Chromosome Abnormalities and
Their Molecular Correlates in
Acute Leukemia
SAI-JUAN CHEN
State Key Laboratory of Medical Genomics
Shanghai Institute of Hematology,Ruijin Hospital
Shanghai Second Medical University(SSMU)
Development of Classification of
Acute Leukemia
1976 French-American-British (FAB) Cooperative
Group proposed classification of acute leukemia
based on morphologic criteria that were
subsequently refined in 1981 and 1985
1986 Morphologic,immunologic and cytogenetic (MIC)
classification was introduced,Recognition of
importance of cytogenetics in the diagnosis and
treatment of acute leukemia
2001 World Health Organization (WHO) classification
recognized new clinically relevant molecular
genetic lesions
4
5
6
Acute leukemia of ambiguous lineage
? Undifferentiated acute leukemia
(HLA-DR,CD34,CD38)
? Bilineal acute leukemia
(dual population of blasts)
? Biphenotypic acute leukemia
(co-expression of myeloid and other
lineage antigen)
Major differences between WHO and FAB
(
Look AT,Science,278,1059
白血病染色体易位
Most of the fusion genes involved in
AL translocations are transcription
factors (TFs),abnormalities in lineage
commitment and differentiation
More study should be done
1,Molecular characterization of
undetectable abnormalities on the
cytogenetic level
2,Characterization of additional
chromosomal or molecular genetic
abnormalities [e.g,t(11;17)] in the well-
defined favorable cytogenetic group
may influence clinical outcome
3,Characterization of cooperating genetic
abnormalities may affect treatment
response in stepwise leukemogenesis
Hematopoiesis,a model of systems biomedicine
Common Myeloid
Progenitor (CMP)
Hematopoietic
Stem Cell (HSC)
Common Lymphoid
Progenitor (CLP)
GMP
EMP
Important players,Hematopoietic stem cells,
cytokines,microenvironment
造血生成调节的关键因子, 转录因子
Platelets
Red cells
TFs are primordial
in regulating the
switch of the
hematopoietic
organ,the lineage
commitment and
the differentiation
of HSCs and
progenitor cells
TF genes related to
hematopoiesis are
evolutionarily well
conserved from the
zebrafish to human
KO animals for some of these TFs exhibit phenotypes of hematological
disorders
Look AT,Science,278,1059
Chromosomal translocations and
gene mutations in leukemias
Most of the fusion genes involved in
AL translocations are transcription
factors (TFs),abnormalities in lineage
commitment and differentiation
Nodal points in controlling
hematopoiesis,tyrosine kinases
PTKs
Hematopoiesis
related PTKs are
essential factors in
determining
growth and survival
of HSCs and
progenitor cells
KO mice for
hematopoiesis
related PTKs
often show
deficiency in
blood formation
Chromosomal translocations and
gene mutations in leukemias
BCR-ABL,a
model of
activation of
protein
tyrosine
kinase (PTK)
in leukemo-
genesis,
growth and
survival
advantages
白血病基因组解剖学计划 (L-GAP)
必要性,约 50％的白血病患者无染色体异常，即使有特异的
基因异常也不足以产生典型的白血病（如 AML-M2b的 AML1-
ETO; CML的 BCR-ABL）；大多数白血病患者无病生存期远不
理想。
大规模系统性候选基因（造血系统转录因子和胞浆信号分子
尤其是酪氨酸激酶）的基因突变检测及蛋白质功能研究。
基于药物靶点的高通量筛选库（传统中药天然药物库，组合
化学合成物库）和基于信号传导途径知识的药物设计 /组合。
Genes re-sequenced,
TFs,AML-1,CBFβ,C/EBPα,GATA1,
GATA1p,GATA2,GATA2p,GATA3,MYB,
PU.1,…
Signaling molecule,PDGFRa,PDGFRb,c-
KIT/SCFR,FLK2/FLT3,NRAS,KRAS,ICSBP,
NOTCH1,NOTCH2,NOTCH3,…
白血病遗传学基础,
白血病基因组解剖学计划 (LGAP)
白血病遗传学基础,
白血病基因组解剖学计划 (LGAP)
Two-hits hypothesis
Acute Myeloid leukemia with
recurrent genetic abnormalities
in classification of WHO
? AML with t(8;21);AML1-ETO
? AML with abnormal marrow
eosinophilia and inv(16) or
t(16;16);CBFb-MYH11
TEL 基因相关的白血病
D i s e a s e
C h r o m o s o m a l
T r a n s l o c a t i o n
