Purpose The purpose of this study was to determine the safety, feasibility, and immunologic responses of treating grade 4 astrocytomas with multiple infusions of anti-CD3 x anti-EGFR bispecific antibody (EGFRBi) armed T cells (EGFR BATs) in combination with radiation and chemotherapy. Methods This phase I study used a 3 + 3 dose escalation design to test the safety and feasibility of intravenously infused EGFR BATs in combination with radiation and temozolomide (TMZ) in patients with newly diagnosed grade 4 astrocytomas (AG4). After finding the feasible dose, an expansion cohort with unmethylated O 6 -methylguanine-DNA methyltransferase (MGMT) tumors received weekly EGFR BATs without TMZ. Results The highest feasible dose was 80 × 10 9 EGFR BATs without dose-limiting toxicities (DLTs) in seven patients. We could not escalate the dose because of the limited T-cell expansion. There were no DLTs in the additional cohort of three patients with unmethylated MGMT tumors who received eight weekly infusions of EGFR BATs without TMZ. EGFR BATs infusions induced increases in glioma specific anti-tumor cytotoxicity by peripheral blood mononuclear cells ( p < 0.03) and NK cell activity ( p < 0.002) ex vivo, and increased serum concentrations of IFN-γ ( p < 0.03), IL-2 ( p < 0.007), and GM-CSF ( p < 0.009). Conclusion Targeting AG4 with EGFR BATs at the maximum feasible dose of 80 × 10 9 , with or without TMZ was safe and induced significant anti-tumor-specific immune responses. These results support further clinical trials to examine the efficacy of this adoptive cell therapy in patients with MGMT-unmethylated GBM. ClinicalTrials.gov Identifier : NCT03344250

PDF file - 296K, Short-time exogenous HGF treatment enhances the in vivo anti-tumor effects of METi

PDF file - 60K, Methods for cell and tissue culture and Reverse Phase Protein Microarray Construction and Analysis

PDF file - 54K, Regression and Spearman (rank-based) correlation analyses between the molecular parameters and cell responsiveness to METi

Supplementary Figure S1. Mutations in CCND3 increase sensitivity to mTOR inhibition. Supplementary Figure S2. Dose-response plots for everolimus and palbociclib. Supplementary Figure S3. mTOR inhibition with everolimus cross-regulates the CDK/Rb pathway. Supplementary Figure S4. Erk activity is linked to CDK4/6 inhibition. Supplementary Figure S5. Palbociclib treatment decreases the phosphorylation of Erk1/2. Supplementary Figure S6. Cells remain attached throughout the Seahorse metabolic assays. Supplementary Figure S7. Immunohistochemistry staining of tumor harboring mouse brains for GIC markers.

Recent studies indicate that signaling molecules traditionally associated with central nervous system function play critical roles in cancer. Dopamine receptor signaling is implicated in various cancers including glioblastoma (GBM) and it is a recognized therapeutic target, as evidenced by recent clinical trials with a selective dopamine receptor D2 (DRD2) inhibitor ONC201. Understanding the molecular mechanism(s) of the dopamine receptor signaling will be critical for development of potent therapeutic options. Using the human GBM patient-derived tumors treated with dopamine receptor agonists and antagonists, we identified the proteins that interact with DRD2. DRD2 signaling promotes glioblastoma (GBM) stem-like cells and GBM growth by activating MET. In contrast, pharmacological inhibition of DRD2 induces DRD2-TRAIL receptor interaction and subsequent cell death. Thus, our findings demonstrate a molecular circuitry of oncogenic DRD2 signaling in which MET and TRAIL receptors, critical factors for tumor cell survival and cell death, respectively, govern GBM survival and death. Finally, tumor-derived dopamine and expression of dopamine biosynthesis enzymes in a subset of GBM may guide patient stratification for DRD2 targeting therapy.

XLSX file - 128K, Reverse Phase Protein Microarray data for the cell lines with high HGF vs. cell lines with low HGF

Journal Article ONC201 and ONC206: Metabolically ClipPing the wings of diffuse midline glioma Get access Benjamin Purow Benjamin Purow Department of Neurology, University of Virginia, Charlottesville, Virginia, USA Corresponding Author: Benjamin Purow, MD, Neuro-Oncology Division, Neurology Department, West Complex Room 6230, University of Virginia, 1300 Jefferson Park Avenue, Charlottesville, VA 22908, USA (bwp5g@virginia.edu). Search for other works by this author on: Oxford Academic PubMed Google Scholar Neuro-Oncology, Volume 24, Issue 9, September 2022, Pages 1452–1453, https://doi.org/10.1093/neuonc/noac103 Published: 23 April 2022 Article history Published: 23 April 2022 Corrected and typeset: 03 May 2022

