Analysis of the Proteasome-Autophagy Crosstalk Under Proteotoxic Stress Conditions in Acute Myeloid Leukemia Rosa Guadalupe Lopez Reyes

Analysis of the proteasome-autophagy crosstalk under proteotoxic stress conditions in acute myeloid leukemia Rosa Guadalupe Lopez Reyes

To cite this version:

Rosa Guadalupe Lopez Reyes. Analysis of the proteasome-autophagy crosstalk under proteotoxic stress conditions in acute myeloid leukemia. Cancer. Université Paul Sabatier - Toulouse III, 2020. English. NNT : 2020TOU30064. tel-03046746

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ACKNOWLEDGEMENT

I want to thank all the people whose assistance was a milestone in the completion of this project. I would like to thank CONACyT for making this research possible. The ITAV and the CNRS for offering me facilities during this project, and Manuel Rodriguez and Pierre Lutz for supervising this thesis. I wish to express my deepest gratitude to Jean Emmanuel Sarry, for always having the time for this research, and to all the members of the 18th team, for all their support, help and patience. Thanks to Clement Larrue for his help during this research, and to Carine Joffre for her help during the completion of this project I finally would like to thank all the members of the UbiCARE team. And of course, the evaluators, for taking the time to evaluate this thesis.

2 SPECIAL THANKS

“Sweet are the uses of adversity which, like the toad, ugly and venomous, wears yet a precious jewel in his head.” William Shakespeare

Thanks to the Supreme Being that has allowed me to be 29 years on earth. My birth is a miracle, and my existence is proof of his mercy. He has given me the strength to continue, thank you for sending me with such great human beings: my parents. Thank you for covering me with your heavenly mantle.

A long time ago I read an African proverb that said: “If you want to go fast, go alone if you want to go far, go accompanied,” I have had the blessing of walking beside my parents: Amanda Reyes Vázquez and Juan López Velázquez. Thank you for fighting battles for and with me, for believing in my dreams (no matter how crazy they sound, no matter how scary they are), for being by my side in times of frustration, and pain, for never letting me give up, because you never cut my wings, on the contrary, they have driven me to fly higher and higher. Because of you I am who I am, because of you I will be, because you are my greatest motivation. Thanks, mom, thanks dad, here is the fruit of teamwork, I love you.

To the rest of my family: Williams, Vianey, Amandita, and Jonathan, for understanding my absence in important family events. Distance is very hard without you.

To my French family, thank you for all the support, and your kind words.

“Guenda nabani xhianga sicarú, ne gasti rú ni Ugaanda laa, Diuxhi biseenda laanu idxi layú, ne laa cuidxi laanu ra nuu” (Life is wonderful, and there is nothing to compare, God sent us to earth and He will call us to his side). To my uncle Wilfrido López Velázquez, “Tío Willy,” because although years ago the circumstances of life snatched you from my side on this earthly plane, I know that you are always with me. Your teachings are still present in me, I live to honor your memory, I always carry your heart with me. It never leaves me and, wherever I go, you are always with me. I know that this moment would have been so special for you, as it is for me. This achievement is also yours, I love you, uncle.

To my husband, Anthony Gallais, thank you for supporting me, for believing in me, for understanding me. Your help has been fundamental and, you have been with me even in the most challenging moments. This project was not easy, but you were always there, motivating me and helping me.

To Dr. Guadalupe Soto Rodriguez, for being crucial in my life to continue on the path of research, for not only being my guide in the degree but for the friendship you have given me these years. Despite the distance you have never left me alone. This dream is possible because of you. Thank you, a paragraph does not summarize all the profound grattitude I have!

To Dr. Marisela Ahumada Santiago, thank you for believing in my dream, thank you for your friendship even in the distance.

3 To my aunt, Lelia López Velasquez, thank you for believing in my dreams and supporting me.

Thanks to all the angels that God has sent me in my everyday life.

Life has taught me that I do not need many friends, but the right ones. Thank you, Miguel Madrid, for all your support, for being beside me in the most challenging times. You have shown me the meaning of loyalty. Thank you for being my friend.

Thank you, Maria Gonzalez Santamarta; in a short period, you have become a very great friend. I want to thank you not only for the help provided but for the good times we have lived. You are a great person, and I love having you by my side as a friend.

Thanks, Clémence Coutelle-Rebut for your kindness, patience and time for reading this thesis.

Thank you, Gabrielle Sueur, for your friendship and kindness, for being there since I have not friends here.

Thank you, Javier Morgado, you have been like a brother, you have supported me, you have never let me down even when I want. Thank you for being my friend.

To Latifa, thank you for your support during these years, thank you for listening to me.

I want to give a special thanks to Claudie, Marie, Estelle, Mathilde, and Emeline for your help and support during these years.

Thank you to the doctoral school for all your support and help during this thesis.

I am pretty sure that I am forgetting too many people who have helped me during all these years but thank you for making this thesis possible.

4 TABLE OF CONTENTS ABBREVIATIONS ...... 7 FIGURES...... 9 LIST OF TABLES ...... 11 SUMMARY ...... 12 RÉSUMÉ ...... 13 PREAMBLE ...... 15 I. INTRODUCTION ...... 17 1. CARCINOGENESIS ...... 17 2. From hematopoiesis to Leukemia ...... 19 2.1 Hematopoietic regulation ...... 19 2.2 Leukemia and leukemic development ...... 21 2.3 Leukemia Classification...... 22 3. Acute Myeloid Leukemia ...... 23 3.1 Epidemiology ...... 23 3.2 AML classification ...... 24 3.2.1 The French-American-British (FAB) classification of AML ...... 24 3.2.2 The World Health Organization (WHO) classification ...... 25 3.3 Cytogenetic characteristics and their prognostic impact in AML ...... 27 3.4 Molecular basis of Acute myeloid leukemia ...... 28 3.5 AML in pathway signaling ...... 29 3.6 Treatment in AML ...... 31 4. Tyrosine Kinases ...... 32 4.1 Tyrosine kinase classification ...... 32 4.2 Mechanism of RTK activation under normal physiological conditions ...... 33 5. The FLT3 receptor ...... 34 5.1 Protein and gene structure of FLT3 ...... 34 5.2 Signal transduction networks activated by FLT3-WT ...... 35 5.3 FLT3 mutations ...... 37 5.4 FLT3-ITD ...... 37 5.5 FLT3-ITD signaling ...... 39 6. Intracellular protein degradation ...... 40 7. Autophagy...... 42 7.1 Types of autophagy ...... 42 7.2 Autophagy induction ...... 45 7.3 Autophagy and its relationship with ubiquitylation ...... 47

5 7.4 LC3/GABARAP and autophagy receptors ...... 48 7.5 The role of Autophagy in cancer ...... 53 7.6 Autophagy inhibitors ...... 56 8. The proteasome ...... 58 8.1 Function, structure and organization ...... 58 8.2 The CP or 20S proteasome ...... 59 8.3 The RP or 19S regulatory complex...... 59 8.3.1 The lid and base subcomplex ...... 60 9. Protein Ubiquitylation...... 61 9.1 Major Ubiquitin receptors for proteasomal degradation ...... 63 9.2 Purification of Ubiquitylated proteins: TANDEM UBIQUITIN BINDING ENTITIES (TUBE) ...... 65 10. Ubiquitin-Proteasome System (UPS) ...... 67 10. 1 UPS inhibitors ...... 68 10.2 The role of the proteasome in acute myeloid leukemia (AML) ...... 71 11. Crosstalk between Autophagy and Ubiquitin Proteasome System ...... 74 11.1 Proteaphagy ...... 76 II. SCIENTIFIC CONTEXT ...... 79 Hypothesis ...... 81 III. MATERIAL AND METHODS ...... 82 IV. RESULTS ...... 90 Setting up conditions for detection of selective autophagy events ...... 90 Apoptosis in FLT3-ITD cells after 8h treatment with inhibitors ...... 95 Proteaphagy in other AML cells ...... 96 Proteaphagy in FLT3-ITD AML cells using an autophagy inhibitor acting on p62 ... 100 Proteaphagy is not constitutively active in FLT3-ITD cells ...... 103 Evidence of proteaphagy and other selective autophagy events by Immunofluorescence in FLT3-ITD cells...... 105 Impact of autophagy inhibition on proteasome activity ...... 111 Identification of ubiquitylated proteins using TANDEM UBIQUITIN BINDING ENTITIES (TUBEs) ...... 113 Ubiquitin-dependent regulation of p62 complexes regulating autophagy activated by Bz 115 Inhibition of UPS and ALS enhances apoptosis in FLT3-ITD AML cells ...... 119 V. GENERAL DISCUSSION ...... 122 VI. BIBLIOGRAPHY ...... 134 VIII. Articles ...... 135

ABBREVIATIONS ALS: Autophagy Lysosome System AML: Acute Myeloid Leukemia Atg: Autophagy-related genes ATP: Adenosine triphosphate BafA: Bafilomycin A1 BB: Boiling buffer BM: Bone Marrow BSA: Bovine serum albumin Bz: Bortezomib CP: Core particle CQ: Chloroquine CMML: Chronic myelomonocytic leukemia CREB: Cyclic adenosine monophosphate response element-binding protein CSF: Colony stimulating factor DUB: Deubiquitinating enzyme DNA: Deoxyribonucleic acid DTT: Dithiothreitol ER: Endoplasmic reticulum Erk: Extracellular signal-regulated kinase FAB: The French-American-British FBS: Fetal bovine serum FDA: Food and Drug Administration FL: FLT3 ligand FLK2: Fetal Liver Tyrosine Kinase 2 FLT3: Fms-Like Tyrosine kinase 3 FLT3-ITD: Fms-Like Tyrosine kinase 3 with Internal Tandem Duplications FLT3L: Fms-Like Tyrosine Kinase 3 with Tyrosine Kinase Domain mutation FOX3 A: Forkhead box 03 GM-CSF: Granulocyte-macrophage colony-stimulating factor GRB2: Growth factor receptor-bound 2 HCQ: Hydroxychloroquine HSCs: Hematopoietic stem cells ITD: Internal Tandem Duplication IP: Immunoprecipitation JAK: Janus Kinase JM: JuxtaMembrane LSCS: Leukemic Stem cells MAPK: Mitogen-Activated Protein kinase MCH: Major complex histocompatibility MDR: Multidrug resistance gene complex MDS: Myelodysplastic syndromes MC: Mantel cell lymphoma MM: Multiple Myeloma MP: Myeloproliferative neoplasms MS: Mass Spectrometry NBR1: Neighbor of BRCA gene-one protein mTOR: Mammalian Target of Rapamycin

7 NF-ĸB: Nuclear factor-B OPTN: Optineurin PAS: Pre-autophagosomal structure PE: Phosphatydiletanolamine PGDFR: Derived growth factor platelet receptor PI: Proteasome inhibitors PI3K: Phosphoinositide 3-kinase PK: Protein Kinase RP: Regulatory particle Rpn: Regulatory particle of non-ATPase subunits Rpt: Regulatory particle of triple-ATPase subunits RTKs: Receptor Tyrosine Kinase SCFR: Stem cell factor receptor SQSTM1: Sequestosome 1 Stat 5: Signal transducer and activator of transcription 5 TBS: Tris-buffered saline TUBE: Tandem Ubiquitin Binding Domain TK: Tyrosine Kinase TKD: Tyrosine Kinase Domain TUBE: Tandem ubiquitin binding entities Ub: Ubiquitin UBA: Ubiquitin associated domain UBD Ubiquitin-Binding domain UBL: Ubiquitin-Like proteins UPS: Ubiquitin-Proteasome System VEGF: Vascular Endothelial Grow Factor VPS34: Vacuolar Protein Sorting 34 VT: Verteporfin WB: Western Blot WHO: World Health Organization WT: Wild Type

8 FIGURES Figure 1. The hallmarks of cancer…………………………………………………..…………..17 Figure 2: A general model of Hematopoiesis…………………………………………….……20 Figure 3: Schematic representation of the FLT3 receptor…………………………….……..35 Figure 4: Ligand independent FLT3 activation…………………………………………….….35 Figure 5: Signaling pathways activated by FLT3-WT………………………………………...36 Figure 6: Constitutive activation of the FLT3 receptor by the ITD mutation………………..38 Figure 7: The Two major protein degradation pathways………………………………….….41 Figure 8: Different Types of Autophagy………………………………………………………..43 Figure 9: The process and regulation of selective autophagy……………………………….44 Figure 10: Assembly and elongation of autophagic membranes are accomplished via sequential action of UPS-like E1-E2-E3 cascades…………………………………………….48 Figure 11: Potential mechanisms of selective macroautophagy……………………………49 Figure 12: Selective autophagy receptors: ubiquitin-mediated cargo recognition…………50 Figure 13: Ubiquitin-dependent and Ubiquitin independent autophagy…………………….51 Figure 14: The autophagy receptor p62………………………………………………………..52 Figure 15: Autophagy has multiple roles during tumorigenesis………………………………55 Figure 16: 26S proteasome structure…………………………………………………………...58 Figure 17: Ubiquitin chains and its major functions…………………………………………....62 Figure 18: Recognition of the ubiquitylated substrates for the 26S proteasome-mediated proteolysis………………………………………………………………………………………….64 Figure 19: The tandem disposition of ubiquitin-associated domains preserves their ubiquitin- binding capacity……………………………………………………………………………………66 Figure 20: Functional representation of the mechanism of ubiquitin conjugation and presentation to the proteasome………………………………………………………………….67 Figure 21: Cellular consequences of proteasome inhibition…………………………………69 Figure 22: The proteasome has several roles in AML………………………………………..73 Figure 23: The compensatory balance between the activities of autophagy and the UPS in order to maintain cellular homeostasis………………………………………………………….75 Figure 24: FLT3-ITD positive AML cells are targeted to apoptosis by Bz-mediated autophagy………………………………………………………………………………………..…80 Figure 25: Immunoprecipitations of p62 in the presencse or absence of TUBEs………….86 Figure 26: Setting up conditions for detection of proteaphagy in MOLM-14 cells using high serum concentration………………………………………………………………………………92 Figure 27: Set up conditions for detection of proteaphagy in MOLM-14 cells using low serum concentration………………………………………………………………………………………94 Figure 28: Cell death evaluation after 8h of treatment with proteasome and autophagy inhibitors in MOLM-14 cells …………………………………………………………………….95 Figure 29: Proteaphagy evaluation in other FTL3-ITD AML cell lines………………………97 Figure 30: Analysis of proteaphagy in FLT3 WT cells……………………………………...... 99 Figure 31: Verteporfin (VT) IC50 in FLT3-ITD AML cells ……………………………………100 Figure 32: Verteporfin blocked proteaphagy in AML cells…………………………………..102 Figure 33: Analysis of basal levels of proteaphagy in MOLM-14 cells…………………….104 Figure 34: Colocalization of β2 proteasome subunit and LC3B after Bz and VT treatment in FLT3-ITD AML cells……………………………………………………………………………...107 Figure 35: Colocalization of α2 proteasome subunit and p62 after Bz treatment in FLT3-ITD AMLcells ………………………………………………………………………………………….108 Figure 36: Colocalization of Rpn1 and LC3B after Bz treatment in FLT3-ITD AMLcells…………………………………………………………………………………………..109

Figure 37: Colocalization of FLT3 and LC3B after Bz and VT treatment in FLT3-ITD AML cells……………………………………………………………………………………………..…109 Figure 38: Colocalization mytophagy indicator COXIV and p62 after Bz and autophagy inhibitors treatment in FLT3-ITD AML cells…………………………………………………...110 Figure 39: Proteasome activity after different doses of Bz in MOLM-14 cells…………...112 Figure 40: The role of protein ubiquitylation in proteaphagy and FLT3-ITD degradation under proteasome inhibition conditions………………………………………………………..114 Figure 41: Setting up conditions for immunoprecipitations of p62 and Beta 2 under proteasome inhibition conditions……………………………………………………………….117 Figure 42: Ubiquitin role in proteaphagy and degradation of FLT3-ITD under proteasome inhibition conditions……………………………………………………………………………...118 Figure 43: Cell death evaluation after 24h treatments, FBS 10% in AML FLT3 wild type, and FLT3-ITD positive ………………………………………………………………………………..120 Figure 44: Proteasome and autophagy inhibitors cooperate to improve the apoptosis of FLt3-ITD expressing cells..……………………………………………………………………...121 Figure 45: FLT3-ITD levels are controlled by the action of UPS and ALS………………….132

LIST OF TABLES

Table 1: Main indicators in 2012 of AML……………………………………………………….24 Table 2: FAB classification for AML…………………………………………………………….25 Table 3: WHO classification for AML…………………………………………………………...26 Table 4: The cytogenetic classification of AML………………………………………………..27 Table 5: Autophagy inhibitors……………………………………………………………………57 Table 6: Mono/multiple monoubiquitylated Proteasomal substrates………………………..62 Table 7: Antibodies used during this study…………………………………………………….82 Table 8: Drugs used during this research..…………………………………………………….83 Table 9: Statistical values………………………………………………………………………..89

11 SUMMARY Acute myeloid leukemia (AML) represents a heterogeneous group of malignant hematopathies characterized by a clonal proliferation of hematopoietic progenitors blocked in their differentiation (blasts), which accumulate in the bone marrow, blood, and other organs. The AML causes more than 3000 death every year in France. Different genetic mutations are found in the AML, among them the ones affecting the kinase receptor activity: KIT and FLT3-ITD. The internal tandem duplication in the FLT3 receptor (FLT3-ITD), represents 30% of cases of AML, and has a poor prognosis, compared to patients expressing the wild-type receptor. Previous studies have demonstrated that autophagy regulates cytotoxicity in FLT3-ITD AML cells after proteasome inhibition. In this study proteotoxic stress conditions generated by Bortezomib (Bz) resulted in the degradation of proteasome subunits in FLT3-ITD MOLM-14 cells but not in FLT3-WT OCI-AML3 cells, suggesting ITD mutation contributes to activate autophagy-mediated proteolysis of the proteasome, known as proteaphagy. In this study, using chemical inhibition of autophagy with Bafilomycin A (BafA) we blocked proteaphagy and accumulated proteasome core subunits into autophagosomes of Bz-treated MOLM-14 cells. To investigate the role of protein ubiquitylation in proteaphagy, we used distinct TUBEs (Tandem Ubiquitin Binding Entities). While TUBE-HHR23 captures p62, proteasome subunits and ubiquitylated forms of FLT3, TUBE-p62 does not. Nevertheless, TUBE-p62 protected the unmodified form of FLT3-ITD from degradation driven by Bz. According to our results, the p62 inhibitor Verteporfin (VT) blocked proteaphagy and reduced the colocalization of p62/2 core subunit but did not affected the one of p62/Rpn1. VT also protected FLT3-ITD from Bz-induced degradation and colocalized within p62 in MOLM-14 cells. Both autophagy inhibitors enhanced Bz-induced apoptosis in MOLM-14 cells suggesting that these combinatorial treatments could be a therapeutic strategy to sensitize FLT3-ITD positive cells. This study allowed us to understand that the proteasome- and autophagy-mediated proteolysis are most likely in a dynamic equilibrium and play an important role when one or the other pathway is impaired.

12 RÉSUMÉ La leucémie myéloïde aiguë (LAM) représente un groupe hétérogène d'hématopathies malignes caractérisées par une prolifération clonale de progéniteurs hématopoïétiques bloqués dans leur différenciation (blastes), qui s'accumulent dans la moelle osseuse, le sang et d'autres organes. Le LAM fait chaque année plus de 3000 morts en France. Différentes mutations génétiques se trouvent dans le LAM, parmi lesquelles celles affectant l'activité des récepteurs de kinase: KIT et FLT3-ITD. La duplication interne en tandem dans le récepteur FLT3 (FLT3-ITD), représente 30% des cas de LAM, et a un mauvais pronostic, par rapport aux patients exprimant le récepteur de type sauvage. Les patientes atteintes de leucémie aiguë myéloïde présentant une duplication interne en tandem dans le récepteur FLT3 (FLT3-ITD), les patients atteints de FLT3-ITD représentent 30% des cas de leucémie aiguë myéloïde (LAM), et ont un mauvais pronostic de survie par rapport aux patients exprimant le récepteur de sauvage. Des études antérieures ont démontré que l'autophagie régule la cytotoxicité induite par l’inhibition du protéasome dans les cellules de LAM FLT3-ITD. Dans cette étude, les conditions de stress protéotoxique générées par le bortézomib (Bz) ont entraîné la dégradation des sous-unités du protéasome dans les cellules FLT3-ITD MOLM-14 mais pas dans les cellules FLT3-WT OCI-AML3, ce qui suggère que la mutation ITD contribue à activer la protéolyse par autophagie du protéasome, connue sous le nom de proteaphagie. L'inhibition chimique de l'autophagie avec la bafilomycine A (BafA) a bloqué la protaphagie et favorisé l’accumulation des sous-unités centrales du protéasome accumulées dans les autophagosomes des cellules MOLM-14 traitées au Bz. Pour étudier le rôle de l'ubiquitylation des protéines dans la protaphagie, nous avons utilisé des TUBE distincts (Tandem Ubiquitin Binding Entities). Alors que TUBE-HHR23 capture p62, les sous-unités de protéasome et les formes ubiquitylées de FLT3, TUBE-p62 ne le fait pas. Néanmoins, TUBE-p62 a protégé la forme non modifiée de FLT3-ITD de la dégradation provoquée par Bz. Selon nos résultats, l'inhibiteur de la p62 Verteporfin (VT) a bloqué la protaphagie et a réduit la colocalisation de la sous-unité centrale p62 avec α2 mais n'a pas affecté celle de p62 avec Rpn1. VT a également protégé FLT3-ITD de la dégradation induite par Bz et augmenté la colocalisation de p62 dans les cellules MOLM-14. Les deux inhibiteurs de l'autophagie ont augmenté l'apoptose induite par le Bz dans les cellules MOLM-14, suggérant que ces traitements combinatoires pourraient être une stratégie thérapeutique

13 pour cibler les cellules positives FLT3-ITD. Ainsi, la protéolyse médiée par le protéasome et l'autophagie est très probablement dans un équilibre dynamique et joue un rôle important lorsque l'une ou l'autre voie est altérée.

14 PREAMBLE Protein homeostasis is necessary for the proper functioning and wellbeing of the cells. In the 19th century, Claude Bernard underlined the need to maintain a stable internal environment - milieu intérieur - which would allow biological processes to take place despite variations in the external environment (Gross, 1998). Bernard’s concept was further explored, developed, and popularized by Walter Cannon, who coined the term “homeostasis” to describe how critical physiological variables are maintained within a predefined range by feedback mechanisms (Cannon, 1929). Nearly two decades after Cannon, James Hardy proposed a model in which homeostatic mechanisms maintain physiological variables within an acceptable range by comparing the actual value of the variable to the desired value or ‘set point,’ (Hardy, 1953). Homeostasis is a unifying theme of modern physiology, and much has been elucidated about molecular mechanisms of homeostatic control. Several disorders occur when cell homeostasis is disrupted, including cancer. Blood cancers encompass an extensive collection of diseases affecting the equilibrium and correct functioning of this tissue. Acute myeloid leukemia (AML) is a malignant disease characterized by differentiation blockage and proliferation of clonal hematopoietic stem or progenitor cells (HSPCs), which rapidly lead to bone marrow (BM) failure and eventually to death if left untreated. Internal tandem duplication (ITD) of the Fms-like tyrosine kinase-3 receptor (FLT3) is found in 30% of acute myeloid leukemia (AML) and is associated with poor outcomes. Multiple approaches based on new pharmacological targets have been considered to tackle this disease, and new treatments are being developed. Among the most promising are autophagy and proteasome inhibitors, which directly affect protein homeostasis and contribute to optimizing the apoptotic response of AML cells. Studies conducted by Larrue and his collaborators showed that AML cell lines and samples from patients bearing FLT3-ITD mutations are more sensitive to the proteasome inhibitor Bortezomib (Bz) than wild-type samples and that this sensitivity is strongly correlated with a higher FLT3-ITD allelic burden. Indeed, FLT3-ITD molecules were detectable within autophagosomes after Bz treatment indicating that autophagy induced by Bz was responsible for the early degradation of FLT3-ITD. This degradation preceded the inhibition of mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (Erk), PI3K/Akt, and Stat5 pathways, as well as the subsequent activation of cell death. Based on this work, we aimed to explore the different molecular mechanisms involved in the crosstalk

15 between the ubiquitin-proteasome system and autophagy in AML, under conditions of proteasome inhibition. In concrete terms, we investigated the participation of selective autophagy events like proteaphagy or aggrephagy activated after Bz treatment. The evidence suggests that chemical inhibitors of autophagy accumulate proteasome subunits into autophagosomes of bortezomib-treated FLT3-ITD AML cells but not in FLT3-WT AML cells. Even if bafilomycin A (BafA) blocks the degradation of the 20S proteasome subunits, 19S subunits appear to be more vulnerable to bortezomib-induced degradation. In particular, the Rpn1 subunit forms high molecular weight complexes with the autophagy receptor p62 after treatment with verteporfin (VT), underlining its implication in proteaphagy. To investigate the role of ubiquitin in this regulatory mechanism, we used a technology previously developed by our laboratory, based on ubiquitin-binding domains of the proteasome adaptor HHR23, or the autophagy adaptor p62. We could observe that ubiquitylated FLT3-ITD or proteasome subunits were captured after Bz+BafA treatment by Tandem Ubiquitin Binding Entities (TUBEs)-HHR23 but not by TUBEs-p62. Nevertheless, both TUBEs protect proteasome subunits, p62 or FLT3-ITD in different proportions. Altogether our evidence supports a role of FLT3-ITD in predisposing the activation of Bz- induced proteaphagy. This route can potentially be targeted using combinatorial approaches with autophagy inhibitors to improve previous results obtained with Bz.

16 I. INTRODUCTION

1. CARCINOGENESIS Over the years, cancer research has provided evidence that tumor development is a multistep process that transforms a normal cell into a malignant derivative. This process was comprehensively schematized (Figure 1) by Hanahan and Weinberg. They constituted the well-established hallmarks of cancer (Hanahan, 2014), where they attempted to organize the dense complexities of cancer biology into six acquired capabilities of cancer cells. These are the major hallmarks: 1. Cancer cells adopt alternative ways to self-sustain proliferation. For example, cell proliferation depends on extracellular stimulus transmitted to the cell by interactions of transmembrane receptors and signaling molecules (growth factors, extracellular matrix components, and cell-cell adhesion molecules). Transmembrane receptors responsive to proliferative signals are deregulated in cancer cells and often contain intracellular tyrosine kinase activity promoting tumor progression.

Figure 1: The hallmarks of cancer. Taken from (Hanahan, 2014).

Cancers acquire ten damaging functional capabilities and facilitators (shown in white ring and indicated by symbols in coloured ring) that collectively manifest successful attacks on the aff ected individual. Each capability can be counteracted by various mechanism-targeted treatments (shown figuratively as explosion shapes) which, generally, do not act as curative magic bullets because of the countervailing development of advanced strategies of resistance. One battlespace plan involves multitargeting of all of these capabilities and facilitators (A). A major challenge, however, is limitation of collateral damage—toxicity to normal tissue and physiological functions. Realistically, tactical variations will involve more selective multitargeting (exemplified in B, C, and D), fine-tuned both by enabling of military intelligence of a patient’s tumour aff orded by increasingly accurate, high-resolution molecular diagnostics, and by the nature of the drugs and the tactical regimens in which they are launched, to optimise eff ectiveness while restricting toxicity

17 2. Simultaneously, to sustaining proliferation, cancer cells acquire the capacity to evade apoptosis. Tissue homeostasis is tightly regulated by the elimination of non-healthy cells originating from infectious and non-infectious insults, a potential cause of oncogenic lesions. In such cases, impairment of tumor suppressors activity enables altered cells to escape apoptosis by acting upon inhibitors of apoptosis. 3. Oncogene signaling can drive angiogenic regulators involved in perpetuating the sprouting of new vessels to give nutrition for the growing tumor. 4. Activation of invasion and metastasis enables the tumor to spread. The multistep process of invasion and metastasis has been schematized as a sequence of discrete steps, often termed the invasion-metastasis cascade (Fidler, 2003; Talmadge & Fidler, 2010). This depiction envisions a succession of cell-biologic changes, beginning with local invasion, then intravasation by cancer cells into nearby blood and lymphatic vessels and the transit of cancer cells through the lymphatic and hematogenous systems. These are followed by the escape of cancer cells from the lumina of such vessels into the parenchyma of distant tissues (extravasation), the formation of small nodules of cancer cells (micrometastases), and finally the growth of micrometastatic lesions into macroscopic tumors. This last step is called ‘‘colonization.’’ 5. Evading growth suppressors: Cancer cells must also circumvent powerful programs that negatively regulate cell proliferation; many of these programs depend on the actions of tumor suppressor genes. 6. Enabling replicative immortality equipping the cancer cells to overcome the Hayflick limit. Tumor cells overcome the finite replicate potential at some point during multistep tumor progression, evolving to premalignant. Cell populations exhaust their endowment of allowed doublings and can only complete their tumorigenic agenda by breaching the mortality barrier and acquiring unlimited replicative potential (Hayflick, 1997).

More recently, Hanahan and Weinberg provided a substantial body of evidence (Hanahan & Weinberg, 2011) to pinpoint new capabilities as emerging hallmarks of cancer, being those related to the involvement of the immune system as a perpetrator of an inflammation condition before tumor development and during its establishment. Furthermore, cancer cells have a selective advantage enabled by specific mutant genotypes, categorized as evading immune destruction and reprogramming energy metabolism (Fouad & Aanei, 2017).

18 2. From hematopoiesis to Leukemia

2.1 Hematopoietic regulation

Hematopoiesis is the process by which all lineages of blood cells are generated in a hierarchical and stepwise manner from immature cells present in the bone marrow (BM) and subsequently released into circulating blood and peripheral organs for further maturation steps and or effector function (Orkin & Zon, 2008). The BM is the primary site of hematopoiesis and normal immature precursors of hematopoietic cells. The hematopoietic system is organized by a pyramidal hierarchy, in the apex of this hierarchy are hematopoietic stem cells (HSCs), which are the only self-renewing cells capable of lifelong production of all lineage of blood cells (Figure 2). Control of cell proliferation, growth, and survival is vital for the maintenance of homeostasis in every tissue. Hematopoiesis is controlled at different levels via the production of growth factors and extracellular cytokines that will control transcription and lead the fate of the cells. Growth factors are required for the survival and proliferation of hematopoietic cells at all stages of development. Among the factors that affect multipotent cells, the best characterized are steel factor, Fms-like tyrosine kinase 3 (FLT3) ligand, granulocyte- macrophage colony-stimulating factor (GM-CSF), interleukin-2, interleukin-3, and interleukin-7. Each of these proteins supports the survival and proliferation of many distinct target cells, and except for interleukin-7 and steel factor, the elimination of any one of them does little harm because of the redundancy in the functions of these early-acting growth factors (Kaushansky, 2006). Growth factors and cytokines secreted by hematopoietic cells or by environmental cells can be recognized by different surface receptors that regulate different pathways, like PI3K, JAK/Stats and MAPKs, involved in proliferation and differentiation.

Figure 2: A general model of Hematopoiesis. Taken from (Kaushansky, 2006).

Blood-cell development progresses from a hematopoietic stem cell (HSC), which can undergo either self- renewal or differentiation into a multilineage committed progenitor cell: a common lymphoid progenitor (CLP) or a common myeloid progenitor (CMP). These cells then give rise to more-differentiated progenitors, comprising those committed to two lineages that include T cells and natural killer cells (TNKs), granulocytes and macrophages (GMs), and megakaryocytes and erythroid cells (MEPs). Ultimately, these cells give rise to unilineage committed progenitors for B cells (BCPs), NK cells (NKPs), T cells (TCPs), granulocytes (GPs), monocytes (MPs), erythrocytes (EPs), and megakaryocytes (MkPs). Cytokines and growth factors that support the survival, proliferation, or differentiation of each type of cell are shown in red. For simplicity, the three types of granulocyte progenitor cells are not shown; in reality, distinct progenitors of neutrophils, eosinophils, and basophils or mast cells exist and are supported by distinct transcription factors and cytokines (e.g., interleukin- 5 in the case of eosinophils, stem-cell factor [SCF] in the case of basophils or mast cells, and G-CSF in the case of neutrophils). IL denotes interleukin, TPO thrombopoietin, M-CSF macrophage colony-stimulating factor, GM-CSF granulocyte-macrophage CSF, and EPO erythropoietin.

