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Combinations of the MEK inhibitor trametinib, and BRAF inhibitor dabrafenib (BRAFi+MEKi) show rapid and sustained responses in patients with BRAF V600E-mutated low-grade glioma, but tumor rebound after treatment discontinuation is frequent. Moreover, a lack of response is common in patient with high-grade glioma raising the need for further research into BRAFi+MEKi effects on tumors. We showed previously that BRAF V600E-mutated glioma cells positive for CD133 (Prominin-1), a marker of brain tumor stem cells, show decreased sensitivity to BRAFi, indicative of their role in promoting therapy resistance. BRAF V600E-mutated murine and patient-derived glioma cell lines (STN-10049, SU-aGBM5) were generated and together with established BRAF V600E-mutated cell lines (DBTRG, AM38) were analyzed for changes in gene expression in response to 48 hrs treatment with BRAFi dabrafenib and MEKi trametinib. Cells were analyzed by RNA-seq and gene enrichment analyses while cell culture supernatant was analyzed for cytokine production using an ELISA. Syngeneic, orthotopic BRAF V600E-mutated tumor allograft-bearing mice were treated with BRAFi+MEKi, with therapeutic antibodies against immune checkpoint molecules (anti-PD-L1 and anti-CTLA-4) and with combination of all four agents, and tumors were analyzed by mass cytometry and immunofluorescence for stem and T cell markers. BRAFi+MEKi treatment induced an interferon gamma (IFNg) response gene signature in BRAF V600E-mutated glioma cells and increased HLA gene expression. The frequency of tumor-infiltrating CD4+ CD8+ T cells in syngeneic BRAF V600E-mutated tumor allografts increased with BRAFi+MEKi treatment. Combining BRAFi+MEKi with anti-PD-L1 and anti-CTLA-4 treatment decreased CD133+ cells more effectively than either therapy alone, and resulted in a T cell-dependent survival benefit of mice with orthotopic BRAF V600E-mutated high-grade glioma. Combination of BRAFi+MEKi with immune checkpoint inhibition should be further explored as a viable option to prevent tumor rebound and therapy resistance in patients with BRAF V600E-mutated glioma.

Novel treatment approaches for pediatric central nervous system (CNS) tumors are urgently needed. A lack of patient-derived tumor cells impedes progress towards developing such new therapies. We intended to overcome this challenge by establishing methods to create a biorepository of viable single cell suspensions of pediatric brain tumor surgical specimens. Quantitative and qualitative comparisons of tissue processing strategies were performed to preserve viability of heterogeneous tumor and immune cells. Novel drug targets were identified by analyzing pathways affected by RNA transcripts that are highly expressed (outliers) in a patients’ tumor; outliers for each patient were determined by RNA-seq data from individual patients’ tumors compared with a compendium of 12,747 pediatric and adult samples harmonized by the Treehouse Childhood Cancer Initiative at the UCSC Genomics Institute. The predicted anti-tumor efficacy of small molecule inhibitors of the outlier pathways was tested in cell viability assays against short-term cultured cells from matched patients. Successful tissue collection required obtaining informed consent, standard operating procedures, and sample recording using a Laboratory Inventory Management Software (LIMS). Since 2020 we have banked 67 pediatric CNS brain tumor specimens at Stanford. Amongst those, 51 cases yielded sufficient tissue for RNA-seq and cryoprotection. The most common tumor histology was low-grade glioma (LGG, 26 of 67), the majority of which were pilocytic astrocytoma (18 of 26). The second and third most common tumor types are embryonal tumors (6 medulloblastoma, 3 AT/RT) and ependymoma (4), respectively. We identified significant differences in cell viability with different preservation media. An outlier pathway previously not implicated in LGG was identified and sensitivity to a small molecule inhibitor of this outlier pathway was demonstrated. Taken together, we established feasibility for validating therapeutic vulnerabilities identified by a genome-guided approach in short-term cultures from surgical specimens. This works facilitates the rapid development of personalized CNS tumor treatment.

Tumor associated microglia and macrophages (TAMs) represent a main cell type of brain malignancies and demonstrate complex interactions with cancerous cells and the tumor microenvironment. These interactions have important implications for the progression and treatment of malignant central nervous system (CNS) tumors. In a non-oncological context, it has been shown that endogenous microglia can be replaced by peripheral hematopoietic cells by genetic or pharmacological intervention. A highly efficient replacement protocol (Shibuya et al, Sci Transl Med 2022, PMID:35294256) utilizing myeloablative bone marrow transplantation (BMT) followed by microglial depletion via pharmacological inhibition of the Colony-stimulating factor-1 receptor (CSF-1R) leads to almost complete repopulation of the CNS myeloid niche by circulation-derived myeloid cells (CDMCs). We investigated if this approach could be utilized to integrate CDMCs into brain tumors and study associated changes in the TAM signature. Microglia replacement in immune-competent mice was followed by intracranial transplantation of syngeneic cell lines of either high-grade glioma (GL261) or breast cancer metastasis (E0771). Lineage tracing proved high intra- and peri-tumoral chimerism of BMT-graft derived cells among CNS myeloid cells in both models two weeks after tumor cell injection. Tumor associated CDMCs (TA-CDMCs) showed a similarly activated morphology as naïve TAMs. We used single cell RNA sequencing of CD45-positive cells to further profile the transcriptomic identity of glioma TA-CDMCs. Pathway enrichment analysis revealed an upregulation of immune response pathways in TA-CDMCs compared to regular TAMs, including the overexpression of genes relevant for the regulation of T cell activation, suggesting possible modification of the glioma immune-environment. Strategies to manipulate the myeloid niche have the potential to provide further understanding of the pathobiology of TAMs and to build the basis for novel cell therapeutic approaches in the neuro-oncological domain.

BACKGROUND Brain metastases from the gastrointestinal tract are a rare event (less than 10% of all brain lesions), but their impact on prognosis is remarkable. Their incidence is increasing due to earlier diagnosis and prolonged survival because of more effective treatment modalities. Because of the lack of evidence-based recommendations, no optimal treatment strategy has been defined. Hope might come from the discovery of new prognostic and predictive factors and their biological inhibitors. The aim of this study was to create a repository of matched primary cancer and brain metastases samples for clinicopathologic and molecular correlations of gastrointestinal tumors and to analyze changes in expression signatures between both samples. The main goal was to study the metastatic process and characterize the immune response by characterizing features such as invasiveness, vascular co-option, proliferation, stemness and cell-type heterogeneity between the matched tissues. METHODS We searched for patients at Stanford hospital with matched brain metastases from gastrointestinal tumors from 2008 to 2020 using the STARR database (The STAnford Research Repository, or STARR, is Stanford Medicine's approved resource for working with clinical data for research purposes.). Matched specimens mean that both brain metastases and gastrointestinal tumors were studied at Stanford University. FFPE blocks from these matched patients were sectioned and sections analyzed for gene expression analysis using nanoString nCounter technology and the immune-oncology panel. RESULTS Expression data from 32 matched patients with primary tumor and brain metastases will be discussed at the meeting. CONCLUSION We believe that analysis of changes in expression signatures between the matched primary gastrointestinal cancer and brain metastases will provide novel insights into the role for tumor factors, such as invasiveness, proliferation and stemness, and the tumor microenvironment in progression.

BACKGROUND Despite successes, clinical MAPK pathway inhibitors show limited anti-tumor activity in the majority of patients with BRAF-mutant high-grade glioma. Because of the presence of higher fraction of CD8+ tumor-infiltrating T cells in MAPK pathway-altered glioma, we explored the possibility that combined BRAF and MEK inhibition with immune checkpoint blockade enhances anti-tumor response. METHODS We engineered mice to carry BRAF V600E expression and CDKN2A deletion in various hemispheric areas. We treated syngeneic tumor-bearing mice with dabrafenib, trametinib, anti-PD-L1 and anti-CTLA-4 antibodies, and analyzed the tumor immune infiltrate by high-dimensional single-cell mass cytometry (CyTOF). RNA sequencing and Gene Set Enrichment Analysis were conducted using patient-derived BRAF-mutant glioma lines upon the inhibitor treatment. RESULTS The transcriptome analysis demonstrated that antigen processing and presentation feature is strongly enriched upon dual MAPK pathway inhibition. Consistent with these molecular changes, dabrafenib and trametinib treatment led to dynamic changes in tumor-infiltrating immune cells, including CD8+ and CD4+ T cells. In line with this, combination of MAPK pathway and immune checkpoint inhibitors elicit a significant survival benefit over MAPK pathway inhibition alone in mice with orthotopic BRAF-mutant glioma. CONCLUSIONS Clinically relevant molecular targeted therapy by dabrafenib and trametinib and immune checkpoint blockade synergize in pre-clinical models.

