Browsing by Subject "Glioma"

Human cancer is a leading cause of morbidity and mortality in the developed world. Contrary to the classical model in which tumors are homogeneously composed of malignant cells, accumulating evidence suggests that subpopulations of highly malignant cells play a dominant role in tumor initiation and growth. These cells have the capacity for prolonged self-renewal and they efficiently generate tumors that phenotypically resemble the parental tumor in transplantation assays. Such characteristics are reminiscent of normal stem cells, and these potently tumorigenic cells have therefore been called cancer stem cells (CSCs). Importantly, studies have shown that CSCs are central mediators of therapeutic resistance, tumor angiogenesis, and metastatic or invasive potential. In the case of malignant glioma, poor patient survival and the paucity of effective therapeutic advances have been attributed to inherent CSC growth potential and treatment resistance, respectively. For this reason, there is great interest in elucidating the molecular features of CSCs, with the ultimate hope of developing CSC-directed therapies.

Given the overlap between the highly malignant characteristics exhibited by CSCs and those promoted by the PI3K/AKT pathway, we hypothesized that AKT activity within CSCs could represent a reasonable therapeutic target for CSC-directed therapies. Indeed, a pharmacological inhibitor of AKT preferentially targeted glioma CSCs versus non-CSCs and was associated with increased apoptosis and impaired tumorigenesis. These data suggest that interventions targeting AKT could effectively target glioma CSCs.

Quite distinct from the PI3K/AKT pathway, we hypothesized that the pro-survival and pro-growth features of nitric oxide (NO) might also operate in glioma CSCs. Our experiments found that glioma CSCs produced more NO than non-CSCs, which is attributed to inducible nitric oxide synthase (iNOS) expression and activity within the CSCs. Interference with iNOS activity or expression, as well as selective NO consumption, attenuated CSC growth and tumorigenicity. The mechanism behind iNOS-mediated survival appears to involve, at least in part, suppression of the cell cycle inhibitor CDA1. iNOS inhibition decreased glioma growth in murine xenografts and human expression studies demonstrate an inverse correlation between iNOS expression and patient survival.

To more fully evaluate the biological effects of NO in CSCs, we designed a novel strategy to consume NO within mammalian cells through heterologous expression of E. coli flavohemoglobin (FlavoHb). This enzyme is a highly specific NO dioxygenase which converts NO to inert nitrate several orders of magnitude faster than iNOS synthesizes NO. Expression of FlavoHb in mammalian cells is therefore a novel and functional tool to interrogate the role of NO in cellular stress and signaling.

In summary, this doctoral thesis focuses on several molecular characteristics that define malignant CSCs and describes a novel strategy for studying NO, which is one of the CSC-specific molecular effectors.

Malignant gliomas are highly lethal cancers characterized by florid angiogenesis. Glioma stem cells (GSCs), enriched through CD133 (Prominin1) selection, are highly tumorigenic and therapy resistance. However, the mechanism through which GSCs promote tumor growth was largely unknown. As we noticed that tumors derived from GSCs contain widespread tumor angiogenesis, necrosis, and hemorrhage, we examined thepotential of GSCs to support tumor angiogenesis. We measured the expression of a panel of angiogenic factors secreted by GSCs. In comparison with matched non-GSC populations, GSCs consistently secreted markedly elevated levels of vascular endothelial growth factor (VEGF), which were further induced by hypoxia. In an in vitro model of angiogenesis, GSC-conditioned medium significantly increased endothelial cell migration and tube formation compared with non-GSC glioma cell-conditioned medium. The proangiogenic effects of GSCs on endothelial cells were specifically abolished by the anti-VEGF neutralizing antibody bevacizumab, which is in clinical use for cancer therapy. Furthermore, bevacizumab displayed potent antiangiogenic efficacy in vivo and suppressed growth of xenografts derived from GSCs but limited efficacy against xenografts derived from a matched non-GSC population. As hypoxia is a key regulator of angiogenesis, I further examined hypoxic responses in GSCs to determine the molecular mechanisms underlying their angiogenic drive. I demonstrated that multiple hypoxia response genes, including the hypoxia-inducible factors (HIFs)-1a and -2a(EPAS-1) were differentially expressed in GSCs in comparison to non-stem glioma cells and normal neural progenitors. GSCs preferentially induced HIF2a; and HIF2a-regulated genes under hypoxia in comparison to non-stem glioma cells. In contrast, neural progenitor/stem cells did not induce HIF2a in response to hypoxia suggesting that the HIF2a hypoxic response is not a general stem cell response. Targeting HIF1a or HIF2a in GSCs using short hairpin RNA (shRNA) inhibited neurosphere formation efficiency, indicating a requirement for HIFs in cancer stem cell self-renewal. HIF1a and HIF2a were also necessary for VEGF expression in GSCs, but HIF2a was not required in matched non-stem glioma cells. In vivo experiments determined that knockdown of HIFs significantly attenuated the tumorigenic capacity of GSCs and increased survival of immunocompromised mice. Together, our work provides the first evidence that that GSCs can be a crucial source of key angiogenic factors in cancers due to their differential hypoxia responses. It also suggests that anti-angiogenic therapies can be designed to target GSC-specific molecular mechanisms of neoangiogenesis, including the expression and/or activity of HIF2a.

