Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

DHODH inhibition impedes glioma stem cell proliferation, induces DNA damage, and prolongs survival in orthotopic glioblastoma xenografts

Oncogene volume 41, pages 5361–5372 (2022)Cite this article

Subjects

Abstract

Glioma stem cells (GSCs) promote tumor progression and therapeutic resistance and exhibit remarkable bioenergetic and metabolic plasticity, a phenomenon that has been linked to their ability to escape standard and targeted therapies. However, specific mechanisms that promote therapeutic resistance have been somewhat elusive. We hypothesized that because GSCs proliferate continuously, they may require the salvage and de novo nucleotide synthesis pathways to satisfy their bioenergetic needs. Here, we demonstrate that GSCs lacking EGFR (or EGFRvIII) amplification are exquisitely sensitive to de novo pyrimidine synthesis perturbations, while GSCs that amplify EGFR are utterly resistant. Furthermore, we show that EGFRvIII promotes BAY2402234 resistance in otherwise BAY2402234 responsive GSCs. Remarkably, a novel, orally bioavailable, blood-brain-barrier penetrating, dihydroorotate dehydrogenase (DHODH) inhibitor BAY2402234 was found to abrogate GSC proliferation, block cell-cycle progression, and induce DNA damage and apoptosis. When dosed daily by oral gavage, BAY2402234 significantly impaired the growth of two different intracranial human glioblastoma xenograft models in mice. Given this observed efficacy and the previously established safety profiles in preclinical animal models and human clinical trials, the clinical testing of BAY2402234 in patients with primary glioblastoma that lacks EGFR amplification is warranted.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: BAY2402234 inhibits glioblastoma neurosphere growth in vitro.
Fig. 2: Growth inhibition by BAY2402234 can be rescued by uridine supplementation.
Fig. 3: BAY2402234 inhibition of de novo pyrimidine synthesis promotes DNA damage in normoxia and hypoxia.
Fig. 4: BAY2402234 induces early S phase cell cycle arrest.
Fig. 5: BAY2402234 inhibits colony growth in NSMC.
Fig. 6: BAY2402234 induces Poly(ADP-ribose) polymerase (PARP) cleavage.
Fig. 7: EGFR signaling promotes BAY2402234 resistance.
Fig. 8: BAY2402234 slows the growth of orthotopic glioblastoma xenografts and prolongs survival.

Similar content being viewed by others

Data availability

All data are available in the main text. The pLenti-PGK-Neo-PIP-FUCCI expression construct is available through Addgene, plasmid # 118616. The pLV[Expression]-mCherry:T2A:Hygro-EF1A>Luc2 construct, generated in this study, is also available at Addgene, plasmid # 174665.

References

  1. Hess KR, Broglio KR, Bondy ML. Adult glioma incidence trends in the United States, 1977–2000. Cancer. 2004;101:2293–9.

    Article  Google Scholar 

  2. Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ, et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med. 2005;352:987–96.

    Article  CAS  Google Scholar 

  3. Woodward WA, Chen MS, Behbod F, Alfaro MP, Buchholz TA, Rosen JM. WNT/beta-catenin mediates radiation resistance of mouse mammary progenitor cells. Proc Natl Acad Sci USA. 2007;104:618–23.

    Article  CAS  Google Scholar 

  4. Wang M, Li X, Qu Y, Xu O, Sun Q. Hypoxia promotes radioresistance of CD133-positive Hep-2 human laryngeal squamous carcinoma cells in vitro. Int J Oncol. 2013;43:131–40.

    Article  CAS  Google Scholar 

  5. Wang J, Wakeman TP, Lathia JD, Hjelmeland AB, Wang XF, White RR, et al. Notch promotes radioresistance of glioma stem cells. Stem Cells. 2010;28:17–28.

    Article  CAS  Google Scholar 

  6. Uribe D, Torres A, Rocha JD, Niechi I, Oyarzun C, Sobrevia L, et al. Multidrug resistance in glioblastoma stem-like cells: Role of the hypoxic microenvironment and adenosine signaling. Mol Asp Med. 2017;55:140–51.

