Retinoic Acid and Arsenic Trioxide sensitize Acute Myeloid Leukemia - - PowerPoint PPT Presentation

SLIDE 1

7th international symposium on Acute Promyelocytic Leukemia September 24-27, 2017

Retinoic Acid and Arsenic Trioxide sensitize Acute Myeloid Leukemia cells to ER stress

SLIDE 2

The Endoplasmic Reticulum

The endoplasmic reticulum (ER) is a multifunctional organelle ✓ lipid biosynthesis ✓ calcium storage ✓ protein folding and processing of membrane and secreted proteins Secretory protein folding and processing

Secretory pathway

BiP BiP

Dobson CM Nature Reviews Drug Discovery 2, 154-160 (February 2003)

SLIDE 3

ER stress triggers the Unfolded Protein Response (UPR)

from Walter and Ron 2011, SCIENCE VOL 334: 1081

PERK

p p

Ire1α ATF6

BiP BiP BiP

p p

✓ physiological and pathological conditions ✓ pharmacological agents ER stress is a condition of imbalance between the folding capacity and the amount of unfolded client proteins.

Signaling cascades to nucleus

SLIDE 4

Rationale

Aggravating ER stress in cancer to shift the UPR balance toward apoptosis UPR

apoptosis survival

SLIDE 5

Rationale

Amounts of ER stress that can be coped with by most cells can be detrimental in cells with altered ER homeostasis ✓ adaptation to increased folding demand ✓ presence of mis-folded or aggregation prone proteins ER intrinsic causes ER extrinsic causes ✓

• xidative stress

✓ presence of mis-folded or aggregation prone proteins ✓ Ca++ unbalance ✓ Ca++ unbalance ✓ impairment of protein degradation systems (proteasome, autophagy) ✓ … ✓ …

SLIDE 6

Rationale

Aggravating ER stress in cancer to shift the UPR balance toward apoptosis AML Characterized by the presence of chimeric or mutant protein, possibly prone to mis-folding and aggregation.

(Data discussed by Ernestina Capuano in poster PO002, 01 APL biology topic)

APL Expression of PML-RARα Granulocytic differentiation induced by all-trans retinoic acid requires increased folding capacity in the ER

Masciarelli et al., Leukemia 2017

UPR apoptosis survival

SLIDE 7

RA-induced granulocytic differentiation sensitizes NB4 cells to ER stress

Treatment of the APL cell line NB4 with physiological doses of RA (10-8) in combination with low doses of Tunicamycin (Tm, 50ng/ml) causes: cell death

***

20 40 60 24 48 72 ctr RA Tm RA-Tm

% PI+ cells

(hrs)

Accumulation of BiP complexes

BiP

75 100 135 180

+

• +
• +
• +
• RA

Tm BiP complexes BiP GAPDH

(72hrs)

5 10 15 20 nil Tm ctr RA relative mRNA expression (24hrs) ** *** 2 4 6 nil Tm ** CHOP BiP

Up-regulation of UPR target genes

ER stress

ER swelling

ctr Tm RA RATm Staining for the ER chaperone calreticulin (72hrs)

ctr Tm RA RATm

n ≥ 3; * p value < 0.05, ** <0.02, ***< 0.005

SLIDE 8

Attenuation of general translation protects differentiating NB4 cells from ER stress-induced death

25 50 75 100

nil GSK2606414 guanabenz

** * +

• +
• +
• +
• RA

Tm % PI+ cells

guanabenz GSK2601414 translation

Activation of the PERK pathway results in attenuation of global translation with consequent reduction of the ER load

n ≥ 3; * p value < 0.05, ** <0.02, ***< 0.005

SLIDE 9

RA sensitizes APL primary blasts to ER stress

APL1 APL2 APL3 BM1 BM2

cell number/colony

• +

+

• +

+

Tm RA

p53 GAPDH

APL4 ctr Tm RA RATm BM1 APL4 APL4 APL5

ATO

• +

+

• + +

Tm RA

• +

+

• + +

+ + + +

• +

+

• + +
• +

+

• + +

+ + + +

• +

+

• + +
• +

+

• + +

+ + + +

PIG3 NOXA p53AIP1

Treatment of APL primary blasts with 10-8 RA in combination with 50ng/ml Tm leads to: Formation of smaller colonies in CFU assay Apoptotic cell death Activation of the TP53 pathway

SLIDE 10

ER stress and ATO exhibit synergistic toxicity in RA-sensitive NB4 and RA-resistant NB4-R4 cells

Disulphide bond-dependent aggregation of PML and PML-RARα

+

• +
• +
• +
• RA

Tm

anti-RARα anti-PML GAPDH

+

• +
• +
• +
• (72hrs)

HMW complexes

ER stress generates oxidative stress that results lethal in cells concomitantly treated with RA or ATO ROS

ATO ER stress

DCF (MFI)

200 400 600 800

+

• +

+ +

• +

RA Tm

• +
• +

+

• +
• +
• +

+

• ATO

cell death (% PI+)

25 50 75 100

nil GSK guanabenz * * +

• +

+ +

• +

RA Tm

• +
• +

+

• +
• +
• +

+

• ATO

10 20 30 40 50

• NAC nil
• NAC GSK

+NAC nil +NAC GSK

cell death (% PI+) +

• +

+

• RA

Tm

• +
• +
• +

+ + ATO

cell death increased levels of ROS Rescue by antioxidant agent

25 50 75 100

nil GSK guanabenz *** ** +

• +

+ +

• +

RA Tm

• +
• +

+

• +
• +
• +

+

• ATO

25 50 75 100

• NAC nil
• NAC nil

+NAC nil +NAC GSK

+

• +

+

• RA

Tm

• +
• +
• +

+ + ATO cell death (% PI+) cell death (% PI+)

cell death Rescue by antioxidant agent

RA-responsive NB4 RA-resistant NB4-R4

RA 10-8 ATO 500nM Tm 50ng/ml

SLIDE 11

Induction of ER stress in combination with RA and/or ATO shifts the balance of the UPR from recovery of homeostasis to apoptosis

APL

+RA

UPR

homeostasis

+RA/ATO

UPR cell death

+low ER stress +low ER stress

UPR

very mild mild strong

CONCLUSION

✓ Low ER stress in combination with RA and/or ATO efficiently targets APL cells, in vitro, without affecting normal hematopoietic progenitors ✓ Doses of RA and ATO below the therapeutical reference range are sufficient to syngergize with Tm ✓ The toxic effect is mediated by activation of the UPR as a pro-apototic response and by generation of

• xidative stress

✓ Pharmacological inhibition of the PERK pathway amplified the toxicity of the combined treatments making it an interesting molecular target

homeostasis

SLIDE 12

Section of Histology and Medical Embryology,

• Dept. of Anatomical, Histological,

Forensic and Orthopedic Sciences, Sapienza University of Rome Francesco Fazi Silvia Masciarelli Ernestina Capuano Claudia Tito

• Dept. of Biomedicine and Prevention,

University of Rome Tor Vergata Francesco Lo Coco Tiziana Ottone Mariadomenica Divona Nelida Noguera Alessandra Picardi Centre for Life Nano Science, Istituto Italiano di Tecnologia of Rome Simone De Panfilis Oncogenomic and Epigenetic Unit Regina Elena National Cancer Institute of Rome Giovanni Blandino Giulia Fontemaggi AIRC