Pathways to hypermutation in high-grade gliomas: Mechanisms, syndromes, and opportunities for immunotherapy.

Despite rapid advances in the field of immunotherapy, including the success of immune checkpoint inhibition in treating multiple cancer types, clinical response in high-grade gliomas (HGGs) has been disappointing. This has been in part attributed to the low tumor mutational burden (TMB) of the majority of HGGs. Hypermutation is a recently characterized glioma signature that occurs in a small subset of cases, which may open an avenue to immunotherapy. The substantially elevated TMB of these tumors most commonly results from alterations in the DNA mismatch repair pathway in the setting of extensive exposure to temozolomide or, less frequently, from inherited cancer predisposition syndromes. In this review, we discuss the genetics and etiology of hypermutation in HGGs, with an emphasis on the resulting genomic signatures, and the state and future directions of immuno-oncology research in these patient populations.

Abrogation of the G2/M checkpoint as a chemosensitization approach for alkylating agents.

BACKGROUND: The cell cycle is tightly regulated by checkpoints, which play a vital role in controlling its progression and timing. Cancer cells exploit the G2/M checkpoint, which serves as a resistance mechanism against genotoxic anticancer treatments, allowing for DNA repair prior to cell division. Manipulating cell cycle timing has emerged as a potential strategy to augment the effectiveness of DNA damage-based therapies. METHODS: In this study, we conducted a forward genome-wide CRISPR/Cas9 screening with repeated exposure to the alkylating agent temozolomide (TMZ) to investigate the mechanisms underlying tumor cell survival under genotoxic stress. RESULTS: Our findings revealed that canonical DNA repair pathways, including the Ataxia-telangiectasia mutated (ATM)/Fanconi and mismatch repair, determine cell fate under genotoxic stress. Notably, we identified the critical role of PKMYT1, in ensuring cell survival. Depletion of PKMYT1 led to overwhelming TMZ-induced cytotoxicity in cancer cells. Isobologram analysis demonstrated potent drug synergy between alkylating agents and a Myt1 kinase inhibitor, RP-6306. Mechanistically, inhibiting Myt1 forced G2/M-arrested cells into an unscheduled transition to the mitotic phase without complete resolution of DNA damage. This forced entry into mitosis, along with persistent DNA damage, resulted in severe mitotic abnormalities. Ultimately, these aberrations led to mitotic exit with substantial apoptosis. Preclinical animal studies demonstrated that the combination regimen involving TMZ and RP-6306 prolonged the overall survival of glioma-bearing mice. CONCLUSIONS: Collectively, our findings highlight the potential of targeting cell cycle timing through Myt1 inhibition as an effective strategy to enhance the efficacy of current standard cancer therapies, potentially leading to improved disease outcomes.

Proteostasis Modulation in Germline Missense von Hippel Lindau Disease.

PURPOSE: Missense mutated von Hippel Lindau (VHL) protein (pVHL) maintains intrinsic function but undergoes proteasomal degradation and tumor initiation and/or progression in VHL disease. Vorinostat can rescue missense mutated pVHL and arrest tumor growth in preclinical models. We asked whether short-term oral vorinostat could rescue pVHL in central nervous system hemangioblastomas in patients with germline missense VHL. PATIENTS AND METHODS: We administered oral vorinostat to 7 subjects (ages 46.0 ± 14.5 years) and then removed symptomatic hemangioblastomas surgically (ClinicalTrials.gov identifier NCT02108002). RESULTS: Vorinostat was tolerated without serious adverse events by all patients. pVHL expression was elevated in neoplastic stromal cells compared with untreated hemangioblastomas from same patients. We found transcriptional suppression of downstream hypoxia-inducible factor (HIF) effectors. Mechanistically, vorinostat prevented Hsp90 recruitment to mutated pVHL in vitro. The effects of vorinostat on the Hsp90-pVHL interaction, pVHL rescue, and transcriptional repression of downstream HIF effectors was independent of the location of the missense mutation on the VHL locus. We confirmed a neoplastic stromal cell-specific effect in suppression of protumorigenic pathways with single-nucleus transcriptomic profiling. CONCLUSIONS: We found that oral vorinostat treatment in patients with germline missense VHL mutations has a potent biologic effect that warrants further clinical study. These results provide biologic evidence to support the use of proteostasis modulation for the treatment of syndromic solid tumors involving protein misfolding. Proteostasis modulation with vorinostat rescues missense mutated VHL protein. Further clinical trials are needed to demonstrate tumor growth arrest.

