Maria Stella Carro

NFKBIA Deletion in Glioblastomas

BACKGROUND Amplification and activating mutations of the epidermal growth factor receptor (EGFR) oncogene are molecular hallmarks of glioblastomas. We hypothesized that deletion of NFKBIA (encoding nuclear factor of κ-light polypeptide gene enhancer in B-cells inhibitor-α), an inhibitor of the EGFR-signaling pathway, promotes tumorigenesis in glioblastomas that do not have alterations of EGFR. METHODS We analyzed 790 human glioblastomas for deletions, mutations, or expression of NFKBIA and EGFR. We studied the tumor-suppressor activity of NFKBIA in tumor-cell culture. We compared the molecular results with the outcome of glioblastoma in 570 affected persons. RESULTS NFKBIA is often deleted but not mutated in glioblastomas; most deletions occur in nonclassical subtypes of the disease. Deletion of NFKBIA and amplification of EGFR show a pattern of mutual exclusivity. Restoration of the expression of NFKBIA attenuated the malignant phenotype and increased the vulnerability to chemotherapy of cells cultured from tumors with NFKBIA deletion; it also reduced the viability of cells with EGFR amplification but not of cells with normal gene dosages of both NFKBIA and EGFR. Deletion and low expression of NFKBIA were associated with unfavorable outcomes. Patients who had tumors with NFKBIA deletion had outcomes that were similar to those in patients with tumors harboring EGFR amplification. These outcomes were poor as compared with the outcomes in patients with tumors that had normal gene dosages of NFKBIA and EGFR. A two-gene model that was based on expression of NFKBIA and O(6)-methylguanine DNA methyltransferase was strongly associated with the clinical course of the disease. CONCLUSIONS Deletion of NFKBIA has an effect that is similar to the effect of EGFR amplification in the pathogenesis of glioblastoma and is associated with comparatively short survival.

Lineage-specific splicing of a brain-enriched alternative exon promotes glioblastoma progression

Tissue-specific alternative splicing is critical for the emergence of tissue identity during development, yet the role of this process in malignant transformation is undefined. Tissue-specific splicing involves evolutionarily conserved, alternative exons that represent only a minority of the total alternative exons identified. Many of these conserved exons have functional features that influence signaling pathways to profound biological effect. Here, we determined that lineage-specific splicing of a brain-enriched cassette exon in the membrane-binding tumor suppressor annexin A7 (ANXA7) diminishes endosomal targeting of the EGFR oncoprotein, consequently enhancing EGFR signaling during brain tumor progression. ANXA7 exon splicing was mediated by the ribonucleoprotein PTBP1, which is normally repressed during neuronal development. PTBP1 was highly expressed in glioblastomas due to loss of a brain-enriched microRNA (miR-124) and to PTBP1 amplification. The alternative ANXA7 splicing trait was present in precursor cells, suggesting that glioblastoma cells inherit the trait from a potential tumor-initiating ancestor and that these cells exploit this trait through accumulation of mutations that enhance EGFR signaling. Our data illustrate that lineage-specific splicing of a tissue-regulated alternative exon in a constituent of an oncogenic pathway eliminates tumor suppressor functions and promotes glioblastoma progression. This paradigm may offer a general model as to how tissue-specific regulatory mechanisms can reprogram normal developmental processes into oncogenic ones.

Functional Role of CLIC1 Ion Channel in Glioblastoma-Derived Stem/Progenitor Cells

