EGFRvIII Confers Sensitivity to Saracatinib in a STAT5-Dependent Manner in Glioblastoma.

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults, with few effective treatments. EGFR alterations, including expression of the truncated variant EGFRvIII, are among the most frequent genomic changes in these tumors. EGFRvIII is known to preferentially signal through STAT5 for oncogenic activation in GBM, yet targeting EGFRvIII has yielded limited clinical success to date. In this study, we employed patient-derived xenograft (PDX) models expressing EGFRvIII to determine the key points of therapeutic vulnerability within the EGFRvIII-STAT5 signaling axis in GBM. Our findings reveal that exogenous expression of paralogs STAT5A and STAT5B augments cell proliferation and that inhibition of STAT5 phosphorylation in vivo improves overall survival in combination with temozolomide (TMZ). STAT5 phosphorylation is independent of JAK1 and JAK2 signaling, instead requiring Src family kinase (SFK) activity. Saracatinib, an SFK inhibitor, attenuates phosphorylation of STAT5 and preferentially sensitizes EGFRvIII+ GBM cells to undergo apoptotic cell death relative to wild-type EGFR. Constitutively active STAT5A or STAT5B mitigates saracatinib sensitivity in EGFRvIII+ cells. In vivo, saracatinib treatment decreased survival in mice bearing EGFR WT tumors compared to the control, yet in EGFRvIII+ tumors, treatment with saracatinib in combination with TMZ preferentially improves survival.

Targetable BRAF and RAF1 Alterations in Advanced Pediatric Cancers.

RAF family protein kinases signal through the MAPK pathway to orchestrate cellular proliferation, survival, and transformation. Identifying BRAF alterations in pediatric cancers is critically important as therapeutic agents targeting BRAF or MEK may be incorporated into the clinical management of these patients. In this study, we performed comprehensive genomic profiling on 3,633 pediatric cancer samples and identified a cohort of 221 (6.1%) cases with known or novel alterations in BRAF or RAF1 detected in extracranial solid tumors, brain tumors, or hematological malignancies. Eighty percent (176/221) of these tumors had a known-activating short variant (98, 55.7%), fusion (72, 40.9%), or insertion/deletion (6, 3.4%). Among BRAF altered cancers, the most common tumor types were brain tumors (74.4%), solid tumors (10.8%), hematological malignancies (9.1%), sarcomas (3.4%), and extracranial embryonal tumors (2.3%). RAF1 fusions containing intact RAF1 kinase domain (encoded by exons 10-17) were identified in seven tumors, including two novel fusions TMF1-RAF1 and SOX6-RAF1. Additionally, we highlight a subset of patients with brain tumor with positive clinical response to BRAF inhibitors, demonstrating the rationale for incorporating precision medicine into pediatric oncology. IMPLICATIONS FOR PRACTICE: Precision medicine has not yet gained a strong foothold in pediatric cancers. This study describes the landscape of BRAF and RAF1 genomic alterations across a diverse spectrum of pediatric cancers, primarily brain tumors, but also encompassing melanoma, sarcoma, several types of hematologic malignancy, and others. Given the availability of multiple U.S. Food and Drug Administration-approved BRAF inhibitors, identification of these alterations may assist with treatment decision making, as described here in three cases of pediatric cancer.

A pilot precision medicine trial for children with diffuse intrinsic pontine glioma-PNOC003: A report from the Pacific Pediatric Neuro-Oncology Consortium.

This clinical trial evaluated whether whole exome sequencing (WES) and RNA sequencing (RNAseq) of paired normal and tumor tissues could be incorporated into a personalized treatment plan for newly diagnosed patients (<25 years of age) with diffuse intrinsic pontine glioma (DIPG). Additionally, whole genome sequencing (WGS) was compared to WES to determine if WGS would further inform treatment decisions, and whether circulating tumor DNA (ctDNA) could detect the H3K27M mutation to allow assessment of therapy response. Patients were selected across three Pacific Pediatric Neuro-Oncology Consortium member institutions between September 2014 and January 2016. WES and RNAseq were performed at diagnosis and recurrence when possible in a CLIA-certified laboratory. Patient-derived cell line development was attempted for each subject. Collection of blood for ctDNA was done prior to treatment and with each MRI. A specialized tumor board generated a treatment recommendation including up to four FDA-approved agents based upon the genomic alterations detected. A treatment plan was successfully issued within 21 business days from tissue collection for all 15 subjects, with 14 of the 15 subjects fulfilling the feasibility criteria. WGS results did not significantly deviate from WES-based therapy recommendations; however, WGS data provided further insight into tumor evolution and fidelity of patient-derived cell models. Detection of the H3F3A or HIST1H3B K27M (H3K27M) mutation using ctDNA was successful in 92% of H3K27M mutant cases. A personalized treatment recommendation for DIPG can be rendered within a multicenter setting using comprehensive next-generation sequencing technology in a clinically relevant timeframe.

