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Introduction: Alterations in the CDK4/6-RB signaling pathway are common causes of cell cycle dysregulation in many cancers, including glioblastoma. Palbociclib is an oral inhibitor of CDK4/6, which leads to phosphorylation of RB1 and cell-cycle arrest. We conducted a two-arm study evaluating efficacy and tissue pharmacokinetics/pharmacodynamics of palbociclib in patients with recurrent glioblastoma., Methods: Eligibility criteria included confirmation of RB1 proficiency by IHC; ≤ 3 relapses; KPS ≥ 60; no limit on prior treatments. Arm 1 received palbociclib for 7 days prior to indicated resection followed by adjuvant palbociclib. Arm 2 received palbociclib without resection. Primary objective was PFS6; secondary included toxicity, OS, and ORR. Exploratory aims included biomarker assessment and pharmacokinetic/pharmacodynamic effects in surgical patients., Results: Total of 22 patients were enrolled; 6 on Arm 1 and 16 on Arm 2. Trial was stopped early secondary to lack of efficacy, with 95% of evaluable patients progressing within 6 months. Median PFS was 5.14 weeks (range 5 days-142 weeks) and median OS was 15.4 weeks (range 2-274 weeks). Two patients (10%) had related grade ≥ 3 AEs. In Arm 1, 5 patients had tissue concentrations of palbociclib felt to be sufficient for biological effect and paired samples available for RB1 IHC. There were no consistent changes in RB1 expression or cell proliferation in the paired tissue., Conclusion: In this trial, despite adequate tissue PK, palbociclib monotherapy was not an effective treatment for recurrent glioblastoma. However, these were heavily pretreated patients and targeting the CDK4/6 pathway may still deserve further exploration.

Mesenchymal (MES) and proneural (PN) are two distinct glioma stem cell (GSC) populations that drive therapeutic resistance in glioblastoma (GBM). We screened a panel of 650 small molecules against patient-derived GBM cells to discover compounds targeting specific GBM subtypes. Arsenic trioxide (ATO), an FDA-approved drug that crosses the blood-brain barrier, was identified as a potent PN-specific compound in the initial screen and follow-up validation studies. Furthermore, MES and PN GSCs exhibited differential sensitivity to ATO. As ATO has been shown to activate the MAPK-interacting kinase 1 (MNK1)-eukaryotic translation initiation factor 4E (eIF4E) pathway and subsequent mRNA translation in a negative regulatory feedback manner, the mechanistic role of ATO resistance in MES GBM was explored. In GBM cells, ATO-activated translation initiation cellular events via the MNK1-eIF4E signaling axis. Furthermore, resistance to ATO in intracranial PDX tumors correlated with high eIF4E phosphorylation. Polysomal fractionation and microarray analysis of GBM cells were performed to identify ATO's effect on mRNA translation and enrichment of anti-apoptotic mRNAs in the ATO-induced translatome was found. Additionally, it was determined that MNK inhibition sensitized MES GSCs to ATO in neurosphere and apoptosis assays. Finally, examination of the effect of ATO on patients from a phase I/II clinical trial of ATO revealed that PN GBM patients responded better to ATO than other subtypes as demonstrated by longer overall and progression-free survival. Implications: These findings raise the possibility of a unique therapeutic approach for GBM, involving MNK1 targeting to sensitize MES GSCs to drugs like arsenic trioxide. Mol Cancer Res; 16(1); 32-46. ©2017 AACR ., (©2017 American Association for Cancer Research.)

BRAF V600E is a common finding in glioma (about 10-60% depending on histopathologic subclassification). BRAF V600E monotherapy shows modest preclinical efficacy against BRAF V600E gliomas and also induces adverse secondary skin malignancies. Here, we examine the molecular mechanism of intrinsic resistance to BRAF V600E inhibition in glioma. Furthermore, we investigate BRAF V600E /MEK combination therapy that overcomes intrinsic resistance to BRAF V600E inhibitor and also prevents BRAF V600E inhibitor induced secondary malignancies. Immunoblotting and Human Phospho-Receptor Tyrosine Kinase Array assays were used to interrogate MAPK pathway activation. The cellular effect of BRAF V600E and MEK inhibition was determined by WST-1 viability assay and cell cycle analysis. Flanked and orthotopic GBM mouse models were used to investigate the in vivo efficacy of BRAF V600E /MEK combination therapy and the effect on secondary malignancies. BRAF V600E inhibition leads to recovery of ERK phosphorylation. Combined BRAF V600E and MEK inhibition prevents reactivation of the MAPK signaling, which correlates with decreased cell viability and augmented cell cycle arrest. Similarly, mice bearing BRAF V600E glioma showed reduced tumor growth when treated with a combination of BRAF V600E and MEK inhibitor compared to BRAF V600E inhibition alone. Additional benefit of BRAF V600E /MEK inhibition was reflected by reduced cutaneous squamous-cell carcinoma (cSCC) growth (a surrogate for RAS-driven secondary maligancies). In glioma, recovery of MAPK signaling upon BRAF inhibition accounts for intrinsic resistance to BRAF V600E inhibitor. Combined BRAF V600E and MEK inhibition prevents rebound of MAPK activation, resulting in enhanced antitumor efficacy and also reduces the risk of secondary malignancy development.

