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Glioblastoma is one of the most devastating of human cancers, with near-uniform fatality within two years of diagnosis. Therapeutic failure is thought to be related to small subpopulations of cells that exhibit the properties of self-renewal and tumorigenicity. Understanding how such subpopulations attain and retain these properties remains a central question in oncology. One fundamental issue is whether tumorigenicity exists within a static population of elite cells or whether the capacity is stochastically acquired. To test these models, we assayed in vitro tumor sphere formation frequencies and in vivo tumor growth of subclones of established glioblastoma lines as well as stem cell lines derived from patients and genetically engineered mouse models. Our findings were best described by a hybrid model that is largely deterministic (elite) but with opportunities for dynamic (stochastic) interchange between non-tumorigenic and tumorigenic states. To identify determinants of tumorigenicity, we performed gene expression profiling of the subclones. Analysis of the data suggested that tumorigenicity in glioblastoma is a dynamic property driven by variations in MYC expression, consistent with the effects of ectopic expression and knockdown. Transitions between tumorigenic and non-tumorigenic cell states were associated with changes in histone modifications at the MYC locus, suggesting epigenetic regulation. The role of MYC as a "tumorigenicity gene" is fundamentally distinct from those previously ascribed to oncogenes. The model suggests the need for therapeutic strategies that disrupt the dynamic transition between cell states.

SapC-DOPS is a novel nanotherapeutic that has been shown to target and induce cell death in a variety of cancers, including glioblastoma (GBM). GBM is a primary brain tumor known to frequently demonstrate resistance to apoptosis-inducing therapeutics. Here we explore the mode of action for SapC-DOPS in GBM, a treatment being developed by Bexion Pharmaceuticals for clinical testing in patients. SapC-DOPS treatment was observed to induce lysosomal dysfunction of GBM cells characterized by decreased glycosylation of LAMP1 and altered proteolytic processing of cathepsin D independent of apoptosis and autophagic cell death. We observed that SapC-DOPS induced lysosomal membrane permeability (LMP) as shown by LysoTracker Red and Acridine Orange staining along with an increase of sphingosine, a known inducer of LMP. Additionally, SapC-DOPS displayed strong synergistic interactions with the apoptosis-inducing agent TMZ. Collectively our data suggest that SapC-DOPS induces lysosomal cell death in GBM cells, providing a new approach for treating tumors resistant to traditional apoptosis-inducing agents.

BACKGROUND: There have been several reports that have linked human cytomegalovirus (CMV) to gliomas. Although clinical trials of immunotherapy against CMV antigens found in gliomas have commenced in multiple institutions, the data linking these tumors to this virus remains controversial. METHODS: To study the controversial role of cytomegalovirus (CMV) in glioblastoma, we assessed the effects of murine CMV (MCMV) perinatal infection in a GFAP-cre; Nf1loxP/+ ; Trp53-/+ genetic mouse model of glioma (Mut3 mice). RESULTS: Early on after infection, MCMV antigen was predominantly localized in CD45+ lymphocytes in the brain with active viral replication and local areas of inflammation, but by 7 weeks there was a generalized loss of MCMV in brain, confirmed by bioluminescent imaging. MCMV-infected Mut3 mice exhibited a shorter survival time from their gliomas, when compared to control Mut3 mice, perinatally infected with mock or with a different neurotropic virus. Animal survival was also significantly shortened when orthotopic gliomas were implanted in mice perinatally infected with MCMV vs. controls. MCMV infection increased phosphorylated STAT3 (P-STAT3) levels in neural stem cells (NSCs) harvested from Mut3 mice SVZ and in vivo there was increased P-STAT3 in NSCs in MCMV-infected compared to control mice. Of relevance, human CMV (HCMV) also increased P-STAT3 and proliferation of patient-derived glioblastoma neurospheres, while a STAT3 inhibitor reversed this effect in vitro and in vivo. CONCLUSIONS: These findings thus associate CMV infection to a STAT3-dependent modulatory role in glioma formation/progression in the context of tumor suppressor mutations in mice and possibly in humans. SECONDARY CATEGORY: Neuropathology & Tumor Biomarkers.

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10° steps over 380°. A rectangular region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10° steps over 380°. A rectangular region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

Purpose: Our goal is to test whether CS1 could be targeted by chimeric antigen receptor (CAR) T cells to treat multiple myeloma (MM). Experimental Design: We generated a retroviral construct of a CS1-specific CAR and engineered primary human T cells expressing the CAR. We then tested the capacity of CS1–CAR T cells to eradicate human MM tumor cells in vitro, ex vivo, and in vivo using orthotopic MM xenograft mouse models. Results: In vitro, compared with mock-transduced T cells, upon recognizing CS1-positive MM cells, CS1–CAR-transduced T cells secreted more IFN-γ as well as interleukin (IL)-2, expressed higher levels of the activation marker CD69, showed higher capacity for degranulation, and displayed enhanced cytotoxicity. Ectopically forced expression of CS1 in MM cells with low CS1 expression enhanced recognition and killing by CAR T cells. Ex vivo, CS1–CAR T cells also showed similarly enhanced activities when responding to primary MM cells. More importantly, in orthotopic MM xenograft mouse models, adoptive transfer of human primary T cells expressing CS1–CAR efficiently suppressed the growth of human MM.1S and IM9 myeloma cells and significantly prolonged mouse survival. Conclusions: CS1 is a promising antigen that can be targeted by CAR-expressing T cells for treatment of MM. Clin Cancer Res; 20(15); 3989–4000. ©2014 AACR.

Neural oncogenesis is currently incurable and invariably lethal. The development of innovative treatments for this devastating cancer will require a deeper molecular understanding of how cancer cells survive, proliferate, and escape from current therapies. In high-grade gliomas (HGGs), glioma stem cells (GSCs) may causally contribute to tumor initiation and propagation, therapeutic resistance, and subsequent recurrence of tumors. Within a tumor mass, GSCs are enriched in a hypoxic niche in which the oxidative stress levels are substantially elevated. Paradoxically, however, recent studies suggest that GSCs appear to generate less reactive oxygen species (ROS), a chemical component responsible for elevation of oxidative stress levels. To date, molecular mechanisms for how GSCs reduce oxidative stress to allow preferential survival in hypoxic areas in tumors remains elusive. This review article summarizes recent studies on the role of ROS-reducing enzymes, including peroxiredoxin 4, in detoxifying oxidative stress preferentially for GSCs in HGGs. In addition, the therapeutic potential of some of the recently identified antioxidant chemotherapeutic agents and avenues for future research in this area are discussed.

