Discovery and Optimization of N-Arylated Tetracyclic Dicarboximides That Target Primary Glioma Stem-like Cells.

We recently discovered a novel N-aryl tetracyclic dicarboximide MM0299 (1) with robust activity against glioma stem-like cells that potently and selectively inhibits lanosterol synthase leading to the accumulation of the toxic shunt metabolite 24(S),25-epoxycholesterol. Herein, we delineate a systematic and comprehensive SAR study that explores the structural space surrounding the N-aryl tetracyclic dicarboximide scaffold. A series of 100 analogs were synthesized and evaluated for activity against the murine glioma stem-like cell line Mut6 and for metabolic stability in mouse liver S9 fractions. This study led to several analogs with single-digit nanomolar activity in Mut6 glioblastoma cells that were metabolically stable in S9 fractions. In vivo pharmacokinetic analysis of selected analogs identified compound 52a (IC50 = 63 nM; S9 T1/2 > 240 min) which was orally available (39% plasma; 58% brain) and displayed excellent brain exposure. Chronic oral dosing of 52a during a 2-week tolerability study indicated no adverse effect on body weight nor signs of hematologic, liver, or kidney toxicity.

Matched three-dimensional organoids and two-dimensional cell lines of melanoma brain metastases mirror response to targeted molecular therapy.

Purpose: Despite significant advances in the treatment paradigm for patients with metastatic melanoma, melanoma brain metastasis (MBM) continues to represent a significant treatment challenge. The study of MBM is limited, in part, by shortcomings in existing preclinical models. Surgically eXplanted Organoids (SXOs) are ex vivo, three-dimensional cultures prepared from primary tissue samples with minimal processing that recapitulate genotypic and phenotypic features of parent tumors and are grown without artificial extracellular scaffolding. We aimed to develop the first matched patient-derived SXO and cell line models of MBM to investigate responses to targeted therapy. Methods: MBM SXOs were created by a novel protocol incorporating techniques for establishing glioma and cutaneous melanoma organoids. A BRAFV600K-mutant and BRAF-wildtype MBM sample were collected directly from the operating room for downstream experiments. Organoids were cultured in an optimized culture medium without an artificial extracellular scaffold. Concurrently, matched patient-derived cell lines were created. Drug screens were conducted to assess treatment response in SXOs and cell lines. Results: Organoid growth was observed within 3-4 weeks, and MBM SXOs retained histological features of the parent tissue, including pleomorphic epithelioid cells with abundant cytoplasm, large nuclei, focal melanin accumulation, and strong SOX10 positivity. After sufficient growth, organoids could be manually parcellated to increase the number of replicates. Matched SXOs and cell lines demonstrated sensitivity to BRAF and MEK inhibitors. Conclusion: Here, we describe the creation of a scaffold-free organoid model of MBM. Further study using SXOs may improve the translational relevance of preclinical studies and enable the study of the metastatic melanoma tumor microenvironment.

Mismatch repair protein mutations in isocitrate dehydrogenase (IDH)-mutant astrocytoma and IDH-wild-type glioblastoma.

Background: Mutations in mismatch repair (MMR) genes (MSH2, MSH6, MLH1, and PMS2) are associated with microsatellite instability and a hypermutator phenotype in numerous systemic cancers, and germline MMR mutations have been implicated in multi-organ tumor syndromes. In gliomas, MMR mutations can function as an adaptive response to alkylating chemotherapy, although there are well-documented cases of germline and sporadic mutations, with detrimental effects on patient survival. Methods: The clinical, pathologic, and molecular features of 18 IDH-mutant astrocytomas and 20 IDH-wild-type glioblastomas with MMR mutations in the primary tumor were analyzed in comparison to 361 IDH-mutant and 906 IDH-wild-type tumors without MMR mutations. In addition, 12 IDH-mutant astrocytomas and 18 IDH-wild-type glioblastomas that developed MMR mutations between initial presentation and tumor recurrence were analyzed in comparison to 50 IDH-mutant and 104 IDH-wild-type cases that remained MMR-wild-type at recurrence. Results: In both IDH-mutant astrocytoma and IDH-wild-type glioblastoma cohorts, the presence of MMR mutation in primary tumors was associated with significantly higher tumor mutation burden (TMB) (P < .0001); however, MMR mutations only resulted in worse overall survival in the IDH-mutant astrocytomas (P = .0069). In addition, gain of MMR mutation between the primary and recurrent surgical specimen occurred more frequently with temozolomide therapy (P = .0073), and resulted in a substantial increase in TMB (P < .0001), higher grade (P = .0119), and worse post-recurrence survival (P = .0022) in the IDH-mutant astrocytoma cohort. Conclusions: These results suggest that whether present initially or in response to therapy, MMR mutations significantly affect TMB but appear to only influence the clinical outcome in IDH-mutant astrocytoma subsets.