20 2.2 Leukemia and leukemic development

The etymological root of the term leukemia comes from the Greek leukos (λευκός), meaning "white,” and haima (αἷμα), meaning "blood." Leukemia is the consequence of stepwise genetic alterations that confer both proliferative and survival advantage, as well as self- renewal capacity to the malignant cells (S. W. Lane et al., 2009). In the case of deregulation of the HSCs processes of self-renewal and differentiation, individuals can develop acute myeloid leukemia, a disease characterized by an accumulation of immature blast cells that fail to differentiate into functional cells. Two types of abnormal events can lead to leukemia: . Firstly, a normal stem cell acquires several mutations, including gene mutation or abnormal expression of the genes/non-coding RNAs (Marcucci et al., 2011). Thus, epigenetic changes alter signaling pathways, affecting growth control, apoptosis and ability to differentiate. . Secondly, partially differentiated cells can restore gene expression patterns, allowing them to reacquire the unique self-renewal properties of stem cells while also interferes with their subsequent ability to differentiate (Testa & Pelosi, 2013). Hematopoietic stem cells (HSCs) are unique, multipotent cells that generate via progenitor and precursor cells of all blood lineages (McCulloch & Till, 2005; Riether et al., 2015). Leukemic hematopoiesis retains process characteristics of normal hematopoiesis. Similar to normal hematopoiesis, leukemia is also hierarchically organized, and a subpopulation, the leukemic stem cells (LSCs), is responsible for disease initiation and maintenance. Indeed, the leukemic clone is organized hierarchically into three different compartments. One of these compartments is composed of immature phenotype leukemic stem cells, mostly quiescent but capable of self-renewal or commitment in the process of differentiation. A more mature compartment of leukemic progenitors (CFU-L) having lost self-renewing capacities, but high proliferation properties. Finally, a major compartment of leukemic cells blocked at a stage of granulo-monocyte differentiation (Bonnet & Dick, 1997; J. C. Y. Wang & Dick, 2005). Leukemogenesis is a process in which multiple events involving independent genetic alterations in proto-oncogene or suppressor genes, together with epigenetic or environmental factors, contribute to the development of the full malignant phenotype (Irons & Stillman, 1996). Genes involved in leukemogenesis are related to various cellular functions, including ligand-receptor interaction, signal transduction, intracellular localization, cell cycle control, and apoptosis. In detail, the oncogenic events that underlie the onset of

21 acute myeloid leukemia are often divided into two classes of mutations, following the two-hit model of leukemogenesis. 1. Class I mutations: Confer a proliferation and survival advantage to blast cells, typically as a result of aberrant activation of signaling pathways. 2. Class II mutations: Lead to an impaired differentiation via interference with transcription factors or co-activators. The cooperation between these two main classes of mutations leads to the emergence of leukemic cells capable of proliferation but not differentiation (Dash & Gilliland, 2001).

2.3 Leukemia Classification

Leukemia is clinically and pathologically divided into different subcategories. It is, therefore, possible to differentiate acute from chronic forms (Daniel A. Arber et al., 2003)  Chronic leukemia (CML): Is characterized by an excessive buildup of relatively mature but still abnormal white blood cells. This form typically takes months or years to progress, the cells are produced at a much higher average rate, resulting in many abnormal white blood cells.  Chronic lymphocytic leukemia (CLL): Is a malignancy of CD5+ B cells that is characterized by the accumulation of small, mature-appearing lymphocytes in the blood, marrow, and lymphoid tissues (Kipps et al., 2017).  Acute lymphoblastic leukemia (ALL): This is a malignant transformation and proliferation of lymphoid progenitor cells in the BM, blood, and extramedullary sites (Terwilliger & Abdul-Hay, 2017).  Acute myeloid leukemia (AML): Is characterized by a rapid increase in the number of immature blood cells. This overcrowding makes the BM unable to produce healthy blood cells. Immediate treatment is then required due to the rapid progression and accumulation of malignant cells, which then spill over into the bloodstream and spread to other organs.

22 3. Acute Myeloid Leukemia Myeloid malignancies are clonal diseases of hematopoietic stem or progenitor cells (Murati et al., 2012). They result from genetic and epigenetic alterations that disrupt vital processes such as self-renewal, proliferation, and differentiation. Myeloid malignancies comprise chronic stages such as myeloproliferative neoplasms (MPN), myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML) and acute stages, i.e., AML. AML is a clonal disorder affecting hematopoietic stem cells or myeloid progenitors, characterized by an accumulation of immature leukemic cells in the BM and peripheral blood, consequently leading to BM failure (Récher et al., 2005). AML is a heterogeneous condition at both the phenotypic and molecular levels, with a variety of distinct genetic alterations giving rise to the disease (S. J. Horton & Huntly, 2012). It is poorly represented in children, but it is the main acute leukemia in adults, among whom it mainly affects people over 60 years of age (the median age for diagnosis is around 70 years). Despite efforts made to improve treatment for several years, overall 5-year survival is only around 40-50% in young people treated with chemotherapy, and much less in the elderly (DiNardo & Cortes, 2016).

3.1 Epidemiology

AML is the most common form of acute leukemia in adults and constitutes approximately 80 percent of cases (Siegel et al., 2016). AMLs are rare diseases but disproportionally have a significant effect on cancer survival statistics (Jemal et al., 2006). The American Cancer Society estimates that 31,500 individuals in the United States are annually diagnosed with a form of leukemia. Approximately 21,500 patients will die of this disease (Greenlee et al., 2001; Hernández et al., 1995). The incidence in France is similar to that observed in Europe with standardized incidence rates on the European population of 3.9 in men and 3.35 in women. These rates are equivalent to the European average and those in central and southern Europe (Gatta et al., 2011). There are around 3,000 new cases of AML per year in France (Data from the National League Against Cancer), including around 200 listed in Occitanie Ouest (formerly Midi- Pyrénées region). AML accounts for about 1 percent of all incident cancers in France and just under 10 percent of all hematological malignancies. The standardized incidence rate of AML for the world population is 2.6 per 100,000 in men and 2.3 per 100,000 in women, i.e.,

23 a male / female ratio of 1.1. In France, in 2012, 2.791 new cases of AML were estimated, of which 49% are in men (Sant et al., 2010). (Table 1). Leading indicators in 2012 of Acute Myeloid Leukemia

Sex Gross Standardized Standardized Number rate rates rates of Europe World cases Incidence Men 4.5 3.5 2.6 1381 Women 4.3 3.0 2.3 1410 Table 1: Main indicators in 2012 of AML

At present, AML can be cured in approximately 40 percent of AML patients who are under 60 years of age. However, the prognosis remains poor in patients who are older than 60 (Saultz & Garzon, 2016). AML is therefore primarily a disease of later adulthood (Forman et al., 2003)

3.2 AML classification

The examination of the myelogram to confirm the diagnosis of AML provides information to characterize AML: line, immunophenotyping and morphological appearance of blasts, genetic and molecular abnormalities. The pooling of this information obtained from many patients over the years has made it possible to develop classifications of subtypes of this disease. These classifications are crucial for developing management recommendations adapted to each patient (Papaemmanuil et al., 2016). Two staging systems are commonly used for AML. The first one proposed by The French- American-British (FAB) classification system is based on morphology to define specific immunotypes. The second one by The World Health Organization (WHO) classification reviews chromosome translocations and evidence of dysplasia (Brunning, 2003).

3.2.1 The French-American-British (FAB) classification of AML

In the 1970s, a group of French, American, and British leukemia experts divided AML into subtypes, from M0 to M7, based on the type of cell leukemia development and how mature the cells are. The FAB classification was mainly based on how the leukemia cells looked under the microscope after routine staining (Table 2).

24 FAB Subtype Name Adult AML patients (%) M0 Undifferentiated acute myeloblastic 5% leukemia M1 Acute myeloblastic leukemia with 15% minimal maturation M2 Acute myeloblastic leukemia with 25% maturation M3 Acute promyelocytic leukemia 10% (APL) M4 Acute myelomonocytic leukemia 20% M4 eos Acute myelomonocytic leukemia 5% with eosinophilia M5 Acute monocytic leukemia 10% M6 Acute erythroid leukemia 5% M7 Acute megakaryoblastic leukemia 5% Table 2: FAB Classification for AML (Bennett et al., 1976; Saultz & Garzon, 2016)

3.2.2 The World Health Organization (WHO) classification

The FAB classification can be useful, but it does not take into account many of the factors that are known to affect prognosis. To allow better stratification of patients, to evaluate prognosis, and to consider specialized treatment of AML, the WHO developed in 2001 a new classification of AML according to the number of genetic and cytogenetic abnormalities (D. A. Arber, 2001). Furthermore, because of the increasing recognition of the importance of genetic events in the diagnosis and treatment of AML, the proposed new WHO classification incorporates genetic aberrations and immunology as major defining features in addition to morphology (Table 3) (Hong & He, 2017).

25 WHO myeloid neoplasm and acute leukemia classification Acute myeloid leukemia (AML) and related neoplasms AML with recurrent genetic abnormalities AML with t(8;21)(q22;q22.1);RUNX1-RUNX1T1 AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22);CBFB-MYH11 APL with PML-RARA AML with t(9;11)(p21.3;q23.3);MLLT3-KMT2A AML with t(6;9)(p23;q34.1);DEK-NUP214 AML with inv(3)(q21.3q26.2) or t(3;3)(q21.3;q26.2); GATA2, MECOM AML (megakaryoblastic) with t(1;22)(p13.3;q13.3);RBM15-MKL1 Provisional entity: AML with BCR-ABL1 AML with mutated NPM1 AML with biallelic mutations of CEBPA Provisional entity: AML with mutated RUNX1 AML with myelodysplasia-related changes Therapy-related myeloid neoplasms AML, NOS AML with minimal differentiation AML without maturation AML with maturation Acute myelomonocytic leukemia Acute monoblastic/monocytic leukemia Pure erythroid leukemia Acute megakaryoblastic leukemia Acute basophilic leukemia Acute panmyelosis with myelofibrosis Myeloid sarcoma Myeloid proliferations related to Down syndrome Transient abnormal myelopoiesis (TAM) Myeloid leukemia associated with Down syndrome Blastic plasmacytoid dendritic cell neoplasm Acute leukemias of ambiguous lineage Acute undifferentiated leukemia Mixed phenotype acute leukemia (MPAL) with t(9;22)(q34.1;q11.2); BCR-ABL1 MPAL with t(v;11q23.3); KMT2A rearranged MPAL, B/myeloid, NOS MPAL, T/myeloid, NOS B-lymphoblastic leukemia/lymphoma B-lymphoblastic leukemia/lymphoma, NOS B-lymphoblastic leukemia/lymphoma with recurrent genetic abnormalities B-lymphoblastic leukemia/lymphoma with t(9;22)(q34.1;q11.2);BCR-ABL1 B-lymphoblastic leukemia/lymphoma with t(v;11q23.3);KMT2A rearranged B-lymphoblastic leukemia/lymphoma with t(12;21)(p13.2;q22.1); ETV6-RUNX1 B-lymphoblastic leukemia/lymphoma with hyperdiploidy B-lymphoblastic leukemia/lymphoma with hypodiploidy B-lymphoblastic leukemia/lymphoma with t(5;14)(q31.1;q32.3) IL3-IGH B-lymphoblastic leukemia/lymphoma with t(1;19)(q23;p13.3);TCF3-PBX1 Provisional entity: B-lymphoblastic leukemia/lymphoma, BCR-ABL1–like Provisional entity: B-lymphoblastic leukemia/lymphoma with iAMP21 T-lymphoblastic leukemia/lymphoma Provisional entity: Early T-cell precursor lymphoblastic leukemia Provisional entity: Natural killer (NK) cell lymphoblastic leukemia/lymphoma Table 3: WHO Classification for AML (Daniel A. Arber et al., 2016).

26 3.3 Cytogenetic characteristics and their prognostic impact in AML

Cytogenetically speaking, AML is a very heterogeneous disease, with more than 160 recurrent structural chromosomal abnormalities. An important mutational analysis of 18 genes, performed by Patel and his collaborators in the Eastern Cooperative Oncology Group (ECOG) E1900 patient population, showed that a more extensive mutational analysis can better discriminate patients with AML into various prognostic groups (Patel et al., 2012). A cytogenetic evaluation of myeloid disorders is useful for diagnosis to identify a proliferation as clonal or not, especially when there is a diagnostic dilemma between a neoplastic or a reactive process to choose the therapy approach (Gupta et al., 2019). Chromosomal abnormalities allow AML to be classified into three subgroups with different prognoses (Table 4): Cytogenetic abnormality Frequency Survival at five years T (15;17) (q22;q21) 70% Favorable T (8;21) (q22;22) 25% Inv (16) (p13;q22)/t (16 ; 16) (p13 ; q22) Normal karyotypes and entities 40% Intermediate not classified as favorable or 50% derogatory Abn (3q) excluding <15% T(3;5)(q2125;q3135) 25% Inv (3) (q21q226)/ t (3;3) (q21; q26) Add (5q), of the (5q), -5, -7add(7q) of the (7q) T(6;11) (q27;q23) Unfavorable T(10;11)(p1113;q23) T(11q23) excluding t(9 ;11)(p2122 ;q23) et t(11 ;19)(q23 ;p13) T(9 ;22)(q34 ;q11) -17/abn(17p) Complexe (>3 abnormalities) Table 4: The cytogenetic classification of AML (Grimwade et al., 2016)

27 3.4 Molecular basis of Acute myeloid leukemia

The molecular pathogenesis of AML has not yet been completely defined (Harada & Harada, 2015; Welch et al., 2012). Current opinions on the molecular basis of leukemia suggest that AMLs derive from at least two critical mutational events (Gilliland & Tallman, 2002).  Tyrosine kinase receptor (class I mutation): Class I mutations confer a proliferation and survival advantage to blast cells, typically as a result of aberrant activation of signaling pathways.  Transcription factor (class II mutation) Class II mutations lead to an impaired differentiation via interference with transcription factors or co-activators (Grafone et al., 2012). The cooperation between these classes of mutations leads to the emergence of leukemic cells capable of proliferation but not differentiation (Gilliland & Tallman, 2002). During the last decade, several studies have shown that the presence or absence of specific gene mutations or changes in gene expression can further classify AML cases and have an effect on the patient's prognosis (Lindsley et al., 2015; Marcucci et al., 2011). Advances in genomic sequencing technologies have helped to identify mutations in AML. Whole genome sequencing analysis has revealed that mutations are common in signaling genes that encode for the tyrosine kinases, FLT3, JAK2, cKIT, for phosphatases, PTPN11, PTPRT, PTPN14, and for Ras GTPases, KRAS and NRAS, and represent 59% of all gene mutations (Cancer Genome Atlas Research Network et al., 2013; Papaemmanuil et al., 2016). Those mutations are often independently associated with poor outcomes (Hatzimichael et al., 2013). The commonality of these mutations, particularly of tyrosine kinases, make them attractive molecular targets. However, as it stands, targeting individual mutations using precision therapies has failed to deliver the anticipated increased survival. Here we will focus on the study of FLT3, in particular the ITD mutation.

. Activating mutations in the FLT3 gene Internal tandem duplication of the FLT3 gene is found in approximately 25- 45% of adult AML and related to adverse prognosis (Gale et al., 2008; Ishikawa et al., 2009; Parcells et al., 2006). Mutations within the FLT3 gene represent one of the most frequently identified genetic alterations that disturb intracellular signaling networks with a role in leukemia pathogenesis. These mutations are required for the genesis of leukemia as they confer survival and or

28 proliferative advantages and impair cell differentiation. Indeed, RAS, FLT3, and c-KIT mutations are exclusive and account for up to 50–60 % of AML cases (D. L. Stirewalt et al., 2001).

. Missense point mutations in the tyrosine kinase domain (TKD) The second most common type of FLT3 mutation is the missense point mutation in exon 20, within the activation loop of the tyrosine kinase domain (TKD), found in 5-10% of AML patients (Yamamoto et al., 2001). These mutations lead to the overexpression or constitutive activation of the tyrosine kinase receptor. Many studies indicate that patients with FLT3 mutations have a worse prognosis than patients without FLT3 alterations. Although most affected AML patients have only one type of FLT3 mutation, some patients have both FLT3- ITD and TKD mutations (Kang et al., 2005; Whitman et al., 2008). Cellular models of AML may differ in FLT3 mutation. For example, MOLM-14 is heterozygous for FLT3-ITD, MV4-11 homozygous for FLT3-ITD, and OCI-AML3 expresses the Wild Type FLT3 receptor.

3.5 AML in pathway signaling

In AML, aberrant signal transduction enhances the survival and proliferation of hematopoietic progenitor cells (Scholl et al., 2008). The activation of signal transduction in AML may occur through a variety of genetic alterations affecting different signaling molecules, such as the FLT3 and KIT receptor tyrosine kinases (RTKs) and members of the RAS family of guanine nucleotide-binding proteins (Scholl et al., 2008). These mutant signaling proteins are attractive therapeutic targets. However, developing targeted therapies for each genotypic variant and determining the relationships between different genotypes and critical functional dependencies of leukemic cells, remain significant challenges. Several mutations lead to the constitutive activation of signaling pathways, which promote the expansion and survival of the leukemic clones. Most patients with AML show constitutive activation of the RAF/MEK/Erk and PI3K/Akt signaling cascades (A. M. Martelli et al., 2006; Platanias, 2003), known as the effector pathways of RTKs and RAS family members (Shaw & Cantley, 2006). Activating JAK2 mutations are less commonly seen, with one large study reporting an incidence of 3% in de novo AML (Sun et al., 2011). Despite this low incidence of JAK mutations, an intriguing report demonstrated that Stat3 activation is common in de novo AML (Steensma et al., 2006). Activating phosphorylation of Stat3 and Stat5a/b has been reported in various malignant cells, including several AML cell lines and a substantial

29 proportion (44-76%) of primary AML samples (Benekli et al., 2002; Gouilleux-Gruart et al., 1997; Spiekermann et al., 2002, 2002, 2003). The involvement of the JAK-Stat pathway was described in leukemia as a hematopoietic defect caused by an amino acid substitution in the Drosophila homolog of the JAK kinase (Luo et al., 1995). Several studies have revealed that other oncogenic events can drive enhanced Stat phosphorylation. For example, Gouilleux- Gruart and her collaborators, studying nuclear extracts derived from patients with AML, examined their content in Stat-DNA binding activity by using specific oligonucleotide probes and antibodies for Stat protein used in bandshift assay (Gouilleux-Gruart et al., 1996). They found that Stat proteins need to be phosphorylated before they translocate to the nucleus and bind to DNA. This statement implied that the Stat proteins detected in nuclear extracts from AML cells were constitutively phosphorylated. FLT3-ITD also leads to enhanced Stat5 signaling. Clinical trials of JAK inhibitor in AML have been initiated based on the fact that the JAK/Stat pathway is activated in many cases of AML and on data from pre-clinical studies, which have shown an anti-tumor effect of JAK-inhibitors. As a result, it has been postulated that aberrant Stat activation might play a central role in the apoptosis, resistance, growth factor independence, and deregulated cell proliferation that characterizes AML (Bar-Natan et al., 2012; Gouilleux-Gruart et al., 1996, 1997; Kirito et al., 2002; Minami et al., 1996).

30 3.6 Treatment of AML

Chemotherapy is the main treatment of AML. For more than ten years, the backbone of AML therapy has remained the same, with an initial remission, induction therapy followed by several months of consolidation (Döhner et al., 2010). There are significant issues in the treatment of AML, as the frequent resistance to available chemotherapeutic agents and the occurrence of relapses following chemotherapy due to resistant leukemic cells localized in the BM. The initial remission induction uses a combination of nucleoside analogs drugs (e.g., cytosine arabinoside) and anthracyclines antibiotics (e.g., idarubicin, daunorubicin), which interfere with DNA replication to induce apoptosis primarily in replicating cells. The consolidation therapy consists of cytosine arabinoside in multiple cycles (Siveen et al., 2017). Remission means that there is no evidence of leukemic cells in the blood and BM. The average blood cell production and normal blood counts are restored. Once a remission has been achieved, more chemotherapy is given to prevent relapse; this is called post-remission or consolidation therapy (Leukemia foundation). Treatments with modern chemotherapy regimens (cytarabine and daunorubicin) usually achieve high remission rates. However, a majority of patients relapse, resulting in only 40–45% overall 5-year survival in young patients and less than 10% in the elderly AML patients (Siveen et al., 2017). With the advent of FLT3 inhibitors, the treatment armamentarium for FLT3-mutated (FLT3+) AML is beginning to expand. Midostaurin, a multikinase inhibitor that targets FLT3, became the first targeted therapy approved by the FDA for FLT3+AML in 2017 (Garcia & Stone, 2017). Nowadays, new therapies have been developed, among which the use of autophagy inhibitors, playing a role in the pathogenesis, differentiation, relapse and drug resistance of AML (Evangelisti et al., 2015; S.-P. Zhang et al., 2013).

31 4. Tyrosine Kinases Tyrosine kinases are a family of enzymes, which catalyze the phosphorylation of select tyrosine residues in target proteins using ATP (Paul & Mukhopadhyay, 2004). Receptor tyrosine kinases (RTKs) are essential components of signal transduction pathways that mediate cell-to-cell communication. These single-pass transmembrane receptors, which bind polypeptide ligands, play critical roles in processes such as cellular growth, differentiation, metabolism, and motility (Hubbard & Miller, 2007). Due to deregulated processes, tyrosine kinases are implicated in several steps of neoplastic development and progression. In normal conditions tyrosine kinases regulate proliferation and cellular survival, when deregulated like in AML, tyrosine kinases help to escape apoptosis, increase proliferation and cellular survival (Staudt et al., 2018).

4.1 Tyrosine kinase classification

The structural organization of the receptor tyrosine kinase exhibits a multidomain extracellular ligand for conveying ligand specificity, a single-pass transmembrane hydrophobic helix, a cytoplasmic portion containing a kinase domain domain, the regulatory sequence both on the N and C terminal end, and the regulator juxtamembrane. Based on their function and structure, tyrosine kinases are primarily classified into two families. As receptor tyrosine kinase (RTK) e.g., EGFR, PDGFR, FGFR, even FLT3, and the IR. And non-receptor tyrosine kinase (NRTK) e.g., SRC, ABL, FAK, and Janus kinase (JAK). RTKs include approximately 21 families; non-receptor tyrosine kinases (NRTKs) include approximately ten groups or families based on their structure, and all share the Src- Homology 2 domain or SH-2 (Sater, 2017).  Receptor tyrosine kinase (RTK): RTK span the cell membrane with three domains: an extracellular receptor domain, a membranous anchoring domain, and an intracellular kinase domain. Ligand binding induces activation typically via structural changes in one or different neighbor receptors. The resulting alterations may lead to the proximity of membranous parts, and thus open inactive domains and turn them on to an active mode, again the “switch analogy.” The resulting process of approximating two receptor structures together is called dimerization (Lemmon & Schlessinger, 2010).

32  Nonreceptor tyrosine kinase (NRTK): NRTK are cytoplasmic proteins, exhibiting considerable structural variability. The NRTK has a kinase domain and often possesses several additional signaling or protein-protein interacting domains such as SH2, SH3, and the PH domain (Jagade et al., 2010). The tyrosine kinase domain spans approximately 300 residues and consists of an N terminal lobe comprising of a five stranded β sheet and one α helix, while the C terminal domain is a large cytoplasmic domain that is mainly α helical. ATP binds in the cleft in between the two lobes, and the tyrosine containing a sequence of the protein substrate interacts with the residues of the C terminal lobe. NRTK activation involves heterologous protein-protein interaction to enable transphosphorylation (Heldin, 1995).

4.2 Mechanism of RTK activation under normal physiological conditions

RTKs are considered as protein platforms, or the starting point for many cellular signaling pathways by recruiting enzymatic effectors (PLCγ, PI3K, Src, etc.). Either directly onto their intra cytoplasmic domain, or indirectly through adapter proteins (Grb2, Shc, etc.), forming complexes capable of activating intracellular enzymes (For example Ras). RTKs usually rest in an inactive mode until a trigger activates them. Growth factor ligands bind to extracellular regions of RTKs, and the receptor is activated by ligand-induced receptor dimerization and or oligomerization (Schlessinger, 2000). For most RTKs, the resultant conformational changes enable trans-autophosphorylation of each TKD and release of the cis-autoinhibition following ligand-induced receptor dimerization (Lemmon & Schlessinger, 2010). This change allows the TKD to assume an active conformation. Autophosphorylation of RTKs recruits and activates a wide variety of downstream signaling proteins, which contain Src homology-2 (SH2) or phosphotyrosine binding (PTB) domains. These domains bind to specific phosphotyrosine residues within the receptor and engage downstream mediators that propagate critical cellular signaling pathways (Du & Lovly, 2018; Pawson et al., 2001).

33 5. The FLT3 receptor

5.1 Protein and gene structure of FLT3

FLT3 is a receptor tyrosine kinase of 130-155 kDa and a member of the type III receptor tyrosine kinase (RTK) family (J. Zhou & Chng, 2018). Six members integrate the RTK family class III: the FLT3 receptor, C-kit, the receptor of Stem Cell Factor (SCFR) , the receptor of CSF (Colony-stimulating factor-1), PGDFR (Derived Growth Factor Platelet Receptor) alpha and beta, and the VEGF receptor (Vascular Endothelial Growth Factor 1 and 2). They are all transmembrane receptors which, like all RTK, have the function of activating cell signaling cascades after interaction with their ligand. They are characterized by the presence of five to seven immunoglobulin-like domains (De Kouchkovsky & Abdul-Hay, 2016; Terman et al., 1992). Although most cell types express the FLT3 ligand (FL), the FLT3 tyrosine kinase is expressed in multipotent hematopoietic stem cells and progenitors, but also in blast cells of most patients with AML (Birg et al., 1992; Carow et al., 1996; Garcia & Stone, 2017, p. 3; Tse et al., 2000). The FLT3 receptor, also known as FLK2 (fetal liver tyrosine kinase 2), STK- 1 (stem cell tyrosine kinase 1) or CD135, is encoded by the FLT3 gene located on chromosome 13q12 (Birg et al., 1992; Carow et al., 1995; Small, 2006). The FLT3 as a gene encodes a tyrosine kinase receptor, which plays a crucial role in controlling the survival, proliferation, and differentiation of hematopoietic cells. The FLT3 structure consists of four regions (Figure 3): i) an N-terminal extracellular region (541aa) consisting of five immunoglobulin-like domains, of which the three most distal from the plasma membrane are involved in ligand binding, while the proximal domains are involved in dimerization of the receptor; ii) a transmembrane portion (21aa); iii) a juxtamembrane (JM) domain, and iv) an intracellular C-terminal region (431aa) with a split kinase domain (S. Takahashi, 2011).

Figure 3: Schematic representation of the FLT3 receptor. Taken from (S. Takahashi, 2011)

General structure of FLT3 receptor. ECD: Extracellular domain; PM, Plasma membrane; CP: cytoplasm, TM: Transmembrane domain; JM: Juxtamembrane domain.

5.2 Signal transduction networks activated by FLT3-WT

Like other class III members, and upon activation, the FLT3 tyrosine kinase promotes the activation of downstream pathways involving phosphatidylinositol-3 kinase (PI3K), Akt, mammalian target of rapamycin (mTOR), RAS, and extracellular signal-related kinase (Erk) (Figure 4).

Figure 4: Ligand independent FLT3 activation. Taken from (El Fakih et al., 2018).

Ligand independent activation of FLT3 activates PI3/AKT, RAS/RAF/MEK, and JAK/STAT5 pathways that activate cell proliferation and anti-apoptotic effects.

35 In normal physiology, after binding to its cognate ligand (FL), FLT3 dimerizes and undergoes a conformational change, which exposes the ATP-binding pocket in the activation loop. Although the activation of the receptor is ordinarily ligand-dependent, mutations also constitutively activate the receptor and the uncontrolled proliferation of cells. Ligand- activated FLT3 next undergoes autophosphorylation and transduces downstream signals, which promotes cell growth and prevents apoptosis through various intermediaries, including RAS/RAF/MEK/extracellular signal-regulated kinase (Erk), signal transducer and activator of transcription 5 (Stat5), and phosphoinositide (PI3)-kinase (Rosnet et al., 1996). FLT3 has a crucial role in many regulatory processes of hematopoietic cells, including phospholipid metabolism, transcription, proliferation, apoptosis, and by establishing a connection with the RAS pathway. Given its apparent pivotal role in normal hematopoiesis, it is perhaps not surprising to find that FLT3 appears to play an essential role in leukemia (Figure 5). FLT3- WT causes the activation of signal transduction networks mainly through phosphatidylinositol-3-kinase (PI3K) and the cascade of RAS, supporting the activation of Akt (protein kinase B, PKB) and signal transducer and activator transcription factor (Stat) (Scholl et al., 2008).

Figure 5: Signaling pathways activated by FLT3-WT. Taken from (Grafone et al., 2012).

This illustration encompasses the activation of signal transduction networks by FLT3-WT, mainly through PI3K and RAS that likewise activates transcription of apoptotic genes, cell survival, and proliferation.

36 Activated FLT3 is associated with the growth factor receptor-bound protein-2 (GRB2). The adaptor protein GRB2 also contains an SH3 domain capable of binding of other proteins, such as SOS (guanine nucleotide exchange factor), stimulating the dissociation of GDP and the subsequent binding of GTP to RAS, leading to the activation of RAS, which stimulates downstream effectors RAF, MAPK/Erk kinase (Belov & Mohammadi, 2012). These effectors activate CREB (cyclic adenosine monophosphate response element-binding protein), ELK, and Stat, leading to the transcription of genes involved in proliferation.

5.3 FLT3 mutations

As previously stated, FLT3 is highly expressed in most cases of AML. The FLT3 mutation results in constitutive activation of the receptor, ligand-independent dimerization, and autophosphorylation leading to uncontrolled proliferation and apoptosis (Gilliland & Griffin, 2002; Small, 2006; Derek L. Stirewalt et al., 2006; Yohe, 2015). Several mutations can affect the FLT3 receptor in AML. Those mutations can be divided into two families: 1. FLT3-TKDs (Tyrosine kinase domain): Mutations in the tyrosine kinase domain (TKD) of FLT3 are less frequent (7%) and currently have no clinically significant impact (Breitenbuecher et al., 2009; El Fakih et al., 2018). 2. FLT3-ITD (Internal Tandem Duplication): Approximately 30% of AML patients harbor FLT3 mutations, with the most frequently occurring as internal tandem duplications (ITDs) of sequences within the juxtamembrane domain (Nakao et al., 1996).

5.4 FLT3-ITD

Several FLT3 activating mutations are found in leukemia patients and human leukemia- derived cell lines. The FLT3-ITD mutation was the first to be identified (Nakao et al., 1996). The FLT3-ITD mutations were discovered through the use of a reverse transcriptase- polymerase chain reaction (RT-PCR) assay to screen a series of primary leukemia specimens for FLT3 expression by Nakao et al. They first reported the presence of internal tandem duplications (ITDs) in the juxtamembrane domain of FLT3 in AML and suggested that these mutations might play an essential role in the pathogenesis of AML. ITD its a duplication of 3 to 400 base pairs in a part of the FLT3 gene sequence coding for the JM domain (exon 14 or 15), not modifying the framework of reading (Nakao et al., 1996). This modification of the JM domain suppresses the receptor’s function of inhibiting the activation of the receptor in the absence of a ligand: the mutated receptor can dimerize and

37 autophosphorylate its TK domains constitutively (H. Kiyoi et al., 1998), and then activate the downstream signaling cell (Figure 6). In the case of a heterozygous mutation, the FLT3-ITD receptor can either homodimerize or form heterodimers with the wild form of the receptor. Indeed, the structural modification of the JM domain on FLT3-ITD makes it possible to lift the auto-inhibition of the wild-type receptor in the absence of FL (Hitoshi Kiyoi et al., 2002). The ITD mutations of the FLT3 receptor found in AML patients are associated with a poor prognosis (J. Fan et al., 2010; Gilliland & Griffin, 2002). The FLT3-ITD modulates cell proliferation, survival and differentiation through constitutive activation of canonical pathways, such as PI3K/Akt, Stat5 or MAP/Erk, and cooperates with other recurrent molecular abnormalities to induce leukemia in preclinical models (Kelly et al., 2002; H.-G. Kim et al., 2007; Ono et al., 2005; Schessl et al., 2005).

Figure 6: The Constitutive activation of the FLT3 receptor by the ITD mutation. Inspired and modified from (Hitoshi Kiyoi et al., 2002) (Suer 2019)

This schema shows the dimerization of FLT3-WT by the ITD Mutation when it loses the self-inhibiting capacity of the JM domain.

Previous studies showed that FLT3-ITD facilitates the abnormal proliferation of hematopoietic cells and hinders the normal differentiation through consecutively activating: PI3K/ Akt/ Stat5 pathway and MAPK pathway (Nogami et al., 2015). A study realized by (Smith et al., 2012) validated FLT3-ITD as a therapeutic target in human AML. Their findings suggested that FLT3-ITD is capable of conferring a state of “oncogene addiction,” whereby cellular survival pathways associated with normal or precancerous cells can become hijacked, leading to a state of reliance upon crucial signaling molecules which can be

38 therapeutically exploited (Smith et al., 2012). FLT3-ITD confers resistance on cells to classic chemotherapy agents by controlling several levels of responses, restraining the entry of the genotoxic agent into the cell; for example, by inhibiting the expression of the transporter responsible for the entry of cytarabine into cells: the protein ENT1 (Equilibrate Nucleoside Transporter 1) (Jin et al., 2009). This drug resistance occurs in approximately 30% of FLT3- ITD-positive AML patients.