Protein Kinase B (PKB/AKT) Protects IDH-Mutated Glioma from Ferroptosis via Nrf2.

PURPOSE: Mutations of the isocitrate dehydrogenase (IDH) gene are common genetic mutations in human malignancies. Increasing evidence indicates that IDH mutations play critical roles in malignant transformation and progression. However, the therapeutic options for IDH-mutated cancers remain limited. In this study, the investigation of patient cohorts revealed that the PI3K/protein kinase B (AKT) signaling pathways were enhanced in IDH-mutated cancer cells. EXPERIMENTAL DESIGN: In this study, we investigated the gene expression profile in IDH-mutated cells using RNA sequencing after the depletion of AKT. Gene set enrichment analysis (GSEA) and pathway enrichment analysis were used to discover altered molecular pathways due to AKT depletion. We further investigated the therapeutic effect of the AKT inhibitor, ipatasertib (Ipa), combined with temozolomide (TMZ) in cell lines and preclinical animal models. RESULTS: GSEA and pathway enrichment analysis indicated that the PI3K/AKT pathway significantly correlated with Nrf2-guided gene expression and ferroptosis-related pathways. Mechanistically, AKT suppresses the activity of GSK3ß and stabilizes Nrf2. Moreover, inhibition of AKT activity with Ipa synergizes with the genotoxic agent TMZ, leading to overwhelming ferroptotic cell death in IDH-mutated cancer cells. The preclinical animal model confirmed that combining Ipa and TMZ treatment prolonged survival. CONCLUSIONS: Our findings highlighted AKT/Nrf2 pathways as a potential synthetic lethality target for IDH-mutated cancers.

IDH mutation and cancer stem cell.

Cancer stem cells (CSCs) are a small population of cells in human malignancies that resemble the biology of human pluripotent stem cells. CSCs are closely related to the critical hallmarks in human cancers, ranging from oncogenesis to disease progression, therapeutic resistance, and overall outcome. Mutations in isocitrate dehydrogenase (IDH) were recently identified as founder mutations for human cancers. An increasing amount of evidence indicates that IDH mutations are closely related to the establishment and maintenance of CSCs. Biosynthesis of oncometabolite, metabolic reprogramming, and epigenetic shifts establish distinctive molecular signatures in IDH-mutated CSCs. Additionally, IDH mutation and IDH-related pathways could be valuable molecular targets to impact the CSC components in human cancers and to improve the disease outcome.

Germline SUCLG2 Variants in Patients With Pheochromocytoma and Paraganglioma.

BACKGROUND: Pheochromocytoma and paraganglioma (PPGL) are neuroendocrine tumors with frequent mutations in genes linked to the tricarboxylic acid cycle. However, no pathogenic variant has been found to date in succinyl-CoA ligase (SUCL), an enzyme that provides substrate for succinate dehydrogenase (SDH; mitochondrial complex II [CII]), a known tumor suppressor in PPGL. METHODS: A cohort of 352 patients with apparently sporadic PPGL underwent genetic testing using a panel of 54 genes developed at the National Institutes of Health, including the SUCLG2 subunit of SUCL. Gene deletion, succinate levels, and protein levels were assessed in tumors where possible. To confirm the possible mechanism, we used a progenitor cell line, hPheo1, derived from a human pheochromocytoma, and ablated and re-expressed SUCLG2. RESULTS: We describe 8 germline variants in the guanosine triphosphate-binding domain of SUCLG2 in 15 patients (15 of 352, 4.3%) with apparently sporadic PPGL. Analysis of SUCLG2-mutated tumors and SUCLG2-deficient hPheo1 cells revealed absence of SUCLG2 protein, decrease in the level of the SDHB subunit of SDH, and faulty assembly of the complex II, resulting in aberrant respiration and elevated succinate accumulation. CONCLUSIONS: Our study suggests SUCLG2 as a novel candidate gene in the genetic landscape of PPGL. Large-scale sequencing may uncover additional cases harboring SUCLG2 variants and provide more detailed information about their prevalence and penetrance.

Cancer stem cells: advances in biology and clinical translation-a Keystone Symposia report.

