Marta Chesi, PhD
Mayo Clinic Arizona
A transgenic mouse model that faithfully
dt
reproduces h
the path
thogenesis, bi
biology
and clinical features of multiple myeloma
Scottsdale, Arizona
Rochester,
Rochester Minnesota
Jacksonville, Florida
Biological and clinical features of MM
MM is an indolent disease of fully differentiated
plasma cells that accumulate over time and:
Secrete high level of monoclonal antibody (>35g/l)
· Easily detectable in the serum as M-protein by SPEP
Have undergone somatic hypermutation of the Ig genes
Have undergone class switch recombination
· IgM myeloma is very rare
Home to and are confined to the bone marrow
· Interaction with BM microenvironment is crucial for survival
Have a very low proliferation index
· Low BrDU incorporation or Ki67 staining
Cause end-organ damage (CRAB)
Current mouse models of myeloma
Currently there are no mouse models that fully recapitulate all the
molecular and biological features of MM.
· Existing Models of Myeloma:
Xenografts -Scid-hu (and HMCLs)
· HMCLs carry additional g
ygenetic abnormalities
· poor tumor-host interaction
· immuno-incompetent
Plasmacytoma in Balb/c
· extramedullary; high proliferation index
Transgenics: MYC, Bcl2, BclXL, IL6,...
· extramedullary disease
· Aggressive phenotype
Spontaneous MG and MM in C57Bl/6 and C57Bl/KaLwRij
· MG in 80% at 2yrs (M-spike <5g/l)
· MM in 0.5% at 2yrs (M-spike >10g/l)
E-XBP1 in C57Bl/6
· Not clearly distinguishable from spontaneous MG in C57Bl/6
Developing the best transgenic mouse model of myeloma
· Build up on C57Bl6 strain
Transgene as secondary event in genetic background predisposed to
dl
develop MG
MG
· Dysregulate transgene expression by physiological mechanism
At the right time, and in the right cell
Somatic hypermutation (and l
c ass switch recombination)
· Choose strong oncogene: MYC
classic oncogene dysregulated in post-germinal center malignancies:
· t(8;14) hallmark of Burkitt's lymphoma
· t(12:15) hallmark of mouse plasmacytoma in Balb/c
· Translocated in 15% MM at diagnosis, 40% in advanced MM and 95% HMCL
Activation of MYC by SHM in C57Bl/6 Vk*MYC mice
VK
JK
iKE
Ck
3'kE
Kappa locus
VK
hMYC
*
*
*
HA
iKE
ex2
ex3
3'kE
Vk*MYC
Wild type HA (HA):
ATG GGA TCG TAC ...
*
Mutated HA (*HA):
ATG GGA TAG TAC...
SH-hotspot (DGYW)
Transgenic MYC transcription occurs constitutively in B cells but translation is aborted
Sporadically, in isolated GC cells, somatic hypermutation will activate MYC translation
Monoclonal spikes in Vk*MYC mice
wt
Vk*MYC
50w
20w
30w
40w
50w
60w
70w
Incidence of spikes over time in Vk*MYC mice
M-spike incidence
Serum IgG1 @70w
100
256
WT
Vk*MYC
128
80
64
60
32
(%)
16
40
8
4
20
2
0
1
Wild Type
VK*MYC
10 20 30 40 50 60 70 80
Median [g/l]:
2.8
38.6
weeks
PC accumulates in the BM of Vk*MYC mice
wt
Vk*MYC
Vk*MYC
H&E
10x
CD138
10x
CD138
40x
0.6
0.5
5.81
0.25
56.7
0.57
CD138
68.9
30
90.7
3.21
28
14.8
B220
PCs in Vk*MYC mice have low proliferation index
wt
Vk*
Vk MYC
H&E
H&E
381
/CD
Ki67
End organ damage in Vk*MYC mice
wt
Vk*MYC
20
tubuli
10
erulim
0
glo
WT
Vk*MYC
wt
Vk*MYC
Bone Mineral Density
MicroCT
BMD (g/cm2)
trabecular number (#/mm2)
wt
0.0727 (+ 0.0025)
1.271 (+ 0.150)
Vk*MYC 0.0595 (+ 0.0011)
0.523 (+ 0.157)
P=0.0016
P=0.0208
Indolent clinical course in Vk*MYC mice
125
wt: 954
100
Vk*MYC: 661
75
50
25
0
0
250
500
750
1000
1250
days
Using Vk*MYC mice to model drug efficacy in MM pts
Drug
Dose
Admin
Note
Max concentration of DMSO in
Placebo (DMSO)
0.5% in NS
IP qd d1-5
other preparations
Dexamethasone
1mg/kg
IP qd d1-5
Extrapolated from human dose
Published in Xenographic MM