Pediatric low-grade glioma (pLGG), the most common brain cancer in children, is difficult to treat especially at recurrence. The BRAF V600E mutation is the second most common mutation in pLGG, and in a high-risk group for progression is associated with deletion of the tumor suppressor CDKN2A. A better understanding of the factors contributing to progression, in particular the role of the immune infiltrate is needed, but studies have been hindered by the lack of low-grade glioma mouse models. We utilized transgenic mice with a cre-activatable (CA) allele of BRAF V600E to generate endogenous models for low-grade gliomas. We found that BRAF V600E expression cooperates with hemizygous CDKN2A deletion to induce low-grade gliomas, with tumors forming at a greater latency than by homozygous deletion. Cell line derivatives from low-grade lesions continue to grow slower upon orthotopic injection than those we previously derived from high-grade tumors. Murine LGG can progress to higher grade tumors within the mouse lifespan and we observe exomic changes and alterations in the tumor immune infiltrate associated with progression, the details of which will be discussed at the meeting. High-grade cells’ phenotypic changes within in vivo passage are accompanied by exomic changes. The high-grade glioma immune infiltrate is altered by dual MAPK pathway inhibition with dabrafenib and trametinib. Adding dual immune checkpoint inhibition by anti-PD-L1 and anti-CTLA-4 antibodies significantly extends survival of dabrafenib-trametinib dual treatment. Human BRAF V600E mutant tumors reportedly have a higher tumor immune infiltrate than that of BRAF wildtype gliomas, consistent with our murine RESULTS: Here we present a novel model for BRAF V600E mutant gliomas in mice that has a frequent rate of progression, similar to human BRAF V600E mutant gliomas, and an active immune infiltrate in high grade tumors which makes them susceptible to the immunostimulatory effects of dual checkpoint inhibition.

Mice lacking the epidermal growth factor receptor (EGFR) develop an early postnatal degeneration of the frontal cortex and olfactory bulbs and show increased cortical astrocyte apoptosis. The poor health and early lethality of EGFR-/- mice prevented the analysis of mechanisms responsible for the neurodegeneration and function of the EGFR in the adult brain. Here, we show that postnatal EGFR-deficient neural stem cells are impaired in their self-renewal potential and lack clonal expansion capacity in vitro. Mice lacking the EGFR in the brain (EGFRΔbrain ) show low penetrance of cortical degeneration compared to EGFR-/- mice despite genetic recombination of the conditional allele. Adult EGFRΔ mice establish a proper blood-brain barrier and perform reactive astrogliosis in response to mechanical and infectious brain injury, but are more sensitive to Kainic acid-induced epileptic seizures. EGFR-deficient cortical astrocytes, but not midbrain astrocytes, have reduced expression of glutamate transporters Glt1 and Glast, and show reduced glutamate uptake in vitro, illustrating an excitotoxic mechanism to explain the hypersensitivity to Kainic acid and region-specific neurodegeneration observed in EGFR-deficient brains.

Oligodendrocyte progenitor cells (OPC) undergo asymmetric cell division (ACD) to generate one OPC and one differentiating oligodendrocyte (OL) progeny. Loss of pro-mitotic proteoglycan and OPC marker NG2 in the OL progeny is the earliest immunophenotypic change of unknown mechanism that indicates differentiation commitment. Here, we report that expression of the mouse homolog of Drosophila tumor suppressor Lethal giant larvae 1 (Lgl1) is induced during OL differentiation. Lgl1 conditional knockout OPC progeny retain NG2 and show reduced OL differentiation, while undergoing more symmetric self-renewing divisions at the expense of asymmetric divisions. Moreover, Lgl1 and hemizygous Ink4a/Arf knockouts in OPC synergistically induce gliomagenesis. Time lapse and total internal reflection microscopy reveals a critical role for Lgl1 in NG2 endocytic routing and links aberrant NG2 recycling to failed differentiation. These data establish Lgl1 as a suppressor of gliomagenesis and positive regulator of asymmetric division and differentiation in the healthy and demyelinated murine brain., Oligodendrocyte progenitor cells (OPCs) undergo asymmetric cell division, and disruption of such mechanism can generate oligodendroglioma precursors. Here, Daynac and colleagues show that Lgl1 regulates asymmetric division and differentiation of OPCs by interfering with the endocytosis pathway, and that Lgl1 knockout can lead to gliomagenesis.

Background BRAF alterations frequently occur in pediatric low-grade gliomas. Previously, we showed that dabrafenib and trametinib (D+T) that target MAPK pathway can mediate the antitumor effect in a preclinical model of BRAF-mutant glioma (PMC5342782). Here, we further investigate the effect of MAPK pathway inhibitors on cancer cells and tumor-infiltrating immune cells to maximize the therapeutic efficacy in malignant gliomas. Methods Drug concentrations in tumor, brain and plasma were assessed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). RNA sequencing and Gene Set Enrichment Analysis were performed using patient-derived BRAF-mutant glioma lines upon D+T treatment. Molecular profiles of drug-resistant clones were assessed for understanding of glioma heterogeneity and exploring new therapeutic targets. Results. BRAF-mutant stem-like glioma cells were particularly resistant to BRAF or MAPK inhibitor, along with aggressive phenotype in mice. LC-MS/MS showed effective D+T drug delivery in tumor regions. The transcriptome analysis demonstrated that D+T upregulate HLA molecules and downregulate immunosuppressive factors in patient-derived BRAF-mutant glioma lines. Consistent with these molecular changes, D+T led to changes in the proportions of tumor-infiltrating immune cells, including CD8+ cytotoxic T lymphocytes and FOXP3+ regulatory T cells. Furthermore, the therapeutic effect of D+T was further enhanced in combination with immune checkpoint inhibition. Conclusions The present study highlights the immunomodulatory activity of MAPK pathway inhibitors in BRAF-mutant gliomas.

Pediatric low-grade gliomas (pLGG) are the most common central nervous system (CNS) tumor in children. Although the majority of pLGG has unregulated MAPK pathway activity, and clinical MAPK pathway inhibitors are available, pLGG treatment remains challenging. Tumor progression, rebound, and therapy resistance limit the success of MAPK pathway inhibitors, especially in high risk subgroups, such as BRAF V600E mutant CDKN2A deleted gliomas.To define mechanisms for progression and overcome resistance, we generated BRAF V600E mutant, CDKN2A deleted murine glioma models of different grades and drug sensitivities. Here, we use genetically engineered mice to target expression of BRAF V600E and deletion of CDKN2A to various germinal and non-germinal areas, assessing neuro-anatomical aspects of oncogene sensitivity. Using neural stem cell (NSC)- and astrocyte-, and oligodendrocyte progenitor cell (OPC)-selective driver line, we investigate the likely origin for pLGG and therapy resistant glioma stem cells, developing models that faithfully recapitulate human tumor development. Our numerous immunocompetent, orthotopic BRAF V600E mutant glioma model derived from endogenous tumors exhibit a range of sensitivities to MAPK inhibitor therapies, allowing us to define mechanisms for therapy resistance. In syngeneic, orthotopic BRAF V600E mutant CDKN2A deleted gliomas, we define MAPK pathway inhibitor-induced-changes in the tumor microenvironment, including the immune infiltrate, and investigate immune modulatory therapy as novel treatment strategy for children with BRAF mutant gliomas. Our numerous models for BRAF mutant glioma will facilitate research on tumor development, progression and the tumor-immune interactions. Collectively, our results describe genetic alteration- and microenvironment-based mechanisms for BRAF mutant tumor initiation and progression, and suggest therapeutic combination therapies for glioma. Citation Format: Claudia K. Petritsch, Stefan Grossauer, Wei Wang, Anne Marie Barrette, Jongwhi Park. Clinically relevant MAPK pathway inhibition reverses stem cell fate defects and sensitize BRAF mutant glioma to immune modulatory therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3072.