F u s i o n g e n e s
A M L t ( 1 ; 1 2 ) ( q 2 1 ; p 1 3 ) T E L - A R N T
A M L t ( 1 ; 1 2 ) ( q 2 5 ; p 1 3 ) T E L - A B L 2
A M L,M D S t ( 3 ; 1 2 ) ( q 2 6 ; q 2 2 ) T E L - M D S 1 - E V I 1
P T C L
( p e r i p h e r a l T - c e l l l y m p h o m a )
t ( 4 ; 1 2 ) ( p 1 6 ; p 1 3 ) T E L - F G F R 3
A M L t ( 4 ; 1 2 ) ( q 1 1 ; p 1 3 ) B T L - T E L
C M L t ( 5 ; 1 2 ) ( q 3 1 ; p 1 3 ) T E L - A C S 2
A M L,M D S t ( 5 ; 1 2 ) ( q 3 3 ; p 1 3 ) T E L - P D G F R β
A L L t ( 6 ; 1 2 ) ( q 2 3 ; p 1 3 ) T E L - S T L
A L L,C M L t ( 9 ; 1 2 ) ( p 2 4 ; p 13) T E L - J A K 2
M D S t ( 9 ; 1 2 ) ( q 2 2 ; p 1 3 ) T E L - S Y K
A L L,C M L,A U L t ( 9 ; 1 2 ) ( q 3 4 ; p 1 3 ) T E L - ABL
A M L,A L L t ( 1 2 ; 1 3 ) ( p 1 3 ; q 1 2 ) T E L - C D X 2
A M L t ( 1 2 ; 1 5 ) ( p 1 3 ; q 2 5 ) T E L - N T R K 3
A M L t ( 1 2 ; 2 1 ) ( p 1 3 ; q 2 2 ) T E L - A M L 1
A M L t ( 1 2 ; 2 2 ) ( p 1 3 ; q 1 1 ) M N 1 - T E L
12-20% of AML
M2b急性髓系白血病靶向治疗研究
AML1-ETO 融合基因
t(8;21) (q22;q22)
40% of AMLM2
t(8;21) 染色体易位 AML-M2患者发生高频率肿瘤基因
c-Kit,功能获得性” 突变 [26/54(48.1%)]
和 /或异常表达（ >80%）
c a s e 1 ( C - K I T,D 8 1 6 Y ; G > T )
d e n o v o 2 m 3, 5 m 1 2 m
G T G T G T G T
K a s
G T
K a s c a s e 1 ( A E )
d e n o v o 2 m 3, 5 m 1 2 m
K a s p a t i e n t 2 p a t i e n t 3
d e n o v o C R d e n o v o C R
A E
K a s c a s e 2 c a s e 3
( T > A ) d e n o v o C R d e n o v o C R
T G T G T G T G
C - K I T N 8 2 2 K ; T > G
T G
mC-kit should be a subsequent event on the basis of t(8;21),
demonstrating a stepwise model in leukemogenesis
In three cases during complete remission,only
AE,but not mC-kit,could be persistent
t r a n s c r i p t i o n f a c t o r
e, g,,A M L 1 - E T O
T G F
m e d i a t o r
P T K a c t i v i t y
C - K I T
d e c r e a s e C - K I T
t r a n s c r i p t
p r o l i f e r a t i v e a n d
s u r v i v a l a d v a n t a g e
G - C S F R
m e d i a t o r
c - J U N,T I S 1 1 b
B C L - 2
e v a d i n g
a p o p t o s i s
N c o R - m S i n 3 - H D A C
P T K m u t a t i o n
C - K I T
m e d i a t o r m e d i a t o r
i n h i b i t i o n o f
d i f f e r e n t i a t i o n
h i t 1 h i t 2
Stepwise leukemogenesis and potential of
targeted therapy in AML with t(8;21)/AML1-ETO
Screening for AML1-ETO
oncoprotein targeted agents
based on natural
compounds from TCM
control SIH10 2μM,24h SIH10 2μM,48h
Oridonin (SIH 10)
AML1-ETO
AML1-ETO
β-Actin
S I H 1 0 ( ? ? ? ? ? ? ? ?T i m e ( h r s ) 0 4 8 1 2 2 4
Procaspase 3
Casp 3,p17
PARP
Casp 9
Control SIH10 2,12h 2 μM,24h
SIH10,extracted from a
TCM herb,targeted
therapy for t(8;21)
leukemia?
Cytoplasmic
SIH10 (?M)
Time (hrs)
cyt C
Stepwise leukemogenesis and potential of
targeted therapy in AML with t(8;21) /AML1-ETO,
Is C-KIT a therapeutic target for Imatinib?
PNAS 2005,102:1104
Apoptosis
inhibition
1,t(8;21)(q22;q22)
AML1-ETO
proliferation
PTK TGFβ
AML1,G-CSF(?),
GM-CSF,
C/EBPα,PU.1
P14(ARF)
2,gene
mutation
t(8;21) leukemia
t(8;21) 白血病,异常转录因子 /酪氨酸激酶双重靶点？
Tyrosine
kinase
SIH10 STI571 HD Ara-C
Upregulation or high level
Downregulation or inhibition
Differentiation
inhibition
HD,high dose
Bcl-2 G-CSF,
G-CSFR
c-JUN TIS11b
Acute Promyelocytic Leukemia with
t(15;17);PML-RARa
Characteristics of 5 Subtypes of APL
Chromosomal t (15;17) t (11;17) t (11;17) t (5;17) dup
aberration (q22;q21) (q23;q21) (q13;q21) (q35;q21) (17) (q21.3q23)
Frequency >95% 1~2% rare rare rare
Fusion gene PML-RARa PLZF-RARa NuMA-RARa NPM-RARa STAT5b-RARa
100% 100% yes yes yes
RARa-PML RARa-PLZF RARa-NuMA RARa-NPM RARa-STAT5b
60~70% 100% no yes no
Nuclear in 100 micro speckles,localized in microspeckle sheet like microspeckle
