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[Preprint]. 2026 Feb 2:2026.01.30.702627. [Version 1] doi: 10.64898/2026.01.30.702627

IDH-mutant inhibitors enhance the sensitivity of IDH1-mutant gliomas to cysteine-methionine deprivation and ferroptosis

Angeliki Mela 1, Abby Brand 1,2, Aayushi Mahajan 3, Athanassios Dovas 1, Nelson Humala 3, Smriti Kanangat 1, Allison C Kleinstein 4, Sandra Leskinen 3, Trang TT Nguyen 1, Qiuqiang Gao 1, Pavan S Upadhyayula 3, Jia Guo 5,6, Brian Gill 3, Markus D Siegelin 1, Peter A Sims 2, Brent R Stockwell 1,7,8,9, Jeffrey N Bruce 3, Peter Canoll 1,3,*
PMCID: PMC12889447  PMID: 41676617

Abstract

Cysteine is essential for synthesizing glutathione, the brain’s main antioxidant, and cysteine deprivation can trigger ferroptosis. Here, using a new mouse model of IDH1-mutant glioma that recapitulates the characteristics of human IDH1-mutant low-grade gliomas, we demonstrate that IDH1-mutant glioma cells are significantly more vulnerable to cysteine deprivation alone or in combination with the ferroptosis inducer RSL3, compared to IDH1-wildtype glioma cells. In addition, treatments with the IDH-mutant inhibitors vorasidenib and ivosidenib further sensitize the cells to ferroptosis. Metabolomics analysis reveals that IDH1-mutant cells have altered cysteine and methionine metabolism with deficiency in transsulfuration, which is further exacerbated by cysteine-methionine deprivation and IDH-mutant inhibitors. Furthermore, dietary cysteine-methionine deprivation alone or in combination with convection-enhanced delivery of RSL3 or ivosidenib in vivo significantly prolongs survival of IDH1-mutant tumor-bearing mice. Our findings suggest that targeting cysteine and methionine metabolism in combination with IDH-mutant inhibition provides promising therapeutic strategies for IDH1-mutant gliomas.

Introduction

Mutations in isocitrate dehydrogenase 1 (IDH1) define a distinct subset of diffuse gliomas characterized by slower progression, diffuse infiltration, and profound metabolic reprogramming1,2,3. The neomorphic activity of mutant-IDH1 converts α-ketoglutarate (α-KG) to the oncometabolite D-2-hydroxyglutarate (2-HG), which inhibits α-KG–dependent dioxygenases and perturbs redox homeostasis and intermediary metabolism4,5,6,7,8,9. Although the epigenetic and signaling consequences of 2-HG accumulation are well characterized, the broader metabolic consequences of mutant-IDH (IDHmut) and their functional implications for tumor growth and therapeutic response remain incompletely defined. Furthermore, the metabolic effects of IDHmut-inhibitors currently used in the treatment of low-grade IDH-mutant gliomas have not yet been fully elucidated10,11,12.

Emerging studies indicate that IDH1-mutant gliomas exhibit a chronic state of oxidative and metabolic stress13,14. Among these changes, alterations in cysteine and methionine metabolism are important, yet understudied features of the IDH1-mutant metabolic landscape13. These pathways play essential roles in maintaining glutathione synthesis, methylation potential, and redox buffering15,16. Dysregulation of transsulfuration and methionine cycle may render IDH1-mutant cells dependent on exogenous cysteine and vulnerable to fluctuations in extracellular nutrient availability17,18,19.

Recent work suggests that alterations in redox state may sensitize cells to ferroptosis, a form of regulated cell death driven by lipid peroxidation and glutathione depletion20,21,22,23. By constraining cysteine and methionine metabolism, mutant-IDH1 may create a permissive context for ferroptotic vulnerability—linking amino acid metabolism, oxidative stress, and cell death sensitivity. Here, we demonstrate that the IDH1 mutation alters cysteine and methionine metabolism to impose a redox constraint that sensitizes glioma cells to ferroptosis, revealing a previously unrecognized metabolic liability with therapeutic potential, that can be further exploited by combining cysteine-methionine deprivation with IDHmut-inhibitors.

Results

IDH1(R132H) significantly delays glioma progression.

To generate an IDH1-mutant glioma model, we performed stereotactic intracranial injections of PDGFA-IRES-CRE expressing retrovirus into the subventricular zone of neonatal (p4) Idh1(R132H)KI/+; p53fl/fl; RiboTag (IDH1-mutant) and +/+; p53fl/fl; RiboTag (IDH1-wildtype) mice (Figure 1A). We monitored tumor progression using MRI (Figure 1B). Our results show that mice of both genotypes formed tumors, however the IDH1 mutation significantly delays glioma progression (Figure 1B, C). Specifically, the IDH1-mutant, PDGFA-overexpressing, p53-deleted mice start developing lesions visible by MRI at 70–190 days post injection (dpi), while IDH1-wildtype mice reach end stage at 50–70 dpi (Figure 1C, D). Histopathological analysis of end-stage primary IDH1-mutant tumors revealed diffuse brain infiltration and hypercellularity, but in contrast to IDH1-wildtype tumors, showed few mitotic figures and an absence of vascular proliferation or necrosis (Figure 1C, Supplementary Figure 1). Immunohistochemistry for HA, the RiboTag reporter, labeled retrovirally infected tumor cells in both the IDH1-mutant and IDH1-wildtype models. In both models, tumor cells robustly expressed Olig2 and displayed a diffusely infiltrative growth pattern, intermingling with GFAP+ astrocytes, NeuN+ neurons, and Iba1+ microglia. Notably, these non-neoplastic cell populations were more prominent in the IDH1-mutant model (Figure 1C). A major distinction between the two models is their Ki67 proliferation index. The IDH1-wildtype mouse model exhibited a Ki67 index of 55.35% at 58 dpi (end stage), whereas the IDH1-mutant model showed markedly lower indices of 5.9% at 60 dpi and 15.45% at 250 dpi (Figure 1E), in line with the diffusely infiltrative, low-grade characteristics of human IDH1-mutant gliomas.

Figure 1. IDH1(R132H) mutation significantly delays glioma progression.

Figure 1.

