No Significant Cytotoxic Effect of the EZH2 Inhibitor Tazemetostat (EPZ-6438) on Pediatric Glioma Cells with Wildtype Histone 3 or Mutated Histone 3.3

Abstract

Background: Glioblastoma multiforme (GBM) and diffuse intrinsic pontine glioma (DIPG) belong to the most aggressive cancers in children with poor prognosis and limited therapeutic options. Therapeutic targeting of epigenetic proteins may offer new treatment options. Preclinical studies identified Enhancer of Zeste Homolog 2 (EZH2) within polycomb repressor complex 2 (PRC2) as a potential epigenetic anti-tumor target in adult GBM cells but similar inhibition studies in pediatric GBM/DIPG were still missing. Moreover, approximately 30% of pediatric high grade gliomas (pedHGG) including GBM and DIPG harbor a lysine 27 mutation (K27M) in histone 3.3 (H3.3) which is correlated with poor outcome and was shown to influence EZH2 function.

Patients, materials and methods: The present study investigated the correlation of expression of EZH2 and other PRC2 genes (EZH1, SUZ12, EED) with overall survival of pediatric GBM patients and the cytotoxic impact of EZH2 inhibition by the novel agent Tazemetostat in pediatric GBM/DIPG cells harboring either a H3.3 mutation or a H3 wildtype.

Results: EZH2 gene expression does not correlate with survival of pedHGG patients, and EZH2 inhibition does not induce significant cytotoxicity in pedHGG cells independently of H3.3 mutations.

Discussion and conclusion: We suggest that EZH2 inhibition might not offer an effective single agent treatment option for paedHGG patients. However, the therapeutic efficacy in combination with cytotoxic and/or other epigenetically active agents still has to be elucidated.

Zusammenfassung

Hintergrund: Glioblastoma multiforme (GBM) und diffus, intrinsische Ponsgliome (DIPG) gehören zu den aggressivsten kindlichen Tumoren mit schlechter Prognose und limitierten Therapiemöglichkeiten. Die Inhibierung epigenetisch relevanter Proteine könnte eine neue Behandlungsoption darstellen. Vorklinische Studien identifizierten das Protein Enhancer of Zeste Homolog 2 (EZH2) des Polycomb Repressor Complex 2 (PRC2) als potentielles epigenetisches Anti-Tumortarget in adulten GBM-Zellen, entsprechende Inhibitionsexperimente in pädiatrischen GBM/DIPG fehlen jedoch bisher. Dabei tragen ca. 30% aller kindlichen hochgradigen Gliome (pädHGG) eine Mutation im Lysin 27 (K27M) des Histons 3.3 (H3.3), die ein schlechteres Überleben zu bedingen scheint und die Funktion von EZH2 beeinflusst.

Patienten, Material und Methoden: Es wurde die Korrelation der Expression von EZH2 und anderer PRC2-Gene (EZH1, SUZ12, EED) mit dem Überleben pädiatrischer GBM-Patienten untersucht sowie die durch den EZH2-Inhibitor Tazemetostat vermittelte Zytotoxizität in pädiatrischen GBM/DIPG-Zellen, die ein mutiertes Histon H3.3 oder ein Wildtyp-Histon H3 tragen.

Ergebnisse: Die EZH2-Genexpression korreliert nicht mit dem Überleben von pädHGG Patienten. EZH2 Inhibition induziert keine wesentliche Zytotoxizität in pädiatrischen GBM/DIPG Zelllinien, und zwar unabhängig von deren Mutationsstatus im Histon H3.3.

Diskussion und Schlussfolgerung: Nach den vorliegenden Ergebnissen scheint die Inhibition von EZH2 keine vielversprechende therapeutische Option für die Monotherapie von pädiatrischen Patienten mit hochgradigen Gliomen darzustellen. Die mögliche Wirksamkeit in Kombination mit zytotoxischen und/oder anderen epigenetisch wirksamen Substanzen sollte dennoch weiter abgeklärt werden.

