Transcriptional diversity of long-term glioblastoma survivors

BACKGROUND: Glioblastoma (GBM) is a highly aggressive type of glioma with poor prognosis. However, a small number of patients live much longer than the median survival. A better understanding of these long-term survivors (LTSs) may provide important insight into the biology of GBM. METHODS: We identified 7 patients with GBM, treated at Memorial Sloan-Kettering Cancer Center (MSKCC), with survival \u3e48 months. We characterized the transcriptome of each patient and determined rates of MGMT promoter methylation and IDH1 and IDH2 mutational status. We identified LTSs in 2 independent cohorts (The Cancer Genome Atlas [TCGA] and NCI Repository for Molecular Brain Neoplasia Data [REMBRANDT]) and analyzed the transcriptomal characteristics of these LTSs. RESULTS: The median overall survival of our cohort was 62.5 months. LTSs were distributed between the proneural (n = 2), neural (n = 2), classical (n = 2), and mesenchymal (n = 1) subtypes. Similarly, LTS in the TCGA and REMBRANDT cohorts demonstrated diverse transcriptomal subclassification identities. The majority of the MSKCC LTSs (71%) were found to have methylation of the MGMT promoter. None of the patients had an IDH1 or IDH2 mutation, and IDH mutation occurred in a minority of the TCGA LTSs as well. A set of 60 genes was found to be differentially expressed in the MSKCC and TCGA LTSs. CONCLUSIONS: While IDH mutant proneural tumors impart a better prognosis in the short-term, survival beyond 4 years does not require IDH mutation and is not dictated by a single transcriptional subclass. In contrast, MGMT methylation continues to have strong prognostic value for survival beyond 4 years. These findings have substantial impact for understanding GBM biology and progression

Phase I/randomized phase II trial of TRC105 plus bevacizumab versus bevacizumab in recurrent glioblastoma: North Central Cancer Treatment Group N1174 (Alliance)

Background Patients with glioblastoma (GBM) have a poor prognosis and limited effective treatment options. Bevacizumab has been approved for treatment of recurrent GBM, but there is questionable survival benefit. Based on preclinical and early clinical data indicating that CD105 upregulation may represent a mechanism of resistance to bevacizumab, we hypothesized that combining bevacizumab with the anti-CD105 antibody TRC105 may improve efficacy in recurrent GBM.Methods Phase I dose-escalation/comparative randomized phase II trial in patients with GBM. During phase I, the maximum tolerated dose (MTD) of TRC105 in combination with bevacizumab was determined. In phase II, patients were randomized 1:1 to TRC105 and bevacizumab or bevacizumab monotherapy. Patients receivedTRC105 (10 mg/kg) weekly and bevacizumab (10 mg/kg) every 2 weeks. Efficacy, as assessed by progression-free survival (PFS), was the primary endpoint; safety, quality of life, and correlative outcomes were also evaluated.Results In total, 15 patients were enrolled in phase I and 101 in phase II; 52 patients were randomized to TRC105 with bevacizumab and 49 to bevacizumab monotherapy. The MTD was determined to be 10 mg/kg TRC105 weekly plus bevacizumab 10 mg/kg every 2 weeks. An increased occurrence of grade >= 3 adverse events was seen in the combination arm, including higher incidences of anemia. Median PFS was similar in both treatment arms: 2.9 months for combination versus 3.2 months for bevacizumab monotherapy (HR = 1.16, 95% CI = 0.75-1.78, P = .51). Quality of life scores were similar for both treatment arms.Conclusions TRC105 in combination with bevacizumab was well tolerated in patients with recurrent GBM, but no difference in efficacy was observed compared to bevacizumab monotherapy

Ibrutinib Unmasks Critical Role of Bruton Tyrosine Kinase in Primary CNS Lymphoma.