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A. Diagram of experimental design of stereotactic intracranial injections of PDGFA-IRES-CRE retrovirus in Idh1(R132H)KI/+; p53fl/fl; RiboTag and +/+; p53fl/fl; RiboTag neonatal (p4) mice, cell line generation and orthotopic injections. B. T2 MRI images showing glioma progression at 50, 70, 190 and 250 dpi in IDH1-mutant (IDH1-PDGFA) and IDH1-wildtype (WT-PDGFA) mice. C. Immunohistochemistry of a WT-PDGFA tumor at 58dpi (end stage), an IDH1-PDGFA tumor at 60dpi (early stage) and an IDH1-PDGFA tumor at 250dpi (end stage) for HA, Ki67, olig2, GFAP, NeuN and Iba1. Anti-HA labels retrovirus-infected cells, hematoxylin was used as nuclear stain. Bar=100mm. D. Survival curves after retrovirus injections in neonatal (p4) mice. Median survival: IDH1-PDGFA undefined, WT-PDGFA 68dpi. Log-Rank (Mantel-Cox) test: p<0.0001****. E. Ki67 labeling index in WT-PDGFA 58dpi, IDH1-PDGFA 60dpi and IDH1-PDGFA 250dpi tumors. Ki67+ cells divided by total (hematoxylin positive) cells counted in 3 mice per group, two 20x fields per mouse (WT-PDGFA 58dpi: 3820 Ki67+ / 6901 total, IDH1-PDGFA 60dpi: 172 Ki67+ / 2914 total, IDH1-PDGFA 250dpi: 768 Ki67+ / 4969 total). t-test WT-PDGFA 58dpi vs IDH1-PDGFA 250dpi p=0.000005****, WT-PDGFA 58dpi vs IDH1-PDGFA 60dpi p=0.000002****, IDH1-PDGFA 60dpi vs IDH1-PDGFA 250dpi p=0.001772**. F. Survival curves after orthotopic injections of IDH1-mutant (IDHm) and IDH1-wildtype (WT) cells into adult B6 mice. Median survival: IDHm 108dpi, WT 50dpi. Log-Rank (Mantel-Cox) test: p<0.0001****.

At end stage, we harvested tumor cells and generated primary cell cultures from IDH1-mutant and IDH1-wildtype tumors. To confirm tumorigenic potential of these cell lines, we performed stereotactic intracranial injections into adult mice (Figure 1A, F). All mice injected with either IDH1-mutant or IDH1-wildtype cell lines form tumors, significantly faster than the primary tumor model that each cell line was generated from. Notably, mice injected with IDH1-mutant cells survive significantly longer than mice injected with IDH1-wildtype cells (Figure 1F). IDH1-mutant cells produce high levels of 2-HG both in vitro and in vivo, as detected by MEGA-PRESS MRS (Supplementary Figure 2A). MEGA-PRESS MRS also detects lower glutathione levels and higher glutamate to GABA ratio in IDH1-mutant mouse tumors in vivo (Supplementary Figure 2B, C), consistent with human IDH1-mutant gliomas24,25. These results suggest that our mouse model recapitulates key characteristics of human IDH1-mutant gliomas and can be used for further in vitro and in vivo studies.

IDH1-mutant glioma cells are sensitive to cysteine-methionine deprivation and ferroptosis in vitro.

Previously we have shown that cysteine-methionine deprivation (CMD) sensitizes IDH-wildtype glioma cells to ferroptosis23. To test this in our IDH1-mutant glioma cells we used RSL3, a GPX-4 inhibitor. We treated both IDH1-mutant and IDH1-wildtype cell lines with different concentrations of RSL3 for 17 hours with or without cysteine and methionine. Our results reveal that IDH1-mutant cells are significantly more sensitive to RSL3-induced ferroptosis in both control and CMD conditions (Figure 2A, B). CMD enhances sensitivity to RSL3, while treatment with the ferroptosis inhibitor Ferrostatin-1 prevents cell death in all conditions. These results were also confirmed by showing RSL3-mediated induction of lipid peroxidation as evidenced by green fluorescence shift in Bodipy-C11, following the addition of RSL3 alone or in combination with CMD (Supplementary Figure 3A). Our results show that IDH1-mutant cells are significantly more sensitive to RSL3 treatment compared to IDH1-wildtype cells and CMD significantly enhances their sensitivity. We also investigated the expression of Slc7a11 and Atf4, which are both transcriptional hallmarks of cellular response to ferroptosis26,27,28. RT-qPCR showed that there are significant increases of Slc7a11 and Atf4 transcripts in both IDH1-mutant and IDH1-wildtype cells after 17-hour treatment with CMD alone or with RSL3 (Supplementary Figure 3B).

Figure 2. Cysteine-methionine deprivation and IDHmut-inhibitors enhance the sensitivity of IDH1-mutant cells to ferroptosis in vitro.

Figure 2.

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A. Dose response curves after 17-hour treatment of IDH1-mutant (IDHm) and IDH1-wildtype (WT) cells with RSL3 (0, 0.5, 1, 5, 10, 15, 31, 62, 125, 250 nM) alone, with CMD or 2mM Ferrostatin-1 (FER) (IC50 WT 52.6, IDHm 5.1, WT-CMD 4.513, IDHm-CMD 0.3573). B. Area under the curve (AUC) plotted from three independent experiments as in A. C. MTS viability assay after 24 hours in control (BFP), CD or CMD conditions in the presence or absence of 2mM Ferrostatin-1. D. Dose response curves of IDH1-mutant cells after 17-hour treatments with RSL3 (0, 0.1, 0.5, 1, 5, 10, 31, 62, 125 nM) in CD conditions with or without 20mM IVO or 2mM Ferrostatin-1 (IC50 IDHm-CD 5.832, IDHm-CD-IVO 2.106). E. Area under the curve (AUC) plotted from three independent experiments as in D. F. Dose response curves after 17-hour treatment of IDH1-mutant cells with IVO (0, 5, 10, 20, 30 mM) in control, CD or CMD conditions (IC50 IDHm-CMD 9.526). G. Area under the curve (AUC) plotted from three independent experiments as in F. H. Dose response curves after 17-hour treatment of IDH1-mutant cells with VOR (0, 0.005, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2 mM) in control, CD or CMD conditions (IC50 IDHm 0.9281, IDHm-CD 0.7768, IDHm-CMD 0.0677). I. Area under the curve (AUC) from three independent experiments as in H. J. MTS viability assay of IDH1-mutant cells grown for 7 days in the absence or presence of 20mM IVO and subsequently treated in control (BFP), CD or CMD conditions for 17 hours with or without 2mM Ferrostatin-1 (FER). Data plotted as mean ± SD. Statistics assessed using t-test. Significance denoted by: *p < 0.05, **p < 0.01, ***p < 0.001, ****p<0.0001, ns: not significant.