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 (

Expression of the constitutively active BRAF(V600E)-mutant kinase occurs in pediatric and predominantly young adult high-grade glioma patients (Cancer Res. 2010, 70:512; Acta Neuropathol. 2011, 121:397). BRAF(V600E) expression frequently coincides with CDKN2A deletion and marks a subgroup of predominantly hemispheric tumors with epigenetic similarities to pleomorphic xanthastrocytoma (PXA) and histologic features of grade IV astrocytoma (Cancer Cell 2018, 33:829). The origin of BRAF(V600E) mutant astrocytoma is under investigation. Here, we are using transgenic mice to assess neuroanatomical aspects and effects on differentiation of BRAF(V600E) expression. In previous studies, expression of BRAF(V600E) in astroglial cells and neural stem cells of the developing brain led to hyperplasia in transgenic mice. To circumvent the early postnatal lethality associated with BRAF(V600E) expression during development, my lab injected adenovirus expressing Cre recombinase into the corpus callosum of BRAFCA Ink4a/Arf floxed young adult mice. In these mice, Cre expression induces BRAF(V600E) expression and deletion of Ink4a/Arf, the mouse locus homologous to human CDKN2A. This approach showed that BRAF(V600E) expression and CDKN2A (Ink4a/Arf) deficient corpus callosum cells form high-grade astrocytoma-like tumors in young adult mice (Proc Natl Acad Sci. 2012, 109:8710). To further investigate the neuroanatomical aspects of tumor formation we directed cre expression to distinct neuroanatomical areas, including the cortex, of the young adult mouse brain. Histo-pathologic and survival analyses showed that each injected area was susceptible to tumor formation albeit with different latency. Induction of BRAF(V600E) expression in Ink4a/Arf-deleted neurospheres in vitro decreased the capacity for neuronal and glial differentiation and led to expansion of oligodendrocyte progenitor-like cells (OPC). Ongoing studies investigate directly the extent to which BRAF(V600E) expression in Ink4a/Arf-deleted OPC in transgenic mice cause tumor formation. Results from cell of origin studies are expected to inform which mouse model most faithfully recapitulates the human diseases.

Background The BRAF V600E mutation occurs in ~ twenty percent of histologically diverse pediatric gliomas and is the second most common mutation in pediatric low-grade gliomas (LGG). BRAF V600E expression in LGG with balanced CDKN2A is associated with a higher rate for progression than for BRAF V600E wildtype tumors, and despite adjuvant therapy, consisting of resection, radiation and chemotherapy. Progression invariably occurs in BRAF V600E mutant CDKN2A deleted gliomas, marking a high-risk group. Here, we aim to overcome the lack BRAF V600E mutant glioma models that allow for studies of stem and progenitor cells and the immune system ability to understand progression. Methods We develop novel immunocompetent, stem and progenitor cell-based mouse models for BRAF mutant gliomas, including genetically engineered mouse models (GEMMs), orthotopic glioma models derived from gliomas in GEMMs as well as in vitro models of those tumors. BRAF mutant mouse brains and cells were analyzed by immunofluorescence staining, flow cytometry, mass cytometry and RNA sequencing. Results Ongoing model development studies indicate that BRAF V600E mutant gliomas in murine brain exhibit very similar neuroanatomical preferences to human gliomas. The BRAF V600E mutation exacerbates the heterogenous cell cycling pattern of normal neural stem and progenitors and expands a symmetrically dividing progenitor population. Cellular plasticity rather than cellular lineage hierarchy drives the generation of a therapy resistant stem cell pool. Transcriptomic analyses of neuroglial stem cells with induced BRAF V600E expression provide insights into mechanisms for neoplastic transformation and progression. Conclusion Analyses of two independent BRAF V600E mutant mouse models provide novel insights into the role for tumor intrinsic factors, such as plasticity and stemness, and the tumor microenvironment in progression.

The determinants of the tumor-associated immune response in brain tumors are poorly understood. Using tumor samples from two molecularly distinct subtypes of lower grade glioma, MAPK-driven glioma with biallelic inactivation of CDKN2A (n=30) and IDH-mutant, 1p/19q-intact astrocytoma (n=29), we demonstrate qualitative and quantitative differences in the tumor-associated immune response and we investigate the molecular mechanisms involved. Histologically the MAPK-driven gliomas were comprised of pleomorphic xanthoastrocytoma (PXA) (n=11) and anaplastic PXA (n=19). Seven patients had paired samples from two sequential surgeries. Immune cell populations and their activity were determined by quantitative multiplex immunostaining and Digital Spatial Profiling and gene expression was analyzed by Nanostring. Functional studies were performed using established cell lines and two new patient-derived lines from MAPK-driven LGGs. MAPK-driven tumors exhibited an increased number of CD8+ T cells and tumor-associated microglial/macrophage (TAMs), including CD163+ TAMs, as compared to IDH-mutant astrocytoma. In contrast, IDH-mutant tumors had increased FOXP3+ immunosuppressive T regulatory cells. Transcriptional and protein level analyses in MAPK-driven tumors suggested an active cytotoxic T cell response with robust expression of granzyme B, present on 27% of CD8+ T cells, increased MHC class I expression, and altered cytokine profiles. Interestingly, MAPK-driven tumors also had increased expression of immunosuppressive molecules, including CXCR4, PD-L1, and VEGFA. Expression differences for cell surface and secreted proteins were confirmed in patient-derived tumor lines and functional relationships between altered chemokine expression and immune cell infiltration was investigated. Our data provide novel insights into the immune contexture of MAPK driven LGGs and suggest MAPK driven gliomas with biallelic inactivation of CDKN2A may be particularly vulnerable to immunotherapeutic modulation

Introduction: Diabetes increases the severity of stroke outcome and impairs the capacity for neuroplasticity. Evidence suggests that neurogenesis, a mechanism of neuroplasticity, is induced after experimental stroke, although how it is confounded by type 2 diabetes mellitus (T2DM) or aging remains to be clarified. Hypothesis: We hypothesize that T2DM exacerbates age-associated reduction in neurogenesis. Methods: Experimental stroke was induced by distal middle cerebral artery occlusion (dMCAO) in young (11-13 weeks-old, n=12) and middle-aged (53-75 weeks-old, n=50) db/+ or db/db mice, as well as in db/+ mice of old age (99-124 weeks-old, n=30). Thymidine analog 5-bromo-2′-deoxyuridine-5′(BrdU) was administered i.p. at 50 mg/kg twice daily from days 4-7 after dMCAO to label proliferating cells. Mice were euthanized 28 days after dMCAO and their brain tissue was processed for immunohistochemistry for BrdU and markers of neuronal or glial lineages. Results: A significant reduction in the number of DG neuroblasts was found in the db/db compared to db/+ mice in both young and middle-aged groups at baseline and after dMCAO. There was a further decline of neurogenesis in the old db/+ mice compared to the middle-aged db/+ mice by 6.2 and 3 folds at baseline and after dMCAO, respectively. dMCAO induces an increase of DCX(+) neuroblasts in all age groups of db/+ mice but not in db/db mice, suggesting that T2DM elicited an early decline of neurogenesis, leading to a failed regeneration after stroke. Large DCX(+) cells without processes were found in the DG of old db/+ and middle-aged db/db mice. Neuroblasts with aberrant morphology do not express markers of astrocytes or oligodendrocytes, although their identity and nature remains unclear. Conclusions: Our data suggest that T2DM accelerates the decline of neurogenesis induced by aging, which may potentially affect the functional outcome of regeneration and post stroke neuroplasticity impacted by this comorbidity. Ongoing investigation will further characterize the functional change of the neuroblasts with altered morphology and determine the effect on differentiation and survival of newborn neurons in the DG of db/db mice.

