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

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)

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

IDH 1/2 mutation in glioma

N Engl J Med. 2009 19;360(8):765-73.

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