Glioblastoma is the most common and aggressive histologic subtype of brain cancer with poor outcomes and limited treatment options. Here, we report the selective overexpression of the protein arginine methyltransferase PRMT5 as a novel candidate theranostic target in this disease. PRMT5 silences the transcription of regulatory genes by catalyzing symmetric dimethylation of arginine residues on histone tails. PRMT5 overexpression in patient-derived primary tumors and cell lines correlated with cell line growth rate and inversely with overall patient survival. Genetic attenuation of PRMT5 led to cell-cycle arrest, apoptosis, and loss of cell migratory activity. Cell death was p53-independent but caspase-dependent and enhanced with temozolomide, a chemotherapeutic agent used as a present standard of care. Global gene profiling and chromatin immunoprecipitation identified the tumor suppressor ST7 as a key gene silenced by PRMT5. Diminished ST7 expression was associated with reduced patient survival. PRMT5 attenuation limited PRMT5 recruitment to the ST7 promoter, led to restored expression of ST7 and cell growth inhibition. Finally, PRMT5 attenuation enhanced glioblastoma cell survival in a mouse xenograft model of aggressive glioblastoma. Together, our findings defined PRMT5 as a candidate prognostic factor and therapeutic target in glioblastoma, offering a preclinical justification for targeting PRMT5-driven oncogenic pathways in this deadly disease. Cancer Res; 74(6); 1752–65. ©2014 AACR.

Somatic inactivation of PTEN occurs in different human tumors including glioblastoma, endometrial carcinoma and prostate carcinoma. Germline mutations in PTEN result in a range of phenotypic abnormalities that occur with variable penetrance, including neurological features such as macrocephaly, seizures, ataxia and Lhermitte-Duclos disease (also described as dysplastic gangliocytoma of the cerebellum). Homozygous deletion of Pten causes embryonic lethality in mice. To investigate function in the brain, we used Cre-loxP technology to selectively inactivate Pten in specific mouse neuronal populations. Loss of Pten resulted in progressive macrocephaly and seizures. Neurons lacking Pten expressed high levels of phosphorylated Akt and showed a progressive increase in soma size without evidence of abnormal proliferation. Cerebellar abnormalities closely resembled the histopathology of human Lhermitte-Duclos disease. These results indicate that Pten regulates neuronal size in vivo in a cell-autonomous manner and provide new insights into the etiology of Lhermitte-Duclos disease.

To study the controversial role of cytomegalovirus (CMV) in glioblastoma, we assessed the effects of murine CMV (MCMV) perinatal infection in a GFAP-cre; Nf1loxP/+; Trp53−/+ genetic mouse model of glioma (Mut3 mice). Early on after infection, MCMV antigen was predominantly localized in CD45+ lymphocytes in the brain with active viral replication and local areas of inflammation, but, by 7 weeks, there was a generalized loss of MCMV in brain, confirmed by bioluminescent imaging. MCMV-infected Mut3 mice exhibited a shorter survival time from their gliomas than control Mut3 mice perinatally infected with mock or with a different neurotropic virus. Animal survival was also significantly shortened when orthotopic gliomas were implanted in mice perinatally infected with MCMV versus controls. MCMV infection increased phosphorylated STAT3 (p-STAT3) levels in neural stem cells (NSC) harvested from Mut3 mice subventricular zone, and, in vivo, there was increased p-STAT3 in NSCs in MCMV-infected compared with control mice. Of relevance, human CMV (HCMV) also increased p-STAT3 and proliferation of patient-derived glioblastoma neurospheres, whereas a STAT3 inhibitor reversed this effect in vitro and in vivo. These findings thus associate CMV infection to a STAT3-dependent modulatory role in glioma formation/progression in the context of tumor suppressor mutations in mice and possibly in humans. Cancer Res; 73(11); 3441–50. ©2013 AACR.

Purpose: Glioblastoma multiforme (GBM) is a devastating disease. Recent studies suggest that the stem cell properties of GBM contribute to the development of therapy resistance. Experimental Design: The expression of Survivin and Ran was evaluated by immunohistochemistry with GBM tissues, and quantitative reverse transcriptase (qRT)-PCR and immunocytochemistry with patient-derived GBM sphere cultures. With a computational structure-based drug design, 11 small-molecule compounds were designed, synthesized, and evaluated as inhibitor candidates for the molecular interaction of Survivin protein. The molecular mechanism of the lead compound, LLP-3, was determined by Western blot, ELISA, in situ proximity ligation assay, and immunocytochemistry. The effects of LLP-3 treatment on GSCs were evaluated both in vitro and in vivo. Quantitative immunohistochemistry was carried out to compare Survivin expression in tissues from 44 newly diagnosed and 31 recurrent post-chemoradiation GBM patients. Lastly, the sensitivities of temozolomide-resistant GBM spheres to LLP-3 were evaluated in vitro. Results: Survivin and Ran were strongly expressed in GBM tissues, particularly in the perivasculature, and also in patient-derived GSC cultures. LLP-3 treatment disrupted the Survivin–Ran protein complex in cancer cells and abolished the growth of patient-derived GBM spheres in vitro and in vivo. This inhibition was dependent on caspase activity and associated with p53 status of cells. Immunohistochemistry showed that Survivin expression is significantly increased in recurrent GBM compared with newly diagnosed tumors, and temozolomide-resistant GBM spheres exhibited high sensitivities to LLP-3 treatment. Conclusions: Disruption of the Survivin–Ran complex by LLP-3 abolishes survival and growth of GSCs both in vitro and in vivo, indicating an attractive novel therapeutic approach for GBM. Clin Cancer Res; 19(3); 631–42. ©2012 AACR.