Genomics-Enabled Precision Medicine for Cancer.

Genomic information is increasingly being incorporated into clinical cancer care. Large-scale sequencing efforts have deepened our understanding of the genomic landscape of cancer and contributed to the expanding catalog of alterations being leveraged to aid in cancer diagnosis, prognosis, and treatment. Genomic profiling can provide clinically relevant information regarding somatic point mutations, copy number alterations, translocations, and gene fusions. Genomic features, such as mutational burden, can also be measured by more comprehensive sequencing strategies and have shown value in informing potential treatment options. Ongoing clinical trials are evaluating the use of molecularly targeted agents in genomically defined subsets of cancers within and across tumor histologies. Continued advancements in clinical genomics promise to further expand the application of genomics-enabled medicine to a broader spectrum of oncology patients.

Propentofylline inhibits glioblastoma cell invasion and survival by targeting the TROY signaling pathway.

Glioblastoma (GBM) is the most common primary tumor of the CNS and carries a dismal prognosis. The aggressive invasion of GBM cells into the surrounding normal brain makes complete resection impossible, significantly increases resistance to the standard therapy regimen, and virtually assures tumor recurrence. Median survival for newly diagnosed GBM is 14.6 months and declines to 8 months for patients with recurrent GBM. New therapeutic strategies that target the molecular drivers of invasion are required for improved clinical outcome. We have demonstrated that TROY (TNFRSF19), a member of the TNFR super-family, plays an important role in GBM invasion and resistance. Knockdown of TROY expression inhibits GBM cell invasion, increases sensitivity to temozolomide, and prolongs survival in an intracranial xenograft model. Propentofylline (PPF), an atypical synthetic methylxanthine compound, has been extensively studied in Phase II and Phase III clinical trials for Alzheimer's disease and vascular dementia where it has demonstrated blood-brain permeability and minimal adverse side effects. Here we showed that PPF decreased GBM cell expression of TROY, inhibited glioma cell invasion, and sensitized GBM cells to TMZ. Mechanistically, PPF decreased glioma cell invasion by modulating TROY expression and downstream signaling, including AKT, NF-κB, and Rac1 activation. Thus, PPF may provide a pharmacologic approach to target TROY, inhibit cell invasion, and reduce therapeutic resistance in GBM.

Loss of Runx2 sensitises osteosarcoma to chemotherapy-induced apoptosis.

BACKGROUND: Osteosarcoma (OS) is the most common bone malignancy in the paediatric population, principally affecting adolescents and young adults. Minimal advancements in patient prognosis have been made over the past two decades because of the poor understanding of disease biology. Runx2, a critical transcription factor in bone development, is frequently amplified and overexpressed in OS. However, the molecular and biological consequences of Runx2 overexpression remain unclear. METHODS: si/shRNA and overexpression technology to alter Runx2 levels in OS cells. In vitro assessment of doxorubicin (doxo)-induced apoptosis and in vivo chemosensitivity studies. Small-molecule inhibitor of c-Myc transcriptional activity was used to assess its role. RESULTS: Loss of Runx2 sensitises cells to doxo-induced apoptosis both in vitro and in vivo. Furthermore, in conjunction with chemotherapy, decreasing Runx2 protein levels activates both the intrinsic and extrinsic apoptotic pathways. Transplanted tumour studies demonstrated that loss of endogenous Runx2 protein expression enhances caspase-3 cleavage and tumour necrosis in response to chemotherapy. Finally, upon doxo-treated Runx2 knockdown OS cells there was evidence of enhanced c-Myc expression and transcriptional activity. Inhibition of c-Myc under these conditions resulted in decreased activation of apoptosis, therefore insinuating a role for c-Myc in dox-induced activation of apoptotic pathways. CONCLUSIONS: Therefore, we have established a novel molecular mechanism by which Runx2 provides a chemoprotective role in OS, indicating that in conjunction to standard chemotherapy, targeting Runx2 may be a new therapeutic strategy for patients with OS.

Two independent regions of simian virus 40 T antigen increase CBP/p300 levels, alter patterns of cellular histone acetylation, and immortalize primary cells.

