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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