Innovative therapy, monoclonal antibodies and beyond

Milan, 22 January 2016

“LXR/LXR ligands and immune mediated control of

tumor growth”

Vincenzo Russo, MD

San Raffaele Scientific Institute

Liver X Receptor(LXR) ligand/Oxysterol

generation

Bjorkhem JCI, 2002;

Pannu et al. Mol and Cell Endocrinol. 2013

LXR ligands/oxysterols are products of cholesterol oxidation

SO3

Inhibition of

COX2, IL-1b, TNFa,

MMP9, MCP-1,

IL-6 transcription

LXR SO3

SULT2B1b

RXR

SO3

Activation of

ABCG1, ABCA1,

SREBP-1c, FASN,

transcription

Active and inactive oxysterols

Cholesterol

Ester (CE)

Oxysterols

Oxidized fatty acids

RXR LXR

ABCG1 gene

ABCG1/ABCA1 Reverse Cholesterol

Transport

LXR/LXR ligand signaling

Functions

• Regulation of cholesterol and fatty acid metabolisms

(Janowsky et al. Nature 1994)

• Modulation of immune responses

(Joseph et al. Nat Med 2003)

2,3;22,23-Diepoxysqualene

Acetyl-CoA

HMG-CoA

HMG-CoA Reductase

Mevalonic Acid

Squalene

2,3-Monoepoxysqualene

Lanosterol

Cholesterol 24(S),25-Epoxylanosterol

24(S),25-Epoxylcholesterol

LXRa

Inactivates oxysterols

by sulfurylation

SULT2B1b

Zaragozic

Acid (ZA)

Bensinger et al. Cell 2008

Cui et al. JCI 2011

iT-cell proliferation

iTh17 polarization

LXR/oxysterols and immune system

Mertk-dependent phagocytosis of

apoptotic cells

iInflammation by transrepressing

COX-2, IL-6, IL-1b, etc.

A-Gonzalez et al. Immunity 2009

Joseph et al. Nat Med 2003

i T cells Macrophages Dendritic cells

Villablanca et al. Nat Med 2010

Geyeregger et al. Blood 2009

iCCR7 chemokine receptor

iAntigen presenting functions

Adapted from York & Bensinger, JEM 2013

LXR/LXR ligands (oxysterols) and tumor

microenvironment

1. Villablanca et al. Nat Med. 2010

2. Russo V. J Leukoc Biol. 2011

3. Traversari C and Russo V. Curr Op Pharmacol. 2012

Pharmacological (ZA) and genetic blockade (SULT2B1b) of

oxysterols prolongs OS of tumor-bearing mice

RMA lymphoma LLC (lung)

AB1 mesothelioma

0 10 20 30 40 50 600

25

50

75

100

Days after tumor infusion

Surv

ival (%

)

WT

Mock

SULT2B1b

RMA lymphoma

0 20 40 60 80 1000

20

40

60

80

100

Days after tumor infusionS

urv

ival (%

)

Mock

SULT2B1b

4T1 breast tumor

Adapted from York & Bensinger, JEM 2013

LXR/LXR ligands (oxysterols) and tumor

microenvironment

1. Raccosta et al. J Exp Med. 2013

2. Raccosta et al. OncoImmunology 2013

3. Traversari et al. Eur J Immunol. 2014

LXR ligands behave as chemoattractants for bone

marrow-purified neutrophils in vitro

Raccosta et al. 2013

Pro-tumor neutrophils

Adapted from Mantovani et al. Nature Rev Immunol, 2011

Adapted from The Biology of Cancer (© Garland Science 2007)

RIP1-Tag2 pancreatic tumor model

Pro-angiogenic

neutrophils

Nozawa et al. PNAS, 2006

Hanahan D. Nature, 1985

Cholesterol hydroxylases generating oxysterols

Bjorkhem JCI, 2002

Cyp46a1 is up-regulated during tumor formation in

the pancreas of RIP1-Tag2 mice

Cyp46a1

Cholesterol

• LXR activation

• Neutrophil recruitment

Cyp46a1

actin

Normal Islets Hyperplastic islets Brain

(Ctrl)

Zaragozic acid (ZA) treatment prevents the

angiogenic switch in Rip1-Tag2 mice

Generation of RIP1-SULT2B1b transgenic mice

Rip1-Tag2 Rip1-SULT2B1b

Rip1-Tag2

Rip1-SULT2B1b Rip1-Tag2 Vs.

Pancreatic tumor

development in the

absence of active

oxysterols

Tumorigenesis in Rip1-Tag2 x Rip1-SULT2B1b mice

11 weeks

Intrinsic effects of SULT2B1b in RIP1-Tag2 x

RIP1-SULT2B1b double transgenic mice

24-Hydroxycholesterol is reduced in RIP1-Tag2 x

RIP1-SULT2B1b pancreata

LC-MS Analyses carried out by Angela Bachi and Umberto Restuccia

Adapted from The Biology of Cancer (© Garland Science 2007)

Analysis of tumor microenvironment conditions

regulating cholesterol hydroxylase expression

Hypoxia

Inducible HIF-1a, Vegf and Cyp46a1 expression

during RIP1-Tag2 tumorigenesis

6 weeks

10 weeks

In collaboration with Rosa Bernardi’s group

Hypoxia and Cyp46a1 expression in vivo

Pimo/Cyp46a1/Dapi

In vitro model to study the induction of Cyp46a1

in RIP1-Tag2 tumor: bTC3 tumor cells

bTC3 tumor cells: tumor cell line derived from insulinomas arising in RIP1-Tag2 mice

(Efrat et al. PNAS: 85,9037-9041, 1988)

HIF-1 silencing reduces expression levels of Cyp46a1 in hypoxic bTC3 cells

In vitro model to study the induction of Cyp46a1

in RIP1-Tag2 tumor: bTC3 tumor cells

bTC3 cells expressing a mutant stable form of HIF-1 (Guarnerio et al. Stem Cell Reports,

2014)

HIF-1 is recruited at Cyp46a1 promoter region of bTC3 cells

HIF-1a

Cyp46a1

Cyp46a1 expression by human pNETs

Graphical Model

Range of activity

of oxysterols

Range of activity

of chemokines

Gr1/Cyp46a1/Dapi

UNIT OF IMMUNO-BIOTHERAPY OF

MELANOMA AND SOLID TUMORS

Raffaella Fontana

Marta Moresco

Daniela Maggioni

Laura Raccosta

Matias Soncini

Gianfranca Corna

MOLMED

Catia Traversari

Claudio Bordignon

Simona Porcellini

Claudia Asperti

Elena Tiziano

PATHOLOGY UNIT

Claudio Doglioni

UNIT OF FUNCTIONAL

PROTEOMICS

IFOM

Angela Bachi

Umberto Restuccia DIVISION OF EXPERIMENTAL ONCOLOGY

Rosa Bernardi

Nadia Coltella

DEPARTMENT OF BIOSCIENCES AND

NUTRITION, KAROLINSKA INSTITUTE

Knut Steffensen

Jan-Ake Gustafsson

DIABETES RESEARCH

INSTITUTE

Lorenzo Piemonti

Valeria Sordi