. 2022 Sep 12;40(9):939-956.e16.

doi: 10.1016/j.ccell.2022.07.011. Epub 2022 Aug 18.

De novo pyrimidine synthesis is a targetable vulnerability in IDH mutant glioma

Diana D Shi¹, Milan R Savani², Michael M Levitt³, Adam C Wang⁴, Jennifer E Endress⁵, Cylaina E Bird⁶, Joseph Buehler³, Sylwia A Stopka⁷, Michael S Regan⁸, Yu-Fen Lin⁹, Vinesh T Puliyappadamba³, Wenhua Gao⁴, Januka Khanal⁴, Laura Evans¹⁰, Joyce H Lee⁴, Lei Guo¹¹, Yi Xiao³, Min Xu³, Bofu Huang⁴, Rebecca B Jennings¹², Dennis M Bonal¹³, Misty S Martin-Sandoval³, Tammie Dang¹⁴, Lauren C Gattie¹⁵, Amy B Cameron¹³, Sungwoo Lee¹⁶, John M Asara¹⁷, Harley I Kornblum¹⁸, Tak W Mak¹⁹, Ryan E Looper²⁰, Quang-De Nguyen¹³, Sabina Signoretti¹², Stefan Gradl²¹, Andreas Sutter²¹, Michael Jeffers²², Andreas Janzer²¹, Mark A Lehrman¹⁴, Lauren G Zacharias³, Thomas P Mathews³, Julie-Aurore Losman⁴, Timothy E Richardson²³, Daniel P Cahill²⁴, Ralph J DeBerardinis²⁵, Keith L Ligon²⁶, Lin Xu²⁷, Peter Ly⁹, Nathalie Y R Agar²⁸, Kalil G Abdullah²⁹, Isaac S Harris³⁰, William G Kaelin Jr³¹, Samuel K McBrayer³²

Affiliations

¹ Department of Radiation Oncology, Dana-Farber/Brigham and Women's Cancer Center, Harvard Medical School, Boston, MA 02215, USA; Children's Medical Center Research Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
² Children's Medical Center Research Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Medical Scientist Training Program, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
³ Children's Medical Center Research Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
⁴ Department of Medical Oncology, Dana-Farber Cancer Institute and Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02215, USA.
⁵ Ludwig Cancer Center, Boston, MA 02115, USA; Harvard Medical School, Boston, MA 02115, USA.
⁶ Department of Neurological Surgery, University of Texas Southwestern Medical Center, Dallas, TX 75235, USA.
⁷ Department of Neurosurgery, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA; Department of Radiology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
⁸ Department of Neurosurgery, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
⁹ Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
¹⁰ Bayer HealthCare Pharmaceuticals, Inc., Cambridge, MA 02142, USA.
¹¹ Quantitative Biomedical Research Center, Department of Population & Data Sciences, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
¹² Department of Medical Oncology, Dana-Farber Cancer Institute and Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02215, USA; Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
¹³ Lurie Family Imaging Center, Center for Biomedical Imaging in Oncology, Dana-Farber Cancer Institute, Boston, MA 02210, USA.
¹⁴ Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
¹⁵ Children's Medical Center Research Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Department of Neurological Surgery, University of Texas Southwestern Medical Center, Dallas, TX 75235, USA.
¹⁶ New Drug Development Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu 41061, Republic of Korea.
¹⁷ Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA; Cancer Center, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.
¹⁸ Department of Molecular and Medical Pharmacology, University of California Los Angeles, Los Angeles, CA 90095, USA; Department of Psychiatry and Behavioral Sciences, and Semel Institute for Neuroscience and Human Behavior, University of California Los Angeles, Los Angeles, CA 90024, USA.
¹⁹ The Campbell Family Institute for Breast Cancer Research, University Health Network, Toronto, ON M5G 2M9, Canada; The Princess Margaret Cancer Centre and Ontario Cancer Institute, University Health Network, Toronto, ON M5G 1L7, Canada; Department of Medical Biophysics, University of Toronto, Toronto, ON M5G 1L7, Canada.
²⁰ Department of Chemistry, University of Utah, Salt Lake City, UT 84112, USA.
²¹ Bayer AG, Muellerstrasse 178, 13353 Berlin, Germany.
²² Bayer HealthCare Pharmaceuticals, Inc., Whippany, NJ 07981, USA.
²³ Department of Pathology, Glenn Biggs Institute for Alzheimer's and Neurodegenerative Diseases, University of Texas Health Science Center, San Antonio, TX 78229, USA.
²⁴ Department of Neurosurgery, Translational Neuro-Oncology Laboratory, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.
²⁵ Children's Medical Center Research Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA; Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX 75235, USA.
²⁶ Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA; Department of Oncologic Pathology, Dana-Farber Cancer Institute, Boston, MA 02115, USA; Department of Pathology, Children's Hospital Boston, Boston, MA 02115, USA.
²⁷ Quantitative Biomedical Research Center, Department of Population & Data Sciences, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX 75235, USA.
²⁸ Department of Neurosurgery, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA; Department of Radiology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA; Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02215, USA.
²⁹ Department of Neurosurgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA; Hillman Comprehensive Cancer Center, University of Pittsburgh Medical Center, Pittsburgh, PA 15232, USA.
³⁰ Wilmot Cancer Institute, University of Rochester Medical Center, Rochester, NY 14642, USA.
³¹ Department of Medical Oncology, Dana-Farber Cancer Institute and Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02215, USA; Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA. Electronic address: william_kaelin@dfci.harvard.edu.
³² Children's Medical Center Research Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX 75235, USA. Electronic address: samuel.mcbrayer@utsouthwestern.edu.