Pleomorphic xantoastrocytoma (PXA) is a typically well circumscribed astrocytic neoplasm that can progress to anaplastic PXA with an aggressive biologic behavior and worse prognosis. Histological features of anaplastic progression include increased proliferation, necrosis, microvascular proliferation, loss of pericellular reticulin, and increased infiltrative growth. Genetically anaplastic PXA is characterized by the combination of CDKN2A biallelic inactivation and oncogenic RAF kinase signaling, as observed in PXA, as well as a relatively small number of additional genetic alterations, with the most common being TERT amplification or promoter mutation (Phillips et al 2018). Common to both PXA and anaplastic PXA is the perivascular accumulation of lymphocytes. Given the potential importance of the immune response in brain tumors we asked whether the immune response in anaplastic PXA was different than that in PXA and in IDH-mutant diffuse glioma. To do this we used quantitative multiplex immunofluorescence of scanned slides and quantified the populations and phenotypes of T cells and microglia/macrophages in tumors identified from the UCSF Brain Tumor Center Biorepository and from the archives of the UCSF Neuropathology Division. All tumors were molecularly characterized by immunostaining and/or comprehensive genomic profiling. A total of 30 PXA, including 6 paired tumors before and after tumor progression, and 30 diffuse glioma were examined. Immunostaining for T cell subsets included CD3, CD8, and FOXP3 and for microglial/macrophages included Iba1, CD204, and CD163. Using quantitative multiplex immunostaining we assess immune cell subsets and their localization within the tissue highlighting the perivascular CD3+ lymphocytes. Our data suggest differences in the quantity and quality of immune cells in anaplastic PXA.

Intratumor heterogeneity (ITH) drives neoplastic progression and therapeutic resistance. We used the bioinformatics tools 'expanding ploidy and allele frequency on nested subpopulations' (EXPANDS) and PyClone to detect clones that are present at a ≥10% frequency in 1,165 exome sequences from tumors in The Cancer Genome Atlas. 86% of tumors across 12 cancer types had at least two clones. ITH in the morphology of nuclei was associated with genetic ITH (Spearman's correlation coefficient, ρ = 0.24-0.41; P0.001). Mutation of a driver gene that typically appears in smaller clones was a survival risk factor (hazard ratio (HR) = 2.15, 95% confidence interval (CI): 1.71-2.69). The risk of mortality also increased when2 clones coexisted in the same tumor sample (HR = 1.49, 95% CI: 1.20-1.87). In two independent data sets, copy-number alterations affecting either25% or75% of a tumor's genome predicted reduced risk (HR = 0.15, 95% CI: 0.08-0.29). Mortality risk also declined when4 clones coexisted in the sample, suggesting a trade-off between the costs and benefits of genomic instability. ITH and genomic instability thus have the potential to be useful measures that can universally be applied to all cancers.

This abstract is being presented as a short talk in the scientific program. A full abstract is printed in the Proffered Abstracts section (PR03) of the Conference Proceedings. Citation Format: Noemi Andor, Trevor A. Graham, Claudia Petritsch, Hanlee P. Ji*, Carlo C. Maley*. Pan-cancer analysis of the etiology and consequences of intratumor heterogeneity. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-54.

Mammalian oligodendrocyte progenitor cells (OPC) divide asymmetrically by dispatching pro-mitotic NG2, EGFR and PDGFRalpha, from here on called the NG2 complex, unequally between daughter cells to establish a cell fate bias and to restrict proliferation. Studies in genetically engineered mouse models have shown that OPC are a putative cellular origin of glioma and have furthermore suggested that asymmetric divisions are tumor suppressive and can be exploited therapeutically. The objective of this project is to test directly if disruption of asymmetric divisions causes premalignant phenotypes and even neoplastic transformation. To reach this objective, we study the hypothesisthat a mammalian gene called Lethal giant Larvae, Lgl1 regulates asymmetric OPC divisions. Lgl1 is frequently inactivated and its expression is lost in glioma. We propose that genetically enforced deletion of Lgl1 will disrupt asymmetric divisions and lead to premalignant changes and perhaps neoplastic transformation. We use a novel FACS based approach to determine if Lethal giant larvae1 (Lgl1) is expressed in OPC. We genetically enforce deletion of Lgl1 in OPC either conventionally with NG2-Cre or conditionally by injecting tamoxifen into triple transgenic mice obtained by breeding Lgl1fl/fl NG2CreERT2 and fluorescence reporter mice. Cre-conditional deleted Lgl1 OPC were analyzed in situ for changes in cell division mode, proliferation rate and differentiation. Whole genome expression analyses were conducted on ex vivo isolated Lgl1 knockout OPC. Lgl1 deletion in embryos leads to tumor formation albeit at low frequency and with long latency. Cre-conditional Lgl1 knockout in adult OPC increases symmetric division and proliferative rate at the expense of asymmetric divisions and differentiation. Whole transcriptome analyses reveal that Lgl1 regulates receptor-mediated endocytosis. Lgl1 loss fails to route NG2 complex to the lysosome for degradation, thereby rapidly increasing NG2 complex levels at the cell membrane and leading to symmetric OPC divisions and immediate premalignant changes such as hyperproliferation.

OBJECTIVE: Within a multi-center feasibility trial, we evaluated whether sequencing of tumor and germline exomes and tumor RNA can be used to develop a personalized treatment plan for children with diffuse intrinsic pontine glioma (DIPG). METHODS: Subjects with newly diagnosed DIPG < 25 years of age were eligible. Xenograft development and cell culture expansion were attempted for each subject. Circulating tumor DNA (ctDNA) was collected at initial diagnosis as well as with each MRI. Genetic alterations were matched to potential therapeutic options using a custom drug-matching pipeline utilizing a pharmacopeia of FDA-approved drugs. A specialized tumor board generated a treatment recommendation with up to four FDA-approved agents. Families retained the option to adhere to the treatment recommendations. Patients were followed for toxicity and clinical outcome. Whole genome sequence analysis of tumor tissue is ongoing. RESULTS: The feasibility portion of this study (n=15) was completed. Whole exome and RNAseq was performed with development of a treatment plan within 21 business days from tissue collection for 14/15 subjects. Average mapped coverage was >249X reads across all exomes and >120M reads for each tumor RNA. Alterations in genes previously implicated in pediatric gliomas were identified, including mutations in histone H3 (H3F3A, HIST1H3B), PDGFRA, TP53, KIT, KDR, MET, ATRX and ACVR1 amongst others. Of the first 15 patients, 9 patients chose to follow the therapy recommendation. Whole genome analysis is ongoing. No unexpected toxicities were identified to date. Five new DIPG cell lines were established and xenograft development is ongoing. CtDNA analysis confirmed the presence of the H3.3K27M mutation and we are currently correlating ctDNA results with imaging data. CONCLUSION: DIPG biopsy is safe and feasible, and allows for construction of a comprehensive tumor profile which is further reflected in patients’ biofluids. A personalized multi-agent therapy plan did not result in unexpected toxicities.

Stem and progenitor cells are characterized by their abilities to self-renew and produce differentiated progeny. The balance between self-renewal and differentiation is achieved through control of cell division mode, which can be either asymmetric or symmetric. Failure to properly control cell division mode may result in premature depletion of the stem/progenitor cell pool or abnormal growth and impaired differentiation. In many tissues, including the brain, stem cells and progenitor cells undergo asymmetric cell division through the establishment of cell polarity. Cell polarity proteins are therefore potentially critical regulators of asymmetric cell division. Decrease or loss of asymmetric cell division can be associated with reduced differentiation common during aging or impaired remyelination as seen in demyelinating diseases. Progenitor-like glioma precursor cells show decreased asymmetric cell division rates and increased symmetric divisions, which suggests that asymmetric cell division suppresses brain tumor formation. Cancer stem cells, on the other hand, still undergo low rates of asymmetric cell division, which may provide them with a survival advantage during therapy. These findings led to the hypotheses that asymmetric cell divisions are not always tumor suppressive but can also be utilized to maintain a cancer stem cell population. Proper control of cell division mode is therefore not only deemed necessary to generate cellular diversity during development and to maintain adult tissue homeostasis but may also prevent disease and determine disease progression. Since brain cancer is most common in the adult and aging population, we review here the current knowledge on molecular mechanisms that regulate asymmetric cell divisions in the neural and oligodendroglial lineage during development and in the adult brain.

