3d culture of braf/kras-mutated erdheim-chester disease ... · giacomo de luca. lorenzo dagna. unit...
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3D culture of BRAF/KRAS-mutated Erdheim-Chester disease tissues
unveils rewired histiocyte metabolism as a new therapeutic target
Marina Ferrarini, MD
B-Cell Neoplasia UnitDivision of Experimental Oncology
IRCCS San Raffaele
7th Annual ECD Medical Symposium
Milan – July 11, 2019
Clinical, imaging and histological findings in patients with Erdheim–Chester disease
Giulio Cavalli et al. Ann Rheum Dis 2013;72:1691-1695
©2013 by BMJ Publishing Group Ltd and European League Against Rheumatism
H&E CD68 CD1a S100
Erdheim-Chester disease: an inflammatory myeloid neoplasm
Dagna et al, 2012 JCO Arnaud et al, 2011 Blood
Stoppacciaro et al, 2006 Arthritis Rheum
Papo et al, 2019 Curr Oncol Rep
CCR1 CCL2 CCR3 CCL5
CCR7 CCL19 IL-6 IFN-γ
CXCR3CXCL10 RANKL
A unifying disease model for Erdheim-Chester disease
Cavalli et al, 2014
IL-6
CCL2
Haroche et al, 2012Blombery et al, 2012Emile et al, 2014 Cangi et al, 2014
Stoppacciaro et al, 2006Dagna et al, 2010Arnaud et al, 2011Dagna et al, 2012
BRAFV600E
p-ERK
Dagna L, Corti A, Langheim S, Guglielmi B, De CobelliF, Doglioni C, Fragasso G, Sabbadini MG, Ferrarini M.
Cytokine inhibition in Erdheim-Chester disease
BRAF inhibition in Erdheim-Chester disease
• not all ECD patients carry a BRAFV600E mutation
• vemurafenib treatment mostly results in partial clinical responses in ECD patients
• vemurafenib treatment is often associated with severe side effects and recurrences upontreatment discontinuation
vemurafenib treatment in ECD: limitations
MEK inhibition in ECD: cobimetinib and trametinib
Aim: to identify• tumor vulnerabilities• pathogenic cues (oncometabolites, immunometabolites..)• cross-talk with microenvironmental components
Improve treatment of cancer patients
Approaching cancer metabolism in 3D culture
Regulation of glucose metabolism by oncoproteins
Hay, Nat Rev Cancer 2016
F Pedica.
Skin Bone Marrow
100X
Renal Cell Carcinoma
RCC
Multiple Myeloma
200X
Hepatocarcinoma
150 3 8days
Renal Cell Carcinoma
Ferrarini et al, PlosOne 2013
Belloni et al, Haematologica 2018
RCCSTM Bioreactor allows long-term culture of tissue explants
0 2 4 6 8 10
VEM
IFX
NT
0 50 100 150 200 250 3000 10 20 30 40 50
TNF-α
IL-8§
IL-6§
IL-1β
0 500 1000 1500 2000
CCL4
CCL2
0 100 200 300 400 500
sTNF-RI
sTNF-RII
*
**
*
*
***
ECD 1Xanthelasma, BRAFV600E
0 100 200 300 400
*
0 500 1000 1500 2000 2500 3000
****
***
*****
**
*
**
0 500 1000 1500 2000 2500 3000
****
0 50 100 150
**
*
pg/ml§ng/ml
**
****
**
ECD 2Xanthelasma, BRAFV600E
ECD 3Pleural lesion, BRAFV600E
pg/ml
pg/ml
ECD tissues retain cytokine production in 3D culture
VEM in vivoIFX in vivo
pErk1/2
Actin
ECD 2 skin biopsy
gluc
ose
(mg/
dl)
0
50
100
150
200
250
0
2
4
6
8
TCM Skin ECD1NT
ECD1Vem
ECD2NT
ECD2Vem
ECD3NT
ECD3Vem
****
*
lact
ate
(mM
/l)
*
Villa et al, ARD 2018
vemurafenib affects histiocyte metabolism but not viabilityN
T
CD68 CD68 p-ERK Glut-1 Casp-3
vem
(6 µ
M)
0
20
40
60
80
ECD 1 ECD 2 ECD 3 Melanoma
NT VEM
LD
H(U
/L)
% P
I+
0
20
40
60
80
100
0 0.1 1 6
LM16
LM16R
vem (µM)
Glut-1
ECD2 HD
Glut-1 Glut-1CD68
Glut-1
HepG2ECD2
100 101 102 103 1040
5
10
15
20
10
310
410
510
60
15
29
44
58
10
310
410
510
60
22
43
65
87
M0 M1
0
20
40
60
80
100
120
IL-1 IL-6 TNF
***
CC
L2
(ng/
ml)
CC
L4
(pg/
ml)
0
5
10
15
20
CCL2 CCL4
NT
Tr*
***
****
IL-1β,
TN
F-α
(pg/
ml)
IL-6
(ng/
ml)
0
2
4
6
0 2 6
NT
tr
days
TN
F-α
(pg/
ml)
0
40
80
120
0 2 6
IL-1β
(pg/
ml)
0
2000
4000
6000
0 2 6
CC
L-2
(pg/
ml)
0
100
200
300
0 2 6
NT
tr
days
CC
L-4
(pg/
ml)
ECD 4BRAFwt, KRASG12D
xanthelasma
trametinib
pErk
Erk
pErk/Erk 0.46 0 0.06
MDMECD+-
pAkt/Akt
pAkt
Akt
0.13 0 0
MDMECD+-
trametinib inhibits cytokine release by KRAS-mutated histiocytes
KRAS c.35 G>A G
A
MG Cangi
manuscript in preparation
Red Oil
NT trametinib (1 nM)
CD68
p-ERK
Glut-1
Casp-3
Glut-1
p-ERK
CD68 Glut-1
p-ERK Glut-1
****
0
4
8
12
16
TCM skin NT tr
***
0
3
6
9
NT tr0
50
100
150
200
250
TCM skin NT tr
lact
ate(
mM
/l)
pH
**
gluc
ose(
mg/
dl)
****
0
4
8
12
16
CD68 pERK Glut-1
NT
tr*
% m