    Article  CAS  Google Scholar 

  7. Rich JN. Cancer stem cells in radiation resistance. Cancer Res. 2007;67:8980–4.

    Article  CAS  Google Scholar 

  8. Osuka S, Van Meir EG. Overcoming therapeutic resistance in glioblastoma: the way forward. J. Clin Investig. 2017;127:415–26.

    Article  Google Scholar 

  9. Nakano RMAI. Glioma stem cells and their therapy resistance. J Carcinogene Mutagene. 2011;1–8.

  10. Kim Y, Kim KH, Lee J, Lee YA, Kim M, Lee SJ, et al. Wnt activation is implicated in glioblastoma radioresistance. Lab Investig. 2012;92:466–73.

    Article  CAS  Google Scholar 

  11. Jialiang W, Timothy PW, Justin DL, Anita BH, Xiao-Fan W, Rebekah RW, et al. Notch Promotes Radioresistance of Glioma Stem Cells. 2009;28:17–28.

  12. Honoki K, Fujii H, Kubo A, Kido A, Mori T, Tanaka Y, et al. Possible involvement of stem-like populations with elevated ALDH1 in sarcomas for chemotherapeutic drug resistance. Oncol Rep. 2010;24:501–5.

    Article  CAS  Google Scholar 

  13. Hardee ME, Marciscano AE, Medina-Ramirez CM, Zagzag D, Narayana A, Lonning SM, et al. Resistance of glioblastoma-initiating cells to radiation mediated by the tumor microenvironment can be abolished by inhibiting transforming growth factor-beta. Cancer Res. 2012;72:4119–29.

    Article  CAS  Google Scholar 

  14. Facchino S, Abdouh M, Chatoo W, Bernier G. BMI1 confers radioresistance to normal and cancerous neural stem cells through recruitment of the DNA damage response machinery. J Neurosci. 2010;30:10096–111.

    Article  CAS  Google Scholar 

  15. Eyler CE, Rich JN. Survival of the fittest: cancer stem cells in therapeutic resistance and angiogenesis. J Clin Oncol. 2008;26:2839–45.

    Article  CAS  Google Scholar 

  16. Diehn M, Cho RW, Lobo NA, Kalisky T, Dorie MJ, Kulp AN, et al. Association of reactive oxygen species levels and radioresistance in cancer stem cells. Nature. 2009;458:780–3.

    Article  CAS  Google Scholar 

  17. Dean M, Fojo T, Bates S. Tumour stem cells and drug resistance. Nat Rev Cancer. 2005;5:275–84.

    Article  CAS  Google Scholar 

  18. Dahan P, Martinez Gala J, Delmas C, Monferran S, Malric L, Zentkowski D, et al. Ionizing radiations sustain glioblastoma cell dedifferentiation to a stem-like phenotype through survivin: possible involvement in radioresistance. Cell Death Dis. 2014;5:e1543.

    Article  CAS  Google Scholar 

  19. Carruthers RD, Ahmed SU, Ramachandran S, Strathdee K, Kurian KM, Hedley A, et al. Replication stress drives constitutive activation of the DNA damage response and radioresistance in glioblastoma stem-like cells. Cancer Res. 2018;78:5060–71.

    Article  CAS  Google Scholar 

  20. Bao S, Wu Q, McLendon RE, Hao Y, Shi Q, Hjelmeland AB, et al. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature. 2006;444:756–60.

    Article  CAS  Google Scholar 

  21. Szeto MD, Chakraborty G, Hadley J, Rockne R, Muzi M, Alvord EC Jr., et al. Quantitative metrics of net proliferation and invasion link biological aggressiveness assessed by MRI with hypoxia assessed by FMISO-PET in newly diagnosed glioblastomas. Cancer Res. 2009;69:4502–9.