To test whether IDH1-mutant cells are more sensitive to cysteine deprivation alone, we treated both IDH1-mutant and IDH1-wildtype cells with media made without cysteine (CD), or without cysteine and methionine (CMD) for 24 hours (Figure 2C). While both cell lines are sensitive to CMD, IDH1-mutant cells are significantly more sensitive than IDH1-wildtype cells to cysteine depletion alone. Both effects were rescued by Ferrostatin-1. To further investigate this sensitivity to cysteine deprivation, we used another ferroptosis inducer, IKE, that blocks cysteine uptake via the Slc7a11/Slc3A2 System Xc− cysteine-glutamate antiporter. We treated IDH1-mutant and IDH1-wildtype cells with different concentrations of IKE in control media (containing normal levels of cysteine and methionine) for 17 hours. Our results show that IDH1-mutant cells are significantly more sensitive to IKE-induced ferroptosis, which is rescued by Ferrostatin-1 (Supplementary Figure 3C, D). Furthermore, we treated IDH1-mutant and IDH1-wildtype cells with RSL3 in cysteine-deprived (CD) media. Our results show that IDH1-mutant cells are significantly more sensitive to CD and ferroptosis compared to IDH1-wildtype cells and these effects are rescued by Ferrostatin-1 (Figure 2D, E; Supplementary Figure 4A, B).

IDHmut-inhibitors enhance the sensitivity to cysteine-methionine deprivation in vitro.

The development of IDHmut-inhibitors and their use as promising therapeutic glioma treatments prompted us to test potential treatment effects in our IDH1-mutant mouse model as well as possible synergistic or antagonistic effects with CMD and ferroptosis inducers. We tested two FDA-approved IDHmut-inhibitors, ivosidenib (IVO) which selectively inhibits mutant IDH1, and vorasidenib (VOR), a dual mutant IDH1/2 inhibitor10,11,12,29.

To assess the effects of IDHmut-inhibitors on cell viability, we treated IDH1-mutant and IDH1-wildtype cells in vitro with ivosidenib (IVO) or vorasidenib (VOR) under control, CD, or CMD conditions for 17 hours (Figure 2F–I). IVO has minimal effect on cell viability of IDH1-wildtype cells across growth conditions (Supplementary Figure 4C, D). In contrast, IVO induced dose-dependent reductions in viability of IDH1-mutant cells specifically when grown in CMD and to a lesser extent in CD conditions, but not in control media (Figure 2F, G). VOR similarly showed minimal effects on IDH1-wildtype cell viability at concentrations below 1 μM (Supplementary Figure 4E, F). However, VOR caused dose-dependent decreases in viability in IDH1-mutant cells at concentrations below 1 μM even in control media, and these effects were significantly enhanced under CD and CMD conditions (Figure 2H, I). At concentrations above 0.05 μM, VOR reduced IDH1-mutant cell viability by approximately 15% in control conditions, ~50% under CD conditions, and ~70% under CMD conditions (Figure 2H). Although these differences between IVO and VOR may reflect differences in potency or the dual targeting of IDH1 and IDH2 by VOR29, both inhibitors markedly reduced IDH1-mutant cell viability when combined with CMD. Together, these findings indicate that cysteine-methionine deprivation sensitizes IDH1-mutant glioma cells to IDH-mutant inhibition, irrespective of the specific inhibitor used.

To investigate whether IDHmut-inhibitors affect the expression of Slc7a11 in our IDH1-mutant glioma cells, we treated IDH1-mutant cells with 20mM IVO or 100nM VOR for 24 and 48 hours and subsequently performed qRT-PCR for Slc7a11 (Supplementary Figure 4G). Both inhibitors significantly increase Slc7a11 transcripts after 24 hours, and IVO continues to do so at 48 hours. These results suggest that combining IDHmut-inhibitors with CMD and ferroptosis inducers may cause an increased dependence on extracellular cysteine and induce ferroptotic cell death.

To further investigate this, we treated IDH1-mutant and IDH1-wildtype cells in CD (normal methionine) conditions with 20mM IVO and various doses of RSL3 (Figure 2D, E; Supplementary Figure 4A, B). Our results show that IDH1-mutant cells are significantly more responsive to triple combination treatment of RSL3 with IVO and CD compared to just RSL3 and CD. In contrast, IDH1-wildtype cells are not significantly affected by the addition of IVO or cysteine deprivation. To assess the effects of more prolonged treatment with IDHmut-inhibitors in vitro, IDH1-mutant cells were grown for 7 days with or without 20mM IVO and subsequently treated with cysteine-deprived or cysteine-methionine deprived media (CD or CMD) (Figure 2J). Our results show that IDH1-mutant cells treated for 7 days with IVO are more sensitive to CD than non-treated cells. CMD in vitro has the same robust effect in both IVO-treated and non-treated cells.

Metabolomic analyses reveal that IDH1-mutant glioma cells have transsulfuration deficiency that is exacerbated by cysteine-methionine deprivation and IDHmut-inhibitors.