Glioblastoma multiforme (GBM), the most common and aggressive primary brain malignancy, is incurable despite the best combination of current cancer therapies. For the development of more effective therapies, discovery of novel candidate tumor drivers is urgently needed. Here, we report that peroxiredoxin 4 (PRDX4) is a putative tumor driver. PRDX4 levels were highly increased in a majority of human GBMs as well as in a mouse model of GBM. Reducing PRDX4 expression significantly decreased GBM cell growth and radiation resistance in vitro with increased levels of ROS, DNA damage, and apoptosis. In a syngenic orthotopic transplantation model, Prdx4 knockdown limited GBM infiltration and significantly prolonged mouse survival. These data suggest that PRDX4 can be a novel target for GBM therapies in the future.

SummaryMalignant astrocytomas are infiltrative and incurable brain tumors. Despite profound therapeutic implications, the identity of the cell (or cells) of origin has not been rigorously determined. We previously reported mouse models based on conditional inactivation of the human astrocytoma-relevant tumor suppressors p53, Nf1, and Pten, wherein through somatic loss of heterozygosity, mutant mice develop tumors with 100% penetrance. In the present study, we show that tumor suppressor inactivation in neural stem/progenitor cells is both necessary and sufficient to induce astrocytoma formation. We demonstrate in vivo that transformed cells and their progeny undergo infiltration and multilineage differentiation during tumorigenesis. Tumor suppressor heterozygous neural stem/progenitor cultures from presymptomatic mice show aberrant growth advantage and altered differentiation, thus identifying a pretumorigenic cell population.

To establish a genetic tool for manipulating the neural stem/progenitor cell (NSC) lineage in a temporally controlled manner, we generated a transgenic mouse line carrying an NSC-specific nestin promoter/enhancer expressing a fusion protein encoding Cre recombinase coupled to modified estrogen receptor ligand-binding domain (ER(T2)). In the background of the Cre reporter mouse strain Rosa26(lacZ), we show that the fusion CreER(T2) recombinase is normally silent but can be activated by the estrogen analog tamoxifen both in utero, in infancy, and in adulthood. As assayed by beta-galactosidase activity in embryonic stages, tamoxifen activates Cre recombinase exclusively in neurogenic cells and their progeny. This property persists in adult mice, but Cre activity can also be detected in granule neurons and Bergmann glia at the anterior of the cerebellum, in piriform cortex, optic nerve, and some peripheral ganglia. No obvious Cre activity was observed outside of the nervous system. Thus, the nestin regulated inducible Cre mouse line provides a powerful tool for studying the physiology and lineage of NSCs.

Despite vigorous efforts in clinics and laboratories, the overall prognosis for patients with glioblastoma multiforme (GBM), the most common and aggressive glioma, has not greatly improved over the last few decades. Innovative new therapies based on detailed molecular mechanisms of glioma biology are highly desired. Mutation or loss of PTEN (phosphatase tensin homolog deleted on chromosome ten), a key phosphatase that antagonizes the PI3K/AKT pathway, is one of the most frequent abnormalities found in high-grade astrocytomas. Whether this genetic alteration is causal or consequential to the tumorigenic process has begun to be elucidated through tumor genetics and new mouse models. This chapter describes the key findings from mouse astrocytoma models involving Pten deficiency or Akt activation, with specific emphasis on the recently reported Pten-heterozygous mouse models. In addition, future directions to inform the design of novel therapeutics for this devastating disease are discussed.

Malignant astrocytomas are highly invasive brain tumors that portend poor prognosis and dismal survival. Mouse models that genetically resemble the human malignancy provide insight into the nature and pathogenesis of these cancers. We previously reported tumor suppressor mouse models based on conditional inactivation of human astrocytoma-relevant genes p53, Nf1, and Pten. These mice develop, with full penetrance, varying grades of astrocytic malignancy that recapitulate the human condition histologically and molecularly. Our studies indicate a central role for neural stem cells and stem-cell-like cancer cells in tumor initiation and progression. These mouse models thus represent powerful tools for investigating various aspects of tumor development that otherwise cannot be explored in humans. Further studies will provide a better understanding of the biology of these tumors and will hopefully pave the way for more effective therapeutic approaches for these devastating diseases.

SUMMARY Adult hippocampal neurogenesis is stimulated by chronic administration of antidepressants (ADs) and by voluntary exercise. Neural progenitor cells (NPCs) in the dentate gyrus (DG) that are capable of continuous proliferation and neuronal differentiation are the source of such structural plasticity. Here we report that mice lacking the receptor tyrosine kinase TrkB in hippocampal NPCs have impaired proliferation and neurogenesis. When exposed to chronic ADs or wheel-running, no increase in proliferation or neurogenesis is observed. Ablation of TrkB also renders these mice behaviorally insensitive to antidepressive treatment in depression- and anxiety-like paradigms. In contrast, mice lacking TrkB only in differentiated DG neurons display typical neurogenesis and respond normally to chronic ADs. Thus, our data establish an essential cell-autonomous role for TrkB in regulating hippocampal neurogenesis and behavioral sensitivity to antidepressive treatments, and support the notion that impairment of the neurogenic niche is an etiological factor for refractory responses to an antidepressive regimen.

Synapse formation requires contact between dendrites and axons. Although this process is often viewed as axon mediated, dendritic filopodia may be actively involved in mediating synaptogenic contact. Although the signaling cues underlying dendritic filopodial motility are mostly unknown, brain-derived neurotrophic factor (BDNF) increases the density of dendritic filopodia and conditional deletion of tyrosine receptor kinase B (TrkB) reduces synapse number in vivo. Here, we report that TrkB associates with dendritic growth cones and filopodia, mediates filopodial motility, and does so via the phosphoinositide 3 kinase (PI3K) pathway. We used genetic and pharmacological manipulations of mouse hippocampal neurons to assess signaling downstream of TrkB. Conditional knock-out of two downstream negative regulators of TrkB signaling, Pten (phosphatase with tensin homolog) and Nf1 (neurofibromatosis type 1), enhanced filopodial motility. This effect was PI3K-dependent and correlated with synaptic density. Phosphatidylinositol 3,4,5-trisphosphate (PIP3) was preferentially localized in filopodia and this distribution was enhanced by BDNF application. Thus, intracellular control of filopodial dynamics converged on PI3K activation and PIP3 accumulation, a cellular paradigm conserved for chemotaxis in other cell types. Our results suggest that filopodial movement is not random, but responsive to synaptic guidance molecules.

