team 3.3

Isocitrate dehydrogenase and Metabolism

in Acute Myeloid Leukemia

Jean-Emmanuel Sarry

Team RESIST@ML – Drug Resistance & Oncometabolism in Acute Myeloid Leukemia

Cancer Research Center of ToulouseUMR1037 INSERM – Université Toulouse 3

Oncopole de Toulouse

Talk KIDF– 14 Janvier 2016 – Paris

Vanden Heiden et al. Science. 2009.

Beyond Warburg: Metabolism is a new Hallmark of AML?

"Warburg effect”

-Etomoxir, avocatin B, CPT1i, CT2 inhibitors: anti-AML activity or increase theapoptotic efficacy of agent arsenic trioxide.Samudio et al. 2010. Estan et al. 2014. Lee et al 2015. Riccardi et al. 2015. Wu et al. 2015. Sriskanthadevan et al 2015.

-Glutamine removal, SLC1A5 transporter knockdown or L-asparaginase:antileukemic activity in AML by altering GSH, inhibiting mTORC1, triggering astrong autophagic response and inducing apoptosis.

Willems et al. 2013; Goto et al. 2014; Jacque et al. 2015.

-2-Deoxy-Glucose: anti-AML activity through the inhibition of both glycolysis and N-linked glycosylation in AML with FLT3-ITD or KIT mutations.

Larrue et al. Mol. Cancer Thr. 2015.

-Dendrogenin A: anti-AML activity through the LXR-dependent modulation of the sterol metabolism.

Segala et al. Submitted. In collaboration with Team 12 (Poirot, INSERM, CRCT).

-Metformin: anti-AML activity through the induction of the Pasteur effect.Scotland et al. Leukemia. 2013.

Metabolism of Acute Myeloid Leukemia cells: new targets?

Effect of Metformin (anti-diabetic drug) on AML cell metabolism

Quantitative metabolomics by LC-MS/MS:getting absolute concentrations

METAToul (INSA, Toulouse)Jean-Charles Portais

Lara GALESLindsay PEYRIGARémy PEYRAUD

Scotland et al. Leukemia. 2013.

LEMM (CEA, Saclay)Christophe JunotFlorence Castelli

Frédéric Theodoro

Metformin induces a metabolic reprogramming that leads to Pasteur effect

Acetyl-CoA

Isocit

Succinyl-CoASuccinate

Fumarate

Mal

Pyruvate

CitrateOAA

Glucose Consumption

Glutamate

GlutamineConsumption

Isocit

Citrate

a-KG

Acetyl-CoA

Malonyl-CoA

Fatty Acids

Glutamate

Acyl-CoA

Sterols

Lactate

a-KG

ACC

Acyl-CoAAcyl-Carnitines

CarnitineCPT1

PDH

SphingolipidsPhospholipids

2H2O ATPO2

VIII

IIIIV

Scotland et al. Leukemia. 2013.

METFORMIN

ATP TriglyceridesCholesterol

esters

Lipid Droplet

Proliferation arrestApoptosis

Vanden Heiden et al. Science. 2009.

Beyond Warburg: Metabolism is a new Hallmark of AML?

Mardis et al. NEJM. 2009.

"Warburg effect”

Van Heiden et al. Science. 2009.

-IDH1/2 interconvert isocitrate and α−KG;

-One of the four NADPH producing enzymes (G6PDH, 6PGDH, ME).

-Generation of the NADPH required to sustain many anabolic pathways (fatty acid elongation, lipid synthesis and cholesterol synthesis) and for the regeneration of reduced glutathione and thioredoxin as cellular defense against oxidative damage.

Citrate

Isocitrate

Glutamine

α-KG

IDH-WT Cells

Succinate

Idh1/2NADPH

NADP+

Key functions of IDH-WT for anabolic metabolism & redox homeostasis

Pyruvate Acetyl-CoA

Isocit

Idh2 Idh1

Succinyl-CoASuccinate

Fumarate

Mal

1,3-DiPG

Pyruvate

GA3P

F6P

G6P

Ribu5P

6-PGG6pd

6-PGL

CitrateOAA

Glucose

Glutamate

Glutamine

Isocit

Citrate

α-KG

Acetyl-CoA

Malonyl-CoA

Fatty Acids

Glutamate

Mal

HMG-CoA SterolsIsoprenes

Lactate

OAA

α-KG

PEP

Idh1IDH2G6pdh6PgdhMe1

NADPHNADP

GSHGSSGGsr

Gpx1H2OH2O2

Me1

Pgd

Fh

Sdh

NADPH-producing enzymes

SphingoLipids

PhosphoLipidsGly-3PDHAP

3-PG Serine

Nucleotides

IDH1 is a crucial enzyme for NADPH production, glutathione regeneration and lipid/sterols synthesis while mitochondria altered

2H2O ATPO2

VIII

IIIIV

HypoxiaAA

METFORMIN

Reductive Glutamine

Metabolism

Metallo et al. 2011Wise et al. 2011

Mullen et al 2012Fendt et al 2013

IDH1-WT overexpressed in AML

✔ ICDH activity 2-fold higher in AML compared to normal cells while G6PDH, 6PGDH, LDH, GAPDH decreased in AML blasts

Belfiore et al. 1975; Rabinowitz 1966; Ghiotto et al. 1963.

✔ IDH1 is over-expressed in AML LSC compared to normal HSC

Gentles et al. 2010; Saito et al. 2010.