Activating mutation of BRAF is a common finding in pediatric gliomas. As many as 14% of high grade and up to 66% of certain subtypes of low grade pediatric glioma have the BRAFV600E mutation. Small molecule inhibitors that selectively target BRAFV600E are FDA approved for melanoma and have shown significant efficacy in treating BRAFV600E glioma in pre-clinical trials. Despite showing initial anti-tumor activity, acquired drug resistance significantly limits the benefit from being treated with BRAFV600E inhibitors. Here, we have identified molecular responses to BRAFV600E inhibitor treatment in human glioma models that have substantial clinical implications. Specifically, we show that BRAFV600E inhibitor resistant cells upregulate pro-survival mediators such as Wnt, and additionally increase receptor tyrosine kinase activity, including EGFR and Axl, promoting resistance to BRAFV600E inhibition. Our results suggest strategies to circumvent acquired resistance to BRAFV600E inhibitor therapy, and thereby improve outcomes for patients with BRAFV600E gliomas.

Inhibitors of BRAFV600E kinase are currently under investigations in preclinical and clinical studies involving BRAFV600E glioma. Studies demonstrated clinical response to such individualized therapy in the majority of patients whereas in some patients tumors continue to grow despite treatment. To study resistance mechanisms, which include feedback activation of mitogen-activated protein kinase (MAPK) signaling in melanoma, we developed a luciferase-modified cell line (2341luc) from a BrafV600E mutant and Cdkn2a- deficient murine high-grade glioma, and analyzed its molecular responses to BRAFV600E- and MAPK kinase (MEK)-targeted inhibition. Immunocompetent, syngeneic FVB/N mice with intracranial grafts of 2341luc were tested for effects of BRAFV600E and MEK inhibitor treatments, with bioluminescence imaging up to 14-days after start of treatment and survival analysis as primary indicators of inhibitor activity. Intracranial injected tumor cells consistently generated high-grade glioma-like tumors in syngeneic mice. Intraperitoneal daily delivery of BRAFV600E inhibitor dabrafenib only transiently suppressed MAPK signaling, and rather increased Akt signaling and failed to extend survival for mice with intracranial 2341luc tumor. MEK inhibitor trametinib delivered by oral gavage daily suppressed MAPK pathway more effectively and had a more durable anti-growth effect than dabrafenib as well as a significant survival benefit. Compared with either agent alone, combined BRAFV600E and MEK inhibitor treatment was more effective in reducing tumor growth and extending animal subject survival, as corresponding to sustained MAPK pathway inhibition. Results derived from the 2341luc engraftment model application have clinical implications for the management of BRAFV600E glioma.

Background: Radiation therapy is the most commonly used postsurgical treatment for primary malignant brain tumors. Consequently, investigating the efficacy of chemotherapeutics combined with radiation for treating malignant brain tumors is of high clinical relevance. In this study, we examined the cyclin-dependent kinase 4/6 inhibitor palbociclib, when used in combination with radiation for treating human atypical teratoid rhabdoid tumor (ATRT) as well as glioblastoma (GBM)., Methods: Evaluation of treatment antitumor activity in vitro was based upon results from cell proliferation assays, clonogenicity assays, flow cytometry, and immunocytochemistry for DNA double-strand break repair. Interpretation of treatment antitumor activity in vivo was based upon bioluminescence imaging, animal subject survival analysis, and staining of tumor sections for markers of proliferation and apoptosis., Results: For each of the retinoblastoma protein (RB)-proficient tumor models examined (2 ATRTs and 2 GBMs), one or more of the combination therapy regimens significantly (P < .05) outperformed both monotherapies with respect to animal subject survival benefit. Among the combination therapy regimens, concurrent palbociclib and radiation treatment and palbociclib treatment following radiation consistently outperformed the sequence in which radiation followed palbociclib treatment. In vitro investigation revealed that the concurrent use of palbociclib with radiation, as well as palbociclib following radiation, inhibited DNA double-strand break repair and promoted increased tumor cell apoptosis., Conclusions: Our results support further investigation and possible clinical translation of palbociclib as an adjuvant to radiation therapy for patients with malignant brain tumors that retain RB expression., (© The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Purpose: Alteration of the BRAF/MEK/MAPK pathway is the hallmark of pediatric low-grade gliomas (PLGGs), and mTOR activation has been documented in the majority of these tumors. We investigated combinations of MEK1/2, BRAF V600E and mTOR inhibitors in gliomas carrying specific genetic alterations of the MAPK pathway., Experimental Design: We used human glioma lines containing BRAF V600E (adult high-grade: AM-38, DBTRG, PLGG: BT40), or wild-type BRAF (pediatric high-grade: SF188, SF9427, SF8628) and isogenic systems of KIAA1549:BRAF-expressing NIH/3T3 cells and BRAF V600E -expressing murine brain cells. Signaling inhibitors included everolimus (mTOR), PLX4720 (BRAF V600E ), and AZD6244 (MEK1/2). Proliferation was determined using ATP-based assays. In vivo inhibitor activities were assessed in the BT40 PLGG xenograft model., Results: In BRAF V600E cells, the three possible doublet combinations of AZD6244, everolimus, and PLX4720 exhibited significantly greater effects on cell viability. In BRAF WT cells, everolimus + AZD6244 was superior compared with respective monotherapies. Similar results were found using isogenic murine cells. In KIAA1549:BRAF cells, MEK1/2 inhibition reduced cell viability and S-phase content, effects that were modestly augmented by mTOR inhibition. In vivo experiments in the BRAF V600E pediatric xenograft model BT40 showed the greatest survival advantage in mice treated with AZD6244 + PLX4720 (P < 0.01)., Conclusions: In BRAF V600E tumors, combination of AZD6244 + PLX4720 is superior to monotherapy and to other combinatorial approaches. In BRAF WT pediatric gliomas, everolimus + AZD6244 is superior to either agent alone. KIAA1549:BRAF-expressing tumors display marked sensitivity to MEK1/2 inhibition. Application of these results to PLGG treatment must be exercised with caution because the dearth of PLGG models necessitated only a single patient-derived PLGG (BT40) in this study. Clin Cancer Res; 22(21); 5312-21. ©2016 AACR., Competing Interests: of potential conflicts of interest None of the authors have any conflict of interest to report., (©2016 American Association for Cancer Research.)