5.5 FLT3-ITD signaling

FLT3-ITD mutations result in the constitutive activation of FLT3 kinase. Mutations in the FLT3 JM domain and activation loop can result in the loss of the autoinhibitory function, with subsequent constitutive activation of FLT3 kinase and its downstream proliferative signaling pathways, including the Ras/MAPK kinase (MEK)/extracellular signal-regulated kinase (Erk) pathway, PI3K/Akt pathway, and Stat5. Besides, and in contrast to wild-type FLT3 signaling, FLT3-ITD potently activates the Stat5 pathway. The MAPK (mitogen-activated protein kinase) pathway is commonly deregulated in human cancer, including childhood AML (J.-D. Zhou et al., 2017). It has been well established that the PI3K/Akt pathway plays a vital role in both standard and malignant hematopoiesis (Alberto M. Martelli et al., 2010; Polak & Buitenhuis, 2012), therefore this pathway represents a promising target. It is frequently activated in various forms of leukemias, including FLT3-mutated AML, and is thought to be correlated with poor prognosis and drug resistance. The transcription factor Stat5 is an essential downstream mediator of many tyrosine kinases (TKs), particularly in hematopoietic cancers. Stat5 is activated by FLT3-ITD, which is a constitutively active TK driving the pathogenesis of AML (Wingelhofer et al., 2018).

39 6. Intracellular protein degradation A dynamic protein turnover through regulated protein synthesis and degradation ensures cellular growth, proliferation, differentiation, and adaption (Schreiber & Peter, 2014). In eukaryotic cells, there are two main intracellular protein degradation pathways, mechanistically distinct but complementary (Nedelsky et al., 2008). One is autophagy, which is a mechanism for bulk protein degradation in lysosomes (Da-wei Wang et al., 2015). The other one, the proteasome pathway, which recognizes individual proteins tagged with polyubiquitin and is known as the Ubiquitin-Proteasome System (UPS) (Cundiff et al., 2019; Hershko & Ciechanover, 1998). The UPS and autophagy-lysosome system (ALS) are two central systems involved in the regulation of protein homeostasis and housekeeping functions. They are involved in maintaining the correct levels of functional proteins, their distribution, and the elimination of damaged/misfolded proteins (Pohl & Dikic, 2019). Both UPS and ALS are involved in inflammatory processes, neurodegeneration, and cancer development, and for this reason, are considered important targets for drug development (White & DiPaola, 2009) (Figure 7). Cellular proteins are classified into “short-lived” (rapidly degraded proteins) and “long-lived” (slowly degraded proteins) (Bradley et al., 1976; Collins & Goldberg, 2017; Dice et al., 1979). The UPS serves as the primary route for the degradation for thousands of short-lived proteins and provides the exquisite specificity and temporal control needed for fine-tuning the steady- state levels of many regulatory proteins (Ciechanover et al., 2000). The UPS mediated catabolism is also essential to maintain amino acid pools in acute starvation and contributes significantly to the degradation of defective proteins (Müller et al., 2015; Vabulas & Hartl, 2005; Wheatley & Inglis, 1980). In contrast, “long-lived” proteins are preferentially degraded by autophagy, and under basal metabolic conditions mostly via microautophagy (Kocaturk & Gozuacik, 2018). Autophagy contributes to maintaining amino acid pools in the setting of chronic starvation, although its contribution to the degradation of defective proteins could be equally crucial than that of the UPS.

Figure 7: The two major protein degradation pathways. Taken from (K. Tanaka & Matsuda, 2014).

Intracellular proteolysis is broadly classified into two distinct pathways consisting of the ubiquitin-proteasome and autophagy-lysosome, which is linked to a variety of physiological and pathological mechanisms in eukaryotic cells. Like quality control, cell cycle, signal transduction, transcription, immunity, stress response, and the starvation response.

ALS and UPS have been considered to be independent and parallel systems. However, recent studies have shown that they interact under many conditions and can share the same substrates, such as the UPS substrate IĸB kinase (IKK), which is also found to be degraded by autophagy (Criollo et al., 2010), this subject will be described later. The first example of this crosstalk is the inhibition of UPS, that leads to a compensatory stimulation of autophagy via several mechanisms, whereas autophagy inhibition activates or impairs proteasomal flux depending on the cellular and environmental conditions (Ji & Kwon, 2017; Kocaturk & Gozuacik, 2018).

41 7. Autophagy Autophagy (from the Greek, “auto” oneself, “phagy” to eat) refers to any cellular degradative pathway involving the delivery of cytoplasmic cargo to the lysosome (Y. Liang, 2019). The autophagy pathway emerges during evolution as an adaptation of the eukaryotic cell to starvation, allowing mobilization of cell-autonomous nutrients by sacrificing parts of the cytosol (King, 2012). ALS is a dynamic process in which intracellular membrane structures sequester proteins and organelles into autophagosomes, which then fuse with lysosomes to form an autolysosome for degradation (Mizushima & Komatsu, 2011; Yoshimori & Noda, 2008).

7.1 Types of autophagy

Autophagy is a generic term of designating all pathways by which cytoplasm materials are delivered to the lysosome in animal cells, or the vacuole in plant and yeast (Y. Liang, 2019; Mizushima & Komatsu, 2011). Autophagy only occurs in the cytoplasm and it is constitutively on-going at low activity in most cells. There are three different types of autophagy in mammals: macroautophagy, microautophagy, and chaperone-mediated autophagy. They differ in terms of physiological functions and modes of cargo delivery to the lysosome (Parzych & Klionsky, 2014) (Figure 8).  Microautophagy: The non-selective lysosomal degradative process, consists of direct engulfment of small volumes of cytosol by lysosomes. The lysosome itself engulfs small components of the cytoplasm by inward invagination of the lysosomal membrane (Li et al., 2012; Sahu et al., 2011).  Autophagy lead by chaperones: Involves a selective, receptor-mediated translocation of proteins into the lysosomal lumen. This class does not involve membrane reorganization; instead, substrate proteins directly translocate across the lysosomal membrane with the help of chaperones (Orenstein & Cuervo, 2010; Tekirdag & Cuervo, 2018).  Macroautophagy: Is the primary type of autophagy and involves membrane formation around the cargos before fusing with the lysosomes. Macroautophagy is the most widely studied form of autophagy. In macroautophagy, a portion of cytoplasm, including organelles, is enclosed by an isolation membrane (also called phagophore) to form an autophagosome. The outer membrane of the autophagosome fuses with the lysosome, and the internal

42 material is degraded in the autolysosome. This type of autophagy will be the subject of our study, and we will refer to it merely as “autophagy.”

Figure 8: Different Types of Autophagy. Taken from (Mizushima & Komatsu, 2011).

Macroautophagy: A portion of cytoplasm, including organelles, is enclosed by an isolation membrane (called phagophore) to form an autophagosome. The outer membrane of the autophagosome fuses with the lysosome, and the internal material is degraded in the autolysosome. Microautophagy: Small pieces of the cytoplasm are directly engulfed by inward invagination of the lysosomal or late endosomal membrane. Chaperone-mediated autophagy: Substrate proteins containing a KFERQ-like pentapeptide sequence are first recognized by cytosolic Hsc70 and cochaperones. Then they are translocated into the lysosomal lumen after binding with lysosomal Lamp-2A. After all three types of autophagy, the resultant degradation products can be used for different purposes, such as new protein synthesis, energy production, and gluconeogenesis.

Autophagy is a regulated multiple-step process that involves the coordinated activity of a family of autophagy-related (Atg) proteins (Klionsky et al., 2003; Mizushima & Komatsu, 2011) (Figure 9). Each step involves different adaptors and regulators and is believed to be cargo specific.

Figure 9: The process and regulation of selective autophagy. Taken from (Mancias & Kimmelman, 2016)

The stages of autophagy (initiation, elongation, closure, maturation, and degradation) are shown in this picture. Cargo is sequestered in selective and bulk degradative manners via a double-membrane phagophore that fuses on itself to form the autophagosome. Subsequently, fuses to the lysosome (autolysosome), where lysosomal enzymes and degradation products, the cargo are recycled to the cytosol by lysosomal transporters. Upon nutrient depletion, the ULK1/2 complex is activated and can promote autophagy initiation. ULK1/2 is also activated at low-energy states (an increased AMP/ATP ratio) by phosphorylation via 5′ AMP-activated protein kinase (AMPK) as well as repression of mTORC1 activity. Activation of the selective autophagy pathway is via multiple specific stimuli and how and whether these stimuli engage the ULK complex in all circumstances is less well understood. HTT was recently identified as a scaffold protein that can activate the ULK complex and bring a selective autophagy receptor into close proximity with activated ULK complex, thereby linking activation of autophagosome formation with cargo destined for degradation. PI (3) KC3 (PI3K complex) composed of Vps34, ATG14, ATG6/Beclin1, and p150 is critical to autophagy initiation. ATG9 containing vesicles contribute membrane to the growing autophagosome and likely participate in the recruitment of other essential autophagy machinery. WIPI2 binds ATG16L1 to localize the ATG5–ATG12–ATG16L1 complex to autophagosomal membranes. This complex act downstream of the ULK and PI (3) KC3 complexes in an E3-like manner to conjugate phosphatidylethanolamine (PE) to LC3-I to produce lipidated LC3-II that then associates with autophagosomal membranes and has roles in autophagosome membrane elongation. LC3-II is present on the outer and inner surfaces of the autophagosome (depicted as a green circle with orange PE moiety) and acts as the physical link between selective autophagy receptors and the autophagosome membrane. Selective autophagic cargos depicted here include ubiquitylated mitochondria, ubiquitylated bacteria (light green rounded rectangle), ubiquitylated protein aggregates recognized by a selective autophagy receptor (brown tangle), and non-ubiquitylated cargo, such as ferritin (blue circle recognized by light blue oval depicting NCOA4).

44 7.2 Autophagy induction

The most typical inducer of autophagy is nutrient starvation, triggered by the lack of essential nutrients (Mizushima, 2007; Sakata et al., 2018). In yeast, nitrogen starvation is the most potent stimulus, but the withdrawal of other essential factors such as carbon, auxotrophic amino acids, nucleic acids, and even sulfate can induce autophagy, albeit less efficiently (Cebollero & Reggiori, 2009; Takeshige et al., 1992). In mammals, the regulation of autophagy appears to be highly complicated. The depletion of total amino acids strongly induces autophagy in many types of cultured cells, but the effects of individual amino acids differ. Leu, Tyr, Phe, Gln, Pro, His, Trp, Met, and Ala suppress autophagy in ex vivo perfused liver (García-Navas et al., 2012; Mortimore & Pösö, 1987). Other form of induce autophagy is through serum starvation. It is well known that serum starvation can induce autophagy in many types of cultured cells, increasing autophagy flux (LC3B-II) (Mizushima et al., 2010). Another example is the hematopoietic growth factor interleukin-3 (IL-3) that suppresses autophagy through the regulation of nutrient availability (Lum et al., 2005). More recently, it was shown that proteotoxic drugs such as chemical inhibitors of the proteasome could also activate autophagy in leukemic cells (Larrue et al., 2016).  Early Events—Initiation and Nucleation of autophagosome The hallmark of autophagy is the formation of the autophagosome, which can vary in size (50-1500 nm) depending on the autophagy-inducing signal, the cargo, and the cell type. Membrane dynamics during autophagy are highly conserved from yeast to plants and animals. In the first step of the autophagosome formation, cytoplasmic constituents, including organelles, are sequestered by a unique membrane called the phagophore or isolation membrane, which is a very flat organelle like a Golgi cisterna (Mizushima, 2007). Total sequestration by the elongating phagophore results in the formation of the autophagosome, which is typically a double-membraned organelle. This step is simple sequestration without degradation. Yeast genetic screens led to the identification of more than 35 Atg genes regulating the different steps of autophagosome formation (Reggiori & Klionsky, 2013; Kuninori Suzuki & Ohsumi, 2007). Many of them gather at a site which can be identified by fluorescence microscopy as a punctate spot very close to the vacuolar membrane. Since autophagosomes are generated from this site, it is called the pre- autophagosomal structure (PAS) (J. Kim et al., 2001; K. Suzuki et al., 2001; Kuninori Suzuki

45 & Ohsumi, 2007). In the PAS, the initiation, nucleation, elongation, and closure of the cup- shaped precursor membrane occur. The initiation of autophagy is controlled by the Atg1-Atg13-Atg17 complex, which is inhibited by the target of rapamycin (TOR) kinase in the presence of nutrients. The mammalian initiation complex is evolutionarily conserved, with ULK1 and ULK2 as homologs of the Ser/Thr kinase Atg1 as well as mATG13 and FIP200 (also known as RB1CC1) representing respectively Atg13 and Atg17. Atg101 is an additional component. Unlike the yeast counterpart, the mammalian ULK1/2- ATG13-FIP200 complex is stable independently of the cell’s nutrition status. Signaling kinases, such as AMP-activated protein kinase (AMPK) and protein kinase A (PKA), directly influence phagophore initiation at the level of the Atg1/ULK1 complex. Nucleation of the phagophore is controlled at PAS by the lipid kinase Vps34 and its regulatory subunits Atg14, Atg6/Vps30, and Vps15. This complex is conserved in human cells with hVPS34, ATG14L (Barkor), Beclin 1 (ATG6), and hVPS15. The net result of the Vps34 kinase activity is the production of phosphatidylinositol 3-phosphate (PI3P), which recruits factors promoting phagophore nucleation.  Elongation and closure- Critical roles of UBLs Phagophore elongation promotes the formation of the typical double-membrane autophagosome. In this step, two pathways are required, Atg7-catalyzed and ubiquitin-like conjugation. The Atg5-Atg12 conjugation is caused first by the ubiquitin-like system that subsequently forms a multimeric complex with Atg16. Next, the Atg5-Atg12-Atg16 complex binds to the external membrane of the extending phagophore (Glick et al., 2010; Kaur & Debnath, 2015). The second ubiquitin-like system results in LC3 processing, which is encoded by the mammalian homolog of the yeast Atg8. When LC3B is induced by autophagy, Atg4 proteolytically cleaves it to create LC3B-I. Atg7 activates this LC3B, which is later conjugated to phosphatidylethanolamine (PE) in the membrane to produce processed LC3B-II. The processed LC3B-II is recruited onto the developing phagophore, and its integration is dependent on Atg5-Atg12 (Y.-K. Lee & Lee, 2016, p. 8). LC3B-II, unlike Atg5-Atg12-Atg16L, is present on the outer and inner surfaces of the autophagosome, where it is needed to expand and complete the autophagic membrane. Once the autophagosomal membrane is closed, the Atg16-Atg5-Atg12 complex detaches membrane. As a result, LC3B-II can be utilized as a marker to track the extent of autophagy in cells (Tanida et al., 2008).

46 Once formed, new autophagosomes move through a stepwise maturation process that culminates with fusion to a lysosome permitting the degradation of the lumenal contents (Figure 9). In mammals, autophagosomes first fuse with endosomes and multivesicular bodies to form amphisomes, which subsequently fuse with lysosomes to create degradative vacuoles termed autolysosomes (Berg et al., 1998; Kondo et al., 2005). Autophagosomes and autolysosomes can be distinguished morphologically, as autophagosomes content has a similar density to the cytosol, while autolysosomes appear as electron-dense material with a hollow rim beneath the limiting membrane.

7.3 Autophagy and its relationship with ubiquitylation

Misfolded proteins degraded by autophagy are ubiquitylated. Hence, ubiquitylation is a common feature of UPS and autophagy substrates (O. Coux & Piechaczyk, 2000; Lamark & Johansen, 2010). Three of the four currently characterized mammalian autophagy receptors (p62, NBR1, and NDP52) contain Ub-binding domains and are therefore thought to select their targets according to their ubiquitylation status (B.-W. Kim et al., 2016; Kraft et al., 2010; Zientara-Rytter & Subramani, 2019). The autophagy core machinery harbors two evolutionary conserved Ub-like conjugation systems, which are recruited to the autophagosomal membranes during their formation, maturation, and expansion (Grumati & Dikic, 2018; J. D. Lane & Nakatogawa, 2013; Nakatogawa, 2013). The process of autophagy is controlled by parallel activation cascades, which involve Ub-like (UBL) protein modification, strikingly similar to the activation cascade that regulates the UPS (Figure 10a). The first one involves ATG7 (E1-like protein) and ATG10 (E2-like protein), and it’s responsible for the formation of the ATG5-ATG12-ATG16 complex (Figure 10b), which has been thought to act as structural support for membrane expansion. More recent work has demonstrated that the Atg12-Atg5 conjugate can function as an E3-like enzyme in the second arm of the Atg conjugation cascade to promote lipidation of Atg8. The second one includes ATG3, ATG4, and ATG7 and catalyzes the activation and membrane conjugation of the autophagy UBL modifiers (ATG8s). The large family of mammalian ATG8 proteins (mATG8s), which can be subdivided into MAP1LC3s (LC3A, LC3B and LC3C) and GABARAPs (GABARAP, GABARAP-L1 and GABARAP-L2) are known as ubiquitin-like modifiers for their homology with the Ub molecule and UBL enzymatic cascade which mediates their covalent conjugation to the lipid phosphatidylethanolamine

47 (PE) known as lipidation. Similar to ubiquitin, the ATG8 conjugation process to PE is covalent and irreversible (Figure 10C). Because of the close relation between the UPS and ALS and the similarity between the Ub molecule and the mATG8s, it is not surprising that ubiquitylation influences the course of autophagy. When the autophagy machinery needs to be shut down to avoid the excessive degradation of cellular components which will lead to apoptosis, the cross-talk between phosphorylation and ubiquitylation signals on the ULK1 and PI3K-III complexes primes the process (Antonioli et al., 2017).

Figure 10: Assembly and elongation of autophagic membranes are accomplished via sequential action of UPS-like E1-E2-E3 cascades. Taken from (Nedelsky et al., 2008).

(a) In the UPS, ubiquitylation of substrates is accomplished by an E1-activating enzyme, E2-conjugating enzyme, and an E3-ligase. (b) In the first arm of the ATG conjugation pathway, ATG 12 associates with the E1-like Atg7, is transferred to the E2-like ATG10 and is subsequently conjugated to ATG5. (c) In the second arm of the ATG conjugation pathway, ATG8 associates with the E1-like ATG7 is transferred to the E2-like ATG3 and is conjugated to PE via the E3-like action of the ATG12- ATG5 complex.

7.4 LC3/GABARAP and autophagy receptors

Selective autophagy can be considered as a surgical instrument used by the cell to eliminate protein aggregates and damaged organelles without affecting any other cytosolic component. Autophagy selectivity can be achieved by several mechanisms which are not necessarily mutually exclusive, but somewhat cooperative. The degradative process is driven and controlled by specialized molecules: the LC3/GABARAP molecules and the autophagy receptors, which physically link the autophagosomal membranes with the cargo for clearance. The first mechanism of selective autophagy is ATG8 dependent (Figure 11a).

48 Many selective substrates have LC3-interacting regions (LIRs, also known as ATG- interacting motifs (AIMs) or GABARAP-interacting motifs (GIMs), which binds to ATG8 (LC3 and GABARAPs in mammals) on autophagic membranes. Alternatively, selective substrates recruit autophagy receptors such as SQSTM1 (p62), NBR1, NDP52, and optineurin, which have LIRs. Interaction with ATG8 explains the specific engulfment of autophagic substrates inside autophagosomes. Autophagic receptors recognize their cargo by binding molecular determinants, such as unfolded regions of a protein or conjugated ubiquitin (Ub), and, via self-oligomerization, contributing to the assembly of molecular platforms on which autophagosomes form.

Figure 11: Potential mechanisms of selective macroautophagy. Taken from (Mizushima, 2018). a) Selective substrates (such as proteins, organelles, and bacteria) have or recruit autophagy receptors (for example, SQSTM1/p62, NBR1, and optineurin), which are recognized by ATG8 (LC3 and GABARAP in mammals) family proteins on the autophagosomal membrane. For recruiting receptors, substrates are often ubiquitylated and bind to the ubiquitin-binding regions in autophagy receptors. b) Autophagy receptors recruit the autophagy initiation complex (ATG1 and ULK complexes in yeast and mammals, respectively) either directly or indirectly to induce autophagy. c) Selective substrates (for example, ubiquitylated proteins, damaged organelles, or bacteria) may accumulate at the site of autophagosome formation and are engulfed by autophagosomes even without direct recognition. d) Autophagic membranes may elongate by wetting on the surface of intracellular condensates (liquid/fluid droplets) generated by liquid-liquid phase separation. In the second mechanism, some selective substrates are known to trigger autophagosome formation by recruiting the ATG1 (ULK in mammals) autophagy-initiation complex (Figure 11 b). The third one is independent of ATG8 and probably any other ATGs (Figure 11 c). Finally, some autophagic substrates might display properties of intracellular condensates (also called liquid or fluid droplets) and provide a surface on which autophagosomal membranes can preferentially elongate. It is known that liquid-liquid phase separation produces intracellular condensates which can wet membranes (Figure 11d). A growing number of subcellular structures are cleared by selective autophagy, and various terms have been

49 coined to reflect the exquisite target specificity of the pathway as autophagic processes: (Figure 12).  Mitophagy: mitochondria  Nucleophagy: portions of the nucleus  Pexophagy: peroxisomes  Reticulophagy: endoplasmic reticulum  Xenophagy: microorganisms  Ribophagy: ribosomes  Aggrephagy: protein aggregates The degradation of the proteasome by autophagy has been more recently discovered and named Proteaphagy, which will be discussed in another section (Marshall et al., 2015; Rogov et al., 2014).

Figure 12: Selective autophagy receptors: ubiquitin-mediated cargo recognition. Taken from (Rogov et al., 2014).

The terms aggrephagy, glycophagy, ribophagy, etc. reflect the recognition and degradation of a particular type of cargo: protein aggregates, glycogen, ribosomes. Established (black) and putative (red) selective autophagy receptors for the respective processes are listed. Question marks indicate as-of-yet unidentified receptor proteins.

There are multiple different types of selective autophagy receptors, but they can be grouped into two categories based on the participation of Ub on cargo recognition. In both cases, receptors harbor an LIR motif to bind the ATG8s, which are linked to the expanding autophagosomal membranes (Figure 13). The two types of selective autophagy are not exclusive, frequently both kinds of receptors are found on the same cargo (Johansen & Lamark, 2019, p. 8; Khaminets et al., 2016; Rogov et al., 2014; Stolz et al., 2014). Even if

50 selective autophagy receptors have been studied in detail in yeast and mammalian systems, much of the core machinery for autophagosome initiation has been conserved and common pathways exist. Nonetheless, there is significantly less conservation between yeast and mammalian cells concerning the selective autophagy machinery (L. Liu et al., 2014). Indeed, the study of selective autophagy pathways in mammalian cells has revealed important clues to their roles in diseased-related processes.

Figure 13: Ubiquitin-dependent and Ubiquitin independent autophagy.Taken from (Mancias & Kimmelman, 2016).

On the left, a prototypical selective autophagy receptor with a UBD recognizes a ubiquitylated substrate (protein aggregate) and physically links the aggregate to the autophagosome via an LIR motif that binds to lipidated and autophagosome membrane associatedATG8 (LC3-II). On the right, FAM134B-mediated-phagy, an ubiquitin-independent selective autophagy pathway, is depicted. Here, FAM134B is an ER-membrane protein with an LIR motif that is recognized by autophagosome associated LC3-II in order to deliver predominantly cisternal ER to autophagosomes for degradation.

. Ubiquitin-dependent selective autophagy With the identification of the original mammalian selective autophagy receptors, it was clear that Ub plays a central role in selective autophagy (Chen et al., 2019). Selective autophagy relies on cargo-specific autophagy receptors, which facilitate cargo sequestration into autophagosomes. The role of Ub-binding proteins in autophagy is just beginning to be unraveled. Several proteins involved in recognition of autophagy targets are Ub-binding proteins, such as p62/SQSTM1, which contains a carboxy-terminal ubiquitin associated

51 domain (UBA). The classic example are aggregates of either aberrantly folded or unused proteins are ubiquitylated and bound by the ubiquitin-binding domain (UBD) containing receptors such as p62/SQSTM1 for subsequent delivery to the autophagosome for lysosomal degradation. It is clear that there is a cooperative function ALS with the UPS to manage the turnover of damaged proteins to maintain the proteome. Indeed, ubiquitin- containing inclusion bodies stain positive for p62 and LC3/Atg8, and their degradation by autophagy depends on the presence of p62 and its ability to interact with LC3 and Ub. Thus, p62 and other autophagy receptors seem to act as adaptors between ubiquitylated protein aggregates and the autophagic machinery (Figure 14).

Figure 14: The autophagy receptor p62. Taken from (Lamark et al., 2017)

(A) Domain architecture. (B) Interactions needed for linking the cargo to a growing phagophore. The C-terminal UBA domain interacts with ubiquitin chains attached to the cargo. The LC3-interacting region (LIR) motif interacts with ATG8 family proteins covalently attached to the inner membrane surface of a growing phagophore. The N-terminal PB1 domain self-interacts into polymeric structures, and this enables a tight interaction between the cargo and the phagophore.

. Ubiquitin-independent selective autophagy Ubiquitin-independent selective autophagy receptors identify a wide variety of protein, lipid, or sugar-based signals. Two recently identified ubiquitin-independent selective autophagy receptors are example of this process: NCOA4 for ferritinophagy (ferritin autophagy), and the FAM134 protein family for ER-phagy. In yeast, Atg11 acts as an adaptor protein for Ub- independent selective autophagy (Mochida et al., 2015).

52 7.5 The role of Autophagy in cancer

Loss of a survival pathway, such as autophagy, undermines tumorigenesis. It is unclear how autophagy can be interpreted in the context of cancer, the autophagy role in cancer, in effect, is paradoxical. On the one hand, its cytoprotective traits could promote the survival of normal and cancer cells (Lum et al., 2005; White, 2015), as well as promote resistance to a variety of therapies (Amaravadi et al., 2007). Whereas on the other hand, autophagy might suppress tumor growth, senescence, and oxidative stress (Maiuri et al., 2009; Mathew et al., 2009; Takamura et al., 2011). This apparent dual role of autophagy can be explained in part because of the same elements that promote tumorigenesis at initial stages (ROS, inflammation, DNA damage) can be deleterious at later stages (Poillet-Perez et al., 2015; Srinivas et al., 2019; H. Zhou et al., 2014). Indeed, the role of autophagy in cancer is dynamic, the pro and anti-tumorigenic potential depends, in part, on tumor type and stage (Mah & Ryan, 2012; Rosenfeldt & Ryan, 2009) (Figure 15). Other role of autophagy in cancer, is inhibiting the initiation of tumorigenesis by limiting damaged cytoplasme and organelles that produce genotoxic stresses such as free radicals, genomic instability, inflammation, and the loss of specific autophagy genes that can lead to cancer. The role for autophagy as a survival mechanism in normal cells and in tumor cells seems to contradict the observation that loss-of-function mutations in the autophagy pathway are associated with tumor progression (Aita et al., 1999; Degenhardt et al., 2006; X. H. Liang et al., 1999; Qu et al., 2003). Viewed just as a signaling pathway, most of the proteins that participate in the regulation of autophagy are either tumor suppressor proteins or oncogenes. Thus, mechanisms involved in the regulation of autophagy largely overlap with signaling pathways implicated in the control of cancer (Ávalos et al., 2014). Tumor suppressor genes that negatively regulate mTOR, such as PTEN, AMPK, LKB1, and TSC1/2 stimulate autophagy while, conversely, oncogenes that activate mTOR, such as class I PI3K, Ras, RHEB, and AKT, inhibit autophagy (Choi et al., 2013). We briefly mention the accumulated evidence from humans and mouse models about the function of ATGs, their direct binding partners, and their roles in either protecting against or promoting cancer:  Beclin 1 (BECN1) The possibility that defective autophagy may play a role in cancer was first recognized through studies focused on Beclin 1 (BECN1) since, at the early stage of tumor development, autophagy functions as a tumor suppressor (Aita et al., 1999; Miracco et al., 2007).

53 Expression of BECN1, a mammalian orthologue of yeast autophagy-related gene Atg6, reduces tumorigenic capacity through induction of autophagy (Kondo et al., 2005). In various murine models, the defects in autophagic machinery caused by the whole-body or tissue- specific, heterozygous or homozygous knockout of essential autophagy genes accelerate oncogenesis. For instance, the role of autophagy in tumor suppression has further been established by the identification and characterization of Beclin 1-interacting proteins. In other studies, done in mice lacking one copy of the gene coding for the BECN1 interactor autophagy/beclin-1 regulator (AMBRA 1) also exhibit a higher rate of spontaneous tumorigenesis than their wild type counterpart (Cianfanelli et al., 2015). Co- immunoprecipitation studies have identified class III phosphoinositide-3-kinase (PI3KIII)/Vps34 as a significant physiological partner of BECN1 in a complex required for autophagy initiation (Petiot et al., 2000) and tumor suppression (Furuya et al., 2005).  ATG5 and ATG7 ATG5 and ATG7 are two critical autophagy regulators that have been deleted in experimental animal models (ARAKAWA et al., 2017, p. 5). A decrease in ATG5 expression, a key regulator of autophagy, reduced survival rate in 158 primary melanoma patients and decreased ATG5 expression promote cancer-cell proliferation and is associated with the progression of early-stage cancer (H. Liu et al., 2013). In mice with knockout of autophagic core proteins, (ATG5 and ATG7) promotes the development of liver cancers due to damaged mitochondria and oxidative stress in hepatocytes (Takamura et al., 2011).  ATG4 The role of ATG4B in cancer has been recently reported. For example, tumor cells of colorectal cancer patients had significantly higher ATG4B expression levels than adjacent normal cells. Suggesting that ATG4B plays a decisive role in colorectal cancer development, although the enhancement for cell proliferation by ATG4B is independent of autophagy (P.- F. Liu et al., 2014).

Figure 15: Autophagy has multiple roles during tumorigenesis. Taken from (E. Y. Liu & Ryan, 2012)

Autophagy can both inhibit and promote cancer formation through different mechanisms, depending on the stage of the tumor. It can limit proteome in the primary tumor. In tumor metastasis, it can promote cancer cell survival against anoikis. It also can inhibit angiogenesis and necrosis. It can assist cells in dealing with metabolic stress.

 UVRAG During autophagy UVRAG interacts with Beclin 1 to promote autophagosome formation. Its role in autophagy is as a tumor suppressor that is commonly mutated in colon and breast cancer. UVRAG expression in human HCT116 colon carcinoma cells reduces the tumorigenic potential of these cells in xenograft studies (Park et al., 2014).  BIF1 BIF1 (SH3GLB1) is another protein that positively regulates autophagy, through interaction with UVRAG and Beclin 1 (Y. Takahashi et al., 2007, p. 1). B1F1-knockout mice are born viable and frequently grow, without any apparent abnormalities except for an enlarged spleen. However, they do have a higher propensity to develop spontaneous cancer (mainly lymphomas and, to a lesser extent, solid tumors) at an average age of 12 months. BIF1 is thus a positive regulator of autophagy and a potential tumor suppressor.

55 7.6 Autophagy inhibitors

An exploitative cancer therapy is altering autophagic, either downregulating or up-regulating it (Cuomo et al., 2019). As autophagy is a survival pathway used by tumors cells to tolerate metabolic stress, then the ability to inhibit autophagy and sensitize apoptosis-resistant tumor cells to metabolic stress is promising for cancer therapy. Given the fact that autophagy may express either cytoprotective or cytotoxic function, it is predictable that ongoing clinical trials involve the combination of pharmacologic autophagy inhibitors. As a result, several autophagy inhibitors have been developed and can be used to study the role of autophagy in cancer development (Pasquier, 2016) (Table 5). Although autophagy is found hyperactivated in many tumor cells, only some cancer cell types are more sensitive to autophagy inhibitors, and this should be taken into account when making clinical decisions. Besides, the timing of therapy is an important criterion to evaluate when constructing a treatment regimen. In patient’s tumors, autophagy inhibition should theoretically enhance tumor cell sensitivity to the radiation or the drugs inducing the autophagic response (Gewirtz, 2016). Under this premise, over the past decade, several Phase I and I/II clinical trials have been published using autophagy inhibitors (Haas et al., 2019; Lotze et al., 2014; Xu et al., 2018). For example, chloroquine or hydroxychloroquine with chemotherapy or radiation in various forms of cancer, generating contradictory, or at the very least equivocal results (Poklepovic & Gewirtz, 2014). Another example is the inhibition + of autophagy by the H -ATP inhibitor bafilomycin A1 (BafA), which increase apoptotic cell death in various cancer cells treated by irradiation or chemotherapy (Sukhai et al., 2013). Protein turnover by lysosomal degradation through the autophagy pathway is functionally coupled to, and compensatory with, the ubiquitin-mediated proteasome protein degradation pathway (Ding et al., 2007; Pandey et al., 2007; Rubinsztein, 2006) and for this reason, there is potential synergy with proteasome inhibitors.