localization which maybe microspeckles pattern aggregation pattern,maybe
localized in be localized
cytoplasm in cytoplasm
Response to
ATRA good no,may respond good yes no
to ATRA+G-CSF
Chemo good no,may respond
to chemo/ATRA
Arsenic good,with no PLZF-RARa not known not known not known
degradation of degradation
PML-RARa
PML,RARa and three distinct isoforms of PML-RAR a gene
P M L - R A R a - L
59
B C D E F
22015388 419 462
A
R A R a
P M L
P L Z F - R A R a
P L Z F
59
B C D E F
22015388 419 462
57 91
B1 B2 C o i l e d c o i l
129 151
204 215
224 361 394
C 3 H C 4
ZnZnP O Z P Zn Zn Zn Zn Zn Zn Zn
673455406
377
120
255
T r a n sT r a n s -- r e g u l a t i o nr e g u l a t i o n
59
B C D E F
22015388 419 462
ZnZnP O Z P
455406
377
120
255
T r a n sT r a n s -- r e g u l a t i o nr e g u l a t i o n
P M L - R A R a - S
A
B
C
D
E
F
57 91
B1 B2 C o i l e d c o i l
129 151
204 215
224 361 394 550
N L S
633
C 3 H C 4 S - P r i c h
59
B C D E F
22015388 419 462
57 91
B1 B2 C o i l e d c o i l
129 151
204 215
224 361 394 550
C 3 H C 4
N L S
Zn Zn Zn Zn Zn Zn Zn
67345559
A
R A R a - P L Z FG
15 der(17) 17 der(15)
Tg mice
Long
latency:12m
low penetrance,
20-30%
急性早幼粒细胞白血病 (APL)-多步骤发病
原理和靶向治疗
d i f f e r e n t i a t i o n
i n h i b i t i o n
P M L
P 5 3
a p o p t o s i s
i n h i b i t i o n
h i t 2
t y r o s i n e k i n a s e
( F L T 3,c - f m s,e t c )
p r o l i f e r a t i o n
R X R,P M L
s e q u e s t r a t i o n
+ + + + +
+ + + + +
+
+
+
+
+
+
P M L N B
f r a g m e n t a t i o n
d e l o c a l i z a t i o n
P M L R A R a
d y s r e g u l a t i o n o f o t h e r
s i g n a l p a t h w a y s
( i e, I F N,e t c )
h i t 1,
t ( 1 5 ; 1 7 )
C o A
C o R C o R
t r a n s c r i p t i o n a l
r e p r e s s i o n
急性早幼粒细胞白血病 (APL)-多步骤发病原理和
靶向治疗
急性早幼粒细胞白血病 (APL)-多步骤发病原理和
靶向治疗
Tg mice with both PML-RARa and FLT3 mutation have
early onset disease in most animals
(number)
(57)
(42)
(5)
(104)
2
17 (40.5%)
2
21 (20.2%)
* 50
6 (5.8%) 77 (74.0%)
24
104
FLT3 mutation in only part of APL cases and its disappearance ahead
of PML-RARa over remission suggests it as an event after PML-RARa
急性早幼粒细胞白血病 (APL)-多步骤发病原理和
靶向治疗
Paradigm shift,from differentiation to
molecular target-based therapy
ATRA differentiation therapy of APL,
? Complete remission rate,85-95%
? Post-remission therapy (with chemo),5 year
disease-free survival (DSF) rate,50%
? Mild side effects
? Targeting PML-RARa
(As)
hUbc 9 hUbc 9
SAE1
SAE2
Ubiquitin-like
SUMO peptide
SUMO
COOH NH2
RING finger B1 B2 coiled-coil NLS
K160 K490
PMLsumolation
a
Enhancement of PML sumoylation
Recruitment of PML and
interacting proteins PMLdegradation
interacting protein
sequestration
ATO b
Arsenic trioxide as treatment
for APL,ancient remedy
performs new trick
As2O3 in the treatment of relapsed
patients after ATRA,
? Complete remission rate 80-90%
? No cross-resistance to ATRA or
chemotherapy
? Mild side effects
? Induction of PML-RARa degradation
维甲酸受体
PML
PML
PML
RARa
RARa SUM
O-
1
PML-RARa
致病蛋白
APL细胞
分化 凋亡
全反式维甲酸
三氧化二砷
ATRA/As2O3对 APL协同靶向治疗,
异常转录因子作为靶点
0
2
4
6
8
10
12
14
16
缓解后 巩固治疗后
ATRA
As2O3
ATRA+As2O3
322824201612840
1, 1
1, 0
.9
.8
.7
.6
.5
.4
.3
.2
.1
.0
ATRA和 As2O3联合治疗初发 APL
取得迄今为止成人急性白血病的最好疗效
无病生存情况（随访期,8-30月,
中位随访期,18月）
治疗后
, 治疗前
(%)
2003年 12月全美血液学大会
(ASH)专题报告，并在会新闻
发布会上作专题介绍
累计生存率
月
ATRA+As2O3(n=20)
As2O3(n=18)
ATRA (n=19)
P=0.00001
Study group
(n=52,MFU:34m)
Historic control
(n=51,MFU:56m)
Historic Control,
1998-2001
No difference from
the study group in
terms of prognostic
factors (age,WBC,
etc)
Tx,ATRA+CT;
ATO used for rescue
in relapsed patients
APL成为第一个可治愈的成人白血病
AML/MDS/ALL with 11p15