Glioblastoma multiforme remains the deadliest malignant brain tumor, with glioma stem cells (GSC) contributing to treatment resistance and tumor recurrence. We have identified MAPK-interacting kinases (MNK) as potential targets for the GSC population in glioblastoma multiforme. Isoform-level subtyping using The Cancer Genome Atlas revealed that both MNK genes (MKNK1 and MKNK2) are upregulated in mesenchymal glioblastoma multiforme as compared with other subtypes. Expression of MKNK1 is associated with increased glioma grade and correlated with the mesenchymal GSC marker, CD44, and coexpression of MKNK1 and CD44 predicts poor survival in glioblastoma multiforme. In established and patient-derived cell lines, pharmacologic MNK inhibition using LY2801653 (merestinib) inhibited phosphorylation of the eukaryotic translation initiation factor 4E, a crucial effector for MNK-induced mRNA translation in cancer cells and a marker of transformation. Importantly, merestinib inhibited growth of GSCs grown as neurospheres as determined by extreme limiting dilution analysis. When the effects of merestinib were assessed in vivo using an intracranial xenograft mouse model, improved overall survival was observed in merestinib-treated mice. Taken together, these data provide strong preclinical evidence that pharmacologic MNK inhibition targets mesenchymal glioblastoma multiforme and its GSC population., Implications: These findings raise the possibility of MNK inhibition as a viable therapeutic approach to target the mesenchymal subtype of glioblastoma multiforme. Mol Cancer Res; 14(10); 984-93. ©2016 AACR., Competing Interests: None, (©2016 American Association for Cancer Research.)

The treatment of glioblastoma (GBM) remains challenging in part due to the presence of stem-like tumor-propagating cells that are resistant to standard therapies consisting of radiation and temozolomide. Among the novel and targeted agents under evaluation for the treatment of GBM are BRAF/MAPK inhibitors, but their effects on tumor-propagating cells are unclear. Here, we characterized the behaviors of CD133(+) tumor-propagating cells isolated from primary GBM cell lines. We show that CD133(+) cells exhibited decreased sensitivity to the antiproliferative effects of BRAF/MAPK inhibition compared to CD133(-) cells. Furthermore, CD133(+) cells exhibited an extended G2-M phase and increased polarized asymmetric cell divisions. At the molecular level, we observed that polo-like kinase (PLK) 1 activity was elevated in CD133(+) cells, prompting our investigation of BRAF/PLK1 combination treatment effects in an orthotopic GBM xenograft model. Combined inhibition of BRAF and PLK1 resulted in significantly greater antiproliferative and proapoptotic effects beyond those achieved by monotherapy (P < 0.05). We propose that PLK1 activity controls a polarity checkpoint and compensates for BRAF/MAPK inhibition in CD133(+) cells, suggesting the need for concurrent PLK1 inhibition to improve antitumor activity against a therapy-resistant cell compartment., (©2015 American Association for Cancer Research.)