The test for the cancer stem cell (CSC) hypothesis is to find a target expressed on all, and only CSCs in a patient tumor, then eliminate all cells with that target that eliminates the cancer. That test has not yet been achieved, but CSC diagnostics and targets found on CSCs and some other cells have resulted in a few clinically relevant therapies. However, it has become apparent that eliminating the subset of tumor cells characterized by self-renewal properties is essential for long-term tumor control. CSCs are able to regenerate and initiate tumor growth, recapitulating the heterogeneity present in the tumor before treatment. As great progress has been made in identifying and elucidating the biology of CSCs as well as their interactions with the tumor microenvironment, the time seems ripe for novel therapeutic strategies that target CSCs to find clinical applicability. On May 19-21, 2021, researchers in cancer stem cells met virtually for the Keystone eSymposium "Cancer Stem Cells: Advances in Biology and Clinical Translation" to discuss recent advances in the understanding of CSCs as well as clinical efforts to target these populations.

Identification of Isocitrate Dehydrogenase 2 (IDH2) Mutation in Carotid Body Paraganglioma.

Carotid body paragangliomas (PGLs) are rare neuroendocrine tumors that develop within the adventitia of the medial aspect of the carotid bifurcation. Carotid body PGLs comprise about 65% of head and neck paragangliomas, however, their genetic background remains elusive. In the present study, we report one case of carotid body PGL with a somatic mutation in the gene encoding isocitrate dehydrogenase 2 (IDH2). The missense mutation in IDH2 resulted in R172G amino acid substitution, which exhibits neomorphic activity and production of D-2-hydroxyglutarate.

D-2-Hydroxyglutarate in Glioma Biology.

Isocitrate dehydrogenase (IDH) mutations are common genetic abnormalities in glioma, which result in the accumulation of an "oncometabolite", D-2-hydroxyglutarate (D-2-HG). Abnormally elevated D-2-HG levels result in a distinctive pattern in cancer biology, through competitively inhibiting α-ketoglutarate (α-KG)/Fe(II)-dependent dioxgenases (α-KGDDs). Recent studies have revealed that D-2-HG affects DNA/histone methylation, hypoxia signaling, DNA repair, and redox homeostasis, which impacts the oncogenesis of IDH-mutated cancers. In this review, we will discuss the current understanding of D-2-HG in cancer biology, as well as the emerging opportunities in therapeutics in IDH-mutated glioma.

Reactive Oxygen Species: A Promising Therapeutic Target for SDHx-Mutated Pheochromocytoma and Paraganglioma.

Pheochromocytoma (PHEO) and paraganglioma (PGL) are rare neuroendocrine tumors derived from neural crest cells. Germline variants in approximately 20 PHEO/PGL susceptibility genes are found in about 40% of patients, half of which are found in the genes that encode succinate dehydrogenase (SDH). Patients with SDH subunit B (SDHB)-mutated PHEO/PGL exhibit a higher likelihood of developing metastatic disease, which can be partially explained by the metabolic cell reprogramming and redox imbalance caused by the mutation. Reactive oxygen species (ROS) are highly reactive molecules involved in a multitude of important signaling pathways. A moderate level of ROS production can help regulate cellular physiology; however, an excessive level of oxidative stress can lead to tumorigenic processes including stimulation of growth factor-dependent pathways and the induction of genetic instability. Tumor cells effectively exploit antioxidant enzymes in order to protect themselves against harmful intracellular ROS accumulation, which highlights the essential balance between ROS production and scavenging. Exploiting ROS accumulation can be used as a possible therapeutic strategy in ROS-scavenging tumor cells. Here, we focus on the role of ROS production in PHEO and PGL, predominantly in SDHB-mutated cases. We discuss potential strategies and approaches to anticancer therapies by enhancing ROS production in these difficult-to-treat tumors.

Genotoxic therapy and resistance mechanism in gliomas.

Glioma is one of the most common and lethal brain tumors. Surgical resection followed by radiotherapy plus chemotherapy is the current standard of care for patients with glioma. The existence of resistance to genotoxic therapy, as well as the nature of tumor heterogeneity greatly limits the efficacy of glioma therapy. DNA damage repair pathways play essential roles in many aspects of glioma biology such as cancer progression, therapy resistance, and tumor relapse. O6-methylguanine-DNA methyltransferase (MGMT) repairs the cytotoxic DNA lesion generated by temozolomide (TMZ), considered as the main mechanism of drug resistance. In addition, mismatch repair, base excision repair, and homologous recombination DNA repair also play pivotal roles in treatment resistance as well. Furthermore, cellular mechanisms, such as cancer stem cells, evasion from apoptosis, and metabolic reprogramming, also contribute to TMZ resistance in gliomas. Investigations over the past two decades have revealed comprehensive mechanisms of glioma therapy resistance, which has led to the development of novel therapeutic strategies and targeting molecules.

Oncometabolites in Cancer: Current Understanding and Challenges.

