Inflammation and cancer
Laura G. Schuettpelz, MD, PhD
Assistant Professor of Pediatrics Division of Pediatric Hematology/OncologyWashington University School of Medicine
Cancer Biology PathwayFebruary 7, 2017
Disclosure: Dr. Schuettpelz has no relevant financial interests to disclose
http://countdowntozerotime.org
Outline
• Role of inflammation in cancer
• Inflammation in hematopoietic malignancy
• Intro to myelodysplastic syndromes; role of aberrant innate immune signaling
• Toll like receptor 2 (TLR2) as a therapeutic target
Coussens and Werb, Nature 2002
In 1863, Rudolph Virchow described the presence of leukocytes in neoplastic tissues and suggested that cancer originates at sites of chronic inflammation.
Inflammation (from Latin inflammatio) is part of the complex biological response to harmful stimuli,such as pathogens, damaged cells, or irritants, and is a protective response involving immune cells, bloodvessels and molecular mediators. The function of inflammation is to eliminate the initial cause of cellinjury, clear out necrotic cells and tissues damaged from the original insult and the inflammatory process,and to initiate tissue repair.
Acute inflammation is the initial response of the body to harmful stimuli and is achieved by theincreased movement of plasma and leukocytes from the blood into the injured tissues. A series ofbiochemical events propagates and matures the inflammatory response, involving the local vascularsystem, the immune system, and various cells within the injured tissue. Prolonged inflammation, known aschronic inflammation, leads to a progressive shift in the type of cells present at the site ofinflammation, such as mononuclear cells, and is characterized by simultaneous destruction and healing ofthe tissue from the inflammatory process.
Wikipedia
Inflammatory Response Pathway
Inducers -microbial-non-microbial (eg, allergens, foreign bodies, products of cellular damage)
Sensors-PRRs (TLRs, NLRs, etc)
Mediators-cytokines, chemokines, vasoactive amines, etc
Effectors-endothelial cells, leukocytes, smooth muscle, etc.
Takizawa et al, Blood 2012
Grivennikov et al; Cell 2010
Karin and Greten 2005
Grivennikov et al; Cell 2010
Types of Inflammation in Cancer
Grivennikov et al; Cell 2010
Kristinsson et al; J Clinical Onc 2011
Myelodysplastic Syndromes
•Clonal hematopoietic stem cell (HSC) disorders characterized by ineffective
hematopoiesis and cytopenias
•Roughly 30% progress to acute leukemia
•Estimated >10,000 new cases/year in the US; incidence increases with age
•May be associated with prior chemotherapy
or radiation, or inherited predisposition
syndromes
•HSCT is only curative therapy;
new therapies are needed
A = binucleate megaloblastoid erythroid precursor
B = megaloblastoid erythroid precursor
C = small megakaryocyte with monolobate nucleus
http://emedicine.medscape.com/article/1976592-overview.
Tefferi A, Vardiman JW. N England J Med 2009
Innate immune and inflammatory signaling deregulation in MDS
Ganan-Gomez et al, Leukemia 2015
Toll Like Receptors
• Family of pattern recognition receptors; recognize pathogen-associated molecular patterns (PAMPs) and damage associated patterns (DAMPs)
• Expressed on immune cells (eg, dendritic cells, macrophages, B cells, T cells, etc) and non-immune cells (endothelium, epithelium)
• TLR signaling leads to activation of NF-kB, MAPKs, IRF3, IRF7 and the production of pro-inflammatory cytokines
TLR6
Endosome
TLR9
MyD88
IRAK1
IRAK4
TRAF6
TRIF
TRAF3
Pro-inflammatorycytokines
TBK1
IRF3
TLR2
Plasma
membrane
Cytoplasm
IRAK2
IKKα
IKKβ
IKKγ
NF-κB
IKKε
IRAK2
IRAK4
IRAK1
TRAF3TRAF6
IRF7
TypeIInterferons,IFNinducible
genes
MKKs
p38 ERK JNK
AP1CREB
Nucleus
NF-κBAP1IRF3IRF7
MyD88
NF-κB
Monlish et al; Frontiers in Immunology 2016
Monlish et al; Frontiers in Immunology 2016
Toll like receptor signaling is enhanced in myelodysplastic syndromes (MDS)
Ganan-Gomez et al, Leukemia 2015
Maratheftis, et al Clin Cancer Res 2007Hoffman, et al Blood 2002Starczynowki, et al Nature Medicine 2010
Wei, et al Leukemia 2013Dimicoli, et al PLoS One 2013Varney, et al J Exp Med 2015
TLR2 expression/signaling is enhanced in the CD34+ cells of patients with MDS
Wei et al; Leukemia 2013
TLR1
TLR2
TLR3
TLR4
TLR4
TLR4
TLR5
TLR6
TLR7
TLR7
TLR8
TLR9
TLR10
TLR10
0
1000
2000
3000
4000
5000
10000
15000
ex
pre
ss
ion
va
lue
control
MDS
**
**
*
Dr. Matt Walter
*Also, TLR2 ligands (DAMPs) are increased in the serum of patients with MDS:• HMGB1 (Velegraki et al; Haematologica 2013)
OPN-305= TLR2-specific monoclonal antibody
What role does TLR2 signaling play in the pathogenesis of MDS?