PMID: 35985343
PMCID: PMC9515386
DOI: 10.1016/j.ccell.2022.07.011

De novo pyrimidine synthesis is a targetable vulnerability in IDH mutant glioma

Diana D Shi et al. Cancer Cell. 2022.

. 2022 Sep 12;40(9):939-956.e16.

doi: 10.1016/j.ccell.2022.07.011. Epub 2022 Aug 18.

Authors

Affiliations

¹ Department of Radiation Oncology, Dana-Farber/Brigham and Women's Cancer Center, Harvard Medical School, Boston, MA 02215, USA; Children's Medical Center Research Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
² Children's Medical Center Research Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Medical Scientist Training Program, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
³ Children's Medical Center Research Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
⁴ Department of Medical Oncology, Dana-Farber Cancer Institute and Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02215, USA.
⁵ Ludwig Cancer Center, Boston, MA 02115, USA; Harvard Medical School, Boston, MA 02115, USA.
⁶ Department of Neurological Surgery, University of Texas Southwestern Medical Center, Dallas, TX 75235, USA.
⁷ Department of Neurosurgery, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA; Department of Radiology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
⁸ Department of Neurosurgery, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
⁹ Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
¹⁰ Bayer HealthCare Pharmaceuticals, Inc., Cambridge, MA 02142, USA.
¹¹ Quantitative Biomedical Research Center, Department of Population & Data Sciences, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
¹² Department of Medical Oncology, Dana-Farber Cancer Institute and Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02215, USA; Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
¹³ Lurie Family Imaging Center, Center for Biomedical Imaging in Oncology, Dana-Farber Cancer Institute, Boston, MA 02210, USA.
¹⁴ Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
¹⁵ Children's Medical Center Research Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Department of Neurological Surgery, University of Texas Southwestern Medical Center, Dallas, TX 75235, USA.
¹⁶ New Drug Development Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu 41061, Republic of Korea.
¹⁷ Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA; Cancer Center, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.
¹⁸ Department of Molecular and Medical Pharmacology, University of California Los Angeles, Los Angeles, CA 90095, USA; Department of Psychiatry and Behavioral Sciences, and Semel Institute for Neuroscience and Human Behavior, University of California Los Angeles, Los Angeles, CA 90024, USA.
¹⁹ The Campbell Family Institute for Breast Cancer Research, University Health Network, Toronto, ON M5G 2M9, Canada; The Princess Margaret Cancer Centre and Ontario Cancer Institute, University Health Network, Toronto, ON M5G 1L7, Canada; Department of Medical Biophysics, University of Toronto, Toronto, ON M5G 1L7, Canada.
²⁰ Department of Chemistry, University of Utah, Salt Lake City, UT 84112, USA.
²¹ Bayer AG, Muellerstrasse 178, 13353 Berlin, Germany.
²² Bayer HealthCare Pharmaceuticals, Inc., Whippany, NJ 07981, USA.
²³ Department of Pathology, Glenn Biggs Institute for Alzheimer's and Neurodegenerative Diseases, University of Texas Health Science Center, San Antonio, TX 78229, USA.
²⁴ Department of Neurosurgery, Translational Neuro-Oncology Laboratory, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.
²⁵ Children's Medical Center Research Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA; Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX 75235, USA.
²⁶ Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA; Department of Oncologic Pathology, Dana-Farber Cancer Institute, Boston, MA 02115, USA; Department of Pathology, Children's Hospital Boston, Boston, MA 02115, USA.
²⁷ Quantitative Biomedical Research Center, Department of Population & Data Sciences, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX 75235, USA.
²⁸ Department of Neurosurgery, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA; Department of Radiology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA; Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02215, USA.
²⁹ Department of Neurosurgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA; Hillman Comprehensive Cancer Center, University of Pittsburgh Medical Center, Pittsburgh, PA 15232, USA.
³⁰ Wilmot Cancer Institute, University of Rochester Medical Center, Rochester, NY 14642, USA.
³¹ Department of Medical Oncology, Dana-Farber Cancer Institute and Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02215, USA; Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA. Electronic address: william_kaelin@dfci.harvard.edu.
³² Children's Medical Center Research Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX 75235, USA. Electronic address: samuel.mcbrayer@utsouthwestern.edu.

