Background Fibroblast growth factor-inducible 14 (Fn14) belongs to the TNFR superfamily. Fn14 overexpression can drive receptor-autonomous signaling, increase both cell invasion and tumor-associated macrophages/microglia (TAMMs) recruitment, and correlates with reduced survival in glioblastoma (GBM) patients and rat gliomas. While prior studies report Fn14 expression in non-tumor cells within the GBM tumor microenvironment (TME), their relative contributions to glioma pathobiology remain unclear.Methods Using tumor-host pairings of Fn14-positive and -knockout (-KO) cells and mice, we examined the role of Fn14 in glioma biology. Mouse glioma and human GBM datasets were analyzed at the cellular, protein, and transcriptomic levels to assess Fn14-associated changes in the glioma TME and survival outcomes.Results Fn14 was found to be highly expressed in tumor cells and TAMMs in human GBM and 2 well-characterized murine glioma models. Fn14 KO in both tumor and host cells increased overall survival. Notably, this survival benefit was greater in the glioma model characterized by a more immunologically activated TME. Immunophenotyping revealed that Fn14 loss reshapes the tumor-immune landscape, reducing the presence of immunosuppressive macrophages and exhausted T-cells, suggesting that Fn14 modulates both innate and adaptive immune responses. These findings were supported by analyses of human GBM datasets, where high Fn14 expression correlated with immunosuppressive shifts and poor patient responses to immune checkpoint inhibitor therapy.Conclusions This study provides the first description of the contributions of both tumor- and host-derived Fn14 expression to tumor immunity and survival and identifies Fn14 as an important mediator of innate and adaptive immune responses in gliomas.

Glioblastoma exhibits profound therapeutic resistance, driven by tumor heterogeneity and highly plastic glioma stem cells (GSCs). This study exploits GSC metabolic dependence on cysteine using systemic Cyst(e)inase, a cysteine-degrading enzyme. In patient-derived GSCs and orthotopic xenograft models, Cyst(e)inase potently inhibited GSC proliferation and extended animal survival by inducing ferroptosis. Mechanistically, Cyst(e)inase triggered elevated reactive oxygen species (ROS), glutathione (GSH) depletion, and significant lipid peroxidation. Crucially, these effects were reversed by N-acetylcysteine (NAC), and lipid peroxidation was abrogated by the iron chelator Deferoxamine (DFX), unequivocally confirming iron-dependent ferroptosis. Characteristic mitochondrial morphological changes further validated ferroptosis induction. Acyl-CoA synthetase long-chain family member-4 (ACSL4) was identified as essential for this process. Critically, Cyst(e)inase synergized with temozolomide (TMZ), markedly enhancing its anti-tumor efficacy and prolonging survival, even in TMZ-resistant xenografts. These findings establish cysteine metabolism as a promising therapeutic target and position Cyst(e)inase, especially with TMZ, as a potent strategy to overcome GBM resistance.

MBNL1 binds the HIF-1α 3'UTR to promote rapid mRNA decay, thereby limiting HIF-1 activity and hypoxia-induced stemness in glioblastoma. Using patient-derived glioma stem cells, we show that MBNL1 loss stabilizes HIF-1α mRNA and increases HIF-1α protein, HRE reporter activity, and target gene expression under hypoxia; MBNL1 knockout also prolongs target gene expression after reoxygenation, indicating enhanced hypoxia "memory." MBNL1 depletion markedly elevates stemness markers (KLF4, SOX2, GLI1) and clonogenic growth, and re-expression of MBNL1 reverses these effects. These results identify a post-transcriptional MBNL1-HIF1α axis that controls hypoxia signaling and stemness, with implications for GBM therapy.

This review explores innovative therapeutic strategies for treating central nervous system (CNS) tumors by targeting their unique metabolic dependencies. This approach marks a significant departure from traditional cytotoxic treatments, focusing instead on the metabolic vulnerabilities created by the tumor's microenvironment and genetic profile. A key area of interest is the de novo pyrimidine synthesis pathway, which is crucial for DNA and RNA synthesis, DNA repair, and protein glycosylation. We highlight the potential of dihydroorotate dehydrogenase (DHODH) inhibitors, which have shown promising anti-tumor activity in preclinical models. The blood-brain barrier, while a challenge for drug delivery, may enhance the efficacy of these inhibitors by maintaining a unique metabolic environment in the brain. Specific brain tumors, such as glioblastoma multiforme, MYC-amplified medulloblastoma, and IDH mutant gliomas, exhibit heightened sensitivity to DHODH inhibition. We suggest that the unique metabolic environment of the brain could make DHODH a more effective therapeutic target for brain tumors compared to other cancer types. Despite the speculative nature of these findings, the compelling preclinical data warrant further investigation into brain-penetrant DHODH inhibitors for CNS malignancies.

