The semaphorin 3A/neuropilin-1 pathway promotes clonogenic growth of glioblastoma via activation of TGF-ß signaling.

Glioblastoma (GBM) is the most lethal brain cancer with a dismal prognosis. Stem-like GBM cells (GSCs) are a major driver of GBM propagation and recurrence; thus, understanding the molecular mechanisms that promote GSCs may lead to effective therapeutic approaches. Through in vitro clonogenic growth-based assays, we determined mitogenic activities of the ligand molecules that are implicated in neural development. We have identified that semaphorin 3A (Sema3A), originally known as an axon guidance molecule in the CNS, promotes clonogenic growth of GBM cells but not normal neural progenitor cells (NPCs). Mechanistically, Sema3A binds to its receptor neuropilin-1 (NRP1) and facilitates an interaction between NRP1 and TGF-ß receptor 1 (TGF-ßR1), which in turn leads to activation of canonical TGF-ß signaling in both GSCs and NPCs. TGF-ß signaling enhances self-renewal and survival of GBM tumors through induction of key stem cell factors, but it evokes cytostatic responses in NPCs. Blockage of the Sema3A/NRP1 axis via shRNA-mediated knockdown of Sema3A or NRP1 impeded clonogenic growth and TGF-ß pathway activity in GSCs and inhibited tumor growth in vivo. Taken together, these findings suggest that the Sema3A/NRP1/TGF-ßR1 signaling axis is a critical regulator of GSC propagation and a potential therapeutic target for GBM.

Transgenic viral expression of PH-20, IL-12, and sPD1-Fc enhances immune cell infiltration and anti-tumor efficacy of an oncolytic virus.

Oncolytic viruses are of significant clinical interest due to their ability to directly infect and kill tumors and enhance the anti-tumor immune response. Previously, we developed KLS-3010, a novel oncolytic virus derived from the International Health Department-White (IHD-W) strain vaccinia virus, which has robust tumoricidal effects. In the present study, we generated a recombinant oncolytic virus, KLS-3020, by inserting three transgenes (hyaluronidase [PH-20], interleukin-12 [IL-12], and soluble programmed cell death 1 fused to the Fc domain [sPD1-Fc]) into KLS-3010 and investigated its anti-tumor efficacy and ability to induce anti-tumor immune responses in CT26.WT and B16F10 mouse tumor models. A single injection of KLS-3020 significantly decreased tumor growth. The roles of the transgenes were investigated using viruses expressing each single transgene alone and KLS-3020. PH-20 promoted virus spread and tumor immune cell infiltration, IL-12 activated and reprogrammed T cells to inflammatory phenotypes, and sPD1-Fc increased intra-tumoral populations of activated T cells. The tumor-specific systemic immune response and the abscopal tumor control elicited by KLS-3020 were demonstrated in the CT26.WT tumor model. The insertion of transgenes into KLS-3020 increased its anti-tumor efficacy, supporting further clinical investigation of KLS-3020 as a novel oncolytic immunotherapy.

Pharmacogenomic profiling reveals molecular features of chemotherapy resistance in IDH wild-type primary glioblastoma.

