Cell Cycle Control of Multiple Myeloma
Pathogenesis
Selina Chen-Kiang, Ph.D.
Weill Medical College of Cornell University

Multiple Myeloma Pathogenesis
Cell Cycle
MGUS
Apoptosis
dysregulation
dysregulation
Smoldering
Proliferation
Active MM
in vivo--Ki67
ex vivo--BrdU
Remission
Proliferation
Relapse
in vivo--Ki67
ex vivo--BrdU

The Cell Cycle
Positive
Negative
Go
p16INK4a
p15INK4b
Cyclin D + CDK4/6
p18INK4c INK
M
p19INK4d
mid-G1
G2
G1
pS-Rb-E2F
checkpoint
p21CIP1 CIP/KIP
S
p27KIP1
Cyclin E + CDK2
p57KIP2
pST-Rb
E2F release
CDK: Cyclin-dependent Kinase
CDKI: Cyclin-dependent kinase inhibitor

Control of G1 to S cell cycle progression
·G1 cell cycle progression is controlled by
the balance between positive (cyclin + Cdk) and
negative (Cdk inhibitors) cell cycle regulators.
·Loss of the mid-G1 cell cycle checkpoint
accelerates G1 to S phase transition.
·S phase entry is further attenuated by the
late G1 Cdk inhibitors (p27, p21).

Why don't we understand cell cycle dysregulation in
myeloma pathogenesis?
1. Cell cycle regulators are regulated at multiple levels;
RNA expression can not predict protein levels.
2. Lack of a functional cell cycle assay
at the single cell level.
3. Lack of understanding of cell cycle control of
normal plasma cell differentiation.

B Cell Terminal Differentiation and Myeloma Pathogenesis
Germinal Center
Ag
T
?
PC
CD40L
CD40
N
?
Bone
MM1
MM2
G0
G1
CD40L/BLyS/IL-6

Cell cycle control of normal plasma cell
differentiation
·Is G1 cell cycle control required for the
generation of normal plasma cells?
·If it is, which are the key cell cycle
regulators?

The Cell Cycle
Positive
Negative
Go
p16INK4a
p15INK4b
CyclinD + CDK4/6
INK
M
p18INK4c
p19INK4d
mid-G1
G2
G1
pS-Rb-E2F
checkpoint
p21CIP1 CIP/KIP
S
CyclinE + CDK2
p27KIP1
p57KIP2
pST-Rb
E2F release

p18 is required for G1-cell cycle arrest in
CD138+ intermediate-plasma cells (iPC)
Syndecan-1/Ki67
p18+/+
Plasma cells
Apoptosis
p18-/-
Bone
Tourigny et al, (2002), Immunity

p18 is essential for the generation of
functional plasma cells
-p18 is required within B cells for CD138+ intermediate plasma cells
(iPCs) to
arrest in G1.
survive,
differentiate to Ig-secreting PCs.
- p18 is dispensable for
differentiation to CD138+ iPC,
homing of iPC to bone.
Morse et al, (1997) Immunity
Franklin et al, (1998) Gene & Dev
Tourigny et al, (2002), Immunity

B Cell Terminal Differentiation and Myeloma Pathogenesis
Germinal Center
p18
p18
Ag
T
iPC
PC
CD40L
CD40
N
?
Bone
MM1
MM2
G0
G1
CD40L/BLyS/IL-6

Why don't we understand cell cycle deregulation in
myeloma pathogenesis?
1. Cell cycle regulators are regulated at multiple levels;
RNA expression can not predict protein levels.
2. Lack of a functional cell cycle assay
at the single cell level.
3. Lack of understanding of cell cycle control of
normal plasma cell differentiation.
p18 is required for normal PC differentiation.

Cell Cycle Control of Myeloma Pathogenesis
Strategy 2
Strategy 1
Functional cell cycle assay
Gene
Mid-G1 checkpoint
Immunohistochemistry
RNA
Protein analysis
Disease correlation
Disease correlation
longitudinal studies
Prediction
RNA
Animal model
Gene
Functional interference
Molecular Targetting

The Cell Cycle
Positive
Negative
Go
p16INK4a
p15INK4b
CyclinD + CDK4/6
p18INK4c INK
M
p19INK4d
mid-G1
G2
G1
pS-Rb-E2F
checkpoint
p21CIP1 CIP/KIP
S
CyclinE + CDK2
p27KIP1
p57KIP2
pST-Rb
E2F release

Dysreguation of early G1 cell cycle regulators
D cyclins
Shaunghnessy et al., 2001. Blood
Zhan et al., 2002. Blood
Zhan et al., 2003. Blood
P18
Kulkarni et al., 2002, Leukemia
p15 and p16 Ng et al., 1997, Blood.
Tasaka et al., 1998, Br J Haematol
However, overexpression of cyclin D1 correlates
With a more favorable clinical course.
Moreau et al, 2002, Blood
Soverini et al, 2003, Blood

Cdk4/6-specific Rb phosphorylation is a functional assay
for the mid-G1 cell cycle checkpoint
a
b
780
807/811
b
pS
i67
pS
K
PT
R
1239
247-1
247-2
c
247-2
+ CA
100
BrdU
247-2
80
Ki67
+ CIP
60
pS780
MCM7
Retina
40
Tumor
MCM2
20
0
PT 329
327
243
246