This report describes results from our analysis of the activity and biodistribution of a novel pan-ERBB inhibitor, NT113, when used in treating mice with intracranial glioblastoma (GBM) xenografts. Approaches used in this investigation include: bioluminescence imaging (BLI) for monitoring intracranial tumor growth and response to therapy; determination of survival benefit from treatment; analysis of tumor IHC reactivity for indication of treatment effect on proliferation and apoptotic response; Western blot analysis for determination of effects of treatment on ERBB and ERBB signaling mediator activation; and high-performance liquid chromatography for determination of NT113 concentration in tissue extracts from animals receiving oral administration of inhibitor. Our results show that NT113 is active against GBM xenografts in which wild-type EGFR or EGFRvIII is highly expressed. In experiments including lapatinib and/or erlotinib, NT113 treatment was associated with the most substantial improvement in survival, as well as the most substantial tumor growth inhibition, as indicated by BLI and IHC results. Western blot analysis results indicated that NT113 has inhibitory activity, both in vivo and in vitro, on ERBB family member phosphorylation, as well as on the phosphorylation of downstream signaling mediator Akt. Results from the analysis of animal tissues revealed significantly higher NT113 normal brain-to-plasma and intracranial tumor-to-plasma ratios for NT113, relative to erlotinib, indicating superior NT113 partitioning to intracranial tissue compartments. These data provide a strong rationale for the clinical investigation of NT113, a novel ERBB inhibitor, in treating patients with GBM., (©2014 American Association for Cancer Research.)

Intratumoral heterogeneity contributes to cancer drug resistance, but the underlying mechanisms are not understood. Single-cell analyses of patient-derived models and clinical samples from glioblastoma patients treated with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) demonstrate that tumor cells reversibly up-regulate or suppress mutant EGFR expression, conferring distinct cellular phenotypes to reach an optimal equilibrium for growth. Resistance to EGFR TKIs is shown to occur by elimination of mutant EGFR from extrachromosomal DNA. After drug withdrawal, reemergence of clonal EGFR mutations on extrachromosomal DNA follows. These results indicate a highly specific, dynamic, and adaptive route by which cancers can evade therapies that target oncogenes maintained on extrachromosomal DNA.

Purpose: Cidofovir (CDV) is an U.S. Food and Drug Administration (FDA)-approved nucleoside antiviral agent used to treat severe human cytomegalovirus (HCMV) infection. Until now, no clear therapeutic effects of CDV have been reported outside of the setting of viral infection, including a potential role for CDV as an antineoplastic agent for the treatment of brain tumors., Experimental Design: We investigated the cytotoxicity of CDV against the glioblastoma cells, U87MG and primary SF7796, both in vitro and in vivo, using an intracranial xenograft model. Standard techniques for cell culturing, immunohistochemistry, Western blotting, and real-time PCR were employed. The survival of athymic mice (n = 8-10 per group) bearing glioblastoma tumors, treated with CDV alone or in combination with radiation, was analyzed by the Kaplan-Meier method and evaluated with a two-sided log-rank test., Results: CDV possesses potent antineoplastic activity against HCMV-infected glioblastoma cells. This activity is associated with the inhibition of HCMV gene expression and with activation of cellular apoptosis. Surprisingly, we also determined that CDV induces glioblastoma cell death in the absence of HCMV infection. CDV is incorporated into tumor cell DNA, which promotes double-stranded DNA breaks and induces apoptosis. In the setting of ionizing radiotherapy, the standard of care for glioblastoma in humans, CDV augments radiation-induced DNA damage and, further, promotes tumor cell death. Combination therapy with CDV and radiotherapy significantly extended the survival of mice bearing intracranial glioblastoma tumors., Conclusion: We have identified a novel antiglioma property of the FDA-approved drug CDV, which heightens the cytotoxic effect of radiotherapy, the standard of care therapy for glioblastoma., (©2013 AACR.)

Purpose: The aim of this study was to identify conserved pharmacodynamic and potential predictive biomarkers of response to anti-VEGF therapy using gene expression profiling in preclinical tumor models and in patients., Experimental Design: Surrogate markers of VEGF inhibition [VEGF-dependent genes or VEGF-dependent vasculature (VDV)] were identified by profiling gene expression changes induced in response to VEGF blockade in preclinical tumor models and in human biopsies from patients treated with anti-VEGF monoclonal antibodies. The potential value of VDV genes as candidate predictive biomarkers was tested by correlating high or low VDV gene expression levels in pretreatment clinical samples with the subsequent clinical efficacy of bevacizumab (anti-VEGF)-containing therapy., Results: We show that VDV genes, including direct and more distal VEGF downstream endothelial targets, enable detection of VEGF signaling inhibition in mouse tumor models and human tumor biopsies. Retrospective analyses of clinical trial data indicate that patients with higher VDV expression in pretreatment tumor samples exhibited improved clinical outcome when treated with bevacizumab-containing therapies., Conclusions: In this work, we identified surrogate markers (VDV genes) for in vivo VEGF signaling in tumors and showed clinical data supporting a correlation between pretreatment VEGF bioactivity and the subsequent efficacy of anti-VEGF therapy. We propose that VDV genes are candidate biomarkers with the potential to aid the selection of novel indications as well as patients likely to respond to anti-VEGF therapy. The data presented here define a diagnostic biomarker hypothesis based on translational research that warrants further evaluation in additional retrospective and prospective trials., (©2013 AACR.)