BACKGROUND: Although temozolomide (TMZ) has been used as a standard adjuvant chemotherapeutic agent for primary glioblastoma (GBM), treating isocitrate dehydrogenase wild-type (IDH-wt) cases remains challenging due to intrinsic and acquired drug resistance. Therefore, elucidation of the molecular mechanisms of TMZ resistance is critical for its precision application. METHODS: We stratified 69 primary IDH-wt GBM patients into TMZ-resistant (n = 29) and sensitive (n = 40) groups, using TMZ screening of the corresponding patient-derived glioma stem-like cells (GSCs). Genomic and transcriptomic features were then examined to identify TMZ-associated molecular alterations. Subsequently, we developed a machine learning (ML) model to predict TMZ response from combined signatures. Moreover, TMZ response in multisector samples (52 tumor sectors from 18 cases) was evaluated to validate findings and investigate the impact of intra-tumoral heterogeneity on TMZ efficacy. RESULTS: In vitro TMZ sensitivity of patient-derived GSCs classified patients into groups with different survival outcomes (P = 1.12e-4 for progression-free survival (PFS) and 3.63e-4 for overall survival (OS)). Moreover, we found that elevated gene expression of EGR4, PAPPA, LRRC3, and ANXA3 was associated to intrinsic TMZ resistance. In addition, other features such as 5-aminolevulinic acid negative, mesenchymal/proneural expression subtypes, and hypermutation phenomena were prone to promote TMZ resistance. In contrast, concurrent copy-number-alteration in PTEN, EGFR, and CDKN2A/B was more frequent in TMZ-sensitive samples (Fisher's exact P = 0.0102), subsequently consolidated by multi-sector sequencing analyses. Integrating all features, we trained a ML tool to segregate TMZ-resistant and sensitive groups. Notably, our method segregated IDH-wt GBM patients from The Cancer Genome Atlas (TCGA) into two groups with divergent survival outcomes (P = 4.58e-4 for PFS and 3.66e-4 for OS). Furthermore, we showed a highly heterogeneous TMZ-response pattern within each GBM patient using in vitro TMZ screening and genomic characterization of multisector GSCs. Lastly, the prediction model that evaluates the TMZ efficacy for primary IDH-wt GBMs was developed into a webserver for public usage ( http://www.wang-lab-hkust.com:3838/TMZEP ). CONCLUSIONS: We identified molecular characteristics associated to TMZ sensitivity, and illustrate the potential clinical value of a ML model trained from pharmacogenomic profiling of patient-derived GSC against IDH-wt GBMs.

Development of a new advanced animal cradle for small animal multiple imaging modalities: acquisition and evaluation of high-throughput multiple-mouse imaging.

The physiological conditions of small animals are an essential component to be considered when acquiring images for pre-clinical studies, and they play a vital role in the overall results of a study. However, several previous studies did not consider these conditions. In this study, a new animal cradle that can be modified and adjusted to suit multiple imaging modalities such as positron emission tomography (PET)/computed tomography (CT) and magnetic resonance imaging (MRI) was developed. Unlike previous cradles where only one mouse can be imaged at a time, a total of four mice can be imaged simultaneously using this new cradle. Additionally, fusion images with high-throughput multiple-mouse imaging (MMI) of PET/MRI and PET/CT images can be acquired using this newly developed cradle. The dynamic brain images were also acquired simultaneously by applying PET dynamic imaging technology to high-throughput MMI methods. The results of this study suggest that the newly developed small animal cradle can be widely used in pre-clinical studies.

Semi-Automated Cell Panning for Efficient Isolation of FGFR3-Targeting Antibody.

Phage display technology is a widely used practical tool for isolating binding molecules against the desired targets in phage libraries. In the case of targeting the membrane protein with its natural conformation, conventional bio-panning has limitations on the efficient screening of the functionally relevant antibodies. To enrich the single-chain variable fragment (scFv) pools for recognizing the natural conformation of the membrane targets, the conventional bio-panning and screening process was modified to include the semi-automated cell panning protocol. Using FGFR3-overexpressing patient-derived cancer cells, biotin-X-DHPE was introduced and coupled to Streptavidin-coated magnetic beads for use in the solution-phage bio-panning procedure. The resulting clones of scFv were compared to the diversity of the binding region, especially on CDR-H3. The clones enriched further by cell-based panning procedure possessed a similar binding site and the CDR-H3 loop structure. The resulting antibodies inhibited cell growth and induced target degradation. This process may be a useful tool for screening biologically related antibodies that recognize natural conformational structure on cell membrane protein. Furthermore, cell-based panning has the potential to further expand to a high-throughput screening (HTS) system and automation process.

Characterization of Gut Microbiome in Korean Patients with Metabolic Associated Fatty Liver Disease.