• How does TLR2 signaling influence normal HSCs?
• Does enhanced TLR2 signaling contribute to the cytopenias and or progression to leukemia in MDS?
https://mutagenetix.utsouthwestern.edu/phenotypic/phenotypic_rec.cfm?pk=214
Toll Like Receptor 2 (TLR2)
Expressed on HSCs (in addition to other immune and non-immune cell types)
Functions as a heterodimer with TLR1 or TLR6
Ligands include a variety of bacteria, viruses, fungi, protozoa/helminths, and non-pathogen DAMPs
Little known about its role in regulating HSCs
KSL S
LAM
CM
PGM
PM
EP
0
10000
20000
30000
1000000
2000000
7000000
8000000
9000000
1×107
cells
/sp
leen
H2O
PAM3CSK4
p<.0001
p<.0001
p<.0001
p<.0001
TLR2 is not necessary for HSC function
WT
Tlr2-/-
0
20
40
60
80
%L
y5
.2
*
Sca-1
CD48
CD34
c-K
it
CD
16
/32
CD
15
0
c-K
it
Lineage
CMP
GMP
MEP
KSL SLAM
WT
Tlr2-/-
0.000
0.005
0.010
0.015
0.020
0.025
% o
f W
BC
s
*
wild-type
TLR2-/-or WT
Ly5.2
Ly5.1
1:1
Ly5.1/Ly5.2
Role of TLR2 signaling in normal hematopoiesis
TLR2 agonist (PAM3CSK4)
or H2O(100ug/dose q48hrs x3)
Analyze HSC cycling, numbers, repopulating
activity
KSL S
LAM
CM
PGM
PM
EP
0.00
0.01
0.02
0.03
0.05
1.05
2.05
% o
f W
BC
s
p=.0149
p=.0001
H 2O
PAM 3
CSK 4
0
100
200
300
CF
U-C
x 1
03/s
ple
en
p=.0004
untreated
PAM3CSK4 or H2O
Ly5.1
Ly5.2
1:1
KSL S
LAM
CM
PGM
PM
EP
0
1000
2000
3000
4000
50005000
127500
250000
ce
lls/f
em
ur
H2O
PAM3CSK4***
KSL S
LAM
CM
PGM
PM
EP
0.00
0.01
0.02
0.03
0.05
1.05
2.05
% o
f W
BC
s
*
***
H 2O
PAM
3CSK4
60
70
80
90
100
%G
0
H 2O
PAM
3CSK4
0
2
4
6
8
10
% S
/G2/M
H2O
PAM3CSK4
0 6 12 18 240
20
40
60
80
Weeks
% 5
.1
0 6 12 18 240
20
40
60
80
Weeks
% 5
.1
*
0 6 12 18 240
20
40
60
80
Weeks
% 5
.1
0 6 12 18 240
20
40
60
80
100
Weeks
% 5
.1 ******
0 6 12 18 240
20
40
60
80
100
Weeks
% 5
.1
** * *
0 6 12 18 240
20
40
60
80
100
Weeks
% 5
.1
**
0 6 12 18 240
20
40
60
80
100
Weeks
% 5
.1
**** **
0 6 12 18 240
20
40
60
80
Weeks
% 5
.1
PAM3CSK4
H2O
A B C
B cells neutrophilsT cellsoverall
Herman et al; Blood Cancer J 2016
Does enhanced TLR signaling cooperate with MDS-associated oncogenes in MDS pathogenesis?