56 Modification of Cancer type Compound Inhibitor cellular References component Breast cancer, (Boya et al., 2005; Inhibits the prostate Bursch et al., 1996; T. 3-MA III-PI3K formation of pre- cancer, colon Kanzawa et al., 2004; autophagosomal cancer, Paglin et al., 2001; structure malignant Petiot et al., 2000) glioma, and cervical cancer Breast cancer, Blocks the prostate (Boya et al., 2005; Bafilomycin H+-ATPase fusion of the cancer, colon Takao Kanzawa et al.,

A1 inhibitor. autophagosome cancer, 2003; Komata et al., and lysosome malignant 2004; Paglin et al., glioma, cervical 2001) cancer Blocks the CQ and Lysosomotropic fusion of the HCQ agent autophagosome Cervical cancer (Boya et al., 2005) and lysosome Proton Blocks the Monensin exchange for fusion of the Cervical cancer (Boya et al., 2005) potassium or autophagosome sodium and lysosome Inhibits autophagy by Formation of blocking Pancreatic Verteporfin high-MW forms autophagosome cancer (Donohue et al., 2011) of p62 that formation at a resist step following denaturation the recruitment of lipidated LC3 3-MA, 3-methyladenine; HCQ, Hydroxychloroquine; PI3K, phosphatidylinositol 3-phosphate kinase Table 5: Autophagy inhibitors

57 8. The proteasome

8.1 Function, structure and organization

The proteasome, also referred to as 26S complex, is a large multiprotein intracellular protease (1500-2000 kDa), with a cylindrical shape, and a molecular weight of approximately 2.5 MDa expressed in the nucleus and cytoplasm of all eukaryotic cells (Bard et al., 2018; Voges et al., 1999). The 26S proteasome is involved in the regulated degradation of ubiquitylated proteins in the cell (Kisselev & Goldberg, 2001). There are several types of proteasome according to its cellular functions, such as the proteasome involved in the immunology response, which is called “immunoproteasome, responsible for the degradation of the peptide presented to cellular surface in the major histocompatibility complex (MCH).The 26S proteasome consists of two distinct sub-complexes: a 20S core particle (CP) and one or two 19S regulatory particles (RP, also termed PA700), which associate with the termini of the barrel-shaped central particle (Bard et al., 2018; Zwickl et al., 1999). The 19S RP provides the link for proteasome-mediated proteolysis with the UPS pathway. That is because the 19S can recognize ubiquitylated proteins, and play a role in their unfolding and translocation into the interior of the 20S CP, which contains catalytic threonine residues on the surface of a chamber formed by two -rings (Olivier Coux et al., 1996; Hershko & Ciechanover, 1998; Hochstrasser, 1996; Voges et al., 1999) (Figure 16).

Figure 16: 26S proteasome structure. Taken from BostonBiochem.

A schematic of the 26S proteasome. The 26S proteasome is a 2000 kDa multiprotein complex comprised of a proteolytically active 20S core particle that is capped by 1 or 2 19S regulatory particles. The 19S regulatory units recognize ubiquitylated proteins and control access to the proteolytic core.

58 8.2 The CP or 20S proteasome

The 20S CP (alias 20S proteasome) is structurally well characterized. When viewed electron microscopically, the 20S proteasome appears as a cylinder-like structure in various eukaryotes, including yeast and mammals (Harris, 1968). It forms a packed particle, a result of axial stacking of two outer α-rings and two inner β-rings, which are made up of seven structurally similar α and β subunits, respectively. The outer α-rings contain seven similar, yet distinct α-subunits (α1-α7), and by forming a pore, they function as a tightly regulated “gate” for the entrance of substrates, and removal of degradation products from the complex. This “gate,” which is made of the N-termini of a subset of α-subunits, blocks the unregulated entrance of substrates into the catalytic chamber. A crucial role in the organization and activation of the “gate” is attributed to the N-terminus of the α3-subunit since its deletion results in a constitutively open pore (Adams, 2003; Groll et al., 2000). The 20S proteasome has at least five peptidase activities. Nevertheless, only three catalytic subunits have been identified. These three β subunits carry out enzymatic activities of the proteasome (Hershko & Ciechanover, 1998; Kisselev et al., 1999)

1. β1 Caspase like activity

2. β2 Trypsin like activity

3. β5 Chymotrypsin like activity

8.3 The RP or 19S regulatory complex

The regulatory particle (RP), 19S RP, is a complex of 700 kDa only found in eukaryotes. The RP recognizes proteins marked by polyubiquitin chains, removes the chain and entraps the protein moiety, unfolds the substrate proteins, opens the α-ring, and transfers the unfolded substrates into the CP for destruction. The enzymatically active proteasome is generally capped on either or both ends of the central 20S proteasomal core by regulatory proteins. The RP can be separated biochemically into the base and lid subcomplexes, the 19S RP comprises approximately 20 different subunits that can be subclassified into two groups:  The regulatory particle of non-ATPase (Rpn) subunits  The regulatory particle of triple-ATPase (Rpt) subunits Both of which contain multiple proteins with molecular masses ranging from 10 to 110 kDa.

59 8.3.1 The lid and base subcomplex

The lid complex is composed of at least nine non-ATPase subunits: Rpn3, Rpn5, Rpn6, Rpn7, Rpn8, Rpn9, Rpn11, Rpn12, and Rpn15. (Dambacher et al., 2016). The primary function of the lid is to deubiquitylate the captured substrates, a process in which the metalloisopeptidase Rpn11 functions to recycle the Ub, process that we will describe later (Wehmer & Sakata, 2016). The base complex is composed of four non-ATPase subunits (Rpn1, Rpn2, Rpn10, and Rpn13). And six homologous AAA-ATPase subunits, (Rpt1–Rpt6) and the base complex of proteasomes has three functional roles: capturing proteins via Ub recognition, promoting substrate unfolding and opening the channel in the α-ring. Recently, a novel functional unit within the lid complex, comprising two subunits, Rpn1 and Rpn2 was proposed; i.e., Rpn2 interfaces with the 20S, whereas Rpn1 sits atop Rpn2, serving as a docking site for a substrate recruitment factor (Rosenzweig et al., 2008). The lid ATPases encircle the Rpn1- Rpn2 stack, covering the remainder of the 20S surface. Both Rpn1-Rpn2 and the ATPases are required for substrate translocation and gating of the proteolytic channel(Pick & Berman, 2013). Rpn1, Rpn10 and Rpn13 function as integral ubiquitin receptors and efficiently trap polyubiquitylated substrates. Rpn1 and ubiquitin receptors of intrinsic proteasome subunits, harbor UBDs specific for Lys48-linked ubiquitin chains, which thus target substrates for proteasome-mediated degradation. Rpn10 and Rpn13 are the major docking subunits harboring one to three docking sites for various UBL-UBA factors. Rpn10 achieves this function via a C-terminal ubiquitin-interacting motif (UIM) (Deveraux et al., 1994), Rpn10 is an exceptional Ub receptor, as it functions both as part of the 26S proteasome complex and as a free entity. More recently, Rpn13 was identified as a second ubiquitin receptor (Husnjak et al., 2008; Schreiner et al., 2008). Both Rpn10 and Rpn13 also bind strongly proteins bearing Ub-like (UBL) domains but have an only weak affinity for free Ub.

60 9. Protein Ubiquitylation Ubiquitin (Ub) is a short 76-amino acid of 8.5 kDa as molecular weight. And bears many potential sites for additional post-translational modifications. Ub exists in cells, either free or covalently linked to other proteins, it is a member of the ubiquitin-like (UBL) protein family and shares sequence and structural homology with proteins like Small Ubiquitin-like Modifier (SUMO) (Mahajan et al., 1997; Matunis et al., 1996; Swatek & Komander, 2016), Interferon Stimulated Gene 15 (ISG15) (Loeb and Haas, 1992), and Neural precursor cell Expressed, Developmentally Down-regulated 8 (NEDD8) also known as RUB (Related to Ubiquitin) in yeast (Kamitani et al., 1997). It is a highly conserved protein from yeast to human, ubiquitously present in eukaryotes (Hershko & Ciechanover, 1998; Smalle & Vierstra, 2004; Vierstra, 2009). Ub is among the most physically stable proteins known and remains folded adequately at temperatures up to 85°C and between a pH of 1 and 13 (Lenkinski et al., 1977). Posttranslational modification of cellular proteins by Ub plays a critical regulatory role in numerous cellular processes, including DNA repair (Schauber et al., 1998; Song & Luo, 2019), transcription (Kaiser et al., 2000), splicing, mRNA export, intracellular trafficking (Strous et al., 1996), signal transduction (Khush et al., 2002) and proteolytic control, but the role of Ub in selective, intracellular protein degradation remains its best-understood function. Post-translational modifications are achieved by E1, E2, and E3 enzymes that sequentially catalyze activation, conjugation, and ligation reactions, respectively, leading to covalent attachment of ubiquitin, usually to lysine residues of substrate proteins (Song & Luo, 2019). In the UPS pathway, ubiquitin modification serves as the recognition motif by the proteasome, which processes proteins into small polypeptides (Cundiff et al., 2019). Ub can be conjugated post-translationally to other proteins via an isopeptide linkage between glycine-76 of Ub and, most typically, the ɛ-amino group of lysine within the target protein. Ub itself contains seven lysine residues, allowing the formation of Ub-Ub polymers with distinct architectures, including homotypic and mixed ubiquitin chains that acquire different conformations (Figure 17). Protein substrates can be modified at specific lysine (monoubiquitylation) or multiple lysines (multiubiquitylation) by a single Ub or by a chain of Ub units linked by one of its seven lysines (polyubiquitylation) to generate a diverse set of Ub signals with distinct functions. Monomeric Ub and Ub chains of different linkage types are functionally distinct signals, for example, whereas monomeric Ub is the dominant signal for

61 intracellular trafficking, the K63-linked ubiquitin chain is involved in DNA repair and signal transduction (Pickart & Fushman, 2004). The predominant Ub signal for UPS appears to be the K48-linked and K11-linked Ub chain with a minimum length of four Ub units (Nathan et al., 2013; Thrower et al., 2000). However, multiple mono-ubiquitylation was as well proposed as a signal for proteasomal degradation (Braten et al., 2016), (Table 6). A significant number of Ub-binding domains (UBDs) are responsible for recognizing the diverse Ub signals and targeting modified proteins to specific cellular processes, including UPS. The majority of proteins that are destined for degradation are marked by the covalent attachment of multiple ubiquitin molecules, which provide a recognition signal for the 26S proteasome.

Figure 17: ubiquitin chains and their primary functions. Taken from (Gómez-Díaz & Ikeda, 2018).

Substrate ubiquitination with eight possible linkage types (Met1/Linear, Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, and Lys 63.

Substrate Type of Function Reference ubiquitylation α-Synuclein Monoubiquitylation Proteasomal (Liani et al., 2004) degradation Pax3 Monoubiquitylation Proteasomal (Boutet et al., 2007) degradation PLD1 Multiple Proteasomal (Yin et al., 2010) monoubiquitylations degradation Cyclin B1 Multiple Proteasomal (Dimova et al., 2012) monoubiquitylation degradation GOT1 Mono/multiple Proteasomal (Braten et al., 2016) monoubiquitylation degradation GRE1 Mono/multiple Proteasomal (Braten et al., 2016) monoubiquitylation degradation Table 6: Mono/multiple monoubiquitylated proteasomal substrates

9.1 Major Ubiquitin receptors for proteasomal degradation Ubiquitylated substrates are primarily recognized by three classes of highly conserved ubiquitin receptors (Callis, 2014). The first one involves intrinsic 26S proteasome base subunits, including Rpn10 (Haririnia et al., 2007), and Rpn13 (Husnjak et al., 2008; Schreiner et al., 2008), which are capable of directly recognizing ubiquitylated substrates. The second class involves the ubiquitin-like domain (UBL)-containing shuttle factors, including Rad23 (in human hHR23A), Dsk2, and Ddi1. These UBL-factors are referred to as shuttle factors because they are capable of targeting ubiquitylated substrates to the 26S proteasome simultaneously using one N-terminal UBL and one to two C-terminal ubiquitin-associated domain (UBA) domains respectively (Figure 18) (Funakoshi et al., 2002; Kaplun et al., 2005; Saeki et al., 2002). The third class involves Cdc48-based complexes, which are primarily involved in endoplasmic reticulum-associated degradation (ERAD) (Elsasser & Finley, 2005; Raasi & Wolf, 2007). The predominant steps in controlling substrate specificity are regulated by post-translational modification or conformational changes in the substrates and by the association between the substrates and their conjugating enzymes (Ravid & Hochstrasser, 2008). However, accumulating evidence indicates that substrate specificity can also be determined at the step of ubiquitylated substrate recognition by ubiquitin receptors. A major challenge is defining the mechanistic details of individual recognition pathways for ubiquitylated substrates and determining the functions and associated substrates of individual recognition pathways. Several major ubiquitin receptors are not only involved in ubiquitin recognition functions but also perform other functions. For example, Rpn10 is also involved in the stable lid-base association of the 26S proteasome (H. Fu et al., 2001; Lin & Fu, 2012), whereas Rpn13 is also involved in recruiting the DUBs, UCH37 (Husnjak et al., 2008; Schreiner et al., 2008). Rad23 has an additional function in DNA repair (Dantuma et al., 2009). Furthermore, the yeast (Saccharomyces cerevisiae) Ddi1 has a function in the late secretory pathway (Gabriely et al., 2008). Differentiating structural elements required for the different functions of these ubiquitin receptors remains a challenge.

Figure 18: Recognition of the ubiquitylated substrates for the 26S proteasome-mediated proteolysis. Taken from (Hongyong Fu et al., 2010).

Schematic diagrams show the primary ubiquitin receptors from yeast, human, and Arabidopsis involved in recognition of the ubiquitylated substrates during UPP. (a) Domain organization of two significant classes of ubiquitin receptors for ubiquitylated UPP substrates. The first class includes intrinsic 26S proteasome base subunits Rpn10 and Rpn13. The second class includes ubiquitin-like (UBL)-containing shuttle factors that contain UBL and ubiquitin-associated (UBA) domains: Rad23, Dsk2 and Ddi1. A third class contains Cdc48 complexes generally involved in endoplasmic reticulum-associated protein degradation (ERAD) and is not depicted here. Annotated sub-regions are various domains and motifs identified using the Simple Modular Architecture Research Tool from the ExPASy proteomics server (Swiss Institute of Bioinformatics, http://www.expasy.ch/). The blue sub-region marked with a question mark designates a potential novel ubiquitin-binding domain in human Ddi1, which is not yet defined. Accession numbers for sequences of various proteins studied in a relisted. (b) Ubiquitylated protein substrates can be recognized directly by intrinsic proteasome base subunits Rpn10 or Rpn13 (red arrow) or indirectly by UBL-UBA shuttle factors (black arrow). Indirect recognition by shuttle factors requires an additional proteasome docking step mediated, primarily, by sites located on Rpn1, Rpn10 or Rpn13(blue arrow). ERAD substrates are recognized indirectly by Cdc48 complexes (gray arrows). Small purple circle chains designate attached ubiquitin polymers. Annotated sub- regions of proteasome subunits are domains involved in receiving substrates and UBL-UBA factors. ASP, retrovirus aspartic protease; LRR, leucine-rich repeat; PRU, pleckstrin-like receptor of ubiquitin; STI, heat shock chaperone in-binding domain; UBA, ubiquitin-associated; UBL, ubiquitin-like; UIM, ubiquitin-interacting motif; and vWA, von Willebrand factor type A domain.

64 9.2 Purification of Ubiquitylated proteins: TANDEM UBIQUITIN BINDING ENTITIES (TUBE) Protein ubiquitylation is a transient posttranslational modification since polyubiquitin chains are continuously removed from proteins by deubiquitylating enzymes (DUBs), but also because of the proteasome degrade many ubiquitylated proteins. For these reasons, it has always been a technical challenge to study post-modification events. Various strategies have been used to isolate ubiquitylated proteins (Aillet et al., 2012), or analyze the ubiquitin proteome or ‘ubiquitome’ by mass spectrometry (MS) (Mattern et al., 2019). MS allows the identification of Ub chains and proteins implicated in a biologic process. The use of tagged forms of Ub (often His-tag) allowed the identification of most known ubiquitylated proteins (Hjerpe et al., 2009). One of the most successful recent methodologies for the detection of ubiquitylated proteins is the use of Ub Lys-ɛ-Gly-Gly (diGly) remnant antibodies allowing to increase the number of identified ubiquitin signatures from hundreds to thousands (Fulzele & Bennett, 2018; Ordureau et al., 2015). In our laboratory, we have developed tools to capture endogenous proteins modified by members of the Ub family. Our tools, which we generically call molecular traps, show high affinity and specificity for Ub or UbL without cross- reaction. Our molecular prototype traps named TUBEs (for Tandem ubiquitin-binding entities) are based on the multiplication of UBDs. TUBEs have a superior affinity for polyubiquitin chains compared to traditional UBDs (up to a 1000-fold increase in affinity). These TUBEs can be used to both stabilize poly-Ub chains and purify ubiquitylated proteins, making them valuable tools. The prototype TUBEs are based in the multiplication of Ubiquitin Associated domains (UBA) from the DNA repair protein hHR23 and Ubiquilin-1 and recognize multiple chain types but in different proportions (Altun et al., 2011) (Figure 19). TUBEs efficiently capture endogenous ubiquitylated proteins from cell cultures, tissues, organs and samples from patients, increasing the value of these tools for the identification of ubiquitylated proteins from distinct cell types and physiological conditions (Lopitz-Otsoa et al., 2012; Yi Shi et al., 2011; Yoshida et al., 2015). Application-specific TUBE adjustments have been introduced, such as Ub chain protection from trypsinization (Ub-Prot) to determine Ub chain length (Tsuchiya et al., 2018) and sensor-based chain-specific TUBEs, like the linear ub-specific M1 specific ub binder (SUB) (Keusekotten et al., 2013).

Figure 19: The tandem disposition of ubiquitin-associated domains preserves their ubiquitin-binding capacity. Taken from (Hjerpe et al., 2009).

Cartoon illustrating the design of TUBEs. A flexible linker separates UBA domains

Last but not least, biochemical measurements often occur post-lysis and can potentially increase the incidence of artifacts. For example, once the proteins of interest are identified, most analyses of ubiquitylated proteins end by using antibodies against the target protein and or the modifier, like immunoblotting, immunofluorescence, or ELISA. These semi- quantitative analyses of protein ubiquitylation offer the possibility to explore physiologic or pathologic events in time and space with high specificity, efficiency, sensitivity and relatively low cost. However, a wrong analysis with inadequate ubiquitin tools or with an inappropriate condition can lead to the misinterpretation of results and or erroneous conclusions (Emmerich & Cohen, 2015).

66 10. Ubiquitin-Proteasome System (UPS) The cellular wellbeing depends on the accurate control of the activity and stability of critical cellular factors. More than 80% of all eukaryotic intracellular protein degradation is controlled by UPS (van Wijk et al., 2019; Zwickl et al., 1999). The UPS is a major proteolytic pathway commonly found in all eukaryotic cells in the cytosol and nucleus and is responsible for the degradation of the redundant or abnormal short-lived protein, it is, therefore, a critical player in maintaining protein homeostasis (Bard et al., 2018; Hershko & Ciechanover, 1986; Lilienbaum, 2013). This pathway includes the machinery implicated in protein ubiquitylation, and the subsequent recognition and degradation by the proteasome (Figure 20). The ubiquitylation is generated by a chain of biochemical events, which will be discussed in the next section.

Figure 20: Functional representation of the mechanism of ubiquitin conjugation and presentation to the proteasome. Taken from (Huang & Dixit, 2016).

Proteins become ubiquitylated by enzymes E1, E2, and E3. E1 activating enzymes form a thioester bond with ubiquitin, followed by subsequent binding of ubiquitin to E2 conjugating enzymes, and ultimately the formation of an isopeptide bond between the carboxyl-terminal glycine of ubiquitin and a lysine residue on the substrate protein, which requires E3 ubiquitin ligases. Multiple intervention nodes in the reaction cascade have been proposed to either block or enhance ubiquitination

Degradation of a protein via the UPS involves two discrete and successive steps: a) Tagging of the substrate by covalent attachment of multiple Ub molecules. b) Degradation of the tagged protein by the 26S proteasome complex with the release of free and reusable Ub. Proteins destined to be degraded by the UPS are tagged for the destruction by conjugation to Ub through the action of three main categories of enzymes (Figure 20):

67 1) E1: The Ub-activating enzyme (UBA) 2) E2: A Ub-carrier protein, also called Ub-conjugating enzyme, (UBC) 3) E3: The Ub ligase. In the first step, the E1 initiates ubiquitin-conjugation by adenylating ubiquitin (Figure 20). One ATP molecule is expended for each E1-ubiquitin linkage. The Ub molecule is transferred to the ubiquitin-conjugating enzyme E2, which transiently carries Ub. E2s work in conjunction with the Ub ligase E3, which is responsible for conferring substrate specificity to the reaction. E3 mediates the transfer of Ub to an internal lysine of the target protein or ubiquitin itself. The first conjugated Ub serves as the nidus for the formation of a polyubiquitin chain, which constitutes the protein degradation signal. The polyubiquitylated target protein undergoes proteolytic cleavage by the proteasome. The products of proteolysis are cleaved into small peptides of the target protein, and Ub molecules can be recycled.

10. 1 UPS inhibitors Because diverse human diseases are linked to the deregulation of UPS-controlled processes, this proteolytic pathway has become an important therapeutic target. In new clinical trials ubiquitin E3 ligase inhibitors, and first-generation deubiquitylating enzyme (DUB) inhibitors are now been used. But it tooks more than a decade after a Nobel Prize was awarded for the discovery of the UPS and the clinical approval (Harrigan et al., 2018). Logically, more lessons have been learned from the use of proteasome inhibitors, even if the mechanism of action/resistance is still under investigation. Proteasome inhibition results in a multitude of cellular responses such as endoplasmic reticulum (ER) stress, unfolded protein response, NFB inhibition, cell cycle arrest, inhibition of angiogenesis, or an increase in proapoptotic factors and tumor suppressors (Crawford et al., 2011) (Figure 21). Proteasome inhibition has been shown to induce autophagy, implying that autophagy can act as a compensatory mechanism upon impairment of proteasomal degradation (Kocaturk & Gozuacik, 2018). Inhibition of the UPS using various compounds (e.g., MG132, bortezomib, lactacystin, among others.) (T. Fan et al., 2018; Selimovic et al., 2013; Wu et al., 2008), or by genetic approaches (Demishtein et al., 2017) resulted in the upregulation of the autophagic activity in cells. For example, inhibition of proteasomal activity by the proteasome inhibitor and chemotherapy agent bortezomib led to an increase in the expression of autophagy genes ATG5 and ATG7, and induced autophagy. The upregulation of the

68 autophagy gene is determined on an ER stress-dependent pathway, which involved eukaryotic translation initiation factor-2 alpha (eIF2a) phosphorylation (Zhu et al., 2010). In another study, proteasome inhibition was associated with an increase in p62 and GABARAPL1 levels by Nrf1-dependent and –independent pathways before autophagy activation (Sha et al., 2018). In other contexts, MG132-mediated proteasome inhibition resulted in a decrease in cell proliferation, cell cycle arrest at the G2/M phase, and stimulation of autophagy through upregulation of Beclin1 and LC3 (Ge et al., 2009; You & Park, 2011, p. 132).

Figure 21: Cellular consequences of proteasome inhibition. Taken from (Dikic, 2017).

Cellular consequences of proteasome inhibition. Inhibiting the proteolytic activity of the proteasome affects almost every cellular process. Signaling networks regulating, for example, cell cycle progression, cell growth, or survival, depend on the proteasomal degradation of effector/inhibitor proteins or require proteasomal processing of precursor proteins for signal propagation. Also, a significant task of proteasomes (in particular, of specialized immunoproteasomes) is the generation of major histocompatibility complex class I antigens through proteolytic cleavage of endogenous proteins. Proteasome inhibition can, therefore, contribute to immunopathology. The most prominent consequence of impaired proteasomal activity is the accumulation of misfolded and aggregated proteins that causes a depletion of essential amino acids and ubiquitin (Ub), which is toxic to multiple organelles and impairs the function of the proteasome itself. Cells react to these threats by upregulating autophagy, stalling protein translation, and activating protein refolding capacity to eliminate aggregates.

The inhibition of the proteasome has emerged as a clinically useful anti-cancer therapeutic approach over the past decade (Orlowski & Kuhn, 2008). Proteasome inhibitors have been demonstrated to be useful therapeutic strategies in several hematologic malignancies, including multiple myeloma (MM) and AML. Although the 26S proteasome exhibits different

69 enzymatic activities, available small molecule inhibitors are directed towards the proteolytic sites of the 20S proteasomes. These inhibitors bind either reversibly or irreversibly to active sites in the core particle and display varying levels of specificity for the proteasome. These proteasome inhibitors are broadly categorized into two groups (Myung et al., 2001): 1. Synthetic analogs 2. Analog products Synthetic inhibitors are peptide-based compounds with diverse pharmacophores, including peptide benzamides, α-peptides keto amides, peptide aldehydes, peptide α-ketoaldehydes, peptide vinyl sulfones, and peptide boronic acids. In contrast, natural product proteasome inhibitors display a variety of scaffolds of core structures and pharmacophores. Known natural product proteasome inhibitors include linear peptide epoxyketones, peptide macrocycles, γ-lactam thiol ester, and epipolythio-dioxopiperazine toxin (Myung et al., 2001). In early studies, proteasome inhibitors were primarily used to uncover and study the proteasome’s catalytic activity (Adams, 2004; Vinitsky et al., 1992). In 1993, Alfred Goldberg, a cell biologist at Harvard Medical School in Boston, Massachusetts, created the first proteasome inhibitor, MG132, which worked by interfering with protein clearing. Myeloma cells, which are already awash with damaged proteins, proved particularly vulnerable to MG132 and would suffocate in their wasted proteins. Nowadays, the proteasome is a highly exciting and long-established drug target with three FDA approved drugs on the market. Those drugs inhibit the proteolytic activity of the proteasome (Cromm and Crews 2017): 1. Bortezomib (Bz) (Velcade), 2. Carfilzomib (Cz) (Kyprolis) 3. Ixazomib (Ninlaro) Proteasome inhibitors (PI), such as Bz and Cz, have become mainstays of treatment for MM and MCL. Bz was the first proteasome inhibitor to be approved in 2003 by the FDA for the treatment of MM. Bz is a reversible dipeptide boronate inhibitor targeting the ChTL-β- subunits β5c and β5i with low nanomolar half-maximal inhibitory concentration (IC50) values of 7 nM and 4nM, respectively while showing a reduced affinity for the β1c subunit (74 nM) and negligible affinity for the remaining β-subunits (Demo et al., 2007). Since its initial approval for multiple myeloma in 2003, Bz has been additionally approved for the treatment of MCL and is currently under investigation in a multitude of clinical trials in combination with various other chemotherapeutic agents (Kane et al., 2007).

70 The second PI, Cz, belongs to the epoxyketone family of proteasome inhibitors and covalently attacks active site Thr1 under the formation of a morpholine ring (Groll et al., 2000; Harshbarger et al., 2015). Showing a broader therapeutic window was approved for the treatment of multiple myeloma by the FDA in 2012 (Moreau, 2014). Like Bz, Cz acts on the chymotrypsin-like protease enzyme, but it is more selective than Bz and does not hit the 26S proteasome's other two enzymes. It should soon receive FDA approval for use on patients who have become resistant to Bz and lenalidomide (Commissioner, 2020). The third proteasome inhibitor approved by the FDA in 2015 is the second-generation peptide boronic acid ixazomib (MLN2238, Millenium Pharmaceuticals). Ixazomib is the first orally available proteasome inhibitor. The only nonpeptidic proteasome inhibitor in advanced clinical trials for multiple myeloma is the natural product salinosporamide A, also known as Marizomib (Mz) (Nereus pharmaceuticals). Mz is orally available and inhibits the proteasome irreversibly via an ester and tetrahydrofuran formation (Cromm & Crews, 2017). Summarizing, due to its success, exploring the role of the proteasome and the effects of the proteasome inhibition in AML represents a new alternative.

10.2 The role of the proteasome in acute myeloid leukemia (AML) Pre-clinical studies have demonstrated that proteasome inhibition disrupts proliferative cell signaling pathways, exhibits cytotoxic synergism with other chemotherapeutics, and induces autophagy of cancer-related proteins (Figure 22) (Csizmar et al., 2016). Malignant cells are highly dependent on increased protein production and degradation, suggesting that they would be sensitive to proteasome inhibition (Adams, 2004; Mani & Gelmann, 2005; Mitsiades et al., 2006). One pathway activated after proteasome inhibition is autophagy. Usually, autophagy acts as an adaptive and protective process that mitigates the deleterious effects of nutrient deprivation, growth factor withdrawal, or metabolic stress. Under conditions of extreme cellular stress, like for example, proteasome inhibition, cells can instead use autophagy to degrade essential components and undergo cell death. Intriguingly, Bz- induced proteasome inhibition has been shown to induce autophagy in both primary AML samples and several cell lines (Figure 22) (Fang et al., 2012; Larrue et al., 2016). For example, Matondo et al. in 2010 demonstrated a correlation between the level of expression of the proteasome 20S and the sensitivity of the cells to Bz and MG-132 (Matondo et al., 2010). Larrue et al. showed that FLT3-ITD+ primary AML samples are more sensitive than

71 wild-type samples to proteasome inhibition by Bz and that this sensitivity correlates to FLT3- ITD allele burden. In this setting, Bz treatment led to inhibition of the mammalian target of rapamycin complex 1 (mTORC1, a potent inhibitor of autophagy), increased conversion of LC3-I–LC3-II, and ultimately, autophagy of FLT3 protein. Furthermore, autophagy of FLT3 occurred in both quizartinib-sensitive and resistant cell lines, indicating that Bz treatment may represent a therapeutic strategy in FLT3-ITD+ AML patients refractory to tyrosine kinase inhibition (Larrue et al., 2016). Indeed, several pre-clinical and early-stage clinical trials investigating the role of the proteasome and proteasome inhibition in AML have shown promising results (Blum et al., 2012; Colado et al., 2008). Regarding the molecular effects of proteasome inhibition in AML, constitutive nuclear factor κB signaling is supported by the proteasome (Hideshima et al., 2009). In AML, NF-κB is constitutively active in leukemic stem cells (LSCs), but not in normal hematopoietic progenitor cells (Guzman et al., 2001). This constitutive NF-κB activity is supported by autocrine signaling via tumor necrosis factor α (TNF-α), which directs the proteasome- mediated degradation of the NF-κB inhibitor IκBα, thereby liberating cytosolic NF-κB. (Kagoya et al., 2014). As NF-kB promotes TNF-α expression, a positive-feedback loop is created between NF-κB and TNF-α, promoting cell survival and progression of leukemia (Figure 22). The importance of the proteasome in this process is underscored by early studies of the effects of proteasome inhibition on NF-κB signaling. For example, treating AML cells with the proteasome inhibitor MG-132 increased the amount of phosphorylated IκBα, leading to potent inhibition of NF-κB activity, decreased expression of NF-κB gene targets and induction of apoptosis in AML cells (including LSCs) (Guzman et al., 2001). As we can notice, the proteasome inhibition is a new target in AML; like most therapies, it is unlikely that proteasome inhibition will be universally efficacious, so the ability to rapidly identify patients who will benefit from this intervention would be immensely helpful and enable the responsible use of these agents.

Figure 22: The proteasome has several roles in AML. Taken from (Csizmar et al., 2016).

The primary function of the proteasome is the proteolytic degradation of ubiquitylated proteins. In AML, phosphorylation of IκBα targets this regulatory protein for ubiquitylation and proteasomal degradation. Degradation of IκBα liberates NF-κB, allowing this transcription factor to translocate to the nucleus and promote the expression of pro-survival and proliferative gene products, including TNFα. Among other actions, TNFα binds to the tumor necrosis factor receptor and drives an autocrine signaling pathway, promoting further IκBα phosphorylation and creating a positive-feedback loop that reinforces NF-κB activity. Inhibition of proteasome activity by agents such as bortezomib or carfilzomib disrupts this cycle, leading to cell death and also inducing other cellular mechanisms of protein degradation, such as autophagy. AML cells treated with bortezomib can sequester cytosolic proteins within membrane-bound vesicles called autophagosomes. These proteins, including the cancer-related proteins FLT3 and TRAF6, are then delivered to the lysosome for oxidative degradation.