Atypical teratoid/rhabdoid tumors (AT/RTs) are among the most malignant brain tumors of childhood. The invariable occurrence of either SMARCB1 or, much less frequently, SMARCA4 mutations in AT/RT underscores the central pathogenic role for SWI/SNF chromatin complex dysfunction. Indeed, molecular profiling studies to date have failed to document any other recurrent genomic alterations that may be driving AT/RT. However, clinically relevant heterogeneity with regard to patient survival and tumor localization implies the existence of endogenous disease subgroups. In a recent Cancer Cell article, Johann et al probed epigenomic landscapes in a large (N1⁄4 192) sample set of AT/RTs. Global DNA methylation arrays revealed three tumor subclasses whose delineation was also supported by mRNA profiling. Their proposed nomenclature reflected these transcriptional distinctions, with AT/RT-TYR tumors exhibiting overexpression of melanosomal markers like MITF, TYR, and DCT, AT/RT-SHH tumors featuring upregulated MYCN and GLI2 reminiscent of SHH-driven medulloblastomas, and AT/RT-MYC tumors remarkable for marked overexpression of MYC. Notably, the three AT/RT subclasses were differentially correlated with supraand infratentorial localization as well as patient age. Moreover, the manner of SMARCB1 inactivation differed by subclass, with AT/RT-TYR tumors commonly featuring broad chromosome 22 loss, whereas AT/RT-SHH and AT/RT-MYC tumors more commonly harbored focal aberrations involving the SMARCB1 gene itself. Consistent with earlier reports, whole-genome sequencing in a subset of the larger AT/RT cohort failed to reveal additional genomic alterations. However, whole-genome bisulfite sequencing demonstrated clear distinctions in global DNA methylation patterns between the aforementioned disease subgroups. Specifically, AT/RT-TYR and AT/RT-SHH tumors exhibited genome-wide hypermethylation particularly in promoter regions with downstream effects on gene expression, whereas AT/RT-MYC tumors instead featured large “partially methylated domains” (PMDs) in association with normally inactive chromatin. Finally, the authors used H3K27acetylation and BRD4 ChIP-seq to identify enhancer regions genome wide and characterize significant differences between AT/RT subclasses. Interestingly, activated enhancers specific to each subclass demonstrated likely regulatory relationships with defining transcription factors, particularly in the case of AT/RT-TYR tumors where both OTX2 and MITF were implicated as master regulators controlling subclass-specific gene expression. Intriguingly, the authors also found that a known MITF inhibitor preferentially reduced viability in an AT/RT cell line with high MITF expression. In summary, these findings establish clinically distinct AT/RT subclasses whose unique biological characteristics point to viable strategies for therapeutic development.

If one had to pick a favorable subset of human diffuse gliomas, one would potentially go for the isocitrate dehydrogenase (IDH) 1 gene mutated, 1p/19q co-deleted “oligodendroglial” subtype. This recently got functionally supported by the discovery of membrane nanotubes being less frequent and shorter in this molecular subtype, leading to a less resistant phenotype. Relevant genes on 1p and 19q are capicua transcriptional repressor gene (CIC on 19q13.2) and far upstream element [FUSE] binding protein 1 (FUBP on 1p31.10) that are mutated in a variable subset of 1p/19 co-deleted gliomas. In a recent issue of Nature Communications, the French Prise en charge des oligodendrogiomes anaplasiques (POLA) network describes heterozygous somatic mutations of the oligodendrocyterelated transcription factor TCF12 on 15q21 in 7.5% of patients with anaplastic oligodendroglioma related to a more aggressive course of the disease. Germline mutations in TCF12 occur in patients with craniosynostosis. TCF12 is a transcription factor and the most common mutations in the basic helix-loop-helix (bHLH) DNA-binding domain abrogate the transcriptional activity of TCF12, interfering with pathways with a role in immune activation or known cancer pathways featuring the tumor suppressor TCF21, enhancer of zeste homolog (EZH)2, and polycomb complex protein (BMI)1. In addition, recent trials proposed combined chemoradiation with procarbazine, lomustine, and vincristine for patients with 1p/19q co-deleted tumors. In daily practice, however, it is surprising to see that patients with exactly this favorable molecular signature, who are treated with the exact guidelineconform chemoradiotherapy, do unexpectedly poorly. In the elegant and comprehensive work from the Sanson group, Gleize et al investigate the clinical and biological impact of CIC gene mutations in oligodendroglial tumors with 1p19q codeletion. Sequencing of the 28 amplicons covering the 20 exons of the CIC gene revealed 63 mutations affecting 60 of 127 patients (109 of 127 tissues had a 1p/19q co-deletion in addition to the oligodendroglial morphology; 70 were WHO grade II and 57 WHO grade III), with almost all 1p19q co-deleted and IDH mutated (59 of 60) tissues affected. Clinically, the patients with the mutation had a worse clinical outcome in the French series, which was confirmed in the TCGA dataset, and CIC mutations were an independent prognosticator of more rapid growth and a shorter interval for the occurrence of contrast enhancement of untreated grade II oligodendroglial tumors. For treated WHO grade II or III gliomas, WHO grade and CIC mutation were independent negative prognostic factors for overall survival. CIC mutations overcame the overall survival advantage conferred by the 1p/19q co-deletion in IDH-mutated gliomas. Molecularly, CIC mutant gliomas show a differential activation of proliferation-related genes and are less differentiated. The authors also demonstrate that CIC renders cells more sensitive to temozolomide, which in turn might explain the poorer course of CIC-mutated tumors with the current alkylator-based chemoradiation. They also propose new target genes of CIC, although direct molecular interaction or delineation of a pathway was not realized. The new targets are the mitogen-activated kinase inhibitors sprouty-related EVH1 domain-containing 1 (SPRED1), dual specificity protein phosphatase 4 (DUSP4), and the promigratory factor Src homology 2 domain-containing transforming protein 3 (SHC3). In addition to these potential new targets, the current work derives its relevance from the addition to the present development of the molecular classification of gliomas. We agree that our aim is to integrate morphology, molecular parameters and prognostic information into an informed diagnosis. The present data help to prevent oversimplification and to understand the mixed bag of 1p/19q co-deleted tumors.