Background Glioblastoma multiforme (GBM) stands as a formidable challenge in oncology because of its aggressive nature and severely limited treatment options. Despite decades of research, the survival rates for GBM remain effectively stagnant. A defining hallmark of GBM is a highly acidic tumor microenvironment, which is thought to activate pro-tumorigenic pathways. This acidification is the result of altered tumor metabolism favoring aerobic glycolysis, a phenomenon known as the Warburg effect. Low extracellular pH confers radioresistant tumors to glial cells. Notably GPR68, an acid sensing GPCR, is upregulated in radioresistant GBM. Usage of Lorazepam, which has off target agonism of GPR68, is linked to worse clinical outcomes for a variety of cancers. However, the role of tumor microenvironment acidification in GPR68 activation has not been assessed in cancer. Here we interrogate the role of GPR68 specifically in GBM cells using a novel highly specific small molecule inhibitor of GPR68 named Ogremorphin (OGM) to induce the iron mediated cell death pathway: ferroptosis. Method OGM was identified in a non-biased zebrafish embryonic development screen and validated with Morpholino and CRISPR based approaches. Next, A GPI-anchored pH reporter, pHluorin2, was stably expressed in U87 glioblastoma cells to probe extracellular acidification. Cell survival assays, via nuclei counting and cell titer glo, were used to demonstrate sensitivity to GPR68 inhibition in twelve immortalized and PDX GBM lines. To determine GPR68 inhibition’s mechanism of cell death we use DAVID pathway analysis of RNAseq. Our major indication, ferroptosis, was then confirmed by western blotting and qRT-PCR of reporter genes including TFRC. This finding was further validated by transmission electron microscopy and liperfluo staining to assess lipid peroxidation. Lastly, we use siRNA and CRISPRi to demonstrate the critical role of ATF4 suppression via GPR68 for GBM survival. Results We used a pHLourin2 probe to demonstrate how glioblastoma cells acidify their microenvironment to activate the commonly over expressed acid sensing GPCR, GPR68. Using our small molecule inhibitor OGM and genetic means, we show that blocking GPR68 signaling results in robust cell death in all thirteen glioblastoma cell lines tested, irrespective of genetic and phenotypic heterogeneity, or resistance to the mainstay GBM chemotherapeutic temozolomide. We use U87 and U138 glioblastoma cell lines to show how selective induction of ferroptosis occurs in an ATF4-dependent manner. Importantly, OGM was not-acutely toxic to zebrafish and its inhibitory effects were found to spare non-malignant neural cells. Conclusion These results indicate GPR68 emerges as a critical sensor for an autocrine pro-tumorigenic signaling cascade triggered by extracellular acidification in glioblastoma cells. In this context, GPR68 suppresses ATF4, inhibition of GPR68 increases expression of ATF4 which leads to ferroptotic cell death. These findings provide a promising therapeutic approach to selectively induce ferroptosis in glioblastoma cells while sparing healthy neural tissue.

Supplementary figures: 1): molecular structure of mibefradil, 2) Mibefradil effects on HIFs, 3) effects of sh-RNA inhibition of Cav3.2 in GSCs, 4) Schematic summary of the findings.

Supplementary table and figures 1-3. Table 1: combined list of screen data identifying 10 genes that commonly sensitize cells to radiation when knocked down. S1: Western blot confirming knockdown of SAT1. S2: Expression of SAT1 in control and shSAT1 knockdown tumors grown orthotopically in nude mice from Figure 3C and D after sacrifice. S3: Flow cytometry sort of control (shGFP) and SAT1 knockdown cells for Figure 5C.

Supplementary methods containing descriptions of PCR primer sequences, Reverse Phase Protein Arrays, RNA-seq, and Rescue Experiments.

PDF file - 127K, Cell cycle analysis was performed in triplicate on HSR-GBM1 XG2-derived neurospheres and analyzed on a Guava PCA.