Human plasma-like medium facilitates metabolic tracing and enables upregulation of immune signaling pathways in glioblastoma explants.

Purpose: Metabolism within the tumor microenvironment (TME) represents an increasing area of interest to understand glioma initiation and progression. Stable isotope tracing is a technique critical to the study of tumor metabolism. Cell culture models of this disease are not routinely cultured under physiologically relevant nutrient conditions and do not retain cellular heterogeneity present in the parental TME. Moreover, in vivo, stable isotope tracing in intracranial glioma xenografts, the gold standard for metabolic investigation, is time consuming and technically challenging. To provide insights into glioma metabolism in the presence of an intact TME, we performed stable isotope tracing analysis of patient-derived, heterocellular Surgically eXplanted Organoid (SXO) glioma models in human plasma-like medium (HPLM). Methods: Glioma SXOs were established and cultured in conventional media or transitioned to HPLM. We evaluated SXO cytoarchitecture and histology, then performed spatial transcriptomic profiling to identify cellular populations and differential gene expression patterns. We performed stable isotope tracing with 15N2-glutamine to evaluate intracellular metabolite labeling patterns. Results: Glioma SXOs cultured in HPLM retain cytoarchitecture and cellular constituents. Immune cells in HPLM-cultured SXOs demonstrated increased transcription of immune-related signatures, including innate immune, adaptive immune, and cytokine signaling programs. 15N isotope enrichment from glutamine was observed in metabolites from diverse pathways, and labeling patterns were stable over time. Conclusion: To enable ex vivo, tractable investigations of whole tumor metabolism, we developed an approach to conduct stable isotope tracing in glioma SXOs cultured under physiologically relevant nutrient conditions. Under these conditions, SXOs maintained viability, composition, and metabolic activity while exhibiting increased immune-related transcriptional programs.

Isocitrate dehydrogenase mutations in gliomas: A review of current understanding and trials.

Isocitrate dehydrogenase (IDH) is a key enzyme in normal metabolism and homeostasis. However, mutant forms of IDH are also defining features of a subset of diffuse gliomas. In this review, we highlight current techniques targeting IDH-mutated gliomas and summarize current and completed clinical trials exploring these strategies. We discuss clinical data from peptide vaccines, mutant IDH (mIDH) inhibitors, and PARP inhibitors. Peptide vaccines have the unique advantage of targeting the specific epitope of a patient's tumor, inducing a highly tumor-specific CD4+ T-cell response. mIDH-inhibitors, on the other hand, specifically target mutant IDH proteins in cancer cell metabolism and thus help halt gliomagenesis. We also explore PARP inhibitors and their role in treating diffuse gliomas, which exploit IDH-mutant diffuse gliomas by allowing the persistence of unrepaired DNA complexes. We summarize various completed and current trials targeting IDH1 and IDH2 mutations in diffuse gliomas. Therapies targeting mutant IDH have significant promise in treating progressive or recurrent IDH-mutant gliomas and may significantly change treatment paradigms in the next decade.

Emerging roles of nucleotide metabolism in cancer.

Nucleotides are substrates for multiple anabolic pathways, most notably DNA and RNA synthesis. Since nucleotide synthesis inhibitors began to be used for cancer therapy in the 1950s, our understanding of how nucleotides function in tumor cells has evolved, prompting a resurgence of interest in targeting nucleotide metabolism for cancer therapy. In this review, we discuss recent advances that challenge the idea that nucleotides are mere building blocks for the genome and transcriptome and highlight ways that these metabolites support oncogenic signaling, stress resistance, and energy homeostasis in tumor cells. These findings point to a rich network of processes sustained by aberrant nucleotide metabolism in cancer and reveal new therapeutic opportunities.

