Inflammation and cancer - Cancer Treatment & Research · Inflammation (from Latin inflammatio) is part of the complex biological response to harmful stimuli, such as pathogens, damaged

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


Types of Inflammation in Cancer


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



Enhance TLR2 signaling:treatment with TLR2 agonist


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?).



Enhance TLR2 signaling:treatment with TLR2 agonist


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

Documents

Inflammation and cancer - Cancer Treatment & Research · Inflammation (from Latin inflammatio) is part of the complex biological response to harmful stimuli, such as pathogens, damaged