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METABOLIC VULNERABILITIES OF CANCEREyal Gottlieb
METABOLIC VULNERABILITIES OF CANCEREyal Gottlieb
glucose glucose-6-phosphate
pyruvate
pyruvate
acetyl-CoA
acetyl-CoA
citrateoxaloacetate
malate
fumarate
succinate
α-ketoglutarate
Glutamate
Aspartate
Alanine
SDH(complex II)
H+
H+
H+H+
NADH
ProlineArginine
Ribose-5-phosphate
NUCLEOTIDES
citrate
FATTY ACIDS
FH
NADPH
ADP + Pi
NADH
NAD+
ATP
NAD+
ATP synthase
FAD
FADH2 O2
H2O
SerineCysteineGlycine
AspartateAsparagine
Glutamate
Glutamine
LIPIDS
Cancer and metabolism: the anabolic angle
Cancer and metabolism: historic perspective
IDH mutationsare shown to be oncogenic
2008
glucose glucose-6-phosphate
pyruvate
pyruvate
acetyl-CoA
citrateoxaloacetate
malate
fumarate
succinate
α-ketoglutarate
SDH(complex II)
H+
H+
H+
H+
NADH
FH
lactate
ADP + Pi
NADH
NAD+
ATP
NAD+
FAD
FADH2
O2
H2O
isocitrate
HLRCC
HPGL
IDH
Glioma/AML
Cancer and metabolism: the bioenergetic angle
HIFα
VHL
Succinate
Fumarate
SDH
Phaeochromocytoma
Paraganglioma
RCC
VHL
HifαPHD
OH
O2
SDH
1) HIF regulation by oxygen
VHL
HifαPHD
OH
O2
SDH
2) HIF regulation by VHL
VHL
HifαPHD
OH
O2
SDH
succinate
α-ketoglutarate
3) HIF regulation by succinate
Germline LoF mutations in SDH, VHL or PHD or GoF mutations in HIF2α can lead to paraganglioma!
Hypoxia is a positive phenotypic modifier of paraganglioma
Dioxygenase
O2
HIF
succinate fumarateO
O
OO
O
O
O
O
SDH FH
IDH1*
O O
OOH
O
2-hydroxyglutarate
Epigenome
α-ketoglutarate
The birth of a new concept: Oncometabolites
Fumarate hydratase deficient (Fh1-/-) cells
Fh1fl/fl
Fh1fl/fl Fh1-/-
ahCRE
Fh1-/- CL1
Fh1-/- CL19
citrat
emala
te
fumara
te
succ
inate
-4
-2
0
2
4
met
abol
itein
tens
ity(lo
g10
,nor
mal
ized
toFh
1fl/fl )
Metabolic adaptation and vulnerabilities of FH-deficient cancers
Zheng et al, Cancer Metab, 1: 12, 2013
Frezza et al, Nature, 477: 225, 2011
Zheng et al, Nature Commun, 6: 6001, 2015
The biosynthetic pathway of succinic-GSH
Electrophilicattack
Glutathione (γGlu-Cys-Gly)
Fumarate
Fumarate attack on GSH induces oxidative stress
Metabolic adaptation and vulnerabilities of FH-deficient cancers
Zheng et al, Cancer Metab, 1: 12, 2013
Frezza et al, Nature, 477: 225, 2011
Zheng et al, Nature Commun, 6: 6001, 2015
Cysts-bearing, FH-deficient mice excrete metabolites (biomarkers) in the urine
Urine metabolic profile
2-succinic cysteine
A new in vitro model for SDH-deficient cancers
SV40 T-Ag
SDH deficient cells depend on Pyruvate Carboxylase for sustaining aspartate levels
Aspartate limitation and PC expression in SDH-deficient tumors
S u c c in a te
0
2 .0×1 0 1 2
4 .0×1 0 1 2
6 .0×1 0 1 2
8 .0×1 0 1 2
1 .0×1 0 1 3
TP
A/t
ota
l c
ell
ula
r v
olu
me
on
we
ll
S D H B fl/ fl
S D H B ∆ /∆
S D H B fl/ fl, H R A S G 1 2 V
S D H B ∆ /∆ , H R A S G 1 2 V
c o lo n y 1
c o lo n y 2
c o lo n y 9
A s p a r ta te
0
1 .0×1 0 1 2
2 .0×1 0 1 2
3 .0×1 0 1 2
4 .0×1 0 1 2
TP
A/t
ota
l c
ell
ula
r v
olu
me
on
we
ll
S D H B fl/ fl
S D H B ∆ /∆
S D H B fl/ fl, H R A S G 1 2 V
S D H B ∆ /∆ , H R A S G 1 2 V
c o lo n y 1
c o lo n y 2
c o lo n y 9
H-RasG12V transform SDH-deficient cells
H-RasG12V H-RasG12V
Metabolic vulnerabilities of SDH-deficient tumours
• Metabolomics is a robust and efficient tool for mechanistic studies of metabolic adaptabilities and vulnerabilities, but also as an important sensor of biomarkers for early prognosis and detection of recurrence disease
• FH or SDH loss of function leads to the accumulation of fumarate or succinate, respectively, and to the inhibition of dioxygenases, including DNA demethylases
• SDH loss of function renders cells more dependent on PC to sustain aspartate and nucleotide biosynthesis