To further explore these metabolic vulnerabilities, we performed targeted metabolite profiling for more than 200 metabolites on our IDH1-mutant and IDH1-wildtype mouse cell lines grown under different conditions. Under control conditions, IDH-mutant cells showed significantly higher levels of 2-HG along with significant alterations in multiple additional metabolites compared to IDH-wildtype cells (Figure 3A, B). Pathway analysis showed that cysteine and methionine metabolism, glutathione metabolism and taurine/hypotaurine metabolism are among the most significantly different pathways (Supplementary Figure 5A). Notably, several molecules of the methionine cycle, transsulfuration and cysteine metabolism (L-methionine, S-adenosylmethionine - SAM, S-adenosylhomocysteine – SAH, L-cystathionine, hypotaurine) are significantly decreased in IDH1-mutant cells (Figure 3A, B). Oxidized glutathione (GSSG) is significantly higher in IDH1-mutant cells, while reduced glutathione (GSH) is significantly lower, as was also seen in end stage tumors by MEGA-PRESS MRS. A redox imbalance is also shown by the significantly lower GSH/GSSG ratio and the significantly lower hypotaurine/taurine ratio (Figure 3B). In line with these results and previous studies in human cell lines30, Seahorse metabolic assays also suggest that IDH1-mutant cells have significantly altered oxidative metabolism (Supplementary Figure 6). In addition, glutamate and glutamine, key molecules in cystine import and glutathione synthesis, are significantly enriched in IDH1-mutant cells, consistent with our in vivo MEGA-PRESS MRS results (Supplementary Figure 7A). Furthermore, our data shows that IDH1-mutant cells have significantly higher SAM/SAH ratio, indicating high methylation potential, and significantly lower cystathionine/SAH ratio (Figure 3B). These results suggest that IDH1-mutant cells have a metabolic state that favors the methionine cycle and methylation potential over transsulfuration, which can account for their increased vulnerability to cysteine deprivation.

Figure 3. IDH1-mutant cells have an altered metabolic profile and are sensitive to cysteine-methionine deprivation.

Figure 3.

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A. Volcano plot (blue and red: FDRq<0.05) of metabolomics analysis comparing IDH1-mutant cells (IDHm) to IDH1-wildtype (WT) cells after 17 hours in control conditions. B. Ratios and values of metabolites of the methionine cycle, transulfuration pathway and glutathione metabolism in untreated and CMD-treated IDHm and WT cells. Statistics assessed using t-test. Significance denoted by: *FDRq < 0.05, **FDRq < 0.01, ***FDRq < 0.001, ****FDRq<0.0001, ns: not significant (SAM: S-adenosylmethionine, SAH: S-adenosylhomocysteine, GSH: Glutathione, GSSG Oxidized glutathione). C. Volcano plot (blue and red: FDRq<0.05) comparing untreated IDHm cells to IDHm cells grown in CMD conditions for 17 hours.

To evaluate the metabolic effects of cysteine-methionine deprivation, we treated our mouse cell lines with CMD for 17 hours and then performed the same targeted metabolite profiling. Our results show that CMD significantly decreases metabolites involved in the methionine cycle, transsulfuration pathway and glutathione metabolism (Figure 3B, C, Supplementary Figures 5B, 7A), as well as the ratios SAM/SAH, GSH/GSSG and hypotaurine/taurine. These results indicate that CMD induces oxidative stress and disrupts transsulfuration in IDH1-wildtype cells, whereas IDH1-mutant cells show a pre-existing metabolic vulnerability due to altered cysteine and methionine metabolism, reduced transsulfuration, and increased oxidative stress, which is further exacerbated by CMD.

To characterize the metabolic effects of IDHmut-inhibitors, alone or in combination with CMD, we treated IDH1-mutant cells with 20mM IVO for 48 hours, CMD for 17 hours or combination of 20mM IVO for 48 hours with CMD for the last 17 hours of treatment (Figure 4, Supplementary Figures 7B, 8). In parallel, cells were treated with 1mM VOR for 48 hours (Supplementary Figure 9). Targeted metabolite profiling on more than 200 metabolites was performed across all conditions. CMD alone had the same effects as previously shown (Figure 3). Both IVO and VOR significantly decreased 2-HG, and altered many metabolites involved in cysteine/methionine metabolism (Figure 4A, Supplementary Figures 9A, B). Pathway analysis showed that IDHmut-inhibition significantly affects cysteine and methionine, glutathione, taurine and hypotaurine metabolism (Supplementary Figures 8, 9C). Specifically, IVO-treated cells have significantly decreased glutathione, as well as GSH/GSSG ratio, indicating higher oxidative stress. In addition, SAM and cystathionine are significantly decreased (Figure 4D). Similarly, in VOR-treated IDH1-mutant cells, SAM, cystathionine, glutathione and hypotaurine are significantly decreased (Supplementary Figure 9B). Combining CMD with IDHmut-inhibitors further exacerbated the effects on glutathione and the GSH/GSSG ratio (Figure 4B-D). Pathway analysis reveals that IDHmut-inhibition combined with CMD significantly affects glutathione, cysteine and methionine metabolism (Supplementary Figure 8). These metabolic changes provide a mechanistic correlation to our cell viability studies, supporting the finding that IDHmut-inhibition further sensitizes IDH1-mutant cells to CMD.

Figure 4. IDHmut-inhibitors enhance the sensitivity of IDH1-mutant glioma cells to cysteine-methionine deprivation.

Figure 4.

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Volcano plots (blue and red: FDRq<0.05) of metabolomics results comparing A. untreated IDH1-mutant cells (IDHm) to treated with 20mM IVO for 48 hours (IDHm IVO), B. IDHm IVO to IDHm treated with 20mM IVO for 48 hours and CMD for 17 hours (IDHm IVO CMD) and C. IDHm IVO CMD to IDHm treated with CMD for 17 hours (IDHm CMD). D. Metabolite ratios and metabolites of the methionine cycle, transulfuration pathway and glutathione metabolism significantly altered by CMD and IVO treatments from experiments presented in A, B, C. Statistics assessed using t-test. Significance denoted by: *FDRq < 0.05, **FDRq < 0.01, ***FDRq < 0.001, ****FDRq<0.0001, ns: not significant (SAM: S-adenosylmethionine, SAH: S-adenosylhomocysteine, GSH: Glutathione, GSSG Oxidized glutathione).

IDHmut-inhibition in combination with cysteine-methionine deprivation diet in vivo, prolongs survival of IDH1-mutant glioma-bearing mice.