Protocol to establish a genetically engineered mouse model of IDH1-mutant astrocytoma.

Lower-grade gliomas exhibit a high prevalence of isocitrate dehydrogenase 1 (IDH1) mutations, but faithful models for studying these tumors are lacking. Here, we present a protocol to establish a genetically engineered mouse (GEM) model of grade 3 astrocytoma driven by the Idh1R132H oncogene. We describe steps for breeding compound transgenic mice and intracranially delivering adeno-associated virus particles, followed by post-surgical surveillance via magnetic resonance imaging. This protocol enables the generation and use of a GEM to study lower-grade IDH-mutant gliomas. For complete details on the use and execution of this protocol, please refer to Shi et al. (2022).1.

Preclinical modeling of lower-grade gliomas.

Models for human gliomas prove critical not only to advancing our understanding of glioma biology but also to facilitate the development of therapeutic modalities. Specifically, creating lower-grade glioma (LGG) models has been challenging, contributing to few investigations and the minimal progress in standard treatment over the past decade. In order to reliably predict and validate the efficacies of novel treatments, however, LGG models need to adhere to specific standards that recapitulate tumor genetic aberrations and micro-environment. This underscores the need to revisit existing models of LGG and explore prospective models that may bridge the gap between preclinical insights and clinical translation. This review first outlines a set of criteria aimed to address the current challenges hindering model development. We then evaluate the strengths and weaknesses of existing preclinical models of LGG with respect to these established standards. To conclude, the review discusses potential future directions for integrating existing models to maximize the exploration of disease mechanisms and therapeutics development.

(R)-2-Hydroxyglutarate Inhibits KDM5 Histone Lysine Demethylases to Drive Transformation in IDH-Mutant Cancers.

Oncogenic mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 occur in a wide range of cancers, including acute myeloid leukemia (AML) and glioma. Mutant IDH enzymes convert 2-oxoglutarate (2OG) to (R)-2-hydroxyglutarate [(R)-2HG], an oncometabolite that is hypothesized to promote cellular transformation by dysregulating 2OG-dependent enzymes. The only (R)-2HG target that has been convincingly shown to contribute to transformation by mutant IDH is the myeloid tumor suppressor TET2. However, there is ample evidence to suggest that (R)-2HG has other functionally relevant targets in IDH-mutant cancers. Here, we show that (R)-2HG inhibits KDM5 histone lysine demethylases and that this inhibition contributes to cellular transformation in IDH-mutant AML and IDH-mutant glioma. These studies provide the first evidence of a functional link between dysregulation of histone lysine methylation and transformation in IDH-mutant cancers. SIGNIFICANCE: Mutant IDH is known to induce histone hypermethylation. However, it is not known if this hypermethylation is functionally significant or is a bystander effect of (R)-2HG accumulation in IDH-mutant cells. Here, we provide evidence that KDM5 inhibition by (R)-2HG contributes to mutant IDH-mediated transformation in AML and glioma. This article is highlighted in the In This Issue feature, p. 1275.

Selective and brain-penetrant lanosterol synthase inhibitors target glioma stem-like cells by inducing 24(S),25-epoxycholesterol production.

Glioblastoma (GBM) is an aggressive adult brain cancer with few treatment options due in part to the challenges of identifying brain-penetrant drugs. Here, we investigated the mechanism of MM0299, a tetracyclic dicarboximide with anti-glioblastoma activity. MM0299 inhibits lanosterol synthase (LSS) and diverts sterol flux away from cholesterol into a "shunt" pathway that culminates in 24(S),25-epoxycholesterol (EPC). EPC synthesis following MM0299 treatment is both necessary and sufficient to block the growth of mouse and human glioma stem-like cells by depleting cellular cholesterol. MM0299 exhibits superior selectivity for LSS over other sterol biosynthetic enzymes. Critical for its application in the brain, we report an MM0299 derivative that is orally bioavailable, brain-penetrant, and induces the production of EPC in orthotopic GBM tumors but not normal mouse brain. These studies have implications for the development of an LSS inhibitor to treat GBM or other neurologic indications.