Abnormality;NUP 98 Rearrangement
NUP98 involved in different translocations
Translocations Partner genes Diseases
t(7;11)(p15;p15) HOXA9 AML,MDS,t- AML / MDS, CML-BC
t(2;11)(q31;p15) HOXD13 AML,t- AML / MDS
t(11;12)(p15;q13) HOXC11 t- AML / MDS
t(1;11)(q23;p15) PMX1 t-AML,CML-BC
t(3;11)(q29q13;p15) NRG Myeloid/T-Lymphoid
inv(11)(p15q22) DDX10 AML,t-MDS/AML
t(11;20)(p15;q11) TOP1 AML,t-MDS/AML
t(9;11)(p22;p15) LEDGF AML-M1
t(5;11)(q35;p15) NSD1 AML-M2
t(8;11)(p11;p15) NSD3 AML
t(4;11)(q21;p15) RAP1GDS1 T-ALL
t(11;17)(p15;q21)? t- AML / MDS
The karyotypes of the patients
46,XY,Ph,t (1;11) 46,XY,t (11;12)
Chr.11
der 11
A B
Fig 1
der(11)
A B
F
C
D F E
Chr 3
Chr 11
b
NUP98-HOXC11
546 aa
Docking site (FG motif) NUP98 1 920
RD
1 Docking site (FG motif)
Docking site (FG motif) 578 aa 1
NUP98-HOXA9
HOXC11 304 1
HOXA9 272
HD
1
PMX1 217 1 HD
HD
HD
HD
Docking site (FG motif) 1 608 aa NUP98-PMX1 HD
HD
750 bp
500 bp
500 bp
300 bp
M P N B M B N P A
B
Fig 2
C
D
NUP98-PMX1
HDAC1
SRE
PMX1
FOS SRE FOS
×
PMX1
SRF SRF
Possible molecular mechanism of NUP98-PMX1 in
leukemogenesis,recruitment of HDAC1 and
transcriptional repression of Fos gene regulated by
SRF/NUP98-PMX1 heterodimer
AML/ALL with 11q23 Abnormality; MLL
Rearrangement
Usually occur in a lineage- specific manner,
suggesting a crucial role for MLL fusion partner in
determining disease phenotype
t(11;19) in an acute monocytic leukemia
MLL
MLL
CBP EEN
MLL
MLL
CBP
R
A
R
N - C o R
S I N 3
H
D
A
C
P L Z F
N - C o R
S I N 3
H
D
A
C
P L Z F - RAR
R
A
R
N - C o R
S I N 3
H
D
A
C
P M L
DD
N - C o R
S I N 3
H
D
A
C
VV
P M L - RAR
V a r i a n t
N - C o R
S I N 3
H
D
A
C
N
U
P
9
8
P M X 1
N U P 9 8 - P M X 1
N - C o R
S I N 3
H
D
A
C
A
M
L
1 ETO
N - C o R
S I N 3
H
D
A
C
A M L 1 - ETO
Transcriptional deregulation in leukemogenesis
Model I,abnormal trans-repression mediated by CoR
MLL
MLL
CBP EEN
MLL
MLL
CBP
Transcriptional deregulation in leukemogenesis
Model II,abnormal trans-activation mediated by CoA
CML/CML-BC/AL with t(9;22);
BCR-ABL
慢粒染色体易位
BCR-ABL,a
model of
activation of
protein
tyrosine
kinase (PTK)
in leukemo-
genesis,
growth and
survival
advantages
慢粒 (CML)治疗：基于结构的配体设计, STI 571竞
争占领 ABL 蛋白激酶结构域的 ATP 结合位点
Hydrogen-bonding:dashed lines
Phosphoryl-
ation site
Action loop
catalytic
segment,
green
Activation
loop,
magenta
STI 571
vicinity
of
Tyr393
Schindler T,et al,Science,
2000; 289,1938
? 对 CP作用好,但对 AP 或 BC疗效不满意
? 即使在 CP,亦不能在所有患者根除白血病克隆，部分患者产生耐药性
? 价格非常昂贵，每年 20－ 30万元
砷剂复合物,与 STI 571合用有协同作用？
STI 571治疗 CML药物, 成绩和需解决的问题
Action loop STI 571
O’Brien SG,et al,NEJM,2003; 348:994
CML的进展,
慢性期 (CP) 加速期 (AP) 急变期 (BC)
STI571和 As4S4联合治疗, 诱导细胞凋亡协同作用
The morphology of K562 cells untreated or treated with 2 μM As4S4 and/or 0.2 μM imatinib for 48 hours,
The K562 cells and CD34+ cells from patients with
CML were treated with As4S4 and/or imatinib for 24 to
72 hours and measured for annexin V positivity
Effects of imatinib and/or As4S4
G2/M phaseof K562 cells
K562 cells CML primary cells
0.00
0.30
0.60
0.90
1.20
1.50
OD
450
24h 48h * * * *
☆
☆
#
▲ ▲
*
* ▲ #
★
▲
STI571/As4S4对 CML协同靶向治疗,
BCR-ABL融合蛋白和异常酪氨酸激酶作为靶点
BCR- ABL
c - ABL
210 kd
185 kd
145 kd
Actin 43 kd
0.2 μ M STI571 － － + +
2 μ M As S 4 － + － + MW 4
STI571, Modulation of genes involved in intrinsic pathway of apoptosis
STI571/As4S4联合治疗 CML基因表达谱
Extrinsic and intrinsic apoptosis pathway activation upon combination treatment
apoptosis
STI571/As4S4联合治疗 CML基因表达谱
STI571/As4S4联合治疗 CML基因表达谱
结论
3.白血病是一组涉及转录因子和信号转导分子
(特别是酪氨酸激酶 )的多步骤发病的遗传学
改变
4.基因产物靶向治疗为 APL和 CML患者带来治愈
的希望,并进一步拓展至其他类型白血病
2.L-GAP是研究白血病发病原理和设计靶向治疗
方法的有力途径
1.WHO 分类方法在白血病诊断和治疗中更加关注
