Transcript IDH mutation in CNS derived cells

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
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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
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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 aKGdependent 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
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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 nontransformed cells
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IDH mutation in non-transformd cells
(differentiation)
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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
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)
IDH mutation in non-transformd cells
(hypermethylation)
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Chromatin immunoprecipitation against
H3K9me3 and H3K27me3
after 4 days induction
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QPCR for promoters of
Cebpa and Adipoq

Detection of H3K9
methylation and H3
acetylation
Effect of IDH mutation
 IDH mutation in non transfromed cells
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 IDH

mutation in CNS derived cells
H3K9 methylation and differentiation in
non-transformed cells
IDH mutations in CNS derived cells
(methylation)
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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)
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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
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 H3K9
methylation and differentiation in
non-transformed cells
H3K9 methylation and differentiation in nontransformed cells
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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 nontransformed cells (differentiation)
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To test H3K9 demethylation is
required in adiocyte
differentiation – block KDM4C
Introduce 3 siRNAs against
KDM4C into 3T3-L1 cells
Conclusion
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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
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MLL gene: H3K4 methyltransferase
 AML or infant leukemia
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KDM3B: H3K9 demethylase, 5q31
 deleted in AML and MDS
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KDM6A: H3K27 demethylase
 deleted in large array cancers
Histone methylation also have role in stem cell
maintainance and differentiation
Conclusion
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Further investigation:
 The sensitivity to 2HG inhibition among
JHDM family
 Cellular feedback mechanisms activated
after defective demethylation