Metabolic associated fatty liver disease (MAFLD) is a new concept where the presence of both fatty liver and metabolic abnormality are necessary for diagnosis. Several studies have reported that altered gut microbiome is closely associated with metabolic diseases and non-alcoholic fatty liver disease. However, the studies on MAFLD population are scarce. This prospective study aimed to identify differences in gut microbiome between patients with MAFLD and healthy controls in Korean population. In this study, patients with MAFLD and age, sex-matched healthy controls were included, and their stool samples were collected. Taxonomic composition of gut microbiota was analyzed using 16S ribosomal ribonucleic acid pyrosequencing. Twenty-two MAFLD patients and 44 healthy controls were included. Taxonomic diversity was lower in patients with MAFLD in the aspect of alpha and beta diversity. The differences were also found at phylum, class, family, and genus levels between the two groups. Phylum Proteobacteria, family Enterobactereriaceae, genus Citrobacter abundance was significantly increased and genus Faecalibacterium was significantly decreased in patients with MAFLD. In addition, butyrate-producing bacteria were decreased and ethanol-producing bacteria were increased in patients with MAFLD. The composition of gut microbiome was different between MAFLD and healthy controls in Korean population. This could offer potential targets for therapeutic intervention in MAFLD.

cIRCR201-dPBD, a Novel Pyrrolobenzodiazepine Dimer-Containing Site-Specific Antibody-Drug Conjugate Targeting c-Met Overexpression Tumors.

c-Met, as a receptor expressed on the cell membrane, contributes to the growth and metastasis of tumors, as well as angiogenesis, mainly through the hepatocyte growth factor (HGF)/c-Met axis during tumor progression. Although several c-Met inhibitors, including small molecules and monoclonal antibody inhibitors, are currently being investigated, their clinical outcomes have not been promising. Development of an antibody-drug conjugate (ADC) against c-Met could be an attractive therapeutic strategy that would provide superior antitumor efficacy with broad-spectrum c-Met expression levels. In the present study, site-specific drug-conjugate technology was applied to develop an ADC using the human-mouse cross-reactive c-Met antibody and a prodrug pyrrolobenzodiazepine (PBD). The toxin payload was uniformly conjugated to the light-chain C-terminus of the native cIRCR201 antibody (drug-to-antibody ratio = 2), as confirmed using LC-MS. Using a high-throughput screening system, we found that cIRCR201-dPBD exhibited varying sensitivities depending on the expression levels of c-Met, and it induced receptor-mediated endocytosis and toxin-mediated apoptosis in 47 different cancer cell lines. cIRCR201-dPBD also showed significant antitumor activity on the MET-amplified cancer cells using in vivo xenograft models. Therefore, cIRCR201-dPBD could be a promising therapeutic strategy for tumors with c-Met expression.

Double-filtered leukoreduction as a method for risk reduction of transfusion-associated graft-versus-host disease.

BACKGROUND: Transfusion-associated graft-versus-host disease (TA-GvHD) is caused by leukocytes, specifically T cells within a transfused blood product. Currently, the prevention of transfusion-associated graft-versus-host disease is performed by irradiation of blood products. With a sufficient reduction of leukocytes, the risk for TA-GvHD can be decreased. With consistent advances in current state-of-the-art blood filters, we herein propose that double filtration can sufficiently reduce leukocytes to reduce the risk for TA-GvHD. MATERIALS: Thirty RBC concentrates were filtered with leukocyte filters, followed by storage at 1-6 oC for 72 hours, and then a second filtration was performed. Residual leukocytes in the double-filtered RBC units (n = 30) were assessed with flow cytometric methods, and an additional assay with isolated peripheral blood mononuclear cells (PBMCs) (n = 6) was done by both flow cytometric methods and an automated hematology analyzer. Quality of the RBCs after filtration was evaluated by hematological and biochemical tests. In vitro T cell expansion was performed using anti-CD3/CD28-coated Dynabeads or anti-CD3 (OKT3). In vivo experiment for GvHD was performed by using NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. RESULTS: Double-filtered blood products showed residual leukocyte levels below detection limits, which calculated to be below 1200-2500 cells per blood unit. In vitro expansion rate of T cells showed that 6x103 and 1x103 cell-seeded specimens showed 60.8±10.6 fold and 10.2±9.7-fold expansion, respectively. Cell expansion was not sufficiently observed in wells planted with 1x102 or 10 cells. In vivo experiments showed that mice injected with 1x105 or more cells cause fatal GvHD. GvHD induced inflammation was observed in mice injected with 1x104 or more cells. No evidence of GvHD was found in mice injected with 103 cells. CONCLUSIONS: Our study suggests that additional removal of contaminating lymphocytes by a second leukodepletion step may further reduce the risk for TA-GvHD.