染色体易位和基因异常
Their Molecular Correlates in
Acute Leukemia
SAI-JUAN CHEN
State Key Laboratory of Medical Genomics
Shanghai Institute of Hematology,Ruijin Hospital
Shanghai Second Medical University(SSMU)
Development of Classification of
Acute Leukemia
1976 French-American-British (FAB) Cooperative
Group proposed classification of acute leukemia
based on morphologic criteria that were
subsequently refined in 1981 and 1985
1986 Morphologic,immunologic and cytogenetic (MIC)
classification was introduced,Recognition of
importance of cytogenetics in the diagnosis and
treatment of acute leukemia
2001 World Health Organization (WHO) classification
recognized new clinically relevant molecular
genetic lesions
4
5
6
Acute leukemia of ambiguous lineage
? Undifferentiated acute leukemia
(HLA-DR,CD34,CD38)
? Bilineal acute leukemia
(dual population of blasts)
? Biphenotypic acute leukemia
(co-expression of myeloid and other
lineage antigen)
Major differences between WHO and FAB
(
Look AT,Science,278,1059
白血病染色体易位
Most of the fusion genes involved in
AL translocations are transcription
factors (TFs),abnormalities in lineage
commitment and differentiation
More study should be done
1,Molecular characterization of
undetectable abnormalities on the
cytogenetic level
2,Characterization of additional
chromosomal or molecular genetic
abnormalities [e.g,t(11;17)] in the well-
defined favorable cytogenetic group
may influence clinical outcome
3,Characterization of cooperating genetic
abnormalities may affect treatment
response in stepwise leukemogenesis
Hematopoiesis,a model of systems biomedicine
Common Myeloid
Progenitor (CMP)
Hematopoietic
Stem Cell (HSC)
Common Lymphoid
Progenitor (CLP)
GMP
EMP
Important players,Hematopoietic stem cells,
cytokines,microenvironment
造血生成调节的关键因子, 转录因子
Platelets
Red cells
TFs are primordial
in regulating the
switch of the
hematopoietic
organ,the lineage
commitment and
the differentiation
of HSCs and
progenitor cells
TF genes related to
hematopoiesis are
evolutionarily well
conserved from the
zebrafish to human
KO animals for some of these TFs exhibit phenotypes of hematological
disorders
Look AT,Science,278,1059
Chromosomal translocations and
gene mutations in leukemias
Most of the fusion genes involved in
AL translocations are transcription
factors (TFs),abnormalities in lineage
commitment and differentiation
Nodal points in controlling
hematopoiesis,tyrosine kinases
PTKs
Hematopoiesis
related PTKs are
essential factors in
determining
growth and survival
of HSCs and
progenitor cells
KO mice for
hematopoiesis
related PTKs
often show
deficiency in
blood formation
Chromosomal translocations and
gene mutations in leukemias
BCR-ABL,a
model of
activation of
protein
tyrosine
kinase (PTK)
in leukemo-
genesis,
growth and
survival
advantages
白血病基因组解剖学计划 (L-GAP)
必要性,约 50％的白血病患者无染色体异常，即使有特异的
基因异常也不足以产生典型的白血病（如 AML-M2b的 AML1-
ETO; CML的 BCR-ABL）；大多数白血病患者无病生存期远不
理想。
大规模系统性候选基因（造血系统转录因子和胞浆信号分子
尤其是酪氨酸激酶）的基因突变检测及蛋白质功能研究。
基于药物靶点的高通量筛选库（传统中药天然药物库，组合
化学合成物库）和基于信号传导途径知识的药物设计 /组合。
Genes re-sequenced,
TFs,AML-1,CBFβ,C/EBPα,GATA1,
GATA1p,GATA2,GATA2p,GATA3,MYB,
PU.1,…
Signaling molecule,PDGFRa,PDGFRb,c-
KIT/SCFR,FLK2/FLT3,NRAS,KRAS,ICSBP,
NOTCH1,NOTCH2,NOTCH3,…
白血病遗传学基础,
白血病基因组解剖学计划 (LGAP)
白血病遗传学基础,
白血病基因组解剖学计划 (LGAP)
Two-hits hypothesis
Acute Myeloid leukemia with
recurrent genetic abnormalities
in classification of WHO
? AML with t(8;21);AML1-ETO
? AML with abnormal marrow
eosinophilia and inv(16) or
t(16;16);CBFb-MYH11
TEL 基因相关的白血病
D i s e a s e
C h r o m o s o m a l
T r a n s l o c a t i o n
F u s i o n g e n e s