Melphalan
2.5mg/kg
IP qd d1-5
mouse models
Published in Xenographic MM
Bortezomib
0.5mg/kg
IP d1+4 x4weeks mouse models
Vincristine
0.5mg/kg
IP x1 dose
MTD for mice
Hydroxyurea
100mg/kg
IP bid d1-
d1 5
Leukopenic dose
Fludarabine
34mg/kg
IP qd d1-5
Published in CLL mouse model
Positive control: drugs active in MM pts are active in Vk*MYC mice
170
M Protein (SPEP)
160
Placebo
150
Dex
e am
a eth
e ason
aso e
140
Melphalan
130
Bortezomib
120
110
100
90
80
70
60
50
40
30
20
10
0
0 2 3 4 5 6 7
0 2 3 4 5 6 7
0 2 3 4 5 6 7
0 2 3 4 5 6 7
Week
Negative control: inactive drugs in pts are inactive in Vk*MYC mice
140
120
100
0= 80
d
60
from%
40
20
0
day
0
14
21
0
14
21
0
14
21
Vincristi
tine
Hd
Hydroxyurea
Fl d
u
b
ara i
bine
PCs growth is restricted to BM in Vk*MYC mice
wt
Vk*
Vk MYC
spleen
BM
spleen
BM
0.37
0.74
0.72
0.56
1.06
1.69
25.2
0.26
83
CD1
44.8
54.1
69.6
29.2
70.9
26.3
68.8
5.66
B220
· BM microenvironment provides crucial signals for PCs survival
acti ti
va on f
o the ncNFkB th
pa way
· Providing PCs with anti-apoptotic signals may bypass requirement for BM growth
crossing Vk*MYC mice with EBcl2 mice
Extra-medullary PCs growth in Vk*MYC x EBCL2 mice
wt
BCL2
MYC
MYC/BCL2
H&E
10x
spleen
CD138
10x
1.15
1.38
2.68
1.63
0.57
0.8
26.7
7.48
26.7
7.48
138D
17.6
CD
55
42.4
20.1
75.6
37 2
.
61 4
31 2
.
34 6
.4
.
B220
70w 90w
40w 60w
70w 90w
20w 30w
Vk*MYC mice recapitulate biological and clinical features of MM
MM is an indolent disease of fully differentiated plasma cells that
accumulate over time and:
· Secrete high level of monoclonal antibody (>35g/l)
· Vk*MYC mice can secrete up to 200g/l of IgG
· Have undergone somatic hypermutation of the Ig genes
· Vk*MYC mice have acquired up to 36 mutations in the VH genes
· Have undergone class switch recombination
· PCs in Vk*MYC mice are always switched
· Home to and are confined to the bone marrow
· PCs in Vk*MYC mice localize exclusively in the BM
· Cross with Bcl2 allows extra-medullary growth
· Have a very low proliferation index
· Low BrDU incorporation or Ki67 staining
· Cause end-organ damage (CRAB)
(CRAB)
· Vk*MYC mice are anemic, show protein deposition in kidney, have bone disease.
· Vk*MYC mice are a valuable tool to test drug efficacy in MM
Contributors
· Hematological Malignancy Lab - MCS
Leif Bergsagel
Keith Stewart
David
ide R bbi
o
i
an
Rf
Raf l
ae Fonseca
Michael Sebag
Jonathan Keats
Stephen Palmer
Roger Tiedemann
St h
ep anie Haas
· Richard Kremer - McGill University - Montreal
· Giorgio Cattoretti - University of Milan - Italy
· Michael Kuehl - NCI
Somatic hypermutation in Vk*MYC mice at IgH and transgenic locus
D
·c NA
DNA from CD138
CD138+ selected BM
BM plasma cellls from Vk*MYC
Vk*MYC i
m ce was PCR
PCR
amplified, subcloned and sequenced.
·SHM was found both at the Ig
fgH and transgenic locus
mouse
condition DNA/RNA VHgene
mut tot TS/TV
clone/seq SPIKES Reversion TS/TV
Vk*MYC11 #33BM
MGUS
cDNA
IGHV1-53
9
9/0
4/5
1 (1)
yes
2/3
Vk*MYC11 #33BM
MGUS
cDNA
IGHV1S2
4
3/1
1/5
yes
Vk*
Vk MYC11 #37BM
MGUS
cDNA
IGHV1-54
10
5/5
4/4
1
yes
4/4
Vk*MYC24 #31BM
MGUS
cDNA
IGHV1S24
27
17/10
4/4
1
ND
ND
Vk*MYC24 #1BM
MGUS
cDNA
IGHV2-3
3
2/1
4/5
1
yes
3/1
Vk*MYC24 #1BM
MGUS
cDNA
IgHV2-9
34
19/15
1/5
yes
Vk*MYC11 #56BM
MGUS
cDNA
IGHV1-53
10
6/4
3/5
3
none
none
Vk*MYC11 #56BM
MGUS
cDNA
IGHV1S2
6
4/2
1/5
Vk*MYC11 #56BM
MGUS
cDNA
IGHV1S56
36
18/18
1/5
Vk*MYC24 #61BM
MGUS
cDNA
IgHV1-55
10
6/4
1/4
4
ND
ND
Vk*MYC24 #61BM
MGUS
cDNA
IgHV10-1
11
3/8
1/4
ND
ND
Vk*MYC24 #61BM
MGUS
cDNA
IGHV1-52
12
10/2
1/4
ND
ND
Vk*MYC24 #61BM
MGUS
cDNA
IGHV1-4
8
5/3
1/4
ND
ND