Despite decades worth of research, glioblastoma remains one of the most lethal cancers. The identification of IDH1 as a major cancer gene in glioblastoma provides an exceptional opportunity for improving our understanding, diagnostics, and treatment of this disease. In addition to mutations in IDH1, recent studies from our laboratory have characterized the genetic landscape of gliomas and have shown the cooperation between IDH1 mutations and other oncogenic alterations such at TP53 mutations. Normally, IDH1 functions in the oxidative decarboxylation of isocitrate to α–ketoglutarate, however the mutant form confers neomorphic enzymatic activity by producing 2–hydroxyglutarate, an oncometabolite responsible for aberrant methylation in IDH1–mutated tumors, among other mutant IDH1–mediated phenotypes. To determine the role of mutant IDH1 in vivo, we generated a conditional knock–in mouse model. This genetically faithful system is both biologically and clinically relevant and will promote the understanding of mutant IDH1–mediated tumorigenesis while offering a route for therapeutic targeting.

We observed that broad expression of mutant IDH1 throughout the brain leads to hydrocephalus in 80% of animals. In assessing the earliest effects of mutant IDH1 on the brain, we determined mutant IDH1 confers a decrease in the proliferative cells of the subventricular zone of the lateral ventricle, the area which houses the neural stem cells in embryonic and adult animals. Additionally, a perturbation to the normal neural stem cell niche was observed in these animals. Combined, this data suggests that mutant IDH1 may be affecting the signaling pathways involved in differentiation in this population of cells. In vivo and in vitro studies will further elucidate mutant IDH1's effects on the differentiation patterns of neural stem cells expressing mutant IDH1.

To express mutant IDH1 in a more restricted manner and harness spatiotemporal control, we crossed mutant animals to a Nestin–CreERT2 strain of mouse that permits expression of floxed alleles upon treatment with tamoxifen. Animals were sacrificed at the onset of symptoms or at 1–year of age. We observed the development of both low– and high–grade gliomas in approximately 15–percent of E18.5 tamoxifen–treated animals. All tumors were found in a TP53–deleted background with mutant IDH1 being detected in only those tumors with the mutant allele. Lastly, to decrease the latency and increase the penetrance of tumor formation, an orthotopic intracranial injection model was generated to allow for visualization of tumor formation and development, as well as investigation of therapeutic modalities. The models generated and the knowledge gained from these studies will offer an understanding of the biological effects of the most common mutations found in the astrocytic subset of gliomas, bringing us strides closer to determining mechanisms and therapeutic targets for IDH1–mutated cancers.

Gliomagenesis is driven by a complex network of genetic alterations and while the glioma genome has been a focus of investigation for many years; critical gaps in our knowledge of this disease remain. The identification of novel molecular biomarkers remains a focus of the greater cancer community as a method to improve the consistency and accuracy of pathological diagnosis. In addition, novel molecular biomarkers are drastically needed for the identification of targets that may ultimately result in novel therapeutics aimed at improving glioma treatment. Through the identification of new biomarkers, laboratories will focus future studies on the molecular mechanisms that underlie glioma development. Here, we report a series of genomic analyses identifying novel molecular biomarkers in multiple histopathological subtypes of glioma and refine the classification of malignant gliomas. We have completed a large scale analysis of the WHO grade II-III astrocytoma exome and report frequent mutations in the chromatin modifier, alpha thalassemia mental retardation x-linked (ATRX), isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2), and mutations in tumor protein 53 (TP53) as the most frequent genetic mutations in low grade astrocytomas. Furthermore, by analyzing the status of recurrently mutated genes in 363 brain tumors, we establish that highly recurrent gene mutational signatures are an effective tool in stratifying homogeneous patient populations into distinct groups with varying outcomes, thereby capable of predicting prognosis. Next, we have established mutations in the promoter of telomerase reverse transcriptase (TERT) as a frequent genetic event in gliomas and in tissues with low rates of self renewal. We identify TERT promoter mutations as the most frequently mutated gene in primary glioblastoma. Additionally, we show that TERT promoter mutations in combination with IDH1 and IDH2 mutations are able to delineate distinct clinical tumor cohorts and are capable of predicting median overall survival more effectively than standard histopathological diagnosis alone. Taken together, these data advance our understanding of the genetic alterations that underlie the transformation of glial cells into neoplasms and we provide novel genetic biomarkers and multi – gene mutational signatures that can be utilized to refine the classification of malignant gliomas and provide opportunity for improved diagnosis.