A M L t ( 1 ; 1 2 ) ( q 2 1 ; p 1 3 ) T E L - A R N T
A M L t ( 1 ; 1 2 ) ( q 2 5 ; p 1 3 ) T E L - A B L 2
A M L,M D S t ( 3 ; 1 2 ) ( q 2 6 ; q 2 2 ) T E L - M D S 1 - E V I 1
P T C L
( p e r i p h e r a l T - c e l l l y m p h o m a )
t ( 4 ; 1 2 ) ( p 1 6 ; p 1 3 ) T E L - F G F R 3
A M L t ( 4 ; 1 2 ) ( q 1 1 ; p 1 3 ) B T L - T E L
C M L t ( 5 ; 1 2 ) ( q 3 1 ; p 1 3 ) T E L - A C S 2
A M L,M D S t ( 5 ; 1 2 ) ( q 3 3 ; p 1 3 ) T E L - P D G F R β
A L L t ( 6 ; 1 2 ) ( q 2 3 ; p 1 3 ) T E L - S T L
A L L,C M L t ( 9 ; 1 2 ) ( p 2 4 ; p 13) T E L - J A K 2
M D S t ( 9 ; 1 2 ) ( q 2 2 ; p 1 3 ) T E L - S Y K
A L L,C M L,A U L t ( 9 ; 1 2 ) ( q 3 4 ; p 1 3 ) T E L - ABL
A M L,A L L t ( 1 2 ; 1 3 ) ( p 1 3 ; q 1 2 ) T E L - C D X 2
A M L t ( 1 2 ; 1 5 ) ( p 1 3 ; q 2 5 ) T E L - N T R K 3
A M L t ( 1 2 ; 2 1 ) ( p 1 3 ; q 2 2 ) T E L - A M L 1
A M L t ( 1 2 ; 2 2 ) ( p 1 3 ; q 1 1 ) M N 1 - T E L
12-20% of AML
M2b急性髓系白血病靶向治疗研究
AML1-ETO 融合基因
t(8;21) (q22;q22)
40% of AMLM2
t(8;21) 染色体易位 AML-M2患者发生高频率肿瘤基因
c-Kit,功能获得性” 突变 [26/54(48.1%)]
和 /或异常表达（ >80%）
c a s e 1 ( C - K I T,D 8 1 6 Y ; G > T )
d e n o v o 2 m 3, 5 m 1 2 m
G T G T G T G T
K a s
G T
K a s c a s e 1 ( A E )
d e n o v o 2 m 3, 5 m 1 2 m
K a s p a t i e n t 2 p a t i e n t 3
d e n o v o C R d e n o v o C R
A E
K a s c a s e 2 c a s e 3
( T > A ) d e n o v o C R d e n o v o C R
T G T G T G T G
C - K I T N 8 2 2 K ; T > G
T G
mC-kit should be a subsequent event on the basis of t(8;21),
demonstrating a stepwise model in leukemogenesis
In three cases during complete remission,only
AE,but not mC-kit,could be persistent
t r a n s c r i p t i o n f a c t o r
e, g,,A M L 1 - E T O
T G F
m e d i a t o r
P T K a c t i v i t y
C - K I T
d e c r e a s e C - K I T
t r a n s c r i p t
p r o l i f e r a t i v e a n d
s u r v i v a l a d v a n t a g e
G - C S F R
m e d i a t o r
c - J U N,T I S 1 1 b
B C L - 2
e v a d i n g
a p o p t o s i s
N c o R - m S i n 3 - H D A C
P T K m u t a t i o n
C - K I T
m e d i a t o r m e d i a t o r
i n h i b i t i o n o f
d i f f e r e n t i a t i o n
h i t 1 h i t 2
Stepwise leukemogenesis and potential of
targeted therapy in AML with t(8;21)/AML1-ETO
Screening for AML1-ETO
oncoprotein targeted agents
based on natural
compounds from TCM
control SIH10 2μM,24h SIH10 2μM,48h
Oridonin (SIH 10)
AML1-ETO
AML1-ETO
β-Actin
S I H 1 0 ( ? ? ? ? ? ? ? ?T i m e ( h r s ) 0 4 8 1 2 2 4
Procaspase 3
Casp 3,p17
PARP
Casp 9
Control SIH10 2,12h 2 μM,24h
SIH10,extracted from a
TCM herb,targeted
therapy for t(8;21)
leukemia?
Cytoplasmic
SIH10 (?M)
Time (hrs)
cyt C
Stepwise leukemogenesis and potential of
targeted therapy in AML with t(8;21) /AML1-ETO,
Is C-KIT a therapeutic target for Imatinib?
PNAS 2005,102:1104
Apoptosis
inhibition
1,t(8;21)(q22;q22)
AML1-ETO
proliferation
PTK TGFβ
AML1,G-CSF(?),
GM-CSF,
C/EBPα,PU.1
P14(ARF)
2,gene
mutation
t(8;21) leukemia
t(8;21) 白血病,异常转录因子 /酪氨酸激酶双重靶点？
Tyrosine
kinase
SIH10 STI571 HD Ara-C
Upregulation or high level
Downregulation or inhibition
Differentiation
inhibition
HD,high dose
Bcl-2 G-CSF,
G-CSFR
c-JUN TIS11b
Acute Promyelocytic Leukemia with
t(15;17);PML-RARa
Characteristics of 5 Subtypes of APL
Chromosomal t (15;17) t (11;17) t (11;17) t (5;17) dup
aberration (q22;q21) (q23;q21) (q13;q21) (q35;q21) (17) (q21.3q23)
Frequency >95% 1~2% rare rare rare
Fusion gene PML-RARa PLZF-RARa NuMA-RARa NPM-RARa STAT5b-RARa
100% 100% yes yes yes
RARa-PML RARa-PLZF RARa-NuMA RARa-NPM RARa-STAT5b
60~70% 100% no yes no
Nuclear in 100 micro speckles,localized in microspeckle sheet like microspeckle