PMID: 35985343
PMCID: PMC9515386
DOI: 10.1016/j.ccell.2022.07.011

Abstract

Mutations affecting isocitrate dehydrogenase (IDH) enzymes are prevalent in glioma, leukemia, and other cancers. Although mutant IDH inhibitors are effective against leukemia, they seem to be less active in aggressive glioma, underscoring the need for alternative treatment strategies. Through a chemical synthetic lethality screen, we discovered that IDH1-mutant glioma cells are hypersensitive to drugs targeting enzymes in the de novo pyrimidine nucleotide synthesis pathway, including dihydroorotate dehydrogenase (DHODH). We developed a genetically engineered mouse model of mutant IDH1-driven astrocytoma and used it and multiple patient-derived models to show that the brain-penetrant DHODH inhibitor BAY 2402234 displays monotherapy efficacy against IDH-mutant gliomas. Mechanistically, this reflects an obligate dependence of glioma cells on the de novo pyrimidine synthesis pathway and mutant IDH's ability to sensitize to DNA damage upon nucleotide pool imbalance. Our work outlines a tumor-selective, biomarker-guided therapeutic strategy that is poised for clinical translation.

Keywords: DHODH; IDH; cancer metabolism; genetically engineered mouse model; glioma; isocitrate dehydrogenase; pyrimidine nucleotides.

PubMed Disclaimer

Conflict of interest statement

Declaration of interests R.J.D., W.G.K., and S.K.M. have served as paid advisors to Agios Pharmaceuticals. W.G.K. receives compensation for roles as an Eli Lilly and LifeMine Therapeutics Board Director, a founder of Tango Therapeutics and Cedilla Therapeutics, and a scientific advisor for Fibrogen, IconOVir Bio, Circle Pharma, Nextext Invest, and Casdin Capital. K.L.L. receives research support from Eli Lilly and Company via the DFCI. S.K.M. and W.G.K. received research funding from Bayer Pharmaceuticals. Bayer had no influence over the design, execution, or interpretation of studies. N.Y.R.A. is key opinion leader for Bruker Daltonics, scientific advisor to Invicro, and receives support from Thermo Finnegan and EMD Serono. S.G., A.S., M.S., A.J., and L.E. are employees at Bayer. S.G., A.S., and A.J. hold stock in Bayer. D.P.C. has consulted for Lilly, GlaxoSmithKline, and Boston Pharmaceuticals and serves on the advisory board of Pyramid Biosciences, which includes an equity interest. All other authors declare no competing interests.