Pharmacogenomic landscape of patient-derived tumor cells informs precision oncology therapy.

Outcomes of anticancer therapy vary dramatically among patients due to diverse genetic and molecular backgrounds, highlighting extensive intertumoral heterogeneity. The fundamental tenet of precision oncology defines molecular characterization of tumors to guide optimal patient-tailored therapy. Towards this goal, we have established a compilation of pharmacological landscapes of 462 patient-derived tumor cells (PDCs) across 14 cancer types, together with genomic and transcriptomic profiling in 385 of these tumors. Compared with the traditional long-term cultured cancer cell line models, PDCs recapitulate the molecular properties and biology of the diseases more precisely. Here, we provide insights into dynamic pharmacogenomic associations, including molecular determinants that elicit therapeutic resistance to EGFR inhibitors, and the potential repurposing of ibrutinib (currently used in hematological malignancies) for EGFR-specific therapy in gliomas. Lastly, we present a potential implementation of PDC-derived drug sensitivities for the prediction of clinical response to targeted therapeutics using retrospective clinical studies.

Innovative Distribution Priorities for the Medical Devices Industry in the Fourth Industrial Revolution.

PURPOSE: This study aimed to set priorities for improving the medical device distribution structure and to suggest an innovative improvement plan for the distribution structure using the analytic hierarchy process (AHP) method, focusing on stakeholders in the medical device industry. METHODS: This study conducted a survey with 35 specialists using the AHP method, which is a multiple-criteria decisionmaking methodology, in order to set priorities for improvement plans to address the problems faced by the medical device distribution structure. RESULTS: The AHP analysis showed that supply stability was the most important factor, followed by greater transparency, efficiency, smart supply, and cost reduction. CONCLUSIONS: It is necessary to establish a stable supply system and manage crises through supply stability, as well as to provide opportunities for fair trade through greater transparency. As steps towards those goals, we propose establishing a unique device identification system, an information disclosure system, online distribution, and a group purchasing organization system in Korea.

Pharmacokinetics, Biodistribution, and Toxicity Evaluation of Anti-SEMA3A (F11) in In Vivo Models.

BACKGROUND/AIM: The aim of our study was to investigate the pharmacokinetics (PK), tissue distribution and toxicity of F11 antibody to semaphorin 3A in mouse models and explore its anti-angiogenic and tumor-inhibitory effect. MATERIALS AND METHODS: Patient-derived xenograft (PDX) models were established via subcutaneous implantation of glioblastoma multiforme (GBM) cells and treated with F11. RESULTS: F11 significantly attenuated tumor growth and angiogenesis in the GBM PDX model. Within the range of administered doses, the PK of F11 in serum demonstrated a linear fashion, consistent with general PK profiles of soluble antigen-targeting antibodies. Additionally, the clearance level was detected at between 4.63 and 7.12 ml/d/kg, while the biological half-life was measured at 6.9 and 9.4 days. Tissue distribution of F11 in kidney, liver and heart was consistent with previously reported antibody patterns. However, the presence of F11 in the brain was an interesting finding. CONCLUSION: Collectively, our results revealed angiogenic and tumor-inhibitory effect of F11 antibody and its potential therapeutic use within a clinical framework based on PK, biodistribution and toxicity evaluation in mouse models.

Anti-SEMA3A Antibody: A Novel Therapeutic Agent to Suppress Glioblastoma Tumor Growth.