Simian virus 40 (SV40) large T antigen (SVT) interferes with normal cell regulation and thus has been used to identify cellular components controlling proliferation and homeostasis. We have previously shown that SVT-mediated transformation requires interaction with the histone acetyltransferases (HATs) CBP/p300 and now report that the ectopic expression of SVT in several cell types in vivo and in vitro results in a significant increase in the steady-state levels of CBP/p300. Furthermore, SVT-expressing cells contain higher levels of acetylated CBP/p300, a modification that has been linked to increased HAT activity. Concomitantly, the acetylation levels of histone residues H3K56 and H4K12 are markedly increased in SVT-expressing cells. Other polyomavirus-encoded large T antigens also increase the levels of CBP/p300 and sustain a rise in the acetylation levels of H3K56 and H4K12. SVT does not affect the transcription of CBP/p300, but rather, alters their overall levels through increasing the loading of CBP/p300 mRNAs onto polysomes. Two distinct regions within SVT, one located in the amino terminus and one in the carboxy terminus, can independently alter both the levels of CBP/p300 and the loading of CBP/p300 transcripts onto polysomes. Within the amino-terminal fragment, a functional J domain is necessary for increasing CBP/p300 and specific histone acetylation levels, as well as for immortalizing primary cells. These studies uncover the action of polyomavirus T antigens on cellular CBP/p300 and suggest that additional mechanisms are used by T antigens to induce cell immortalization and transformation.

Genomic and Transcriptomic Analysis of Relapsed and Refractory Childhood Solid Tumors Reveals a Diverse Molecular Landscape and Mechanisms of Immune Evasion.

Children with treatment-refractory or relapsed (R/R) tumors face poor prognoses. As the genomic underpinnings driving R/R disease are not well defined, we describe here the genomic and transcriptomic landscapes of R/R solid tumors from 202 patients enrolled in Beat Childhood Cancer Consortium clinical trials. Tumor mutational burden (TMB) was elevated relative to untreated tumors at diagnosis, with one-third of tumors classified as having a pediatric high TMB. Prior chemotherapy exposure influenced the mutational landscape of these R/R tumors, with more than 40% of tumors demonstrating mutational signatures associated with platinum or temozolomide chemotherapy and two tumors showing treatment-associated hypermutation. Immunogenomic profiling found a heterogenous pattern of neoantigen and MHC class I expression and a general absence of immune infiltration. Transcriptional analysis and functional gene set enrichment analysis identified cross-pathology clusters associated with development, immune signaling, and cellular signaling pathways. While the landscapes of these R/R tumors reflected those of their corresponding untreated tumors at diagnosis, important exceptions were observed, suggestive of tumor evolution, treatment resistance mechanisms, and mutagenic etiologies of treatment. SIGNIFICANCE: Tumor heterogeneity, chemotherapy exposure, and tumor evolution contribute to the molecular profiles and increased mutational burden that occur in treatment-refractory and relapsed childhood solid tumors.

A Novel Signaling Complex between TROY and EGFR Mediates Glioblastoma Cell Invasion.

Glioblastoma is the most frequent primary brain tumor in adults and a highly lethal malignancy with a median survival of about 15 months. The aggressive invasion of the surrounding normal brain makes complete surgical resection impossible, increases the resistance to radiation and chemotherapy, and assures tumor recurrence. Thus, there is an urgent need to develop innovative therapeutics to target the invasive tumor cells for improved treatment outcomes of this disease. Expression of TROY (TNFRSF19), a member of the tumor necrosis factor (TNF) receptor family, increases with increasing glial tumor grade and inversely correlates with patient survival. Increased expression of TROY stimulates glioblastoma cell invasion in vitro and in vivo and increases resistance to temozolomide and radiation therapy. Conversely, silencing TROY expression inhibits glioblastoma cell invasion, increases temozolomide sensitivity, and prolongs survival in an intracranial xenograft model. Here, a novel complex is identified between TROY and EGFR, which is mediated predominantly by the cysteine-rich CRD3 domain of TROY. Glioblastoma tumors with elevated TROY expression have a statistically positive correlation with increased EGFR expression. TROY expression significantly increases the capacity of EGF to stimulate glioblastoma cell invasion, whereas depletion of TROY expression blocks EGF stimulation of glioblastoma cell invasion. Mechanistically, TROY expression modulates EGFR signaling by facilitating EGFR activation and delaying EGFR receptor internalization. Moreover, the association of EGFR with TROY increases TROY-induced NF-κB activation. These findings substantiate a critical role for the TROY-EGFR complex in regulation of glioblastoma cell invasion.Implications: The TROY-EGFR signaling complex emerges as a potential therapeutic target to inhibit glioblastoma cell invasion. Mol Cancer Res; 16(2); 322-32. ©2017 AACR.

The TNF receptor family member Fn14 is highly expressed in recurrent glioblastoma and in GBM patient-derived xenografts with acquired temozolomide resistance.