Glioma is a heterogeneous disease process with differential histology and treatment response. It was previously thought that the histological features of glial tumors indicated their cell of origin. However, the discovery of continuous neuro-gliogenesis in the normal adult brain and the identification of brain tumor stem cells within glioma have led to the hypothesis that these brain tumors originate from multipotent neural stem or progenitor cells, which primarily divide asymmetrically during the postnatal period. Asymmetric cell division allows these cell types to concurrently self-renew whilst also producing cells for the differentiation pathway. It has recently been shown that increased symmetrical cell division, favoring the self-renewal pathway, leads to oligodendroglioma formation from oligodendrocyte progenitor cells. In contrast, there is some evidence that asymmetric cell division maintenance in tumor stem-like cells within astrocytoma may lead to acquisition of treatment resistance. Therefore cell division mode in normal brain stem and progenitor cells may play a role in setting tumorigenic potential and the type of tumor formed. Moreover, heterogeneous tumor cell populations and their respective cell division mode may confer differential sensitivity to therapy. This review aims to shed light on the controllers of cell division mode which may be therapeutically targeted to prevent glioma formation and improve treatment response.

Stem and progenitor cells are characterized by their ability to self-renew and produce differentiated progeny. A fine balance between these processes is achieved through controlled asymmetric divisions and is necessary to generate cellular diversity during development and to maintain adult tissue homeostasis. Disruption of this balance may result in premature depletion of the stem/progenitor cell pool, or abnormal growth. In many tissues, including the brain, dysregulated asymmetric divisions are associated with cancer. Whether there is a causal relationship between asymmetric cell division defects and cancer initiation is as yet not known. Here, we review the cellular and molecular mechanisms that regulate asymmetric cell divisions in the neural lineage and discuss the potential connections between this regulatory machinery and cancer.

Glioblastoma, a malignant brain cancer, is characterized by abnormal activation of receptor tyrosine kinase signalling pathways and a poor prognosis. Extracellular proteoglycans, including heparan sulfate and chondroitin sulfate, play critical roles in the regulation of cell signalling and migration via interactions with extracellular ligands, growth factor receptors and extracellular matrix components, as well as intracellular enzymes and structural proteins. In cancer, proteoglycans help drive multiple oncogenic pathways in tumour cells and promote critical tumour-microenvironment interactions. In the present review, we summarize the evidence for proteoglycan function in gliomagenesis and examine the expression of proteoglycans and their modifying enzymes in human glioblastoma using data obtained from The Cancer Genome Atlas (http://cancergenome.nih.gov/). Furthermore, we demonstrate an association between specific proteoglycan alterations and changes in receptor tyrosine kinases. Based on these data, we propose a model in which proteoglycans and their modifying enzymes promote receptor tyrosine kinase signalling and progression in glioblastoma, and we suggest that cancer-associated proteoglycans are promising biomarkers for disease and therapeutic targets.

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, BRAFV600E and mTOR inhibitors in gliomas carrying specific genetic alterations of the MAPK pathway. Experimental Design: We used human glioma lines containing BRAFV600E (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 BRAFV600E-expressing murine brain cells. Signaling inhibitors included everolimus (mTOR), PLX4720 (BRAFV600E), 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 BRAFV600E cells, the three possible doublet combinations of AZD6244, everolimus, and PLX4720 exhibited significantly greater effects on cell viability. In BRAFWT 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 BRAFV600E pediatric xenograft model BT40 showed the greatest survival advantage in mice treated with AZD6244 + PLX4720 (P < 0.01). Conclusions: In BRAFV600E tumors, combination of AZD6244 + PLX4720 is superior to monotherapy and to other combinatorial approaches. In BRAFWT 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.

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.

Glioma stem cells (GSC) are postulated to provide a reservoir of cells with selfrenewal capabilities, sustain tumor growth by generating differentiated nonstem tumor cells and may be responsible for recurrences after chemo-, radioand surgical therapy. Elimination of the GSC population is regarded as a key to successful treatment of glioma, but high resistance of GSC to conventional glioma therapy remains a therapeutic challenge. Preclinical studies show now that immune therapy for glioma using immune checkpoint inhibitors can achieve unprecedented efficacy that translates into significantly prolonged survival. Although these studies show a high anti-glioma efficacy of immune checkpoint inhibitors, especially when multiple pathways are inhibited, their impact on GSC is rarely addressed and the mechanisms behind it are far from fully elucidated. Nevertheless, recent evidence revealed that GSCs contribute more to tumor development than the non-GSC population by a more pronounced attenuation of immune surveillance. Therefore, abrogation of gliomainduced immunesuppression with immune checkpoint inhibitors seems to be a promising rationale to enhance anti-GSC efficacy. Clinical trials studying immune checkpoint blockage in high-grade gliomas have been recently designed with some of them already enrolling patients.

Defective asymmetric cell divisions of stem and progenitor cells are associated with tumorigenesis by a largely unknown mechanism. A signalling axis involving Snail, microRNA-146a and Numb is now shown to regulate the switch between symmetric and asymmetric cell division in colorectal cancer stem cells.

BRAFV600E mutations are frequent in pediatric gliomas. We previously showed antitumor activity when using BRAF and/or MEK inhibitors against BRAFV600E glioma syngeneic grafts (Grossauer et al., Oncotarget 2016;7:775839). While early clinical trial reports suggest an encouraging response rate to BRAFV600E inhibitors alone (Burger et al., Oncol Rep 2017;38:3291) and in combination with MEK inhibition (Brown et al., CNS Oncol 2017:6:291), resistance has been reported in BRAFV600E mutant melanoma. Moreover, we reported that resistance to BRAFV600E inhibitor monotherapy occurs in mouse models and is driven by stem-like cells (Lerner et al., Cancer Res 2015;75:5355). Rapid reactivation of MEK pathway and prosurvival signaling via EGFR have been implicated as mechanisms for acquired resistance (Yao et al., Oncotarget 2017;8:583). It is just unclear why stem-like cells survive treatment better than non-stem like tumor cells and how they respond to additional therapies, such as MEK inhibition but also immune checkpoint inhibition. Immunocompetent, syngeneic FVB/N mice were intracranial-engrafted with luciferase-modified BRAFV600E mutant CDKN2A-deficient murine glioma cell line 2341luc and treated with BRAFV600E and/or MEK inhibitors, either alone or in combination, and either with or without PD-L1 (Clone 10F.9G2) and CTLA-4 (Clone 9D9) mAbs. While the combination of BRAF and MEK inhibitors significantly increased the median survival from 46 to 70 days (P Citation Format: Stefan Grossauer, Katharina Koeck, Malek Chouchane, Joanna J. Phillips, Claudia K. Petritsch, Theodore Nicolaides. MAPK pathway blockade effects on glioma stem cells and immunotherapy in BRAFV600E mutant gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 143.

