Retinoic Acid and Arsenic Trioxide sensitize Acute Myeloid Leukemia cells to ER stress
7th international symposium on Acute Promyelocytic Leukemia! September 24-27, 2017! The Endoplasmic Reticulum
Secretory protein folding and processing
The endoplasmic reticulum (ER) is a multifunctional organelle
BiP
BiP ✓ lipid biosynthesis
✓ calcium storage
✓ protein folding and processing of membrane and secreted proteins
Secretory pathway
Dobson CM Nature Reviews Drug Discovery 2, 154-160 (February 2003) ER stress triggers the Unfolded Protein Response (UPR)
BiP
ATF6
BiP
BiP Ire1α PERK
p p p p
Signaling cascades to nucleus
ER stress is a condition of imbalance between the folding capacity and the amount of unfolded client proteins. ✓ physiological and pathological conditions from Walter and Ron 2011, SCIENCE VOL 334: 1081 ✓ pharmacological agents Rationale
Aggravating ER stress in cancer to shift the UPR balance toward apoptosis
UPR
survival
apoptosis Rationale
Amounts of ER stress that can be coped with by most cells can be detrimental in cells with altered ER homeostasis
ER intrinsic causes
✓ adaptation to increased folding demand ✓ presence of mis-folded or aggregation prone proteins ✓ Ca++ unbalance ✓ …
ER extrinsic causes
✓ oxidative stress ✓ Ca++ unbalance
✓ presence of mis-folded or aggregation prone proteins
✓ impairment of protein degradation systems (proteasome, autophagy) ✓ … Rationale
UPR Aggravating ER stress in cancer to shift the UPR balance toward apoptosis survival
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 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 ER stress 60 ctr *** RA ER swelling Tm 40 RA-Tm ctr RA Tm RATm
20 % PI+ cells cells %PI+
0 24 48 72 (hrs) Staining for the ER chaperone calreticulin (72hrs)
ctr Tm Up-regulation of UPR target genes Accumulation of BiP complexes CHOP BiP ** Tm - - + + 20 ctr 6 ** RA - + - + 15 RA BiP 4 10 BiP 180 135 complexes *** 2 100 5 75 BiP
RA RATm relativemRNA GAPDH expression (24hrs) expression(24hrs) 0 0 nil Tm nil Tm (72hrs) n ≥ 3; * p value < 0.05, ** <0.02, ***< 0.005 Attenuation of general translation protects differentiating NB4 cells from ER stress-induced death
100 guanabenz nil * GSK2606414 75 guanabenz
50 GSK2601414 **
% PI+ cells cells %PI+ 25
translation 0 Tm - - + + RA - + - + 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 RA sensitizes APL primary blasts to ER stress
Treatment of APL primary blasts with 10-8 RA in combination with 50ng/ml Tm leads to:
APL1 APL2 APL3 BM1 BM2
Formation of smaller colonies in CFU assay cellnumber/colony
ctr Tm RA RATm APL4
APL4
Apoptotic cell death
BM1
APL4 APL5 Tm - - + + PIG3 NOXA p53AIP1 RA - + - + Activation of the TP53
p53 pathway
GAPDH RA - + - + - + - + - + - + - + - + - + - + - + - + Tm - - + + - - + + - - + + - - + + - - + + - - + + ATO - - - - + + + + - - - - + + + + - - - - + + + + ER stress and ATO exhibit synergistic toxicity in RA-sensitive NB4 and RA-resistant NB4-R4 cells
ER stress generates oxidative stress that results lethal in cells concomitantly treated with RA or ATO RA-responsive NB4 RA-resistant NB4-R4 cell death nil GSK guanabenz 100 * ER ) * cell death ATO + stress 75 50 nil GSK guanabenz 25 ** ) *** + 100 0 75 RA - + - + - + - + 50 Tm - - + + - - + + 25 cell death (%PI ATO - - - - + + + + 0 RA - + - + - + - + increased levels of ROS Tm - - + + - - + +
ROS 800 cell death (%PI ATO - - - - + + + + 600 400 Rescue by antioxidant agent 200 Disulphide bond-dependent -NAC nil -NAC nil DCF (MFI) DCF(MFI) 0 ) aggregation of PML and PML-RARα + 100 +NAC nil +NAC GSK RA - + - + - + - + 75 Tm - - + + - - + + Tm - - + + - - + + RA - + - + - + - + ATO - - - - + + + + 50 25 0 HMW Rescue by antioxidant agent RA - + - - + complexes Tm - + - + + α anti-RAR -NAC nil -NAC GSK ) anti-PML anti-PML cell death (%PI + ATO - - + + + 50 +NAC nil +NAC GSK 40 30 RA 10-8 20 10 ATO 500nM 0 Tm 50ng/ml GAPDH RA - + - - + (72hrs) cell death (%PI Tm - + - + + ATO - - + + + Induction of ER stress in combination with RA and/or ATO shifts the balance of the UPR from recovery of homeostasis to apoptosis
CONCLUSION
UPR homeostasis ✓ Low ER stress in combination with RA and/or ATO efficiently targets APL cells, +RA! in vitro, without affecting normal hematopoietic progenitors +low ER ✓ Doses of RA and ATO below the stress! APL UPR homeostasis therapeutical reference range are sufficient to syngergize with Tm
+RA/ATO! ✓ The toxic effect is mediated by +low ER activation of the UPR as a pro-apototic UPR cell response and by generation of stress! death oxidative stress
✓ Pharmacological inhibition of the PERK very mild! pathway amplified the toxicity of the combined treatments making it an mild! interesting molecular target strong! 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 AIRC!
Oncogenomic and Epigenetic Unit Regina Elena National Cancer Institute of Rome Giovanni Blandino Giulia Fontemaggi