Unlabelled: Acquired resistance to tyrosine kinase inhibitors (TKI) represents a major challenge for personalized cancer therapy. Multiple genetic mechanisms of acquired TKI resistance have been identified in several types of human cancer. However, the possibility that cancer cells may also evade treatment by co-opting physiologically regulated receptors has not been addressed. Here, we show the first example of this alternate mechanism in brain tumors by showing that EGF receptor (EGFR)-mutant glioblastomas (GBMs) evade EGFR TKIs by transcriptionally de-repressing platelet-derived growth factor receptor β (PDGFRβ). Mechanistic studies show that EGFRvIII signaling actively suppresses PDGFRβ transcription in an mTORC1- and extracellular signal-regulated kinase-dependent manner. Genetic or pharmacologic inhibition of oncogenic EGFR renders GBMs dependent on the consequently de-repressed PDGFRβ signaling for growth and survival. Importantly, combined inhibition of EGFR and PDGFRβ signaling potently suppresses tumor growth in vivo. These data identify a novel, nongenetic TKI resistance mechanism in brain tumors and provide compelling rationale for combination therapy., Significance: These results provide the fi rst clinical and biologic evidence for receptor tyrosinekinase (RTK) "switching" as a mechanism of resistance to EGFR inhibitors in GBM and provide a molecular explanation of how tumors can become "addicted" to a non amplified, nonmutated, physiologically regulated RTK to evade targeted treatment.

The majority of newly diagnosed brain tumors are treated with surgery, radiation, and the chemotherapeutic temozolomide. Development of additional therapeutics to improve treatment outcomes is complicated by the blood-brain barrier (BBB), which acts to protect healthy tissue from chemical insults. The high pressure found within brain tumors adds a challenge to local delivery of therapy by limiting the distribution of bolus injections. Here we discuss various drug delivery strategies, including convection-enhanced delivery, intranasal delivery, and intrathecal delivery, as well as pharmacological strategies for improving therapeutic efficacy, such as blood-brain barrier disruption., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The prognosis for diffuse infiltrating pontine gliomas (DIPG) remains extremely poor, with the majority of patients surviving less than 2 years. Here, we have adapted standard xenograft techniques to study glioma growth in the mouse brainstem, and have utilized the mouse model for studying a relevant therapeutic for treating DIPGs. bioluminescence imaging monitoring revealed a progressive increase in signal following the injection of either of two tumor cell types into the brainstem. Mice with orthotopic GS2 tumors, and receiving a single 100 mg/kg dose of temozolomide showed a lengthy period of decreased tumor luminescence, with substantially increased survival relative to untreated mice (P < 0.001). A small molecule inhibitor that targets cdk4/6 was used to test AM-38 brainstem xenograft response to treatment. Drug treatment resulted in delayed tumor growth, and significantly extended survival. Our results demonstrate the feasibility of using an orthotopic brainstem tumor model in athymic mice, and for application to testing therapeutic agents in treating DIPG.

The phosphatidylinositol-3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway is activated in more than88% of glioblastomas (GBM). New drugs targeting this pathway are currently in clinical trials. However, noninvasive assessment of treatment response remains challenging. By using magnetic resonance spectroscopy (MRS), PI3K/Akt/mTOR pathway inhibition was monitored in 3 GBM cell lines (GS-2, GBM8, and GBM6; each with a distinct pathway activating mutation) through the measurement of 2 mechanistically linked MR biomarkers: phosphocholine (PC) and hyperpolarized lactate.(31)P MRS studies showed that treatment with the PI3K inhibitor LY294002 induced significant decreases in PC to 34 %± 9% of control in GS-2 cells, 48% ± 5% in GBM8, and 45% ± 4% in GBM6. The mTOR inhibitor everolimus also induced a significant decrease in PC to 62% ± 14%, 57% ± 1%, and 58% ± 1% in GS-2, GBM8, and GBM6 cells, respectively. Using hyperpolarized (13)C MRS, we demonstrated that hyperpolarized lactate levels were significantly decreased following PI3K/Akt/mTOR pathway inhibition in all 3 cell lines to 51% ± 10%, 62% ± 3%, and 58% ± 2% of control with LY294002 and 72% ± 3%, 61% ± 2%, and 66% ± 3% of control with everolimus in GS-2, GBM8, and GBM6 cells, respectively. These effects were mediated by decreases in the activity and expression of choline kinase α and lactate dehydrogenase, which respectively control PC and lactate production downstream of HIF-1. Treatment with the DNA damaging agent temozolomide did not have an effect on either biomarker in any cell line. This study highlights the potential of PC and hyperpolarized lactate as noninvasive MR biomarkers of response to targeted inhibitors in GBM.