Oncometabolites are pathognomonic hallmarks in human cancers, including glioma, leukemia, neuroendocrine tumors, and renal cancer. Oncometabolites are aberrantly accumulated from disrupted Krebs cycle and affect the catalytic activity of α-ketoglutarate-dependent dioxygenases. Oncometabolites indicate distinct cancer-related patterns ranging from oncogenesis and metabolism to therapeutic resistance. Here we discuss the current understanding of oncometabolites as well as the controversies and challenges associated with oncometabolite-driven cancers. New insights into the relationship between cancer and oncometabolites will elucidate novel therapeutic avenues for improved cancer treatment.

IDH1 mutations induce organelle defects via dysregulated phospholipids.

Infiltrating gliomas are devastating and incurable tumors. Amongst all gliomas, those harboring a mutation in isocitrate dehydrogenase 1 mutation (IDH1mut) acquire a different tumor biology and clinical manifestation from those that are IDH1WT. Understanding the unique metabolic profile reprogrammed by IDH1 mutation has the potential to identify new molecular targets for glioma therapy. Herein, we uncover increased monounsaturated fatty acids (MUFA) and their phospholipids in endoplasmic reticulum (ER), generated by IDH1 mutation, that are responsible for Golgi and ER dilation. We demonstrate a direct link between the IDH1 mutation and this organelle morphology via D-2HG-induced stearyl-CoA desaturase (SCD) overexpression, the rate-limiting enzyme in MUFA biosynthesis. Inhibition of IDH1 mutation or SCD silencing restores ER and Golgi morphology, while D-2HG and oleic acid induces morphological defects in these organelles. Moreover, addition of oleic acid, which tilts the balance towards elevated levels of MUFA, produces IDH1mut-specific cellular apoptosis. Collectively, these results suggest that IDH1mut-induced SCD overexpression can rearrange the distribution of lipids in the organelles of glioma cells, providing new insight into the link between lipid metabolism and organelle morphology in these cells, with potential and unique therapeutic implications.

NRF2 in human neoplasm: Cancer biology and potential therapeutic target.

Nuclear factor-erythroid 2-related factor 2 (NRF2) is a master regulator of a series of cytoprotective genes, which protects cells from stress conditions such as reactive oxygen species (ROS) and electrophiles. Besides its pivotal role in cellular physiology and stress response, several recent advances revealed that NRF2-governed pathways are extensively exploited in cancer cells, and play critical roles in granting growth advantage, supporting cancer metabolism, and promoting malignant transformation. In the present review, we focus on the regulatory mechanisms of NRF2 and its roles in cancer biology. We also discuss the value of targeting NRF2-associated pathways as a means of developing future cancer therapeutics.

Isocitrate Dehydrogenase Mutations in Glioma: Genetics, Biochemistry, and Clinical Indications.

Mutations in isocitrate dehydrogenase (IDH) are commonly observed in lower-grade glioma and secondary glioblastomas. IDH mutants confer a neomorphic enzyme activity that converts α-ketoglutarate to an oncometabolite D-2-hydroxyglutarate, which impacts cellular epigenetics and metabolism. IDH mutation establishes distinctive patterns in metabolism, cancer biology, and the therapeutic sensitivity of glioma. Thus, a deeper understanding of the roles of IDH mutations is of great value to improve the therapeutic efficacy of glioma and other malignancies that share similar genetic characteristics. In this review, we focused on the genetics, biochemistry, and clinical impacts of IDH mutations in glioma.

Metabolic Landscape of a Genetically Engineered Mouse Model of IDH1 Mutant Glioma.

Understanding the metabolic reprogramming of aggressive brain tumors has potential applications for therapeutics as well as imaging biomarkers. However, little is known about the nutrient requirements of isocitrate dehydrogenase 1 (IDH1) mutant gliomas. The IDH1 mutation involves the acquisition of a neomorphic enzymatic activity which generates D-2-hydroxyglutarate from α-ketoglutarate. In order to gain insight into the metabolism of these malignant brain tumors, we conducted metabolic profiling of the orthotopic tumor and the contralateral regions for the mouse model of IDH1 mutant glioma; as well as to examine the utilization of glucose and glutamine in supplying major metabolic pathways such as glycolysis and tricarboxylic acid (TCA). We also revealed that the main substrate of 2-hydroxyglutarate is glutamine in this model, and how this re-routing impairs its utilization in the TCA. Our 13C tracing analysis, along with hyperpolarized magnetic resonance experiments, revealed an active glycolytic pathway similar in both regions (tumor and contralateral) of the brain. Therefore, we describe the reprogramming of the central carbon metabolism associated with the IDH1 mutation in a genetically engineered mouse model which reflects the tumor biology encountered in glioma patients.

