A novel patient stratification strategy to enhance the therapeutic efficacy of dasatinib in glioblastoma.

BACKGROUND: Glioblastoma is the most common primary malignancy of the central nervous system with a dismal prognosis. Genomic signatures classify isocitrate dehydrogenase 1 (IDH)-wildtype glioblastoma into three subtypes: proneural, mesenchymal, and classical. Dasatinib, an inhibitor of proto-oncogene kinase Src (SRC), is one of many therapeutics which, despite promising preclinical results, have failed to improve overall survival in glioblastoma patients in clinical trials. We examined whether glioblastoma subtypes differ in their response to dasatinib and could hence be evaluated for patient enrichment strategies in clinical trials. METHODS: We carried out in silico analyses on glioblastoma gene expression (TCGA) and single-cell RNA-Seq data. In addition, in vitro experiments using glioblastoma stem-like cells (GSCs) derived from primary patient tumors were performed, with complementary gene expression profiling and immunohistochemistry analysis of tumor samples. RESULTS: Patients with the mesenchymal subtype of glioblastoma showed higher SRC pathway activation based on gene expression profiling. Accordingly, mesenchymal GSCs were more sensitive to SRC inhibition by dasatinib compared to proneural and classical GSCs. Notably, SRC phosphorylation status did not predict response to dasatinib treatment. Furthermore, serpin peptidase inhibitor clade H member 1 (SERPINH1), a collagen-related heat-shock protein associated with cancer progression, was shown to correlate with dasatinib response and with the mesenchymal subtype. CONCLUSION: This work highlights further molecular-based patient selection strategies in clinical trials and suggests the mesenchymal subtype as well as SERPINH1 to be associated with response to dasatinib. Our findings indicate that stratification based on gene expression subtyping should be considered in future dasatinib trials.

PERK-mediated expression of peptidylglycine α-amidating monooxygenase supports angiogenesis in glioblastoma.

PKR-like kinase (PERK) plays a significant role in inducing angiogenesis in various cancer types including glioblastoma. By proteomics analysis of the conditioned medium from a glioblastoma cell line treated with a PERK inhibitor, we showed that peptidylglycine α-amidating monooxygenase (PAM) expression is regulated by PERK under hypoxic conditions. Moreover, PERK activation via CCT020312 (a PERK selective activator) increased the cleavage and thus the generation of PAM cleaved cytosolic domain (PAM sfCD) that acts as a signaling molecule from the cytoplasm to the nuclei. PERK was also found to interact with PAM, suggesting a possible involvement in the generation of PAM sfCD. Knockdown of PERK or PAM reduced the formation of tubes by HUVECs in vitro. Furthermore, in vivo data highlighted the importance of PAM in the growth of glioblastoma with reduction of PAM expression in engrafted tumor significantly increasing the survival in mice. In summary, our data revealed PAM as a potential target for antiangiogenic therapy in glioblastoma.

Targeting atypical protein kinase C iota reduces viability in glioblastoma stem-like cells via a notch signaling mechanism.

In a previous study, Protein Kinase C iota (PRKCI) emerged as an important candidate gene for glioblastoma (GBM) stem-like cell (GSC) survival. Here, we show that PKCι is overexpressed and activated in patient derived GSCs compared with normal neural stem cells and normal brain lysate, and that silencing of PRKCI in GSCs causes apoptosis, along with loss of clonogenicity and reduced proliferation. Notably, PRKCI silencing reduces tumor growth in vivo in a xenograft mouse model. PKCι has been intensively studied as a therapeutic target in non-small cell lung cancer, resulting in the identification of an inhibitor, aurothiomalate (ATM), which disrupts the PKCι/ERK signaling axis. However, we show that, although sensitive to pharmacological inhibition via a pseudosubstrate peptide inhibitor, GSCs are much less sensitive to ATM, suggesting that PKCι acts along a different signaling axis in GSCs. Gene expression profiling of PRKCI-silenced GSCs revealed a novel role of the Notch signaling pathway in PKCι mediated GSC survival. A proximity ligation assay showed that Notch1 and PKCι are in close proximity in GSCs. Targeting PKCι in the context of Notch signaling could be an effective way of attacking the GSC population in GBM.

Kinome-wide shRNA screen identifies the receptor tyrosine kinase AXL as a key regulator for mesenchymal glioblastoma stem-like cells.

Glioblastoma is a highly lethal cancer for which novel therapeutics are urgently needed. Two distinct subtypes of glioblastoma stem-like cells (GSCs) were recently identified: mesenchymal (MES) and proneural (PN). To identify mechanisms to target the more aggressive MES GSCs, we combined transcriptomic expression analysis and kinome-wide short hairpin RNA screening of MES and PN GSCs. In comparison to PN GSCs, we found significant upregulation and phosphorylation of the receptor tyrosine kinase AXL in MES GSCs. Knockdown of AXL significantly decreased MES GSC self-renewal capacity in vitro and inhibited the growth of glioblastoma patient-derived xenografts. Moreover, inhibition of AXL with shRNA or pharmacologic inhibitors also increased cell death significantly more in MES GSCs. Clinically, AXL expression was elevated in the MES GBM subtype and significantly correlated with poor prognosis in multiple cancers. In conclusion, we identified AXL as a potential molecular target for novel approaches to treat glioblastoma and other solid cancers.

Recurrent coamplification of cytoskeleton-associated genes EMS1 and SHANK2 with CCND1 in oral squamous cell carcinoma.

Chromosomal band 11q13 is frequently amplified in oral squamous cell carcinoma (OSCC) and assumed to be critically involved in tumor initiation and progression by proto-oncogene activation. Though cyclin D1 (CCND1) is supposed to be the most relevant oncogene, several additional putative candidate genes are inside this chromosomal region, for which their actual role in tumorigenesis still needs to be elucidated. To characterize the 11q13 amplicon in detail, 40 OSCCs were analyzed by comparative genomic hybridization to DNA microarrays (matrix-CGH) containing BAC clones derived from chromosomal band 11q13. This high-resolution approach revealed a consistent amplicon about 1.7 Mb in size including the CCND1 oncogene. Seven BAC clones covering FGF3, EMS1, and SHANK2 were shown to be frequently coamplified inside the CCND1 amplicon. Subsequent analysis of tissue microarrays by FISH revealed amplification frequencies of 36.8% (88/239) for CCND1, 34.3% (60/175) for FGF3, 37.4% (68/182) for EMS1, and 36.3% (61/168) for SHANK2. Finally, quantitative mRNA expression analysis demonstrated consistent overexpression of CCND1 in all tumors and of EMS1 and SHANK2 in a subset of specimens with 11q13 amplification, but no expression of FGF3 in any of the cases. Our study underlines the critical role of CCND1 in OSCC development and additionally points to the functionally related genes EMS1 and SHANK2, both encoding for cytoskeleton-associated proteins, which are frequently coamplified with CCND1 and therefore could cooperatively contribute to OSCC pathogenesis.