MDS miceNUP98-HOXD13
Enhance TLR2 signaling:treatment with TLR2 agonists
Reduce TLR signaling:cross with TLR2 or MyD88
deficient mice
Evaluate disease progression and survival, hematopoietic stem and
progenitor cycling, apoptosis, differentiation
NUP98-HOXD13 (NHD13) mice:
- NUP98-HOXD13 fusion expressed from vav-1 promoter
- Mice develop cytopenias early in life, die by 14 mos of age from MDS, leukemia (average about 9 mos).
TLR2
% o
f m
ax
Tlr2-/-
Wild-type
NHD13
Lin, et al; Blood 2005
-Monthly CBC, PB flow-Follow until illness/death
NHD13+
x
MyD88-/--
Tlr2-/- NHD13+ NHD13; Tlr2-/- Tlr2-/- WT
NHD13+ NHD13; MyD88-/- MyD88-/- WT
Does loss of TLR2 impact disease course or survival of NHD13 mice?
0 100 200 300 400 500 600 7000
10
20
30
40
50
60
70
80
90
100
110
Days
Pe
rce
nt s
urv
iva
l
NHD+;TLR+/+
NHD+;TLR2-/-
NHD-TLR2-/-
NHD-;TLR2+/+
NHD+;
TLR2+
/+
NHD+;T
LR2-
/-
NHD-;TLR
2+/+
NHD-;TLR
2-/-
7.5
10.0
12.5
15.0
17.5
20.0
22.5
He
mo
glo
bin
(g
/dL
)
NHD+;T
LR2+
/+
NHD+;T
LR2-
/-
NHD-;TLR
2+/+
NHD-;TLR
2-/-
0
5
10
15
20
80
105
130
WB
Cs
/ul (x
10
3)
Cause of Death
NHD+;TLR2-/- NHD+;TLR2+/+ NHD-;TLR2-/- NHD-;TLR2+/+
MPD-like leukemia
5 3
Leukemia 5 7
T-ALL 1
Erythroid Leukemia
1
AML
MDS 1
Unknown 8 2
Other 2
p=.019
Dr. Eric Duncavage
NHD+;T
LR2+
/+
NHD+;
TLR2-
/-
NHD-;TLR
2+/+
NHD-;TLR
2-/-
0
200
400
600
800
1000
1200
1400
pla
tele
ts/u
l (x
10
3)
**
****
Loss of TLR2 is associated with earlier transformation of NHD13 mice
TLR2 expression correlates with increased apoptosis and lower-risk disease
Wei et al; Leukemia 2013 Zeng et al; Exp Cell Res 2016
Loss of TLR2 is associated with reduced cell death in NHD13 HSPCs
Some TLR2 signaling may be protective (via promotion of death of premalignant cells?)WT
TLR2-
/-
NHD+
NHD+;
TLR2-
/-0
10
20
30
% A
nn
exin
V+
(K
SL
)
** **
**
WT
TLR2-
/-
NHD+
NHD+, T
LR2-
/-0.00
0.05
0.10
0.15
0.20
KS
L f
req
uen
cy
WT
TLR2-
/-
NHD+
NHD+, T
LR2-
/-0
2000
4000
6000
8000
1000010000
20000
30000
40000
50000
KS
L c
ells
/ fe
mu
r
c-K
it
Sca-1
WT NHD13+ NHD13+; Tlr2-/-
Ki-
67
DAPI
WT
TLR2-
/-
NHD+
NHD+,
TLR
2-/-
0
20
40
60
80
%G
0
**
**
**
*
WT
TLR2-
/-
NHD+
NHD+,
TLR
2-/-
0
10
20
30
40
%G
1
*
WT
TLR2-
/-
NHD+
NHD+,
TLR
2-/-
0
5
10
15
20
%S
/G2/M
***
WT NHD13 NHD13; Tlr2-/-
Mechanisms of cell death in MDS
Sallman et al; Frontiers in Oncology 2016Basiorka et al; Blood 2016
Does enhanced TLR signaling cooperate with MDS-associated oncogenes in MDS pathogenesis?
