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St. Jude Graduate School of Biomedical SciencesA Role for ABC Transporters and Porphyrin Metabolism Leukemia ?

John D. Schuetz

St. Jude Childern’s Research HospitalMemphis, TN 38105

September 2019Solvo Biotechnology Transporter Conference

Cambridge, MA

Outline

• Intro-ABC transporters

• ABCG2 in acute myeloid leukemia (AML)

• Heme/porphyrin synthesis

• PPIX mechanism of death

• Identification of dual function ABCG2 inhibitors

ABC transporters have two primary functional domains

Fukuda & Schuetz Bioch Pharm 2012

48 Human ABC Genes

1 2 3 4 5 6 7 8 9 10 11 12 13

14 15 16 17 18 19 20 21 22 X Y

A

B

C

D

E

F

G

Acute myeloid leukemia (AML) is a disease of altered hematopoiesis in myeloid lineage

LT-HSC

ST-HSC

MPP

CD34+

CD34+

CD34+

Myeloid Lymphoid

CD34-

CMP CLP

B cell T cell NK cell

Lin-Sca1+cKit+

MEP(CD34-) GMP (CD34+)

Erythrocyte

Megakaryocyte

Monocyte

Neutrophil

Basophil

Eosinophil

HSC

Multipotent

progenitor

Committed

progenitor

ABCG2 is highly expressed among AML patients with poor disease-free and overall survival

DFS

OS

Blood 2006

ABCG2 alone is a poor prognostic factor in AML

MYCN is a transcription factor highly expressed in pediatric

AML

Kawagoe et al., Cancer Res. 2007

No

rmal

BM MYCN

MYC

Nex

pre

ssio

n r

elat

ive

to H

PR

T

MYCN is a poor prognostic factor in AML

www.oncomine.org

Does MYCN produce metabolic effects?

GSEA reveals upregulated hememetabolism in pediatric AML with high

MYCN expression

High MYCN Low MYCN

UROD was the most elevated heme pathway gene in High MYCN adult and pediatric AML

Fukuda et al JCI-Insight, 2017

UROD

ABCB6

Succinyl-CoA +glycine

ALA

ALA

PPIXHeme

MYCN

Porphyrin biosynthesis is a poor prognostic factor in adult AML

UROD

www.oncomine.org

UROD

High MYCN and UROD expression increases unfavorable outcome in adult AML

Oncomine.org

Favorable: complete remission

Unfavorable: relapse and refractory disease Fukuda et al JCI-Insight, 2017

AML patients have increased heme synthesis

1967 Cancer TR Walters et al pg 1117

What is the purpose of increased heme biosynthesis ?

In Leukemic Progenitors MYCN upregulates heme/porphyrin synthesis, UROD and ABCG2 expression

Fukuda et al JCI-Insight, 2017

MYCNtransduction HPC

Heme

synthesis

inhibitors

Heme synthesis is required for maximal mitochondrial oxygen consumption (OCR) in MYCN-Leukemic Progenitors

Fukuda et al JCI-Insight, 2017

N-MPP

Hypothesized that upregulated heme biosynthetic pathway would be a metabolic vulnerability in

MYCN driven AML

ALA

PPIXHeme

ABCG2

ALA

MYCN

Heme

biosynthesis

Leukemogenesis

RATE-LIMITING

A mouse model shows that loss of Abcg2increases MYCN driven myeloid leukemia

survival

0 50 1000

50

100

WT

Abcg2KO

Days

Pe

rce

nt su

rviv

al

*

Fukuda et al JCI-Insight, 2017

PPIX

WT or Abcg2KO

donor mouse

HPCs

MYCN transduction

(Leukemic progenitors)

Transplant into lethally

irradiated congenic

recipient mice

UROD

ABCB6

Succinyl-CoAglycine

ALA

ALA

PPIXHeme

ABCG2

PPIX

N-MYC

ABCG2

PPIX

O2.

P53

Does PPIX activate P53?

MYCN

>Median expression

genes

Higher MYCN correlates with p53 activation in SJ AML patients

High MYCNLow MYCN

P53 is activated in MYCN leukemic progenitors

P53 coreN-M

YC

Vec

tor

MYCNtransduction HPC

P53 deletion mostly rescues self-renewal of porphyrin treated Abcg2KO MYCN cells

p53 null mice- Martine Roussel and Frederique Zindy

UROD

ABCB6

Succinyl-CoAglycine

ALA

ALA

PPIXHeme

ABCG2

PPIX

MYCN

ABCG2

PPIX

O2.