The rodent barrel cortex is a useful system to study the role of genes and neuronal activity in the patterning of the nervous system. Several genes encoding either intracellular signaling molecules or neurotransmitter receptors are required for barrel formation. Neurofibromin is a tumor suppressor protein that has Ras GTPase activity, thus attenuating the MAPK (mitogen-activated protein kinase) and and PI-3 kinase (phosphatidylinositol 3-kinase) pathways, and is mutated in humans with the condition neurofibromatosis type 1 (NF1). Neurofibromin is widely expressed in the developing and adult nervous system, and a common feature of NF1 is deficits in intellectual development. In addition, NF1 is an uncommonly high disorder among individuals with autism. Thus, NF1 may have important roles in normal CNS development and function. To explore roles for neurofibromin in the development of the CNS, we took advantage of a mouse conditional allele. We show that mice that lack neurofibromin in the majority of cortical neurons and astrocytes fail to form cortical barrels in the somatosensory cortex, whereas segregation of thalamic axons within the somatosensory cortex appears unaffected.

Conditional deletion of Pten (phosphatase and tensin homolog on chromosome ten) in differentiated cortical and hippocampal neurons in the mouse results in seizures, macrocephaly, social interaction deficits and anxiety, reminiscent of human autism spectrum disorder. Here we extended our previous examination of these mice using electroencephalogram/electromyogram (EEG/EMG) monitoring and found age-related increases in spontaneous seizures, which were correlated with cellular dispersion in the hippocampal dentate gyrus. Increased spontaneous locomotor activity in the open field on the first and the second day of a 3-day continuous study suggested heightened anxiety in Pten mutant mice. In contrast, the mutants exhibited decreased wheel running activity, which may reflect reduced adaptability to a novel environment. Synchronization to the light-dark cycle was normal, but for up to 28 days under constant darkness, the Pten mutants maintained a significantly lengthened and remarkably constant free-running period of almost exactly 24 h. This result implies the involvement of Pten in the maintenance of circadian rhythms, which we interpret as being due to an effect on the phosphatidylinositol 3-kinase (PI3K) signaling cascade.

Glioblastoma is one of the most devastating of human cancers, with near-uniform fatality within two years of diagnosis. Therapeutic failure is thought to be related to small subpopulation of cells that exhibit the properties of self-renewal and tumorigenicity. It remains unclear whether tumorigenicity exists as a static property of a few cells (“elite” model) or as a dynamically acquired property (“stochastic” model). We used tumor-sphere and xenograft formation as assays for tumorigenicity and examined subclones isolated from established and primary patient-derived glioblastoma (PDX) lines. In multiple PDX lines, glioblastoma subclones that are isogenic (within the limits of the current DNA sequencing technology) nevertheless display varying tumorigenicity. Moreover, the property can be gained or lost at low frequencies. The results indicated that while glioblastoma tumorigenicity largely conforms to the “elite” model, there is dynamic interplay between tumorgenic and non-tumorgenic cell states. Using independent Genetically Engineered Murine Models (GEMMs) of glioblastoma, PDX glioblastoma models, and single cell analysis of clinical glioblastoma specimens, we demonstrate that these dynamic transitions are governed by epigenetic reprogramming through the lysine-specific demethylase 1 (LSD1). LSD1 depletion increases H3K4me3 accumulation at the MYC locus, which elevates MYC expression. MYC, in turn, regulates OLIG2, SOX2 and POU3F2, a core set of transcription factors required for reprogramming glioblastoma cells into stem-like states. Altered tumorgenic potential was not accompanied by a change in the molecular subtype (as defined by the TCGA) and was therefore not related to subtype plasticity. Importantly, complete inhibition or silencing of LSD1 led to suppression of MYC expression and glioblastoma tumorigenicity in vitro and in vivo. However, partial inhibition of LSD1 lead to epigenetic reprogramming resulting in heritable increase MYC expression and glioblastoma tumorigenicity. Our model suggests epigenetic regulation of key transcription factors governs transitions between tumorigenic states and provides a framework for glioblastoma therapeutic development.

Glioblastoma is one of the most devastating of human cancers, with near-uniform fatality within two years of diagnosis. Therapeutic failure is thought to be related to small subpopulation of cells that exhibit the properties of self-renewal and tumorigenicity. Understanding how such subpopulations attain and retain these properties remains a central question in oncology. One fundamental issue is whether tumorigenicity exists within a static population of elite cells or whether the capacity is stochastically acquired. To test these models, we assayed the tumorigenicity of single-cell subclones derived from long-terms passaged and primary patient-derived xenograft (PDX) glioblastoma lines. Our findings were best described by a hybrid model that is largely deterministic (elite) but with opportunities for dynamic (stochastic) interchange between non-tumorigenic and tumorigenic states. To identify molecular determinants of tumorigenicity, we performed gene expression profiling of the subclones. Analysis of the data suggested that tumorigenicity in glioblastoma is a dynamic property driven by variation in MYC expression, which in turn regulates Olig2 expression, a neural stem cell marker. Ectopic expression of MYC conferred tumorigenicty and MYC silencing abolished tumorigenicity in vitro and in vivo for multiple PDX and GEMM models. Transition between tumorigenic and non-tumorigenic cell states was associated with changes in histone modification at the MYC locus mediated by expression of lysine-specific demethylase 1 (LSD1). The model suggests a critical LSD1-MYC-OLIG2 axis that regulates the dynamic transition between glioblastoma cell states of differing tumorigenicity and unveils a novel framework for glioblastoma therapeutic development. Citation Format: Clark Chen, David Kozono, Jie Li, Masayuki Nitta, Oltea Sampetrean, Kimberly Ng, David Gonda, Deepa S. Kushwaha, Dmitry Merzon, Valya Ramakrishnan, Shan Zhu, Kaya Zhu, Hiroko Matsui, Olivier Harismendy, Wei Hua, Ying Mao, Chang-Hyuk Kwon, Keith L. Ligon, Hideyuki Saya, Bob S. Carter, Donald P. Pizzo, Scott R. VandenBerg, Frank Furnari, Webster Cavenee. Dynamic epigenetic regulation of glioblastoma tumorigenicity through a LSD1-MYC-OLIG2 axis. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr PR02.