Background: Glioblastoma (GBM) is a difficult to treat brain cancer that nearly uniformly recurs, and recurrent tumors are largely therapy resistant. Our prior work has demonstrated an important role for the tumor necrosis factor-like weak inducer of apoptosis (TWEAK) receptor fibroblast growth factor-inducible 14 (Fn14) in GBM pathobiology. In this study, we investigated Fn14 expression in recurrent GBM and in the setting of temozolomide (TMZ) resistance. Methods: Fn14 mRNA expression levels in nonneoplastic brain, primary (newly diagnosed) GBM, and recurrent GBM (post-chemotherapy and radiation) specimens were obtained from The Cancer Genome Atlas data portal. Immunohistochemistry was performed using nonneoplastic brain, patient-matched primary and recurrent GBM, and gliosarcoma (GSM) specimens to examine Fn14 protein levels. Western blot analysis was used to compare Fn14 expression in parental TMZ-sensitive or matched TMZ-resistant patient-derived xenografts (PDXs) established from primary or recurrent tumor samples. The migratory capacity of control and Fn14-depleted TMZ-resistant GBM cells was assessed using the transwell migration assay. Results: We found that Fn14 is more highly expressed in recurrent GBM tumors than their matched primary GBM counterparts. Fn14 expression is also significantly elevated in GSM tumors. GBM PDX cells with acquired TMZ resistance have higher Fn14 levels and greater migratory capacity than their corresponding parental TMZ-sensitive cells, and the migratory difference is due, at least in part, to Fn14 expression in the TMZ-resistant cells. Conclusions: This study demonstrates that the Fn14 gene is highly expressed in recurrent GBM, GSM, and TMZ-resistant GBM PDX tumors. These findings suggest that Fn14 may be a valuable therapeutic target or drug delivery portal for treatment of recurrent GBM and GSM patients.

EGFRvIII-Stat5 Signaling Enhances Glioblastoma Cell Migration and Survival.

Glioblastoma multiforme (GBM) is the most common brain malignancies in adults. Most GBM patients succumb to the disease less than 1 year after diagnosis due to the highly invasive nature of the tumor, which prevents complete surgical resection and gives rise to tumor recurrence. The invasive phenotype also confers radioresistant and chemoresistant properties to the tumor cells; therefore, there is a critical need to develop new therapeutics that target drivers of GBM invasion. Amplification of EGFR is observed in over 50% of GBM tumors, of which half concurrently overexpress the variant EGFRvIII, and expression of both receptors confers a worse prognosis. EGFR and EGFRvIII cooperate to promote tumor progression and invasion, in part, through activation of the Stat signaling pathway. Here, it is reported that EGFRvIII activates Stat5 and GBM invasion by inducing the expression of a previously established mediator of glioma cell invasion and survival: fibroblast growth factor-inducible 14 (Fn14). EGFRvIII-mediated induction of Fn14 expression is Stat5 dependent and requires activation of Src, whereas EGFR regulation of Fn14 is dependent upon Src-MEK/ERK-Stat3 activation. Notably, treatment of EGFRvIII-expressing GBM cells with the FDA-approved Stat5 inhibitor pimozide blocked Stat5 phosphorylation, Fn14 expression, and cell migration and survival. Because EGFR inhibitors display limited therapeutic efficacy in GBM patients, the EGFRvIII-Stat5-Fn14 signaling pathway represents a node of vulnerability in the invasive GBM cell populations.Implications: Targeting critical effectors in the EGFRvIII-Stat5-Fn14 pathway may limit GBM tumor dispersion, mitigate therapeutic resistance, and increase survival. Mol Cancer Res; 16(7); 1185-95. ©2018 AACR.

Integrated mapping of pharmacokinetics and pharmacodynamics in a patient-derived xenograft model of glioblastoma.

Therapeutic options for the treatment of glioblastoma remain inadequate despite concerted research efforts in drug development. Therapeutic failure can result from poor permeability of the blood-brain barrier, heterogeneous drug distribution, and development of resistance. Elucidation of relationships among such parameters could enable the development of predictive models of drug response in patients and inform drug development. Complementary analyses were applied to a glioblastoma patient-derived xenograft model in order to quantitatively map distribution and resulting cellular response to the EGFR inhibitor erlotinib. Mass spectrometry images of erlotinib were registered to histology and magnetic resonance images in order to correlate drug distribution with tumor characteristics. Phosphoproteomics and immunohistochemistry were used to assess protein signaling in response to drug, and integrated with transcriptional response using mRNA sequencing. This comprehensive dataset provides simultaneous insight into pharmacokinetics and pharmacodynamics and indicates that erlotinib delivery to intracranial tumors is insufficient to inhibit EGFR tyrosine kinase signaling.