Isocitrate dehydrogenase (IDH) mutant gliomas: A Society for Neuro-Oncology (SNO) consensus review on diagnosis, management, and future directions.

Isocitrate dehydrogenase (IDH) mutant gliomas are the most common adult, malignant primary brain tumors diagnosed in patients younger than 50, constituting an important cause of morbidity and mortality. In recent years, there has been significant progress in understanding the molecular pathogenesis and biology of these tumors, sparking multiple efforts to improve their diagnosis and treatment. In this consensus review from the Society for Neuro-Oncology (SNO), the current diagnosis and management of IDH-mutant gliomas will be discussed. In addition, novel therapies, such as targeted molecular therapies and immunotherapies, will be reviewed. Current challenges and future directions for research will be discussed.

DNA damage in IDH-mutant gliomas: mechanisms and clinical implications.

PURPOSE: Since the discovery of IDH mutations in glioma over a decade ago, significant progress has been made in determining how these mutations affect epigenetic, transcriptomic, and metabolic programs in brain tumor cells. In this article, we summarize current understanding of how IDH mutations influence DNA damage in glioma and discuss clinical implications of these findings. METHODS: We performed a thorough review of peer-reviewed publications and provide an overview of key mechanisms by which IDH mutations impact response to DNA damage in gliomas, with an emphasis on clinical implications. RESULTS: The effects of mutant IDH on DNA damage largely fall into four overarching categories: Gene Expression, Sensitivity to Alkylating Agents, Homologous Recombination, and Oxidative Stress. From a mechanistic standpoint, we discuss how mutant IDH and the oncometabolite (R)-2HG affect each of these categories of DNA damage. We also contextualize these mechanisms with respect to ongoing clinical trials. Studies are underway that incorporate current standard-of-care therapies, including radiation and alkylating agents, in addition to novel therapeutic agents that exert genotoxic stress specifically in IDH-mutant gliomas. Lastly, we discuss key unanswered questions and emerging data in this field that have important implications for our understanding of glioma biology and for the development of new brain tumor therapies. CONCLUSION: Mounting preclinical and clinical data suggest that IDH mutations alter DNA damage sensing and repair pathways through distinct mechanisms. Future studies are needed to deepen our understanding of these processes and provide additional mechanistic insights that can be leveraged for therapeutic benefit.

Oncometabolite d-2HG alters T cell metabolism to impair CD8+ T cell function.

Gain-of-function mutations in isocitrate dehydrogenase (IDH) in human cancers result in the production of d-2-hydroxyglutarate (d-2HG), an oncometabolite that promotes tumorigenesis through epigenetic alterations. The cancer cell-intrinsic effects of d-2HG are well understood, but its tumor cell-nonautonomous roles remain poorly explored. We compared the oncometabolite d-2HG with its enantiomer, l-2HG, and found that tumor-derived d-2HG was taken up by CD8+ T cells and altered their metabolism and antitumor functions in an acute and reversible fashion. We identified the glycolytic enzyme lactate dehydrogenase (LDH) as a molecular target of d-2HG. d-2HG and inhibition of LDH drive a metabolic program and immune CD8+ T cell signature marked by decreased cytotoxicity and impaired interferon-γ signaling that was recapitulated in clinical samples from human patients with IDH1 mutant gliomas.

Chromosomal instability in adult-type diffuse gliomas.

Chromosomal instability (CIN) is a fundamental property of cancer and a key underlying mechanism of tumorigenesis and malignant progression, and has been documented in a wide variety of cancers, including colorectal carcinoma with mutations in genes such as APC. Recent reports have demonstrated that CIN, driven in part by mutations in genes maintaining overall genomic stability, is found in subsets of adult-type diffusely infiltrating gliomas of all histologic and molecular grades, with resulting elevated overall copy number burden, chromothripsis, and poor clinical outcome. Still, relatively few studies have examined the effect of this process, due in part to the difficulty of routinely measuring CIN clinically. Herein, we review the underlying mechanisms of CIN, the relationship between chromosomal instability and malignancy, the prognostic significance and treatment potential in various cancers, systemic disease, and more specifically, in diffusely infiltrating glioma subtypes. While still in the early stages of discovery compared to other solid tumor types in which CIN is a known driver of malignancy, the presence of CIN as an early factor in gliomas may in part explain the ability of these tumors to develop resistance to standard therapy, while also providing a potential molecular target for future therapies.