Triptolide suppresses IDH1-mutated malignancy via Nrf2-driven glutathione metabolism.

Isocitrate dehydrogenase (IDH) mutation is a common genetic abnormality in human malignancies characterized by remarkable metabolic reprogramming. Our present study demonstrated that IDH1-mutated cells showed elevated levels of reactive oxygen species and higher demands on Nrf2-guided glutathione de novo synthesis. Our findings showed that triptolide, a diterpenoid epoxide from Tripterygium wilfordii, served as a potent Nrf2 inhibitor, which exhibited selective cytotoxicity to patient-derived IDH1-mutated glioma cells in vitro and in vivo. Mechanistically, triptolide compromised the expression of GCLC, GCLM, and SLC7A11, which disrupted glutathione metabolism and established synthetic lethality with reactive oxygen species derived from IDH1 mutant neomorphic activity. Our findings highlight triptolide as a valuable therapeutic approach for IDH1-mutated malignancies by targeting the Nrf2-driven glutathione synthesis pathway.

mTORC2/Rac1 Pathway Predisposes Cancer Aggressiveness in IDH1-Mutated Glioma.

Isocitrate dehydrogenase (IDH) mutations are common genetic abnormalities in lower grade gliomas. The neomorphic enzyme activity of IDH mutants leads to tumor formation through epigenetic alteration, dysfunction of dioxygenases, and metabolic reprogramming. However, it remains elusive as to how IDH mutants regulate the pathways associated with oncogenic transformation and aggressiveness. In the present study, by using unbiased transcriptomic profiling, we showed that IDH1 mutations result in substantial changes in the gene sets that govern cellular motility, chemotaxis, and invasion. Mechanistically, rapamycin-insensitive companion of mammalian target of rapamycin (Rictor)/Ras-related C3 botulinum toxin substrate 1 (Rac1) signaling plays an essential role in the motility and proliferation of IDH1-mutated cells by prompting cytoskeleton reorganization, lamellipodia formation, and enhanced endocytosis. Targeting the Rictor/Rac1 pathway suppresses IDH1-mutated cells by limiting endocytosis and cell proliferation. Overall, our findings indicate a novel metabolic reprogramming mechanism of IDH1-mutated cells by exploiting metabolites from the extracellular milieu. Targeting the Rictor/Rac1 pathway could be an alternative therapeutic strategy for IDH1-mutated malignancies.

IDH mutation in glioma: molecular mechanisms and potential therapeutic targets.

Isocitrate dehydrogenase (IDH) enzymes catalyse the oxidative decarboxylation of isocitrate and therefore play key roles in the Krebs cycle and cellular homoeostasis. Major advances in cancer genetics over the past decade have revealed that the genes encoding IDHs are frequently mutated in a variety of human malignancies, including gliomas, acute myeloid leukaemia, cholangiocarcinoma, chondrosarcoma and thyroid carcinoma. A series of seminal studies further elucidated the biological impact of the IDH mutation and uncovered the potential role of IDH mutants in oncogenesis. Notably, the neomorphic activity of the IDH mutants establishes distinctive patterns in cancer metabolism, epigenetic shift and therapy resistance. Novel molecular targeting approaches have been developed to improve the efficacy of therapeutics against IDH-mutated cancers. Here we provide an overview of the latest findings in IDH-mutated human malignancies, with a focus on glioma, discussing unique biological signatures and proceedings in translational research.

A novel germline gain-of-function HIF2A mutation in hepatocellular carcinoma with polycythemia.

Hypoxia-inducible factors (HIFs) regulate oxygen sensing and expression of genes involved in angiogenesis and erythropoiesis. Polycythemia has been observed in patients with hepatocellular carcinoma (HCC), but the underlying molecular basis remains unknown. Liver tissues from 302 HCC patients, including 104 with polycythemia, were sequenced for HIF2A mutations. A germline HIF2A mutation was detected in one HCC patient with concurrent polycythemia. Three additional family members carried this mutation, but none exhibited polycythemia or were diagnosed with HCC. The gain-of-function mutation resulted in a HIF-2α protein that was transcribed normally but resistant to degradation. HIF-2α target genes EDN1, EPO, GNA14, and VEGF were significantly upregulated in the tumor bed but not in the surrounding liver tissue. Polycythemia resolved upon total resection of the tumor tissue. This newly described HIF2A mutation may promote HCC oncogenesis.