Glioblastoma is the most common form of primary brain cancer and remains one of the deadliest of human cancers with near-uniform fatality. Increasing evidence suggests that the lethality of glioblastoma is driven by small subpopulations of cells with self-renew ability and tumorigenicity, termed as tumor initiating cells. The mechanism how the tumor initiating cells maintain and gain tumorigenicity in glioblastoma still remains unclear. Here, we used sphere formation and tumor propagating potential to measure the tumorigenicity in established cell line and primary glioblastoma cells. The results indicated that glioblastoma tumorigenicity appears largely deterministic, though spontaneous gain and loss of this property occur at low frequency. Mechanically, this dynamic transition in tumorigenicity was governed by MYC level which was modulated epigenetically by the lysine-specific demethylase 1 (LSD1). Elevated MYC expression, in turn, regulates OLIG2, SOX2 and POU3F2, a core set of transcription factors required for reprogramming glioblastoma cells into stem-like states. Our model suggests epigenetic regulation of key transcription factors facilitates transitions between tumorigenic states and provides a framework for glioblastoma therapeutic development. Importantly, the effect of LSD1 on tumorigenity is “Janus”-like; partial depletion of LSD1 caused increased MYC expression and a pro-tumorigenic state. In contrast, complete suppression of LSD1 induced cell death. As such, therapeutic strategies targeting LSD1 and other targets manifesting similar “Janus” effect should be designed to prevent unintended induction of tumorigenesis during treatment. Citation Format: Jie Li, David Kozono, Masayuki Nitta, Oltea Sampetrean, David Gonda, Deepa S. Kushwaha, Dmitry Merzon, Valya Ramakrishnan, Shan Zhu, Kaya Zhu, Hiroko Matsui, Olivier Harismendy, Wei Hua, Ying Mao, Chang-Hyuk Kwon, Hideyuki Saya, Bob S. Carter, Donald P. Pizzo, Scott R. VandenBerg, Clark C. Chen. Dynamic epigenetic regulation of glioblastoma tumorigenicity through LSD1 modulation of MYC expression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 979. doi:10.1158/1538-7445.AM2015-979

Glioblastoma is the most common form of brain cancer and remains a devastating disease. Recent studies revealed significant intra-tumoral heterogeneity in the genetic and epigenetic make-up of glioblastomas. One level of heterogeneity involves subpopulations of cells capable of tumor initiation (TI). Here we provide data suggesting that the transition between TI- and non-TI is a dynamic process governed by spontaneous fluctuation in the level of MYC. In vitro culturing of sub-clones derived from long-term passaged and primary glioblastoma lines revealed that only a subset of the sub-clones possess TI capacity. The property of TI for each individual sub-clone appeared stable through serial passages. However, a small fraction of the non-TI clones will spontaneously acquire the TI ability. This phenomenon was observed both in vitro and in vivo. Transcriptome profiling of the sub-clones with varied TI capacity revealed a gene signature enriched for genes regulated by MYC. Among various sub-clones, MYC expression levels correlated with TI capacity tightly. More direct evidence was provided by MYC overexpression which augmented TI capacity in both xenograft and genetic murine models. Reversely, MYC silencing abolished glioblastoma capacity for TI. Importantly, the sub-clones that spontaneously acquired capacity exhibited enhanced MYC expression. In freshly resected glioblastoma specimens, overall MYC expression levels of the specimens correlated with their xenograft-forming ability. When these specimens were sub-fractionated by A2B5, a cell surface marker enriched in TI glioblastoma populations, the MYC level was significantly elevated in the A2B5+ fraction relative to the A2B5- fraction. In The Cancer Genome Atlas (TCGA) glioblastoma specimens, the expression level of a MYC signature directly correlated with mRNA signatures associated with the Cancer Stem Cell states. In dual immunofluorescence staining of clinical glioblastoma specimens, C-MYC co-stained with MIB1, suggesting that MYC expression support in vivo tumor proliferation. Since the various sub-clones were genetically identical based on SNP array profiling, we hypothesized that fluctuations in MYC expression was regulated through epigenetic regulation. Supporting our hypothesis, the primary glioblastoma tumor lines that demonstrated high MYC levels showed higher ratios of H3K4me3 to H3K27me3 at the MYC locus. Culturing conditions that enhanced TI capacity of glioblastoma cell lines also increased the ratio of H3K4me3 to H3K27me3 and induced MYC expression. In sum, our results suggest a threshold model in which TI capacity is driven by epigenetic regulation of MYC. MYC inhibition constitutes an attractive therapeutic target since this inhibition reduces dynamic cell state transition and reduce the complexity of the tumor heterogeneity. This abstract is also presented as Poster B75. Citation Format: David Kozono, Jie Li, Masayuki Nitta, Oltea Sampetrean, Kimberly Ng, David Gonda, Deepa Kushwaha, Matsui Hiroko, Olivier Harismendy, Oren Becher, Chang-Hyuk Kwon, Keith L. Ligon, Hideyuki Saya, Bob S. Carter, Donald Pizzo, Scott Vandenberg, Clark C. Chen. Epigenetic regulation of MYC drives dynamic transition between tumor initiating states in glioblastoma. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr PR13. doi:10.1158/1538-7445.CHTME14-PR13

Von Recklinghausen's neurofibromatosis is a dominantly inherited cancer syndrome. Its gene encodes neurofibromin, a protein with ras GTPase-activating function (rasGAP) and, therefore, all NF1-associated pathology is thought to originate from selective deregulation of the ras pathway. We have constructed a variety of mouse models for NF1 that permit recapitulation of the most common tumors seen in patients. In addition, these mouse models offer insights into tumor origin and into paracrine interactions. Given the molecular and pathological fidelity of the mouse tumors to the human counterparts, it is hoped that these mouse strains will serve as effective tools for therapeutic discovery.