Figures

**Figure 1.. *De novo* pyrimidine synthesis inhibitors preferentially kill IDH1-mutant glioma cells**
(A) Schema of the multifunctional approach to pharmacologic screening (MAPS) platform drug screen. Percent difference in area under curve (AUC) values (%AUC_Diff) in HOG-EV and HOG-R132H cells were calculated for all drugs. (B) Top hits and related pathways. (C) Waterfall plot of all drugs ranked by %AUC_Diff. (D) Schematic of *de novo* and salvage pyrimidine synthesis pathways and targets of indicated hits. (E–G) Cell death assays of HOG-EV or HOG-R132H cells treated with the indicated drugs (n = 5). (H–J) Cell death assays of HOG-R132H cells treated with indicated drugs with or without 100 μM uridine (n = 3). (K) Cell death assays of GSC lines treated with brequinar for two population doublings (n ≥ 3). (L) Correlation between 2HG levels and sensitivity of GSC lines to brequinar. Symbols and colors are as in (K). For all panels, data presented are means ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001. For (L), p value was determined by simple linear regression analysis. For all others, two-tailed p values were determined by unpaired t test.

**Figure 2.. The DHODHi BAY 2402234 is brain-penetrant in mice and selectively kills IDH1-mutant glioma cells**
(A and B) Volcano plot of metabolites in heart (A) and liver (B) of mice treated with BAY 2402234 or vehicle (n = 9 per cohort). Carbamoyl asp = carbamoyl aspartate. (C) OCAR in tissues of mice treated with BAY 2402234 or vehicle as in (A) (n = 9 per cohort). (D) Weight changes in mice treated with BAY 2402234 or vehicle. (E) Cell death assays of HOG-EV or HOG-R132H cells treated with BAY 2402234 with or without 100 μM uridine (n = 3). (F) Representative photomicrographs of cells in (E). Scale bars, 100 μm. (G) Cell death assays of GSC lines treated with BAY 2402234 or DMSO for two population doublings (n ≥ 3). (H) Correlation between 2HG levels and sensitivity of GSC lines to BAY 2402234. Symbols and colors are as in (G). (I) Cell death assays of HOG-R132H cells expressing EV, DHODH WT, or DHODH A58T treated with BAY 2402234 or DMSO (n = 3). (J) UTP and dihydroorotate levels in HOG-R132H stable lines treated with 50 nM BAY 2402234 or DMSO (n = 3). For panels (A–E and G), data are means ± SEM; for panels (I and J), data are means ± SD; *p < 0.05, **p < 0.01, ***p < 0.001. For (H), the p value was determined by simple linear regression analysis. For all others, two-tailed p values were determined by unpaired t test.

**Figure 3.. Treatment with BAY 2402234 improves survival of mice bearing IDH1-mutant glioma orthotopic xenografts**
(A) Brain sections from mice bearing MGG152 xenografts and treated with BAY 2402234 or vehicle (n = 5 per cohort). Top: stain: hematoxylin and eosin. Relative orotate (middle) and carbamoyl aspartate (bottom) levels as determined by matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI). Scale bar, 3 mm. (B and C) MALDI-MSI-based quantification of orotate, carbamoyl aspartate, and OCAR (B) and BAY 2402234 (C) in tumor tissues in (A). n.d., not detected. (D and E) Kaplan-Meier curves of mice bearing MGG152 xenografts treated with BAY 2402234 or vehicle (D) or cranial radiation or sham (E). Red arrows in (E) indicate irradiation. (F) Kaplan-Meier curves of mice bearing TS516 xenografts treated with BAY 2402234 or vehicle. (G and H) OCAR in MGG152 (n = 8 per cohort) (G) and TS516 (n = 6 per cohort) (H) tumor tissues. (I) DHODH activity in MGG152 or TS516 tumor tissues after BAY 2402234 or vehicle treatment. (J and K) Kaplan-Meier curves of mice bearing HOG-R132H xenografts expressing either EV (J) or DHODH-A58T (K). For (B), Tukey plots are shown. For (C), data are means ± SD. For all other panels, data are means ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001. In (D–F), (J), and (K), p values were calculated by log rank test. Grubbs’ test was used to detect and exclude outliers in (J and K) (a = 0.05). In (B and G–I), two-tailed p values were determined by unpaired t test.

