of 14.6 months and a ve-year survival rate of 5.5-6.8% 2,3,5. Unbiased genome-wide functional genomic screens have provided insights into genes and pathways regulating tumor cell survival, invasion, and sensitivity to TMZ in primary pre-treatment tumor cells 6-9. However, these studies do not examine changes at post-treatment tumor recurrence, and thus cannot explain treatment failure in ~70% of GBM patients 10. Here, we conduct a genome-scale comparison between patient-matched pre-and post-treatment GBM cells at the functional, transcriptomic, and proteomic levels. We uncover a therapeutic vulnerability for protein tyrosine phosphatase 4A2 (PTP4A2) at tumor recurrence, and introduce a modulatory role for PTP4A2 on axonal guidance proteins. Comparing primary and recurrent GBM We derived a pair of patient-matched GBM cell lines, one from a tumor specimen obtained at initial diagnosis prior to chemoradiotherapy (BT594, primary tumor cells), and a second specimen obtained at rst disease recurrence post-therapy (BT972, recurrent tumor cells) (Figure 1A, Table S1). Consistent with previous observations 11,12 , recurrent tumor cells showed a 25-fold increase in in vitro self-renewal capacity (P = 5.0e-09
The integrated landscape of driver genomic alterations in glioblastoma
Nature Genetics, 2013
nature genetics advance online publication 1 a r t i c l e s Recurrent and oncogenic gene fusions are hallmarks of hematological malignancies and have also been uncovered in solid tumors . We recently reported that a small subset of GBMs harbor FGFR-TACC gene fusions and provided data to suggest that individuals with FGFR-TACCpositive tumors would benefit from targeted FGFR kinase inhibition 9 . It remains unknown whether gene fusions involving other receptor tyrosine kinase (RTK)-coding genes exist and produce oncogene addiction in GBM. Here we analyze a large RNA-sequencing (RNA-seq) data set of primary GBMs and glioma sphere cultures (GSCs) and report the global landscape of in-frame gene fusions in human GBM.
Neuro-Oncology
Background. A high heterogeneity and activation of multiple oncogenic pathways have been implicated in failure of targeted therapies in glioblastoma (GBM). Methods. Using The Cancer Genome Atlas data, we identified subtype-specific prognostic core genes by a combined approach of genome-wide Cox regression and Gene Set Enrichment Analysis. The results were validated with 8 combined public datasets containing 608 GBMs. We further examined prognostic chromosome aberrations and mutations. Results. In classical and mesenchymal subtypes, 2 receptor tyrosine kinases (RTKs) (MET and IGF1R), and the genes in RTK downstream pathways such as phosphatidylinositol-3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR), and nuclear factor-kappaB (NF-kB), were commonly detected as prognostic core genes. Classical subtype-specific prognostic core genes included those in cell cycle, DNA repair, and the Janus kinase/signal transducers and activators of transcription (JAK-STAT) pathway. Immune-related genes were enriched in the prognostic genes showing negative promoter cytosine-phosphate-guanine (CpG) methylation/expression correlations. Mesenchymal subtype-specific prognostic genes were those related to mesenchymal cell movement, PI3K/Akt, mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), Wnt/β-catenin, and Wnt/Ca 2+ pathways. In copy number alterations and mutations, 6p loss and TP53 mutation were associated with poor and good survival, respectively, in the classical subtype. In the mesenchymal subtype, patients with PIK3R1 or PCLO mutations showed poor prognosis. In the glioma CpG island methylator phenotype (G-CIMP) subtype, patients harboring 10q loss, 12p gain, or 14q loss exhibited poor survival. Furthermore, 10q loss was significantly associated with the recently recognized G-CIMP subclass showing relatively low CpG methylation and poor prognosis. Conclusion. These subtype-specific alterations have promising potentials as new prognostic biomarkers and therapeutic targets combined with surrogate markers of GBM subtypes. However, considering the small number of events, the results of copy number alterations and mutations require further validations.
Emerging targets for glioblastoma stem cell therapy
Journal of biomedical research, 2015
Glioblastoma multiforme (GBM), designated as World Health Organization (WHO) grade IV astrocytoma, is a lethal and therapy-resistant brain cancer comprised of several tumor cell subpopulations, including GBM stem cells (GSCs) which are believed to contribute to tumor recurrence following initial response to therapies. Emerging evidence demonstrates that GBM tumors are initiated from GSCs. The development and use of novel therapies including small molecule inhibitors of specific proteins in signaling pathways that regulate stemness, proliferation and migration of GSCs, immunotherapy, and non-coding microRNAs may provide better means of treating GBM. Identification and characterization of GSC-specific signaling pathways would be necessary to identify specific therapeutic targets which may lead to the development of more efficient therapies selectively targeting GSCs. Several signaling pathways including mTOR, AKT, maternal embryonic leucine zipper kinase (MELK), NOTCH1 and Wnt/β-caten...
Glioblastomas derived from genetically modified pluripotent stem cells recapitulate pathobiology
Glioblastoma (GBM) is the most common malignant brain tumor, and particularly difficult to treat due to its inherent heterogeneity, which is promoted by a variety of genetic drivers. A lack of models that robustly recapitulate heterogeneity has been a major obstacle for research progress on this disease. Here we show that neural progenitor cells derived from human induced pluripotent stem cells, CRISPR/Cas9 engineered with different combinations of authentic GBM-related genetic drivers give rise to GBM models that recapitulate the pathobiology of this tumor, including inter- and intra-tumor heterogeneity, differential drug sensitivity, extrachromosomal DNA amplifications, and rapid clonal evolution. Different models established with this approach could serve as a platform for longitudinal assessment of drug treatment sensitivity governed by subtype-specific driver mutations.
Somatic CRISPR/Cas9-mediated tumour suppressor disruption enables versatile brain tumour modelling
Nature Communications, 2015
In vivo functional investigation of oncogenes using somatic gene transfer has been successfully exploited to validate their role in tumorigenesis. For tumour suppressor genes this has proven more challenging due to technical aspects. To provide a flexible and effective method for investigating somatic loss-of-function alterations and their influence on tumorigenesis, we have established CRISPR/Cas9-mediated somatic gene disruption, allowing for in vivo targeting of TSGs. Here we demonstrate the utility of this approach by deleting single (Ptch1) or multiple genes (Trp53, Pten, Nf1) in the mouse brain, resulting in the development of medulloblastoma and glioblastoma, respectively. Using whole-genome sequencing (WGS) we characterized the medulloblastoma-driving Ptch1 deletions in detail and show that no off-targets were detected in these tumours. This method provides a fast and convenient system for validating the emerging wealth of novel candidate tumour suppressor genes and the generation of faithful animal models of human cancer.