Somatic mutations of PTEN are found in many types of cancers including glioblastoma, the most malignant astrocytic tumor. PTEN mutation occurs in 25 to 40% of glioblastomas but is rarely observed in low-grade glial neoplasms. To determine the role of Pten in astrocytes and glial tumor formation, we inactivated Pten by a Cre-loxP approach with a GFAP-cre transgenic mouse that induced Cre-mediated recombination in astrocytes. Pten conditional knockout mice showed a striking progressive enlargement of the entire brain. Increased nuclear and soma size was observed in both astrocytes and neurons, which contributed in part to the increase in brain size. Pten-deficient astrocytes showed accelerated proliferation in vitro and aberrant ongoing proliferation in adult brains in vivo. In contrast, neurons lacking Pten did not show alterations in proliferation. This study shows cell-type dependent effects of Pten loss in the adult brain, including increased astrocyte proliferation that may render astroglial cells susceptible to neoplastic transformation or malignant progression.

The mechanisms that regulate mammalian cell size during development and homeostatic maintenance are poorly understood. The tumor suppressor Pten is required for correct maintenance of mammalian neuronal soma size. Selective inactivation of Pten in postnatal granule neurons of the cerebellum and dentate gyrus in mouse causes cell-autonomous hypertrophy as well as more complex phenotypes, including progressive macrocephaly, seizures, and premature death. To determine the contribution of mTor signaling to Pten-mediated growth regulation in the mammalian nervous system, we treated Pten conditional knockout mice with CCI-779, a specific mTor inhibitor. mTor inhibition decreased the seizure frequency and death rate in Pten mutant mice, prevented the increase in Pten-deficient neuronal soma size in young mice, and reversed neuronal soma enlargement in adult mice. mTor inhibition did not decrease the size of wild-type adult neurons. Thus, mTor is required for neuronal hypertrophy downstream of Pten deficiency, but is not required for maintenance of normal neuronal soma size. mTOR inhibitors may be useful therapeutic agents for diseases in brain resulting from PTEN deficiency such as Lhermitte–Duclos disease or glioblastoma multiforme.

Piwi-interacting RNAs (piRNAs) are a class of small non-coding RNAs that regulate differentiation in primordial germ cells, found mainly in the nucleus and cytoplasm. However, despite the essentiality of mitochondria to intrinsic apoptosis and energy metabolism, the presence of piRNAs (and their associated Piwi proteins) in this organelle remains largely unknown. In this study, a public database comparison of mitochondrial RNA-seq data from human normal (HEK293) and cancer (HeLa and 143B) cells revealed the presence of 29 mature piRNA sequences, with 12 of those piRNA sequences perfectly matching 7 tRNAs (Phe, Val, Leu, His, Ser, Glu, and Thr), suggesting piRNAs and tRNAs derive from a common precursor mitochondrial polycistronic transcript. Consistent with their known interaction with piRNAs, further examination revealed the presence of PIWI proteins in mitochondria. Moreover, the majority of the 29 piRNA sequences overlapped multiple longer sequences (expressed sequence tags, ESTs) that uniquely mapped to the mitochondrial genome. To our knowledge, this is the first report of Piwi and piRNAs in the mitochondria of mammalian normal and cancer cells. Citation Format: ChangHyuk Kwon, Hyosun Tak, Mina Rho, Hae Ryung Chang, Yon Hui Km, Kyung Tae Kim, Eun Kyung Lee, Curt Balch, Seungyoon Nam. Detection of PIWI-piRNAS in the mitochondria of human normal and cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 523. doi:10.1158/1538-7445.AM2014-523

Over the last decade, cytomegalovirus (CMV) has been suggested to promote the development of glioblastoma multiforme (GBM). Recent evidence demonstrates that CMV contributes to the progression of GBM in the context of oncosuppressor gene mutations. This finding provides further insights into the mechanisms whereby CMV exacerbates the malignancy of GBM.

Stomach cancer is the most prevalent cancer in Eastern Asia(1). Given the diverse route to oncognesis, it is highly attractive to develop targeted therapy based on each patient's biology(2). Several molecular targeting agents that show survival advantage in other cancer types are being under clinical investigation in gastric cancer (GC) (3-6). These agents focus on a few known oncogenic genes, yet its true efficacy of their biologically targeted therapies in gastric cancer is still unknown. To identify high-quality therapeutic targets, we utilized PATHOME (pathway and genome information), a computational method to identify therapeutic target through sub-pathway employing simple statistic-based approach (unpublished). PATHOME design had two stages: 1) discovery stage aimed at selecting target signaling pathway, and 2) validation stage aimed at identifying small number of reproducible genes from sub-pathways by using independent dataset to provide a novel theragnostic strategy in GC patients. Four sets of GEO database datasets were used: GSE13861 (84 Korean GC), GSE15081 (56 Japanese GC) for the discovery stage, and GSE36968 (25 Korean GC), and GSE27342 (160 Chinese GC) were added for validation. Using Vogelstein et al. as the gold standard, DAVID and GSEA vs. PATHOME were applied for comparison of potency in prediction of signaling pathways(7). Previous study showed that the energy sensing mechanism by AMPKα is involved in Asian GC(8). Metformin is a drug used to treat diabetes, which affects AMPKα in the cell. We treated GC cell line with metformin and saw the expression level of proteins that were selected by PATHOME to test its predictability of biological context. 13 up-regulated genes were identified using 3 datasets (GSE 13861, GSE 15081 and GSE36968), and 5 (GENE1, GENE2, GENE3, GENE4 and GENE5) from all 4 datasets using PATHOME. Transcription factor binding site analysis showed that two of the 5 genes contained GENE6 binding sites. Implications of AMPKα as a potential therapeutic target has been published(9). Metformin is shown to increase PRKAA2 level, which encodes AMPKα, also inhibits HNF4α(8). To see if metformin has any effect on the genes with HNF4α binding site, we observed the expression level of GENE4 and related proteins. Metformin treated GC cell lines (NCI-N87 and AGS) result in decreased viability compared to untreated cells. On Western-blot analysis, metformin treated cells show a decrease of GENE4, GENE6, but not GENE7 or GENE8. Utilizing PATHOME, which showed greater consistency compared to DAVID and GSEA in terms of cancer-related pathways, we showed Wnt pathway to have a potential impact on GC in East Asia. Therefore, our approach has simplified insight of multiplexed signals apply in molecular targeted therapies. We report a cross-talk between AMPKα and Wnt pathway in gastric cancer, which is known to be involved in other cancer types. This finding will lead to other potential drug targets for GC. Citation Format: Hae Ryung Chang, Seungyoon Nam, Hae Rim Jung, ChangHyuk Kwon, Hye-Hyun Seo, Frances S. Sung, Hee Seo Park, Taesung Park, Yon Hui Kim. Crosstalk between Wnt and AMPK pathway in Asian gastric cancer by metformin. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5540. doi:10.1158/1538-7445.AM2013-5540