Four-and-a-half LIM protein2 (FHL2) is a member of the LIM-only protein family, which plays a critical role in tumorigenesis. We previously reported that FHL2 is upregulated and plays an oncogenic role in glioblastoma (GBM), the most common and aggressive brain tumor. GBM is also marked by amplification of the epidermal growth factor receptor (EGFR) gene and its mutations, of which EGFRvIII is the most common and functionally significant. Here we report that FHL2 physically interacts with the wild-type EGFR and its mutated EGFRvIII form in GBM cells. Expression of FHL2 caused increased EGFR and EGFRvIII protein levels and this was due to an increase in protein stability rather than an increase in EGFR mRNA expression. In contrast, FHL2 knockdown using RNA interference reduced EGFR and EGFRvIII protein expression and the phosphorylation levels of EGFR and AKT. Consistent with these features, EGFR expression was significantly lower in mouse FHL2-null astrocytes, where reintroduction of FHL2 was able to restore EGFR levels. Using established GBM cell lines and patient-derived neurosphere lines, FHL2 silencing markedly induced cell apoptosis in EGFRvIII-positive cells. Targeting FHL2 significantly prevented EGFRvIII-positive GBM tumor growth in vivo. FHL2 expression also positively correlated with EGFR expression in GBM samples from patients. Taken together, our results demonstrate that FHL2 interacts with EGFR and EGFRvIII to increase their levels and this promotes glioma growth, representing a novel mechanism that may be therapeutically targetable.