**Figure 4.. *IDH1* and *PIK3R1* oncogenes cooperate to transform immortalized astrocytes**
(A) Alterations in the indicated genes in the Brain Lower Grade Glioma TCGA dataset. (B) Kaplan-Meier curves of glioma patients with the indicated genotypes. Note that the apparent tail of the black curve is driven by a single patient. (C and D) Immunoblot analyses of NHA cell lines expressing HA-tagged IDH1-R132H (or EV) and FLAG-tagged WT or mutant (D560_S565del, R574fs, or T576del) p85 (protein product of *PIK3R1* gene) or the corresponding EV. (E) Representative anchorage independence assays of NHA cells used in (C) and (D) (n = 5). Scale bar, 100 μm. (F and G) Bioluminescence imaging (F) and Kaplan-Meier curves (G) of mice after intracranial injection of EV/EV (control), IDH1^R132H/EV (IDH1mut), EV/p85^D560_S565del (PIK3R1mut), IDH1^R132H/p85^D560_S565del (IDH1mut + PIK3R1mut), or H-Ras-V12 (positive control) NHA lines. *p < 0.05. n.s., not significant. P values were determined by the log rank test.

**Figure 5.. Creation of GEM models of lower grade astrocytoma**
(A) Mouse injection scheme. Indicated AAV was intracranially injected into mouse strains (***PIC***, IC, PC, and C). LSL, *loxP*-stop-*loxP* cassette. Mice were monitored for tumor initiation with serial monthly MRI scans. (B) Representative MRI images of a ***PIC*** mouse after intracranial AAV injection, as in (A). (C) Kaplan-Meier curves of AAV-injected mice, as in (A). Time 0 = day of injection. (D) Cumulative glioma incidence in mice (as determined by MRI, histopathologic analysis, or both), as in (A), in the 12 months after AAV injection. Mice that developed injection-site sarcomas were censored. (E and F) Hematoxylin and eosin (E) and immunohistochemical (F) stained sections of the brain from a representative AAV-injected ***PIC*** mouse. In (E), the black box indicates the region in the right panel; in all panels, scale bars, 200 μm. (G) Representative MRI images from an AAV-injected ***PIC*** mouse. Arrows indicate tumor. (H and I) Photomicrograph (H) of DF-AA27 GEM model (GEMM) GSCs and immunoblot analysis (I) of DF-AA27 GEMM GSCs, human neural stem cells (NSC), and primary human astrocytes. In (H), scale bar, 100 μm. GFAP, glial fibrillary acidic protein. (J) Kaplan-Meier tumor-free survival curve of mice intracranially injected with DF-AA27 cells. Time 0 = day of injection. (n = 15). For (B and G), MRI images are coronal slices of the entire mouse brain. For all panels, data are means ± SD; *p < 0.05, **p < 0.01. In (C), the p value was determined by log rank test. In (D), the p value was determined by one-way ANOVA.

**Figure 6.. Organoid and genetically engineered allograft models of IDH-mutant glioma respond to DHODH inhibition**
(A) Cell death assay in DF-AA27 GEMM GSCs treated with BAY 2402234 or DMSO with or without 100 μM uridine (n = 3). (B) OCAR in DF-AA27 orthotopic allografts in mice treated with BAY 2402234 or vehicle (n = 6 per cohort). (C and D) Tumor volumes (C) and representative MRI images (D) of mice with DF-AA27 orthotopic allografts treated continuously following tumor formation with BAY 2402234 or vehicle. (E) Histology, immunohistochemistry, and quantification of cleaved caspase 3 (CC3) in glioma SXOs treated with 30 nM BAY 2402234 or DMSO. Scale bars, 50 μm. Gr, tumor grade, A, astrocytoma, O, oligodendroglioma, GBM, glioblastoma. For (A–C), data are means ± SEM. For (D), MRI images are coronal slices of the entire mouse brain. For (E), data are means ± SD. *p < 0.05, ***p < 0.001, n.s., not significant. Two-tailed p values were determined by unpaired t test.