We previously discovered that oligodendrocyte progenitor cells (OPC) undergo asymmetric cell division and symmetric, self-renewing divisions at an equal rate, whereby they may establish homeostasis. The proteoglycan and OPC marker NG2 plays a critical role in establishing asymmetric cell division (1). Studies in genetically engineered mouse models identified OPC as a putative cellular origin of human glioma (2,3). Studies also showed that premalignant OPC (aka glioma precursor) increase rates of symmetric, self-renewing divisions at the expense of asymmetric division, suggesting that tipping the balance towards symmetric divisions might disrupt homeostasis and lead to cancer. The objective of this project is to gain functional insights into asymmetric division to assess its role in maintaining tissue homeostasis and tumor suppression. Using our novel FACS-based approach we were able to identify that several conserved regulators of asymmetric division, including the WD40 protein Lethal giant larvae1 (Lgl1) protein, are upregulated in asymmetric versus symmetric dividing OPC. We genetically enforced deletion of Lgl1 in OPC conditionally by injecting tamoxifen into triple transgenic mice obtained by breeding Lgl1fl/fl NG2CreERT2 and red fluorescence Cre reporter mice. Cre-conditional deleted Lgl1 OPC were analyzed for changes in cell division mode, proliferation rate and differentiation in vivo and ex vivo. Whole-transcriptome analyses of Lgl1-deleted OPC followed by Ingenuity Pathway Analyses revealed significant changes in several pathways, including in receptor-mediated endocytosis. Functional analyses were conducted to test if Lgl1 regulates endocytosis in OPC. Our results show that Lgl1 deletion cooperates with CDKN2A homozygous deletion to form glioma from OPC, albeit with long latency. Lgl1-deleted OPC show increased rates of symmetric, self-renewing division and proliferative rate at the expense of asymmetric divisions and differentiation. Lgl1 regulates endocytosis of NG2 and by routing NG2 complex to the lysosome for degradation, Lgl1 may establish the asymmetric NG2 distribution, which is critical for asymmetric division. Taken together, our data identified a conserved regulator of asymmetric OPC division and showed that loss of such a regulator not only disrupts asymmetric divisions but also leads to premalignant changes and neoplastic transformation and the accumulation of glioma precursor. Investigations into the mechanistic details of asymmetric division and the potential role for endocytosis in cell fate determination of OPC and gliomagenesis are ongoing. References: 1. Sugiarto et al., Cancer Cell 2011;20:328. 2. Persson et al., Cancer Cell 2010:18:229. 3. Liu et al., Cell 2011:146:209. Citation Format: Mathieu Daynac, Malek Chouchane, Alex Valenzuela, Claudia K. Petritsch. Tumor suppression by regulator of asymmetric cell division in glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 149.

SummaryDevelopment of hypoxic regions is an indicator of poor prognosis in many tumors. Here, we demonstrate that HIF1α, the direct effector of hypoxia, partly through increases in SDF1α, induces recruitment of bone marrow-derived CD45+ myeloid cells containing Tie2+, VEGFR1+, CD11b+, and F4/80+ subpopulations, as well as endothelial and pericyte progenitor cells to promote neovascularization in glioblastoma. MMP-9 activity of bone marrow-derived CD45+ cells is essential and sufficient to initiate angiogenesis by increasing VEGF bioavailability. In the absence of HIF1α, SDF1α levels decrease, and fewer BM-derived cells are recruited to the tumors, decreasing MMP-9 and mobilization of VEGF. VEGF also directly regulates tumor cell invasiveness. When VEGF activity is impaired, tumor cells invade deep into the brain in the perivascular compartment.

Stem-like glioma cells, frequently referred to as tumor-propagating cells or cancer stem cells are culprits for recurrence due to their intrinsic resistance to standard therapy and their ability to regrow the parental tumor in xenografts. Similar to normal neural stem cells CD133+ tumor-propagating cells undergo asymmetric, self-sustaining cell divisions (Gomez-Lopez S. et al, Cell Mol Life Sci, 2014, 71(4):575-97). Cancer-associated changes in cell division mode contribute to neoplastic transformation of glioma precursors (Sugiarto S. et al, Cancer Cell, 2011 20(3):328-40). While increased symmetric, self-renewing divisions of glioma precursors correlate with therapy sensitivity, asymmetric self-sustaining divisions potentially maintain the pool of tumor-propagating cells and contribute to therapy evasion. How pharmacological MAPK pathway inhibitors clinically evaluated for the treatment of GBM affect the division mode of tumor-propagating cells is unknown. We investigate CD133+ tumor-propagating cell responses to novel targeted therapies, in specifically, small molecule inhibitors of BRAFV600E, a mutant kinase frequently found in pediatric malignant astrocytoma. Our investigations showed that CD133+ tumor-propagating cells have higher asymmetric divisions than progenitor-like glioma cells. CD133+ stem-like GBM subpopulation exhibit decreased sensitivity to the anti-proliferative effects of MAPK pathway inhibition and show extended G2/M phase. We find that the mitotic checkpoint kinase and polarity regulator Plk1 is more active in CD133+ tumor-propagating cells. Plk1 activity links asymmetric division and mitotic entry. In an orthotopic GBM xenograft model, combined MAPK-pathway and PLK1 inhibition showed increased anti-proliferative effects and cell death frequency towards CD133+ cells beyond that achieved by either inhibitor alone. Ongoing work is investigating if Plk1 controls a polarity checkpoint, the integrity of which is especially important in the therapy–evasive compartment in GBM and that provides a rationale for combination therapy.

INTRODUCTION: Targeted monotherapy against BRAFV600E displays efficacy in preclinical models of BRAFV600E-mutant gliomas, however xenograft tumors adapt rapidly and escape from the growth-inhibitory effects. The question emerges whether a more durable anti-tumor effect will be achieved by combining BRAF inhibitors with immune-modulatory agents such as anti-CTLA-4 and anti-PD-L1. The lack of an immunocompetent preclinical model of BRAFV600E-mutant astrocytoma however has been prohibitive to addressing this question. METHODS: A BRAFV600E-mutant murine tumor cell line (2341) was isolated from tumors developed in transgenic mice with Cre-conditional alleles of BRAFV600E and CDKN2A. Multiple cohorts of syngeneic FVBN mice, as well as athymic mice were injected orthotopically with the BRAFV600E-mutant tumor cell line and evaluated for the occurrence of neurological symptoms. Symptomatic animals were sacrificed and brains were harvested and subjected to a detailed immunohistopathological characterization. Tumor growth was studied employing bioluminescence (BLI). The presence of tumor fluorescence following in vivo injection of 5-aminolevulinic acid (5-ALA) was investigated as well. RESULTS: Injected animals developed tumors in the right striatum exhibiting all histopathological diagnostic features of human glioblastomas. Neurological free survival was significantly longer in immunocompetent FVBN mice (mean 56 days) compared to similarly treated athymic mice (mean 15 days). Additionally, 5-ALA injected mice exhibited strong fluorescence of tumor cells, when exposed to ultraviolet light compared to normal brain tissue. Results from ongoing work regarding T-cell regulation in the immunocompetent glioma model will be presented and discussed at the meeting. CONCLUSIONS: A BRAFV600E-mutant glioma model that recapitulates the typical genetical, histopathological and behavioral features of human glioblastoma multiforme can be established in fully immunocompetent FVBN mice. These tumors also exhibit 5-ALA induced tumor fluorescence, a typical feature of human malignant astrocytomas. Further studies will reveal this modelś ability to reliably test the anti-glioma efficacy of immune-modulatory drugs in combination with BRAFV600E inhibition.

Despite aggressive standard and novel targeted therapies, malignant gliomas recur invariably and develop therapy resistance. Our previous studies have shown that oligodendrocyte progenitor cells (OPCs) undergo asymmetric cell division whereby they generate self-renewing and differentiating cells in a single division by unequally segregating cell fate determinants. We found that premalignant OPC, the origin of glioma in genetically engineered mouse models, exhibit disrupted asymmetric divisions, together with other hallmarks of cancer, including hyper-proliferation (Sugiarto et al., 2011, Cancer Cell 20:328-40). Yet, it is not clear whether disruption of asymmetric division of OPCs is a contributing factor or a consequence of neoplastic transformation. Lethal giant larvae 1 (Lgl1), a gene that was initially identified as a tumor suppressor in Drosophila, has been implicated in the asymmetric localization of cell fate determinants in neural progenitor cells (Klezovitch et al., 2004, Genes Dev 18:559-571). We investigated the effects of Lgl1 depletion on distinct hallmarks of glioma. We find that in murine OPC carrying conditional null alleles of Lgl1, depletion of Lgl1 resulted in reduced rates of asymmetric cell division and other defects previously associated with glioma precursors. Our data suggest that loss of asymmetric divisions contribute to neoplastic transformation. Asymmetric cell divisions of stem and progenitor cells are a point of disruption to which novel therapies can be targeted. Underlying mechanisms for the phenotypes of Lgl1 knockout and their relevance for malignant glioma will be discussed.