P53

UROD

ABCB6

Succinyl-CoAglycine

ALA

ALA

PPIXHeme

ABCG2

PPIX

MYCN

ABCG2

PPIX

Do additional PPIX-protein interactionsproduce cytotoxicity?

Targeting the heme biosynthetic pathway in leukemic progenitors

Leukemic cell death in in vivo?

HemePPSuccinyl-CoA ALA

Precursors (porphyrins)Toxic

HemeCrucial

Loss of Abcg2P53 activationOther targets?

PP

Does increasing PPIX cure AML?

ALA-treatment of MYCN Leukemic Progenitors cures ABCG2 KO AML

Summary

ALA

PPIXHeme

ABCG2

ALA

MYCN

Heme

biosynthesis

Leukemogenesis

ALA

PPIXHeme

ABCG2

ALA

MYCN

Heme

biosynthesis

Leukemogenesis

ALA

PPIXHeme

ABCG2

MYCN

Heme

biosynthesis

Leukemogenesis

ALA ALA

PPIXHeme

ABCG2

ALA

MYCN

Heme

biosynthesis

Leukemogenesis

ABCG2 substrates are used in conventional AML therapy

Rubnitiz et al. 2010 Lancet Oncol

Induction

Daunorubicin

etoposide

cytarabine

Consolidation

C1: cytarabine & cladribine

cytarabine & etoposide

cytarabine & mitoxantrone

C2: cytarabine & L-asparaginase

C3: cytarabine & mitoxantrone

ABCG2 role?

AML02 treatment scheme

Screening for ABCG2 and Cancer liability inhibitors

ABCG2 inhibitors with cytotoxic properties

High throughput screening to identify ABCG2 inhibitors

Screen 11297 (5000 unique, 10 mM) FDA-approved bioact ive

compounds for inhibition of pheophorbide a ef flux from WT MEFs.

Use lapatinib as a positive control.

PhA accumulation >80% of lapatinib

Potential inhibitors- further validation.

n=401 (274 unique)Not inhibitors

Dose response of 274 compounds for PhA efflux

inhibition (46.7 mM - 2.37 nM).

Curve-fitting to calculate IC50

and assign priorities.

Validate hit compounds for ABCG2

inhibition and test whether they

also act as substrates.

High priority:

A Active

Low IC50

Chemotherapeutic drugs

YES NO

Z-p

rim

e

-0.5

0.0

0.5

1.0

0 10 20 30 40

Plates in order tested

0.63 ± 0.1 (0.4 to 0.81)

Figure 1

Ra

w a

ctivity

0

50

100

0 5,000

150

10,000 15,000

Compounds in order tested

positive control

test compounds (above or below cut-off)

negative control-50

Lapatinib

10 mM

20 mM 1:2

serial dilutions

High throughput screening for ABCG2 inhibitors identified novel inhibitors

Screen 11297 (5000 unique, 10 mM) FDA-approved bioact ive

compounds for inhibition of pheophorbide a ef flux from WT MEFs.

Use lapatinib as a positive control.

PhA accumulation >80% of lapatinib

Potential inhibitors- further validation.

n=401 (274 unique)Not inhibitors

Dose response of 274 compounds for PhA efflux

inhibition (46.7 mM - 2.37 nM).

Curve-fitting to calculate IC50

and assign priorities.

Validate hit compounds for ABCG2

inhibition and test whether they

also act as substrates.

High priority:

A Active

Low IC50

Chemotherapeutic drugs

YES NO

Z-p

rim

e

-0.5

0.0

0.5

1.0

0 10 20 30 40

Plates in order tested

0.63 ± 0.1 (0.4 to 0.81)

Figure 1

Ra

w a

ctivity

0

50

100

0 5,000

150

10,000 15,000

Compounds in order tested

positive control

test compounds (above or below cut-off)

negative control-50

Lapatinib

10 mM

20 mM 1:2

serial dilutions

SJ000831433

Unpublished 2019

Wijaya, J. et al., Int. J. Mol. Sci., 2017; 18 (12):pii: E2544

Inhibition of ABCG2 at the blood-brain barrier (BBB)

drugs

Wijaya, J. et al., Int. J. Mol. Sci., 2017; 18 (12):pii: E2544

ABCG2 inhibition may allow the drugs accumulation in the tumor

“Teamwork to make the Dream work”

J Schuetz Lab-2018

MartineRousselTumor Cell Biol

JiyangYuComp. Bio

Funding• P30 Cancer Center Support Grant • ALSAC• NIH R01s

TaoshengChenChem. Bio.

JunminPengStruc.Biol

St Jude CollaboratorsYuFukuda

APNarenCCHMC

JinZhangUCSD