Background Chloride channels are physiologically involved in cell division and motility. Chloride intracellular channel 1 (CLIC1) is overexpressed in a variety of human solid tumors compared with normal tissues, suggesting a potential involvement of CLIC1 in the regulation of tumorigenesis. This led us to investigate the role of CLIC1 in gliomagenesis. Methods We used the neurosphere system to isolate stem/progenitor cells from human glioblastomas (GBMs). CLIC1 targeting in GBM neurospheres was achieved by both lentiviral-mediated short-hairpin RNA transduction and CLIC1 antibody treatment, and its effect on stem-like properties was analyzed in vitro by proliferation and clonogenic assays and in vivo by orthotopic injection in immunocompromised mice. Channel activity was studied by perforated patch clamp technique. Differences in expression were analyzed by analysis of variance with Tamhane’s multiple comparison test. Kaplan–Meier analyses and log-rank test were used to assess survival. All statistical tests were two-sided. Results CLIC1 was statistically significantly overexpressed in GBMs compared with normal brain tissues (P < .001) with a better survival of patients with CLIC1 low-expressing tumors (CLIC1low vs CLIC1high survival: χ2 = 74.35; degrees of freedom = 1; log-rank P < .001). CLIC1 was variably expressed in patient-derived GBM neurospheres and was found enriched in the stem/progenitor compartment. CLIC1 silencing reduced proliferative (P < .01), clonogenic (P < .01), and tumorigenic capacity (P < .05) of stem/progenitor cells. The reduction of CLIC1 chloride currents with a specific CLIC1 antibody mirrored the biological effects of CLIC1 silencing in GBM patient–derived neurospheres. Conclusions Reduced gliomagenesis after CLIC1 targeting in tumoral stem/progenitor cells and the finding that CLIC1 expression is inversely associated with patient survival suggest CLIC1 as a potential target and prognostic biomarker.

Comprehensive analysis of PD-L1 expression in glioblastoma multiforme

Glioblastoma multiforme are highly malignant brain tumours with frequent genetic and epigenetic alterations. The poor clinical outcome of these tumours necessitates the development of new treatment options. Immunotherapies for glioblastoma multiforme including PD1/PD-L1 inhibition are currently tested in ongoing clinical trials. The purpose of this study was to investigate the molecular background of PD-L1 expression in glioblastoma multiforme and to find associated pathway activation and genetic alterations. We show that PD-L1 is up-regulated in IDH1/2 wildtype glioblastoma multiforme compared to lower-grade gliomas. In addition, a strong association of PD-L1 with the mesenchymal expression subgroup was observed. Consistent with that, NF1 mutation and corresponding activation of the MAPK pathway was strongly connected to PD-L1 expression. Our findings may explain different response to PD-L1 inhibition of patients in ongoing trials and may help to select patients that may profit of immunotherapy in the future.

Integrative Network-based Analysis of Magnetic Resonance Spectroscopy and Genome Wide Expression in Glioblastoma multiforme

The goal of this study was to identify correlations between metabolites from proton MR spectroscopy and genetic pathway activity in glioblastoma multiforme (GBM). Twenty patients with primary GBM were analysed by short echo-time chemical shift imaging and genome-wide expression analyses. Weighed Gene Co-Expression Analysis was used for an integrative analysis of imaging and genetic data. N-acetylaspartate, normalised to the contralateral healthy side (nNAA), was significantly correlated to oligodendrocytic and neural development. For normalised creatine (nCr), a group with low nCr was linked to the mesenchymal subtype, while high nCr could be assigned to the proneural subtype. Moreover, clustering of normalised glutamine and glutamate (nGlx) revealed two groups, one with high nGlx being attributed to the neural subtype, and one with low nGlx associated with the classical subtype. Hence, the metabolites nNAA, nCr, and nGlx correlate with a specific gene expression pattern reflecting the previously described subtypes of GBM. Moreover high nNAA was associated with better clinical prognosis, whereas patients with lower nNAA revealed a shorter progression-free survival (PFS).

