Browsing by Author "Yan, Hai"

Gliomas are the most common tumors of the central nervous system. Our lab recently identified mutations in IDH1 and IDH2 as occurring frequently in progressive gliomas. We applied a series of biochemical and genetic approaches to explore the roles of the mutations in tumors and generate models for study.

IDH1/2 mutations have the potential to impact a number of metabolic pathways. IDH1/2 convert isocitrate to α-ketoglutarate while simultaneously converting NADP+ to NADPH. To assess changes in metabolism, we completed metabolic profiling and complementary studies in cell lines with and without mutant IDH1 or mutant IDH2. We identified a decrease in hypoxia signaling and a decrease in global 5-hydroxymethylcytosine in cell lines with mutant IDH1/2 .

Having observed mutations in IDH1/2 in a large fraction of progressive gliomas, we asked if the mutations were either 1) advantageous for growth in brain parenchyma, or 2) advantageous in a particular cell-of-origin. Sequencing of a series of metastases to the brain from non-central nervous system tumors identified no mutations in IDH1/2, lending less credence to the first hypothesis. To elucidate whether mutations in IDH1/2 can initiate glioma progression and explore the potential cell-of-origin for progressive gliomas, we generated mice in which we induced expression of mutant IDH2 in different populations of cells in the brain, either alone or in combination with TP53 deletion, another frequently altered gene in progressive gliomas. Mice with broad expression of mutant IDH2 developed hydrocephalus and encephalomalacia early in life, but did not develop tumors. Therefore, we restricted expression, and two brain tumors were identified in mice with both IDH2 mutation and TP53 deletion. While this suggests that both mutations might be required for the development of tumors, this is too small a number to draw significant conclusions. Further research with an expanded cohort of mice, utilization of additional drivers of expression, and further characterization of identified tumors will help in elucidating the role of mutant IDH2 and the cell-of-origin for progressive gliomas.

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.

Primary glioblastoma (GBM) is the most common and lethal primary malignant brain tumor, with a median patient survival of only 15 months from the time of diagnosis. GBM is particularly challenging to treat due to its aggressive and invasive nature, and has proven resistant to therapeutic advances, with no significant improvement in outcomes over the past several decades. Understanding of the molecular characteristics of GBM, however, has improved dramatically, with genetic, epigenetic, and transcriptomic classifications now able to divide GBM into subtypes that provide prognostic information and guide the organization of clinical trials. One of the most frequent genetic alterations that has been identified in GBM is homozygous deletion of the methylthioadenosine phosphorylase (MTAP) gene, which occurs in 50% of all GBM cases. Despite its common occurrence, it is unclear what contribution MTAP loss makes in the pathogenesis of GBM or whether this genetic alteration can be used as a therapeutic target.

MTAP is a metabolic enzyme in the salvage pathway of adenine and methionine and its absence results in the accumulation of its metabolic substrate, methylthioadenosine (MTA), within and around tumor cells. MTA is known to inhibit activity of methyltransferases, raising the possibility that MTA accumulation is interfering with regulatory processes within the cell.

We utilized patient-derived GBM cell lines in vitro and GBM xenografts in vivo, to characterize consequence of MTAP deletion in GBM through analysis of DNA methylation, gene expression, and response to therapeutic agents. We show that MTAP loss promotes the formation of glioma stem-like cells through epigenomic dysregulation. We show these epigenetic changes influence gene expression patterns and alter the sensitivity to epigenome-modifying drugs. We also demonstrate that MTAP-null GBM cells are more tumorigenic in experimental models and that patients with MTAP deletion have poor disease outcomes. Finally, we show that targeting metabolic liabilities of MTAP-null cells through inhibition of de novo purine synthesis specifically depletes the therapy-resistant, stem-like cell subpopulation of GBM.

As the final component of this work, we explore the impact of MTA accumulation in the tumor microenvironment. We found that MTA alters the function of immune cells through adenosine receptor signaling, suggesting that modulation of adenosine receptor signaling in GBM may improve the native immune response and the efficacy of immunotherapeutics in the treatment of this disease.

This work thus establishes MTAP deletion as a pathogenic genetic alteration in the process of gliomagenesis by illustrating it’s contribution to the formation of the cancer cell epigenomic landscape, stemness characteristics, growth, and response to therapeutic agents.

Gliomas are tumors of the central nervous system for which improvements in treatment are critically needed. Mutations in IDH1 and IDH2, which encode the cytosolic and mitochondrial NADP+-dependent isocitrate dehydrogenases, respectively, are frequent in gliomas. Here, we summarize recent literature concerning gliomas, the normal cellular functions of IDH1/2, the epidemiology of IDH1/2 mutations, and the understanding of the function of IDH1/2 mutations in cancer. We then show in vitro using liquid chromatography-mass spectrometry that a function of many IDH1/2 mutations is to produce 2-hydroxyglutarate. Next, we use a mass spectrometry based platform to characterize metabolic changes in a glioma cell line expressing IDH1/2 mutants and show that the IDH mutants are associated with lowered N-acetylated amino acids both in this cell line model and in primary tumor tissue. Finally, we develop and characterize a Drosophila melanogaster (fruit fly) model of IDH1/2-mutated cancer by expressing the mutated Drosophila homolog of IDH1 in fly tissues using the UAS-Gal4 binary expression system. These results delineate downstream molecular players that likely play a role in IDH1/2-mutated cancer and provide a model organism for interrogation of genetic networks that interact with IDH1/2 mutation. These findings refine our understanding of glioma pathogenesis and may inform the design of new glioma therapies.

