Targeted Overexpression of the Transcription Factor
XBP-1 in
B cells Promotes Plasma cell N
l
eop asms in Transgenic
Mice
Genetic Model of Multiple Myeloma
Ruben D. Carrasco, M.D., Ph.D.
Medical Oncology, Dana-Farber Cancer Institute
Pathology Department, BWH
Harvard Medical School
Boston, MA
Multiple Myeloma (MM)
B-cell neoplasm characterized by bone marrow infiltration with
malignant plasma cells, which synthesize and secrete monoclonal
immunoglobulin (IG) fragments
Current Mouse Models of MM
Human tumors or cell lines in immunodeficient mice.
SCID mousehuman bone model
Sb
Subcutaneous implantatition of human f t
e al bone it
into SC
SCID
mice, followed by injection with human MM tumor cells
(Yaccoby,1998 and Tassone et al., 2005).
Spontaneous multiple myeloma in mice
At two years of age, 0.5% of C57BL/KaLwRij mice develop
tumors similar to MM (Radl et al. (1990)).
Genetic mouse models
Genetic Mouse Models of Human Plasma Cell Neoplasms
Model system
Type
c-
c myc activation
Reference
--------------------------------------------------------------------------------------------------------------------------------
Peritoneal PCTs
Plasmacytoma
Yes
Potter M. (1977)
5T (C57BL/Ka)
MGUS, MM
No
Radl J. (1981)
E-v-abl transgenic
Plasmacytoma
Yes
Rosenbaum H. (1990)
IL-6 Transgenic
Plasmacytoma
Yes
Kovalchuk A. (2002)
NPM-ALK Transgenic
Plasmacytoma
No
Chiarle R. (2003)
ABL/MYC retrovirus
Plasmacytoma
Yes
Largaespada D. (1992)
E-bcl-2 transgenic
Plasmacytoma
Yes
Strasser A. (1993)
Bcl-X(L) transgenic
Plasmacytoma
Yes
Fang W.
W (1996)
MYC-Bcl-X(L) transgenic Plasmacytoma
Yes
Cheung W. (2004)
Vk* myc mice
MGUS, MM
Yes
Sebag M. (2006)
E-XBP-1s transgene
MGUS, MM
No
Carrasco D. (2007)
Plasmacytoma, Plasma cell leukemia, Immunoblastic DLBCL with plasmacytoid differentiation, Burkitt
lymphoma with plasmacytoid differentiation.
XBP 1s
-
Transgenic Mice
Plasma Cell Differentiation Requires
the Transcription Factor XBP-1
Shaffer et al., 2002
X-box Binding Protein 1 (XBP-1)
bZIP Transcription
factor (IL6...)
Essential for
differentiation of PC
Unf
U ol
o ded prot
o ein
e
response
Upregulated in MM
Two splice variants:
Spliced (
p(54kDa)
Unspliced (30kDa)
Spliced form predominates in MM
Generation of Em-XBP-1s Transgenic
Mice
Immunoglobulin VH promoter
gp
and E enhancer (pE
(p )
elements
RT-PCR
WT
S1
S2
S3
-XBP-1(U)
-XBP-1(S)
Southern blot
GFP
Ponceau
Clinical Presentation
Summary of E-XBP-1s mice
Cutaneous autoimmune condition
dermal immune infiltration and
vascular proliferation (POEMs)
60% between 7-24 months
MGUS
WT
28% between 11-20 months
Multiple Myeloma
20% between 14-24 months
XBP-1(S)
XBP-1s expression in human plasma
cells and MM
IHC
0/10
10/20 (50%)
50/70 (70%)
WB
Increased numbers of plasma
cells in XBP-1s transgenic mice.
80x
H&E
200x
Immunostained sections
using anti-CD138
antibodies (plasma cell
80x
specific)
Single B cell suspensions
Anti-
Anti B220 (B cell
specific) and anti-
CD138 antibodies.