Despite not being considered oncogenic, cytomegalovirus (CMV) has been detected in an increasing number of malignancies. However, recent studies have suggested mechanisms through which CMV may modulate the tumor environment, encouraging its study as a positive modifier of tumorigenesis. In this study, we investigated the effects of CMV infection in Trp53 heterozygous mice. Animals were infected with murine CMV (MCMV) after birth at 2 days (P2) or 4 weeks of age via intraperitoneal injection and then monitored for tumor formation. Additional control cohorts were mock-infected (saline vehicle) or infected with HSV-1, a similar herpes virus. Following MCMV infection, widespread systemic infection ensued followed by immune clearance of the virus resulting in seroconversion. Mice infected at P2 developed tumors at a high frequency (43%) by 9 months of age. In contrast, only 3% of mock-infected or mice infected at 4 weeks developed tumors. The majority of tumors from P2 MCMV-infected mice were pleomorphic rhabdomyosarcomas (RMS) as evidenced by MyoD1, Desmin, and Myogenin staining. Importantly, MCMV-infected tumors contained MCMV DNA, RNA, and protein. An examination of clinical cases revealed that human RMS (embryonal, alveolar, and pleomorphic) harbored human CMV IE1 and pp65 protein as well as viral RNA. All of the cases (n=18) tested positive for CMV genetic material. CMV protein was found throughout the tumor including pleomorphic cells. Additionally, perivascular areas demonstrated intense staining consistent with other reports of linking CMV with angiogenesis. To confirm the selective presence of HCMV DNA in human RMS, quantitative PCR analysis showed that tumors expressed an average of 81.1 copies/ng of HCMV-UL83 and of 22.2 copies/ng of HCMV-UL146 compared to 0 and 0.49 copies/ng in normal muscle, respectively. The amplified products’ nucleotide sequence was greater than 95% identical to that of a clinical strain Taken together, our findings offer support for the hypothesis that CMV contributes to the development of pleomorphic RMS in the context of Trp53 mutation, a situation which occurs with high frequency in human RMS. Citation Format: Richard L. Price, Katherine Bingmer, Lualhati Harkins, Hans Iwenofu, Chang-Hyuk Kwon, Charles Cook, Christopher Pelloski, E Antonio Chiocca. Cytomegalovirus infection leads to pleomorphic rhabdomyosarcomas in Trp53 +/- mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4777. doi:10.1158/1538-7445.AM2013-4777

The goal of this study is to evaluate SapC-DOPS, a novel cancer nanotherapeutic, for glioblastoma multiforme (GBM). SapC-DOPS delivered intravenously (i.v) was found to specifically target intracranial tumors in mice bearing spontaneous brain tumor, as well in nude mice intracranially implanted with human GBM cells. Treatment of tumor bearing mice with SapC-DOPS (i.v.) significantly increased survival: 25% and 75% long-term survivors in U87ΔEGFR-Luc and X12v2 implanted mice, respectively (P0.05). In contrast, SapC-DOPS treatment increased levels of an autophagic marker LC3-II via western blot. Autophagosome formation was also confirmed through transmission electron microscopy. Utilizing a stable GBM cell line expressing a GFP-LC3 fusion protein, we observed punctuated GFP expression following treatment, indicative of autophagosome formation. Quantification of GFP punctated cells showed a significant increase in SapC-DOPS treated cells compared to control (P Citation Format: Jeffrey Wojton, Naduparambil K. Jacob, Nicholas Denton, Nina Dmitrieva, Hiroshi Nakashima, Chang-Hyuk Kwon, Lionel Chow, Ennio A. Chiocca, Arnab Chakravarti, Balveen Kaur, Xiaoyang Qi. SapC-DOPS induces lethal mitophagy in glioblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2169. doi:10.1158/1538-7445.AM2013-2169

Aminoacyl-tRNA synthetases (ARSs) and ARS-interacting multifunctional proteins (AIMPs) exhibit remarkable functional versatility beyond their catalytic activities in protein synthesis. Their non-canonical functions have been pathologically linked to cancers. Here we described our integrative genome-wide analysis of ARSs to show cancer-associated activities in glioblastoma multiforme (GBM), the most aggressive malignant primary brain tumor. We first selected 23 ARS/AIMPs (together referred to as ARSN), 124 cancer-associated druggable target genes (DTGs) and 404 protein-protein interactors (PPIs) of ARSs using NCI's cancer gene index. 254 GBM affymetrix microarray data in The Cancer Genome Atlas (TCGA) were used to identify the probe sets whose expression were most strongly correlated with survival (Kaplan-Meier plots versus survival times, log-rank t-test