Glioblastoma (GBM) is the most common and lethal primary malignant brain tumor in humans. Because the phosphatidylinositol-3-kinase (PI3K) signaling pathway is activated in more than 88% of GBM, new drugs which target this pathway, such as the mTOR inhibitor Everolimus, are currently in clinical trials. Early tumor response to molecularly targeted treatments remains challenging to assess non-invasively, because it is often associated with tumor stasis or slower tumor growth. Innovative neuroimaging methods are therefore critically needed to provide metabolic or functional information that is indicative of targeted therapeutic action at early time points during the course of treatment. In this study, we demonstrated for the first time that hyperpolarized (HP) 13C magnetic resonance spectroscopic imaging (MRSI) can be used on a clinical MR system to monitor early metabolic response of orthotopic GBM tumors to Everolimus treatment through measurement of the HP lactate-to-pyruvate ratios. The study was performed on a highly invasive non-enhancing orthotopic GBM tumor model in rats (GS-2 tumors), which replicates many fundamental features of human GBM tumors. Seven days after initiation of treatment there was a significant drop in the HP lactate-to-pyruvate ratio from the tumor tissue in treated animals relative to day 0 (67%±27% decrease). In the control group, no significant changes in the HP lactate-to-pyruvate ratios were observed. Importantly, at the 7 day time point, conventional MR imaging (MRI) was unable to detect a significant difference in tumor size between control and treated groups. Inhibition of tumor growth by conventional MRI was observed from day 15 of treatment. This implies that the decrease in the HP lactate-to-pyruvate ratio could be detected before any treatment-induced inhibition of tumor growth. Using immunohistochemical staining to further examine tumor response to treatment, we found that the decrease in the HP lactate-to-pyruvate ratio was associated with a drop in expression of lactate dehydrogenase, the enzyme that catalyzes pyruvate to lactate conversion. Also evident was decreased staining for carbonic anhydrase IX (CA-IX), an indicator of hypoxia-inducible factor 1α (HIF-1α) activity, which, in turn, regulates expression of lactate dehydrogenase. To our knowledge, this study is the first report of the use of HP 13C MRSI at a clinical field strength to monitor GBM response to molecularly targeted treatments. It highlights the potential of HP lactate-to-pyruvate ratio as an early biomarker of response, thereby supporting further investigation of this non-invasive imaging approach for eventual clinical application., (Published by Elsevier Inc.)

Elevated epidermal growth factor receptor (EGFR) and mammalian target of rapamycin (mTOR) signaling are known to contribute to the malignant properties of glioblastoma multiforme (GBM), which include uncontrolled cell proliferation and evasion of apoptosis. Small molecule inhibitors that target these protein kinases have been evaluated in multiple clinical trials for cancer patients, including those with GBM. Here we have examined the cellular and molecular effects of a combined kinase inhibition of mTOR (rapamycin) and EGFR (EKI-785) in U87 and U251 GBM cells. Simultaneous treatment with rapamycin and EKI-785 results in synergistic antiproliferative as well as proapoptotic effects. At a molecular level, rapamycin alone significantly decreases S6 phosphorylation, whereas EKI-785 alone promotes substantially reduced signal transducer and activator of transcription (STAT3) phosphorylation. Treatment with rapamycin alone also increases Akt phosphorylation on Ser-473, but this effect is blocked by a simultaneous administration of EKI-785. Individually, EKI-785 diminishes while rapamycin promotes the binding of the translation inhibitor eukaryotic initiation factor 4E binding protein (4EBP1) to the eukaryotic translation initiation factor 4E (eIF4E). In spite of these opposing effects, the highest level of 4EBP1-eIF4E binding occurs with the combination of the two inhibitors. These results indicate that the inhibition of EGFR and mTOR has distinct as well as common signaling consequences and provides a molecular rationale for the synergistic antitumor effects of EKI-785 and rapamycin administration.

Although the coincidental amplification and accompanying overexpression of bystander genes that neighbor oncogene targets occur frequently during the development of human tumors, little has been done to investigate the functional or biological consequences of amplified bystander gene overexpression. LANCL2 (LANC-like 2) is a bystander gene that is coamplified and overexpressed with epidermal growth factor receptor in approximately 20% of all glioblastomas. This gene has also been designated as Testis Adriamycin Sensitivity Protein because it is most highly expressed in testis and its expression has been noted to increase cellular sensitivity to Adriamycin. Because of the latter association, we have examined potential relationships between LANCL2 and the expression of multidrug-resistance (MDR)1, as well as its cognate protein, P-glycoprotein (P-gp), because elevated expression of P-gp is known to increase cell resistance to many cytotoxic drugs, including Adriamycin. Using the Dx5 derivative of MES-SA cells in which P-gp is overexpressed, we show that the level of endogenous P-gp decreases with increased expression of exogenous LanCl-2 and that cells with reduced P-gp show increased sensitivity to Adriamycin. Results from reverse transcription-PCR and MDR1 promoter activity analyses suggest that LanCl-2 transcriptionally suppresses MDR1, and this interpretation of LanCl-2 function is consistent with results from immunofluorescence analysis, which shows that LanCl-2 resides in the nucleus, as well as at the plasma membrane. With respect to this study, our data indicate that LanCl-2 increases cellular sensitivity to Adriamycin by decreasing the expression of P-gp, but more generally, these results indicate that the identification of bystander gene amplification in human tumors can have clinical implications.