73 11. Crosstalk between Autophagy and Ubiquitin Proteasome System The UPS and ALS are the two major and evolutionarily conserved degradation and recycling systems in eukaryotes. Although their activities are interdependent there is accumulating evidence in the literature about connections, and even a crosstalk between the UPS and autophagy. Ub is involved with many aspects of autophagy (Grumati & Dikic, 2018; Kwon & Ciechanover, 2017). For example, aggregated proteins, bacteria (exclusively Salmonella), and damaged mitochondria tagged with Ub chains can be brought to the autophagosomal membrane via autophagy receptor (López-Montero et al., 2016), and thus represent cargos targeted for autophagy-dependent degradation. Mitophagy constitutes another prominent example connecting these two degradative systems, yet several other examples exist (Kocaturk & Gozuacik, 2018). Ub tags on autophagy regulators, influences protein folding and interactomes. As both proteasomal and autophagy-mediated protein degradation use Ub as an essential substrate recognition signal, these major cellular protein degradation pathways share the Ub conjugation machinery and downstream Ub recognition modules. It has been suggested that the UPS and ALS function in parallel during the cell’s life. Growing evidence has indicated the existence of multiple points of communication between the two systems at different layers of regulation (Dikic, 2017). As the UPS and autophagy are interconnected, and inhibition of one system affects the other, therefore to maintain homeostasis, cellular materials that accumulate following inhibition of one degradative system needs to be cleared, at least in part, by the other system (Figure 23). Autophagy primarily compensates for the reduced degradative capacity of the proteasome and eliminates the threat resulting from damaged organelles or the accumulation of potentially toxic protein aggregates. In contrast, there are evidence for an opposite autophagy-to-proteasome shift activated upon impairment of autophagy. Initial observations about functional connections between the UPS and autophagy systems revealed that inhibition of one led to a compensatory upregulation of the proteolytic system. Inhibition of the UPS using chemical compounds, for example, Bz or MG132 (T. Fan et al., 2018; Wu et al., 2008; Yan et al., 2016); or by genetic approaches (Demishtein et al., 2017) resulted in the upregulation of the autophagic activity in cells. Nevertheless, this mechanism of compensation is also observed when using proteasome inhibition that leads to autophagy induction. For example, inhibition of proteasomal activity by the PI Bz increases the

74 expression of autophagy genes ATG5 and ATG7, inducing autophagy (Jaganathan et al., 2014). Another example using a PI is the case of MG132-mediated proteasome inhibition, that resulted in a decrease in cell proliferation, cell cycle arrest at the G2/M phase, and stimulation of autophagy through upregulation of BECN1 and LC3 (Ge et al., 2009). Similarly, impairment of autophagy correlated with the activation of the UPS. For example, in colon cancer cells, chemical inhibition of autophagy and small RNA mediated knockdown of ATG genes resulted in the upregulation of proteasomal subunit levels, including the 5 proteasome subunit, leading to increased UPS activity (Da-wei Wang et al., 2015). There are proteins associated as critical steps in the choice between the UPS and autophagy, proteins that possess UBA and LIR domains, like p62. Although p62 can attach both K48 and K63-linked Ub chains through its UBA domain, the binding affinity of the protein for K63- linked chains seems to be higher (Long et al., 2008; Wooten et al., 2008). However, not only p62 has this dual-action, other examples are NBR1, HDA6, Alfy (Kato et al., 2014; Kirkin, Lamark, et al., 2009; M. Tanaka et al., 2008). But also there are new proteins that possess UBA and LIR domains, like Rpn10 in Arabidopsis, and Cue5 in Saccharomyces cerevisiae (Marshall et al., 2015, 2016). There is increasing evidence about how proteasomes are identified as autophagic degradation targets (proteaphagy), enhanced proteasome peptidase activity following autophagy inhibition might be associated with the accumulation of proteasomes (Marshall et al., 2015), this proteaphagy subject will be discussed in the below chapter.

Figure 23: The compensatory balance between the activities of autophagy and the UPS in order to maintain cellular homeostasis. Taken from (Kocaturk & Gozuacik, 2018). When cellular homeostasis is disrupted by UPS impairment, an enhanced autophagy activity id developed, however if UPS is up regulated, there is and autophagy impairment.

75 11.1 Proteaphagy The selective autophagy process that clears 26S proteasomes is called proteaphagy. Proteaphagy is a homeostatic mechanism within the ubiquitin system that modulates proteolytic capacity and eliminates damaged particles. The term proteaphagy was coined by studies in Arabidopsis demonstrating that the turnover of proteasomes is mediated by autophagy, as shown in the studies made by Marshall et al., 2015, collectively they propose that Arabidopsis RPN10 acts as a selective autophagy receptor that targets inactive 26S proteasomes by concurrent interactions with ubiquitylated proteasome subunits/targets and lipidated ATG8 lining the enveloping autophagic membranes. Proteaphagy can proceed via nonselective and selective routes, depending on the initial trigger. Marshall and his collaborators further demonstrated that both nitrogen starvation and chemical or genetic inhibition of the proteasome could induce autophagic degradation of the 26S proteasome by proteaphagy. Starvation leads to nonselective proteaphagy, whereas PI results in selective proteasome degradation, which requires the proteasome subunit RPN10 as a specific receptor; RPN10 simultaneously interacts with ubiquitylated proteasome subunits and lipidated ATG8 on the phagophore (Marshall et al., 2015). Continue with the investigation on proteaphagy Marshall et al. in 2016 found that 26S proteasomes are degraded by autophagy in yeast, a process stimulated by inactivation of nitrogen starvation. Proteasome inhibition is accompanied by both Hsp42-mediated aggregation and ubiquitylation of the complex, which is then targeted to autophagic membranes by the Ub binding autophagy receptor Cue5 (Marshall et al., 2016). Selective proteaphagy is induced by inhibition of the proteasome and requires the ubiquitylation of inactive proteasomes, which is not the case in nonselective proteaphagy occurring upon starvation (Waite et al., 2016). Marshall and his collaborators have shown that proteasome subunit levels are elevated in a variety of autophagy-deficient backgrounds, but that is not accompanied by concomitant increases in transcript levels or total proteasome activity. Therefore, they speculated that this increase might reflect the accumulation of inactive 26s proteasome generally degraded by autophagy. The observation that proteasome can be degraded by autophagy raised several questions, for example, how inactive proteasomes are recognized, and the mechanism(s) that deliver them to autophagic vesicles, were unknown. One clue emerged from prior proteomic studies showing that proteasomes become extensively ubiquitylated upon MG132 inhibition (Marshall et al.,

76 2015). Their current work confirmed this finding and, besides, showed that proteasome inactivation is also accompanied by an increased association of Rpn10, a component of the proteasome regulatory lid, and one of its major receptors for ubiquitylated substrates. Rpn10 is unusual among core proteasome subunits, as it is often present at substoichiometric levels and can also exist in free form (Wen & Klionsky, 2016, p. 10). This increased association is reduced by treatment with a deubiquitylating enzyme, suggesting it results from Rpn10 binding Ub moieties present on the proteasome, rather than from increased incorporation into the particle. This evidence made Rpn10 an attractive prospect as a receptor for inhibitor- induced proteaphagy. The confirmation that Rpn10 is involved was shown by the fact that inhibitor-induced proteaphagy is blocked in rpn10-1 plants, which lack the C-terminal portion of RPN10 containing the UIMs. Those results are consistent with the hypothesis that Rpn10 acts as a receptor to tether inactive proteasomes to expanding autophagic membranes (Marshall & Vierstra, 2015) Similar to other selective autophagy pathways, the sequestration of ubiquitylated proteasomes into the autophagosome critically depends on autophagy receptors that can simultaneously bind to Ub attached to the cargo and Atg8/LC3, a Ub-like modifier exposed on autophagosomal membranes (Marshall et al., 2015). The discovery of proteaphagy adds yet another example to the ever-expanding repertoire of quality control and recycling processes for which autophagy is responsible. While Rpn10 has now been identified as a proteaphagy receptor, its existence as a free protein, plus its ability to bind ubiquitin and ATG8, indicates that it might play a broader role as a general autophagy receptor for ubiquitylated substrates. Support for proteaphagy as an evolutionarily conserved mechanism for proteasome clearance has been inferred from proteomic studies, which identified proteasome subunits as abundant contents within purified yeast and mammalian autophagic vesicles. Waite and his collaborators conducted other studies about proteaphagy, they show that in yeast, upon nitrogen starvation, proteasomes are targeted for vacuolar degradation through autophagy. Using GFP-tagged proteasome subunits, they observed that autophagy core particle (CP) subunit depends on the deubiquitinating enzyme Ubp3, although a regulatory particle (RP) subunit does not. All their data suggest a regulated process for the removal of proteasomes upon nitrogen starvation. This process involves CP and RP dissociation, nuclear export and, independent vacuolar targeting of CP and RP.

77 Further investigations realized in mammals by Cohen-Kaplan and her collaborators in 2016 demonstrated that amino acid starvation enhances polyubiquitylation on specific sites of the proteasome, a modification essential for its targeting to the autophagic machinery. They suggested that its interaction with p62 mediates the uptake of the ubiquitylated proteasome. In sum, in stressed mammalian cells, the proteasome is targeted by autophagy, which requires site-specific ubiquitylation of its Ub receptors (Cohen-Kaplan et al., 2016). As we can realize until this moment, proteaphagic mechanism likely differs between species. One plausible function of proteaphagy could be to dispose of nonfunctional proteasome subunits, which may interfere with proteasome activity or other cellular functions. Regulatory proteins shared by UPS and autophagy also seem to play a crucial role in selecting the degradation route, for example, p62, an autophagic adaptor that can bind Ub attached to cargoes and shuttle them to UPS or autophagy by binding to ATG8 (Cohen-Kaplan et al., 2016). This ‘tug of war’ between autophagy and UPS for p62 could potentially limit its availability for either proteolytic pathway. It is tempting to speculate that proteaphagy may encompass Ub chains linked by K63, as in yeast Ub-proteasomes first aggregate and then embark on proteaphagy (Marshall et al., 2016). However, there is a current need for further investigations in mammalian cells about the proteaphagy system under not stressfully conditions.

78 II. SCIENTIFIC CONTEXT

o Scientific context Internal tandem duplication of the FLT3 gene (FLT3-ITD) is found in approximately 25- 45% of adult AML and is related to an adverse prognosis. Mutations within the FMS-like tyrosine kinase 3 (FLT3) gene represent one of the most frequently identified genetic alterations which disturb intracellular signaling networks with a role in leukemia pathogenesis in comparison with the wild type receptor. AML cells expressing FLT3-ITD AML tend to relapse after conventional treatments. It is, therefore, necessary to study in more detail the biology of this subtype of AML, in order to improve results obtained with current treatments. Previous studies (Larrue et al., 2016) demonstrated that Akt, Stat5, and Erk, activated by FLT3-ITD, were significantly down-regulated by Bz treatment in MOLM-14 cells expressing FLT3-ITD (Figure 24A). Interestingly, the FLT3-ITD homozygous cell line MV4-11, and heterozygous cell line MOLM-14 are both sensitive to bortezomib, suggesting that FLT3-ITD determine susceptibility to Bz-induced cell death. Sensitivity to Bz was observed in a panel of FLT3-ITD positive primary AML samples but not in WT phenotypes (Figure 24B). Larrue et al showed that autophagy inhibition by chemical approach or by silencing using lentiviral vectors encoding inducible shRNA against Atg5 or Atg13, (two autophagy key factors), reverted the Bz-induced apoptosis. Altogether, these results demonstrated that Bz induces autophagy, which affects the stability of crucial cellular factors resulting in increased cell death in cells expressing FLT3-ITD. In this document, we present new evidence indicating that various selective autophagy events are activated after Bz treatment in FLT3-ITD cells, including proteaphagy. In this model the autophagy receptor p62 appears to participate in many of these proteolytic events.

Figure 24: FLT3-ITD positive AML cells are targeted to apoptosis by Bz-mediated autophagy. Taken from (Larrue et al., 2016).

A) MOLM-14 cells were treated 24 hours, with 10 nM bortezomib and then analyzed by Western blot with the indicated specific antibodies. B) Primary cells expressing FLT3-ITD are more sensitive to Bz-drive apoptosis. C) MV4-11 and MOLM-14 cells were treated with 10nM Bz or Bz + 3MA 5 mM in MV4-11 ATG5, or ATG13 were also silenced using lentiviral vectors. shRNAs were induced with 1 µg/mL doxycycline treatment during 72 hours, and then cells were incubated for 24 hours in the presence of 10 nM bortezomib.

80 Hypotheses

According to the current scientific context, we proposed the following hypothesis.

 A combination of proteasome and autophagy inhibition could affect stability of the 19s proteasome subunits than the 20s. Little is known about the mechanisms regulating proteaphagy. However, a publication suggests that the mechanisms for degradation of the 19S proteasome subunit are distinct to the one of the 20S subunit (Waite et al., 2016). This difference could be in part because the 19S regulates the ubiquitin-dependent part of the process. By controlling the 19S degradation in priority, proteaphagy might not need to degrade 20S subunits. We expect that the combination of bortezomib with distinct autophagy inhibitors would help us to reveal those differences.

 A combination of proteasome and autophagy inhibition could lead to an accumulation of crucial signaling factors contributing to the cell-killing response of the FLT3-ITD AML cells Chemical inhibitors of the proteasome and autophagy are known to affect the stability and activity of crucial signaling factors leading to apoptosis. By using combined treatments, we expect to improve the stability of these factors, and improve the apoptosis observed with individual treatments.

 Protein ubiquitylation might contribute to regulate the UPS-ALS autophagy crosstalk. Although many evidences exist in the literature supporting the role of protein ubiquitylation in the UPS-ALS crosstalk, we expect to accumulate evidence supporting that proteaphagy and other selective autophagy events also require this post-translational modification to drive proteasome degradation under proteasome inhibition conditions in AML cells.

81 III. MATERIAL AND METHODS The material and methods used during this thesis are now detailed. You will find a short version of these in the first version of the paper adjoined.  Antibodies and reagents In order to study the different pathways related to ALS, UPS, and some of the proteasome subunits by several approaches, the antibodies here below were used. Antibody Company Reference Host Dilution Technique Akt Cell signaling 9272 Rabbit 1:1000 WB P44/42 MAPK Cell signaling 9102 Rabbit 1:1000 WB (Erk1/2) Stat5 Cell signaling 94205 Rabbit 1:1000 WB LC3B-II Cell signaling 2775 Rabbit 1:1000 WB, IP, IF COX IV Cell signaling 4850 Rabbit 1:1000 SQSTM1 (p62) Santa Cruz Sc-28359 Mouse 1:1000 WB, IP, IF SQSTM1 (p62) Abcam Ab91526 Rabbit 1:200 IF GAPDH Sigma G8795-100UL Mouse 1:2000 WB Ubiquitin (P4D1) Cell signaling 3936 Mouse 1:1000 WB PSMA6 (Alpha 6) Invitrogen PA5-22288 Rabbit 1:1000 WB PSMB6 (Beta 1) Enzo MCP421 Mouse 1:1000 WB Beta 2 Santa Cruz Sc-515066 Mouse 1:100 WB, IP, IF PSMB5 (Beta 5) Cell signaling 12919 Rabbit 1:1000 WB PSMD2 (Rpn1) Santa Cruz Sc-271775 Mouse 1:1000, WB, IF 1:100 PSMD3 (RPN3) Thermo Fisher PA5-27665 Rabbit 1:1000 WB Rpn10 Enzo BML-PW9250 Mouse 1:1000 WB FLT3 Santa Cruz Sc-479 Rabbit 1:500 WB Aberrior Star Aberrior ST635P Mouse 1:500 IF 635p Alexa Fluor 594 Abcam Ab150120 Rabbit 1:500 IF

Table 7: Antibodies used in this study

82 Bortezomib was used to inhibit the proteasome. Bafilomycin A was used to block autophagy by preventing the maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes. Finally, Verteporfin was used to block autophagy but at an upstream level. Drug Company Reference Stock Working Cellular Vehicle solution solution Doses Bortezomib Sigma CAS 179324- 1 mM 10 µM 10 nM DMSO (Bz) Aldrich 69-7 Bafilomycin InvivoGen tlrl-baf1 100 µM 10 µM 10 nM DMSO A (BafA) Verteporfin Sigma 129497-78-5 2 mM 2 mM 1 µM DMSO Aldrich Table 8: Drugs used during this research

 Cell culture For all the experiments, to study the human myeloid leukemia, cells were purchased from the ATCC collection. AML cell lines were maintained in RPMI supplemented with 10% fetal calf serum in the presence of 100 U/mL of penicillin and 100 µg/ml of streptomycin. Cells were incubated at 37°C with 5% CO2. For autophagy cell analysis, 2% calf serum concentration was used during cell treatments for a maximum time of 8 hours. The AML cells with positive FLT3-ITD used were:  MOLM-14: MOLM-14 cells are heterozygous for the FLT3-ITD mutation (two out of three alleles, trisomy of chromosome 13) and carrying a t (9; 11) translocation (rearrangement of MLL with the AF9 gene). Human leukemia cell line established from the peripheral blood of the patient at relapse of AML, FAB M5, which had evolved from myelodysplastic syndrome (MDS).  MV4-11: MV4-11 is an AML line (FAB M5), homozygous for the FLT3-ITD mutation, and carrying a t (4; 11) (q21; q23) translocation (rearrangement of MLL with the AF4 gene). The MV4-11 cell line was established by Rovera and associates from the blast cells of a 10-year-old male with biphenotypic B-myelomonocytic leukemia. The wild type AML cell line used was:  OCI-AML3: A myelomonocytic AML line (FAB M4), expressing the wild-type FLT3 receptor. OCI-AML3 was established from the peripheral blood of a 57-year-old man with acute myeloid leukemia (AML FAB M4) at diagnosis in 1987; cells carry an NPM1 gene mutation (type A) and the DNMT3A R882C mutation.

83  TUBES purification TUBEs were produced according to (Hjerpe et al., 2009), with some modifications. TUBEs were produced in Escherichia coli, strain bacteria C41-DE3, and plate in AMP LB. Bacteria were cultured in LB medium with AMP at 37ºC with shaking, to reach an OD600 of 0.4-0.6 before IPTG induction. The addition of 1 mM IPTG induced TUBEs expression. The bacteria were grown during 4h at 37ºC, or overnight at 25°C for TUBEp62. Then, bacteria pellet was centrifuged, at 6000 rpm/ 20 min at 4ºC, and kept at -80ºC, for long term storage. Bacteria pellet was thaw on ice and resuspended in Lysis Buffer (10 mL of cold PBS supplemented with 0.5 mM NaCl, 2 mM benzamidine, and 1mM PMSF). In the case of GST- TUBEp62, 0.8 mM of NaCl was used. Cells were lysed on ice by sonication and always kept on ice to avoid overheating. Lysates were supplemented with Triton to a final concentration of 1% (for GST-TUBEp62 2%); and clarified by centrifugation at 20K rpm/ 1.5-2 hrs at 4°C. Afterward, the clarified lysate was incubated with 2 mL of dry glutathione-agarose (GST) beads (Sigma) per liter of LB culture. Agarose beads were loaded into a column and were kept rotating at 4ºC overnight. The next day, Flow Throw (FT) was collected, and beads were washed with PBS supplemented with 0.5-0,8 mM NaCl and Triton 0.1%. TUBES were eluted 7-10 times by adding 50 mM Tris pH 9.5, 0.5-0.8 mM NaCl, Triton 0.1%, and 10 mM of reduced glutathione, verifying the protein peak of proteins eluted by Bradford assay. Buffer exchange was done, by dialysis against PBS, and 0.5-0.8M of NaCl Triton 0.05%, overnight at 4°C to eliminate the presence of glutathione. Concentration was checked then by Nano- Drop at 280 nM. If the concentration was too low, proteins were concentrated up to 3-10 mg/mL using Amicon ultra with 3 or 10 KDa MWCO columns (Millipore). Finally, glycerol was added to a final concentration of 10%, and aliquots of 50-100 L were prepared and kept at -80°C.

 TUBES capture In order to capture ubiquitylated proteins, 100L dried of glutathione-agarose beads were used per point. Beads were equilibrated with TUBE buffer and supplemented with one mM DTT before use. Five hundred microliters of TUBE lysis buffer, containing one mM PMSF, 20µM MG123, complete protease inhibitors cocktail and TUBE (100 µg/sample) were used for the sample. Twenty million cells were used for each condition (TUBE p62, TUBE HHR23, or GST Control). Cells were resuspended in 500 µL of Lysis buffer, kept 10 minutes at 4ºC,

84 and spin down at 15500 g. A fraction of this supernatant was diluted in 3X BB (50 mM Tris- HCl, pH 6.8, 10% glycerol, 2% SDS, bromophenol blue, 10% β-mercaptoethanol) and considered as Input. The supernatant was transferred to the glutathione-agarose beads with TUBEs or GST control, and samples were incubated overnight in rotation at 4ºC. The next day, samples were spin down at 500g at 4ºC, a fraction recollected, diluted in BB, and considered as Flow-throw (FT). Beads were washed three times, with 1 mL of PBS Tween 0.05%. Finally, beads were resuspended in 100 µL of BB and boiled 10 min before protein electrophoresis.

 Antibody crosslinking To have antibodies attached to magnetic beads and get less background noise that will allow better studies in the presence or absence of TUBEs p62 or HHR23, antibodies p62 and Beta two were crosslinked. Thirty microliters per point of magnetic beads protein A (EMD Millipore LSKMAGA10) were washed with PBS and equilibrated in binding buffer (50mM Tris pH 8.5, 150mM NaCl, + 0.5% NP40), using a magnetic holder. Antibodies were added to beads and rotated overnight. Next day 10-20 µL of supernatant was kept to controlling antibody binding. Beads were washed twice with PBS and once with 500 µL of coupling buffer (200mM Borate, 3M NaCl pH9). Fifty millimolar DMP were added to the coupling buffer, and samples were rotated for 30 min with this crosslinking solution (CS). The supernatant was discarded, replaced with fresh CS and incubated at 4ºC for 30 min. Beads were washed twice with a coupling buffer before blocking with 20 mM ethanolamine pH 8.2. The supernatant was discarded and replaced by fresh ethanolamine and incubated during 1h. Beads were washed twice with PBS. Non-coupled antibodies were removed with two washes of 1M NaCl/binding buffer. A PBS equilibration was done before washing three times with 200 mM glycine pH 2.5. Beads were blocked with 0.1% BSA in the binding buffer for 90 min. Magnetic beads were equilibrated in binding buffer and kept in PBS until used. A fraction of these beads (10-20 µL) were analyzed by electrophoresis, followed by Coomassie blue staining to compare antibodies before and after crosslinking. This process is illustrated in Figure 25.

Figure 25: Immunoprecipitations of p62 in the presence or absence of TUBEs.

1st step: Antibodies p62 or Beta 2 were crosslinked to a protein A using dimethyl pimelidate (DMP), DMP contains an imido ester at each end of a 7-carbon spacer arm and forms an amidine bond with amino groups at alkaline pH. 2nd step: Protection assay plus Immunoprecipitation. The antibodies crosslinked to magnetic beads were used to precipitate proteins from AML cells in the presence or absence of TUBEs. TUBEs-protect ubiquitylated proteins from degradation. 3rd step: Magnetic beads were washed, and eluted proteins analyzed by Western blot with distinct recognizing specific proteins of interest

 Immunoprecipitation in the presence of TUBEs Twenty millions of cells were spun down at 1300 rpm/ 10 min, and the dry pellet was resuspended in 500 L of TUBE lysis buffer, including 100 g of TUBE-p62 or TUBE-HHR23. Cell lysates were homogenized using a Dounce homogenizer (each sample was homogenized with 40 strokes, always on ice). The Dounce was carefully washed with large volumes of distilled water between samples to avoid contamination. The whole sample was centrifuged at 13,000 rpm/5min, and the supernatant was used for immunoprecipitation. A fraction (1/20) of the supernatant was recovered for the INPUT sample (25 µL). The crosslinked antibody was divided and distributed in each Eppendorf with the lysate. The Eppendorf was incubated in rotation in the cold room during 1h30. Samples were disposed of in a magnetic holder and never spun down. Unbound supernatant was recollected in a new Eppendorf, and 50 µL for the FT sample was recovered. Magnetic beads were washed with PBS/tween 3-5 times and finally resuspended in 30 µL BB 3x to be analyzed by Western blot.

86  Proteasome assay A test of proteasome activity in MOLM-14 cells was performed to detect the intracellular proteasome activity after proteasome or autophagy inhibition. MOLM-14 cells were seeded at a density of 0, 3 x 106 cells per mL in 10% BFS, and plated the next morning at 2 x 106 cells per well in 2% BFS like in the autophagy cell analysis conditions. Then cells were stimulated with different concentrations of Bortezomib or Bafilomycin A. After treatments of 8h; cells were washed in PBS and then resuspended it in 50 L of homogenization buffer

[50mM TRIS-HCl, 250mM Sucrose, 5mM MgCl2, 2mM ATP (add fresh), one mM DTT (add fresh), 0.5mM EDTA (add fresh), 0.025% digitonin (add fresh)], then incubated during 5 min on ice, and spun down at 13000 rpm during 30 min at 4°C. Therefore, a sample of supernatant was taken, and total protein concentration was measured by BSA Bradford.

Supernatant was diluted in Assay buffer (pH= 7.5, 50mM TRIS-HCl, 5mM MgCl2, 0.5 mM ATP (add fresh), 1 mM DTT (add fresh), 40mM KCl and 0.05 mg/mL BSA (add fresh)), to get an enzymatic solute of total protein concentration between 0.1 mg/mL. For each point, 50 L of enzymatic solute, 100 L of substrate SUC (0.2 mM diluted in assay buffer), and 50 L of Marizomib concentration 10 M were add-in control wells, to avoid nonspecificity activity. Afterward, the plate was cover with aluminum and incubated for 20 min, and each measurement was done at least three times. The time of reading per sample was 500 ms, 380 mM excitation, and 460 mM emission was made using the VICTOR Nivo™ system.

 Western blot analyses With the intention of characterized proteins after the different treatments, AML cells were lysed with BB or TUBE Buffer. Then, proteins were resolved using 8 to 12% polyacrylamide gel electrophoresis PAGE and electro transferred (300 mA) to Polyvinylidene difluoride (PVDF) membranes during 2h at 4°C. Unsaturated binding sites were blocked in Tris- buffered saline (TBS) milk 5% solution during 1h. Unbound antibodies were washed three times with TBS. Membranes were incubated overnight at 4°C with the correspondent primary antibodies. The following day, membranes were washed two times before been incubated with an anti-rabbit or anti-mouse IgG antibody, diluted 1:5000 in TBS 2,5% milk solution, then three washes with TBS were done. Immunoreactive bands were visualized by enhanced chemiluminescence using ECL REVELBLOT® PLUS: SUBSTRATE CHIMIOLUMINESCENT by Ozyme, and SuperSignal™ West Femto Maximum Sensitivity

87 Substrate by Thermofisher; with PXi Multi-Application Gel Imaging System. Finally, images were analyzed with the software ImageJ.

 Apoptosis Assay In order to measure cell death in AML cells, an apoptosis assay by flow cytometry was performed. AML cell lines were treated at different times and concentrations with Bortezomib, Bafilomycin A and Verteporfin. During the treatments, cells were maintained at 2, 5, or 10% FBS. One million cells were washed in PBS and resuspended in 100 L of Annexin-V binding buffer (BD pharmigen), containing 2 L of Annexin-V-FITC (BD pharmigen). All samples were analyzed by a fluorescence-activated cell sorter (FACS) caliber flow cytometer (BD pharmingen). Flowjo software was used to analyze the apoptosis assay.

 Immunofluorescence microscopy Intending to evaluate proteaphagy and aggrephagy events in AML cells, immunofluorescence assays were realized. AML cells samples were treated 8h with 10 nM Bortezomib, 20 nM BafA, or 1 M Verteporfin. Samples were transferred onto poly-L-Lysine- coated slides and incubated for 15 minutes. Cells were fixed with 4% PFA for 10 min, then permeabilized 5 min at 4ºC with 100% methanol. Therefore, cells were washed and blocked during 1h in PBS containing 10% bovine serum albumin (BSA) and 0.1% Triton at room temperature. This process was followed by overnight incubation with the indicated primary antibodies that were diluted in 5% BSA. The next day, unbounded antibodies were washed and incubated for 30 min with secondary antibodies, either goat anti-mouse Abberior Star 635 or donkey anti-rabbit Alexa fluor 594; diluted 1:500 in blocking solution (5% FBS 0,1% triton X100 in PBS) at room temperature. Nuclei were stained with DAPI (P36931, Invitrogen). Images were acquired with the Zeiss Observer Z.1 microscope implemented with the Zeiss LSM 510, C-apochromatic objective 40X/1.20 W (UV-V15-IR, Torr). Pictures were acquired using Zen 2009 software package (Zeiss). Images were not modified other than adjustments of levels, brightness, and magnification. Colocalization analyses were performed using the Image J software.

88  Statistical analysis For the statistical analysis, data from 4 independent experiments were reported as the mean ± standard error of the mean (SEM). Statistical analyses using ANOVA one way with a Tukey posthoc were performed with Prism 6 software.

Symbol Meaning ns P > 0.05 * P ≤ 0.05 ** P ≤ 0.01 *** P ≤ 0.001 **** P ≤ 0.0001 Red stars Reference values (Bz reduction and combined treatment increase) Table 9: Statistical values

89 IV. RESULTS  Setting up conditions for detection of selective autophagy events Cell culture conditions are crucial to detect molecular events regulated by autophagy since this process is inhibited when cells are grown under rich nutrient conditions and activated upon starvation (He et al., 2018; Lauzier et al., 2019; Mizushima et al., 2004). We started using the previously reported cell growing conditions, RPMI with 10% FBS, where autophagy was activated after proteasome inhibition in FLT3-ITD AML cells (Larrue et al., 2016). In this project, we used MOLM-14 as main cellular model. We inhibited the proteasome using bortezomib (Bz) in a concentration range from 10 to 15 nM during 8h. In most reported studies (Albornoz et al., 2019; Vink et al., 2006), proteasome inhibition promotes the accumulation of ubiquitylated proteins that are degraded by the proteasome. According to Larrue et al., in AML expressing FLT3-ITD, Bz induces degradation of crucial signaling factors, including FLT3-ITD (Larrue et al., 2016). These proteolytic events were blocked by autophagy inhibition, indicating that Bz was promoting autophagy-mediated degradation (Larrue et al., 2016). In our experimental settings we verified Bz-mediated autophagy activation using two markers for this process, the autophagy receptor p62 and LC3B (Figure 26A). When a stimulus promotes autophagy, reduction of target proteins is often difficult to observe, unless autophagy inhibitors acting before the fusion with the lysosome are used. This difficulty is in part due to the highly dynamic regulation of these processes, where post-translational modifications regulate stability, synthesis, and function of implicated molecules. Western blot analysis of cell extracts showed that both autophagy markers are affected after Bz treatment, but those changes were not significant when using the statistical test. However, both markers were accumulated after Bz and Bafilomycin A (BafA) treatment, indicating that these settings were correct. In the case of BafA treatments in comparition with the control, both autophagy markers were not statistical significant. For this reason, we considered as referent values the reduction after Bz treatment and the accumulation after the combined treatment. These changes were indicated with red stars in all statistical analyses. Furthermore, a form of LC3B (LC3B-II) corresponding to a lipidated form of this ubiquitin-like molecule was also observed under the same experimental conditions -with BafA and Bz+BafA treaments- (Figure 26A). Then, we explored if proteaphagy was indeed activated after Bz treatment. Proteaphagy is a complicated process to analyze since not all proteasomes are directly concerned by this

90 type of degradation. Nuclear proteasomes will not be immediately affected since proteaphagy is a cytoplasmic event. According to the literature in mammalian cells, only 20- 50% of the proteasomes are regulated by proteaphagy depending on the time, intensity, and type of stimuli (Cohen-Kaplan et al., 2016; Marshall et al., 2016; Waite et al., 2016). Our results showed that after Bz treatment, a slight reduction in the levels of the α6. No change in β1, and a modest increase of the β2 core subunits were observed under the same conditions. Those changes were not statistically significant when analyzed individually. However, when we analyzed all together, 20S subunits showed a statistically significant accumulation when combining Bz and BafA compared to Bz treatment alone (Figure 26B). The reduction of 19S subunits (Rpn1 and Rpn3) after Bz treatment, or its accumulation after BafA was not statistically significant (Figure 26C). For this reason, we decided to change our experimental conditions to improve proteaphagy detection. Serum concentration was reduced to 2% during the treatment with distinct chemical inhibitors. We used on MOLM-14 cells the same concentration and treatment time for Bz and or BafA (Figure 27). Under these conditions the accumulation of both autophagy markers (LC3B and p62), after Bz and BafA treatments compared to Bz alone indicated a correct autophagy activation (Figure 27A).

When analyzing individually the core proteasome subunits -α6 and β5-, we found a statistically significant reduction after Bz treatment. A modest and consistent accumulation of the same 20S subunits was observed after Bz+Bafa treatment. The analysis of both subunits together showed an accumulation with more statistic value than the one of individual core proteasome subunits. (Figure 27B). Regulatory particle subunits, Rpn1 and Rpn3 were reduced after Bz treatment and increased after BafA. The combined treatment Bz+Bafa favored the accumulation of Rpn1 and Rpn3, even if values were not statistically significant (Figure 27C). In summary, under these conditions, our data were less dispersed, and we observed a better recovery of 20S proteasome subunits after Bz+Bafa treatment (Figure 27C).

Figure 26: Setting up conditions for the detection of proteaphagy in MOLM-14 cells using high serum concentrations.

