Kirsi J. Granberg

Genomically amplified Akt3 activates DNA repair pathway and promotes glioma progression

Significance Glioblastoma is the most common and aggressive type of glioma, with a median survival of 15 mo. A major obstacle to effective treatment is de novo or acquired resistance to standard-care therapies, including radiation and temozolomide. Enhanced DNA repair can allow damaged or mutated cells to survive, contributing to resistance and tumor recurrence. We have identified Akt3 as the dominant Akt isoform that robustly stimulates glioma progression. We also discovered key roles for Akt3 in activating DNA repair pathways, which led to enhanced survival of human glioblastoma cells following radiation or temozolomide treatment. Our work has potential broad application to multiple cancer types in which Akt3 is expressed. Blocking this pathway may help prevent or alleviate DNA repair-mediated therapeutic resistance. Akt is a robust oncogene that plays key roles in the development and progression of many cancers, including glioma. We evaluated the differential propensities of the Akt isoforms toward progression in the well-characterized RCAS/Ntv-a mouse model of PDGFB-driven low grade glioma. A constitutively active myristoylated form of Akt1 did not induce high-grade glioma (HGG). In stark contrast, Akt2 and Akt3 showed strong progression potential with 78% and 97% of tumors diagnosed as HGG, respectively. We further revealed that significant variations in polarity and hydropathy values among the Akt isoforms in both the pleckstrin homology domain (P domain) and regulatory domain (R domain) were critical in mediating glioma progression. Gene expression profiles from representative Akt-derived tumors indicated dominant and distinct roles for Akt3, consisting primarily of DNA repair pathways. TCGA data from human GBM closely reflected the DNA repair function, as Akt3 was significantly correlated with a 76-gene signature DNA repair panel. Consistently, compared with Akt1 and Akt2 overexpression models, Akt3-expressing human GBM cells had enhanced activation of DNA repair proteins, leading to increased DNA repair and subsequent resistance to radiation and temozolomide. Given the wide range of Akt3-amplified cancers, Akt3 may represent a key resistance factor.

IGFBP2 potentiates nuclear EGFR-STAT3 signaling

Insulin-like growth factor binding protein 2 (IGFBP2) is a pleiotropic oncogenic protein that has both extracellular and intracellular functions. Despite a clear causal role in cancer development, the tumor-promoting mechanisms of IGFBP2 are poorly understood. The contributions of intracellular IGFBP2 to tumor development and progression are also unclear. Here we present evidence that both exogenous IGFBP2 treatment and cellular IGFBP2 overexpression lead to aberrant activation of epidermal growth factor receptor (EGFR), which subsequently activates signal transducer and activator of transcription factor 3 (STAT3) signaling. Furthermore, we demonstrate that IGFBP2 augments the nuclear accumulation of EGFR to potentiate STAT3 transactivation activities, via activation of the nuclear EGFR signaling pathway. Nuclear IGFBP2 directly influences the invasive and migratory capacities of human glioblastoma cells, providing a direct link between intracellular (and particularly nuclear) IGFBP2 and cancer hallmarks. These activities are also consistent with the strong association between IGFBP2 and STAT3-activated genes derived from The Cancer Genome Atlas database for human glioma. A high level of all three proteins (IGFBP2, EGFR and STAT3) was strongly correlated with poorer survival in an independent patient data set. These results identify a novel tumor-promoting function for IGFBP2 of activating EGFR/STAT3 signaling and facilitating EGFR accumulation in the nucleus, thereby deregulating EGFR signaling by two distinct mechanisms. As targeting EGFR in glioma has been relatively unsuccessful, this study suggests that IGFBP2 may be a novel therapeutic target.

Twist predicts poor outcome of patients with astrocytic glioma

Aims and methods Epithelial-mesenchymal transition (EMT) has previously been linked to glioma invasion and progression. To determine whether EMT regulators, Twist and Zeb1, had clinical significance in astrocytic gliomas, the association of Twist and Zeb1 with clinicopathological and molecular factors was studied in 269 astrocytoma samples. Results Twist and Zeb1 were widely expressed in astrocytic gliomas, but the expression of the former did not correlate with that of the latter. Stronger Twist expression levels were associated with higher WHO grades (p=0.001), whereas Zeb1 did not correlate with WHO grades. We found no association between Twist and proliferation activity (Ki67/MIB-1), p53 status, epidermal growth factor receptor (EGFR) amplification or neural cell adhesion molecule (NCAM) expression. There was no significant difference in Twist or Zeb1 expression when primary and secondary gliomas were analysed. Tumours with high Twist expression were IDH1 negative (p=0.009). High hypoxia-inducible factor-1α expression correlated significantly with positive Twist expression (p<0.001), whereas it was not associated with Zeb1 expression. Zeb1 expression did not correlate with proliferation, EGFR or IDH1. Nevertheless, we did find a correlation between high Zeb1 expression and low p53 expression levels (p=0.027). Positive NCAM expression was significantly associated with Zeb1 positivity (p=0.022). Zeb1 had no association with patient survival, whereas positive Twist expression predicted poor survival for patients in both univariate (p<0.001) and multivariable analyses (p=0.027). Conclusions EMT regulators, Twist and Zeb1, are common features of infiltrating astrocytomas, and Twist is upregulated in glioblastomas in particular. Twist may be a novel marker for poor prognosis in glioma patients.

