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

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 ✓ lipid biosynthesis BiP ✓ 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 Ire1 α 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-RAR α 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 % PI+ cells 40 ctr RA Tm RATm RA-Tm 20 0 (hrs) 24 48 72 Staining for the ER chaperone calreticulin (72hrs) ctr Tm Accumulation of BiP complexes Up-regulation of UPR target genes CHOP BiP Tm - - + + ** 20 6 ** - - RA + + expression (24hrs) ctr relative mRNA RA 15 BiP 4 BiP 10 180 complexes 2 135 *** 100 5 BiP 75 RA RATm GAPDH 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 % PI+ cells 50 ** GSK2601414 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 cell number/colony Formation of smaller colonies in CFU assay APL4 ctr Tm RA RATm APL4 Apoptotic cell death BM1 APL5 APL4 PIG3 NOXA p53AIP1 Tm - - + + - + - + Activation of the TP53 RA pathway p53 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 * ER 100 * cell death ATO cell death (% PI + ) 75 stress nil GSK guanabenz 50 ** 25 *** 100 cell death (% PI + ) 0 75 - + - + - + - + RA 50 Tm - - + + - - + + 25 ATO - - - - + + + + 0 - + - + - + - + RA increased levels of ROS Tm - - + + - - + + ROS - - - - + + + + ATO DCF (MFI) 800 600 Rescue by antioxidant agent 400 200 -NAC nil -NAC nil Disulphide bond-dependent 0 +NAC nil +NAC GSK cell death (% PI + ) aggregation of PML and PML-RAR α 100 - + - + - + - + RA 75 Tm - - - - Tm - - + + - - + + + + + + 50 - - - - RA + + + + - - - - + + + + ATO 25 0 - + - - + Rescue by antioxidant agent RA HMW Tm - + - + + complexes -NAC nil -NAC GSK - - + + + ATO cell death (% PI + ) anti-RAR α +NAC nil +NAC GSK anti-PML 50 40 RA 10 -8 30 20 ATO 500nM 10 Tm 50ng/ml 0 - + - - + GAPDH RA Tm - + - + + (72hrs) - - + + + 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 stress � ✓ Doses of RA and ATO below the APL UPR homeostasis therapeutical reference range are sufficient to syngergize with Tm +RA/ATO � ✓ The toxic effect is mediated by +low ER cell activation of the UPR as a pro-apototic UPR stress � response and by generation of death oxidative stress ✓ Pharmacological inhibition of the PERK very mild � pathway amplified the toxicity of the mild � combined treatments making it an 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