• SDH loss in the kidney leads to hyperproliferative benign cyst formation in-vivo (in GEMMs)
• HRas activation in the kidney does not demonstrate any phenotype, but it dramatically accelerate cyst formation and size in SDHB ablated genotype
Conclusions
Normal haemopoiesis
HSC
GMP
Chronic myeloid leukaemia
LSCDifferentiation
MPP
CLP CMP
MEP
Granulocytes
Macrophages
Platelets
Erythrocytes
T cells
B cells
Self-renewal
CD34
CD38
Survival andProliferation
BCR ABL Imatinib
CML is a stem cells disease
TKIs do not target CML stem cells
CD34Deat
h
TKIs do not target CML stem cells
(LTC-IC)
Untreated Imatinib0
50
100
150
num
ber c
olon
ies
(% o
f unt
reat
ed)
Untreated Imatinib
Progenitors cellsShort-term colony formation assay
CML Stem cellsLong- term colony formation assay
0
50
100
150
num
ber c
olon
ies
(% o
f unt
reat
ed)
Feeder cells 5 weeks in culture
Remaining cells plated into
colony assay
Colonies count after 2weeks
CML CD34+ cells
Single drug treatment
Measures drug effect on stem cells
Metabolomics in CML CD34+ cells versus CML CD34- cells
MNC
CML Patient at diagnosis
CD34+
CD34 -
Stem+ Progenitorscells
Differentiated cells
Fatty acid oxidation is increased in CML CD34+ cells
Acetyl-CoA
Citrate
Isocitrate
α-KetoglutarateSuccynil-CoA
Fumarate
Malate
Oxaloacetate
Glutamate Glutamine
CO2
CO2
Succinate
CitricAcidCycle
13C16 Palmitate13C (m+1)12C (m)
Aspartate
Fatty acid oxidation is increased in CML CD34+ cells
CML CD34+ cells have an increase in oxidative metabolism
PCAcetyl-CoA
Citrate
Isocitrate
α-KetoglutarateSuccynil-CoA
Fumarate
Malate
Oxaloacetate
Pyruvate
Glutamate
CO2
CO2
CO2
Succinate
CitricAcidCycle
13C6 Glucose
PDH
Aspartate
CML Patientat diagnosis
Healthy donor
CD34+
CD34+
increased oxidative metabolism in CML CD34+ Vs. normal CD34+ cells
increased oxidative metabolism in CML CD34+ Vs. normal CD34+ cells
increased oxidative metabolism in CML stem cells
PHARMACOLOGICAL INHIBITION OF MITOCHONDRIAL METABOLISM WITHTIGECYCLINE
• FDA approved antibiotic
• Inhibitor of bacterial protein synthesis
• Inhibitor of mitochondrial protein synthesis
Inhibitor of mitochondrial oxidative metabolism decreases proliferation of CD34+ CML cells
Inhibition of mitochondrial oxidative metabolism targets CML stem cells
Sub-lethalirradiation
Tail vein injectionCML CD34+ cells
6 weeks engraftment
CD45 Human
Confirmation of engraftment
Vehicle(n=6)
TIG(n=6)
IM(n=5)
IM+TIG(n=6)
In vivo drug treatment for 4 weeks
In vivo model to study drug effect on CML Stem Cells
Imatinib
Tigecycline
NSG MICENOD scid gamma
N=24
Inhibition of mitochondrial oxidative metabolism in-vivo is tolerated in treated mice
Inhibition of mitochondrial oxidative metabolism eradicates CML stem cells in-vivo
Inhibition of mitochondrial oxidative metabolism delays disease relapse after Imatinib withdrawal
Vehicle TIG IM TIG+IM0
20
40
60
Num
ber
of h
uman
CD
34+ c
ells
(x10
3 )
Vehicle TIG IM TIG+IM0
20
40
60
Num
ber
of h
uman
CD
34+ c
ells
(x10
3 )
Experiment 2Experiment 1
After 3 weeks of treatment
After 2-3 weeks of treatment discontinuation
2 or 3 weeks of treatment discontinuation
Gottlieb LabCurrent membersElaine MacKenzieSimone CardaciHenry DäbritzJiska van der ReestElodie KuntzJohan Vande Voorde
Past membersMary SelakChristian FrezzaLeon Zheng
MetabolomicsGillian McKayNiels van den Broek
Acknowledgements
Gottlieb LabCurrent membersElaine MacKenzieSimone CardaciHenry DäbritzJiska van der ReestElodie KuntzJohan Vande Voorde
MetabolomicsGillian McKayDavid Sumpton
Acknowledgements
WOLFSON WOHL CANCER RESEARCH CENTREVignir Helgason
PAUL O’GORMAN LEUKAEMIA RESEARCH CENTRETessa Holyoake