Inflammation and Cancer

Inflammation and cancer

Laura G. Schuettpelz, MD, PhD

Assistant Professor of Pediatrics Division of Pediatric Hematology/Oncology Washington University School of Medicine

Cancer Biology Pathway February 7, 2017 http://countdowntozerotime.org

Disclosure: Dr. Schuettpelz has no relevant financial interests to disclose 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 In 1863, Rudolph Virchow described the presence of leukocytes in neoplastic tissues and suggested that cancer originates at sites of chronic inflammation.

Coussens and Werb, Nature 2002 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, blood vessels and molecular mediators. The function of inflammation is to eliminate the initial cause of cell injury, 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 the increased movement of plasma and leukocytes from the blood into the injured tissues. A series of biochemical events propagates and matures the inflammatory response, involving the local vascular system, the immune system, and various cells within the injured tissue. Prolonged inflammation, known as chronic inflammation, leads to a progressive shift in the type of cells present at the site of inflammation, such as mononuclear cells, and is characterized by simultaneous destruction and healing of the 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 TLR2 TLR6 Toll Like Receptors

• Family of pattern recognition receptors; recognize pathogen-associated molecular patterns (PAMPs) and damage associated MyD88 Plasma patterns (DAMPs) IRAK4 membrane

Cytoplasm IRAK1 IRAK2

TRAF6 TLR9 IKKγ IKKα • TLR signaling leads to activation of NF- IKKβ kB, MAPKs, IRF3, IRF7 and the MKKs production of pro-inflammatory TRIF Endosome p38 ERK JNK NF-κB cytokines TRAF3 TBK1 CREB MyD88 IKKε AP1

IRAK4 • Expressed on immune cells (eg, dendritic IRF7 IRF3 AP1 NF-κB IRAK1 IRAK2 IRF3 cells, macrophages, B cells, T cells, etc) Type I Pro-inflammatory TRAF3 Interferons, cytokines and non-immune cells (endothelium, TRAF6 IFN inducible genes epithelium) IRF7 Nucleus

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)

Maratheftis, et al Clin Cancer Res 2007 Wei, et al Leukemia 2013 Hoffman, et al Blood 2002 Dimicoli, et al PLoS One 2013 Ganan-Gomez et al, Leukemia 2015 Starczynowki, et al Nature Medicine 2010 Varney, et al J Exp Med 2015 TLR2 expression/signaling is enhanced in the CD34+ cells of patients with MDS

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*Also, TLR2 ligands (DAMPs) are increased in the serum of patients with MDS: • HMGB1 (Velegraki et al; Haematologica 2013) Wei et al; Leukemia 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? 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

https://mutagenetix.utsouthwestern.edu/phenotypic/phenotypic_rec.cfm?pk=214 Role of TLR2 signaling in normal hematopoiesis TLR2 is not necessary for HSC function

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numbers, repopulating .

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Does enhanced TLR signaling cooperate with MDS-associated oncogenes in MDS pathogenesis?

Enhance TLR2 signaling: treatment with TLR2 agonists Evaluate disease progression and survival, hematopoietic stem and progenitor cycling, Reduce TLR signaling: apoptosis, differentiation MDS mice cross with TLR2 or MyD88 NUP98-HOXD13 deficient mice NUP98-HOXD13 (NHD13) mice: Tlr2-/- Wild-type - NUP98-HOXD13 fusion expressed from vav-1 promoter NHD13

- Mice develop cytopenias early in life, die by 14 mos of age from MDS, leukemia (average about 9 mos). of % max

TLR2

Lin, et al; Blood 2005 Does loss of TLR2 impact disease course or survival of NHD13 mice?

x Tlr2-/- NHD13+ NHD13; Tlr2-/- Tlr2-/- WT

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-Monthly CBC, PB flow -Follow until illness/death Loss of TLR2 is associated with earlier transformation of NHD13 mice

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Wei et al; Leukemia 2013 Zeng et al; Exp Cell Res 2016 Loss of TLR2 is associated with reduced cell death in NHD13 HSPCs

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Sallman et al; Frontiers in Oncology 2016 Basiorka et al; Blood 2016 Does enhanced TLR signaling cooperate with MDS-associated oncogenes in MDS pathogenesis?

Enhance TLR2 signaling: treatment with TLR2 agonist

Evaluate disease progression and survival, hematopoietic stem and progenitor cycling, Reduce TLR signaling: MDS mice apoptosis, differentiation cross with TLR2 or MyD88 NUP98-HOXD13 deficient mice

• decreased cell death • accumulation of HSPCs • faster transformation to leukemia Does stimulation of TLR2 impact disease course or survival of NHD13 mice?

H20 PAM3CSK4 (TLR1/2 agonist) 25 mcg PAM2CSK4 (TLR2/6 agonist) 1 mcg

WT or NHD13+

3x/wk; starting at 8wks of age -monthly CBC, PB flow -treat until illness/death

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 Treatment of NHD13 mice with TLR2/6 (PAM2) agonist (but not TLR1/2 agonist, PAM3) leads to earlier death

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20r e 30 NHD-;PAM2 10P 20 P=.019 010 00 100 200 300 400 500 600 0 100 200 300 400 500 600 Days Days What are the differences in the effects of TLR1/2 vs TLR2/6 stimulation on HSPCs?

H20 PAM3CSK4 (TLR1/2 agonist) 25 mcg PAM2CSK4 (TLR6/2 agonist) 1 mcg

WT or NHD13+ treat x 2 wks (6 doses total)  flow for HSPC #s, cycling, apoptosis, serum cytokines, and sort KSL cells for RNA profiling water PAM2 PAM3

Enriched in NHD13 PAM2 vs water *Excessive TLR2/6 signaling enhances leukemogenesis (via activation of MEK/ERK/Myc?).

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K 0.05 0.00 r 2 3 r 2 3 te M M te M M a A A a w A A ; ;P ;P ;w ;P ;P + + + T T T D D D W H H H W W N N N Does enhanced TLR signaling cooperate with MDS-associated oncogenes in MDS pathogenesis?

• faster death with TLR2/6, but not TLR1/2 stimulation • activated Myc signature with TLR2/6

Enhance TLR2 signaling: treatment with TLR2 agonist

Reduce TLR signaling: cross with TLR2 or MyD88 MDS mice deficient mice NUP98-HOXD13

• faster transformation to leukemia • decreased cell death • accumulation of HSPCs Myeloid differentiation Mobilization Repopulating activity

Cell death Myc activation (TLR2/6) Acknowledgements

Schuettpelz Lab Darlene Monlish, PhD Sima Bhatt, MD Luke Keller

Zev Greenberg Molly Romine Thanh-Nga Le Sabrina Zippel

Link Lab Daniel Link, MD

Walter Lab Funding Matt Walter, MD Children’s Discovery Institute Eric Duncavage, MD, PhD R01 HL134896-01 SPORE in Leukemia, CEP, Wash U American Cancer Society-IRG, Wash U