HSC

LSC

HSC

LSC

HSC

LSC

HSC

LSC

HSC

LSC

HSC

LSC

HSC

LSC

10

100

1000

10000

N AML N AML N AML N AML N AML N AML N AML

IDH1 IDH2 G6PD PGD ME1 ME2 GAPDHre

lati

ve m

RN

A e

xpre

ssio

n

MNCs AML cell lines

IDH1

Actin

NH

V01

NH

V02

NH

V03

MV4

-11

MO

LM14

HL-

60KG

1aKG

1U

937

Citrate

Isocitrate

Glutamine

α-KG

IDH-WT Cells

Succinate

Idh1/2NADPH

NADP+

Citrate

Isocitrate

Glutamine

α-KG

NADPH

Succinate

NADP+

Idh1/2

Idh1/2mut

IDHmut Cells

15% in AML with mono-allelic point mutation R132/R172

one wt allele retains in tumors

2-HG: Oncometabolite

Dang et al. Nature. 2009Ward et al. Cancer cell. 2010Gross et al. J.Exp.Med. 2010

As a biomarker for Diagnosis & Minimal Residual Disease follow-up

IDH mutations lead to the production of an oncometabolite!

Citrate

Isocitrate

Glutamine

α-KG

NADPH

Succinate

NADP+

Idh1/2

Idh1/2mut2-HG

IDHmut Cells

Patients UPenn (M. Carroll)

Patients IUCT (C. Récher)

Ion-exchange chomatography / Q-trap

LEMM (CEA, Saclay)Christophe JunotFlorence Castelli

Frédéric TheodoroWT R132 WT R132

How can we use IDH mutation-induced metabolic flexibility to uncover an alternative way to specifically target these AML?

Metabolic DependencyRedox Changes

Compound X

IncreasedChemosensitivity

Citrate

Isocitrate

Glutamine

α-KG

NADPH

Succinate

NADP+

Idh1/2

Idh1/2mut 2-HG

IDHmut Cells

How can we use IDH mutation-induced metabolic flexibility to uncover an alternative way to specifically target these AML?

Citrate

Isocitrate

Glutamine

α-KG

NADPH

Succinate

NADP+

Idh1/2

Idh1/2mut2-HG

IDHmut Cells

Actin

total IDH1

IDH1 R132H

R13

2H

WT

CTL

cloneCTL WT R132H

11 14 15 2 4 7 3 5 11

HL60 MOLM14

HL60 MOLM14

2-HG

α-KG

WT R132H WT R132H

WT R132H WT R132H

LEMM (CEA, Saclay)Christophe JunotFlorence Castelli

Frédéric Theodoro

Metabolic Reprogramming of IDH1 mutant AML cells

13C-Fluxomics/Metabolic Flux Analysis (LC-MS/MS & NMR)Isotopomer Analysis + Flux Balance Analysis + Computer-based Modeling Software

Glucose

g6p

rib5p

pga pep

oaa

accoa

mal

pyr

akgsuc

rib5p

e4p s7pgap

Acetate

f6p

gap

100

98.5

180168 121

118

445123

4219

43

1.4

51

1.492

icit

4

∆zwfNo phenotype

6pg

G6PDH C precursorcompensation

NADPH compensation

LUCILLE STUANI

METAToul (INSA, Toulouse)Jean-Charles Portais

Lara GALES – Tony PalamaFlorent Belvert – Maud Heuillet

First results on intracellular metabolic fluxes in IDH1 mutant

WT R132H

WT MUT WT MUT

fumarate

WT MUT WT MUT

PEP

WT MUT WT MUT

malate

WT MUT WT MUT

citrate

cis-aconitate

WT MUT WT MUT

α-KGWT MUT WT MUT

2-HG

WT MUT WT MUT

Acetyl-CoA

Pyruvate

Suc

OAA

Mal

Fum αKG

Cis-aco

Cit

GLUTAMINE

Citrate

IDH2

IDH1

2-HG

GLUCOSE

αKGIdh1/2mut

First results on intracellular metabolic fluxes in IDH1 mutant

WT R132H

Summary - IDH mutations impact AML cell metabolism

Citrate

Isocitrate

Glutamine

α-KG

NADPH

Succinate

NADP+

Idh1/2

Idh1/2mut2-HG

IDHmut Cells -Origin of 2-HG and α-KG compartmentatingcytosolic redirection?

-Modified oxidative part of the TCA cycle increased oxidative glutamine/glucose incorporation

-Modified glycolytic and PPP capacitiescompensatory NADPH/GSH reducing source?

Oncometabolites vs. Tumor suppressor metabolites

α-KG

2-HGα-KG

2-HG

Normal Cells Tumor Cells

Sarry/Récher LabFrancois VergezLucille StuaniNizar SerhanEstelle SalandThomas FargeMarine FraisseMarie-Laure TraversHéléna BoutzenClément LarrueNesrine ArouaGabriel LemercierSonia Zaghdoudi

Past MembersFabienne de Toni Sarah Scotland Camille Fialin

Acknowledgments

Network “Metabo”M. Selak

(UPenn, Philadelphia)C. Joffre

(INSERM, Toulouse)A. Bouloumié

(INSERM, Toulouse)A. Mayral

(INSERM, Toulouse)T. Levade

(INSERM, Toulouse)M. Poirot, S. Silvente(INSERM, Toulouse)

N. Mazure(CNRS, Nice)

L. Le Cam, L. Linares(INSERM, Montpellier)

METAToul (INSA, Toulouse)Jean-Charles Portais

Lara GALESLindsay PEYRIGARémy PEYRAUDFlorent BellvertMaud Heuillet

Edern CahoreauTony Palama

Justine Bertrand MichelFabien Riols

Fabien JourdanNathalie Poupin

LEMM (CEA, Saclay)Christophe JunotFlorence Castelli

Frédéric Theodoro

Lunt and Vander Heiden, 2010