Integrative Modeling Reveals Annexin A2-mediated Epigenetic Control of Mesenchymal Glioblastoma

Glioblastomas are characterized by transcriptionally distinct subtypes, but despite possible clinical relevance, their regulation remains poorly understood. The commonly used molecular classification systems for GBM all identify a subtype with high expression of mesenchymal marker transcripts, strongly associated with invasive growth. We used a comprehensive data-driven network modeling technique (augmented sparse inverse covariance selection, aSICS) to define separate genomic, epigenetic, and transcriptional regulators of glioblastoma subtypes. Our model identified Annexin A2 (ANXA2) as a novel methylation-controlled positive regulator of the mesenchymal subtype. Subsequent evaluation in two independent cohorts established ANXA2 expression as a prognostic factor that is dependent on ANXA2 promoter methylation. ANXA2 knockdown in primary glioblastoma stem cell-like cultures suppressed known mesenchymal master regulators, and abrogated cell proliferation and invasion. Our results place ANXA2 at the apex of a regulatory cascade that determines glioblastoma mesenchymal transformation and validate aSICS as a general methodology to uncover regulators of cancer subtypes.

Expression differences of programmed death ligand 1 in de-novo and recurrent glioblastoma multiforme

The biology of recurrent glioblastoma multiforme (GBM) is a dynamic process influenced by selection pressure induced by different antitumoural therapies. The poor clinical outcome of tumours in the recurrent stage necessitates the development of effective therapeutic strategies. Checkpoint-inhibition (PD1/PD-L1 Inhibition) is a hallmark of immunotherapy being investigated in ongoing clinical trials. The purpose of this study was to analyse the PD-L1 expression in de-novo and recurrent glioblastoma multiforme and to explore associated genetic alterations and clinical traits. We show that PD-L1 expression was reduced in recurrent GBM in comparison to de-novo GBM. Additionally, patients who received an extended dose of temozolomide (TMZ) chemotherapy showed a significantly reduced level of PD-L1 expression in the recurrence stage compared to the corresponding de-novo tumour. Our findings may provide an explanation for potentially lower response to immunotherapy in the recurrent stage due to the reduced expression of the therapeutic target PD-L1.The biology of recurrent glioblastoma multiforme (GBM) is a dynamic process influenced by selection pressure induced by different antitumoural therapies. The poor clinical outcome of tumours in the recurrent stage necessitates the development of effective therapeutic strategies. Checkpoint-inhibition (PD1/PD-L1 Inhibition) is a hallmark of immunotherapy being investigated in ongoing clinical trials. The purpose of this study was to analyse the PD-L1 expression in de-novo and recurrent glioblastoma multiforme and to explore associated genetic alterations and clinical traits. We show that PD-L1 expression was reduced in recurrent GBM in comparison to de-novo GBM. Additionally, patients who received an extended dose of temozolomide (TMZ) chemotherapy showed a significantly reduced level of PD-L1 expression in the recurrence stage compared to the corresponding de-novo tumour. Our findings may provide an explanation for potentially lower response to immunotherapy in the recurrent stage due to the reduced expression of the therapeutic target PD-L1.

c-Jun-N-terminal phosphorylation regulates DNMT1 expression and genome wide methylation in gliomas.

High-grade gliomas (HGG) are the most common brain tumors, with an average survival time of 14 months. A glioma-CpG island methylator phenotype (G-CIMP), associated with better clinical outcome, has been described in low and high-grade gliomas. Mutation of IDH1 is known to drive the G-CIMP status. In some cases, however, the hypermethylation phenotype is independent of IDH1 mutation, suggesting the involvement of other mechanisms. Here, we demonstrate that DNMT1 expression is higher in low-grade gliomas compared to glioblastomas and correlates with phosphorylated c-Jun. We show that phospho-c-Jun binds to the DNMT1 promoter and causes DNA hypermethylation. Phospho-c-Jun activation by Anisomycin treatment in primary glioblastoma-derived cells attenuates the aggressive features of mesenchymal glioblastomas and leads to promoter methylation and downregulation of key mesenchymal genes (CD44, MMP9 and CHI3L1). Our findings suggest that phospho-c-Jun activates an important regulatory mechanism to control DNMT1 expression and regulate global DNA methylation in Glioblastoma.