MOLM-14 cells were grown on RPMI containing 10% FBS. Cell treatments were realized during 8h with a Bz range concentration from 10-15 nM and BafA 20 nM. Western blot analyses of cell extract were performed using specific antibodies: A) Autophagy markers: LC3B-II and p62, B) 20s proteasome α6, β1, and β2, C) 19s: Rpn1 and Rpn3. N= 3 biological replicates. Statistical analyses and graphs were performed using an ANOVA test with tukey posthoc with Prism 6 software. P<0.05 was regarded as significant. *, **, ***, **** correspond to P<0.05, P<0.01, P<0.001, P<0.0001 respectively. Red stars indicate reference values.

92 As other autophagy events appear to be activated after Bz treatment, and since several crucial signaling factors were reported to be stabilized after autophagy inhibition (Larrue et al., 2016). We investigated, if under our experimental conditions, we were able to observe the same proteolytic events. Considering that FLT3-ITD modulates cell proliferation, survival, and differentiation (Cauchy et al., 2015; Alberto M. Martelli et al., 2010), canonical signaling pathways were evaluated as reported by Larrue and collaborators (Larrue et al., 2016). Our Western blot analyses confirmed that Flt3 was significantly reduced after Bz treatment in MOLM-14 cells just as much as Erk, Akt, and Stat5. However, the reduction of all these factors was not efficiently blocked by BafA (Figure 27D). Altogether these results suggested that our experimental conditions were only correct to observe Bz-mediated reduction. These observations encourage us to test distinct autophagy inhibitors acting at a distinct level of this pathway (see Figure 33). These results will be developed in another section of this document.

Figure 27: Set up conditions for detection of proteaphagy in MOLM-14 cells using low serum concentrations.

MOLM-14 cells were grown on RPMI containing 10% FBS. When MOLM-14 cells were treated, cells were put it on RPMI containing 2% FBS and Bz 10-15 nM and BafA 20 nM during 8h. Then Western blot analyses of cell extract were realized using specific antibodies A) Autophagy markers LC3B-II and p62, B) 20s proteasome: α6 and β5, C) 19s proteasome: Rpn1 and Rpn3, D) Pathway markers: Flt3, Erk, Akt and Stat 5. N= 4 biological replicates. Statistical analyses and graphs were performed using an ANOVA test with tukey posthoc with Prism 6 software. P<0.05 was regarded as significant. *, **, ***, **** correspond to P<0.05, P<0.01, P<0.001, P<0.0001 respectively. Red stars indicate reference values.

94  Apoptosis in FLT3-ITD cells after 8h treatment with inhibitors Once the experimental conditions were set for the analysis of Bz-mediated proteaphagy, we controlled that no significant apoptosis was observed. Annexin-V staining was performed using the indicated concentrations of Bz and BafA after 8h treatment (Figure 28). Our results indicated that individual treatments showed low apoptosis levels (below 10%), even if the combined treatment induced statistically significant apoptosis, it remains below 20%. These results indicated that these experimental conditions do not induce massive cell killing in MOLM-14 cells.

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40 % Cell death Cell % ** * 20

T z A A N B f af a B B + z B Treatment

Figure 28: Cell death evaluation after 8h of treatment with proteasome and autophagy inhibitors in MOLM-14 cells

MOLM-14 cells were treated on RPMI containing 2% FBS using Bz 15 nM and BafA 10 nM during 8h before Annexin-V staining and flow cytometry (see methods), to validate Western blot conditions N= 12, 3 biological replicates. Statistical analyses and grapsh were performed using an ANOVA test with tukey posthoc with Prism 6 software. P<0.05 was regarded as significant. *, **, ***, **** correspond to P<0.05, P<0.01, P<0.001, P<0.0001 respectively. Red stars indicate reference values.

95  Proteaphagy in other AML cell lines After observing that Bz-mediated proteasome degradation was blocked with BafA in MOLM- 14 cells, we could refer this process as proteaphagy. We investigated if this process could be detected in other AML cell lines with or without FLT3-ITD. To accomplish it, we explored MV4-11 cells, which is homozygote for FLT3-ITD mutation (Figure 29). Proteasome was inhibited in MV4-11 cells using Bz 10 nM during 8h; autophagy was blocked using BafA at 20 nM concentration. LC3B-II and p62 were used as markers for autophagy activation mediated by Bz and BafA (Figure 29A). Western blot analysis of MV4-11 cells extracts showed that both autophagy markers do not significantly change their levels after Bz treatment. However, BafA or the Bz+BafA combination accumulate both autophagy markers even if in the case of the p62 this accumulation shows little statistical significance.

Using the same experimental conditions, the proteasome subunits α6 and β5 were not reduced after Bz treatment either with BafA. We did not find an accumulation of these core subunits using the combined Bz+BafA treatment (Figure 29B). When analyzing individual 19S proteasome subunits, -Rpn1 and Rpn3- after Bz treatment, both subunits showed poor reduction. The accumulation in the presence of both inhibitors was modest in the case of the Rpn1 subunit, and we did not recover Rpn3 levels (Figure 29C). Nevertheless, when analyzing both 19S subunits together, we find a reduction after Bz treatment and no significant recovery of protein levels after combined treatments. To investigate the impact of both inhibitors on the FLT3-ITD level, Western blot analysis with specific Flt3 antibodies were performed. A reduction of Flt3 levels was observed after Bz treatment and this process was blocked with BafA even if recovery was not statistically significant (Figure 29D). Among the other analyzed signaling factors, only Erk showed an accumulation in the presence of both proteolytic inhibitors (Figure 29D). All these results show that proteaphagy observed in MV4- 11 was modest compared to MOLM-14 cells, suggesting that the presence of FLT3-ITD might not be sufficient to favor an optimal activation of this proteolytic process. Other AML FLT3-ITD positive cells and/or approaches should be used to reach clear conclusions on the role of this translocation in proteaphagy.

Figure 29: Proteaphagy evaluation in other FLT3-ITD AML cell lines.

MV4-11 cells were treated on RPMI containing 2% FBS, Bz 10 nM, and BafA 20 nM during 8h. Then Western blot analyses of cell extract were realized using specific antibodies A) Autophagy markers LC3B-II and p62, B) 20s proteasome: α6 and β5, C) 19s proteasome: Rpn1 and Rpn3, D) Pathway markers. N= 4 biological replicates. Statistical analyses and graphs were performed using an ANOVA test with tukey posthoc with Prism 6 software. P<0.05 was regarded as significant. *, **, ***, **** correspond to P<0.05, P<0.01, P<0.001, P<0.0001 respectively. Red stars indicate reference values.

Another way to evaluate the contribution of FLT3-ITD in the Bz-induced autophagy-activation in AML cells is to compare the response in a FLT3 wild-type cell line. For this purpose, we used OCI-AML3 cells to evaluate the autophagy activation after Bz treatment (Figure 30). Contrary to MOLM-14 or MV4-11, OCI-AML3 cells showed both LC3B and p62 autophagy markers increased after Bz treatments, even if this increase was not statistically significant (Figure 30A). In the case of BafA treatments we observed an increased of autophagy marker levels, that indicates a correct autophagy inhibition in OCI-AML3 cells. For the combined Bz+BafA treatment statistically increased the levels of p62 autophagy marker compared to the Bz treatment. Regarding the other autophagy marker, under the same conditions, LC3B increased, but this accumulation was not statistically significant (Figure 30A).

In OCI-AML3 cells, when analyzing core 20S subunits (α6 and β5) together or individually, neither analyzes showed significant changes after Bz, nor under combined Bz+BafA treatment (Figure 30B). In the case of 19S subunits (Rpn1 and Rpn3) (Figure 30C), both showed increased levels after Bz treatment but, without statistical significance. Only the Rpn3 subunit showed a modest recovery after combined Bz+BafA treatment. When analyzing together both subunits of the 19S, we found a poor increase in protein levels after Bz treatments and a decrease when combining treatments (Figure 30C). Those results are essentially different compared to the one from MOLM-14 cells expressing FLT3-ITD. When analyzing signaling factors, only Erk and Stat5 showed an increase with the double Bz+BafA treatment; however, these changes showed little statistical significance (Figure 30D). These results may suggest 26S proteasome is not affected by proteaphagy in FLT3 wild type cells compared to FLT3-ITD expressing AML cells under these experimental conditions.

Figure 30: Analysis of proteaphagy in FLT3 WT cells

OCI-AML3 cells were treated on RPMI containing 2% FBS, Bz 10 nM, and BafA 20 nM during 8h. Then Western blot analyses of cell extract were realized using specific antibodies A) Autophagy markers: LC3B-II and p62 B) 20s proteasome: α6 and β5, C) 19s proteasome: Rpn1 and Rpn3, D) Pathway Flt3, Erk, Akt and Stat 5. N= 4 biological replicates. Statistical analyses and grapsh were performed using an ANOVA test with tukey posthoc with Prism 6 software. P<0.05 was regarded as significant. *, **, ***, **** correspond to P<0.05, P<0.01, P<0.001, P<0.0001 respectively. Red stars indicate reference values.

99  Proteaphagy in FLT3-ITD AML cells using an autophagy inhibitor acting on p62 To investigate the autophagy dependency of the Bz-mediated proteolysis, we tested other autophagy inhibitors acting at the level of autophagy receptor p62 such as Verteporfin (VT) (Schmidt-Erfurth & Hasan, 2000). In contrast to BafA, VT acts at the level of p62 by blocking its activity. Before using VT in different experiments, we evaluated the cytotoxicity at 24h in two AML models: MOLM-14 and MV4 (Figure 31). VT requires one micromolar dose to induce 50% apoptosis, in other words, the half-maximal inhibitory concentration (IC50) to affect this cellular process in these AML cell lines (Figure 31A and B). This cytotoxicity measure was essential to consider the settings of distinct cell biology and biochemical experiments, therefore avoiding data interpretation with dead cells. For this reason, in several experiments, the time treatment with one micromolar VT was reduced to 8 hours.

Figure 31: Verteporfin (VT) IC50 in FLT3-ITD AML cells

FLT3-ITD AML cells were treated for 24h with different doses of VT before Annexin-V staining and flow cytometry analysis. A) MOLM-14 cells, B) MV4-11 cells. Apoptosis was analyzed in (a) and (b) by FACS (see methods). The percentage of cell death was measured from 4 biological replicates.

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The inhibition of Bz-mediated proteaphagy with BafA showed active proteaphagy in MOLM- 14 cells. However, BafA could block other autophagy-dependent processes as it is not a specific inhibitor of proteaphagy. Since no specific inhibitors of proteaphagy exist, we decided to tackle the autophagy receptor p62, because its stability is affected with Bz/BafA treatment (Figures 27 to 30), and because p62 receptor has been previously implicated in the proteaphagy pathway (Cohen-Kaplan et al., 2016). As VT is one of the few molecules known to inhibit p62 activity, when using the calculated IC50 dose for VT (1 µM) in MOLM- 14 cells, high molecular weight forms of p62 were detected by Western blot analyses (Figure 32A). Under these conditions we did not find lipidated forms of LC3B after VT treatments, suggesting that this drug act upstream in this pathway and reduce the autophagy flux. The next step to explore was the Bz-mediated reduction of the 20S or 19S proteasome subunits in MOLM-14 cells. Both α6 and β5 subunits show modest reductions after Bz alone and VT alone treatment. Only β5 accumulated protein levels after combined Bz+VT treatment in comparison with the Bz treatment alone. In the case of the 19S subunits, only Rpn1 showed statistically significant accumulation with the combo Bz+VT treatment (Figure 32B and C). However, when analyzing the assembly of 19S proteasome versus 20S proteasome, the 19S proteasome showed higher and statistical accumulation than the 20S proteasome. With respect to Flt3, VT treatment (alone or in combination with Bz), resulted in the formation of high molecular weight forms of FLT3-ITD that could represent ubiquitylated forms of this protein (Figure 32D). In the case of other relevant signaling factors like Stat5, Bz treatment was not able to reduce the protein levels, in contrast, combination with VT reduced Stat5 levels. Other factors such as Akt and Erk, were poorly reduced by Bz, but this reduction was not efficiently reverted by VT, suggesting that p62 might not be involved in their autophagy- mediated degradation (Figure 32D).

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Figure 32: Verteporfin blocked proteaphagy in AML cell lines.

MOLM-14 cells were treated on RPMI containing 2% FBS, Bz 10 nM and VT 1µM during an 8h. Then Western blot analyses of cell extract were realized using specific antibodies A) Autophagy markers: LC3B-II and p62 B) 20s proteasome: α6 and β5, C) 19s proteasome: Rpn1 and Rpn3, D) Pathway Flt3, Erk, Akt and Stat 5. N= 4 biological replicates. Statistical analyses and graphics were performed using an ANOVA test with tukey posthoc with Prism 6 software. P<0.05 was regarded as significant. *, **, ***, **** correspond to P<0.05, P<0.01, P<0.001, P<0.0001 respectively. Red stars indicate reference values.

102  Proteaphagy is not constitutively active in FLT3-ITD cells Since proteaphagy was more easily detected in cells expressing FLT3-ITD, we explored the possibility of having basal levels of proteaphagy that could justify the better response to Bz in MOLM-14 cells. Hence, we decided to use two autophagy inhibitors acting at two distinct levels of the pathway. One of them, BafA, blocks at the level of the fusion with the lysosomes, the other one VT that inhibits at p62 level and inhibits the formation of mature autophagosomes (Figure 33). We first verified if autophagy was well activated by testing LC3B and p62 levels (Figure 33A). Both autophagy markers were accumulated in a statistically significant way after BafA treatment, even if in the case of p62 this accumulation was not highly significant. In contrast, the lipidated form of LC3B was reduced after VT exposition, and p62 showed high molecular aggregated forms that are typical of the VT mechanism of action (Donohue et al., 2011, 2014). The combined treatment BafA and VT accumulated both autophagy markers.

Under these experimental conditions, the 20S ( 6 and β5) and 19S (Rpn1 and Rpn3) proteasome subunits did not show a significant increase after BafA treatment alone (Figure 33B and C). Under VT treatment, only 19s proteasome, Rpn1, and Rpn3 were accumulated (Figure 33C). This accumulation was improved in both subunits after VT compared to not treatment, and Rpn1 significantly increased when both autophagy inhibitors were present. Altogether these results might indicate that both inhibitors have different mechanisms of action and that VT alone accumulates 19s proteasome in the absence of proteasome inhibition. These observations also suggest that Rpn1 could be involved in the mechanism of VT action regulating proteaphagy. When analyzing signaling factors under the same experimental conditions, we found that Flt3 and Erk accumulated after VT treatments, however, this accumulation was not statistically significant. When combining VT with BafA, an accumulation was only observed on Flt3 suggesting a possible cooperative effect of these inhibitors to accumulate (Figure 33D). Some factors like Stat5 were decreased after BafA, VT, or combined treatments. To summarize, only 19S proteasome and Flt3 were accumulated after VT treatment by an unknown mechanism. The fact that 19S proteasome are affected by VT or BafA, in particular Rpn1 suggest an implication of proteaphagy in MOLM-14 cells, through this subunit. However further investigations need to be done to study Rpn1’s role.

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Figure 33: Analysis of basal levels of proteaphagy in MOLM14 cells.

MOLM-14 cells were treated on RPMI containing 2% FBS, BafA 20 nM and VT 1 µM during an 8h. Then Western blot analyses of cell extract were realized using specific antibodies A) Autophagy markers: LC3B-II and p62 B) 20s proteasome: α6 and β5, C) 19s proteasome: Rpn1 and Rpn3, D) Pathway Flt3, Erk, Akt and Stat5. N= 4 biological replicates. Statistical analyses and graphics were performed using an ANOVA test with tukey posthoc with Prism 6 software. P<0.05 was regarded as significant. *, **, ***, **** correspond to P<0.05, P<0.01, P<0.001, P<0.0001 respectively. Red stars indicate reference values.

104  Evidence of proteaphagy and other selective autophagy events by Immunofluorescence in FLT3-ITD cells We investigated by immunofluorescence the colocalization of proteasome subunits with autophagosome markers to confirm if proteaphagy was correctly activated after proteasome inhibition in MOLM-14 cells (Figure 34-38). The chemical inhibition of this process with BafA colocalizes proteasome subunits with autophagosome markers. This colocalization was observed as cytoplasmic dots called “autophagy puncta” (Pugsley, 2017). The colocalization suggests a possible interaction between two visualized proteins, for example, the proteasome subunit 2 and the autophagosome marker LC3, supporting the activation of proteaphagy. The colocalization of LC3B and 2 subunits into autophagy puncta was well observed and had statistically significant after Bz+BafA treatment compared to untreated or Bz treatment of

MOLM-14 cells (Figure 34A). We also investigated the LC3B/2 distribution after VT and Bz+VT exposition, and we found a statistically significant colocalization when using VT and in combined treatments (Bz+VT) compared to untreated conditions (Figure 34B). However,

LC3B/2 showed a diffused distribution under these conditions, and not in puncta, contrary to the case after Bz+BafA treatment. To confirm the participation of p62 in proteaphagy, we explored the colocalization between p62 and the proteasome subunit α2 (Figure 35). A significant, but a modest reduction of p62/α2 colocalization was observed after Bz treatment, suggesting a Bz-mediated degradation. The colocalization of p62/α2 was recovered with the Bz+BafA treatment confirming that proteaphagy was activated in MOLM-14 under these conditions (Figure 35A). In remarkable contrast, the p62/α2 colocalization was modestly but consistently reduced with VT or combined treatments (Bz+VT), indicating that blocking of p62 could inhibit proteaphagy through a completely distinct mechanism than BafA (Figure 35B). Indeed, VT is known to induce aggregation of p62 that could affect the interaction between p62 and the proteasome subunit

α2, explaining the observed colocalization reduction (Donohue et al., 2014). Surprisingly, VT treatment has a negative impact on the p62/α2 colocalization and rather increases the LC3B/2 colocalization, suggesting the existence of a p62-independent proteaphagy. Alternatively, VT could change the localization of LC3B, but more studies need to be done to reach any conclusion. Using the Bz+VT treatment conditions, we also explored colocalization between Rpn1 and LC3B into diffused structures (Figure 36). The colocalization did not significantly change when

105 compared to Bz alone. These results might suggest that the Rpn1 subunit is affected diferently than the 20S subunits tested. Other 19S proteasome subunits should be tested to know if this is a particularity of Rpn1 or other 19S subunits could also be affected in the same manner. Altogether our results suggest that the distribution of colocalized autophagy markers/proteasome subunits is not the same when using both autophagy inhibitors. However, we can conclude that Bz treatment promotes the activation of proteaphagy in MOLM-14 cells. The colocalization of proteasomes with autophagy markers depends most likely on the mechanism of BafA or VT action. Immunofluorescence analyses were also performed to evaluate the role of LC3B in the Bz-dependent autophagy degradation of FLT3- ITD (Figure 37). To achieve this, we look for the colocalization of FLT3-ITD/LC3B applying the experimental conditions used in figure 32. The results suggested that Bz-driven colocalization of FLT3-ITD/ LC3B is significantly decreased when using Bz compared to untreated conditions and that when combined with VT we found a statistical increase in colocalization compared to Bz alone (Figure 37). However when comparing the combined treatment (Bz+VT) with the control, we did not find any increased in the colocalization signal. These results suggest that FLT3-ITD could be degraded through bortezomib-induced autophagy. Further investigations need to be done to confirm that p62 is the autophagy receptor mediating the degradation of FLT3-ITD. To investigate if other selective autophagy pathways could potentially be activated by Bz, we performed immunofluorescence assays under the same conditions. We analyzed if mitophagy was activated in MOLM-14 cells by looking at the colocalization of the mitochondrial protein COXIV and the autophagy receptor p62 (Figure 38). After Bz exposition, no changes in the colocalization of COXIV/p62 were found compared to the untreated condition. A reduction in COXIV/p62 colocalization rather than an accumulation was observed after BafA and Bz+BafA treatment. Our results indicate that under the tested experimental conditions, no mitophagy activation was found.

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Figure 34: Colocalization of 2 proteasome subunit and LC3B after Bz and autophagy inhibitors treatment in FLT3-ITD AML cells.

Indirect immunofluorescence staining of LC3B/β2 positive structures in MOLM-14 cells treated 8h with (A) Bz 10 nM and 20 nM BafA or (B) Bz 10 nM and one µM VT. Analyzes were performed using a Zeiss microscope. Statistical analyses and graphs were performed using unpaired one-way ANOVA, with a Tukey posthoc with Prism 6 software. P<0.05 was regarded as significant. *, **, ***, **** correspond to P<0.05, P<0.01, P<0.001, P<0.0001 respectively. Red stars indicate reference values.

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Figure 35: Colocalization of α2 proteasome subunit and p62 after Bz and autophagy inhibitors treatment in FLT3-ITD AML cells.

Indirect immunofluorescence staining of p62/α2 positive structures in MOLM-14 cells treated 8h with (A) Bz 10 nM and 20 nM BafA or (B) Bz 10 nM and one µM VT. Analyzes were performed using a Zeiss microscope. Statistical analyses and graphs were performed using unpaired one-way ANOVA, with a Tukey posthoc with Prism 6 software. P<0.05 was regarded as significant. *, **, ***, **** correspond to P<0.05, P<0.01, P<0.001, P<0.0001 respectively. Red stars indicate reference values.

108

Figure 36: Colocalization of Rpn1 and LC3B after Bz and VT treatment in FLT3-ITD AML cells.

Indirect immunofluorescence staining of LC3B/Rpn1 positive structures in MOLM-14 cells treated 8h with Bz 10 nM and 20 nM BafA. Analyses were performed using a Zeiss microscope. Statistical analyses and graphs were performed using unpaired one-way ANOVA, with a Tukey posthoc with Prism 6 software. P<0.05 was regarded as significant. *, **, ***, **** correspond to P<0.05, P<0.01, P<0.001, P<0.0001 respectively. Red stars indicate reference values.

Figure 37: Colocalization of FLT3 and LC3B after Bz and VT treatment in FLT3-ITD AML cells.

Indirect immunofluorescence staining of LC3/FLT3 positive structures in MOLM-14 cells treated 8h with Bz 10 nM one µM VT. Analyses were performed using a Zeiss microscope. Statistical analyses and graphs were performed using unpaired one-way ANOVA, with a Tukey posthoc with Prism 6 software. P<0.05 was regarded as significant. *, **, ***, **** correspond to P<0.05, P<0.01, P<0.001, P<0.0001 respectively. Red stars indicate reference values. Red stars indicate reference values.

109

Figure 38: Colocalization of mytophagy indicator COXIV and p62 after Bz and autophagy inhibitors treatment in FLT3-ITD AML cells.

Indirect immunofluorescence staining of COXIV/p62 positive structures in MOLM-14 cells treated 8h with Bz 10 nM and 20 nM BafA. Analyses were performed using a Zeiss microscope. Statistical analyses and graphs were performed using unpaired one-way ANOVA, with a Tukey posthoc with Prism 6 software. P<0.05 was regarded as significant. *, **, ***, **** correspond to P<0.05, P<0.01, P<0.001, P<0.0001 respectively. Red stars indicate reference values.

110  Impact of autophagy inhibition on proteasome activity The experiments presented in the previous section showed that the inhibition of autophagy recovered protein levels of different proteasome subunits degraded after Bz treatment in MOLM-14. To understand what the impact of these stabilized subunits on the proteasome activity would be, we first investigated if the “recovered proteasomes” would be active or inactive. To address this question, the chymotrypsin-like activity of the proteasome was measured using the fluorogenic substrate SUC (Suc-LLVY-AMC). The difficulty of this assays resides in the fact that proteaphagy is activated by Bz, meaning that proteasome inhibitors will always be present in our assay. Measuring proteasome activity in the presence of proteasome inhibitors is difficult and imposed the use of low doses of Bz to be able to evaluate the recovery of the activity after autophagy inhibition. MOLM-14 cells were treated or not during 8 hours with Bz, BafA, or combined treatments (Figure 39). The subtraction of nonspecific proteolytical activity was evaluated using Marizomib (Mz), an irreversible PI that potently inhibits the three 20S proteasome activities (Potts et al., 2011). Marizomib values were subtracted to measured values of chymotrypsin-like activity that contains both nonspecific and specific proteasome activities. To interpret these results, comparisons with and without Mz subtraction were made in all cases. In our model system MOLM-14, proteaphagy was induced using three doses of Bz: 15 nM (Figure 39A), Bz 5 nM (Figure 39B), and Bz 2.5 nM (Figure 39C), alone or in combination with BafA 10 nM. Results showed that BafA alone does not affect in a significant way proteasome activity (Figure 39A, B, and C). The dose of Bz 15 nM reduced more than 90% proteasome activity compared to untreated conditions, however, this dose was too strong to be reverted by BafA treatment (Figure 39A). When using Bz 5 nM, 80% of the activity is reduced compared to basal levels. A small recovery of proteasome activity was observed after BafA treatment, but it was still insufficient to detect any significant impact of this autophagy inhibitor (Figure 39B). The lowest dose tested, Bz, 2.5 nM, was not very efficient in blocking all proteasome activity (around 60%). However, it was the best dose to observe the recovery of the proteasome activity after incubation with BafA (Figure 39C). Altogether our results indicated that the activity of proteasomes degraded by autophagy could be potentially recovered if the degradation is stopped before phagosomes fuse with lysosomes. However, it is not known if there is any biological situation where this phenomenon could be possible.

111

Figure 39: Proteasome activity after different doses of Bz in MOLM-14 cells.

MOLM-14 cells were treated 8h with Bz a) 15 nM, b) 5 nM and c) 2.5 nM and BafA 20 nM. A-B) 7 technical replicates and C) 2 biological replicates. Statistical analyses and graphs were performed using unpaired one- way ANOVA, with a Tukey posthoc with Prism 6 software. P<0.05 was regarded as significant. *, **, ***, **** correspond to P<0.05, P<0.01, P<0.001, P<0.0001 respectively. Red stars indicate reference values.

112  Identification of ubiquitylated proteins using TANDEM UBIQUITIN BINDING ENTITIES (TUBEs)

According to our results, Bz treatment induces several autophagy-mediated degradation events (Figures 27 to 33). We decided to investigate the role of ubiquitin in these proteolytic changes. To explore that, we took advantage of the ubiquitin-traps developed in our laboratory, also known as tandem-ubiquitin binding entities (TUBEs). TUBEs are based on the repetition of ubiquitin-binding domains to increase the affinity of these tools for ubiquitylated proteins. We used TUBEs based in the UBA domain of HHR23 or p62, to capture total ubiquitylated proteins from MOLM-14 cells after treatments with Bz alone or in combination with BafA (Figure 40). In untreated cells, ubiquitylated proteins were captured by both TUBEs, however, TUBE HHR23 was a more efficient tool for enrichment. This result could be explained by the fact that TUBEs-HHR23 recognizes multiple chain types, while TUBE-p62 captures mainly K63 chains (Altun et al., 2011; Hjerpe et al., 2009; Long et al., 2008; Xolalpa et al., 2016). Bz-treatment alone or, in combination with BafA, enriched the capture of total ubiquitylated proteins in comparison with basal levels (Figure 40A). Under these conditions, the GST control did not capture any proteins with specific antibodies tested. Interestingly the BafA treatment alone did not favor the capture of total ubiquitylated proteins with any of the two TUBEs suggesting that blocking autophagy at late stages does not accumulate ubiquitylated proteins targeted to autophagy. We aimed to investigate if the autophagy receptor p62 was well captured under the same conditions (Figure 40). According to the Western blot results, only the TUBE HRR23 captured p62, the combination of Bz+BafA enriched this protein compared to Bz treatment alone, indicating that both proteolytic pathways contribute to regulate p62 levels. To investigate which proteins could integrate the ubiquitin proteome under those conditions, we tested proteins involved in the proteaphagy such as 5 and Rpn1. These proteasome subunits were only captured by the TUBE-HHR23 under Bz and Bz+BafA treatment (Figure 40A). Those results indicate that these two proteasome subunits integrate the ubiquitome after proteasome inhibition. TUBE-HHR23 also captured ubiquitylated forms of FLT3-ITD under all conditions, but these forms increased with individual or combined treatments of Bz and BafA. Unexpectedly, TUBE p62 that is meant to participate in recognition of ubiquitylated proteins targeted to autophagy degradation failed to capture all proteins tested. The reason why TUBE-p62 failed to capture the same cellular factors than TUBE-HHR23 is unclear and

113 these aspects will be developed in the final discussion. Some experiments aiming to understand the role of p62 in these proteolytic events regulated by autophagy are reported in the following section. Altogether these results indicate that protein ubiquitylation plays a role in the proteolysis of proteins implicated in proteaphagy and FLT3-ITD in MOLM-14 cells. The role of protein ubiquitylation in this proteolytic crosstalk was further investigated in the section below.

Figure 40: The role of protein ubiquitylation in proteaphagy and FLT3-ITD degradation under proteasome inhibition conditions MOLM-14 cells were treated or not during 8h with Bz 10 nM, BafA 20 nM, or both drugs, and cells were lysed as reported by Hjerpe et al. 2009. Ubiquitylated proteins were captured using TUBEs-HHR23, TUBEs-p62, or GST control. (A) Captured proteins were resolved in SDS-PAGE and immunoblotted with the indicated antibodies. Input and flow-through (FT) fractions were also analyzed with anti-ub antibody. GAPDH was used as the loading control. (B) MOLM-14 cells were treated or not with Bz/BafA with the same doses as “A.” IP captured P62-bound proteins with a specific p62 antibody in the presence or absence of TUBE-HHR23 or TUBE-p62. Western-blot analyzed precipitated material with the indicated antibodies. Input fraction was analyzed for the indicated proteins.

114  Ubiquitin-dependent regulation of p62 complexes regulating autophagy activated by Bz The data presented in this document suggest that p62 could be playing a role in the autophagy-mediated degradation of proteasomes and FLT3 in MOLM-14 cells. To better evaluate the role of ubiquitin in the regulation of p62-complexes formed after Bz treatment, we set up conditions for the detection of proteins interacting with p62 in the presence of TUBEs. We were particularly interested in analyzing proteins integrated into autophagosomes containing proteasomes and FLT3-ITD. Therefore, we performed two types of immunoprecipitations (IP): one using the antibody p62 (IP p62) and the other one with 2 antibodies (IP Beta 2) (Figure 41, and see materials and methods Figure 25). The presence of TUBE-HHR23 was expected to protect ubiquitylated proteins targeted to degradation by the proteasome (Hjerpe et al., 2009). In the presence of TUBE-p62, it was expected to protect proteins targeted to autophagy considering the K63 chain preference of its UBA domain. However, this new tool is still under characterization. In the case of IP p62, isolated proteins could come from the pre-autophagosome or autophagosomes. In the case of IP 2, captured proteasomes and associated proteins could come from autophagosomes, but also from other cellular compartments, not directly linked to autophagy. Our results indicate that the amount of LC3 and p62 associated with 2 were low (IP Beta 2), suggesting that only a minor proportion of the total 2 proteasome subunits present in the cell were engaged in autophagy events. In comparison, the amount of 5 bound to 2 was higher, indicating that these two molecules were privileged partners (Figure 41). Interestingly the presence of TUBE-HHR23 accumulated a higher molecular weight form of

5 that could represent an ubiquitylated form of this proteasome subunit. Furthermore, FLT3- ITD is also protected in the presence of both TUBEs, but TUBE-HHR23 better protects this cellular factor. Considering that p62 better precipitate LC3B (IP p62) and reasonable interacts with 5, we decided to continue our experiments using only IP p62. These settings appeared to be more appropriate to explore autophagy-related events (Figure 41) - furthermore, the IP p62 also preserve interactions with FLT3-ITD-. Since our experiments suggested that p62 could be the autophagy receptor implicated in the degradation of the proteasome and FLT3-ITD (Figures 32, 33, 35, 41), we decided to continue investigating these interactions in conditions where proteasome and autophagy are inhibited. The IP experiments using a specific p62 antibody were performed using MOLM-

115 14 cells treated or not with Bz/BafA (Figure 42). IP was performed in the presence or absence of TUBE-HHR23A or TUBE-p62 (Figure 42). The results suggest in the absence of TUBEs and any treatment, p62 is well immunoprecipitated comparing to the Bz+BafA treatment, which reduces the level of immunoprecipitated p62. In the presence of each TUBEs, p62 is well detected after Bz+BafA treatment, indicating that both tools protect p62. However, it is remarkable that in the presence of the TUBE p62 the combined treatment showed an increase of p62 levels, suggesting that this tool accumulates better this autophagy receptor under the tested conditions. Compared to the situation without any TUBEs or TUBE-HHR23, the presence of TUBE-p62 allow a better immunoprecipitation of proteasome subunits 2 and Rpn1 in the absence of Bz+BafA treatment. Nevertheless, high molecular weight forms, most likely represented ubiquitylated forms of Rpn1, were immunoprecipitated in presence of both TUBES, but preferable immunoprecipitated in the presence of TUBE-HHR23 under Bz+BafA condition. Coherent with these observations, FLT3-ITD is also well protected by TUBE-p62 when Bz+BafA was used. Nevertheless, putative ubiquitylated forms of this protein were better immunoprecipitated in the presence of TUBE-HHR23, suggesting that the UPS contributes to the FLT3-ITD degradation (Figure 42). Altogether these results indicate that p62 is implicated in proteaphagy and FLT3-ITD degradation. High molecular weight forms of FLT3-ITD and Rpn1 indicate that these factors are also degraded in a ubiquitin-dependent manner. In the case of FLT3-ITD, it is clear that the UPS plays an important role in its degradation. Targeting the same proteins to degradation by UPS or ALS most likely occur in situations where the activity of one or both proteolytic systems are compromised. More information about the pharmacological and clinical relevance of this information will be explored in the following section.