    Article  CAS  Google Scholar 

  22. Fuller GN. The WHO classification of tumours of the central nervous system, 4th edition. Arch Pathol Lab Med. 2008;132:906.

    Article  Google Scholar 

  23. Heddleston JM, Li Z, McLendon RE, Hjelmeland AB, Rich JN. The hypoxic microenvironment maintains glioblastoma stem cells and promotes reprogramming towards a cancer stem cell phenotype. Cell Cycle. 2009;8:3274–84.

    Article  CAS  Google Scholar 

  24. Bar EE, Lin A, Mahairaki V, Matsui W, Eberhart CG. Hypoxia increases the expression of stem-cell markers and promotes clonogenicity in glioblastoma neurospheres. Am J Pathol. 2010;177:1491–502.

    Article  CAS  Google Scholar 

  25. Pistollato F, Chen HL, Rood BR, Zhang HZ, D’Avella D, Denaro L, et al. Hypoxia and HIF1alpha repress the differentiative effects of BMPs in high-grade glioma. Stem Cells. 2009;27:7–17.

    Article  CAS  Google Scholar 

  26. Li Z, Bao S, Wu Q, Wang H, Eyler C, Sathornsumetee S, et al. Hypoxia-inducible factors regulate tumorigenic capacity of glioma stem cells. Cancer Cell. 2009;15:501–13.

    Article  CAS  Google Scholar 

  27. Seidel S, Garvalov BK, Wirta V, von Stechow L, Schanzer A, Meletis K, et al. A hypoxic niche regulates glioblastoma stem cells through hypoxia inducible factor 2 alpha. Brain. 2010;133:983–95.

    Article  Google Scholar 

  28. Soeda A, Park M, Lee D, Mintz A, Androutsellis-Theotokis A, McKay RD, et al. Hypoxia promotes expansion of the CD133-positive glioma stem cells through activation of HIF-1alpha. Oncogene. 2009;28:3949–59.

    Article  CAS  Google Scholar 

  29. Spina R, Voss DM, Yang X, Sohn JW, Vinkler R, Schraner J, et al. MCT4 regulates de novo pyrimidine biosynthesis in GBM in a lactate-independent manner. Neurooncol Adv. 2020;2:vdz062.

    Google Scholar 

  30. Bar EE, Chaudhry A, Lin A, Fan X, Schreck K, Matsui W, et al. Cyclopamine-mediated hedgehog pathway inhibition depletes stem-like cancer cells in glioblastoma. Stem Cells. 2007;25:2524–33.

    Article  CAS  Google Scholar 

  31. Kahlert UD, Bender NO, Maciaczyk D, Bogiel T, Bar EE, Eberhart CG, et al. CD133/CD15 defines distinct cell subpopulations with differential in vitro clonogenic activity and stem cell-related gene expression profile in in vitro propagated glioblastoma multiforme-derived cell line with a PNET-like component. Folia Neuropathol. 2012;50:357–68.

    Article  CAS  Google Scholar 

  32. Chu Q, Orr BA, Semenkow S, Bar EE, Eberhart CG. Prolonged inhibition of glioblastoma xenograft initiation and clonogenic growth following in vivo Notch blockade. Clin Cancer Res. 2013;19:3224–33.

    Article  CAS  Google Scholar 

  33. Lim KS, Lim KJ, Price AC, Orr BA, Eberhart CG, Bar EE. Inhibition of monocarboxylate transporter-4 depletes stem-like glioblastoma cells and inhibits HIF transcriptional response in a lactate-independent manner. Oncogene. 2014;33:4433–41.

    Article  CAS  Google Scholar 

  34. Kahlert UD, Suwala AK, Raabe EH, Siebzehnrubl FA, Suarez MJ, Orr BA, et al. ZEB1 promotes invasion in human fetal neural stem cells and hypoxic glioma neurospheres. Brain Pathol. 2015;25:724–32.