MDS miceNUP98-HOXD13
Enhance TLR2 signaling:treatment with TLR2 agonist
Reduce TLR signaling:cross with TLR2 or MyD88
deficient mice
Evaluate disease progression and survival, hematopoietic stem and
progenitor cycling, apoptosis, differentiation
• decreased cell death• accumulation of HSPCs• faster transformation to leukemia
WT or NHD13+
3x/wk; starting at 8wks of age-monthly CBC, PB flow-treat until illness/death
H20PAM3CSK4 (TLR1/2 agonist) 25 mcgPAM2CSK4 (TLR2/6 agonist) 1 mcg
Does stimulation of TLR2 impact disease course or survival of NHD13 mice?
https://mutagenetix.utsouthwestern.edu/phenotypic/phenotypic_rec.cfm?pk=214
High TLR6 expression (but not TLR2) is associated with higher-risk disease
Higher TLR2 expression= lower risk disease (Wei et al, 2013, also Zeng et al, 2015)
Higher TLR6 expression= higher risk disease
0 100 200 300 400 500 6000
10
20
30
40
50
60
70
80
90
100
110
Days P
erc
en
t s
urv
iva
l
NHD+;PAM3
NHD+;PAM2
NHD+;water
NHD-;PAM3
NHD-;water
NHD-;PAM2
Treatment of NHD13 mice with TLR2/6 (PAM2) agonist (but not TLR1/2 agonist, PAM3) leads to earlier death
0 100 200 300 400 500 6000
10
20
30
40
50
60
70
80
90
100
110
Days
Pe
rce
nt s
urv
iva
l
NHD+;PAM3
NHD+;PAM2
NHD+;water
NHD-;PAM3
NHD-;water
NHD-;PAM2P=.019
What are the differences in the effects of TLR1/2 vs TLR2/6 stimulation on HSPCs?
WT or NHD13+
H20PAM3CSK4 (TLR1/2 agonist) 25 mcgPAM2CSK4 (TLR6/2 agonist) 1 mcg
treat x 2 wks (6 doses total) flow for HSPC #s, cycling, apoptosis, serum cytokines, and sort KSL cells for RNA profiling
NHD+;
wat
er
NHD+;
PAM
2
NHD+;
PAM
3
WT;w
ater
WT;P
AM
2
WT;P
AM
3
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
KS
L f
req
ue
nc
y
****
*
**
****
****
water PAM2 PAM3
WT
NHD13
Sca-1
c-K
it
Enriched in NHD13 PAM2 vs water*Excessive TLR2/6 signaling enhances leukemogenesis (via activation of MEK/ERK/Myc?).
Does enhanced TLR signaling cooperate with MDS-associated oncogenes in MDS pathogenesis?
MDS miceNUP98-HOXD13
Enhance TLR2 signaling:treatment with TLR2 agonist
Reduce TLR signaling:cross with TLR2 or MyD88
deficient mice
• faster transformation to leukemia• decreased cell death• accumulation of HSPCs
• faster death with TLR2/6, but not TLR1/2 stimulation
• activated Myc signature with TLR2/6
Myeloid differentiation
Mobilization
Repopulating activity
Cell death
Myc activation (TLR2/6)
Schuettpelz LabDarlene Monlish, PhDSima Bhatt, MDLuke Keller
Zev GreenbergMolly RomineThanh-Nga LeSabrina Zippel
Link LabDaniel Link, MD
Walter LabMatt Walter, MDEric Duncavage, MD, PhD
Acknowledgements
FundingChildren’s Discovery InstituteR01 HL134896-01SPORE in Leukemia, CEP, Wash UAmerican Cancer Society-IRG, Wash U