To assess whether IDH1-mutant glioma cells are more sensitive to CMD and ferroptosis in vivo, we tested the effects of RSL3 treatment in combination with a cysteine-deprived, methionine-restricted diet (CMD, 0.0% cysteine, 0.15% methionine) in mice with IDH1-mutant brain tumors. Notably, we treat cells with 0.0% cysteine and 0.0% methionine for CMD in vitro. For in vivo studies, methionine, as an essential amino acid, can be reduced but not completely removed from the diet. At 15dpi, mice were switched to either control diet or CMD and were maintained on these diets until end stage. Mice were treated with RSL3 or vehicle through convection-enhanced delivery (CED) for 7 days, starting at 30 dpi. Kaplan-Meier survival analysis showed a significant survival benefit for CMD or RSL3 treated mice over untreated mice. Notably, mice co-treated with both RSL3 and CMD survived significantly longer than control or CMD-treated mice (Figure 5A, B).

Figure 5. IDHmut-inhibition and cysteine-methionine deprivation prolong survival of IDH1-mutant tumor-bearing mice in vivo.

Figure 5.

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A. Schematic representation of survival studies after intracranial injections of IDH1-mutant cells into adult mice. At 15 days post injection (dpi), mice were transferred to cysteine-deprived, methionine-restricted (CMD) or control diet. At 30dpi osmotic pumps were placed for intratumoral convection enhanced delivery (CED) for 7 days with 500nM RSL3 (RSL3 CED) or 0.01% DMSO (Control CED). In a second study, mice were treated for 7 days with 20mM IVO (IVO CED) or 0.01% DMSO. B. Survival curves after treatment with 500nM RSL3 and CMD in adult mice. Median survival: CTL 87dpi, CMD 101dpi, RSL3 108dpi, RSL3-CMD 127dpi. Log-Rank (Mantel-Cox) test: CTL vs RSL3 p=0.0050**, CTL vs CMD p=0.0280*, CMD vs RSL3 p=0.1563, RSL3 vs RSL3-CMD p=0.0743, CMD vs RSL3-CMD p=0.0104*, CTL vs RSL3-CMD p<0.0001****. C. Survival curves after treatment with 20mM IVO and CMD in adult mice. Median survival: CTL 97dpi, CMD 127dpi, IVO 114dpi, IVO-CMD 182dpi. Log-Rank (Mantel-Cox) test: CTL vs IVO p=0.0311*, CTL vs CMD p=0.0049**, CMD vs IVO p=0.9415, CMD vs IVO-CMD p=0.0417*, IVO vs IVO-CMD p=0.0449*, CTL vs IVO-CMD p=0.0001***.

To further explore the therapeutic potential of our findings, we tested the effects of IVO in combination with CMD diet in mice with IDH1-mutant brain tumors (Figure 5A, C). We selected ivosidenib (IVO) for these studies because it is highly selective for mutant IDH112,29. Although clinical trials12 have shown that systemically administered vorasidenib (VOR) achieves superior brain penetrance compared to IVO, in our studies we employ CED, which bypasses the blood–brain barrier and permits direct intratumoral drug administration at substantially higher local concentrations than are achievable through systemic dosing. This approach allows us to maximize on-target inhibition within the tumor while minimizing systemic exposure and potential off-target or isoform-nonspecific effects. Using the same approach as in the CMD-RSL3 survival study above, at 15dpi mice were switched to either control or CMD diet and maintained on those diets for 7 months, until 230dpi (Figure 5A, C) when the remaining long-term survivors were switched from CMD to control diet. At 30dpi and for 7 days, mice were treated with CED of 20mM IVO or vehicle. Kaplan-Meier survival analysis showed a significant survival benefit for CMD or IVO treated mice over control mice. Notably, mice co-treated with both IVO and CMD survived significantly longer than control, IVO or CMD treated mice.

Discussion

In this study we present a new mouse model of IDH1-mutant glioma, which successfully recapitulates the pathological characteristics of human low-grade IDH1-mutant gliomas, including diffuse infiltration, slow progression, and high levels of 2-HG. In addition, we identified significant metabolic alterations that were further exacerbated by cysteine-methionine deprivation and IDH1mut-inhibition. Previous studies have highlighted the limitations of establishing efficient IDH1-mutant glioma models in vitro and in vivo, that make studying the effects of the IDH1 mutation on metabolism and other cellular functions, as well as testing different therapeutic treatments, very challenging31,32. Here, comparing our IDH1-mutant model with an IDH1-wildtype model allowed us to identify key metabolic differences due to the presence of the IDH1 mutation. Our results reveal that IDH1-mutant cells are significantly more sensitive than IDH1-wildtype cells to cysteine deprivation and ferroptosis, and have significant alterations in glutathione, cysteine and methionine metabolism. Previous studies have focused on the epigenetic and metabolic alterations caused by 2-HG on glucose metabolism and the TCA cycle33,34, while studies on cysteine and methionine metabolism have shown that cystathionine, the transsulfuration intermediate, has low abundance in IDH1-mutant human glioma tissue and PDX models13,35,36,37,38. Furthermore, human IDH1-mutant glioma cells are sensitive to cysteine deprivation and can be rescued by the addition of cystathionine35. Our results demonstrate that cystathionine is significantly depleted in our mouse IDH1-mutant cells, recapitulating the human data and identifying deficiency in transsulfuration as a potential metabolic vulnerability for IDH1-mutant gliomas.

Our results reveal that, in addition to transsulfuration, IDH1-mutant gliomas have a dysfunction in the methionine cycle, as SAM is significantly lower in IDH1-mutant compared to IDH1-wildtype cells, and that is further exacerbated after treatments with CMD and IDHmut-inhibitors. It has previously been shown that elevated SAM levels directly promote transsulfuration39,40,41 and inhibit remethylation of homocysteine to methionine17,42,43,44. Compared to IDH1-wildtype, IDH1-mutant glioma cells have significantly lower levels of SAM and cystathionine, indicating disruption of the methionine cycle and transsulfuration pathway (Supplementary Figure 10). SAM levels decrease even further in CMD conditions, as well as after IDHmut-inhibition and further still after combination of both treatments. This coincides with further reduction of transsulfuration and diminished antioxidant defense, as demonstrated by the reduced ratios of GSH/GSSG and Hypotaurine/Taurine45. These effects render the IDH1-mutant cells more vulnerable to ferroptosis. Consistent with our results, a previous study showed that human IDH1-mutant glioma cells are sensitive to cysteine deprivation and depend on exogenous cysteine rather than transsulfuration for glutathione synthesis35. In our IDH1-mutant mouse model, exploiting this vulnerability in cysteine metabolism by combining IDHmut-inhibition and dietary CMD in vivo, significantly prolongs survival of tumor-bearing mice. Notably, while IDHmut-inhibitors significantly reduce 2-HG towards IDH1-wildtype levels, other metabolites remain altered or are further disturbed.