BRAFV600E is a common finding in glioma (about 10-60% depending on histopathologic subclassification). BRAFV600E monotherapy shows modest preclinical efficacy against BRAFV600E gliomas and also induces adverse secondary skin malignancies. Here, we examine the molecular mechanism of intrinsic resistance to BRAFV600E inhibition in glioma. Furthermore, we investigate BRAFV600E/MEK combination therapy that overcomes intrinsic resistance to BRAFV600E inhibitor and also prevents BRAFV600E inhibitor induced secondary malignancies. Immunoblotting and Human Phospho-Receptor Tyrosine Kinase Array assays were used to interrogate MAPK pathway activation. The cellular effect of BRAFV600E and MEK inhibition was determined by WST-1 viability assay and cell cycle analysis. Flanked and orthotopic GBM mouse models were used to investigate the in vivo efficacy of BRAFV600E/MEK combination therapy and the effect on secondary malignancies. BRAFV600E inhibition leads to recovery of ERK phosphorylation. Combined BRAFV600E and MEK inhibition prevents reactivation of the MAPK signaling, which correlates with decreased cell viability and augmented cell cycle arrest. Similarly, mice bearing BRAFV600E glioma showed reduced tumor growth when treated with a combination of BRAFV600E and MEK inhibitor compared to BRAFV600E inhibition alone. Additional benefit of BRAFV600E/MEK inhibition was reflected by reduced cutaneous squamous-cell carcinoma (cSCC) growth (a surrogate for RAS-driven secondary maligancies). In glioma, recovery of MAPK signaling upon BRAF inhibition accounts for intrinsic resistance to BRAFV600E inhibitor. Combined BRAFV600E and MEK inhibition prevents rebound of MAPK activation, resulting in enhanced antitumor efficacy and also reduces the risk of secondary malignancy development.

Activating mutation of BRAF is a common finding in pediatric gliomas. As many as 14% of high grade and up to 66% of certain subtypes of low grade pediatric glioma have the BRAFV600E mutation. Small molecule inhibitors that selectively target BRAFV600E are FDA approved for melanoma and have shown significant efficacy in treating BRAFV600E glioma in pre-clinical trials. Despite showing initial anti-tumor activity, acquired drug resistance significantly limits the benefit from being treated with BRAFV600E inhibitors. Here, we have identified molecular responses to BRAFV600E inhibitor treatment in human glioma models that have substantial clinical implications. Specifically, we show that BRAFV600E inhibitor resistant cells upregulate pro-survival mediators such as Wnt, and additionally increase receptor tyrosine kinase activity, including EGFR and Axl, promoting resistance to BRAFV600E inhibition. Our results suggest strategies to circumvent acquired resistance to BRAFV600E inhibitor therapy, and thereby improve outcomes for patients with BRAFV600E gliomas.

ObjectiveHead and neck squamous cell carcinoma (HNSCC) accounts for more than 5% of all cancers worldwide. The mortality rate of HNSCC has remained unchanged (approximately 50%) over the last few decades. Ubiquitous overexpression of wild type EGFR in many solid tumors has led to the development of EGFR targeted therapies. EGFR can be constitutively activated via several mechanisms including the truncated, EGFR variant III isoform (EGFRvIII). EGFRvIII lacks exons 2-7 and has been reported to be present in up to 20-40% of HNSCC. EGFRvIII has been shown to contribute to cetuximab resistance. The mechanisms leading to EGFRvIII expression in HNSCC are unknown. The present investigation was undertaken to determine the etiology of EGFRvIII in HNSCC.Materials and methodsFixed HNSCC and glioma tissues were analyzed by fluorescence in situ hybridization for EGFR amplification. DNA and RNA from fresh frozen specimens were used to determine the presence of EGFRvIII transcripts and the mechanisms of expression via PCR, RT-PCR and RNA sequencing.ResultsUnlike glioma, EGFRvIII expression in HNSCC did not correlate with EGFR amplification. We found evidence of genomic deletion of the exon 2-7 in 6 of 7 HNSCC cases examined, however, the presence of genomic deletion did not always result in mRNA expression of EGFRvIII. RNA sequencing with automated alignment did not identify EGFRvIII due to microhomology between intron 1 and exon 8. RNA sequencing analyzed by manual alignment methods did not correlate well with RT-PCR and PCR findings.ConclusionThese findings suggest that genomic deletion as well as additional regulatory mechanisms may contribute to EGFRvIII expression in HNSCC. Further, large scale automated alignment of sequencing are unlikely to identify EGFRvIII and an assay specifically designed to detect EGFRvIII may be necessary to detect this altered form of EGFR in HNSCC tumors.

