D-2-HG Inhibits IDH1mut Glioma Growth via FTO Inhibition and Resultant m6A Hypermethylation.

IDH1mut gliomas produce high levels of D-2-hydroxyglutarate (D-2-HG), an oncometabolite capable of inhibiting α-ketoglutarate-dependent dioxygenases critical to a range of cellular functions involved in gliomagenesis. IDH1mut gliomas also exhibit slower growth rates and improved treatment sensitivity compared with their IDH1wt counterparts. This study explores the mechanism driving apparent reduced growth in IDH1mut gliomas. Specifically, we investigated the relationship between IDH1mut and the RNA N6-methyladenosine (m6A) demethylases FTO and ALKBH5, and their potential for therapeutic targeting. We investigated the role of D-2-HG and m6A in tumor proliferation/viability using glioma patient tumor samples, patient-derived gliomaspheres, and U87 cells, as well as with mouse intracranial IDH1wt gliomasphere xenografts. Methylation RNA immunoprecipitation sequencing (MeRIP-seq) RNA sequencing was used to identify m6A-enriched transcripts in IDH1mut glioma. We show that IDH1mut production of D-2-HG is capable of reducing glioma cell growth via inhibition of the m6A epitranscriptomic regulator, FTO, with resultant m6A hypermethylation of a set of mRNA transcripts. On the basis of unbiased MeRIP-seq epitranscriptomic profiling, we identify ATF5 as a hypermethylated, downregulated transcript that potentially contributes to increased apoptosis. We further demonstrate how targeting this pathway genetically and pharmacologically reduces the proliferative potential of malignant IDH1wt gliomas, both in vitro and in vivo. Our work provides evidence that selective inhibition of the m6A epitranscriptomic regulator FTO attenuates growth in IDH1wt glioma, recapitulating the clinically favorable growth phenotype seen in the IDH1mut subtype. SIGNIFICANCE: We show that IDH1mut-generated D-2-HG can reduce glioma growth via inhibition of the m6A demethylase, FTO. FTO inhibition represents a potential therapeutic target for IDH1wt gliomas and possibly in conjunction with IDH1mut inhibitors for the treatment of IDH1mut glioma. Future studies are necessary to demonstrate the role of ATF5 downregulation in the indolent phenotype of IDH1mut gliomas, as well as to identify other involved gene transcripts deregulated by m6A hypermethylation.

dCas9/CRISPR-based methylation of O-6-methylguanine-DNA methyltransferase enhances chemosensitivity to temozolomide in malignant glioma.

BACKGROUND: Malignant glioma carries a poor prognosis despite current therapeutic modalities. Standard of care therapy consists of surgical resection, fractionated radiotherapy concurrently administered with temozolomide (TMZ), a DNA-alkylating chemotherapeutic agent, followed by adjuvant TMZ. O-6-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme, removes alkylated lesions from tumor DNA, thereby promoting chemoresistance. MGMT promoter methylation status predicts responsiveness to TMZ; patients harboring unmethylated MGMT (~60% of glioblastoma) have a poorer prognosis with limited treatment benefits from TMZ. METHODS: Via lentiviral-mediated delivery into LN18 glioma cells, we employed deactivated Cas9-CRISPR technology to target the MGMT promoter and enhancer regions for methylation, as mediated by the catalytic domain of the methylation enzyme DNMT3A. Methylation patterns were examined at a clonal level in regions containing Differentially Methylation Regions (DMR1, DMR2) and the Methylation Specific PCR (MSP) region used for clinical assessment of MGMT methylation status. Correlative studies of genomic and transcriptomic effects of dCas9/CRISPR-based methylation were performed via Illumina 850K methylation array platform and bulk RNA-Seq analysis. RESULTS: We used the dCas9/DNMT3A catalytic domain to achieve targeted MGMT methylation at specific CpG clusters in the vicinity of promoter, enhancer, DMRs and MSP regions. Consequently, we observed MGMT downregulation and enhanced glioma chemosensitivity in survival assays in vitro, with minimal off-target effects. CONCLUSION: dCas9/CRISPR is a viable method of epigenetic editing, using the DNMT3A catalytic domain. This study provides initial proof-of-principle for CRISPR technology applications in malignant glioma, laying groundwork for subsequent translational studies, with implications for future epigenetic editing-based clinical applications.