PURPOSE: Glioblastoma (GBM) is classified as one of the most aggressive and lethal brain tumor. Great strides have been made in understanding the genomic and molecular underpinnings of GBM, which translated into development of new therapeutic approaches to combat such deadly disease. However, there are only few therapeutic agents that can effectively inhibit GBM invasion in a clinical framework. In an effort to address such challenges, we have generated anti-SEMA3A monoclonal antibody as a potential therapeutic antibody against GBM progression. MATERIALS AND METHODS: We employed public glioma datasets, Repository of Molecular Brain Neoplasia Data and The Cancer Genome Atlas, to analyze SEMA3AmRNA expression in human GBM specimens. We also evaluated for protein expression level of SEMA3A via tissue microarray (TMA) analysis. Cell migration and proliferation kinetics were assessed in various GBM patient-derived cells (PDCs) and U87-MG cell-line for SEMA3A antibody efficacy. GBM patient-derived xenograft (PDX) models were generated to evaluate tumor inhibitory effect of anti-SEMA3A antibody in vivo. RESULTS: By combining bioinformatics and TMA analysis, we discovered that SEMA3A is highly expressed in human GBM specimens compared to non-neoplastic tissues. We developed three different anti-SEMA3A antibodies, in fully human IgG form, through screening phage-displayed synthetic antibody library using a classical panning method. Neutralization of SEMA3A significantly reduced migration and proliferation capabilities of PDCs and U87-MG cell line in vitro. In PDX models, treatment with anti-SEMA3A antibody exhibited notable tumor inhibitory effect through down-regulation of cellular proliferative kinetics and tumor-associated macrophages recruitment. CONCLUSION: In present study, we demonstrated tumor inhibitory effect of SEMA3A antibody in GBM progression and present its potential relevance as a therapeutic agent in a clinical framework.

Antitumor activity, pharmacokinetics, tumor-homing effect, and hepatotoxicity of a species cross-reactive c-Met antibody.

The receptor tyrosine kinase c-Met plays critical roles in promoting tumor growth, invasion, metastasis, and angiogenesis in various types of cancer and is a promising therapeutic target. The development of a species cross-reactive therapeutic antibody could provide useful to comprehensive preclinical assessment in animal models. Towards this goal, we developed human/mouse cross-reactive c-Met antibodies using an antibody phage library. IRCR201, a c-Met antibody with species cross-reactivity, successfully inhibited the HGF/c-Met signaling pathway via degradation of c-Met and disruption of the binding with its partners, and demonstrated strong in vivo antitumor activity. In pharmacokinetic analysis, IRCR201 exhibited a nonlinear pharmacokinetic profile and showed rapid serum clearance at low dosage. Ex vivo fluorescence imaging and immunohistochemistry demonstrated strong tumor accumulation of IRCR201. Hepatotoxicity analysis revealed that IRCR201 does not significantly affect primary human and mouse hepatocytes. Serum chemistry analysis demonstrated that the alanine aminotransferase serum level was elevated in mice treated with 30 mg/kg IRCR201 than in PBS-treated mice, whereas the levels of aspartate aminotransferase and blood urea nitrogen did not significantly differ. Thus, IRCR201 is a potent therapeutic antibody that can disrupt the HGF/c-Met signaling axis and its species cross-reactivity would enable to evaluate precise biological activity in animal models.

Tumor Inhibitory Effect of IRCR201, a Novel Cross-Reactive c-Met Antibody Targeting the PSI Domain.

Hepatocyte growth factor receptor (HGFR, c-Met) is an essential member of the receptor tyrosine kinase (RTK) family that is often dysregulated during tumor progression, driving a malignant phenotypic state and modulating important cellular functions including tumor growth, invasion, metastasis, and angiogenesis, providing a strong rationale for targeting HGF/c-Met signaling axis in cancer therapy. Based on its protumorigenic potentials, we developed IRCR201, a potent antagonistic antibody targeting the plexin-semaphorin-integrin (PSI) domain of c-Met, using synthetic human antibody phage libraries. We characterized and evaluated the biochemical properties and tumor inhibitory effect of IRCR201 in vitro and in vivo. IRCR201 is a novel fully-human bivalent therapeutic antibody that exhibits cross-reactivity against both human and mouse c-Met proteins with high affinity and specificity. IRCR201 displayed low agonist activity and rapidly depleted total c-Met protein via the lysosomal degradation pathway, inhibiting c-Met-dependent downstream activation and attenuating cellular proliferation in various c-Met-expressing cancer cells. In vivo tumor xenograft models also demonstrated the superior tumor inhibitory responsiveness of IRCR201. Taken together, IRCR201 provides a promising therapeutic agent for c-Met-positive cancer patients through suppressing the c-Met signaling pathway and tumor growth.