The synergy between CMD and IDHmut-inhibition may reflect the fact that pharmacologic inhibition of mutant IDH1 reverses some, but not all, metabolic consequences of the IDH1-mutant state. While IDHmut-inhibitors rapidly suppress 2-HG production, multiple studies have shown that broader metabolic, epigenetic, and redox-associated alterations persist or normalize only incompletely following treatment46,47,48. Concurrently, CMD acutely restricts cysteine availability and glutathione synthesis36, whereas restoration of redox homeostasis likely requires slower metabolic and transcriptional rewiring, including adjustments in transsulfuration and one-carbon metabolism, which can take several days49,50. Persistent or incompletely reversible alterations in sulfur metabolism or redox buffering may further enhance this vulnerability. Together, these findings suggest that combining dietary sulfur amino acid restriction with IDHmut-inhibition may exploit a transient metabolic liability in IDH1-mutant gliomas, providing a rationale for integrating CMD with IVO as a therapeutic strategy.

In conclusion, here we present a mouse model of IDH1-mutant glioma that can be used as a powerful tool to study the effects of both the IDH1 mutation and IDHmut-inhibitors in vitro and in vivo. Our results show that the IDH1-mutant cells have a disruption in cysteine and methionine metabolism and are sensitive to ferroptosis. IDHmut-inhibitors further exacerbate this vulnerability and, in combination with cysteine-methionine deprivation, induce oxidative stress and ferroptotic cell death of glioma cells in vitro and significantly prolong survival of tumor-bearing mice in vivo. Our study elucidates the benefits of combining IDHmut-inhibitors with a diet that targets specific metabolic vulnerabilities, in this case cysteine and methionine metabolism. Further studies are required to demonstrate efficiency in a clinical setting with the goal of improving therapeutic strategies for IDH1-mutant gliomas.

Methods

Mouse strains

Two mice (male and female) that harbor a knock-in (KI) R132H mutation of the mouse Idh1 gene in heterozygous condition1 were crossed to generate homozygous mice. These were then crossed with p53fl/fl; RiboTag2 mice and offspring were bred to Idh1(R132H)KI, p53fl/fl, and RiboTag homozygosity. These mice are viable and fertile as homozygotes. Since the Idh1(R132H) appears in heterozygous condition in human gliomas, for all necessary in vivo experiments we crossed Idh1(R132H)KI; p53fl/fl; RiboTag mice with +/+; p53fl/fl; RiboTag mice. The offspring have Idh1(R132H)KI in heterozygous condition and maintain p53 and RiboTag as homozygotes3,4.

All experimental procedures involving mice were reviewed and approved by the Columbia University Institutional Animal Care and Use Committee (IACUC). The mice were monitored daily and euthanized when they exhibited signs that demonstrate that they are symptomatic from tumor burden including any of the following: decreased level of alertness and activity, seizures, periorbital hemorrhages, epistaxis (nose bleeds), impaired motor function, and/or impaired ability to feed secondary to decreased motor function, paresis or coma.

Isolation of primary tumor cells and generation of cell lines

Murine glioma cell lines were generated from retrovirus-induced orthotopic murine glioma models. Neonatal (p4) Idh1(R132H)KI/+; p53fl/fl; RiboTag mice were anesthetized using hypothermia and orthotopically injected with 1μL of PDGFA or PDGFB–internal ribosomal entry site (IRES)–cyclization recombination (Cre) retrovirus with titer of 106 cfu/μL and stereotactic coordinates relative to bregma: 1mm anterior, 1mm lateral, 1mm deep, aiming for subcortical white matter, using a Hamilton syringe at a flow rate of 0.3 μL/min. End-stage tumors were harvested and primary cells were isolated as follows: mice were sacrificed by cervical dislocation and decapitation, brains were acutely harvested and tumors were surgically resected. Tissue was minced and then dissociated in 10mL of 3% TrypLE (ThermoFisher Scientific, 12604013) in PBS, at 37°C under shaking for 10 minutes. Tissue was triturated using 1mL tips and subsequently filtered through a 70μm mesh. An equal volume of media was added to neutralize trypsin, and the suspension was centrifuged at 1200 rpm for 5 min. The supernatant was discarded, and the cell pellet was resuspended in media. The cell suspension was cultured in one well of a PLL-coated 6-well plate.

IDH1-wildtype tumor cells are cultured in basal media (BFP), containing DMEM (Gibco™ 11965–092) with 0.5% FBS (Gibco™ 16000044), 1% antibiotic-antimycotic (Gibco™ 15240–062), 1% N2 supplement (Gibco™ 17502–048), 10 ng/ml of recombinant human PDGF-AA (Peprotech 100–13A) and 10 ng/ml of recombinant human FGFb (Peprotech 100–18B-50UG).

IDH1-mutant tumor cells were isolated and cultured in RH media, an enriched form of BFP, based on media used for isolation of OPCs5,6, containing DMEM (Gibco™ 11965–092) with 0.25% FBS (Gibco™ 16000044), 1% antibiotic-antimycotic (Gibco™ 15240–062), 1.5% N2 supplement (Gibco™ 17502–048), 15 ng/ml of recombinant human PDGF-AA (Peprotech 100–13A), 15 ng/ml of recombinant human FGFb (Peprotech 100–18B-50UG), 2.5% Insulin-Transferrin-Selenium (Gibco™ 41400–045), 0.5% Sodium Pyruvate (SIGMA S8636), 0.5% GlutaMax™ (Gibco™ 35050–061), 0.5% B27 Supplement (Gibco™ 17504–044), 5 ng/ml biotin (SIGMA B4501). IDH1-mutant cells express high levels of 2-HG and grow better in RH medium (Supplementary Figure 11A, B).