Malignant gliomas account for more than 80% of all primary brain malignancies and 14,000 deaths in the U.S. annually. Despite aggressive treatment, malignant gliomas are largely fatal, as their invasive nature renders them prone to rapid recurrence. Gliomas are classified according to histopathologic criteria which are critical to treatment planning, as certain subtypes show increased sensitivity to particular therapeutic agents. However, gliomas often exhibit extensive tumor heterogeneity and ambiguity among histologic features, leading to subjectivity in diagnosis and low concordance rates among neuropathologists.

Recently, a number of large-scale genomic studies identified mutations in the TERT promoter and IDH1/2 in ~80% of all gliomas. Based on the occurrence of these mutations, gliomas can be classified into objective molecular subtypes that stratify patients into clear prognostic subgroups more effectively than by histology alone. However, current sequencing-based methods to identify these alterations are limited by low sensitivity (40% tumor cellularity), a major constraint on their clinical utility in the context of diffusely infiltrative gliomas. Importantly, this work also revealed that 20% of glioblastomas lack these alterations, delineating a subset of tumors known as the TERT promoter wildtype-IDH wildtype (TERTpWT-IDHWT) glioblastomas.

Preliminary studies indicate that TERT promoter and IDH mutations can effectively stratify the majority (80%) of patients into clinically-relevant genetic subtypes, however current mutation detection methods lack sensitivity (Sanger sequencing) or are overly time-consuming (next-generation sequencing). Here, we report the development of a qPCR-based approach which can provide more sensitive and rapid detection of these mutations and practical utility in glioma diagnosis by detecting low-abundance mutations (e.g., poorly sampled tumors). Finally, we report the genetic landscape of TERTpWT-IDHWT glioblastomas using whole exome and whole genome sequencing, revealing that these tumors harbor a unique set of genetic alterations and exhibit distinct genetic mechanisms of telomere maintenance from other known subgroups of GBM, including recurrent SMARCAL1 mutations and rearrangements upstream of TERT. Using cell-based assays and markers of alternative lengthening of telomeres (ALT), we provide evidence showing that SMARCAL1 acts as a tumor and ALT suppressor and that loss of function cancer-associated mutations are involved in ALT mechanism of telomere maintenance.

These studies have identified the key underlying genetic alterations that characterize TERTpWT-IDHWT glioblastomas, and can serve as biomarkers for more accurate diagnosis and treatment of this glioma subgroup. By developing a sensitive diagnostic for the critical TERTp and IDH alterations, we facilitate accurate diagnosis and prognostication of glioma patients.

Point mutations in the active site of isocitrate dehydrogenases 1 and 2 (\textit{IDH}) occur in the majority of WHO grade II and III gliomas, resulting in a unique milieu of signaling and metabolism. IDH1/2 active site mutations confer a gain-of-function activity to the enzyme, which results in the production of the oncometabolite D-2-hydroxyglutarate (D-2HG). D-2HG accumulation in turn promotes tumor formation through competitive inhibition of $\alpha$-ketoglutarate dependent ($\alpha$-KG) enzymes. Inhibition of $\alpha$-KG-dependent enzymes, such as histone demethylases and DNA demethylases, is sufficient to induce tumor-promoting epigenetic changes, but can also impose situational constraints on cell proliferation. To develop better therapies for mutant IDH1-bearing gliomas, it is essential to determine whether the epigenetic changes induced by the mutant IDH proteins actively require the mutation after tumor formation. Furthermore, it is imperative to decode the molecular mechanisms that promote tumor cells’ fitness under IDH mutation-dependent constraints in representative models. Here, we describe and characterize CRISPR-Cas9 based isogenic cell line models using patient-derived IDH1$^{R132H/WT}$ glioma cell lines. We uncover that these models show persistent DNA hypermethylation in CpG loci of the glioma CpG island methylator phenotype even after D-2HG production has been abolished. We also report a genome wide pattern of DNA demethylation in CpG sites outside of CpG islands, which reflect the acquisition of a G-CIMP-low like state after loss of D-2HG production. Then, using these cell line tools, we performed an unbiased sub-genomic CRISPR-library screening to identify genes whose functions supported the growth of glioma cells bearing endogenous IDH1 mutations. This work thus provides new patient derived models for exploring novel therapeutic opportunities for IDH1 mutant tumors, and uncovers the extent to which IDH mutation linked hypermethylation profiles in glioma depend upon D-2HG production from the IDH mutation.