Bruton tyrosine kinase (BTK) links the B-cell antigen receptor (BCR) and Toll-like receptors with NF-κB. The role of BTK in primary central nervous system (CNS) lymphoma (PCNSL) is unknown. We performed a phase I clinical trial with ibrutinib, the first-in-class BTK inhibitor, for patients with relapsed or refractory CNS lymphoma. Clinical responses to ibrutinib occurred in 10 of 13 (77%) patients with PCNSL, including five complete responses. The only PCNSL with complete ibrutinib resistance harbored a mutation within the coiled-coil domain of CARD11, a known ibrutinib resistance mechanism. Incomplete tumor responses were associated with mutations in the B-cell antigen receptor-associated protein CD79B

Radiotherapy combined with nivolumab or temozolomide for newly diagnosed glioblastoma with unmethylated MGMT promoter: An international randomized phase III trial

BACKGROUND: Addition of temozolomide (TMZ) to radiotherapy (RT) improves overall survival (OS) in patients with glioblastoma (GBM), but previous studies suggest that patients with tumors harboring an unmethylated MGMT promoter derive minimal benefit. The aim of this open-label, phase III CheckMate 498 study was to evaluate the efficacy of nivolumab (NIVO) + RT compared with TMZ + RT in newly diagnosed GBM with unmethylated MGMT promoter. METHODS: Patients were randomized 1:1 to standard RT (60 Gy) + NIVO (240 mg every 2 weeks for eight cycles, then 480 mg every 4 weeks) or RT + TMZ (75 mg/m2 daily during RT and 150-200 mg/m2/day 5/28 days during maintenance). The primary endpoint was OS. RESULTS: A total of 560 patients were randomized, 280 to each arm. Median OS (mOS) was 13.4 months (95% CI, 12.6 to 14.3) with NIVO + RT and 14.9 months (95% CI, 13.3 to 16.1) with TMZ + RT (hazard ratio [HR], 1.31; 95% CI, 1.09 to 1.58; P = .0037). Median progression-free survival was 6.0 months (95% CI, 5.7 to 6.2) with NIVO + RT and 6.2 months (95% CI, 5.9 to 6.7) with TMZ + RT (HR, 1.38; 95% CI, 1.15 to 1.65). Response rates were 7.8% (9/116) with NIVO + RT and 7.2% (8/111) with TMZ + RT; grade 3/4 treatment-related adverse event (TRAE) rates were 21.9% and 25.1%, and any-grade serious TRAE rates were 17.3% and 7.6%, respectively. CONCLUSIONS: The study did not meet the primary endpoint of improved OS; TMZ + RT demonstrated a longer mOS than NIVO + RT. No new safety signals were detected with NIVO in this study. The difference between the study treatment arms is consistent with the use of TMZ + RT as the standard of care for GBM.ClinicalTrials.gov NCT02617589

First-line treatment and outcome of elderly patients with primary central nervous system lymphoma (PCNSL)—a systematic review and individual patient data meta-analysis

Evidence for prognosis and treatment of elderly patient with primary central nervous system is limited. High-dose methotrexate should be applied whenever possible, especially combination with oral alkylating agents is a promising approach. Further combinations with other intravenous drugs do not seem to improve outcome. More prospective trials designed for elderly PCNSL patients are warrante