GC1118, an Anti-EGFR Antibody with a Distinct Binding Epitope and Superior Inhibitory Activity against High-Affinity EGFR Ligands.

The EGFR-targeted monoclonal antibodies are a valid therapeutic strategy for patients with metastatic colorectal cancer (mCRC). However, only a small subset of mCRC patients has therapeutic benefits and there are high demands for EGFR therapeutics with a broader patient pool and more potent efficacy. In this study, we report GC1118 exhibiting a different character in terms of binding epitope, affinity, mode of action, and efficacy from other anti-EGFR antibodies. Structural analysis of the EGFR-GC1118 crystal complex revealed that GC1118 recognizes linear, discrete N-terminal epitopes of domain III of EGFR, critical for EGF binding but not overlapping with those of other EGFR-targeted antibodies. GC1118 exhibited superior inhibitory activity against high-affinity EGFR ligands in terms of EGFR binding, triggering EGFR signaling, and proliferation compared with cetuximab and panitumumab. EGFR signaling driven by low-affinity ligands, on the contrary, was well inhibited by all the antibodies tested. GC1118 demonstrated robust antitumor activity in tumor xenografts with elevated expression of high-affinity ligands in vivo, whereas cetuximab did not. Considering the significant role of high-affinity EGFR ligands in modulating tumor microenvironment and inducing resistance to various cancer therapeutics, our study suggests a potential therapeutic advantage of GC1118 in terms of efficacy and a range of benefited patient pool. Mol Cancer Ther; 15(2); 251-63. ©2015 AACR.

USP1 targeting impedes GBM growth by inhibiting stem cell maintenance and radioresistance.

BACKGROUND: Clinical benefits from standard therapies against glioblastoma (GBM) are limited in part due to intrinsic radio- and chemoresistance of GBM and inefficient targeting of GBM stem-like cells (GSCs). Novel therapeutic approaches that overcome treatment resistance and diminish stem-like properties of GBM are needed. METHODS: We determined the expression levels of ubiquitination-specific proteases (USPs) by transcriptome analysis and found that USP1 is highly expressed in GBM. Using the patient GBM-derived primary tumor cells, we inhibited USP1 by shRNA-mediated knockdown or its specific inhibitor pimozide and evaluated the effects on stem cell marker expression, proliferation, and clonogenic growth of tumor cells. RESULTS: USP1 was highly expressed in gliomas relative to normal brain tissues and more preferentially in GSC enrichment marker (CD133 or CD15) positive cells. USP1 positively regulated the protein stability of the ID1 and CHEK1, critical regulators of DNA damage response and stem cell maintenance. Targeting USP1 by RNA interference or treatment with a chemical USP1 inhibitor attenuated clonogenic growth and survival of GSCs and enhanced radiosensitivity of GBM cells. Finally, USP1 inhibition alone or in combination with radiation significantly prolonged the survival of tumor-bearing mice. CONCLUSION: USP1-mediated protein stabilization promotes GSC maintenance and treatment resistance, thereby providing a rationale for USP1 inhibition as a potential therapeutic approach against GBM.

Natural killer (NK) cells inhibit systemic metastasis of glioblastoma cells and have therapeutic effects against glioblastomas in the brain.

