Presented by R5 李霖昆 Supervised by VS 顏厥全 大夫 報告日期 : | nature | vol 483 | 22 March 2012

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

Mutation in IDH 1/2  First described in CRC, also noted in glioblastoma, glioma, 2 nd 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

IDH 1/2 mutation in glioma N Engl J Med ;360(8):

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

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

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

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

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

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

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

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

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

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

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

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

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

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)

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

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

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

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

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

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