**Figure 7.. Glioma cells use divergent routes for pyrimidine and purine nucleotide synthesis under physiologic conditions**
(A) Steady-state quantification of the indicated metabolites in BT054 GSCs treated with 10 nM BAY 2402234, 5 μM lometrexol, or DMSO (n = 8 per condition). (B) Pathways targeted by BAY 2402234 (BAY) and lometrexol (Lom). (C) ¹⁵N stable isotope tracing assays in BT054 and TS516 GSC lines (n = 3). (D) Select pathways using intermediates derived from *de novo* pyrimidine nucleotide synthesis. (E and F) Volcano plots of metabolites in HOG-EV (E) or HOG-R132H (F) cells treated with 10 nM BAY 2402234 relative to DMSO (n = 4). (G) Ratio of pyrimidine (dTTP, dCTP) to purine (dATP) deoxynucleotide triphosphate (dNTP) pools in cells shown in (E and F). For all panels, data are means ± SEM; *p < 0.05, ***p < 0.001. n.s., not significant. Two-tailed p values were determined by unpaired t test.

**Figure 8.. The *IDH1-R132H* mutation enhances DNA damage caused by DHODH inhibition**
(A and B) Immunoblot analysis of HOG-EV and HOG-R132H cells (A) or GSC lines (B) treated with BAY 2402234 (HOGs: 1, 3, or 10 nM; GSCs: 3 or 10 nM) or DMSO. (C) Immunoblot analysis of HOG-EV and HOG-R132H cells treated with 10 nM BAY 2402234, 5 μM lometrexol, or DMSO. (D) Quantification of 53BP1 foci formation in HOG-EV and HOG-R132H cells treated with BAY 2402234 (n ≥ 325 cells per condition per time point) and representative images. Scale bar, 5 μm; dashed lines show individual nuclei. (E–H) Immunoblot analysis and cell death assays in HOG-EV and HOG-R132H cells treated with BAY 2402234, DMSO, 15 μM dC, and/or 15 μM dT (n = 3) (E and F), or pre-treated with 1 μM palbociclib or DMSO (G and H), then treated with BAY 2402234 or DMSO (n = 3). (I and J) Representative hematoxylin and eosin, anti-γH2A.X IHC staining (I), and γH2A.X quantification (J) of MGG152 orthotopic xenografts treated with BAY 2402234 (n = 5) or vehicle (n = 2). Scale bar, 100 μm. (K and L) MALDI-MSI ion images (K) and quantified relative changes (L) of brains from mice with MGG152 orthotopic glioma xenografts treated with BAY 2402234 or vehicle. Scale bar, 3 mm. (M) Schema depicting model by which BAY 2402234 (BAY) treatment preferentially kills IDH-mutant glioma cells. For (D), data are means and variance is not displayed. For (L), Tukey plots are shown. For all other panels, data are means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, n.s., not significant. Two-tailed p values were determined by unpaired t test.

See this image and copyright information in PMC

Comment in

Gliomas lean on pyrimidines.
Willson J. Willson J. Nat Rev Cancer. 2022 Nov;22(11):606-607. doi: 10.1038/s41568-022-00515-9. Nat Rev Cancer. 2022. PMID: 36131135 No abstract available.

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De novo pyrimidine synthesis is a targetable vulnerability in IDH mutant glioma

Affiliations

De novo pyrimidine synthesis is a targetable vulnerability in IDH mutant glioma

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

Comment in

References

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources

Full Text Sources

Medical

Molecular Biology Databases

Research Materials

Miscellaneous