A PDGFA-driven cell line made from one tumor with the IDH1R143HKI/+ ; p53fl/fl; RiboTag genetic background (IDHm or RHA), a PDGFB-expressing cell line made from one tumor with the same IDH1R143HKI/+ ; p53fl/fl; RiboTag background (IDHmB or RHB) and a PDGFB-expressing cell line from a tumor with +/+ ; p53fl/fl background (WTB) were used for this study. A PDGFA-expressing +/+ ; p53fl/fl cell line (WT, labeled MG37 or APCL8 in previous studies) was also used. Similar results are observed with cell lines isolated from PDGFA-induced and PDGFB-induced primary tumors (IDHmB and WTB cell lines, Supplementary Figure 12).

Orthotopic tumor implantation, diet allocation and convection enhanced delivery

Adherent cells were lifted using TrypLE (ThermoFisher Scientific, 12604013) and a cell suspension was made with a concentration of 7 × 104 cells in 1μL. Mice were injected at 7–8 weeks old. Mice were anesthetized with Ketamine/Xylazine (100mg/kg and 10mg/kg, respectively) and assessed for lack of reflexes by toe pinch. Hair was shaved and scalp skin was incised. A burr hole was made with a 17-gauge needle 2mm lateral and 2mm anterior to bregma. Intracranial injection of 1μL of cell suspension was performed under stereotactic guidance, 2mm deep into the brain parenchyma aiming for subcortical white matter, using a Hamilton syringe at a flow rate of 0.3 μL/min. Tumor growth was assessed by MRI monitoring using a Bruker BioSpec 9.4 Tesla Small Animal MR Imager (CUIMC Oncology Precision Therapeutics and Imaging Core, OPTIC).

For in vivo studies with CMD diet, mice were transitioned to the diet fifteen days post tumor implantation. Special diets were created by LabTestDiet (W.F. Fisher and Sons): a control diet with 0.43% methionine and 0.33% cystine (w/w, catalog no. 5WVL) and a cystine-deprived, methionine-restricted diet with 0.15% methionine and 0.0% cystine (w/w, catalog no. 5WVM).

Similarly to what we have previously shown in the IDH-wildtype (WT) glioma model7, the diet was well tolerated throughout the duration of the survival studies, although notably CMD mice maintained lower weights than control mice. Investigators were not blinded to the diet allocation during experiments or outcome assessments.

After randomization to control or CMD diets, mice were further randomized into control or treatment groups using convection enhanced delivery. Pump implantation was performed at 30 days post tumor implantation. A total of 36 mice, 9 per group (5 female, 4 male), were included in the RSL3-CMD study and a total of 28 mice, 7 per group (4 female, 3 male), were included for the IVO-CMD study. Mini osmotic 7-day pumps (Alzet, model 1007D) were filled with 100μL of either 0.1% DMSO in PBS (control), 500nM RSL3 in PBS or 20mM ivosidenib in PBS. These pumps infuse 100 μL of fluid over 7 days. Mice were anesthetized with Ketamine/Xylazine (100mg/kg and 10mg/kg, respectively), hair was shaved and scalp skin incised. The pump was inserted subcutaneously on the back of the mouse, and a burr hole was made 1mm posterior to the tumor implantation site. Then, the CED catheter was inserted 3mm into the tumor and stabilized with instant adhesive (Loctite 454, Alzet #0008670). Pumps were explanted after 7 days of infusion (at 37dpi) and mice were monitored until they developed signs of tumor morbidity.

Tissue processing

Mice were assessed daily for signs of tumor morbidity. At end stage, following cervical dislocation, the brain was surgically removed and processed according to the experiment. For histological analysis, the whole brain was placed in 4% PFA. After 24 hours, the brain was placed in PBS and sent to the Molecular Pathology Shared Resource (CUIMC MPSR) for paraffin embedding, sectioning and Hematoxylin-Eosin staining. For primary cell isolation, the largest anterior part of the tumor was used, and a small portion of the back was placed in 4% PFA to fix for histological analysis.

Immunohistochemistry and antibodies

Paraffin-embedded 5μm tissue sections were processed for immunohistochemistry. Briefly, following deparaffinization with xylene and ethanol, sections were boiled in Sodium Citrate buffer, pH 6.0, for 10min at high pressure in a pressure cooker. After cooling down, sections were treated with 1% hydrogen peroxide to block endogenous peroxidases, then washed in PBS and incubated in blocking solution (0.5% Triton-X in PBS with 5% Horse Serum), at room temperature for 30–60 minutes. Following overnight (16–19 hours) incubation with primary antibody in blocking solution at 4oC, slides were washed in PBS and incubated with biotinylated secondary antibody for 1 hour and ABC reagent for 30–45 minutes according to the Vectastain Elite ABC kit instructions (Vector Laboratories). DAB (Dako) incubation was used to visualize staining, followed by counterstain with hematoxylin. Sections were then dehydrated and mounted with Permount. Primary antibodies for olig2 (Millipore AB9610, rabbit 1:200), Ki67 (Cell Signaling 9129, rabbit 1:200), GFAP (Dako Z0334, rabbit 1:500), NeuN (Millipore ABN78, rabbit 1:200), Iba1 (Cell Signaling 17198, rabbit 1:200), HA (Biolegend 901513, Covance MMS-101R clone 16B12, mouse IgG1, 1:500) were used.

Cell culture and in vitro treatments

All cells were grown at 37°C with 5% CO2 in poly-L-lysine coated plates. All IDH1-wildtype cell lines were grown in BFP media. IDH1-mutant cells were grown in RH media. For all in vitro treatments, IDH1-mutant cell lines were transferred and grown into BFP media for 3 days before the experiment. Cysteine-methionine deprived (CMD) media was made from basal DMEM without cysteine, methionine, and glutamine (Thermo Fisher Scientific, 21013024) that was supplemented with L-glutamine to a final concentration of 4mM (as is the concentration in DMEM, Gibco™ 11965–092, used to make BFP). In vitro treatments of IDH1-mutant and IDH1-wildtype cells for 2 days with different concentrations (0–4mM) of glutamine, show that IDH1-mutant cells are significantly more dependent on glutamine (Supplementary Figure 11C). Cysteine deprived (CD) media was made from CMD media, supplemented with L-methionine to a final concentration of 30mg/L. All other components of the media were the same for control (BFP) and CMD media. Thus, for all experiments the only difference between CMD, CD and control conditions for each cell line was the concentration of cysteine and methionine in the media used. Murine glioma cells were plated in triplicate at a density of 7000 cells per well in a 96-well plate, then, 17 hours after plating, media was removed and treatment was added. We treated IDHm and WT cells in vitro with different concentrations of RSL3 (Stockwell lab), ivosidenib (MedChemExpress, AG-1205) or vorasidenib (MedChemExpress, AG-881) in control, CD or CMD media. Cell viability was assessed after treatment using a colorimetric reaction (MTS assay) with CellTiter 96 Aqueous One Solution reagent (Promega G3580). Absorbance at 490nm was detected with a BioRad Microplate Reader. Values were normalized to control or zero treatment conditions. Averages across 3 independent experiments are reported.