Supplementary Figures 1-8 S1. MBNL1 expression in Glioblastoma by subtype S2. MBNL1 target gene splicing S3. MBNL1 expression in cultured GSCs S4. MBNL1 activity is monitored with RG6 splicing reporter minigene S5. Effect of induced MBNL1 expression on GSC proliferation S6. Self-renewal assays in 622 and 08-387 neurospheres S7. Self-renewal assays in 913-luciferase neurospheres S8. Survival analysis for RTTA control cells

Supplementary Figures S1-S3; Supplementary Tables S1-S2.

The Warburg Effect is a common feature of cancer cells characterized by increased glucose uptake and fermentation of glucose to lactate even in the presence of oxygen. While it is commonly accepted that Warburg Effect promotes the growth, survival, proliferation, and long-term maintenance of cancers, its precise function and its downstream mediators remain unclear. A key physiological consequence of the Warburg effect is lactate secretion, which acidifies the tumor milieu, thought to promote oncogenesis and confer tumor resistance to chemotherapy and radiotherapy. Glioblastoma multiforme (GBM) is one of the most aggressive and deadly cancers, characterized by cellular heterogeneity and plasticity, which are thought to drive extreme therapeutic resistance. Despite their heterogeneity, common hallmarks of GBM tumors are high levels of aerobic glycolysis (“Warburg Effect”) and a resultant acidic tumor microenvironment (TME), which promotes tumor progression. In an in vivo zebrafish developmental screen, we identified ogremorphin (OGM), a small molecule inhibitor of GPR68/OGR-1, a G-protein coupled receptor (GPCR) which is activated by extracellular protons. Using ogremorphin and pHluorin2-GPI, a novel sensor of extracellular acidification, we demonstrate that glioblastoma cells acidify their own environment in vitro and activate GPR68, and visualize, for the first time, the establishment of the acidic extracellular microenvironment during the formation of GBM spheroids in vitro. Selective inhibition of GPR68 causes robust cell death in all 12 glioblastoma cell lines tested to date, despite genetic and molecular heterogeneity, without toxicity on healthy cells in whole animals. Mechanistically, GPR68 inhibition activates ferroptosis, a programmed cell death characterized by lipid peroxidation, in an ATF4 (activating transcription factor 4)-dependent manner. Finally, in GBM cells, ogremorphin treatment demonstrates strong synergistic effects with the frontline therapeutics temozolomide and ionizing radiation. Our results indicate that GPR68 activation by extracellular acidification is a key cancer survival pathway downstream of the Warburg Effect, and that GPR68 inhibition, either alone or in combination with temozolomide and radiation therapy, is a promising therapeutic approach to selectively induce ferroptosis in GBM tumors. Citation Format: Charles H. Williams, Leif R. Neitzel, Jessica Cornell, Samantha Rea, Ian Mills, Maya Silver-Isenstadt, Jovanni D. Ahmad, Henry Brem, Betty Tyler, Eli E. Bar, Charles C. Hong. Therapeutic targeting of GPR68 activated by acidic extracellular microenvironment induces ferroptosis in glioblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 443.

Supplementary Figures S1-S3 from BRAF Activation Induces Transformation and Then Senescence in Human Neural Stem Cells: A Pilocytic Astrocytoma Model

Although an acidic microenvironment is a hallmark of cancer that promotes cancer progression, the underlying signaling pathways are not well understood. Here, in a chemical genetic screen for compounds that perturb zebrafish neural crest development, we identified ogremorphin, a novel small molecule inhibitor of the extracellular proton-sensing G protein-coupled receptor GPR68 that blocks all known modes of GPR68 activation, including acidity, flow and mechanical stretch. Using ogremorphin and genetic tools, we demonstrate that activation of GPR68 promotes cell migration and invasion in vitro and in vivo, by modulating formation of filipodia and focal adhesions. Next, we show that, even in globally neutral pH conditions, like the zebrafish embryo, there exists dynamic extracellular zones of local acidification, which we term proton flares. At the individual cell level, the transient proton flares are associated with new focal adhesions, and in tumor spheroids, a larger and longer lasting acidic domains are observed, recapitulating acidic tumor milieu in vitro. Using genetic and chemical biological approaches, we demonstrate that GPR68 signaling promotes glioblastoma cell survival and activates two important pathogenic pathways: O6-methylguanine-DNA methyltransferase, which confers resistance to temozolomide, and prosurvival and immune evasion chemokine interleukin 8 (IL8). Thus, GPR68 mediates the pathogenic signaling of the acidic tumor microenvironment, indicating ogremorphin and its analogs represent an attractive, novel therapeutic class.