PML-RARa interferes with Nrf2 function in Acute

Promyelocytic Leukemia Cells

Nelida I. Noguera, Gianfranco Catalano, Serena Travaglini, Cristina

Banella, Maria Teresa Voso, Francesco Lo Coco

! NRF2 is a transcription factor, involved in the cellular

response to oxidative stress.

!  In normal conditions, it is maintained at low levels via

Keap-1 binding, constitutive ubiquitylation and

proteasomal degradation.

!  Electrophiles and oxidants inhibit NRF2 degradation,

enabling its accumulation in the nucleus and initiates a

genetic program to allow cellular adaptation to stress

Keap1-Nrf2 regulation System

Sumoylation and Nuclear degradation of NRF2

JBiolChem2013Malloyetal.

Inhibition of NRF2 by nuclear receptors

Bioch Bioph Acta 2014 Namani et al.

STUDY AIMS

!  APL blasts are more sensitive to treatment with oxidants than

AML blasts

!  We hypothesized that NRF2 function is impaired in APL

METHODS

!  NRF2 expression in primary APL and AML blasts by Q-RT-PCR and WB

!  The effects of PML-RARa on NRF2 expression and its transcriptional activity, by Q-RT-PCR and CHIP analysis

!  Expression of its principal regulator Keap-1 by WB

!  The effects of PML-RARa expression on NRF2 localization, by confocal microscopy and nuclear / cytoplasmic fractionation

!  NRF2 half life in presence of PML-RARa

PML-RARa expression impairs cellular response to ROS Production

ROS

0.0

0.5

1.0

1.5Mock PR9

Zn - + - +

p=0.01

RO

S R

elat

ive

Sign

alin

g

!  PR9 is a PML/RARa Zn-inducible cell-line

!  Addition of Zn induces NRF2 expression

RESULTS

NRF2 protein level is lower in APL compared to other AML samples

NRF2

B-Actine

APL AML

NRF2

APL AML

B-Actine

APLAML

mRNA Protein

0

1

2

3 p= 0.01

NR

F2 /

B-A

ctin

e

0.0

0.5

1.0

1.5 p= 0.03

n.s.

p= 0.003

NR

F2 /

AB

L

APLAMLNBMCD34+

Keap-1 is expressed at similar levels in APL and AML patients’ samples

B-Actine

APL AML

Keap1 Keap1

APL AML

B-Actine

0.0

0.5

1.0

1.5

2.0

n.s.

APL AML

KEAP1/B-Actin

In PR9 cells, expression of PML-RARa inhibits the Zn-related increase of NRF2 protein

NRF2 Protein NRF2 mRNA

NRF2

B-Ac7n

Mock PR9

0362403624

PML PML-RARa

0 3 6 9 12 15 18 21 240

1

2

3

4

Mock

PR9

Hs

mRNA

NR

F2 /

AB

L

0 3 6 9 12 15 18 21 240

1

2

3

Mock

PR9

0.008

NR

F2 /

B-A

ctin

e

PR9 + Zn: PML-RARa induction impairs HO-1 upregulation

0 3 6 9 12 15 18 21 240

20

40

60

Mock

PR9

p=0.003

Hs

mRNAHO-1/ABL

!  HO-1 is a NRF2 target gene

Expression of NRF2 target genes in primary blasts from APL and AML samples

APLn=13AMLn=12NBMn=5CD34+=1

0

2

4

6

8

p=0.01

p= 0.02p= 0.004

AK

R1C

1 / A

BL

APLAMLNBMCD34+

APLAMLNBMCD34+0

50

100

150

200p= 0.002

p= 0.005

p= 0.002

NQ

O-1

/ A

BL

APLAMLNBMCD34+0

30

60

90

120

n.sp= 0.0007

p= 0.01

HO

1 / A

BL

PML-RARa inhibits NRF2 binding to HO-1 promoter (CHIP: PR9 + Zn)

GAPDH

0.00

0.05

0.10

0.15

0.20

NRF2 IgG NRF2 IgG Zn - + - + - + - +

PR9 Mock

Fold

vs

Inpu

t0.00

0.05

0.10

0.15

0.20HO-1

NRF2 IgG NRF2 IgG Zn - + - + - + - +

PR9 Mock

Fold

vs

Inpu

t

MERGE PML NRF2 NRF2-PML

Moc

k 8H

PR

9 +

Zn 8

H

NRF2 is delocalized to the cytoplasm in the presence of PML-RARa

PML-RARa induces NRF2 degradation in the cytoplasm, but not in the nucleus

B-Actin

NRF2

H3

Zn - + + - + +

Mock PR9 MG132 - - + - - +

Nucleus

Ctrl Zn

Zn + MG13

2Ctrl Zn

Zn + MG13

20.0

0.5

1.0

1.5 Mock

PR9

NR

F2/ H

3

Cytoplasm

Ub-NRF2

Ctrl Zn

Zn + MG13

2Ctrl Zn

Zn + MG13

20.0

0.5

1.0

1.5

2.0PR9

Mock

NR

F2 /

B-A

ctin

B-Actin

NRF2

H3

Zn - + + - + +

Mock PR9 MG132 - - + - - +

PML-RARa reduces NRF2 Half Life

0 1 2 3 4 50.0

0.3

0.6

0.9

1.2

1.5

Mock

PR9

H

NRF2/B-Actin

B-Actin

NRF2

Mock PR9

0 0 1 3 5 0 0 1 3 5 hours Zn - + + + + - + + + + CHX - - + + + - - + + +

Conclusions

"  NRF2 protein is significantly downregulated in APL compared with other AML

"  PML-RARa protein induces translocation of NRF2 to the cytoplasm, and increases its proteosomal degradation, reducing its half-life

"  In APL, impairment of Nrf2 is associated to deregulation of redox cell metabolism, and probably sensitizes cells to the cytotoxic effects of oxidants

"  PML-RARa inhibits NRF2 transcriptional activity.

Acknowledgments

TorVergata University, Rome -Francesco Lo Coco -Maria Teresa Voso -Sergio Amadori - Gianfranco Catalano - Claudia Ciardi - Laura Cicconi - Mariadomenica Divona - Emiliano Fabiani - Giulia Falconi - Licia Iaccarino - Serena Lavorgna - Tiziana Otone

Santa Lucia Foundation, Rome

- Luca Battistini - Cristina Banella - Maria Liliana Piredda - Serena Travaglini

Institute of Molecular Oncology Foundation (IFOM), Milan -Emanuela Colombo