    Article  CAS  Google Scholar 

  35. Fan X, Khaki L, Zhu TS, Soules ME, Talsma CE, Gul N, et al. NOTCH pathway blockade depletes CD133-positive glioblastoma cells and inhibits growth of tumor neurospheres and xenografts. Stem Cells. 2010;28:5–16.

    Article  CAS  Google Scholar 

  36. Sun P, Xia S, Lal B, Eberhart CG, Quinones-Hinojosa A, Maciaczyk J, et al. DNER, an epigenetically modulated gene, regulates glioblastoma-derived neurosphere cell differentiation and tumor propagation. Stem Cells. 2009;27:1473–86.

    Article  CAS  Google Scholar 

  37. Ying M, Tilghman J, Wei Y, Guerrero-Cazares H, Quinones-Hinojosa A, Ji H, et al. Kruppel-like factor-9 (KLF9) inhibits glioblastoma stemness through global transcription repression and integrin alpha6 inhibition. J Biol Chem. 2014;289:32742–56.

    Article  CAS  Google Scholar 

  38. Sun P, Xia S, Lal B, Shi X, Yang KS, Watkins PA, et al. Lipid metabolism enzyme ACSVL3 supports glioblastoma stem cell maintenance and tumorigenicity. BMC Cancer. 2014;14:401.

    Article  Google Scholar 

  39. Tilghman J, Wu H, Sang Y, Shi X, Guerrero-Cazares H, Quinones-Hinojosa A, et al. HMMR maintains the stemness and tumorigenicity of glioblastoma stem-like cells. Cancer Res. 2014;74:3168–79.

    Article  CAS  Google Scholar 

  40. Wu Y, Richard JP, Wang SD, Rath P, Laterra J, Xia S. Regulation of glioblastoma multiforme stem-like cells by inhibitor of DNA binding proteins and oligodendroglial lineage-associated transcription factors. Cancer Sci. 2012;103:1028–37.

    Article  CAS  Google Scholar 

  41. Wang SD, Rath P, Lal B, Richard JP, Li Y, Goodwin CR, et al. EphB2 receptor controls proliferation/migration dichotomy of glioblastoma by interacting with focal adhesion kinase. Oncogene. 2012;31:5132–43.

    Article  CAS  Google Scholar 

  42. Li Y, Li A, Glas M, Lal B, Ying M, Sang Y, et al. c-Met signaling induces a reprogramming network and supports the glioblastoma stem-like phenotype. Proc Natl Acad Sci USA. 2011;108:9951–6.

    Article  CAS  Google Scholar 

  43. Ying M, Wang S, Sang Y, Sun P, Lal B, Goodwin CR, et al. Regulation of glioblastoma stem cells by retinoic acid: role for Notch pathway inhibition. Oncogene. 2011;30:3454–67.

    Article  CAS  Google Scholar 

  44. Ying M, Sang Y, Li Y, Guerrero-Cazares H, Quinones-Hinojosa A, Vescovi AL, et al. Kruppel-like family of transcription factor 9, a differentiation-associated transcription factor, suppresses Notch1 signaling and inhibits glioblastoma-initiating stem cells. Stem Cells. 2011;29:20–31.

    Article  CAS  Google Scholar 

  45. Lal B, Goodwin CR, Sang Y, Foss CA, Cornet K, Muzamil S, et al. EGFRvIII and c-Met pathway inhibitors synergize against PTEN-null/EGFRvIII+ glioblastoma xenografts. Mol Cancer Ther. 2009;8:1751–60.

    Article  CAS  Google Scholar 

  46. Galli R, Binda E, Orfanelli U, Cipelletti B, Gritti A, De Vitis S, et al. Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma. Cancer Res. 2004;64:7011–21.

    Article  CAS  Google Scholar 

  47. Lee J, Kotliarova S, Kotliarov Y, Li A, Su Q, Donin NM, et al. Tumor stem cells derived from glioblastomas cultured in bFGF and EGF more closely mirror the phenotype and genotype of primary tumors than do serum-cultured cell lines. Cancer Cell. 2006;9:391–403.