116

Figure 41: Setting up conditions for immunoprecipitations of p62 and Beta 2 under proteasome inhibition conditions

MOLM-14 cells were treated during 8h with both drugs Bz 10 nM, and BafA 20 nM, cells were lysed as reported (Hjerpe et al., 2009). Immunoprecipitation tests using p62 or Beta 2 antibodies were performed. Finally, ubiquitylated proteins were captured using TUBEs-HHR23, or TUBEs-p62. Then cell extracts were analysed by Western blot with the indicated antibody.

117

Figure 42: Ubiquitin role in proteaphagy and degradation of FLT3-ITD under proteasome inhibition conditions.

MOLM-14 cells were treated or not during 8h with Bz 10 nM, BafA 20 nM, or both drugs, and cells were lysed as reported. Ubiquitylated proteins were captured using TUBEs-HHR23, TUBEs-p62. IP captured P62-bound proteins with a specific p62 antibody in the presence or absence of TUBE-HHR23 or TUBE-p62. Precipitated material was analysed by Western blot with the indicated antibodies. Input fraction was analysed for the indicated proteins.

118  Inhibition of UPS and ALS enhances apoptosis in FLT3-ITD AML

cells After the analysis of the molecular events regulating the Bz-induced autophagy-mediated degradation, we wanted to understand the pharmacologic and clinical potential of these treatments. To perform this evaluation, we compared Bz-mediated cell-killing response in OCI-AML3 (FLT3 wild type) and MOLM-14 (FLT3-ITD) AML cells after 24h treatment (Figure 43). To better observe positive or negative cell-killing effects in both models, we set up conditions to obtain a maximum of 40% of apoptosis induced by Bz. Even when in both cell lines Bz treatment increased the percentage of cell death, this was significantly higher in MOLM-14 cells, supporting a correlation between FLT3-ITD and higher susceptibility to Bz treatments, as it was reported (Larrue et al., 2016). Autophagy inhibition using BafA increased apoptosis in both cell lines, but the effect was more prominent in MOLM-14 cells. When combining both drugs, the effect is quite similar to the one observed with Bz and did not revert apoptosis induced by proteasome inhibition, contrary to previously reported results (Larrue et al., 2016). This may come for the fact that some of the experiments demonstrating the autophagy-dependent apoptosis induced by Bz used conditioned silencing approaches in which the expression of autophagy factors was compromised- (Larrue et al., 2016). To mimic this approach, we decided to block first autophagy using distinct autophagy inhibitors, and in a second time, Bz was used to promote autophagy-mediated apoptosis (Figure 44A). Under these experimental conditions, 6-8 hours of pretreatment with BafA cooperate with Bz treatment to enhance apoptosis in MOLM-14 cells. These results were reproducible when using two distinct doses of Bz (6 and 7.5 nM), confirming that a pretreatment inhibiting autophagy with BafA improved the apoptosis induced by 16 hours of Bz treatment. Therefore, we decided to explore if other autophagy inhibitors could have the same effects, and for this reason, we did experiments with VT (Figure 44B). Considering the efficacy of VT, Bz and VT were simultaneously added to MOLM-14 cell cultures, resulting in an additional 8h of Bz compared to the situation of Figure 43A. Under conditions where 24 hours of Bz alone promoted a right apoptosis level, the cooperative effects with VT were more difficult to evaluate. For this reason, lower doses of VT were used to better observe cooperative effects. Altogether our results clearly show that inhibition of both proteolytic pathways could improve results obtained by inhibition of each individual route.

119 These results have an interesting potential to be tested in future in vivo experiments and clinical studies.

A) B)

O C I-A M L 3 M O L M -1 4

1 0 0 1 0 0 **** 8 0 8 0 *** **** **** *** 6 0 6 0 **** **** **** 4 0 **** 4 0 % Cell death % Cell death 2 0 2 0

0 0

A A NT B z NT B z B a f B a fA B a f B a fA

B z + B z + T re a tm e n t T re a tm e n t

Figure 43: Cell death evaluation after 24h treatments, FBS 10% in AML FLT3 wild type, and FLT3 positive.

Cells were treated on RPMI containing 10% FBS using Bz 10 nM and BafA 10nM before Annexin-V staining and flow Cytometry Analysis (see methods). N= 24, 3 biological replicates. A) FLT3 wild type cells OCI-AML3. B) FLT3-ITD cells MOLM-14. Statistical analyses and graphs were performed using unpaired one-way ANOVA, with a Tukey posthoc with Prism 6 software. P<0.05 was regarded as significant. *, **, ***, **** correspond to P<0.05, P<0.01, P<0.001, P<0.0001 respectively. Red stars indicate reference values.

120

Figure 44: Proteasome and autophagy inhibitors cooperate to improve the apoptosis of FLT3-ITD expressing cells.

Cells were treated on RPMI containing 5% FBS. (A) MOLM-14 cells were treated 8h with the indicated doses of BafA. Bz was added at 6 or 7.5 nM concentration for an additional 16h treatment. (B) MOLM-14 cells were treated with a fixed concentration of Bz 7.5 nM, and two distinct doses of Verteporfin as indicated. Cell death was analyzed in (A) and (B) by cytometry (see methods). The percentage of cell death was measured from 4 biological replicates. Statistical analyses and graphs were performed using unpaired one-way ANOVA, with a Tukey posthoc with Prism 6 software. P<0.05 was regarded as significant. *, **, ***, **** correspond to P<0.05, P<0.01, P<0.001, P<0.0001 respectively. Red stars indicate reference values.

121 V. GENERAL DISCUSSION

 The importance of proteostasis in health and disease The regulation of cell fate, by opposing death and survival pathways, is essential for homeostasis (Gómez-Díaz & Ikeda, 2018; Tang et al., 2019). The UPS and the ALS pathway are crucial to maintaining protein homeostasis by controlling the degradation of essential cellular factors. Dysregulation of these pathways leads to a wide variety of diseases: inflammation, neurodegeneration, and cancer (Ramesh & Pandey, 2017). Understanding the crosstalk of UPS and ALS during cancer development has not been an easy task. Research efforts of many groups have contributed to making clear some aspects of the role of this crosstalk in cancer development. Nevertheless, many questions remain open and are controversial. To understand how this proteolytic connection is regulated under conditions of proteasome inhibition we used MOLM-14, an AML FLT3-ITD cell model previously shown to activate autophagy after proteasome inhibition (Larrue et al., 2016). The susceptibility of malignant cells to proteasome inhibition might be primarily explained by the fact that the inhibition of proteasome activity could bypass some mutational effects in cell-cycle and apoptotic checkpoints that led to tumorigenesis. Furthermore, proteasome inhibitors can overcome various types of drug resistance in cancer cells (Almond et al., 2001; Pahler et al., 2003). Although in our studies we have not explored combinatorial approaches of Bz with chemotherapeutic agents used to treat AML: e.g., Cytarabine, daunorubicin or Fludarabine (Attar et al., 2012, 2013; T. M. Horton et al., 2014; Mateos et al., 2009), this remains an exciting possibility. Nevertheless, in this work, we have explored the autophagy mechanisms activated after proteasome inhibition using Bz, alone or in combination with BafA. Only a few published evidence indicate that proteasome inhibition could activate autophagy in cancerous cells: Yao and collaborators demonstrated that autophagy was induced after proteasome inhibition by combining chemical inhibitors of both pathways in human breast cancer MCF-7 cells (Yao et al., 2012). In their experimental settings, they used Bz and 3-MA as autophagy inhibitor and found that autophagy was activated after incubation with Bz, revealed by the increased percentage of GFP-LC3-positive cells (Yao et al., 2012). Yao’s study, together with the evidence generated in AML by Larrue et al., illustrates that proteasome impairment can induce active autophagy in cancerous cells.

122 Other biologic models and diseases support the interconnection between the UPS and ALS, for example, the genetic ablation of autophagy results in the accumulation of ubiquitylated proteins (Pankiv et al., 2007), proteasome inhibition has been associated with an increase in p62 and GABARAPL1 levels by Nrf1-dependent and -independent pathways before to autophagy activation (Sha et al., 2018). Our work contributes to the definition of one specific selective autophagy pathway, proteaphagy, driving the degradation of the proteasome when its activity is impaired. Proteaphagy defines the nature of the interconnections established between UPS and ALS since, in the absence of an efficient proteasome, autophagy can degrade proteasomes. Our results suggest p62 as a major proteaphagy receptor in MOLM-14 cells. p62 also contributes to the degradation of FLT3-ITD, but it is not clear if both processes occur in the same autophagosomes. These evidences indicate that UPS and ALS function as two complementary, reciprocally regulated protein degradation systems,that contribute to regulate proteostasis. However, it remains to be defined how damaged proteasome is recognized and if any chaperons/cofactors are regulating this event.

 Can the UPS and the ALS share proteins that are targeted to degradation? The UPS and ALS have been viewed as distinct degradation systems, but recent studies suggest that these pathways are mechanistically linked. The UPS and ALS are interconnected, and inhibition of one or the other system leads to the accumulation of damaged proteins or organelles that need to be cleared. Protein ubiquitylation is an important signal for both systems since it directs substrates to degradation and contributes to the UPS- autophagy crosstalk (Dikic, 2017; Korolchuk et al., 2010, p.). According to the current dogmatic view, proteins predominantly linked to K48-ubiquitin chains are directed for proteasomal degradation, and aggregates that are linked to K63-ubiquitin chains are targeted for autophagic degradation. p62 binding capacity to K63-ubiquitin chains has been considered as one of the critical steps in the choice between the UPS and ALS (Long et al., 2008; Tan et al., 2008; Wooten et al., 2008). In the case of chains that have mixed composition, this choice might not be easy unless the intervention of DUBs to remodel these chains favor one or the other system (Clague et al., 2019; Hospenthal et al., 2015). It has been reported that p62 binds non-covalently to polyubiquitylated proteins and then oligomerizes to form cytosolic and lysosomal aggregates (Lamark et al., 2003; Lippai & Lőw, 2014; W. J. Liu et al., 2016; Seibenhener et al., 2004). p62 also interacts with LC3 and acts

123 as a bridge connecting ubiquitylated protein aggregates and autophagosomes (Komatsu et al., 2007). In the case of large ubiquitylated protein-aggregates, these are excluded from proteasomal degradation and are targeted to autophagy-mediated proteolysis (Lu et al., 2017). Depending on the type of cargo, this process requires various autophagy receptors like: p62/SQSTM1, the neighbor of BRCA1 gene 1 (NBR1) protein (Wilde et al., 2011), optineurin (OPTN) among other receptors (Johansen & Lamark, 2011; Leyva-Paredes et al., 2016). Sometimes these autophagy receptors cooperate, making it difficult to define their participation in a single autophagy event. For instance, direct interaction of p62 and NBR1 with ubiquitylated protein aggregates can efficiently deliver these cargoes to autophagosomes (Ichimura et al., 2008; Lamark & Johansen, 2012). Several transcription factors that are regulated by the UPS, including p53, NFĸB, HIF1α, and NRF2, FOXO, have been implicated in the control of proteasome and autophagy (Kocaturk & Gozuacik, 2018). These transcription factors activate key genes regulating proteolysis under stress and disease conditions, directly affecting UPS-ALS crosstalk and the response to chemotherapy (Juenemann et al., 2013). During this research work, we have shown that when the proteasome is inhibited, the UPS-ALS crosstalk is affected, favoring the reduction of crucial cellular factors and macromolecular complexes via autophagy in MOLM-14 cells. As the pharmacological inhibition of autophagy limits the ability of cells to cope with stress and restores homeostasis (Kroemer et al., 2010; Mizushima et al., 2008). In this study, we combined several autophagy inhibitors to show that this pathway plays an essential role in response to Bz in FLT3-ITD cells. We found that proteaphagy was activated after proteasome inhibition, but also other autophagy events were activated to drive the reduction of crucial cellular factors typically regulated by ubiquitylation: FLT3 (Buchwald et al., 2010; Taylor et al., 2015), Stat5 (C.-S. Liu et al., 2017), and Akt (Noguchi et al., 2014; Yang et al., 2010).

 Role of FLT3-ITD translocation in the predisposition of the Bz-induced autophagy-mediated degradation of proteasome subunits To explore the molecular mechanisms regulating the crosstalk between UPS and ALS, we first evaluated the impact of Bz in combination with BafA in AML cells harboring or not the FLT3-ITD mutation. We used as models: MOLM-14 (FLT3-ITD+/-), MV4-11 (FLT3-ITD+/+) (Figure 27, Figure 29) compared to a FLT3 wild type cell line OCI-AML3 (Figure 30). The first evaluated aspect was the activation of autophagy using two markers that integrate

124 autophagosomes: p62 and LC3B-I/LC3B-II (Figure 27A, 29A, Figure 30A). In other studies performed in distinct cell lines, increased cellular levels of LC3B-II were found after Bz treatment, including human glioblastoma U251 and U87 (X. Zhang et al., 2014), melanoma (Selimovic et al., 2013) or osteosarcoma cells (HOS) (Lou et al., 2013). Previously published results showed that distinct AML models also accumulate LC3B-II in most cases (Larrue et al., 2016). In our experiments using AML cell models, the autophagy marker LC3B-II decreased after Bz treatment, these differences could be due to the experimental conditions used during the treatment (2% serum). As it was previously argued, rich nutrient culture conditions block autophagy, while poor nutrient medium conditions favor autophagy. Therefore, the presence of high serum concentrations (5-10% used in most common cell lines) would be comparable to have autophagy inhibitors. This fact could explain why in our experiments, we do not accumulate LC3B-II but is instead reduced. The reason why Bz-induced autophagy is more pronounced in FLT3-ITD expressing cells is not clear. In our experiments, variations on the capacity to activate autophagy after proteasome inhibitions were observed in distinct cell lines expressing FLT3-ITD. This evidence was also valid with other AML cell lines or primary samples from patients expressing FLT3-ITD (Heydt et al., 2018; Larrue et al., 2016; Qiu et al., 2019), suggesting that other mutations expressed in these cells could have a positive or negative impact on autophagy activation. Since the proteasome was shown to be a target for autophagic degradation after Bz treatment (Albornoz et al., 2019; Pitcher et al., 2015; Dawei Wang et al., 2019), here we analyzed key 20S and 19S proteasome subunits, in the presence or absence of distinct autophagy inhibitors. In our work, we found that FLT3-ITD predisposes AML cells to activate proteaphagy (Figures 27 and 29), and depending on the cell line the degradation of 19S subunits appeared more pronounced than the 20S subunits. A recently published work indicates that the mechanism targeting autophagy degradation of the 20S and 19S subunits is not the same (Waite et al., 2016). This published work could explain why in our experiments, the 20S and 19S were not degraded in the same proportion (Figures 27B and C, Figures 29B and C). Furthermore, these differences could also be explained by the genetic background present in distinct cell lines that could affect positively or negatively their degradation. One important aspect that we did not explore was the contribution of transcription to determine the level of distinct proteasome subunits. Since some differences could exist on the expression of proteasome subunits depending on the genetic context, it would be

125 essential to study the mRNA expression of distinct proteasome subunits in distinct AML cell lines. Another aspect we did not explore, was the impact of cell cycle on the activation of proteaphagy since all experiments were performed in asynchronous cells. This could explain, at least in part, the low percentage of proteaphagy observed in our analyses. Trying to understand the role of FLT3-ITD translocation in the predisposition of the Bz- induced autophagy, we further analyzed proteaphagy with other methods such as the colocalization of proteasome subunits within autophagosomes by indirect immunofluorescence (Figure 34-38). We observed an increased colocalization of LC3 and

2 in MOLM-14 cells after Bz+BafA and Bz+VT treatment (Figures 34 and 36), indicating an active proteaphagy in MOLM-14. When treating MOLM-14 cells with Bz+BafA, the colocalization of p62/α2 increased. However, when using Bz+VT this colocalization was reduced, suggesting that the autophagy receptor p62 is implicated in the proteaphagy activated by Bz in MOLM-14 cells. Other autophagy receptors should be tested under the same conditions to evaluate if they could also participate in proteaphagy. Although it is clear that proteasome impairment switches on proteaphagy, it is not clear how and which mechanisms recognize inactivated proteasomes within a population of the 20S, 26S, or 30S complexes. In our model, the proteaphagy mechanism in FLT3-ITD positive cells is not permanently activated since BafA alone does not accumulate proteasome subunits in the absence of Bz treatment (Figure 33 and 34A). In contrast, VT alone accumulates 19S subunits, Rpn1, and Rpn3 (Figure 33C) but does not improve the colocalize Rpn1 in LC3B positive bodies (Figure 36). However, LC3B/2 colocalization was improved by VT alone indicating that this treatment was not equally affecting all proteasome subunits, most likely due to their different mechanisms of action. Indeed, VT treatment does not accumulate p62 and Rpn1 in the absence of Bz (Figure 32), our first interpretation was that VT was supporting a permanently activated proteaphagy in AML cells expressing FLT3-ITD. However, VT- induced accumulation of Rpn1 could be related to the mechanisms of action of VT since Rpn1 is a well-known interactor of p62 (W. J. Liu et al., 2016). Therefore, Rpn1 could be directly affected by the aggregation of p62 induced by VT (Maitra et al., 2019), this information and other aspects of p62 are discussed below. We aimed to understand if during these conditions the recovery proteasomes are active or not. We observed that proteasome activity could be recovered after BafA treatment if low doses of Bz are used (Figure 39). In concrete, our results suggest that Bz 2.5 nM is enough

126 to observe an inhibition of proteasome activity, and this effect can be reverted when a standard dose of BafA is used (Figure 39C). Higher concentrations of Bz block proteasome activity more efficiently but reverting this inhibition with autophagy inhibitors is more challenging. This proteasome activity assay allowed us to confirm, with a distinct approach, that the inhibited proteasome was targeted to degradation via autophagy. Regarding the autophagic degradation of FLT3-ITD (Figure 37), our results suggest that Bz- driven colocalization of FLT3-ITD/LC3B is significantly decreased when using Bz and increased when combined with VT. This result indicated that FLT3-ITD is degraded through a bortezomib-induced autophagy process. Although some of the collected evidence indicate that p62 is implicated in the autophagic degradation of FLT3-ITD, more experiments are required to confirm or exclude the participation of other autophagy receptors in this proteolysis. FLT3-ITD activates different signaling pathways like Stat5, Erk, MAPK, among others. Since translocations and mutations on tyrosine kinase receptors are found in multiple cancer (Regad, 2015), we started by analyzing AML expressing FLT3-ITD. The activation of Stat5 by FLT3-ITD is controversial, it seems that FLT3-ITD does not activate JAK (Stat5 principal regulator). Choudhary and his collaborators proposed a direct Stat5 regulation by FLT3-ITD (Choudhary et al., 2007). The serine-threonine kinase Akt plays a central role in the regulation of cell survival in a variety of human neoplastic diseases. A series of studies have revealed a connection between Akt signaling and protein degradation by the UPS and the ALS (Noguchi et al., 2014). Two distinct ubiquitylation systems have been reported to regulate Akt signaling: K63 ubiquitin chains (which promote Akt oncogenic activation), and K48 ubiquitylation that triggers the proteasomal degradation of phosphorylated Akt (Noguchi et al., 2014). Akt K48 ubiquitylation was observed in breast cancer, where the BRCT domain of BRCA1 (breast cancer susceptibility gene 1) binds to phospho-T308 and phospho-S473 of Akt and triggers their degradation, presumably in the nucleus (Xiang et al., 2008). The mechanism of Stat5 protein activation by FLT3 is poorly understood, and the role of JAKs in FLT3 signaling remains controversial (Murata et al., 2003; Tse et al., 2000; S. Zhang et al., 2000). Our results in FLT3-ITD AML cells show that Erk and Stat5 are also degraded by autophagy after Bz-treatment in MOLM-14 and MV4-11 cells (Figure 27D and 32D). However, this is not exclusive of FLT3-ITD cells since it is also observed in OCI-AML3 (Figure 33D). Proteolysis of all these factors could contribute to increased apoptosis in AML cells after Bz treatment (Larrue et al., 2016).

127  Is p62 the only autophagy receptor involved in Proteaphagy? A crucial point to consider when blocking autophagy with drugs currently used in clinical trials is that these are not specified for one selective autophagy pathway and can even affect functions unrelated to autophagy. For example, BafA inhibits autophagy when it alters lysosomal pH, and it accumulates cargoes into autophagosomes. However, pH modification could also alter the drug bioavailability and decrease treatment efficacity depending on the dose and time treatment (Juhász, 2012; Pellegrini et al., 2014). For this reason, we explored other autophagy inhibitors such as VT, which blocks the activity of p62 (Konstantinou et al., 2017). To try to answer this question, we investigated the role of p62 in the Bz-induced proteaphagy using VT. In this research, we worked with VT at concentrations equal or below the estimated IC50 for MOLM-14 (Figure 31). The use of VT in MOLM-14 cells showed that the inactivation of p62 stops Bz-induced proteaphagy, supporting a significant role of this autophagy receptor (Figure 32). This autophagy inhibitor induces high molecular forms of p62 that we detected using specific antibodies (Donohue et al., 2011) (Figure 32). However even when working with VT at doses below the IC50, we cannot discard known side effects reported for this drug. Like ROS production that affects proteasome activity, enhances protein modification by ubiquitin family members, or activate signaling cascades (Finkel, 2011; Lefaki et al., 2017). According to Donohue’s team, the p62 aggregation induced by VT required the disposition of PB1 domains (Moscat et al., 2007), in a front-to-back mode to form heterodimers or homo- oligomers (Ito et al., 2001; Terasawa et al., 2001). Interestingly, our results showed high molecular forms of FLT3 induced after VT treatment. However, in the case of VT-induced FLT3 multimerization, nothing has been published (Figure 32D). If both proteaphagy and FLT3-ITD use the same p62 autophagy receptor to drive their proteolysis, our results could explain why Bz-induced autophagy degradation of FLT3-ITD is blocked after VT treatment. Selective autophagy typically employs bridging receptor proteins that bind both, a condemned cargo and a LC3/GABARAP molecule on the expanding autophagosome membrane. For example, NBR1 binds both ubiquitin and LC3 for a limited number of ubiquitylated targets (Kirkin, McEwan, et al., 2009). Among the autophagy selective pathways, we found that proteaphagy was playing a role in response to Bz of FLT3-ITD positive AML cells. Proteaphagy is a process that was recently discovered in distinct biologic models, suggesting that this is a selective autophagy route well preserved in evolution. Until now, proteaphagy is known to be activated in two ways: 1) after nutrient starvation or 2)

128 proteasome inhibition. Depending on the biologic model, distinct forms of nutrient starvation have been used including amino-acid, nitrogen, or limitation of other nutrients (Marshall et al., 2015, 2016). Among the approaches used to study proteaphagy we can find: GFP tagged proteasomes used in Arabidopsis thaliana to demonstrate that autophagy is required for the delivery of proteasome into the plant vacuole(Marshall et al., 2015). Chemical or genetic inhibition of the proteasome can also induce autophagic degradation of the 26S proteasome in Arabidopsis thaliana (Marshall et al., 2015). These investigations in Arabidopsis thaliana revealed that proteaphagy requires the proteasome subunit Rpn10 as a specific receptor. In this model, Rpn10 simultaneously interacts with ubiquitylated proteasome subunits and lipidated ATG8 (LC3 equivalent) residing in the phagophore (Marshall et al., 2015). Critical molecules in selective autophagy events are autophagy receptors that often have other functions (B.-W. Kim et al., 2016). The evidence presented here demonstrates that autophagy receptor p62 is implicated in proteaphagy activated by Bz in MOLM-14 (Figures 27, 33, and 35). This is not an isolated finding, similar results were obtained in HeLa cells after aminoacid starvation, where p62 participates in proteaphagy (Cohen-Kaplan et al., 2016), indicating that both starvation and proteasome inhibition can use the same autophagy receptor. In other biologic models such as Saccharomyces cerevisiae or Arabidopsis thaliana, the ubiquitin receptor Cue5 (Marshall et al., 2016, p. 26) and the chaperone Hsp40 (Kaushik & Cuervo, 2012; Orenstein & Cuervo, 2010), mediate proteaphagy. Many open questions remain without a clear answer: Can other autophagy receptors participate in proteaphagy? Is there any functional redundancy or cooperation between autophagy receptors? How many LCB3/GABARAP molecules are implicated in a selective autophagy event? These and other aspects would be interesting to explore in this field in future studies. The development of more specific p62 inhibitors would help to separate p62-specific action from other effects. Considering the previously mentioned side effects of VT, we cannot exclude the possibility that these could also contribute to the cell-killing capacity of this drug.

 Role of Ub in the Bz-induced selective autophagy? UBDs with different amino acid composition and structure recognize ubiquitylated proteins. Various proteasome subunits have this capacity and contribute to present target proteins for proteasomal degradation. It is known that Rpn1, Rpn10, and Rpn13 subunits of the 19S complex, harbor UBDs that specifically recognize K48 linked Ub chains (Husnjak et al., 2008; Yuan Shi et al., 2016). Even if each of these proteins has a role in a particular physiologic or

129 pathologic situation, studies performed in yeast demonstrated that these molecules have functional redundancy and in the absence of one of them, another subunit can do the job (Gomez et al., 2011; Husnjak et al., 2008). This evidence suggested that UBDs are promiscuous and can recognize multiple protein targets if their intervention is required. With this idea in mind, our group developed a technology-based in distinct UBDs to capture ubiquitylated proteins (Hjerpe et al., 2009). Here we used two different types of TUBEs: one based in the UBA domain of the proteasome adaptor HHR23, and another based on the UBA domain of p62. Unexpectedly, TUBE-HHR23 captured proteins implicated in the proteaphagy process: p62, Rpn1, or 5. These proteins are only captured after Bz or Bz/BafA treatment, but not with BafA treatment (Figure 40), indicating that this autophagy inhibitor does not accumulate ubiquitylated forms of these factors in the absence of an activator of autophagy. Since Bz activates proteaphagy in these cells, this explains why p62, Rpn1, or

5 are captured by TUBE-HHR23 under proteasome inhibition conditions. Interestingly, p62 is the only factor that is more enriched in the presence of both Bz and BafA, underlining its role in the UPS-ALS crosstalk. TUBE-HHR23 also captures FLT3-ITD under all tested conditions, even if only the Bz treatment increases the amount of ubiquitylated FLT3. It was surprising the fact that we did not capture p62, Rpn1, or 5 proteins when using TUBE p62. Several possibilities could explain these results. The incapacity of TUBE-p62 to compete with endogenous p62-ubiquitylated protein complexes, considering that we do not know the affinity of the new TUBE-p62 for K63 ubiquitin chains. If the affinity of TUBE-p62 for K63 chains is too low, then it will be more challenging to compete with naturally formed complexes. Another possibility is that since the TUBE-p62 is restricted to capture K63 chains, proteins with other chain types cannot be captured with this tool. This second interpretation is based on the fact that the amount of total ubiquitylated proteins captured by TUBE-p62 is reduced compared to the one captured by TUBE-HHR23 (Figure 40). Since TUBE-HHR23 recognizes all chain types, this could facilitate the capture of substrates that are targeted to both proteolytic systems (Cabe et al., 2018; Hjerpe et al., 2009; Long et al., 2008; Xolalpa et al., 2016). In order to investigate these possibilities, we performed protection/competition assays with endogenous p62-ubiquitylated protein complexes using IP with specific p62 antibodies, in the presence or absence of both TUBEs (Figure 42). These protection assays

(Hjerpe et al., 2009), revealed that TUBE-p62 protects unmodified forms of p62, Rpn1, 2, or FLT3-ITD from Bz-driven degradation that has been blocked with BafA (Figure 41). TUBE-

130 HHR23 also protects unmodified forms of these factors, but less well than TUBE-p62. However, TUBE-HHR23 better protects ubiquitylated forms of Rpn1 or FLT3-ITD (Figure 42), explaining why these proteins were better detected in TUBE HHR23 capture. These results indicate that the tandem disposition of UBA domains of TUBE-p62 is not sufficient to capture ubiquitylated factors implicated in proteaphagy, but it is sufficient to protect them from Bz- driven degradation. Altogether our data suggest that when both proteolytic pathways are blocked, cellular factors might shuttle between UPS and ALS. The failure to find fully active degradation machinery could explain the accumulation of these factors. Fundamental investigations in the future should explore the role of other autophagy inhibitors acting at other levels of this pathway (e.g. signaling pathways) and the identification of the molecular sensors determining the shuttling of cargoes between these two major proteolytic systems.

 Regulation of UPS-ALS crosstalk in AML cells expressing FLT3-ITD after Bz- treatment. One significant challenge is understanding how the UPS and the ALS communicate when one or the other proteolytic system is blocked, therefore here we focused on the proteasome inhibition situation. Our investigations underlined that under Bz treatment conditions and in the absence of a fully active proteasome, typical substrates of the UPS are driven to ALS- mediated degradation (C.-D. Fan et al., 2013; M.-S. Lee et al., 2015; Nguyen et al., 2013; Taylor et al., 2015; Weisberg et al., 2017; Yang et al., 2010). However, at this stage, it is not clear if the different proteolytic events analyzed here could be linked to the same or distinct selective autophagy events. This question could be answered using cell biology, and biochemical approaches to investigate if these cellular factors can be found in the same autophagosomes. It is clear that the impairment of both pathways with Bz and autophagy inhibitor BafA or VT contributes to the accumulation of crucial cellular factors that could contribute to improve apoptosis in MOLM-14 cells (Figure 33). Some discrepancies have been observed with published data where genetic or chemical inhibition of autophagy reverted the Bz-induced apoptosis (Larrue et al., 2016). We can speculate that the genetic inhibition of autophagy could lead to an adaptive response of the cell, which may contribute to a different Bz-mediated cell-killing response. This speculation is based on the fact that multiple functionally-redundant molecules such as autophagy receptors or LC3/GABARAP family members contribute to regulate autophagy, and some of these molecules could “do the job” in the absence of the usual molecular machinery. Concerning the different results

131 obtained using distinct chemical inhibitors, it might depend on the distinct chemical characteristics of these molecules, the used doses, and the mechanisms they tackle. In future investigations, it would be interesting to test other autophagy inhibitors acting at other levels of this pathway. Altogether, our results suggest four possible scenarios occurring in FLT3-ITD expressing cells: In basal conditions, FLT3-ITD levels are controlled by the action of both proteolytic pathways (Figure 45, 1). When blocking the UPS pathway with Bz, proteaphagy, and degradation of crucial factors involved in signaling pathways like Erk and Stat5 is observed together with increased apoptosis. A third scenario is observed after autophagy inhibition that drives the degradation of some cellular factors to proteasome- mediated degradation. Finally, when both proteolytic pathways are impaired, proteaphagy is inhibited, resulting in the accumulation of proteasome subunits and factors that might contribute to increase apoptosis in FLT3-ITD positive cells (Figure 45).

Figure 45: FLT3-ITD levels are controlled by the action of UPS and ALS. 1) In basal states, the turnover of important cellular factors is ensured by equilibrated proteolytic pathways. 2) The inhibition of UPS by Bz drives to an increase in ALS. 3) Inhibition of autophagy drives the degradation of some cellular factors to proteasome-mediated degradation 4) when both proteolytic pathways are impaired, both UPS and ALS contribute to accumulating proteasome subunits that will favour the accumulation of cellular factors and increase apoptosis in FLT3-ITD positive cells.

132  Biological and clinical relevance As AML is a rapidly progressing hematopoietic malignancy with a dismal response. Understanding its biology is needed for the development of new and more effective treatments. One prognostic feature associated with an inadequate response is the presence of mutations activating FMS-like tyrosine kinase 3 (FLT3) (Weis et al., 2019). FLT3 mutations occur in about 30% of AML patients; the most frequent mutation in this gene occurs via an internal tandem duplication (FLT3-ITD). The unfortunate outcome associated with FLT3-ITD mutations (Kayser et al., 2009) has generated considerable interest in the development of specifying tyrosine kinase treatments. Drug resistance in AML has been particularly challenging to obtain successful treatments. Several studies have tried to understand the role of autophagy in AML and suggest that inhibiting autophagy sensitizes particular subgroups of AML to chemotherapies (Bosnjak et al., 2014; Piya et al., 2016). Nevertheless, the potential role of selective autophagy mechanisms activated in AML cells expressing FLT3-ITD is still under intense investigation. During this study we tested the proteasome inhibitor Bz, which has previously demonstrated having clinical activity in AML patients, alone or in combination in chemotherapy (Attar et al., 2013; Sarlo et al., 2013). When blocking the proteasome, we induced proteaphagy (Figure 27), and BafA to blocks proteaphagy with a consequent accumulation of proteasome subunits (Figure 27 and 35). Interestingly, under these conditions, crucial signaling factors such as Erk, Akt, and Stat5 are also accumulated not only in FLT3-ITD but also in other AML phenotypes. Altogether our results suggest that the FLT3-ITD phenotype is favoring but might not be required to activate proteaphagy. Nevertheless, proteaphagy might contribute to sensitize these cells to the action of both autophagy and proteasome inhibitors. Autophagy-mediated degradation of proteins involved in essential pathways like Akt, Stat5, and Erk may occur in parallel and be independent of proteaphagy. Further studies are necessary to clarify how proteaphagy could be enhanced in FLT3-ITD AML cells and how this process is regulated in other AML subtypes. Future research should consider the use of combined treatments blocking FLT3-ITD (by drugs like Quizartinib) and proteaphagy. These investigations would expand the impact of our results on a wide diversity of phenotypes present in AML, where protein homeostasis is also affected.