CLASP2 links Reelin to the cytoskeleton during neocortical development

Published in final edited form as: Neuron. 2017 March 22; 93(6): 1344–1358.e5. doi:10.1016/j.neuron.2017.02.039.INTRODUCTION The complex architecture of the brain requires precise control over the timing of neurogenesis, neuron migration, and differentiation. These three developmental processes are exquisitely controlled during the expansion of the mammalian neocortex. The six morphologically distinct layers of the neocortex form in an “inside-out” pattern with early-born neurons forming deeper layers and later-born neurons migrating past them to form superficial layers of the cortical plate (Rakic, 1974). The Reelin signaling pathway plays a crucial role in cortical lamination. Reelin is a secreted glycoprotein that exerts its function by binding to the lipoprotein receptors ApoER2 and VLDLR and inducing tyrosine phosphorylation of the intracellular adaptor protein Disabled (Dab1) (Howell et al., 2000, Bock and Herz, 2003). Phosphorylated Dab1 then recruits downstream signaling molecules to promote cytoskeletal changes necessary for neuronal migration, final positioning, and morphology (D’Arcangelo, 2005). Mutations of Reelin, the dual ApoER2/VLDLR receptor, or Dab1 lead to an inversion of the normal inside-out pattern of cortex development (D’Arcangelo et al., 1995, Howell et al., 1997, Trommsdorff et al., 1999). In addition, a number of mutations in cytoskeletal-encoded genes produce deficits in neuron migration and cortical lamination phenotypically similar to Reelin mutants, firmly establishing a mechanistic and developmentally critical connection between Reelin and the cytoskeleton. For example, human mutations in lissencephaly-1, doublecortin, and tubulin, integral components of the microtubule cytoskeleton, cause severe cortical lamination defects with later-born neurons failing to migrate past previously born neurons (Reiner et al., 1993, Gleeson et al., 1998, Romaniello et al., 2015). The culmination of these genetic studies indicates that several signaling pathways, including the Reelin pathway, converge on downstream cytoskeletal proteins to affect proper neuronal migration and brain development. However, the molecular effectors of these pathways have not been fully characterized. CLASPs (cytoplasmic linker associated proteins) belong to a heterogeneous family of plus-end tracking proteins (+TIPs) that specifically accumulate at the growth cone. This localization strategically places them in a position to control neurite growth, directionality, and the crosstalk between microtubules and the actin cytoskeleton (Akhmanova and Hoogenraad, 2005, Basu and Chang, 2007, Akhmanova and Steinmetz, 2008). Previous evidence showed that CLASPs accumulate asymmetrically toward the leading edge of migrating fibroblasts, indicating a role for CLASPs in cell polarity and movement (Akhmanova et al., 2001, Wittmann and Waterman-Storer, 2005). We found that CLASP2 protein levels steadily increase throughout neuronal development and are specifically enriched at the growth cones of extending neurites. In particular, short-hairpin RNA (shRNA)-mediated knockdown of CLASP2 in primary mouse neurons decreases neurite length, whereas overexpression of human CLASP2 causes the formation of multiple axons, enhanced dendritic branching, and Golgi condensation (Beffert et al., 2012). These results implicate a role for CLASP2 in neuronal morphogenesis and polarization; however, the function of CLASP2 during brain development is unknown. Here we demonstrate that CLASP2 is a modifier of the Reelin signaling pathway during cortical development. In vivo knockdown experiments demonstrate that CLASP2 plays significant roles in neural precursor proliferation, neuronal migration, and morphogenesis. In addition, we show that GSK3β-mediated phosphorylation of CLASP2 controls its binding to the Reelin adaptor protein Dab1, a required molecular step governing CLASP2’s regulatory effects on neuron morphology and movement. RESULTS CLASP2 Expression Is Functionally Associated with the Reelin Signaling Pathway To identify novel genes downstream of Reelin signaling, we examined the expression of mRNA transcripts by microarray between adult brain cortices from mice deficient in either Reelin, the double ApoER2/VLDLR receptor mutant, or Dab1 and compared Affymetrix gene expression profiles against age-matched, wild-type mice. Importantly, each of these mutant mouse models present a similar phenotype that includes severe neuronal migration defects (D’Arcangelo et al., 1995, Howell et al., 1997, Trommsdorff et al., 1999). We defined a large network of genes perturbed above a threshold of 1.5-fold in response to deficient Reelin signaling, identifying 832 upregulated and 628 downregulated genes that were common to all three mouse models (Figure 1A). Ingenuity Pathway Analysis revealed a network of genes that is functionally related to cytoskeleton organization, microtubule dynamics, neurogenesis, and migration of cells (Figure 1B). Of the few cytoskeletal candidate genes identified, CLASP2 was the only microtubule +TIP. Specifically, CLASP2 mRNA expression was increased in all three Reelin mutant phenotypes, while CLASP1 mRNA expression remained unchanged (Figure 1B). Consistent with the microarray data, CLASP2 protein levels were ∼2.8-fold higher in Dab1 knockout mice (Figure 1C). These findings suggest that Reelin signaling controls CLASP2 expression and establishes the first molecular link between a plus-end, microtubule binding protein downstream of extracellular Reelin signaling.We thank Drs. Thomas C. Sudhof, Joachim Herz, Santos Franco, and Torsten Wittmann for plasmids and antibodies. We thank Alicia Dupre, Elias Fong, and Christine Learned for technical support. This work was supported by grants from the National Institutes of Health (R21 MH100581 to T.F.H., U.B., and A.H.). (R21 MH100581 - National Institutes of Health)Accepted manuscrip