    Article  CAS  Google Scholar 

  48. Singh SK, Clarke ID, Terasaki M, Bonn VE, Hawkins C, Squire J, et al. Identification of a cancer stem cell in human brain tumors. Cancer Res. 2003;63:5821–8.

    CAS  Google Scholar 

  49. Pollard SM, Yoshikawa K, Clarke ID, Danovi D, Stricker S, Russell R, et al. Glioma stem cell lines expanded in adherent culture have tumor-specific phenotypes and are suitable for chemical and genetic screens. Cell Stem Cell. 2009;4:568–80.

    Article  CAS  Google Scholar 

  50. Christian S, Merz C, Evans L, Gradl S, Seidel H, Friberg A, et al. The novel dihydroorotate dehydrogenase (DHODH) inhibitor BAY 2402234 triggers differentiation and is effective in the treatment of myeloid malignancies. Leukemia. 2019;33:2403–15.

    Article  CAS  Google Scholar 

  51. Sakaue-Sawano A, Kurokawa H, Morimura T, Hanyu A, Hama H, Osawa H, et al. Visualizing spatiotemporal dynamics of multicellular cell-cycle progression. Cell. 2008;132:487–98.

    Article  CAS  Google Scholar 

  52. Grant GD, Kedziora KM, Limas JC, Cook JG, Purvis JE. Accurate delineation of cell cycle phase transitions in living cells with PIP-FUCCI. Cell Cycle. 2018;17:2496–516.

    Article  CAS  Google Scholar 

  53. Rouleau M, Patel A, Hendzel MJ, Kaufmann SH, Poirier GG. PARP inhibition: PARP1 and beyond. Nat Rev Cancer. 2010;10:293–301.

    Article  CAS  Google Scholar 

  54. Lord CJ, Ashworth A. PARP inhibitors: synthetic lethality in the clinic. Science. 2017;355:1152–8.

    Article  CAS  Google Scholar 

  55. Mashimo M, Onishi M, Uno A, Tanimichi A, Nobeyama A, Mori M, et al. The 89-kDa PARP1 cleavage fragment serves as a cytoplasmic PAR carrier to induce AIF-mediated apoptosis. J Biol Chem. 2021;296:100046.

    Article  CAS  Google Scholar 

  56. Robinson AD, Eich ML, Varambally S. Dysregulation of de novo nucleotide biosynthetic pathway enzymes in cancer and targeting opportunities. Cancer Lett. 2020;470:134–40.

    Article  CAS  Google Scholar 

  57. Wang X, Yang K, Wu Q, Kim LJY, Morton AR, Gimple RC, et al. Targeting pyrimidine synthesis accentuates molecular therapy response in glioblastoma stem cells. Sci Transl Med. 2019;11:1–14.

  58. Quemeneur L, Gerland LM, Flacher M, Ffrench M, Revillard JP, Genestier L. Differential control of cell cycle, proliferation, and survival of primary T lymphocytes by purine and pyrimidine nucleotides. J Immunol. 2003;170:4986–95.

    Article  CAS  Google Scholar 

  59. Henricks LM, Jacobs BAW, Meulendijks D, Pluim D, van den Broek D, de Vries N, et al. Food-effect study on uracil and dihydrouracil plasma levels as marker for dihydropyrimidine dehydrogenase activity in human volunteers. Br J Clin Pharmacol. 2018;84:2761–9.

    Article  CAS  Google Scholar 

  60. van Wijk N, Slot RER, Duits FH, Strik M, Biesheuvel E, Sijben JWC, et al. Nutrients required for phospholipid synthesis are lower in blood and cerebrospinal fluid in mild cognitive impairment and Alzheimer’s disease dementia. Alzheimers Dement. 2017;8:139–46.