Molecular and Microenvironmental Determinants of Glioma Stem-Like Cell Survival and Invasion.

Glioblastoma multiforme (GBM) is the most frequent primary brain tumor in adults with a 5-year survival rate of 5% despite intensive research efforts. The poor prognosis is due, in part, to aggressive invasion into the surrounding brain parenchyma. Invasion is a complex process mediated by cell-intrinsic pathways, extrinsic microenvironmental cues, and biophysical cues from the peritumoral stromal matrix. Recent data have attributed GBM invasion to the glioma stem-like cell (GSC) subpopulation. GSCs are slowly dividing, highly invasive, therapy resistant, and are considered to give rise to tumor recurrence. GSCs are localized in a heterogeneous cellular niche, and cross talk between stromal cells and GSCs cultivates a fertile environment that promotes GSC invasion. Pro-migratory soluble factors from endothelial cells, astrocytes, macrophages, microglia, and non-stem-like tumor cells can stimulate peritumoral invasion of GSCs. Therefore, therapeutic efforts designed to target the invasive GSCs may enhance patient survival. In this review, we summarize the current understanding of extrinsic pathways and major stromal and immune players facilitating GSC maintenance and survival.

Identification of aurintricarboxylic acid as a selective inhibitor of the TWEAK-Fn14 signaling pathway in glioblastoma cells.

The survival of patients diagnosed with glioblastoma (GBM), the most deadly form of brain cancer, is compromised by the proclivity for local invasion into the surrounding normal brain, which prevents complete surgical resection and contributes to therapeutic resistance. Tumor necrosis factor-like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis factor (TNF) superfamily, can stimulate glioma cell invasion and survival via binding to fibroblast growth factor-inducible 14 (Fn14) and subsequent activation of the transcription factor NF-κB. To discover small molecule inhibitors that disrupt the TWEAK-Fn14 signaling axis, we utilized a cell-based drug-screening assay using HEK293 cells engineered to express both Fn14 and a NF-κB-driven firefly luciferase reporter protein. Focusing on the LOPAC1280 library of 1280 pharmacologically active compounds, we identified aurintricarboxylic acid (ATA) as an agent that suppressed TWEAK-Fn14-NF-κB dependent signaling, but not TNFα-TNFR-NF-κB driven signaling. We demonstrated that ATA repressed TWEAK-induced glioma cell chemotactic migration and invasion via inhibition of Rac1 activation but had no effect on cell viability or Fn14 expression. In addition, ATA treatment enhanced glioma cell sensitivity to both the chemotherapeutic agent temozolomide (TMZ) and radiation-induced cell death. In summary, this work reports a repurposed use of a small molecule inhibitor that targets the TWEAK-Fn14 signaling axis, which could potentially be developed as a new therapeutic agent for treatment of GBM patients.

SGEF Is Regulated via TWEAK/Fn14/NF-κB Signaling and Promotes Survival by Modulation of the DNA Repair Response to Temozolomide.

UNLABELLED: Glioblastoma (GB) is the highest grade and most common form of primary adult brain tumors. Despite surgical removal followed by concomitant radiation and chemotherapy with the alkylating agent temozolomide, GB tumors develop treatment resistance and ultimately recur. Impaired response to treatment occurs rapidly, conferring a median survival of just fifteen months. Thus, it is necessary to identify the genetic and signaling mechanisms that promote tumor resistance to develop targeted therapies to combat this refractory disease. Previous observations indicated that SGEF (ARHGEF26), a RhoG-specific guanine nucleotide exchange factor (GEF), is overexpressed in GB tumors and plays a role in promoting TWEAK-Fn14-mediated glioma invasion. Here, further investigation revealed an important role for SGEF in glioma cell survival. SGEF expression is upregulated by TWEAK-Fn14 signaling via NF-κB activity while shRNA-mediated reduction of SGEF expression sensitizes glioma cells to temozolomide-induced apoptosis and suppresses colony formation following temozolomide treatment. Nuclear SGEF is activated following temozolomide exposure and complexes with the DNA damage repair (DDR) protein BRCA1. Moreover, BRCA1 phosphorylation in response to temozolomide treatment is hindered by SGEF knockdown. The role of SGEF in promoting chemotherapeutic resistance highlights a heretofore unappreciated driver, and suggests its candidacy for development of novel targeted therapeutics for temozolomide-refractory, invasive GB cells. IMPLICATION: SGEF, as a dual process modulator of cell survival and invasion, represents a novel target for treatment refractory glioblastoma.