BACKGROUND: Glioblastoma multiforme (GBM) is characterized by extensive local invasion, which is in contrast with extremely rare systemic metastasis of GBM. Molecular mechanisms inhibiting systemic metastasis of GBM would be a novel therapeutic candidate for GBM in the brain. METHODS: Patient-derived GBM cells were primarily cultured from surgical samples of GBM patients and were inoculated into the brains of immune deficient BALB/c-nude or NOD-SCID IL2Rgamma(null) (NSG) mice. Human NK cells were isolated from peripheral blood mononucleated cells and expanded in vitro. RESULTS: Patient-derived GBM cells in the brains of NSG mice unexpectedly induced spontaneous lung metastasis although no metastasis was detected in BALB/c-nude mice. Based on the difference of the innate immunity between two mouse strains, NK cell activities of orthotopic GBM xenograft models based on BALB/c-nude mice were inhibited. NK cell inactivation induced spontaneous lung metastasis of GBM cells, which indicated that NK cells inhibit the systemic metastasis. In vitro cytotoxic activities of human NK cells against GBM cells indicated that cytotoxic activity of NK cells against GBM cells prevents systemic metastasis of GBM and that NK cells could be effective cell therapeutics against GBM. Accordingly, NK cells transplanted into orthotopic GBM xenograft models intravenously or intratumorally induced apoptosis of GBM cells in the brain and showed significant therapeutic effects. CONCLUSIONS: Our results suggest that innate NK immunity is responsible for rare systemic metastasis of GBM and that sufficient supplementation of NK cells could be a promising immunotherapeutic strategy for GBM in the brain.

Spatiotemporal Evolution of the Primary Glioblastoma Genome.

Tumor recurrence following treatment is the major cause of mortality for glioblastoma multiforme (GBM) patients. Thus, insights on the evolutionary process at recurrence are critical for improved patient care. Here, we describe our genomic analyses of the initial and recurrent tumor specimens from each of 38 GBM patients. A substantial divergence in the landscape of driver alterations was associated with distant appearance of a recurrent tumor from the initial tumor, suggesting that the genomic profile of the initial tumor can mislead targeted therapies for the distally recurred tumor. In addition, in contrast to IDH1-mutated gliomas, IDH1-wild-type primary GBMs rarely developed hypermutation following temozolomide (TMZ) treatment, indicating low risk for TMZ-induced hypermutation for these tumors under the standard regimen.

In vivo RNAi screen identifies NLK as a negative regulator of mesenchymal activity in glioblastoma.

Glioblastoma (GBM) is the most lethal brain cancer with profound genomic alterations. While the bona fide tumor suppressor genes such as PTEN, NF1, and TP53 have high frequency of inactivating mutations, there may be the genes with GBM-suppressive roles for which genomic mutation is not a primary cause for inactivation. To identify such genes, we employed in vivo RNAi screening approach using the patient-derived GBM xenograft models. We found that Nemo-Like Kinase (NLK) negatively regulates mesenchymal activities, a characteristic of aggressive GBM, in part via inhibition of WNT/ß-catenin signaling. Consistent with this, we found that NLK expression is especially low in a subset of GBMs that harbors high WNT/mesenchymal activities. Restoration of NLK inhibited WNT and mesenchymal activities, decreased clonogenic growth and survival, and impeded tumor growth in vivo. These data unravel a tumor suppressive role of NLK and support the feasibility of combining oncogenomics with in vivo RNAi screen.

Targeting the epithelial to mesenchymal transition in glioblastoma: the emerging role of MET signaling.

Glioblastoma multiforme (GBM) is the most common human primary brain malignancy and has a dismal prognosis. Aggressive treatments using maximal surgical resection, radiotherapy, and temozolomide result in median survival of only 14.6 months in patients with GBM. Numerous clinical approaches using small molecule inhibitors have shown disappointing results because of the genetic heterogeneity of GBM. The epithelial to mesenchymal transition (EMT) is a crucial biological process occurring in the early development stages of many species. However, cancer cells often obtain the ability to invade and metastasize through the EMT, which triggers the scattering of cells. The hepatocyte growth factor (HGF)/MET signaling pathway is indicative of the EMT during both embryogenesis and the invasive growth of tumors, because HGF potently induces mesenchymal transition in epithelial-driven cells. Activation of MET signaling or co-overexpression of HGF and MET frequently represents aggressive growth and poor prognosis of various cancers, including GBM. Thus, efforts to treat cancers by inhibiting MET signaling using neutralizing antibodies or small molecule inhibitors have progressed during the last decade. In this review, we discuss HGF/MET signaling in the development of diseases, including cancers, as well as updates on MET inhibition therapy.