METABOLIC VULNERABILITIES OF...

41
METABOLIC VULNERABILITIES OF CANCER Eyal Gottlieb

Transcript of METABOLIC VULNERABILITIES OF...

Page 1: METABOLIC VULNERABILITIES OF CANCERbioinformatics.cesb.uky.edu/pub/RCSIRM/WorkshopSymposium2017/... · The biosynthetic pathway of succinic-GSH. Electrophilic attack. Glutathione

METABOLIC VULNERABILITIES OF CANCEREyal Gottlieb

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METABOLIC VULNERABILITIES OF CANCEREyal Gottlieb

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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

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Cancer and metabolism: historic perspective

IDH mutationsare shown to be oncogenic

2008

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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

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HIFα

VHL

Succinate

Fumarate

SDH

Phaeochromocytoma

Paraganglioma

RCC

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VHL

HifαPHD

OH

O2

SDH

1) HIF regulation by oxygen

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VHL

HifαPHD

OH

O2

SDH

2) HIF regulation by VHL

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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

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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

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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 )

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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

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The biosynthetic pathway of succinic-GSH

Electrophilicattack

Glutathione (γGlu-Cys-Gly)

Fumarate

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Fumarate attack on GSH induces oxidative stress

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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

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Cysts-bearing, FH-deficient mice excrete metabolites (biomarkers) in the urine

Urine metabolic profile

2-succinic cysteine

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A new in vitro model for SDH-deficient cancers

SV40 T-Ag

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SDH deficient cells depend on Pyruvate Carboxylase for sustaining aspartate levels

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Aspartate limitation and PC expression in SDH-deficient tumors

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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

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Metabolic vulnerabilities of SDH-deficient tumours

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• 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

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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

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TKIs do not target CML stem cells

CD34Deat

h

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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

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Metabolomics in CML CD34+ cells versus CML CD34- cells

MNC

CML Patient at diagnosis

CD34+

CD34 -

Stem+ Progenitorscells

Differentiated cells

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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

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Fatty acid oxidation is increased in CML CD34+ cells

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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

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CML Patientat diagnosis

Healthy donor

CD34+

CD34+

increased oxidative metabolism in CML CD34+ Vs. normal CD34+ cells

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increased oxidative metabolism in CML CD34+ Vs. normal CD34+ cells

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increased oxidative metabolism in CML stem cells

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PHARMACOLOGICAL INHIBITION OF MITOCHONDRIAL METABOLISM WITHTIGECYCLINE

• FDA approved antibiotic

• Inhibitor of bacterial protein synthesis

• Inhibitor of mitochondrial protein synthesis

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Inhibitor of mitochondrial oxidative metabolism decreases proliferation of CD34+ CML cells

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Inhibition of mitochondrial oxidative metabolism targets CML stem cells

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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

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Inhibition of mitochondrial oxidative metabolism in-vivo is tolerated in treated mice

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Inhibition of mitochondrial oxidative metabolism eradicates CML stem cells in-vivo

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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

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Gottlieb LabCurrent membersElaine MacKenzieSimone CardaciHenry DäbritzJiska van der ReestElodie KuntzJohan Vande Voorde

Past membersMary SelakChristian FrezzaLeon Zheng

MetabolomicsGillian McKayNiels van den Broek

Acknowledgements

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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