Tumors with high glycolytic or hypoxic properties must produce sufficient levels of acetyl-CoA to maintain cellular survival and proliferation under adverse nutrient conditions. Acetyl-CoA can be generated by oxidation of glucose, glutamine, or fatty acids. There are principal differences between the metabolism of normal, well-fed cells and tumor cells, especially under nutrientdeprived conditions. Whereas the former consume glucose/ pyruvate/acetyl-CoA-derived citrate to synthesize acetyl-CoA, which is then used for fatty acid and cholesterol synthesis, generation of nucleotides and amino acids, and histone acetylation, the latter divert the main metabolite of glucose into aerobic glycolysis from pyruvate-derived mitochondrial acetylCoA to lactate. The “bioenergetic substrate gap” develops when under oxygen-limiting conditions the ability of a cell to make acetyl-CoA is impaired, and the question arises how sufficient amounts of citrate via ATP citrate lyase are produced. In the brain, glial cells are capable of oxidizing acetate. In an extremely elegant study, integrating observations from clinical experiments, ex vivo data, and preclinical mouse and cell culture studies, Mashimo and Pichumani et al from the Bachoo and Maher laboratories showed that primary and metastatic tumors growing in the brain have the capacity to oxidize acetate while simultaneously oxidizing glucose, providing an explanation for the yet unknown carbon source for the generation of the critical metabolite acetyl-CoA. Previously, C-nuclear magnetic resonance analysis showed that upon infusion of C-glucose during surgery for primary or metastatic brain tumors, ,50% of carbon in the acetyl-CoA pool was derived from glucose. The principal hypothesis by Mashimo and Pichumani et al is that glioma cells retain the acetatemetabolizing ability of the “healthy” glial cells. For metastases, it had been speculated that the microenvironment provokes a change in cells from non-acetate-metabolizing organs, like lung and breast, to allow them to metabolize acetate. In their work, relevant human orthotopic tumor mouse models of glioblastoma and brain metastases with remarkable preservation of the hallmark histopathological and molecular features (ER-/PR-negative/Her2-positive breast cancer, EGFR/ALK/KRAS wild-type non-small cell lung cancer, VHL-nil clear cell renal carcinoma, or BRAF melanoma) were used in C-NMR tissue examinations with very clear co-infusion experiments of C-glucose and C-acetate. Each of the metastatic cell lines oxidized glucose but showed a relevant bioenergetics carbon substrate gap. In co-infusion experiments with C-glucose and C-acetate, which produce distinct labeling patterns in the citric acid cycle intermediates, in six glioblastoma human orthotopic lines derived from patients prior to treatment and one line derived from the progression of one initial line a considerable, tumor-specific and variable consumption of acetate was demonstrated. The finding that the human tumors oxidize acetate was validated in the clinical setting by infusing C-acetate during glioblastoma and brain metastases operations. In contrast, glutamine is taken up by primary and metastatic brain tumors but not metabolized in the citric acid cycle in vivo. In a study published back-to-back with the Mashimo and Pichumani et al paper, Comerford and colleagues demonstrate that the nucleocytosolic acetyl-CoA synthetase enzyme (ACSS2) is expressed and has a critical role in hepatocellular carcinoma, gliomas, and breast and lung cancer for acetate utilization. Immunoreactivity to ACSS2 is correlated with glioma WHO grade (II-IV) and reduced survival in grade II/III gliomas. ACSS2 expression was also correlated with accumulation of mutation in diverse genetic mouse glioma models. Reintroduction of ACSS2 in ACSS2-knockout mouse embryonic fibroblasts enabled these cells to increase acetate incorporation into glutamate. The authors conclude that despite extensive molecular alterations in glioblastoma the ability to oxidize acetate, which is typical for glial cells, is at least maintained. The unexpected finding that metastases to the CNS can metabolize acetate may be discussed as an adaptation to the brain microenvironment or rather a more general property of tumor cells. ACSS2 upregulation is a prerequisite for the tumor cell to convert actetate to acetyl-CoA. Therefore, ACSS2 might be a vulnerability specific to the tumor metabolism. Further, the studies summarized provide nice examples of the relevance of in vivo models for research in cancer metabolism and also the strength of clinical cross-validation.

TPS2104 Background: Bevacizumab is the standard of care for patients with recurrent high-grade glioma (HGG). However, with current treatment options the median duration of response is only approximately 4 months. Potential mechanisms of resistance include upregulation of fibroblast growth factor (FGF) and increased pericyte coverage mediated by platelet-derived growth factor (PDGF). Nintedanibis an oral, small-molecule tyrosine kinase inhibitor of PDGFR α/β, FGFR 1/3, and vascular endothelial growth factor receptor (VEGFR) 1-3 that may overcome the problem of resistance to prior anti-VEGF therapy. Methods: This is an open-label, phase II trial in adults with first or second recurrence of HGG, stratified by prior therapy with bevacizumab. The primary endpoint is 6-month progression-free survival (PFS6) in the bevacizumab-naive arm and PFS3 in the post-bevacizumab arm. A Simon two-stage design is employed. Although the glioblastoma (GBM) comparison is the one of primary concern, 10 anaplastic glioma (AG) participants will be accrued to each arm in exploratory cohorts. Results: Nine of 40 GBM patients and 10 of 10 AG patients have been accrued in the bevacizumab-naive arm. Data in this arm are maturing. In the post-bevacizumab arm, 14 patients have been accrued, 10 of whom had GBM (71%). There were 11 men (79%), median age was 52 years (range 32-70), and median KPS was 90 (range, 60-100). One patient with anaplastic astrocytoma was not evaluable for response analysis because of early withdrawal of consent. There have been no responses. Two patients (1 with GBM and 1 with anaplastic oligodendroglioma) achieved stable disease. Median PFS was 28 days (95% CI: 27-28), and maximum PFS was 56 days. Median OS was 57 days (95% CI: 29-155). The post-bevacizumab arm was stopped after stage 1. Treatment was well tolerated, with limited Grade 3 liver function test abnormalities (n = 3), abdominal pain (n = 1), and hypertension (n = 1). Grade 1-2 adverse events also included diarrhea, nausea/vomiting, fatigue, and bleeding. Conclusions: Despite limited toxicity, nintedanibis ineffective in the cohort of recurrent HGG patients who failed bevacizumab. Updated results in the bevacizumab-naive arm will be presented. Clinical trial information: NCT01380782.