Key words

EZH2 inhibition - Tazemetostat (EPZ-6438) - cancer epigenetics - histone 3 mutation - pediatric high-grade glioma (pedHGG)

Schlüsselwörter

EZH2 Inhibition - Tazemetostat (EPZ-6438) - Tumor Epigenetik - Histon 3 Mutation - Pädiatrische hochgradige Gliome (pädHGG)

• References

• 1 Adhikary G, Grun D, Balasubramanian S et al. Survival of skin cancer stem cells requires the Ezh2 polycomb group protein. Carcinogenesis 2015; 36: 800-810
• 2 Bax DA, Little SE, Gaspar N et al. Molecular and phenotypic characterisation of paediatric glioma cell lines as models for preclinical drug development. PloS one 2009; 4: e5209
• 3 Castel D, Philippe C, Calmon R et al. Histone H3F3A and HIST1H3B K27M mutations define two subgroups of diffuse intrinsic pontine gliomas with different prognosis and phenotypes. Acta neuropathologica 2015;
• 4 Chan KM, Fang D, Gan H et al. The histone H3.3K27M mutation in pediatric glioma reprograms H3K27 methylation and gene expression. Genes & development 2013; 27: 985-990
• 5 Conway E, Healy E, Bracken AP. PRC2 mediated H3K27 methylations in cellular identity and cancer. Current opinion in cell biology 2015; 37: 42-48
• 6 Crea F, Hurt EM, Farrar WL. Clinical significance of Polycomb gene expression in brain tumors. Molecular cancer 2010; 9: 265
• 7 Crea F, Fornaro L, Bocci G et al. EZH2 inhibition: targeting the crossroad of tumor invasion and angiogenesis. Cancer metastasis reviews 2012; 31: 753-761
• 8 de Vries NA, Hulsman D, Akhtar W et al. Prolonged Ezh2 Depletion in Glioblastoma Causes a Robust Switch in Cell Fate Resulting in Tumor Progression. Cell reports 2015; 10: 383-397
• 9 Fisher ML, Adhikary G, Grun D et al. The Ezh2 polycomb group protein drives an aggressive phenotype in melanoma cancer stem cells and is a target of diet derived sulforaphane. Molecular carcinogenesis 2015;
• 10 Khan AA, Lee AJ, Roh TY. Polycomb group protein-mediated histone modifications during cell differentiation. Epigenomics 2015; 7: 75-84
• 11 Khuong-Quang DA, Buczkowicz P, Rakopoulos P et al. K27M mutation in histone H3.3 defines clinically and biologically distinct subgroups of pediatric diffuse intrinsic pontine gliomas. Acta neuropathologica 2012; 124: 439-447
• 12 Kim KH, Roberts CW. Targeting EZH2 in cancer. Nature medicine 2016; 22: 128-134
• 13 Kim SH, Joshi K, Ezhilarasan R et al. EZH2 Protects Glioma Stem Cells from Radiation-Induced Cell Death in a MELK/FOXMI-Dependent Manner. Stem Cell Rep 2015; 4: 226-238
• 14 Knutson SK, Warholic NM, Wigle TJ et al. Durable tumor regression in genetically altered malignant rhabdoid tumors by inhibition of methyltransferase EZH2. Proceedings of the National Academy of Sciences of the United States of America 2013; 110: 7922-7927
• 15 Knutson SK, Kawano S, Minoshima Y et al. Selective inhibition of EZH2 by EPZ-6438 leads to potent antitumor activity in EZH2-mutant non-Hodgkin lymphoma. Molecular cancer therapeutics 2014; 13: 842-854
• 16 Knutson SK, Warholic NM, Johnston LD et al. Synergistic Anti-Tumor Activity of EZH2 Inhibitors and Glucocorticoid Receptor Agonists in Models of Germinal Center Non-Hodgkin Lymphomas. PloS one 2014; 9: e111840
• 17 Kramm CRU, Butenhoff S, Kühnöl C et al. Gliome im Kindes- und Jugendalter. Monatsschrift Kinderheilkunde 2008; 156: 1201-1207
• 18 Sharma V, Purkait S, Takkar S et al. Analysis of EZH2: micro-RNA network in low and high grade astrocytic tumors. Brain tumor pathology 2016;
• 19 Sturm D, Witt H, Hovestadt V et al. Hotspot mutations in H3F3A and IDH1 define distinct epigenetic and biological subgroups of glioblastoma. Cancer cell 2012; 22: 425-437
• 20 Trent J, Meltzer P, Rosenblum M et al. Evidence for rearrangement, amplification, and expression of c-myc in a human glioblastoma. Proceedings of the National Academy of Sciences of the United States of America 1986; 83: 470-473
• 21 Veringa SJ, Biesmans D, van Vuurden DG et al. In vitro drug response and efflux transporters associated with drug resistance in pediatric high grade glioma and diffuse intrinsic pontine glioma. PloS one 2013; 8: e61512
• 22 Yin Y, Qiu S, Peng Y. Functional roles of enhancer of zeste homolog 2 in gliomas. Gene 2016; 576: 189-194
• 23 Zhang P, de Gooijer MC, Buil LC et al. ABCB1 and ABCG2 restrict the brain penetration of a panel of novel EZH2-Inhibitors. International journal of cancer Journal international du cancer 2015; 137: 2007-2018