Overexpression of SNORD114-3 marks acute promyelocytic leukemia

partial in vitro and in vivo efficacy in del17p CLL patients. In CLL patients of advanced age and with a high burden of comorbidities, the feasibility of an association with chlorambucil has been tested for obinutuzumab. Recently, a very promising approach with the Bruton’s Tyrosine Kinase inhibitor, ibrutinib, in combination with rituximab has been shown to be safe and well tolerated for highrisk CLL patients, inducing very high early response rates. These encouraging data emphasize the need for the rapid further development of ‘chemofree’ combinations for high-risk CLL patients, including those with ublituximab.

Strong FGFR3 staining is a marker for FGFR3 fusions in diffuse gliomas

Abstract Background Inhibitors of fibroblast growth factor receptors (FGFRs) have recently arisen as a promising treatment option for patients with FGFR alterations. Gene fusions involving FGFR3 and transforming acidic coiled-coil protein 3 (TACC3) have been detected in diffuse gliomas and other malignancies, and fusion-positive cases have responded well to FGFR inhibition. As high FGFR3 expression has been detected in fusion-positive tumors, we sought to determine the clinical significance of FGFR3 protein expression level as well as its potential for indicating FGFR3 fusions. Methods We performed FGFR3 immunohistochemistry on tissue microarrays containing 676 grades II–IV astrocytomas and 116 grades II–III oligodendroglial tumor specimens. Fifty-one cases were further analyzed using targeted sequencing. Results Moderate to strong FGFR3 staining was detected in gliomas of all grades, was more common in females, and was associated with poor survival in diffuse astrocytomas. Targeted sequencing identified FGFR3-TACC3 fusions and an FGFR3-CAMK2A fusion in 10 of 15 strongly stained cases, whereas no fusions were found in 36 negatively to moderately stained cases. Fusion-positive cases were predominantly female and negative for IDH and EGFR/PDGFRA/MET alterations. These and moderately stained cases show lower MIB-1 proliferation index than negatively to weakly stained cases. Furthermore, stronger FGFR3 expression was commonly observed in malignant tissue regions of lower cellularity in fusion-negative cases. Importantly, subregional negative FGFR3 staining was also observed in a few fusion-positive cases. Conclusions Strong FGFR3 protein expression is indicative of FGFR3 fusions and may serve as a clinically applicable predictive marker for treatment regimens based on FGFR inhibitors.

ESTOOLS Data@Hand: human stem cell gene expression resource

MiTCR modules are checked by more than 80 comprehensive unit tests, which improved the reliability and correctness of the code. The MiTCR API package can be used in Java projects through Maven and in Groovy scripts using Groovy Grapes. MiTCR is regularly updated; Windows installer, cross-platform binaries and source code are available from http://mitcr.milaboratory.com/ under the terms of the GNU GPL v3 license.

Abstract 4148: Different immune cell responses are associated with glioblastoma subclassification and typical genetic alterations

Interactions between various components in the tumor microenvironment and dysregulated immune responses are thought to play important roles in cancer development. To better understand the role of immune cells in tumor pathogenesis and destruction, we computationally model the microenvironment of an aggressive brain tumor glioblastoma multiforme (GBM). We downloaded GBM patient RNA-seq data from the Cancer Genome Atlas (TCGA). Using cluster analysis, we identified 16 clusters, each containing 10 to 933 genes that show a statistical enrichment of immune response related gene ontology terms. Utilizing a panel of RNA-seq data from normal cell types, we constructed global and cluster specific regression models to characterize the expression profiles of GBM samples in the clusters of interest as linear combinations of normal cell and reference GBM expression profiles. Simulated data was used to validate that the regression model coefficients accurately reflect the contributions of normal cell types to the expression profiles of tumor samples. Based on the regression analysis, we were able to uncover high variability in the composition of microenvironment across the TCGA cohort, suggesting diverse immune responses in tumors. The results from global regression analysis were then associated with common genetic alterations in GBM and with GBM subclassification. Especially the estimated macrophage, granulocyte, and CD8+ T lymphocyte proportions show significant differences between different GBM subgroups. Accumulation of immune cells was increased in the mesenchymal and neural subtype compared to other subtypes. Furthermore, estimated immune cell proportions were associated with alterations in EGFR and NF1. Taken together, our analysis provided a characterization of the immunomicroenvironment in GBM and linked immune cell responses to typical GBM alterations and GBM subtypes. More detailed characterization of diverse immune responses will facilitate patient stratification and might provide tools for personalized immunotherapy in the future. Citation Format: Suvi Luoto, Juha Kesseli, Matti Nykter, Kirsi J. Granberg. Different immune cell responses are associated with glioblastoma subclassification and typical genetic alterations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4148.