Expression of cyclin D1 and D3 in MM is mutually exclusive and
insufficient to promote Rb phosphorylation or proliferation
100
b
PT 254
338
3655
282
a
%D1+/CD138+ Cells
80
0-10
D1
60
11-70
40
>70
ts
20
en
D3
0
Pati
of
%D3+/CD138+ Cells
100
pS807/811
% 80
60
Ki67
40
20
0
CD20
NormalMGUSnew MMTx MM
c 1.
1.
0
0
0.
0.
Cells+
Cells
8
8
+
38
8 0.
0.
1
31 6
6
/CD+
/CD 0.
0.
+
4
4
D1
Ki67
0.
0.
2
2
0
0
251 Multiple Myeloma
Patients

Cyclin D1 expression does not vary during
the clinical course
1.0
0.8
lls
Ce+ 0.6
138D
/C+
0.4
0.4
%D1
0.2
0.2
0.0
0.0
1 2 3 4 5 6 7 8 9
1 2 3 4 5 6 7 8 9
1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 6 7 8 9 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 6 7 1 2 3 4 5 6 7 8 9
1 2 3 4 5 6 7 1 2 3 4 5 6 7 8 9
1 2 3 4 5 6 1 2 3 4 5 6 7 1 2 3 4 5 1 2 3 4 5 6 7 8 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 1 2 3 4 5 6 7
10 11
10
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
10
55- 55- 55- 55- 55- 55- 55-
6
123- 123- 123- 123- 123- 123- 123- 123- 123-
812- 812- 812- 812- 812- 812- 812- 812- 812-
330- 330- 330- 330- 330- 330- 330-
331- 331- 331- 331- 331-
114- 114- 114- 114- 114-
158- 158- 158- 158- 158- 158- 158- 158- 158-
235- 235- 235- 235- 235-
194- 194- 194- 194- 194- 194-
163- 163- 163- 163- 163- 163- 163-
123- 123-
812- 2649- 2649- 2649- 2649- 2649- 2649- 2649- 2649-
3306- 3306- 3306- 3306- 3306-
2456- 2456- 2456- 2456- 2456- 2456- 2456- 2456- 2456-
3862- 3862- 3862- 3862- 3862- 3795- 3795- 3795- 3795- 3795- 3795- 3795-
158- 158- 158- 158- 158- 158- 158- 158- 158- 158- 158- 158- 158- 158- 158- 158- 158-
1818- 1818- 1818- 1818- 1818- 1818- 1818- 1818- 1818-
5033- 5033- 5033- 5033- 5033- 5033- 2781- 2781- 2781- 2781- 2781- 2781- 2781-
4672- 4672- 4672- 4672- 4672- 4672- 4672- 4672- 2219- 2219- 2219- 2219- 2219- 2219-
4337- 4337- 4337- 4337- 4337-
1818-
BM
1-11
1-10
1-8
1-7
1-5
1-5
1-9
1-5
1-5
1-7
1-2
1-7
1-10
1-6
1-7
1-5
1-8
1-6
1-6
1-5
1-7
PT
1
2
3 4 5 6 7 8 9 10
11
12 13 14 1516171819 20 21

Summary -1
Overexpression of cyclin D1 and cyclin D3 in MM is
mutually exclusive;
Overexpression of cyclin D1 does not vary in disease
progression;
Cyclin D1 overexpression is insufficient to promote G1
cell cycle progression.

Rb is selectively phosphorylated by cyclin D1-Cdk4 or
cyclin D2-Cdk6/4 in MM cells.
137-2
a PT 336 346 137-1
b
pS780
Rb
Cdk6
Ki67
D1
D3

Selective phosphorylation of Rb by cyclin D2-Cdk6/4 is
enhanced in advanced myeloma

Summary-2
One, and only one cyclin D, is overexpressed in each
myeloma patient.
Cyclin D2 and Cdk6 are coordinately regulated at the
transcriptional level.
Co-expression of cyclin D2 and Cdk6 leads to Rb
phosphorylation (loss of the mid-G1 cell cycle checkpoint),
and promotes proliferation.
The levels of cyclin D2 and Cdk6 expression correlates
with myeloma progression.

The Cell Cycle
Positive
Negative
Go
p16INK4a
p15INK4b
CyclinD + CDK4/6
p18INK4c INK
M
p19INK4d
mid-G1
G2
G1
pS-Rb-E2F
checkpoint
p21CIP1 CIP/KIP
S
CyclinE + CDK2
p27KIP1
p57KIP2
pST-Rb
E2F release

Thanks
Cornell Medical College
Maurizio Di Liberto
Scott Ely
Eunice Hatada
Ruben Niesvizky
Hearn Cho
Birmingham Medical School
Ian MacLennan
Adam Cummingham
Supported by a Specialized Center of Research for Multiple Myeloma
Grant fromthe Leukemia and Lymphoma Society and NIH

Multiple Myeloma
Asymptomatic
Symptomatic
100
Active
Myeloma
(g/l)
50
Relapse
Refractory
ProteinM
Relapse
MGUS or
Smoldering
Myeloma
20
Plateau
Remission
Therapy
Therapy
Therapy
~15,000
~45,000
~11,000
New cases
Annual patients in U.S
Annual
in U.S.
deaths in
U.S
1. Adapted from International Myeloma Foundation; 2001. Reprinted with permission.
2. American Cancer Society. Cancer Facts & Figures; 2003. 3. Millennium Pharmaceuticals, Inc., 2003.

Document Outline

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