Retrospective examination of pseudoprogression in IDH mutant gliomas.

Background: Tumor surveillance of isocitrate dehydrogenase (IDH) mutant gliomas is accomplished via serial contrast MRI. When new contrast enhancement (CEnew) is detected during postsurgical surveillance, clinicians must assess whether CEnew indicates pseudoprogression (PsP) or tumor progression (TP). PsP has been better studied in IDH wild-type glioblastoma but has not been well characterized in IDH mutant gliomas. We conducted a retrospective study evaluating the incidence, predictors, natural history, and survival of PsP patients in a large cohort of IDH mutant glioma patients treated at a single institution. Methods: We identified 587 IDH mutant glioma patients treated at UCLA. We directly inspected MRI images and radiology reports to identify CEnew and categorized CEnew into TP or PsP using MRI or histopathology. Results: Fifty-six percent of patients developed CEnew (326/587); of these, 92/326 patients (28% of CEnew; 16% of all) developed PsP and 179/326 (55%) developed TP. All PsP patients had prior radiation, chemotherapy, or chemoradiotherapy. PsP was associated with longer overall survival (OS) versus TP patients and similar OS versus no CEnew. PsP differs from TP based on earlier time of onset (median 5.8 vs 17.4 months from treatment, P < .0001) and MRI features that include punctate enhancement and enhancement location. Conclusion: PsP patients represented 28% of CEnew patients and 16% of all patients; PsP patients demonstrated superior outcomes to TP patients, and equivalent survival to patients without CEnew. PsP persists for <1 year, occurs after treatment, and differs from TP based on time of onset and radiographic features. Poor outcomes after CEnew are driven by TP.

A single-institution retrospective analysis of pathologically determined malignant transformation in IDH mutant glioma patients.

Background: Lower-grade IDH mutant glioma patients frequently undergo malignant transformation (MT), with apparent worse prognosis. Many studies examine MT in mixed IDH status cohorts and define MT using imaging, not histopathology. Our study examines the timing, predictors, and prognostic implications of pathologically determined MT in a large, exclusively IDH mutant cohort. Methods: We identified 193 IDH mutant lower-grade glioma patients at UCLA who received multiple surgeries. We examined the outcomes of pathologically determined MT patients. Results: Time to MT is longer in grade 2 oligodendroglioma (G2 Oligo) than in grade 2 astrocytoma (G2 Astro) (HR = 0.46, P = .0007). The grade 3 astrocytoma (G3 Astro) to grade 4 astrocytoma (G4 Astro) interval is shorter in stepwise MT (G2 to G3 to G4 Astro) patients than in initial G3 Astro patients (P = .03). Novel contrast enhancement had 65% positive predictivity, 67% negative predictivity, 75% sensitivity, and 55% specificity in indicating pathologically defined MT. In G2 Astro, initial gross total resection delayed MT (HR = 0.50, P = .02) and predicted better overall survival (OS) (HR = 0.34, P = .009). In G2 Oligo, spontaneous MT occurred earlier than treated MT (HR = 11.43, P = .0002), but treatment did not predict improved OS (P = .8). MT patients (n = 126) exhibited worse OS than non-MT patients (n = 67) in All (HR = 2.54, P = .0009) and G2 Astro (HR = 4.26, P = .02). Conclusion: Our study expands the understanding of MT to improve IDH mutant lower-grade glioma management.

Prognostic value of O 6-methylguanine-DNA methyltransferase methylation in isocitrate dehydrogenase mutant gliomas.