Cell death is essential for eliminating excess cells during development as well as removing damaged cells. While multiple conserved apoptosis pathways involving different cascades of caspases, which are cysteine proteases, have been identified, their regulation in the context of a developing organism is not very well understood. Expression of the Drosophila caspase-9 homolog, DRONC, can be induced by ecdysone, a steroid hormone, which induces metamorphosis. To elucidate the functional role of DRONC during metamorphosis and for cell death during development we have generated and analyzed two loss-of-function alleles of DRONC. We report that DRONC is required for developmentally induced neuroblast cell death and apoptosis in response to X irradiation. DRONC mutants show reduced pupariation even in the presence of high levels of ecdysone and impaired cell death of larval midgut. The levels of ecdysone-inducible transcripts such as E75A and Reaper (Rpr) are normal in the absence of DRONC, suggesting that DRONC acts downstream of these genes. In addition, Reaper and Grim, but not Hid induced apoptosis is sensitive to a reduction of DRONC levels. Our study places DRONC at a central point of convergence for multiple cell death pathways and for the ecdysone pathway regulating metamorphosis.

Much attention has focused on dendritic translational regulation of neuronal signaling and plasticity [1, 2]. For example, long-term memory in adult Drosophila requires Pumilio (Pum) [3], an RNA binding protein that interacts with the RNA binding protein Nanos (Nos) to form a localized translation repression complex essential for anterior-posterior body patterning in early embryogenesis [4]. Whether dendrite morphogenesis requires similar translational regulation is unknown. Here we report that nos and pum control the elaboration of high-order dendritic branches of class III and IV, but not class I and II, dendritic arborization (da) neurons. Analogous to their function in body patterning, nos and pum require each other to control dendrite morphogenesis, a process likely to involve translational regulation of nos itself. The control of dendrite morphogenesis by Nos/Pum, however, does not require hunchback, which is essential for body patterning. Interestingly, Nos protein is localized to RNA granules in the dendrites of da neurons, raising the possibility that the Nos/Pum translation repression complex operates in dendrites. This work serves as an entry point for future studies of dendritic translational control of dendrite morphogenesis.

In the past, we demonstrated that adult oligodendrocyte progenitor cells (OPC) undergo asymmetric cell division (ACD) to self-renew and generate functional cells by unequally distributing active EGFR and the OPC-specific marker NG2. Moreover, premalignant OPC switch from an asymmetric to symmetric, self-renewing division mode at premalignant stages, and exhibit aberrant proliferation and impaired differentiation (Sugiarto S et al Cancer Cell 2011). OPC are the cell-of-origin of glioma in genetically engineered mouse models (Persson A et al Cancer Cell 2010) and OPC-like glioma cells have high tumor-initiating potential in some glioma types. These data contribute to the emerging evidence that asymmetric cell division (ACD) has tumor suppressive activity. Whether the loss of ACD directly promotes tumor initiation is unclear, because the detailed mechanisms of mammalian ACD have not been elucidated (Gomez-Lopez S et al. Cell Mol Life Sci 2014). Lethal giant larvae 1 (Lgl1) was initially identified as a tumor suppressor in Drosophila and has been implicated in the asymmetric localization of cell fate determinants in mammalian neural progenitor cells (Klezovitch et al., Genes Dev, 2004). Others have shown also that human Lgl1 is inhibited by PTEN loss, which is common in glioblastoma, and Lgl1 depletion in human glioblastoma cells increases their invasive properties (Gont et al, Oncotarget, 2013 and 2014). Whether Lgl1 restricts proliferation, promotes differentiation and prevents neoplastic transformation of OPC, by regulating their asymmetric division, is unknown. We developed a flow cytometry method to isolate proliferative and differentiation-committed OPC (COP) and showed that Lgl1 expression is elevated in differentiation. Using a Cre-conditional knock-out allele (Klezovitch et al., Genes Dev, 2004), we show that Lgl1 knockout OPC proliferate more and differentiate less when compared with Lgl1 wildtype OPC. Lgl1 depletion increases symmetric, self-renewing divisions and reduces the frequency of ACD. Lgl1 appears to control the expression of several conserved ACD regulators, as revealed by comparative Taqman expression analyses of Lgl1 knockout versus control OPC. Ongoing studies are aimed to investigate the link of Lgl1 depletion, altered ACD regulator expression and observed OPC phenotypes. Data derived from this work are expected to provide further insights into the extent to which defective ACD contributes to neoplastic transformation in all cancers. Citation Format: Mathieu Daynac, Claudia K. Petritsch. Asymmetric cell division regulator prevents hyperproliferation in glioma cell-of-origin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 916. doi:10.1158/1538-7445.AM2017-916

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. Cancer Res; 75(24); 5355–66. ©2015 AACR.

BACKGROUND: Studies show that the expression of an activating mutation of BRAF (BRAFV600E) in conjunction with Ink4a/Arf deletion, but not each alteration by itself, lead to formation of malignant astrocytoma (MA)-like tumors. Importantly, BRAFV600E occurs in 39% of pediatric MA and in 7.7% of adult glioblastomas with a preference for young adult patients, and the majority coincides with homozygous deletion of cyclin-dependent kinase inhibitor 2A (CDKN2A) encoding Ink4a/Arf. Responses to pharmacologic inhibition of BRAFV600E in melanoma patients were stunning but led to rapid resistance. A better understanding of the molecular mechanism by which activated Raf signaling transforms adult neural progenitors into high-grade MA cells is needed to overcome resistance. APPROACH AND RESULTS: We hypothesize that BRAFV600E transforms Ink4a/Arf-depleted neural progenitor cells, by disrupting their asymmetric cell division, thereby initiating a cascade of events that fails to restrict cell fate determinants during division, alters cell fate, enhances proliferation and allows for other changes associated with neoplastic transformation. We use a Cre inducible mouse model of BRAFV600E, the BRafCA allele, and of Ink4a/ARf (Ink4a/Arf-loxP) in combination with fluorescence-activated cell sorting to isolate neural progenitor cells from the adult mouse brain subventricular zone. Enriched neural progenitor populations are subjected in vitro modified to express BRAFV600E and deleted Ink4a/Arf by adenovirus-Cre infection. Cell-culture-based assays determine their rate of asymmetric cell division, self-renewal, proliferation and their differentiation capacity. CONCLUSION: BRAFV600E expression causes a cell-type specific decrease in asymmetric cell division in oligodendrocyte progenitor cells (OPC). Asymmetry-defective in vitro modified OPC and OPC-like cells isolated from a tumor cell line are capable of forming tumors when injected into mice. We will present data from our ongoing search for critical molecular regulators and pathway(s) associated with BRAFVE-mediated disruption of asymmetric cell division and malignant transformation.

Balancing quiescence, self-renewal, and differentiation in adult stem cells is critical for tissue homeostasis. The underlying mechanisms, however, remain incompletely understood. Here we identify Fezf2 as a novel regulator of fate balance in adult zebrafish dorsal telencephalic neural stem cells (NSCs). Transgenic reporters show intermingledfezf2-GFPhiquiescent andfezf2-GFPloproliferative NSCs. Constitutive or conditional impairment offezf2activity demonstrates its requirement for maintaining quiescence. Analyses of genetic chimeras reveal a dose-dependent role offezf2in NSC activation, suggesting that the difference infezf2levels directionally biases fate. Single NSC profiling coupled with genetic analysis further uncovers afezf2-dependent gradient Notch activity that is high in quiescent and low in proliferative NSCs. Finally,fezf2-GFPhiquiescent andfezf2-GFPloproliferative NSCs are observed in postnatal mouse hippocampus, suggesting possible evolutionary conservation. Our results support a model in whichfezf2heterogeneity patterns gradient Notch activity among neighbors that is critical to balance NSC fate.