    Google Scholar 

  61. Wurtman RJ, Regan M, Ulus I, Yu L. Effect of oral CDP-choline on plasma choline and uridine levels in humans. Biochem Pharmacol. 2000;60:989–92.

    Article  CAS  Google Scholar 

  62. Ashour OM, Al Safarjalani ON, Naguib FN, Goudgaon NM, Schinazi RF, el Kouni MH. Modulation of plasma uridine concentration by 5-(phenylselenenyl)acyclouridine, an inhibitor of uridine phosphorylase: relevance to chemotherapy. Cancer Chemother Pharmacol. 2000;45:351–61.

    Article  CAS  Google Scholar 

  63. Williams MG, Palandra J, Shobe EM. Rapid determination of rat plasma uridine levels by HPLC-ESI-MS utilizing the Captiva plates for sample preparation. Biomed Chromatogr. 2003;17:215–8.

    Article  CAS  Google Scholar 

  64. Cansev M, Watkins CJ, van der Beek EM, Wurtman RJ. Oral uridine-5’-monophosphate (UMP) increases brain CDP-choline levels in gerbils. Brain Res. 2005;1058:101–8.

    Article  CAS  Google Scholar 

  65. Louis DN, Perry A, Wesseling P, Brat DJ, Cree IA, Figarella-Branger D, et al. The 2021 WHO classification of tumors of the central nervous system: a summary. Neuro Oncol. 2021;23:1231–51.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We would like to acknowledge Drs. Vescovi, Nakano, Rich, and Sarkaria. The GSC and PDX models they established were used in our studies.

Funding

This study is funded by National Institutes of Health grant R01CA187780 (EEB), National Institutes of Health grant R21NS106553 (EEB), National Cancer Institute - Cancer Center Support Grant (CCSG) - P30CA134274, and Maryland Department of Health’s Cigarette Restitution Fund Program.

Author information

Author notes

  1. These authors contributed equally: Raffaella Spina, Ian Mills.

Authors and Affiliations

  1. Department of Pathology, University of Maryland School of Medicine, Baltimore, MD, USA

    Raffaella Spina, Ian Mills, Fahim Ahmad, Heather M. Ames & Eli E. Bar

  2. Department of Neurosurgery, University of Maryland School of Medicine, Baltimore, MD, USA

    Chixiang Chen, Jeffrey A. Winkles, Graeme F. Woodworth & Eli E. Bar

  3. Department of Epidemiology & Public Health, University of Maryland School of Medicine, Baltimore, MD, USA

    Chixiang Chen

  4. University of Maryland, Marlene and Stewart Greenebaum Comprehensive Cancer Center, Baltimore, MD, USA

    Heather M. Ames, Jeffrey A. Winkles, Graeme F. Woodworth & Eli E. Bar

  5. Department of Surgery, University of Maryland School of Medicine, Baltimore, MD, USA

    Jeffrey A. Winkles

  6. Center for Vascular and Inflammatory Diseases, University of Maryland School of Medicine, Baltimore, MD, USA

    Jeffrey A. Winkles

Authors
  1. Raffaella Spina
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ian Mills
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Fahim Ahmad
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Chixiang Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Heather M. Ames
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jeffrey A. Winkles
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Graeme F. Woodworth
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Eli E. Bar
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Conceptualization: EEB. Methodology: RS, IM. Investigation: RS, IM, FA. Statistics: CC. Visualization: EEB, RS, IM, CC. Funding acquisition: EEB. Supervision: RS, EEB. Writing – original draft: RS, EEB. Writing – review & editing: EEB, GFW, JAW.

Corresponding author

Correspondence to Eli E. Bar.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Spina, R., Mills, I., Ahmad, F. et al. DHODH inhibition impedes glioma stem cell proliferation, induces DNA damage, and prolongs survival in orthotopic glioblastoma xenografts. Oncogene 41, 5361–5372 (2022). https://doi.org/10.1038/s41388-022-02517-1

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41388-022-02517-1

Search

Advanced search

Quick links