Abstract 3338: IGFBP2 promotes EGFR/STAT3 nuclear crosstalk in glioma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Insulin-like growth factor binding protein 2 (IGFBP2) is a pleiotropic secreted protein that is overexpressed in various cancers including glioma. Extracellular IGFBP2 binds and modulates the activity of insulin growth factors I and II (IGFI and IGFII) while cytoplasmic IGFBP2 interacts with signaling pathways essential for cell proliferation and survival. The versatility of IGFBP2 as a secreted or cytoplasmic signaling effector is widely characterized; however, the functions of nuclear IGFBP2 remain largely unknown. Through genomic and functional analyses of glioma, we mechanistically elucidated the role of nuclear IGFBP2 and discovered a functional interaction of IGFBP2 with epidermal growth factor receptor (EGFR) and signal transducer and activator of transcription 3 (STAT3) proteins. Our findings established a signaling hierarchy of IGFBP2-EGFR-STAT3; IGFBP2 not only upregulates EGFR/STAT3 signaling activity, but also facilitates EGFR nuclear translocation and subsequent nuclear EGFR/STAT3 activity. Furthermore, mutation of the nuclear localization signal of IGFBP2 demonstrated that nuclear translocation of IGFBP2 is critical to promote the nuclear activity of EGFR. By identifying the role of IGFBP2 in perpetuating nuclear crosstalk between EGFR/STAT3, our study further reveals the importance of exploiting IGFBP2 as a target for glioma therapy. Citation Format: YingXuan Chua, Yuexin Liu, Limei Hu, Kirsi Granberg, Maartje Verploegen, Wei Zhang. IGFBP2 promotes EGFR/STAT3 nuclear crosstalk in glioma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3338. doi:10.1158/1538-7445.AM2014-3338

Abstract LB-92: Using immunohistochemistry to evaluate FGFR3 gene fusion recurrence and clinical associations in astrocytomas

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Aberrant activation of fibroblast growth factor receptor 3 (FGFR3) has been reported to be tumorigenic in various cancer types. When FGFR3 is fused with another gene, most commonly with transforming acidic coiled coil 3 (TACC3), it can escape miRNA-mediated regulation and remain constitutively active most likely due to the dimerization via the domains of TACC3 or another fusion partner. Among brain cancers, gene fusions involving FGFR3 have been detected in glioblastoma and certain low grade gliomas. Furthermore, FGFR3-TACC3 fusion positive glioma cells have responded well to targeted therapies. The endogenous FGFR3 protein levels are extremely low in brain partly due to miR-99a-mediated regulation and gene fusions lead to prominent protein expression. In previous studies, FGFR3 fusion recurrence and fusion partners have been analyzed only in limited patient cohorts and there are no reports about their clinical associations. In this study, we have performed immunohistochemistry for FGFR3 on tissue microarrays with over 750 clinical astrocytoma samples (including grade 1-3 astrocytomas and glioblastomas). FGFR3 positive staining has been detected in subpopulations of all included tumor grades. The observed recurrences (1.8%-6.2%, depending on the grade) are consistent with previous sequencing-based studies. To validate immunohistochemistry-based approach for FGFR3 fusion detection, we are using frozen tumor material to confirm the presence of fusion with Western blotting and with PCR followed by Sanger sequencing. Furthermore, we have associated FGFR3 staining scores with clinical records for all the cases. FGFR3 staining is more common in females than males both in astrocytoma (7.6% vs 2.2%, pearson chi-square p-value 0.004, n=752) and glioblastoma cohorts (8.9% vs 2.1%, pearson chi-square p-value 0.008, n=494). In addition, FGFR3-staining negatively associates with aberrant p53 staining indicating p53 mutation (FGFR3-positive cases: 5.8% vs 1.8% in p53 low vs high groups, respectively, pearson chi-square p-value 0.030, n=399). Altogether, this study evaluates the suitability of immunohistochemical methods for FGFR3 fusion detection as well as investigates their recurrence and clinical significance in astrocytomas. Citation Format: Kirsi J. Granberg, Birgitta Lehtinen, Matti J. Annala, Hannu Haapasalo, Matti Nykter, Wei Zhang. Using immunohistochemistry to evaluate FGFR3 gene fusion recurrence and clinical associations in astrocytomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-92. doi:10.1158/1538-7445.AM2014-LB-92

DBComposer: An R package for integrative analysis and management of gene expression microarray data

DBComposer is an R package with a graphical user interface (GUI) to analyze and integrate human gene expression microarray data. With DBComposer, the data can be easily annotated, preprocessed and analyzed in several ways. DBComposer can also serve as a personal expression microarray database allowing users to store multiple datasets together for later retrieval or data analysis. It takes advantage of many R packages for statistics and visualizations, and provides a flexible framework to implement custom workflows to extend the data analysis capabilities.