BACKGROUND: Diffuse intrinsic pontine gliomas (DIPGs) in children carry a dismal prognosis despite the use of aggressive multi-modality treatment. Recent results have identified a somatic mutation in the H3F3A gene, resulting in replacement of lysine 27 by methionine in its encoded histone H3.3 protein (H3.3-K27M), which occurs primarily in DIPGs. The K27M mutation results in both increased and decreased gene expression. This specific mutation is an attractive therapeutic target for the treatment of DIPGs. METHODS: Using cell lines and tissues from two pediatric DIPGs and one pediatric GBM tumor obtained from surgical biopsies, H3.3-K27M mutation status was determined by direct sequencing. Histone H3.3 lysine 27 (H3K27) methylation status was evaluated by western blotting with antibodies specific for mono-, di-, and trimethylated H3K27. A selective inhibitor of the H3K27 demethylase JMJD3, GSK-J4, was used to examine cell proliferation and survival. GSK-J4 was found to inhibit JMJD3-induced H3K27 demethylation, resulting in an increase of H3K27 methylation. RESULTS: The H3.3-K27M mutation was identified in the two DIPG cell lines, but not in the pediatric GBM cells, nor in other adult glioma cells. H3.3-K27M mutant DIPG cells showed rapid growth in vitro, with a doubling time of approximately 30 hrs. In contrast, a pediatric GBM cell line with wild-type H3.3 grew much more slowly, with a doubling time of 72 hrs. H3.3-K27M mutant DIPG cells showed elevated H3K27 methylation in comparison to H3.3 wild-type glioma cells. GSK-J4 induced a marked dose-dependent inhibition of growth in H3.3-K27M mutant DIPG cells while H3.3 wild-type glioma cells showed no response to GSK-J4. GSK-J4 appears to have a potent and specific effect on tumor cells with the K27M mutation. CONCLUSIONS: Altered histone H3.3 K27 methylation is associated with rapid tumor cell growth in vitro, and is associated withn increased tumor cell sensitivity to GSK-J4. SECONDARY CATEGORY: Tumor Biology.

On TGF-β binding, the TGF-β receptor directly phosphorylates and activates the transcription factors Smad2/3, leading to G1 arrest. Here, we present evidence for a second, parallel, TGF-β-dependent pathway for cell cycle arrest, achieved via inhibition of p70s6k. TGF-β induces association of its receptor with protein phosphatase-2A (PP2A)-Bα. Concomitantly, three PP2A-subunits, Bα, Aβ, and Cα, associate with p70s6k, leading to its dephosphorylation and inactivation. Although either pathway is sufficient to induce G1 arrest, abrogation of both, the inhibition of p70s6k, and transcription through Smad proteins is required for release of epithelial cells from TGF-β-induced G1 arrest. TGF-β thereby modulates the translational and posttranscriptional control of cell cycle progression.

Motivation: Several cancer types consist of multiple genetically and phenotypically distinct subpopulations. The underlying mechanism for this intra-tumoral heterogeneity can be explained by the clonal evolution model, whereby growth advantageous mutations cause the expansion of cancer cell subclones. The recurrent phenotype of many cancers may be a consequence of these coexisting subpopulations responding unequally to therapies. Methods to computationally infer tumor evolution and subpopulation diversity are emerging and they hold the promise to improve the understanding of genetic and molecular determinants of recurrence. Results: To address cellular subpopulation dynamics within human tumors, we developed a bioinformatic method, EXPANDS. It estimates the proportion of cells harboring specific mutations in a tumor. By modeling cellular frequencies as probability distributions, EXPANDS predicts mutations that accumulate in a cell before its clonal expansion. We assessed the performance of EXPANDS on one whole genome sequenced breast cancer and performed SP analyses on 118 glioblastoma multiforme samples obtained from TCGA. Our results inform about the extent of subclonal diversity in primary glioblastoma, subpopulation dynamics during recurrence and provide a set of candidate genes mutated in the most well-adapted subpopulations. In summary, EXPANDS predicts tumor purity and subclonal composition from sequencing data. Availability and implementation: EXPANDS is available for download at http://code.google.com/p/expands (matlab version - used in this manuscript) and http://cran.r-project.org/web/packages/expands (R version). Contact: claudia.petritsch@ucsf.edu Supplementary information: Supplementary data are available at Bioinformatics online.

We show here using synchronized Swiss mouse 3T3 fibroblasts that p70 S6 kinase (p70S6k) and mitogen-activated protein kinases (p42mapk/p44mapk) are not only activated at the G0/G1 boundary, but also in cells progressing from M into G1. p70S6k activity increases 20-fold in G1 cells released from G0. Throughout G1, S, and G2 it decreases constantly, so that during M phase low kinase activity is measured. The kinase is reactivated 10-fold when cells released from a nocodazole-induced metaphase block enter G1 of the next cell cycle. p42mapk/p44mapk in G0 cells are activated transiently early in G1 and are reactivated late in mitosis after nocodazole release. p70S6k activity is dependent on permanent signaling from growth factors at all stages of the cell cycle. Immunofluorescence studies showed that p70S6k and its isoform p85S6k become concentrated in localized spots in the nucleus at certain stages in the cell cycle. Cell cycle-dependent changes in p70S6k activity are associated with alterations in the phosphorylation state of the protein. However, examination of the regulation of a p70S6k mutant in which the four carboxyl-terminal phosphorylation sites are changed to acidic amino acids suggests that a mechanism independent of these phosphorylation sites controls the activity of the enzyme during the cell cycle.

Cancers are a mosaic of clones of varying population sizes. Any single cancer sample encodes a tumor-metagenome, since it represents the aggregate genomes of diverse clones that coexist within the sample. We quantified genomic instability as the fraction of the tumor-metagenome affected by copy number variations (CNVs) and leveraged two tumor mixture separation algorithms, EXPANDS and PyClone, to quantify genetic intra-tumor heterogeneity (ITH) from single cancer samples. We tested the potential of measures of genomic instability and ITH as prognostic biomarkers across 1,165 exome sequenced primary tumors from 12 cancer types at TCGA. Our results suggest that a tradeoff between the costs and adaptive benefits of genomic instability governs differential radiotherapy sensitivity. Between 1 and 18 clones were estimated to coexist per tumor sample at >10% cell frequency (median = 4). Clone number varied considerably within and between cancer types, with melanomas representing the most heterogeneous group. 86% all analyzed tumor samples contained at least 2 clones. Across cancer types, the presence of >2 clones was associated with worse overall survival as compared to tumors in which < = 2 clones were detected (Log-rank test: P = 8.6E-4, HR = 1.49). An exceptionally favorable outcome was observed when >75% genomic instability was shared among < = 2 clones. The highest risk was observed among individuals with an intermediate number of 4 clones - additional diversification beyond 4 clones did not impart further risk. The highest risk was also observed among individuals with 50-75% genomic instability, in both the original exome sequencing dataset and an independent SNP array dataset. Genomic instability 75% predicted reduced risk (HR = 0.15, 95% CI: 0.08-0.29). We analyzed the relation between radiotherapy intensity and overall survival among 242 individuals (21%) treated with radiotherapy and found that not all individuals did benefit equally from therapy. In order to achieve the same benefit from therapy, individuals with 25-50% genomic instability required higher therapy intensity (regression slope = 1.83; P = 0.009) than individuals with 50-75% genomic instability (slope = 2.09; P = 0.005). In contrast, individuals with Radiotherapy may be particularly effective against tumors with intermediate CNV burden, by pushing them past the limit of ‘tolerable’ genomic instability. Our results from two independent pan-cancer cohorts suggest that this limit is exceeded when >75% of a tumor's metagenome is affected by CNVs. This upper limit of tolerable genomic instability may be responsible for the non-linear association we observed between genetic ITH and survival. Leveraging a clone's distance to the upper limit of tolerable genomic instability may represent a new strategy to optimize therapy intensity. Citation Format: Noemi Andor, Trevor A. Graham, Marnix Jansen, Li C. Xia, Athena Aktipis, Claudia Petritsch, Hanlee P. Ji, Carlo C. Maley. Pan-cancer analysis of clonal evolution reveals the costs and adaptive benefits of genomic instability. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2387.