Microenvironment-Derived Regulation of HIF Signaling Drives Transcriptional Heterogeneity in Glioblastoma Multiforme

The evolving and highly heterogeneous nature of malignant brain tumors underlies their limited response to therapy and poor prognosis. In addition to genetic alterations, highly dynamic processes, such as transcriptional and metabolic reprogramming, play an important role in the development of tumor heterogeneity. The current study reports an adaptive mechanism in which the metabolic environment of malignant glioma drives transcriptional reprogramming. Multiregional analysis of a glioblastoma patient biopsy revealed a metabolic landscape marked by varying stages of hypoxia and creatine enrichment. Creatine treatment and metabolism was further shown to promote a synergistic effect through upregulation of the glycine cleavage system and chemical regulation of prolyl-hydroxylase domain. Consequently, creatine maintained a reduction of reactive oxygen species and change of the α-ketoglutarate/succinate ratio, leading to an inhibition of HIF signaling in primary tumor cell lines. These effects shifted the transcriptional pattern toward a proneural subtype and reduced the rate of cell migration and invasion in vitro. Implications: Transcriptional subclasses of glioblastoma multiforme are heterogeneously distributed within the same tumor. This study uncovered a regulatory function of the tumor microenvironment by metabolism-driven transcriptional reprogramming in infiltrating glioma cells. Mol Cancer Res; 16(4); 655–68. ©2018 AACR.

Abstract 3202: PTBP1-mediated aberrant splicing of a brain-specific exon in ANXA7 promotes brain tumorigenesis.

Glioblastoma multiforme (GBM) is the most common and malignant type of brain tumor in adults and is nearly uniformly fatal, with a median survival of one year. Diffuse infiltration, extensive neo-angiogenesis, uncontrolled cellular proliferation, presence of necrotic regions and resistance to apoptosis are all hallmark features of these tumors. The complex nature of GBMs is worsened by an incomplete understanding of its cellular origin and of the molecular determinants of its aggressiveness. This has contributed to make this cancer one of the most difficult to treat. Alternative splicing of pre-mRNA is an important mechanism to increase the diversity of protein functions; its dysregulation leads to the aberrant expression of specific protein isoforms, often associated with cancer. A relatively small number of splicing factors regulates the production of different splice variants of many proteins. In cancer, alterations in the expression of few splicing factors may lead to the aberrant splicing of several targets including oncogenes and tumor suppressors, which may contribute to the transformed cellular phenotype. Here, we investigate the tumorigenic role of the heterogeneous nuclear ribonucleoprotein PTBP1, a splicing factor commonly overexpressed in GBM. In the present study we used RNA immunoprecipitation and Exon Trap assay to describe how PTBP1 drives the aberrant splicing of a brain-specific exon in the membrane-binding tumor suppressor Annexin A7 (ANXA7). By differential overexpression of ANXA7 splice variants in tumor cell lines, we showed that expression of ANXA7 variant B, normally expressed in brain, controls EGFR pathway; conversely, the tumor variant ANXA7-vA does not stop EGFR activation cascade. Immunostaining experiments demonstrated that, compared to ANXA7-vB, ANXA7-vA reduces the endosomal targeting of EGFR and prolonging the activation of its signaling pathway. Knocking down PTBP1 in tumor cell lines restored the expression of ANXA7-vB and mimicked the effect of ANXA7-vB overexpression on EGFR trafficking and pathway activation. Furthermore, we performed in vivo experiments to show that PTBP1 increased expression has a general effect on tumor aggressiveness as it drives angiogenesis and promotes invasiveness. Finally, we assessed the association of PTBP1 expression and clinical outcome and we found that patients with high PTBP1 expression had a significantly worse survival rate. Our study highlights the important role the splicing factors play in cancer. In fact, the mechanism we describe here could represent a common paradigm of malignant transformation which could possibly lead to the development of new therapeutic strategies for GBM treatment. Citation Format: Roberto Ferrarese, Eva Bug, Daniel Maticzka, Wilfried Reichardt, Maria Stella Carro, Markus Bredel. PTBP1-mediated aberrant splicing of a brain-specific exon in ANXA7 promotes brain tumorigenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3202. doi:10.1158/1538-7445.AM2013-3202