Recently several groups have demonstrated cytomegalovirus (CMV) protein and DNA in a large majority of glioblastomas (GBMs), though the exact role of CMV in tumors remains controversial. Although CMV is capable of activating several oncogenic pathways, it has never been shown to be a transforming virus. Because of this, we hypothesize that CMV infection can modify the rate of gliomagenesis in the context of genetic mutations known to predispose to tumor formation. To test this, we used the Smith strain mouse CMV (MCMV) to infect Mut3 (GFAP-cre; Nf1loxP/+; Trp53-/+) mice that develop normally but eventually develop spontaneous gliomas at an adult age After intraperitoneal viral inoculation, mice exhibited multisystemic infection throughout the body, including the brain. Mut3 mice infected perinatally (103 plaque forming units) developed gliomas significantly sooner than mock-infected Mut3 mice. MCMV-infected mice developed GBMs (WHO grade IV) at a higher rate than less severe tumors, whereas, mock-infected mice developed less severe gliomas (WHO grade III). Since MCMV preferentially infected the neural stem cell (NSC) niche, an area shown to generate gliomas, we interrogated changes in signaling in this population via microarray of cultured NSCs in MCMV- versus Mock-infected mice. Ingenuity Pathway Analysis identified a PDGF-B network as the highest scoring differentially regulated network between MCMV-and Mock-infected neurospheres. In vitro infection of mouse tumorspheres and human GBM tumorspheres with CMV validated the observed upregulation of PDGF-B, a molecule shown to cause gliomas de novo. Additionally, in vitro infection of human glioma stem cells with HCMV (Towne strain) or exogenous PDGF-B activated STAT3, a key regulator of gliomas. In mice infected with MCMV, PDGF-B is upregulated in CA2/3 neurons and STAT3 is activated in the subventricular zone and dentate gyrus suggesting a paracrine effect of MCMV-induced PDGF-B. Also, infected human brain tumor stem cells demonstrated an increase in proliferation via flow cytometry. Taken together, our data suggest that CMV in gliomas may accelerate GBM progression by activating PDGF-B/STAT3 signaling in the NSC population. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4815. doi:1538-7445.AM2012-4815

Introduction: Malignant glioma remains one of the most devastating diseases despite recent advances in therapies. Standard of care for malignant gliomas consists of maximal surgical resection, followed by external radiation with concurrent chemotherapy with temozolomide. Unfortunately, tumor recurrence is essentially inevitable and most of the therapies have only limited effects on the survival of patients experiencing tumor recurrence or progression. Emerging evidence has indicated that Brain Tumor Stem Cells (BTSC) can be the specific target of therapy and that elimination of BTSC will most likely result in the growth arrest of the entire tumor mass and the prevention of recurrence. The current therapies, however, preferentially target non-stem tumor cells. Thus, identification of BTSC-specific drugs is highly recommended in order to eradicate the entire tumor cells. To discover the prospective compounds from a large number of candidates efficiently, we have developed here the screening system for BTSC-targeting chemotherapeutic agents. Candidate compounds are screened in a stepwise manner based on their inhibitory potential against BTSC by using several in vitro and in vivo preclinical assays. Being different from the conventional compound discovery procedure, we combine multiple assays aiming at detection of the drugs directly targeting BTSC. Thus, this system functions as a rational agent-prioritization process essential for future progress in developing effective novel cancer treatments. Experimental Approach: We use several human BTSC cultures derived from surgical specimens. These samples have been confirmed to be clonogenic and multipotent in culture, and tumorigenic by xenotransplantation in immunodeficient mice brains. To assess the effect of compounds, the initial step is to use clonogenic assay, cell survival and migration assays with slice cultures of mouse brains, and xenotransplantation of pretreated BTSC. Potential compounds are then processed to the in vivo efficiency and efficacy validation assays using the following animal models, 1. BTSC-derived mouse brain tumor model: patient-derived BTSC transplanted in the mouse brain, 2. Genetically-engineered spontaneous mouse brain tumor model, and 3. Subcutaneous xenograft model of surgical specimens of brain tumor tissues: tumor tissue from patient directly transplanted subcutaneously into immunodeficient mice. Effect of the treatment on tumor progression is monitored by several imaging modalities including microMRI, SPECT/CT, and PET/CT. We also carry out pharmacokinetics profiling to determine the serum levels of the tested compounds and the maximum tolerated dose evaluation to determine their toxicities in comparison to their antitumor activities. In this presentation, we describe the strengths and limitations of each of our assays and also our data with the leading compounds from the previous screening. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5504.

High grade astrocytomas are aggressive brain tumors that are associated with a dismal prognosis and are incurable with a median survival of less than 15 months despite intensive multimodal therapy. The linability to effectively target grade III and grade IV (glioblastoma multiforme, GBM) astrocytomas highlights the need for novel therapeutic approaches. Recent studies have shown that epigenetic regulation of chromatin plays a central role in the control of cell growth, differentiation, and survival. Chromatin remodeling enzymes like histone deacetylases, DNA methyltransferases and protein arginine methyltransferase 5 (PRMT5) are involved in silencing tumor suppressor gene (TSG) expression and may contribute towards cellular transformation. PRMT5 silences the transcription of key regulatory genes by symmetric di-methylation (S2Me) of arginine (R) residues on histone proteins (H4R3 and H3R8) and works more efficiently when associated with other co-repressor enzymes. Eight patient-derived GBM cell lines and 45 primary GBM tumors showed abundant expression of PRMT5 protein. Confocal microscopy and immunohistochemical staining showed the PRMT5 signal to localize primarily to the nucleus. Normal brain tissue, normal human astrocytes and low/intermediate grade astrocytomas failed to show PRMT5 expression. The degree of PRMT5 over expression inversely correlated with survival of GBM patients (r=-0.57, p=0.0001) and correlated with proliferation of GBM cell lines (r=0.81, p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1584.

Malignant astrocytomas are infiltrative and incurable brain tumors. Despite profound therapeutic implications, the identity of the cell (or cells) of origin has not been rigorously determined. We previously reported mouse models based on conditional inactivation of the human astrocytoma-relevant tumor suppressors Nf1, p53, and Pten, wherein through somatic loss of heterozygosity, mutant mice develop tumors with 100% penetrance. In the present study, we show that tumor suppressor inactivation in neural stem cells induces malignant astrocytoma formation. Genetic targeting of lineage-restricted progenitors alone is also sufficient to initiate astrocytomas. We demonstrate in vivo that transformed cells and their progeny undergo infiltration and multi-lineage differentiation in situ during tumor development. Analysis of mutant mice several months prior to advanced disease shows defects in proliferation, differentiation, and migration, suggesting that these are requisite processes prior to tumor formation. Gene expression profiling in pre-symptomatic mutant mice provides insight into the molecular events involved during brain tumor development. Citation Information: Cancer Res 2009;69(23 Suppl):C10.