localization which maybe microspeckles pattern aggregation pattern,maybe
localized in be localized
cytoplasm in cytoplasm
Response to
ATRA good no,may respond good yes no
to ATRA+G-CSF
Chemo good no,may respond
to chemo/ATRA
Arsenic good,with no PLZF-RARa not known not known not known
degradation of degradation
PML-RARa
PML,RARa and three distinct isoforms of PML-RAR a gene
P M L - R A R a - L
59
B C D E F
22015388 419 462
A
R A R a
P M L
P L Z F - R A R a
P L Z F
59
B C D E F
22015388 419 462
57 91
B1 B2 C o i l e d c o i l
129 151
204 215
224 361 394
C 3 H C 4
ZnZnP O Z P Zn Zn Zn Zn Zn Zn Zn
673455406
377
120
255
T r a n sT r a n s -- r e g u l a t i o nr e g u l a t i o n
59
B C D E F
22015388 419 462
ZnZnP O Z P
455406
377
120
255
T r a n sT r a n s -- r e g u l a t i o nr e g u l a t i o n
P M L - R A R a - S
A
B
C
D
E
F
57 91
B1 B2 C o i l e d c o i l
129 151
204 215
224 361 394 550
N L S
633
C 3 H C 4 S - P r i c h
59
B C D E F
22015388 419 462
57 91
B1 B2 C o i l e d c o i l
129 151
204 215
224 361 394 550
C 3 H C 4
N L S
Zn Zn Zn Zn Zn Zn Zn
67345559
A
R A R a - P L Z FG
15 der(17) 17 der(15)
Tg mice
Long
latency:12m
low penetrance,
20-30%
急性早幼粒细胞白血病 (APL)-多步骤发病
原理和靶向治疗
d i f f e r e n t i a t i o n
i n h i b i t i o n
P M L
P 5 3
a p o p t o s i s
i n h i b i t i o n
h i t 2
t y r o s i n e k i n a s e
( F L T 3,c - f m s,e t c )
p r o l i f e r a t i o n
R X R,P M L
s e q u e s t r a t i o n
+ + + + +
+ + + + +
+
+
+
+
+
+
P M L N B
f r a g m e n t a t i o n
d e l o c a l i z a t i o n
P M L R A R a
d y s r e g u l a t i o n o f o t h e r
s i g n a l p a t h w a y s
( i e, I F N,e t c )
h i t 1,
t ( 1 5 ; 1 7 )
C o A
C o R C o R
t r a n s c r i p t i o n a l
r e p r e s s i o n
急性早幼粒细胞白血病 (APL)-多步骤发病原理和
靶向治疗
急性早幼粒细胞白血病 (APL)-多步骤发病原理和
靶向治疗
Tg mice with both PML-RARa and FLT3 mutation have
early onset disease in most animals
(number)
(57)
(42)
(5)
(104)
2
17 (40.5%)
2
21 (20.2%)
* 50
6 (5.8%) 77 (74.0%)
24
104
FLT3 mutation in only part of APL cases and its disappearance ahead
of PML-RARa over remission suggests it as an event after PML-RARa
急性早幼粒细胞白血病 (APL)-多步骤发病原理和
靶向治疗
Paradigm shift,from differentiation to
molecular target-based therapy
ATRA differentiation therapy of APL,
? Complete remission rate,85-95%
? Post-remission therapy (with chemo),5 year
disease-free survival (DSF) rate,50%
? Mild side effects
? Targeting PML-RARa
(As)
hUbc 9 hUbc 9
SAE1
SAE2
Ubiquitin-like
SUMO peptide
SUMO
COOH NH2
RING finger B1 B2 coiled-coil NLS
K160 K490
PMLsumolation
a
Enhancement of PML sumoylation
Recruitment of PML and
interacting proteins PMLdegradation
interacting protein
sequestration
ATO b
Arsenic trioxide as treatment
for APL,ancient remedy
performs new trick
As2O3 in the treatment of relapsed
patients after ATRA,
? Complete remission rate 80-90%
? No cross-resistance to ATRA or
chemotherapy
? Mild side effects
? Induction of PML-RARa degradation
维甲酸受体
PML
PML
PML
RARa
RARa SUM
O-
1
PML-RARa
致病蛋白
APL细胞
分化 凋亡
全反式维甲酸
三氧化二砷
ATRA/As2O3对 APL协同靶向治疗,
异常转录因子作为靶点
0
2
4
6
8
10
12
14
16
缓解后 巩固治疗后
ATRA
As2O3
ATRA+As2O3
322824201612840
1, 1
1, 0
.9
.8
.7
.6
.5
.4
.3
.2
.1
.0
ATRA和 As2O3联合治疗初发 APL
取得迄今为止成人急性白血病的最好疗效
无病生存情况（随访期,8-30月,
中位随访期,18月）
治疗后
, 治疗前
(%)
2003年 12月全美血液学大会
(ASH)专题报告，并在会新闻
发布会上作专题介绍
累计生存率
月
ATRA+As2O3(n=20)
As2O3(n=18)
ATRA (n=19)
P=0.00001
Study group
(n=52,MFU:34m)
Historic control
(n=51,MFU:56m)
Historic Control,
1998-2001
No difference from
the study group in
terms of prognostic
factors (age,WBC,
etc)
Tx,ATRA+CT;
ATO used for rescue
in relapsed patients
APL成为第一个可治愈的成人白血病
AML/MDS/ALL with 11p15