Spleen, 20 wks
Em-XBP-1s Transgenic Mouse Immunoglobulin Profi
Increased serum Ig
Monoclonal Spike
Clonality by
yy IHC
IHC
Bone Marrow
Hypermutation analysis
Kidney Pathology
WT
XPB1s
PAS
H&E
PAS
H&E
Tubular cast deposition
Glomerular changes characterized by mesangial widening and
deposition of PAS positive material
Electron microscopy showed mesangial and subendothelial
deposition of electron-
electron dense
-
material
IF
Bone Lytic Lesions in XBP-1s
Control
XBP-1s
Bone
Bone
XBP-1s
Only one tumor did show
t(
rearrangements t(12
(t(12 X
; )
X)
d
an
others)...
...also at the aCGH, that in the other
cases did not
not show any change
change.
Cyclin D1
Cyclin D2
MAF
700
1500
900
WT
WT
WT
600
800
B XBP1
B XBP1
B XBP1
700
500
MM XBP1
MM XBP1
MM XBP1
1000
600
400
500
300
400
300
500
300
200
200
100
100
0
0
0
WT
B XBP1
MM XBP1
WT
B XBP1
MM XBP1
WT
B XBP1
MM XBP1
Cyclin D1
Cyclin D2
MAF
MAFB
IL6RA
IL6ST
800
300
700
WT
WT
WT
700
600
B XBP1
B XBP1
B XBP1
600
MM XBP1
MM XBP1
500
MM XBP1
200
500
400
400
300
300
100
200
200
100
100
0
0
0
WT
B XBP1
MM XBP1
WT
B XBP1
MM XBP1
WT
B XBP1
MM XBP1
MAFB
interleukin 6 receptor
interleukin 6 receptor
Bergsagel et al., Blood, 2005
Thymidylate synthase
Thymidine Kinase 1
Cyclin B1
100
WT
600
700
B cel s XBP1
)
600
75
MM XBP1s
500
raw
500
(
400
no 50
400
300
essi
300
Expr
25
200
200
100
100
0
s
0
0
WT
P1
B
P1
WT
B XBP1s
MM XBP1s
WT
B XBP1s
MM XBP1s
XB
ells X
c
MM
thymidine kinase 1
Cyclin B1
B
ki67
GINS4
CKS1B
2500
250
2500
2000
200
2000
1500
150
1500
1000
100
1000
500
0
50
500
T
W
1s
1s
0
0
XBP
XBP
B
M
WT
B XBP1s
MM XBP1s
WT
B XBP1s
MM XBP1s
M
Ki67
GINS4
CKS1B
UBE2C
ZWINT
TRIP13
900
300
400
800
700
300
600
200
500
200
400
300
100
200
100
100
0
0
0
WT
B XBP1s
MM XBP1s
WT
B XBP1s
MM XBP1s
WT
B XBP1s
MM XBP1s
UBE2C
ZWINT
TRIP13
KIF11
TOP2A
500
2000
400
300
1000
200
100
0
0
WT
B XBP1s
MM XBP1s
WT
B XBP1s
MM XBP1s
KIF11
TOP2A
Shared in the 2 lists
Zhan et al., Blood, 2006
BIRC5
cyclin B2
Nek2
1000
750
350
300
750
250
500
200
500
150
250
100
250
50
0
0
0
WT
B XBP1s
MM XBP1s
WT
B XBP1s
MM XBP1s
WT
B XBP1s
MM XBP1s
BIRC5
Cyclin B2
Nek2
ANAPC7
Aurora Kinase A
BUB1
450
400
750
400
350
300
300
500
250
200
200
150
250
100
100
50
0
0
0
WT
B XBP1s
MM XBP1s
WT
B XBP1s
MM XBP1s
WT
B XBP1s
MM XBP1s
ANAPC7
Aurora Kinase A