A HIF-independent, CD133-mediated mechanism of cisplatin resistance in glioblastoma cells

Purpose Glioblastoma Multiforme (GBM) is the commonest brain tumour in adults. A population of cells, known as cancer stem cells (CSCs), is thought to mediate chemo/radiotherapy resistance. CD133 is a cell surface marker to identify and isolate CSCs. However, its functional significance and the relevant microenvironment in which to study CD133 remain unknown. We examined the influence of hypoxia on CD133 expression and the potential functional significance of CD133 in glioblastoma chemoresistance. Methods Gene expression was analysed by qRT-PCR. siRNA technique was used to downregulate genes and confirmed by flow cytometry. IC50 values was evaluated with the Alamar blue assay. Results CD133 expression was upregulated in hypoxia in 2D and 3D models. There was increased resistance to chemotherapeutics, cisplatin, temozolomide and etoposide, in cells cultured in hypoxia compared to normoxia. siRNA knockdown of either HIF1a or HIF2a resulted in reduced CD133 mRNA expression with HIF2a having a more prolonged effect on CD133 expression. HIF2a downregulation sensitized GBM cells to cisplatin to a greater extent than HIF1a but CD133 knockdown had a much more marked effect on cisplatin sensitisation than knockdown of either of the HIFs suggesting a HIF-independent mechanism of cisplatin resistance mediated via CD133. The same mechanism was not involved in temozolomide resistance since downregulation of HIF1a but not HIF2a or CD133 sensitized GBM cells to temozolomide. Conclusion Knowledge of the mechanisms involved in the novel hypoxia-induced CD133-mediated resistance to cisplatin observed might lead to identification of new strategies that enable more effective use of current therapeutic agents

A novel chalcone derivative which acts as a microtubule depolymerising agent and an inhibitor of P-gp and BCRP in in-vitro and in-vivo glioblastoma models

<p>Abstract</p> <p>Background</p> <p>Over the past decades, in spite of intensive search, no significant increase in the survival of patients with glioblastoma has been obtained. The role of the blood-brain barrier (BBB) and especially the activity of efflux pumps belonging to the ATP Binding Cassette (ABC) family may, in part, explain this defect.</p> <p>Methods</p> <p>The <it>in-vitro </it>activities of JAI-51 on cell proliferation were assessed by various experimental approaches in four human and a murine glioblastoma cell lines. Using drug exclusion assays and flow-cytometry, potential inhibitory effects of JAI-51 on P-gp and BCRP were evaluated in sensitive or resistant cell lines. JAI-51 activity on <it>in-vitro </it>microtubule polymerization was assessed by tubulin polymerization assay and direct binding measurements by analytical ultracentrifugation. Finally, a model of C57BL/6 mice bearing subcutaneous GL26 glioblastoma xenografts was used to assess the activity of the title compound <it>in vivo</it>. An HPLC method was designed to detect JAI-51 in the brain and other target organs of the treated animals, as well as in the tumours.</p> <p>Results</p> <p>In the four human and the murine glioblastoma cell lines tested, 10 μM JAI-51 inhibited proliferation and blocked cells in the M phase of the cell cycle, via its activity as a microtubule depolymerising agent. This ligand binds to tubulin with an association constant of 2 × 10⁵M^-1, overlapping the colchicine binding site. JAI-51 also inhibited the activity of P-gp and BCRP, without being a substrate of these efflux pumps. These <it>in vitro </it>studies were reinforced by our <it>in vivo </it>investigations of C57BL/6 mice bearing GL26 glioblastoma xenografts, in which JAI-51 induced a delay in tumour onset and a tumour growth inhibition, following intraperitoneal administration of 96 mg/kg once a week. In accordance with these results, JAI-51 was detected by HPLC in the tumours of the treated animals. Moreover, JAI-51 was detected in the brain, showing that the molecule is also able to cross the BBB.</p> <p>Conclusion</p> <p>These <it>in vitro </it>and <it>in vivo </it>data suggest that JAI-51 could be a good candidate for a new treatment of tumours of the CNS. Further investigations are in progress to associate the title compound chemotherapy to radiotherapy in a rat model.</p