Role of IDH Mutations in Cancer: Genetic and Metabolic Implications

1. Presented by R5 李霖昆 Supervised by VS 顏厥全 大夫 報告日期:2012-03-04 474 | nature | vol 483 | 22 March 2012

2. Introduction • Malignant transformation • A series of genetic, epigenetic and post-transcriptional events • Metabolic adaption • Certain metabolites as regulators or cofactor of enzymes in @ Chromatin remodeling @ Mitochondrial respiration @ Angiogenesis @ Migration • Proto-oncogene and tumor suppressor genes • Fumarate hydratase (FH), succinate dehydrogenase (SDH)

3. Mutation in IDH 1/2 • First described in CRC, also noted in glioblastoma, glioma, 2nd GBM and AML (16~17%) • IDH1/IDH2 mutation • Unable to effectively catalyze the oxidative decarboxylation of isocitrate (loss of enzyme activity) • Novel enzymatic activity – 2-hydroxyglutarate (2-HG) • 2-HG and tumorigenesis ?? Oncogene (2010) 29, 6409–6417

4. IDH 1/2 mutation in glioma N Engl J Med. 2009 19;360(8):765-73.

5. IDH 1/2 mutation in malignancy Oncogene (2010) 29, 6409–6417

6. Effect of IDH mutation • IDH mutation in non transfromed cells • IDH mutation in CNS derived cells • H3K9 methylation and differentiation in non-transformed cells

7. Effects of IDH mutations • Gr II-III oligodendroglioma • Microarray analysis • Total 41 samples • 33 had R132 IDH1 mutation • 2 had R172 IDH2 mutation • 6 had wild type IDH 1/2 The gene signatures were independent of tumor grade and recurrence status

8. IDH mutation in AML Cancer Cell 18, 553–567, December 14, 2010

9. IDH in glioma cells • DNA hypermethylation was associated with IDH 1 mutation • No TET family mutation in glioma cells  IDH mutation may affect the regulation of cell differentiation Cancer Cell 17, 510–522, May 18, 2010

10. 2-HG effect on histone demethylase In vitro study • 2-HG inhibit a family of aKG-dependent Jumonji-C domain histone demethylase • 2-HG occupies the same space as a-KG Cancer Cell 19, 17–30, January 18, 2011

11. IDH and histone change in cells • Ectopically expressed IDH1/2 mutation in 293T cells • 2-HG levels • Histone methylation (H3K9 as marker of methylation) • Immunohistochemistry analysis of the samples for methylation marker  IDH mutations might affect the regulation of repressive histone methylation markers in vivo

12. Effect of IDH mutation • IDH mutation in non transfromed cells • IDH mutation in CNS derived cells • H3K9 methylation and differentiation in non-transformed cells

13. IDH mutation in non-transformd cells (differentiation) • Differentiation stimulation of murine 3T3-L1 cells into adipocyte • Transduced either wild type IDH2, mutant IDH2 or vector alone into 3T3-L1 cells • 7 days differentiation induction • Synthetic cell-permeable octyl-2HG

14. IDH mutation in non-transformd cells (gene expression) • Gene expression analysis of the transcription factors essential for executing adipogenesis (Cebpa&Pparg) and adipocytic lineage specific gene (Adipoq)

15. IDH mutation in non-transformd cells (hypermethylation) • Chromatin immuno-precipitation against H3K9me3 and H3K27me3 after 4 days induction • QPCR for promoters of Cebpa and Adipoq • Detection of H3K9 methylation and H3 acetylation

16. Effect of IDH mutation • IDH mutation in non transfromed cells • IDH mutation in CNS derived cells • H3K9 methylation and differentiation in non-transformed cells

17. IDH mutations in CNS derived cells (methylation) • Transduce wild type or R132 mutant IDH1 into normal human astrocyte (NHA) • Western blot for methylation marker and neural marker (nestin) • Examine the temporal relationship of histone and DNA methylation

18. IDH mutations in CNS derived cells(Differentiation) • Brains from p16/p19-/- mice introduced with R132 mutant, wild IDH1 or vector • Re-plated under conditions for astrocyte differentiation • Retinoic acid induction • Astrocyte marker (GFAP) • Neural marker (β3-tubulin)

19. Effect of IDH mutation • IDH mutation in non transfromed cells • IDH mutation in CNS derived cells • H3K9 methylation and differentiation in non-transformed cells

20. H3K9 methylation and differentiation in non-transformed cells • KDM4C (H3K9 specific JHDM), induced in 3T3-L1 cells during differentiation • In vitro demethylase assay with GST-tagged KDM4C • 2HG inhibited demethylation in dose dependent manner • Increase aKG concertration reverse 2HG effects

21. H3K9 methylation and differentiation in non-transformed cells (differentiation) • To test H3K9 demethylation is required in adiocyte differentiation – block KDM4C • Introduce 3 siRNAs against KDM4C into 3T3-L1 cells

22. Conclusion • 2HG is a universal inhibitor of JHDM family members • H3K9 methylation seemed to be more sensitive to mutant IDH induced suppression than others • H3K9 demethylase more sensitive to 2HG inhibition • H3K27 methylation may crosstalk with H3K9 methylation • Other marker with delayed change may be the result of differentiation block, • aKG-dependent demethylase in cell differentiation can be impaired through cellular accumulation of 2HG induced by IDH mutation

23. Conclusion • MLL gene: H3K4 methyltransferase  AML or infant leukemia • KDM3B: H3K9 demethylase, 5q31  deleted in AML and MDS • KDM6A: H3K27 demethylase  deleted in large array cancers • Histone methylation also have role in stem cell maintainance and differentiation

24. Conclusion • Further investigation: • The sensitivity to 2HG inhibition among JHDM family • Cellular feedback mechanisms activated after defective demethylation