Aurora Kinase A
CDC2
CEP55 [C10orf3]
ASPM
1000
700
300
600
750
500
200
400
500
300
100
200
250
100
0
0
0
WT
B XBP1s
MM XBP1s
WT
B XBP1s
MM XBP1s
WT
B XBP1s
MM XBP1s
CDC2
CEP55 [C10orf3]
CEP55 [C10orf3]
CDCA1
350
300
250
200
150
100
50
0
WT
B XBP1s
MM XBP1s
CDCA1
Zhan et al., Blood, 2006
Topo2A
BIRC5
CCNB2
NEK2
2500
4000
3000
400
**
*
* *
**
*
*
**
2000
ls
ls
3000
e
NS
e
ls
ls
e
*
v
e
300
e
ve
v
v
e
2000
e
l
le
1500
l
le
n
n
o
2000
ion
ion
200
io
si
ss
s
s
1000
e
s
s
res
pr
1000
re
1000
x
pre
p 100
x
Exp 500
E
Ex
E
0
0
0
0
T
s
s
T
s
s
s
s
1s
1s
WT
1s
1
1s
1
W
WT
P
W
WT
P
P
P1
P1
P1
P1
B
BP
B
X
X
X
XB
XB
XB
ls
rs
s XB
ls
ors XB
lls
ors
ors
cel
m
ce
cell
m
cel
m
B
Tu
B
Tumo
B
Tu
B
Tu
ANAPC7
AURKA
BUB1
CDC2
700
2000
1100
400
**
**
1000
600
**
**
NS
**
*
*
*
900
ls
ls
350
ls
ls
e
1500
e
500
800
v
v
ve
e
e
eve
700
l
l
le
l
300
400
n
n
n
n 600
o
o 1000
io
si
s
500
sio
300
s
ssi
250
400
re
res
res
re
200
p
p
p
p
300
500
x
x
x
200
E
E
Ex
00
E
200
100
100
150
0
0
0
s
s
T
1s
s
s
WT
W
1s
1s
P
WT
P
P1
WT
P1s
P1
B
B
XBP
X
XB
XB
X
ls XBP1
rs XBP1
ls
ls
ors XB
ors
ells
ors
cel
cel
m
cel
c
B
Tumo
B
Tu
B
Tum
B
Tum
CEPP55
ASP
AS M
CDCA1
CEPP55
CDCA
900
700
650
NS
NS
NS
*
= p < 0.05
600
800
600
*
*
*
550
ls
ls
ls
700
500
e
e
500
ve
v
v
450
600
e
le
l
400
le
n
400
= p < 0.01
500
n
350
o
**
ion
io
300
s
s 400
300
s
s
ssi
250
25
e
e
re
300
r
200
p
pr
200
150
200
Ex
Ex
Exp
100
100
** * = p < 0.001
100
50
0
0
0
s
T
1s
WT
W
1s
1s
P
P
WT
P1s
P
BP1s
B
B
XB
X
XB
s X
lls X
rs XBP1
lls
ls
ors
or
ce
mo
ce
cel
B
Tu
B
Tum
B
Tum
BCL2
MCL1
400
3000
WT
WT
B XBP1
B XBP1
300
MM XBP1
MM XBP1
2000
200
1000
100
0
0
WT
B XBP1
MM XBP1
WT
B XBP1
MM XBP1
BCL2
MCL1
XBP-1s Myeloma Model possesses some clinical and
pathological features observed in human MM
Human Di
Di
t
agnos itic C irtit i
er a:
Monoclonal spike
Lytic Bone lesions
MAb spike on SPEP
Plasma cell infiltration of BM
Bone Lytic Lesions
Kidney Subendotheilial HUMP
Plasma cell infiltration
Renal lesions
Acknowledgments
Ron DePinho
Kenneth Anderson
Giovanni Tonon
Nikhil Munshi
Marina Protopopova
Kumar Sukhdeo
Joel Henderson
Mala Mani
Funding
It
Int
t
erna i
ti
l
ona Myeloma Foundt
da i
tion
Fund to Cure Myeloma
Multiple Myeloma SPORE
Leukemia and Lymphoma Society
Sidney Kimmel Foundation
Doris Duke Foundation
NIH and NCI