BackgroundGlioblastoma multiforme (GBM) contains a population of cells that exhibit stem cell phenotypes. These cancer stem cells (CSCs) may be a source of therapeutic resistance, although support for this important concept is limited.MethodsWe determined whether early-passage GBM CSCs respond differently than patient-matched, genotypically similar non-CSCs to clinically relevant single or serial doses of temozolomide (TMZ), radiation therapy (XRT), or alternating TMZ treatment and XRT, which is the standard of care for GBM patients.ResultsDespite the phenotypic differences, including the presence of stem cell markers and formation of intracranial tumors, the CSCs and matched non-CSCs were equally resistant to TMZ in a majority of patients, using 2 independent assays. TMZ response was consistent with methylated O(6)-DNA methylguanine-methyltransferase (MGMT) and MGMT protein levels in both culture types. In contrast, CSCs were unexpectedly more responsive to XRT compared with matched non-CSCs from 2 patients despite having relatively equal resistance to TMZ. However, for the majority of culture pairs from individual patients, responses in CSCs were indistinguishable from non-CSC cultures.ConclusionsIn our patient-matched primary cultures, response to TMZ was tightly linked to the individual tumor's MGMT status and independent of their phenotypic differences. TMZ and XRT together revealed no additive benefit compared with monotherapy for either culture type, in contrast to the notion that the CSC population is more resistant to XRT. If the tumor cell response in vitro mirrors therapeutic response in larger patient cohorts, these rapid assays in primary cultures could allow -empirical selection of efficacious therapeutic agents on a patient-specific basis.

BackgroundLiposomal drug packaging is well established as an effective means for increasing drug half-life, sustaining drug activity, and increasing drug efficacy, whether administered locally or distally to the site of disease. However, information regarding the relative effectiveness of peripheral (distal) versus local administration of liposomal therapeutics is limited. This issue is of importance with respect to the treatment of central nervous system cancer, for which the blood-brain barrier presents a significant challenge in achieving sufficient drug concentration in tumors to provide treatment benefit for patients.MethodsWe compared the anti-tumor activity and efficacy of a nanoliposomal formulation of irinotecan when delivered peripherally by vascular route with intratumoral administration by convection-enhanced delivery (CED) for treating intracranial glioblastoma xenografts in athymic mice.ResultsOur results show significantly greater anti-tumor activity and survival benefit from CED of nanoliposomal irinotecan. In 2 of 3 efficacy experiments, there were animal subjects that experienced apparent cure of tumor from local administration of therapy, as indicated by a lack of detectable intracranial tumor through bioluminescence imaging and histopathologic analysis. Results from investigating the effectiveness of combination therapy with nanoliposomal irinotecan plus radiation revealed that CED administration of irinotecan plus radiation conferred greater survival benefit than did irinotecan or radiation monotherapy and also when compared with radiation plus vascularly administered irinotecan.ConclusionsOur results indicate that liposomal formulation plus direct intratumoral administration of therapeutic are important for maximizing the anti-tumor effects of irinotecan and support clinical trial evaluation of this therapeutic plus route of administration combination.

The tumor-selective, proapoptotic, death receptor ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a mediator of antitumor drug activity and in itself is a promising agent for the treatment of human malignancies. Like many tumors, however, glioblastoma multiforme (GBM), the most fatal form of glioma, exhibits a range of TRAIL sensitivity, and only a small percentage of GBM tumors undergo TRAIL-induced apoptosis. We here show that TRAIL resistance in GBM is a consequence of overexpression of the short isoform of the caspase-8 inhibitor, c-FLICE inhibitory protein (FLIPS), and that FLIPS expression is in turn translationally enhanced by activation of the Akt-mammalian target of rapamycin (mTOR)-p70 S6 kinase 1 (S6K1) pathway. Conversely, pharmacologic or genetic inhibition of mTOR, or the mTOR target S6KI, suppresses polyribosomal accumulation of FLIPS mRNA, FLIPS protein expression, and TRAIL resistance. In archived material from 12 human GBM tumors, PTEN status was a predictor of activation of the Akt-mTOR-S6K1 pathway and of FLIPS levels, while in xenografted human GBM, activation status of the PTEN-Akt-mTOR pathway distinguished the tumors inherently sensitive to TRAIL from those which could be sensitized by the mTOR inhibitor rapamycin. These results define the mTOR pathway as a key limiter of tumor elimination by TRAIL-mediated mechanisms, provide a means by which the TRAIL-sensitive subset of GBM can be identified, and provide rationale for the combined use of TRAIL with mTOR inhibitors in the treatment of human cancers. [ABSTRACT FROM AUTHOR]

Pediatric brainstem gliomas often harbor oncogenic K27M mutation of histone H3.3. Here we show that GSKJ4 pharmacologic inhibition of K27 demethylase JMJD3 increases cellular H3K27 methylation in K27M tumor cells and demonstrate potent antitumor activity both in vitro against K27M cells and in vivo against K27M xenografts. Our results demonstrate that increasing H3K27 methylation by inhibiting K27 demethylase is a valid therapeutic strategy for treating K27M-expressing brainstem glioma. [ABSTRACT FROM AUTHOR]