2-HG detection

To detect D-2-HG, 2×106 IDH1-mutant cells were plated per 10cm plate coated with poly-L-lysine. After 48 hours, cells were lifted using TryPLE, and cell pellets were collected after centrifugation at 1200rpm for 5 minutes at room temperature. Cell pellets were frozen at −80°C and processed for 2-HG detection assay according to the instructions of D-2-Hydroxyglutarate Assay Kit (Abcam ab211070).

Flow cytometric analysis of Lipid Peroxidation

IDH1-mutant and IDH1-wildtype cells were treated for 4 hours with either control or CMD media, followed by RSL3 treatment for 30 minutes at two different concentrations (50nM and 125nM). After treatment in 6-well plates, cells were lifted using TrypLE (ThermoFisher, 12604013). Cell pellets were resuspended in 1mL PBS with Bodipy-C11 (ThermoFisher, D3861) added to a final concentration of 2mM and incubated for 10min at 37°C. Cells were then washed once in PBS, centrifuged at 400 g for 5min, resuspended in flow buffer (PBS, 0.5% FBS, 1 μg/mL DAPI) and filtered in polystyrene flow tubes (Fisher Scientific #352008). Data were collected on a BD LSRIII Fortessa Cell Analyzer using Pacific Blue, FITC and PE filters as described9 and analyzed using FlowJo v10.

Mitochondrial stress assay

The process using a Seahorse XFe24 analyzer is described in depth elsewhere10. A mitochondrial stress assay was performed based on Agilent Technologies manual. Murine glioma cell lines were seeded in XFe24 cell culture microplates (Agilent Technologies) at 18,000 cells per well in 250μL of BFP media. After 17 hours, media was aspirated and replaced with assay medium (Agilent Technologies). Mitochondrial stress tests were run in the following order: 2 μM oligomycin, 2 μM trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP), and 0.5 μM rotenone/antimycin A.

Metabolomics

To prepare samples for Metabolomics, 2×106 cells were plated per 10cm plate coated with poly-L-lysine (3 plates per condition) and treated according to the experiment (BFP, CMD, BFP with IVO, BFP with IVO and CMD). Then, plates were washed 2 times with ice-cold PBS, aspirated, placed on dry ice and 1mL of 100% HPLC grade methanol was added to the dish. Cells were scraped and transferred with methanol into cold Eppendorf tubes. Samples were then centrifuged at 14,000 × g for 20min at 4°C. Pellets were used to extract protein using cell extraction buffer with protease and phosphatase inhibitors. A colorimetric Bradford assay was read at 740 nm for evaluation of total protein content. Supernatants were frozen at −80°C and sent in dry ice for mass spectrometric analysis at the Weill Cornell Medicine Proteomics and Metabolomics Core Facility. Samples were normalized by total metabolite counts and FDRq value was calculated in GraphPad Prism 10.0 with the two-stage step-up method of Benjamini, Krieger and Yekutieli11. Pathway analysis was performed using MetaboAnalyst 6.0 (https://www.metaboanalyst.ca/).

Real time Quantitative PCR method

Primer sequences used for Slc7a11, Atf4 and Actin were as described previously7. Cells were treated into 6-well plates for 17 hours. Based on our in vitro viability studies, we used a prolonged treatment of 5nM RSL3 (close to IC50) to avoid cell death and detect possible treatment effects on mRNA transcripts. We treated IDH1-mutant and IDH1-wildtype cells with 5nM RSL3 with or without CMD for 17 hours. Cells were scraped into ice-cold PBS and cell pellets were collected after centrifugation at 14000rpm at 4°C and stored at −80°C. Total RNA was extracted from the cell pellets using the RNeasy Mini kit (QIAGEN, 74106). Up to 2.5 μg of RNA was used with the SuperScript Vilo cDNA synthesis kit (Thermo Fisher, 117540–50). cDNA was diluted to a concentration of 250 ng/μL and the RT-qPCR reactions were conducted with Thermo Scientific ABsolute Blue qPCR SYBR (ThermoFisher, AB4323A). Duplicate samples per condition were analyzed on an Applied Biosystems Quant Studio 3 qPCR instrument with all experiments being repeated 3 independent times. beta-Actin was used as reference and log fold change was calculated using the ddCT method comparing treatments to a control (BFP with 0.01% DMSO) sample.

MR Spectroscopy

MRS MEGA-PRESS for 2-HG, Glx (glutamate and glutamine), glutathione and GABA was performed as described previously12,13.

Supplementary Material

Supplement 1
media-1.pdf (1.7MB, pdf)

Acknowledgments

This study was supported by the NIH/NCI (U54CA274504 and P01CA291697), the NIH/NINDS (R01NS103473), the Emerson Health Collective Cancer Research Fund and generous support from the Summer family. We thank Dr. Tak Wah Mak for the IDH1(R132H)KI mouse. We thank Trine Giaever for her artistic help with the figures. Metabolomics mass spectrometric analysis was performed at the Weill Cornell Medicine Proteomics and Metabolomics Core Facility. The MRI imaging was carried out in the MR Facility of the Oncology Precision Therapeutics and Imaging Core (OPTIC) Shared Resource, supported by the Columbia University Medical Center NIH/NCI Cancer Center Support grant (P30CA013696). This study used the CCTI Flow Cytometry Core and the Molecular Pathology Shared Resource (MPSR) core, supported in part by the Office of the Director, NIH (S10OD020056). This work was supported by NIH Shared Instrumentation Grants (S10OD012351, S10OD021764 and S10OD032433), NIH/NCI Cancer Center Support grants (P30CA013696 and P30CA008748) and the Columbia University Digestive and Liver Disease Research Center grant (NIH 5P30DK132710).

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