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