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.

Gliomas of the brainstem and closely surrounding structures are a devastating form of brain cancers with some of the highest mortality rates among all cancers. The delicate structures surrounding these tumors make surgical resection rare due to the risk of perioperative mortality or paralysis. A lack of available tissue has created a dearth of genomic information regarding gliomas effecting specific structures in the brainstem impacting options for intervention.

This work comprises two major projects of brainstem gliomas. The first part is to utilize next generation sequencing technique to discover novel oncogenes, and to use epigenetic features to characterize brainstem gliomas. The second part is to utilize integrated analysis of whole genome sequencing, methylation microarray, RNA sequencing data to establish the classification of brainstem gliomas.

In the first part we completed exomic and Sanger sequencing on 33 brainstem gliomas and 17 thalamic gliomas to reveal major findings regarding their respective oncogenomes. We reported the presence of IDH1 mutations occurring in brain- stem gliomas exclusive of H3.3 mutations. We further revealed somatic mutations to PPM1D, the gene encoding for the Protein phosphatase 1D in 37.5% of brainstem gliomas harboring H3.3 mutations. We determined that PPM1D mutations are mu- tually exclusive with TP53 mutations in brainstem gliomas. This was the largest body of genomic work completed on specific loci within the brainstem providing new insights into their respective oncogenomes and presents PPM1D as a new oncotarget.

In the second part we then performed a comprehensive study incorporating com- bined epigenetic and genomic analyses with clinical features, aiming to understand the genetics and molecular biology of this disease. From DNA methylation data, we identified four distinct clusters termed H3-Pons, H3-Medulla, IDH, and PA-like, each associated with unique genomic and clinical profiles. The majority of tumors within H3-Pons and H3-Medulla harbored H3F3A mutations, but showed distinct methy- lation patterns that correlated with anatomical localization of these tumors within the pons or medulla, respectively. Clinical data also showed significantly different overall survival between these clusters, and pathway analysis demonstrated different mechanisms in these samples. In summary, we performed a comprehensive molecular profiling study on a large brainstem glioma cohort, including Diffuse Intrinsic Pon- tine Gliomas. Our findings indicate that the integration of DNA methylation data may facilitate a better understanding of brainstem glioma classification and guide future studies for the development of novel treatments for this disease.

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.

Glioma development and progression are driven by complex genetic alterations, including point mutations and gain or loss of genomic copy number, as well as epigenetic aberrations, including DNA methylation and histone modifications. However, the molecular mechanisms underlying the causes and effects of these alterations are poorly understood, and improved treatments are greatly needed. Here, we report a comprehensive evaluation of the recurrent genomic alterations in gliomas and further dissect the molecular effects of the most frequently-occurring genomic events. First, we performed a multifaceted genomic analysis to identify genes targeted by copy number alteration in glioblastoma, the most aggressive malignant glioma. We identify EGFR negative regulator, ERRFI1, as a glioblastoma-targeted gene within the minimal region of deletion in 1p36.23. Furthermore, we demonstrate that Aurora-A kinase substrate, TACC3, displays gain of copy number on 4p16.3 and is overexpressed in a grade-specific pattern. Next, using a gene targeting approach, we knocked-in a single copy of the most frequently observed point mutation in gliomas, IDH1R132H/WT, into a human cancer cell line. We show that heterozygous expression of the IDH1R132H allele is sufficient to induce the genome-wide alterations in DNA methylation characteristic of these tumors. Together, these data provide insight on genetic and epigenetic alterations which drive human gliomas.

Medulloblastoma is the most common malignant brain tumor in children. The understanding of the genetic alterations in this tumor is emergent, and many such genetic driver events have yet to be functionally-characterized. Our studies have sought to understand the causes and consequences of OTX2 dysregulation in established medulloblastomas and in its putative cellular origins. Using a tumor genetic approach, we have uncovered frequent OTX2 copy number gains driving expression of this oncogene in a subset of medulloblastomas. However, OTX2 is frequently expressed in medulloblastomas independent of genomic copy number gain, and we thus sought to understand the transcriptional regulation of this gene in these tumors. We have found that chromatin accessibility, promoter DNA methylation, and activity of a distal downstream enhancer is distinct between OTX2-expressing and -nonexpressing medulloblastomas. Notably, autoregulation serves to maintain OTX2 expression in some medulloblastomas, whereas DNA methylation actively suppresses OTX2 in tumors not expressing this gene. Finally, we describe the effect of expressing Otx2 (the mouse homolog of OTX2) aberrantly in the developing mouse hindbrain, revealing that Otx2 disrupts spatiotemporal restrictions of neuronal progenitor cell proliferation. The effect of Otx2 in vivo is transient, with ectopically-proliferating cells give way to differentiated neurons. We found that OTX2 expression was not able to give rise to high penetrance medulloblastoma when combined with P53 deletion or double heterozygosity for P53 and PTEN. Thus, although Otx2 alters migration and proliferation dynamics of hindbrain neuronal progenitor cells, further studies are needed to identify the genetic alterations that cooperate with this oncogene to give rise to medulloblastoma.