Abnormality;NUP 98 Rearrangement
NUP98 involved in different translocations
Translocations Partner genes Diseases
t(7;11)(p15;p15) HOXA9 AML,MDS,t- AML / MDS, CML-BC
t(2;11)(q31;p15) HOXD13 AML,t- AML / MDS
t(11;12)(p15;q13) HOXC11 t- AML / MDS
t(1;11)(q23;p15) PMX1 t-AML,CML-BC
t(3;11)(q29q13;p15) NRG Myeloid/T-Lymphoid
inv(11)(p15q22) DDX10 AML,t-MDS/AML
t(11;20)(p15;q11) TOP1 AML,t-MDS/AML
t(9;11)(p22;p15) LEDGF AML-M1
t(5;11)(q35;p15) NSD1 AML-M2
t(8;11)(p11;p15) NSD3 AML
t(4;11)(q21;p15) RAP1GDS1 T-ALL
t(11;17)(p15;q21)? t- AML / MDS
The karyotypes of the patients
46,XY,Ph,t (1;11) 46,XY,t (11;12)
Chr.11
der 11
A B
Fig 1
der(11)
A B
F
C
D F E
Chr 3
Chr 11
b
NUP98-HOXC11
546 aa
Docking site (FG motif) NUP98 1 920
RD
1 Docking site (FG motif)
Docking site (FG motif) 578 aa 1
NUP98-HOXA9
HOXC11 304 1
HOXA9 272
HD
1
PMX1 217 1 HD
HD
HD
HD
Docking site (FG motif) 1 608 aa NUP98-PMX1 HD
HD
750 bp
500 bp
500 bp
300 bp
M P N B M B N P A
B
Fig 2
C
D
NUP98-PMX1
HDAC1
SRE
PMX1
FOS SRE FOS
×
PMX1
SRF SRF
Possible molecular mechanism of NUP98-PMX1 in
leukemogenesis,recruitment of HDAC1 and
transcriptional repression of Fos gene regulated by
SRF/NUP98-PMX1 heterodimer
AML/ALL with 11q23 Abnormality; MLL
Rearrangement
Usually occur in a lineage- specific manner,
suggesting a crucial role for MLL fusion partner in
determining disease phenotype
t(11;19) in an acute monocytic leukemia
MLL
MLL
CBP EEN
MLL
MLL
CBP
R
A
R
N - C o R
S I N 3
H
D
A
C
P L Z F
N - C o R
S I N 3
H
D
A
C
P L Z F - RAR
R
A
R
N - C o R
S I N 3
H
D
A
C
P M L
DD
N - C o R
S I N 3
H
D
A
C
VV
P M L - RAR
V a r i a n t
N - C o R
S I N 3
H
D
A
C
N
U
P
9
8
P M X 1
N U P 9 8 - P M X 1
N - C o R
S I N 3
H
D
A
C
A
M
L
1 ETO
N - C o R
S I N 3
H
D
A
C
A M L 1 - ETO
Transcriptional deregulation in leukemogenesis
Model I,abnormal trans-repression mediated by CoR
MLL
MLL
CBP EEN
MLL
MLL
CBP
Transcriptional deregulation in leukemogenesis
Model II,abnormal trans-activation mediated by CoA
CML/CML-BC/AL with t(9;22);
BCR-ABL
慢粒染色体易位
BCR-ABL,a
model of
activation of
protein
tyrosine
kinase (PTK)
in leukemo-
genesis,
growth and
survival
advantages
慢粒 (CML)治疗：基于结构的配体设计, STI 571竞
争占领 ABL 蛋白激酶结构域的 ATP 结合位点
Hydrogen-bonding:dashed lines
Phosphoryl-
ation site
Action loop
catalytic
segment,
green
Activation
loop,
magenta
STI 571
vicinity
of
Tyr393
Schindler T,et al,Science,
2000; 289,1938
? 对 CP作用好,但对 AP 或 BC疗效不满意
? 即使在 CP,亦不能在所有患者根除白血病克隆，部分患者产生耐药性
? 价格非常昂贵，每年 20－ 30万元
砷剂复合物,与 STI 571合用有协同作用？
STI 571治疗 CML药物, 成绩和需解决的问题
Action loop STI 571
O’Brien SG,et al,NEJM,2003; 348:994
CML的进展,
慢性期 (CP) 加速期 (AP) 急变期 (BC)
STI571和 As4S4联合治疗, 诱导细胞凋亡协同作用
The morphology of K562 cells untreated or treated with 2 μM As4S4 and/or 0.2 μM imatinib for 48 hours,
The K562 cells and CD34+ cells from patients with
CML were treated with As4S4 and/or imatinib for 24 to
72 hours and measured for annexin V positivity
Effects of imatinib and/or As4S4
G2/M phaseof K562 cells
K562 cells CML primary cells
0.00
0.30
0.60
0.90
1.20
1.50
OD
450
24h 48h * * * *
☆
☆
#
▲ ▲
*
* ▲ #
★
▲
STI571/As4S4对 CML协同靶向治疗,
BCR-ABL融合蛋白和异常酪氨酸激酶作为靶点
BCR- ABL
c - ABL
210 kd
185 kd
145 kd
Actin 43 kd
0.2 μ M STI571 － － + +
2 μ M As S 4 － + － + MW 4
STI571, Modulation of genes involved in intrinsic pathway of apoptosis
STI571/As4S4联合治疗 CML基因表达谱
Extrinsic and intrinsic apoptosis pathway activation upon combination treatment
apoptosis
STI571/As4S4联合治疗 CML基因表达谱
STI571/As4S4联合治疗 CML基因表达谱
结论
3.白血病是一组涉及转录因子和信号转导分子
(特别是酪氨酸激酶 )的多步骤发病的遗传学
改变
4.基因产物靶向治疗为 APL和 CML患者带来治愈
的希望,并进一步拓展至其他类型白血病
2.L-GAP是研究白血病发病原理和设计靶向治疗
方法的有力途径
1.WHO 分类方法在白血病诊断和治疗中更加关注
染色体易位和基因异常