Background: Patients with isocitrate dehydrogenase (IDH) mutant gliomas have been associated with longer survival time than those that are IDH wild-type. Previous studies have shown the prognostic value of O 6 -methylguanine-DNA methyltransferase (MGMT) promoter methylation for glioblastoma multiforme (GBM), which are predominantly IDH wild-type. Little is known of the prognostic value of MGMT methylation status for IDH mutant gliomas. Methods: We retrospectively identified IDH mutant gliomas patients between 2011 and 2020 that were tested for MGMT promoter methylation. We generated Kaplan-Meier estimator curves and performed Cox proportional hazard models for overall survival (OS) and progression-free survival (PFS) to compare the outcomes of MGMT promoter methylated versus MGMT unmethylated patients. Results: Of 419 IDH mutant gliomas with MGMT promoter methylation testing, we identified 54 GBMs, 223 astrocytomas, and 142 oligodendrogliomas. 62.3% patients had MGMT methylated tumors while 37.7% were MGMT unmethylated. On Kaplan-Meier analysis, median OS for all MGMT methylated patients was 17.7 years and 14.6 years for unmethylated patients. Median PFS for all MGMT methylated patients was 7.0 years and for unmethylated patients 5.2 years. After univariate subgroup analysis, MGMT methylation is only prognostic for OS and PFS in GBM, and for OS in anaplastic oligodendroglioma and anaplastic oligodendroglioma for OS. In multivariate analysis, MGMT unmethylated GBM patients carry a higher risk of death (HR 7.72, 95% CI 2.10-28.33) and recurrence (HR 3.85, 95% CI 1.35-10.96). Conclusions: MGMT promoter methylation is associated with better OS and PFS for IDH mutant GBM. MGMT promoter methylation testing for other IDH mutant glioma subtypes may not provide additional information on prognostication.

Sequential logic of polarity determination during the haploid-to-diploid transition in Saccharomyces cerevisiae.

In many organisms, the geometry of encounter of haploid germ cells is arbitrary. In Saccharomyces cerevisiae, the resulting zygotes have been seen to bud asymmetrically in several directions as they produce diploid progeny. What mechanisms account for the choice of direction, and do the mechanisms directing polarity change over time? Distinct subgroups of cortical "landmark" proteins guide budding by haploid versus diploid cells, both of which require the Bud1/Rsr1 GTPase to link landmarks to actin. We observed that as mating pairs of haploid cells form zygotes, bud site specification progresses through three phases. The first phase follows disassembly and limited scattering of proteins that concentrated at the zone of cell contact, followed by their reassembly to produce a large medial bud. Bud1 is not required for medial placement of the initial bud. The second phase produces a contiguous bud(s) and depends on axial landmarks. As the titer of the Axl1 landmark diminishes, the third phase ultimately redirects budding toward terminal sites and is promoted by bipolar landmarks. Thus, following the initial random encounter that specifies medial budding, sequential spatial choices are orchestrated by the titer of a single cortical determinant that determines whether successive buds will be contiguous to their predecessors.

Flow cytometry-based purification of S. cerevisiae zygotes.

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) (1). S. cerevisiae zygotes result from the fusion of haploid cells of distinct mating type (MATa, MATalpha) and give rise to corresponding stable diploids that successively generate as many as 20 diploid progeny as a result of their strikingly asymmetric mitotic divisions (2). Zygote formation is orchestrated by a complex sequence of events: In this process, soluble mating factors bind to cognate receptors, triggering receptor-mediated signaling cascades that facilitate interruption of the cell cycle and culminate in cell-cell fusion. Zygotes may be considered a model for progenitor or stem cell function. Although much has been learned about the formation of zygotes and although zygotes have been used to investigate cell-molecular questions of general significance, almost all studies have made use of mating mixtures in which zygotes are intermixed with a majority population of haploid cells (3-8). Many aspects of the biochemistry of zygote formation and the continuing life of the zygote therefore remain uninvestigated. Reports of purification of yeast zygotes describe protocols based on their sedimentation properties (9); however, this sedimentation-based procedure did not yield nearly 90% purity in our hands. Moreover, it has the disadvantage that cells are exposed to hypertonic sorbitol. We therefore have developed a versatile purification procedure. For this purpose, pairs of haploid cells expressing red or green fluorescent proteins were co-incubated to allow zygote formation, harvested at various times, and the resulting zygotes were purified using a flow cytometry-based sorting protocol. This technique provides a convenient visual assessment of purity and maturation. The average purity of the fraction is approximately 90%. According to the timing of harvest, zygotes of varying degrees of maturity can be recovered. The purified samples provide a convenient point of departure for "-omic" studies, for recovery of initial progeny, and for systematic investigation of this progenitor cell.