arke
r de
nsity
trametinib counteracts metabolic reprogramming in histiocytes
CD
68P-
ER
K
manuscript in preparation
E. Casiraghi
Metabolomic analysis of ECD tissues cultured in bioreactor
manuscript in preparation
Glycolisis
TCA cycle
GSH homeostasis
FA metabolism
itaconate
NT tr
TC
MRCCS
NT tr
histio
• activation of the glycolysis pathway• accumulation of TCA metabolites• accumulation of citrate, suggestive of activation of the cholesterol synthesis
pathway• activation of tryptophan metabolism, most likely by activation of IDO1• high induction of itaconate• overall, the metabolic profile resembles that of activated monocytes and
macrophages• all these noted metabolic changes were counteracted by culture of tissue
samples with trametinib
Travis NemkovAngelo D'AlessandroUniversity of Colorado, Denver
Metabolomics analysis of ECD tissues
Minhas et al, 2018 Nat Immunol
Energy metabolism in ECD histiocytes and activated macrophages
Amino acids
Glycolisis
TCA cycle
GSH homeostasis
FA metabolism
itaconate
NT tr
TC
M
RCCS
NT tr
histio
G. Dell’Antonio
ECD: a disease of “foamy” histiocytes
Uber lipoidgranulomatoseChester W, 1930
Wherry & Kurachi, Nat Rev Immunol. 2015
Overview of mechanisms of T cell exhaustion
0
2
4
6
8
0
40
80
120
160
200
0
20
40
60
80
100
120
vem 6µM +2-DG 1mM + +
+gl
ucos
e(m
g/dl
)la
ctat
e(m
M/l)
LD
H(U
/L)
TCM
TCM
TCM
Muñoz-Pinedo C et al, Cell Death and Disease 2012
Targeting ECD metabolism in 3D culture: 2-DG
ECD 5Xanthelasma, BRAFV600E
0
2
4
6
8
10
0
40
80
120
160
200
0
20
40
60
80
100
120
vem 6µM +2-DG 1 mM + +
+
gluc
ose
(mg/
dl)
lact
ate
(mM
/l)L
DH
(U/L
)
Targeting ECD metabolism in 3D culture: metformin
Reduction in lipid storage increased fatty acid oxidation inhibition of fatty acid and cholesterol synthesis
Regulation of carbohydrate metabolismincreased GLUT1-dependent glucose uptakecell-type-specific increased glycolysis
Steinberg GR & Carling D, Nat Rev Drug Discovery 2019
TCM
TCM
TCM
0
2
4
6
8
10
0
20
40
60
80
100
120
0
40
80
120
160
200
vem 6µM +Met 100 µM + +
+
gluc
ose
(mg/
dl)
lact
ate
(mM
/l)L
DH
(U/L
)
ECD 5Xanthelasma, BRAFV600E
metformin
Conclusions
• Dynamic 3D culture in bioreactor is suitable for pathogenic studies and fordrug testing in ECD
• The technology allowed us to define outcomes down-stream oncogenicmutations, and specifically to identify rewired metabolism as a peculiarfeature of ECD histiocytes.
• Our model can be further exploited to design new therapeutic strategies forECD and conceivably for other forms of histiocytosis.
Rewired metabolism
Acknowledgements
Giulio CavalliAlvise BertiCorrado CampochiaroGiacomo De LucaLorenzo DagnaUnit of Medicine and Clinical ImmunologySan Raffaele Scientific Institute, Milano
Giulia CangiClaudio DoglioniPathology UnitSan Raffaele Scientific Institute, Milano
Barbara VerganiAntonello VillaConsorzio MIAUniversity of Milano-Bicocca
Travis NemkovAngelo D'AlessandroDepartment of Biochemistry and Molecular Genetics, University of Colorado, Denver
Kathy BrewerECD Global Alliance, DeRidder, LA
Daniela BelloniSilvia HeltaiElisabetta Ferrero Marina FerrariniB-cell Neoplasia UnitSan Raffaele Scientific Institute, Milano
Riccardo BiavascoEugenio MontiniSR-TigetSan Raffaele Scientific Institute, Milano
Elisabetta CasiraghiDepartment of Computer ScienceUniversity of Milano.
ECD patients and families
Acknowledgements
Giulio CavalliAlvise BertiCorrado CampochiaroGiacomo De LucaLorenzo DagnaUnit of Medicine and Clinical ImmunologySan Raffaele Scientific Institute, Milano
Giulia CangiClaudio DoglioniPathology UnitSan Raffaele Scientific Institute, Milano
Barbara VerganiAntonello VillaConsorzio MIAUniversity of Milano-Bicocca
Travis NemkovAngelo D'AlessandroDepartment of Biochemistry and Molecular Genetics, University of Colorado, Denver
Kathy BrewerECD Global Alliance, DeRidder, LA
Daniela BelloniSilvia HeltaiElisabetta Ferrero Marina FerrariniB-cell Neoplasia UnitSan Raffaele Scientific Institute, Milano
Riccardo BiavascoEugenio MontiniSR-TigetSan Raffaele Scientific Institute, Milano
Elisabetta CasiraghiDepartment of Computer ScienceUniversity of Milano.