A druggable addiction to de novo pyrimidine biosynthesis in diffuse midline glioma.

Diffuse midline glioma (DMG) is a uniformly fatal pediatric cancer driven by oncohistones that do not readily lend themselves to drug development. To identify druggable targets for DMG, we conducted a genome-wide CRISPR screen that reveals a DMG selective dependency on the de novo pathway for pyrimidine biosynthesis. This metabolic vulnerability reflects an elevated rate of uridine/uracil degradation that depletes DMG cells of substrates for the alternate salvage pyrimidine biosynthesis pathway. A clinical stage inhibitor of DHODH (rate-limiting enzyme in the de novo pathway) diminishes uridine-5'-phosphate (UMP) pools, generates DNA damage, and induces apoptosis through suppression of replication forks-an "on-target" effect, as shown by uridine rescue. Matrix-assisted laser desorption/ionization (MALDI) mass spectroscopy imaging demonstrates that this DHODH inhibitor (BAY2402234) accumulates in the brain at therapeutically relevant concentrations, suppresses de novo pyrimidine biosynthesis in vivo, and prolongs survival of mice bearing intracranial DMG xenografts, highlighting BAY2402234 as a promising therapy against DMGs.

EGFR ligand shifts the role of EGFR from oncogene to tumour suppressor in EGFR-amplified glioblastoma by suppressing invasion through BIN3 upregulation.

The epidermal growth factor receptor (EGFR) is a prime oncogene that is frequently amplified in glioblastomas. Here we demonstrate a new tumour-suppressive function of EGFR in EGFR-amplified glioblastomas regulated by EGFR ligands. Constitutive EGFR signalling promotes invasion via activation of a TAB1-TAK1-NF-κB-EMP1 pathway, resulting in large tumours and decreased survival in orthotopic models. Ligand-activated EGFR promotes proliferation and surprisingly suppresses invasion by upregulating BIN3, which inhibits a DOCK7-regulated Rho GTPase pathway, resulting in small hyperproliferating non-invasive tumours and improved survival. Data from The Cancer Genome Atlas reveal that in EGFR-amplified glioblastomas, a low level of EGFR ligands confers a worse prognosis, whereas a high level of EGFR ligands confers an improved prognosis. Thus, increased EGFR ligand levels shift the role of EGFR from oncogene to tumour suppressor in EGFR-amplified glioblastomas by suppressing invasion. The tumour-suppressive function of EGFR can be activated therapeutically using tofacitinib, which suppresses invasion by increasing EGFR ligand levels and upregulating BIN3.

Semi-Automated Computational Assessment of Cancer Organoid Viability Using Rapid Live-Cell Microscopy.

The creation of patient-derived cancer organoids represents a key advance in preclinical modeling and has recently been applied to a variety of human solid tumor types. However, conventional methods used to assess in vivo tumor tissue treatment response are poorly suited for the evaluation of cancer organoids because they are time-intensive and involve tissue destruction. To address this issue, we established a suite of 3-dimensional patient-derived glioma organoids, treated them with chemoradiotherapy, stained organoids with non-toxic cell dyes, and imaged them using a rapid laser scanning confocal microscopy method termed "Apex Imaging." We then developed and tested a fragmentation algorithm to quantify heterogeneity in the topography of the organoids as a potential surrogate marker of viability. This algorithm, SSDquant, provides a 3-dimensional visual representation of the organoid surface and a numerical measurement of the sum-squared distance (SSD) from the derived mass center of the organoid. We tested whether SSD scores correlate with traditional immunohistochemistry-derived cell viability markers (cellularity and cleaved caspase 3 expression) and observed statistically significant associations between them using linear regression analysis. Our work describes a quantitative, non-invasive approach for the serial measurement of patient-derived cancer organoid viability, thus opening new avenues for the application of these models to studies of cancer biology and therapy.

Global DNA methylation profiling reveals chromosomal instability in IDH-mutant astrocytomas.

Diffusely infiltrating gliomas are among the most common central nervous system tumors in adults. Over the past decade, the subcategorization of these tumors has changed to include both traditional histologic features and more recently identified molecular factors. However, one molecular feature that has yet to be integrated is the presence/absence of chromosomal instability (CIN). Herein, we use global methylation profiling to evaluate a reference cohort of IDH-mutant astrocytomas with and without prior evidence of CIN (n = 42), and apply the resulting methylation-based characteristics to a larger test cohort of publicly-available IDH-mutant astrocytomas (n = 245). We demonstrate that IDH-mutant astrocytomas with evidence of CIN cluster separately from their chromosomally-stable counterparts. CIN cases were associated with higher initial histologic grade, altered expression patterns of genes related to CIN in other cancers, elevated initial total copy number burden, and significantly worse progression-free and overall survival. In addition, in a grade-for-grade analysis, patients with CIN-positive WHO grade 2 and 3 tumors had significantly worse survival. These results suggest that global methylation profiling can be used to discriminate between chromosomally stable and unstable IDH-mutant astrocytomas, and may therefore provide a reliable and cost-effective method for identifying gliomas with chromosomal instability and resultant poor clinical outcome.

Establishment of patient-derived organoid models of lower-grade glioma.

BACKGROUND: Historically, creating patient-derived models of lower-grade glioma (LGG) has been challenging, contributing to few experimental platforms that support laboratory-based investigations of this disease. Although organoid modeling approaches have recently been employed to create in vitro models of high-grade glioma (HGG), it is unknown whether this approach can be successfully applied to LGG. METHODS: In this study, we developed an optimized protocol for the establishment of organoids from LGG primary tissue samples by utilizing physiologic (5%) oxygenation conditions and employed it to produce the first known suite of these models. To assess their fidelity, we surveyed key biological features of patient-derived organoids using metabolic, genomic, histologic, and lineage marker gene expression assays. RESULTS: Organoid models were created with a success rate of 91% (n = 20/22) from primary tumor samples across glioma histological subtypes and tumor grades (WHO Grades 1-4), and a success rate of 87% (13/15) for WHO Grade 1-3 tumors. Patient-derived organoids recapitulated stemness, proliferative, and tumor-stromal composition profiles of their respective parental tumor specimens. Cytoarchitectural, mutational, and metabolic traits of parental tumors were also conserved. Importantly, LGG organoids were maintained in vitro for weeks to months and reanimated after biobanking without loss of integrity. CONCLUSIONS: We report an efficient method for producing faithful in vitro models of LGG. New experimental platforms generated through this approach are well positioned to support preclinical studies of this disease, particularly those related to tumor immunology, tumor-stroma interactions, identification of novel drug targets, and personalized assessments of treatment response profiles.

Integrated Metabolic Profiling and Transcriptional Analysis Reveals Therapeutic Modalities for Targeting Rapidly Proliferating Breast Cancers.

Metabolic dysregulation is a prominent feature in breast cancer, but it remains poorly characterized in patient tumors. In this study, untargeted metabolomics analysis of triple-negative breast cancer (TNBC) and patient with estrogen receptor (ER)-positive breast cancer samples, as well as TNBC patient-derived xenografts (PDX), revealed two major metabolic groups independent of breast cancer histologic subtypes: a "Nucleotide/Carbohydrate-Enriched" group and a "Lipid/Fatty Acid-Enriched" group. Cell lines grown in vivo more faithfully recapitulated the metabolic profiles of patient tumors compared with those grown in vitro. Integrated metabolic and gene expression analyses identified genes that strongly correlate with metabolic dysregulation and predict patient prognosis. As a proof of principle, targeting Nucleotide/Carbohydrate-Enriched TNBC cell lines or PDX xenografts with a pyrimidine biosynthesis inhibitor or a glutaminase inhibitor led to therapeutic efficacy. In multiple in vivo models of TNBC, treatment with the pyrimidine biosynthesis inhibitor conferred better therapeutic outcomes than chemotherapeutic agents. This study provides a metabolic stratification of breast tumor samples that can guide the selection of effective therapeutic strategies targeting breast cancer subsets. In addition, we have developed a public, interactive data visualization portal (http://brcametab.org) based on the data generated from this study to facilitate future research. SIGNIFICANCE: A multiomics strategy that integrates metabolic and gene expression profiling in patient tumor samples and animal models identifies effective pharmacologic approaches to target rapidly proliferating breast tumor subtypes.