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189

VIII. Articles

1. Analysis of defective protein ubiquitylation associated to adriamycin resistant cells. Published as co authour

2. Proteasome inhibition induces proteaphagy in FLT3-ITD acute myeloid leukemia cells. Sent to publication

190 DR. MANUEL S. RODRIGUEZ (Orcid ID : 0000-0001-5445-4495)

Received Date : 27-May-2020 Revised Date : 15-Jul-2020 Accepted Date : 12-Aug-2020 Article type : Research Article

Inhibition of the proteasome and proteaphagy enhances apoptosis in FLT3-ITD-driven acute myeloid leukemia Rosa G Lopez-Reyes1,2, Grégoire Quinet1, Maria Gonzalez-Santamarta1, Clément Larrue 2, Jean-Emmanuel Sarry2, Manuel S. Rodriguez 1. 1Institute of Advanced Technology and Life Sciences (ITAV), IPBS-Centre de la Recherche Scientifique (CNRS), and Université Toulouse III Paul Sabatier, Toulouse France. 2Cancer Research Center of Toulouse Unité Mixte de Recherche (UMR) 1037 INSERM, ERL 5294 Centre de la Recherche Scientifique (CNRS), Toulouse, France. Corresponding authors: Manuel S. Rodriguez, UbiCARE, Institute of Advanced Technology and Life Sciences, 1 Place Pierre Potier, 31000 Toulouse. E-mail: [email protected]

Running heading: Proteaphagy-inhibition and FLT3-ITD AML apoptosis Abbreviations: AML, acute myeloid leukaemia; Atg, autophagy; ALS, autophagy-lysosome system; BafA, Bafilomycin A; Bz, Bortezomib; FLT3, fms-like tyrosine kinase 3; LC3B, microtubule-associated proteins 1A/1B light chain 3B; ITD, internal tandem duplication; PI, proteasome inhibitor; TUBEs, tandem ubiquitin bidning entities; UPS, ubiquitin-proteasome system, VT, Verteporfin.

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/2211-5463.12950 FEBS Open Bio (2020) © 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.Accepted Article This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Abstract: Acute myeloid leukaemia (AML) is a clonal disorder that affects hematopoietic stem cells or myeloid progenitors. One of the most common mutations that results in AML occurs in the gene encoding fms-like tyrosine kinase 3 (FLT3). Previous studies demonstrated that AML cells expressing FLT3-ITD are more sensitive to the proteasome inhibitor (PI) Bortezomib (Bz), than FLT3 wild-type cells, and this cytotoxicity is mediated by autophagy. Here we show that proteasome inhibition with Bz results in modest but consistent proteaphagy in MOLM-14 leukemic cells expressing the FLT3-ITD mutation, but not in OCI-AML3 leukemic cells with wild type FLT3. Chemical inhibition of autophagy with Bafilomycin A (BafA) simultaneously blocked proteaphagy and resulted in accumulation of the p62 autophagy receptor in Bz-treated MOLM-14 cells. The use of ubiquitin traps (TUBEs) revealed that ubiquitin plays an important role in proteasome-autophagy crosstalk. The p62 inhibitor Verteporfin (VT) blocked proteaphagy and, importantly, resulted in accumulation of high molecular weight forms of p62 and FLT3-ITD in Bz-treated MOLM-14 cells. Both autophagy inhibitors enhanced Bz-induced apoptosis in FLT3- ITD-driven leukemic cells, underlining the therapeutic potential of these treatments. Keywords: Proteaphagy, Bortezomib, Ubiquitin, AML, FLT3-ITD, Leukaemia 1. Introduction

Acute myeloid leukaemia (AML) is a clonal disorder that affects hematopoietic stem cells or myeloid progenitors. It is characterized by an accumulation of immature leukemic cells in the bone marrow and peripheral blood and leads to bone marrow failure [1]. AML is a heterogeneous disease with a variety of distinct genetic alterations [2]. One of the most common mutations occurs in the gene encoding fms-like tyrosine kinase 3 (FLT3) [3]. This type III receptor tyrosine kinase regulates the normal growth and differentiation of hematopoietic cells via the activation of multiple signalling including Akt, MAPK and Stat5 [4,5]. FLT3 cooperates with other recurrent molecular abnormalities to induce acute leukaemia in preclinical models. Internal tandem duplication (ITD) mutations in the FLT3 gene are found in approximately 30% of AML patients and are associated with a poor clinical outcome. Previous studies demonstrated that AML cells expressing FLT3-ITD are more sensitive to the proteasome inhibitor (PI) Bortezomib (Bz), than FLT3 wild-type cells. This cytotoxicity is mediated by autophagy [6]. Furthermore, the genetic inhibition of early autophagy steps or of autophagosome formation block FLT3-ITD, Stat5 and Akt degradation induced by Bz, [6]. The proteasome has been envisioned as a promising target for the development of anticancer Accepted Article

FEBS Open Bio (2020) © 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd. therapeutic drugs [7]. The 26S proteasome is a large multi-subunit protease (1500-2000 kDa), formed by the 20S proteolytic core, and one or two 19S regulatory particles [8]. Three proteolytically active subunits integrate the 20S core: β1 with a caspase-like activity, β2 with a trypsin-like activity, and β5 with a chymotrypsin-like activity. β5 is the primary target for most PIs that reached a clinical phase [8,9]. Cancer therapy also targets the Autophagy-Lysosome System (ALS), another proteolytic process that is responsible for the bulk degradation of cytoplasmic components. Amino acid deficiency activates autophagy by regulating signalling cascades controlling this proteolytic pathway [10]. During selective autophagy, phagophores engulf cytoplasmic material, and then fuse to form double-membrane autophagosomes. Cargo recruitment occurs through a family of autophagy receptors including p62, OPTN or NBR1 that are often used as markers for autophagy activation together with the ubiquitin-like molecule Atg8 [11]. The autophagy (Atg) machinery was first identified in yeast and equivalent molecules reported in mammalian cells [11–13]. Distinct autophagy events drive the degradation of organelles or aggregated proteins such as mitophagy (mitochondria), aggrephagy (protein aggregates) or proteasome (proteaphagy), to name a few [14]. Many of the current autophagy inhibitors act at a late stage of the system, such as the V-ATPase inhibitor Bafilomycin A (BafA). BafA inhibits autophagy in a non-selective way, by neutralizing acidic pH of lysosomal hydrolases which drive autophagic degradation [15]. Verteporfin (VT) is an FDA-approved drug that was identified in a screen for chemicals that prevent autophagosome formation [16]. Unlike BafA, VT inhibits autophagy at an early stage, and does not allow autophagosome accumulation[16]. A better understanding of the proteolytic regulation mechanism and interplay will allow exploring alternative/combined treatments to tackle cancer development and/or drug resistance. Here we aimed to better understand the proteolytic crosstalk connecting proteasome with autophagy after Bz treatment in FLT3-ITD positive MOLM-14 AML cells. Using a chemical approach to block autophagy at distinct levels together with ubiquitin traps (known as TUBEs [17]), immunoprecipitation (IP) and immunofluorescence, we found that proteaphagy was activated after Bz treatment. Although proteaphagy is a process preserved in distinct species [18– 20], we found that the presence of FLT3-ITD predisposes MOLM-14 cells to activate it.

2. Material and Methods Cell lines Accepted Article

FEBS Open Bio (2020) © 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd. Human myeloid leukaemia cell lines MOLM-14 and OCI-AML-3 were purchased from the ATCC collection. AML cell lines were maintained in RPMI supplemented with 10% foetal calf serum in the presence of 100 U/mL of penicillin and 100 µg/ml of streptomycin. Cells were incubated at

37°C with 5% CO2[6]. To facilitate autophagy analysis, 2% calf serum was used during chemical inhibitors treatment for a maximum time of 8 hours. Antibodies and reagents Antibodies anti-LC3B, anti-Erk 1/2, anti-Stat5, anti-Akt, anti-Ubiquitin (P4D1), anti-PSMB5, were purchased from Cell Signalling Technology (Beverly, MA, USA). Anti-FLT3, anti- p62/SQSTM1 and anti-Beta 2 were obtained from Santa Cruz Biotechnology (Dallas, Texas, USA), anti-PSMA6 from Invitrogen. Anti-PSMB6 and anti-Rpn10 were purchased from Enzo, anti-PSMD3 from Thermofisher and anti-GAPDH from Sigma. Bortezomib and Chloroquine were purchased from Sigma-Aldrich and Bafilomycin A from InvivoGen, and Verteporfin from Sigma Aldrich. Western blot analysis Proteins were resolved using 8 to 12% PAGE and electro-transferred to PVDF membranes. Membranes were then blocked with 5% skimmed powdered milk in TBS, or 5% bovine serum albumin in TBS. Membranes were immunostained with appropriate antibodies and horseradish peroxidase-conjugated secondary antibodies and visualized with enhanced chemiluminescence system. Apoptosis assay Cells line were cultured in RPMI 5%, treated at different times with Bortezomib, Bafilomycin A and Verteporfin. Then 5x105 cells were washed with PBS and resuspended in 100 µL of Annexin- V binding buffer. Two microliters of Annexin-V-FITC were added at room temperature. All samples were analysed by a fluorescent-activated cell sorter FACS Calibur flow cytometer [6]. TUBES capture TUBEs were produced according to Hjerpe et al., [17,21]. 20 millions of cells were used for each condition (TUBE p62, TUBE HHR23 or GST Control). Cells were resuspended in 500 µL of TUBE lysis buffer, kept 10 minutes at 4ºC and spin down at 15500 g. A fraction of this supernatant was diluted in 3X boiling buffer (50 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, bromophenol blue, 10% β-mercaptoethanol) and considered as Input. Supernatant was transferred to the glutathione-agarose beads with TUBEs or GST control, and samples were incubated overnight in rotation at 4ºC. On the next day, samples were spin down at 500g at 4ºC, a fraction Accepted Article

FEBS Open Bio (2020) © 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd. recollected, diluted in BB and considered as Flow-throw (FT). Beads were washed 3 times with 1 mL of PBS Tween 0.05%. Beads were resuspended in 100 µL of BB and boiled 5 minutes before protein electrophoresis. Antibody crosslinking

Thirty microliters per point of magnetic beads protein A (Milipore) were washed with PBS and equilibrated in binding buffer (50mM Tris pH 8.5, 150mM NaCl, + 0.5% NP40), using a magnetic holder. Antibodies were added to beads and rotated overnight. On the next day 10-20 µL of supernatant were kept to control antibody binding. Beads were washed twice with PBS and once with 500 µL of coupling buffer (200mM Borate, 3M NaCl pH9). Fifty millimolar DMP were added to coupling buffer, and samples were rotated during 30 minutes with this crosslinking solution (CS). Supernatant was discarded, replaced with fresh CS and incubated at 4ºC during 30 minutes. Beads were washed twice with coupling buffer before blocking with 20 mM ethanolamine pH 8.2. Supernatant was discarded and replaced by fresh ethanolamine and incubated for an hour. Beads were washed twice with PBS. Non-coupled antibodies were removed with 2 washes of 1M NaCl/binding buffer. A PBS equilibration was done before washing 3 times with 200 mM glycine pH 2.5. Beads were blocked with 0.1% BSA in binding buffer during 90 minutes. Magnetic beads were equilibrated in binding buffer and kept in PBS until used. A fraction of these beads (10-20 µL) were analyzed by electrophoresis followed by Coomassie blue staining to compare antibodies before and after crosslinking. Immunoprecipitation in presence of TUBEs Twenty millions of cells were spin down at 300g/10min, the dry pellet was resuspended in 500 L of TUBE lysis buffer including 100 g of TUBE p62 or TUBE HHR23[17]. Cell lysates were homogenized with 40 strokes at 4ºC using a dounce homogenizer. The whole sample was centrifuged at 200g/5min and supernatant was recovered for immunoprecipitation. A fraction (1/20) of the supernatant was considered as Input. The crosslinked antibody was incubated with cell lysates in rotation during 1h30 at 4ºC. Samples were disposed in magnetic holder to separate bound from unbound material. Proteins unbound to crosslinked antibodies were considered as flow-through (FT) fraction. Magnetic beads were washed with PBS/tween 0.05% 3-5 times and resuspended in 30 µL BB 3x to be analyzed by Western blot. Statistical analysis Data from 4 independent experiments were reported as the mean ± standard error of the mean. Accepted Article

FEBS Open Bio (2020) © 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd. Statistical analyses were performed using unpaired two-tailed Student t-tests with Prism 4 software. P0.05 was regarded as significant. *, **, ***, **** correspond to P0.05, P0.01, P0.001 and P0.0001 respectively. 3. Results Proteaphagy activation after proteasome inhibition in FLT3-ITD mutant driven MOLM-14 leukemic cells To mechanistically understand the regulation of the crosstalk between UPS and ALS in leukaemia, we investigated the role of proteaphagy which is known to be activated after proteasome inhibition [22]. The impact of Bz was assessed in MOLM-14 cells with the FLT3-ITD mutation and compared with the FLT3 wild-type cell line OCI-AML3 (Fig. 1). Bz treatment does not always promote an obvious degradation of proteasome subunits since proteolysis can be compensated by the novo synthesis of these subunits as previously reported [23,24]. For this reason, proteaphagy was evaluated by the degradation of 20S and 19S proteasomal subunits after Bz treatment and their accumulation with autophagy inhibitors. BafA treatment resulted in the accumulation of autophagy markers p62 and LC3B in the presence or absence of Bz, indicating that autophagy was activated under these experimental conditions in both cell lines (Fig. 1A). Nevertheless, lipidated forms of LC3B were only observed after BafA treatment in MOLM-14 but not in OCI-AML3. The low levels of apoptosis observed after 8h of individual or combined Bz/BafA treatment excluded the possibility that differences could be due to massive death of MOLM-14 cells (Fig. S1). Our results showed a modest but consistent Bz-mediated degradation of 20S proteasome subunits 6s and 5 and 19S subunits Rpn1 and Rpn3 that was blocked by BafA in MOLM-14 (Fig. 1B and C). However, the combination of BafA with Bz did not significantly accumulate proteasome subunits in OCI-AML3 as it was the case in MOLM-14 cells (Fig. 1B and C low panels), suggesting that a predisposition for degradation of 26S proteasome could be linked to the presence of FLT3-ITD.

These results were also confirmed by immunofluorescence where proteasomes subunit 2 or 2 colocalized with autophagosomes (Atg8 equivalent LC3B or p62 staining respectively) afterα Bz/BafA treatment of MOLM-14 cells (Fig. 2A and B). This Bz-induced degradation of proteasome subunits is blocked by BafA, indicating that proteaphagy mediated these proteolytic events. Role of p62 and ubiquitin in Bz-induced autophagy To analyse the role of p62 in the Bz-induced autophagic degradation of the proteasomes and Accepted Article

FEBS Open Bio (2020) © 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd. FLT3-ITD, the interaction of p62 with proteasome subunits and FLT3-ITD was investigated. IP experiments using a specific p62 antibody were performed using MOLM-14 cells treated or not with Bz/BafA. Taking advantage of the protective effects of TUBEs that block the action of proteases, accumulate ubiquitylated proteins and interact with multiple chain types [17,25], IPs were done in the presence or absence of TUBEs based in the UBA domain of HHR23 (TUBE- HHR23) or the UBA domain of p62 (TUBE-p62) (Fig. 3A). In the absence of TUBEs, p62 was immunoprecipitated without treatment and Bz/BafA reduced the level of precipitated p62. However, in the presence of both TUBEs, p62 was protected from proteasome and/or autophagy mediated degradation under Bz/BafA conditions (Fig. 3A). Compared to the situation without any TUBEs or TUBE-HHR23, the presence of TUBE-p62 allows a better coimmunoprecipitation of p62-bound proteasome subunits 2 and but not RPN1. High molecular weight forms most likely representing ubiquitylated forms of RPN1 were better immunoprecipitated in the presence of TUBE-HHR23 (Fig. 3A). Consistent with these observations, FLT3-ITD was also protected by TUBE-p62 but putative ubiquitylated forms of this protein were better immunoprecipitated in the presence of TUBE-HHR23. Since ubiquitin is a major coordinator of UPS and ALS, we investigated its role in the crosstalk of these pathways induced after Bz and BafA treatments. In particular, we were interested in investigating whether the high molecular weight forms of FLT3 co-immunoprecipitated with p62 corresponded to ubiquitylated FLT3. TUBE-HHR23 was used to capture total ubiquitylated proteins from MOLM-14 cells treated or not with individual and combined Bz/BafA treatment (Fig. 3B). Total ubiquitylated proteins were efficiently trapped by TUBE-HHR23. BafA treatment alone did not significantly enrich ubiquitylated proteins captured by TUBE-HHR23 compared to

Bz or Bz/BafA. Proteasome subunits 5 and Rpn1 are captured by TUBE-HHR23 (Fig. 3B). The p62 receptor was also captured under the same conditions but the combination of Bz/BafA enriched this protein compared to Bz alone (Fig. 3B). Interestingly, ubiquitylated forms of FLT3- ITD were captured by TUBE-HHR23 under all conditions but best enriched when Bz/BafA treatment was used (Fig. 3B). p62 drives proteaphagy and autophagic degradation of FLT3-ITD To further assess the role of the autophagy receptor p62 in proteaphagy, VT was used to treat MOLM-14 cells and Western blot analyses were performed to detect distinct proteasome subunits. High molecular weight forms of p62 were detected after VT treatment (Fig. 4A). Interestingly, the lipidated form of LC3B decreases after VT treatment, indicating that this drug reduced the Accepted Article

FEBS Open Bio (2020) © 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd. autophagy flux. Bz-mediated degradation of 20S or 19S subunits was blocked by VT even if the Bz-mediated degradation of 19S subunits was more prominent than 20S subunits (Fig. 4B and C).

The colocalization of p62/ 2 was significantly reduced with VT or Bz/VT indicating that blocking of p62 could inhibit proteaphagy through a mechanism distinct from BafA (Fig. 4D). To α investigate if the high molecular weight forms of p62 observed in Fig 4A were aggregated and/or ubiquitylated, IP were performed with p62 antibodies in the presence or absence of TUBE-p62 (Fig 4E) and TUBE-capture of ubiquitylated proteins (Fig. 4F). Interestingly, the aggregated forms of p62 generated after the Bz/VT treatment do not interact with the lipidated form of LC3B indicating that this treatment negatively affected this interaction (Fig. 4E). The lipidated forms of LCB3 were also reduced in the input fraction indicating that VT stops the autophagy flux as it was observed in Figure 4A. In order to explore the ubiquitylation status of p62 after VT treatment we captured ubiquitylated proteins using TUBE-p62 (Figure 4F). VT has a negative impact on the total ubiquitylated proteins as it can be observed in the input fraction. Nevertheless, the levels of ubiquitylation could be recovered when VT was combined with Bz. These results were also observed in the TUBE capture of total ubiquitylated proteins. Under these experimental conditions, p62 increased its ubiquitylated levels after Bz treatment and decreased when cells were treated with VT (Figure 4F). Altogether these evidences indicated that after VT treatment, aggregated forms of p62 are accumulated and these forms are less ubiquitylated. The reduction of the interaction p62/lipidated LC3B indicate that this p62 was not integrated into autophagosomes after Bz/VT treatment. In a similar manner, VT reduced the localization of p62 with proteasome subunits, supporting the notion that proteaphagy is hampered by this treatment (Figure 4D). Thus, these data show that p62 plays an important role in proteaphagy observed in MOLM-14 cells.

Inhibition of UPS and ALS pathways enhances apoptosis of FLT3-ITD cells Individual or combined treatments were used to investigate if Bz-induced degradation of FLT3- ITD was blocked by VT in MOLM-14 cells (Fig 5A). Our results indicate that FLT3-ITD and FLT3 were degraded up to 25% after Bz treatment in MOLM-14 cells (Fig 5A). FLT3-ITD degradation was blocked by VT while high molecular weight forms of FLT3 were formed under the same conditions in MOLM-14 suggesting a ubiquitin driven autophagy-mediated proteolysis event (Fig. 5A). Interestingly, in OCI-AML3 cells, under the same experimental conditions FLT3 was not degraded by Bz and VT treatment rather accumulated high mobility forms of this protein (Fig. 5B). The double Bz/VT treatment does not significantly accumulate FLT3 in OCI-AML3 Accepted Article

FEBS Open Bio (2020) © 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd. (Fig. 5B), supporting the notion that proteolytic pathways are not efficiently activated in these cells. Finally, we investigated the consequences of accumulated proteasome and FLT3-ITD after inhibition of UPS and ALS pathways in MOLM-14 cells and compared to OCI-AML3 cells. Apoptosis was measured by analysing Annexin-V positive cells after single agent or combined treatments (Fig. 5). Bz-induced cell death was always kept below 50% to differentiate positive or negative effects of BafA on the Bz treatments. To improve the efficiency of autophagy inhibition, cells were pre-treated 8h with BafA before adding Bz for an additional 16h at the indicated doses (Fig. 5C and D). Our results showed that the toxicity of the individual BafA treatment was around 20% (MOLM-14) or below 5% (OCI-AML3) and both drugs efficiently cooperated to enhance the cell killing effect on MOLM-14 (Fig. 5C) but this was not in the case in OCI-AML3 (Fig. 5D). Apoptosis induced by VT also improved the results observed with Bz alone in MOLM-14 cells (Fig. 5E) but not in OCI-AML3 cells (Fig. 5F). In this case, Bz and VT were simultaneously added to cell cultures to work below the VT IC50. This results in an additional 8h of Bz compared to Fig. 5A, explaining the higher apoptosis observed with Bz treatment only. Cooperative effects observed with the double Bz/VT treatment were statistically significant in MOLM-14 cells but not in OCI-AML3 (Fig. 5E and 5F). Altogether, our results show that the inhibition of both proteolytic pathways markedly enhances apoptosis levels in MOLM-14 that express FLT3-ITD (Fig. 6) but not in OCI-AML3 that express WT FLT3.

4. Discussion Multiple mechanisms of interplay between the UPS and ALS have been documented over the last ten years. Here we identified proteaphagy as part of the selective autophagic events, which are activated after Bz treatment in FLT3-ITD positive leukemic cells. Our data showed that this tyrosine kinase translocation facilitates the Bz-mediated proteolysis of 20S and 19S subunits and their colocalization within autophagosomes. FLT3-ITD can potentially predispose to proteaphagy due to its capacity to activate multiple signalling cascades that have an impact on autophagy activation. Among these pathways are the phosphatidylinositol-3 kinase (PI3K) [26,27], Akt [28], mammalian target of rapamycin (mTOR) [29], RAS, and extracellular signal-related kinase (Erk), MAPK and Stat5 [5,30]. It remains to be demonstrated if any or various of these signalling pathways have a positive or negative impact on proteaphagy. Proteaphagy is a complicated process to analyse since not all proteasomes are directly concerned Accepted Article

FEBS Open Bio (2020) © 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd. by this type of degradation. Nuclear proteasomes will not be immediately affected since proteaphagy is a cytoplasmic event. According to the literature, only 20-50% of the proteasomes are regulated by proteaphagy depending on the time, intensity and type of stimuli in distinct biologic models [18–20]. Our results demonstrate that the autophagy receptor p62 is implicated in the proteaphagy activated by Bz in MOLM-14 cells. However, our data do not exclude the participation of other autophagy receptors in this process [14]. For instance, both the ubiquitin receptor Cue5 and the chaperon Hsp40 [19] or the proteasome subunit RPN10 [18], respectively mediate proteaphagy in Saccharomyces cerevisiae or Arabidopsis thaliana. Nevertheless, the use of VT in leukemic cells showed that the inactivation of p62 stops Bz-induced proteaphagy supporting a major role of p62 in this process. Interestingly, VT favours the formation of high molecular weight aggregates of p62 [31], which are also observed with FLT3-ITD with the same treatment. However, other effects have been reported for VT including the activation of ROS that could affect other processes [32,33] making difficult to attribute VT effects only to its action on p62.

The IP/protection assays [17] revealed that TUBE-p62 protects p62, RPN1, 2 or FLT3-ITD from Bz-driven degradation blocked with BafA in MOLM14 cells. TUBE-HHR23 also protects these factors but to a lesser extent than TUBE-p62. However, TUBE-HHR23 better accumulates ubiquitylated forms of RPN1 or FLT3-ITD. Similar IP experiments performed with Bz/VT in MOLM-14 showed that high molecular weight forms of p62 did not interact with the lipidated form of LC3B, indicating that p62 was not integrated into autophagosomes under those conditions. This could be associated to the reduction of total ubiquitylated forms observed after VT treatment that might have a negative impact in ubiquitin-regulated events. The ubiquitin proteome captured with TUBE-HHR23 includes several proteins implicated in proteaphagy such as p62, RPN1 or 5 after Bz or Bz/BafA treatments but not with BafA alone, indicating that this autophagy inhibitor does not accumulate ubiquitylated forms of these factors in the absence of proteasome inhibition. TUBE-p62 captures significantly less ubiquitylated proteins than TUBE-HHR23 and specific ubiquitylated forms can only be seen when overexposing. Nonetheless, in this way we found that while Bz accumulated ubiquitylated forms of p62, VT reduces these forms most likely interfering with the integration of this receptor into autophagosomes. The use of both TUBEs to investigate UPS and ALS regulated events has the interest to keep all possibilities open to find which pathway will be playing a role under distinct experimental settings. While TUBE-HHR23 recognises virtually all types of chains [17,34,35], the Accepted Article

FEBS Open Bio (2020) © 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd. UBA domain of p62 recognises mainly K63 ubiquitin chains explaining the observed differences [36,37]. Altogether our data indicate that when both proteolytic pathways are blocked, accumulation of cellular factors occurs due to the functional absence of these degradation machineries (Fig. 6). This contributes to enhance apoptosis in MOLM-14 but not OCI-AML3 cells under the same experimental conditions. In conclusion, targeting protein homeostasis could be an alternative to improve current treatments of FLT3-ITD AML cells. Although the crosstalk of these complex proteolytic mechanisms remains to be fully elucidated, our results open new possibilities for the treatment of this AML phenotype.

Acknowledgments We thank Clémence Coutelle-Rebut for the proofreading of this manuscript. MSR and MGS are funded by UbiCODE a network supported by the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 765445. MSR also receives funding from the Institut National du Cancer, France (PLBIO16-251), LASSERLAB- EUROPE under the grant No 654148, CONACyT-SRE (Mexico) under the grant No 0280365 and the Occitanie region, France, through the REPERE and Prematuration programs. GQ and RGLR are respectively fellows from The French Ministry of Higher Education and Research and CONACYT (Mexico). Author Contributions RGLP, MSR, JES conceived and designed the project, RGLP, GQ and MGS acquired the data, RGLP, GQ, MGS and CL analysed and interpreted the data, MSR and RGLP wrote the paper, all authors contributed to proofread this manuscript. Data Accessibility Data will be available from the corresponding author upon reasonable request. Conflict of interest Authors declare no conflict of interest.

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Figure legends Fig. 1. Bz-driven proteaphagy is enhanced in FLT3-ITD phenotype. MOLM-14 (FLT3-ITD+/- ) or OCI-AML3 (FLT3-WT) cells were treated 8h with Bz 10 nM, and 20 nM Bafilomycin. Total cell lysates were resolved by SDS-PAGE and immunoblotted with the indicated antibodies recognizing the autophagy receptor p62 (A), proteasome core subunits α 6 and 5 (B) or 19S subunits Rpn1 and Rpn3 (C). Protein expression levels were quantified by densitometry analysis (Image J software). Statistical analyses were performed using unpaired two-tailed Student t-tests with Prism 4 software. P0.05 was regarded as significant. *, **, ***, **** correspond to P0.05, P0.01, P0.001, P0.0001 respectively, the data are the s.e.m, n=4. Fig. 2. Colocalization of proteasome and autophagy markers after Bz and autophagy inhibitors treatment in FLT3-ITD AML cells. Indirect immunofluorescence staining LC3B/β2

(A) or p62/ 2 (B) positive structures in MOLM-14 cells treated 8h with Bz 10 nM and 20 nM

BafA. Imagesα were captured by confocal microscopy. Scale bar 10 m Immunofluorescence images were quantified from 3 replicates. Statistical analyses were performed using unpaired two- tailed Student t-tests with Prism 6 software. P<0.05 was regarded as significant. *, **, ***, **** correspond to P<0.05, P<0.01, P<0.001, P<0.0001 respectively, the data are the s.e.m.

Fig. 3. Ubiquitin role in proteaphagy and degradation of FLT3-ITD under proteasome inhibition conditions. MOLM-14 cells were treated or not during 8h with Bz 10 nM, BafA 20 nM Accepted Article

FEBS Open Bio (2020) © 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd. or both drugs and cells were lysed as reported [17]. Ubiquitylated proteins were captured using TUBEs-HHR23, TUBEs-p62 or GST control (A). Captured proteins were resolved is SDS-PAGE and immunoblotted with the indicated antibodies. Input and flow through (FT) fractions were also analysed with anti-ub antibody. GAPDH was used as loading control. (B) MOLM-14 cells were treated or not with Bz/BafA under the same conditions than “A”. p62-bound proteins were captured by IP with a specific p62 antibody in the presence or absence of TUBE-HHR23 or TUBE-p62. Precipitated material was analysed by Western-blot with the indicated antibodies. Input fraction was analysed for the indicated proteins.

Fig. 4. p62 drives proteaphagy in FLT3-ITD AML cells. MOLM-14 cells were treated 8h with Bz 10 nM and VT 1µM. Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against autophagy markers LCB3 and p62 (A), proteasome core subunits 6 and 5

(B) and 19 subunits Rpn1 and Rpn3 (C). Protein expression levels were quantified by densitometry analysis (Image J software). (D) Indirect immunofluorescence staining of p62/ 2 positive structures in MOLM-14 cells treated 8h with Bz 10 nM and 1 µM VT. Images were α captured by confocal microscopy. Scale bar 10 m. Immunofluorescence images were quantified from 3 replicates. Statistical analyses were performed using unpaired two-tailed Student t-tests with Prism 6 software. P<0.05 was regarded as significant. *, **, ***, **** correspond to P<0.05, P<0.01, P<0.001, P<0.0001 respectively, the data are the s.e.m. n=5. (E) Immunoprecipitation of p62 from MOLM-14 cells treated or not with Bz 10nM and VT 1M. Experiments were performed in the presence or absence of TUBE-p62. Precipitated material was analysed by Western blot with specific p62 and LC3B antibodies. Input fraction was analysed with the indicated antibodies. (F) Capture of ubiquitylated proteins using TUBE-p62 trap. GST was used as negative control. Captured proteins were analysed by Western blot with anti-ubiquitin or p62 antibodies. Input and flow through (FT) fractions were analysed with the indicated antibodies.

Fig. 5. Proteasome and autophagy inhibitors cooperate to improve apoptosis of FLT3-ITD expressing cells. MOLM-14 (A) and OCI-AML3 (B) cells were treated 8h with Bz 10 nM and VT 1µM. Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against FLT3 (A and B). Protein expression levels were quantified by densitometry analysis (Image J software). Statistical analyses were performed using unpaired two-tailed Student t-tests with Prism Accepted Article

FEBS Open Bio (2020) © 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd. 6 software. P<0.05 was regarded as significant. *, **, ***, **** correspond to P<0.05, P<0.01, P<0.001, P<0.0001 respectively, the data are the s.e.m. (C and D) MOLM-14 and OCI-AML3 cells were treated 8h with BafA 15nM. Bz was added at 6 or 7.5 nM concentration for additional 16h. (E and F) MOLM-14 and OCI-AML3 cells were treated with a fixed concentration of Bz 7.5 nM and two distinct doses of VT (0.5 and 1 M) as indicated. Apoptosis was analysed in by FACS. Percentage of cell death was measured from 4 biological replicates. Statistical analyses were performed using unpaired two-tailed Student t-tests with Prism 4 software. P0.05 was regarded as significant. *, **, ***, **** correspond to P0.05, P0.01, P0.001, P0.0001 respectively, the data are the s.e.m, n=3.

Fig. 6. UPS and ALS crosstalk under proteasome and/or autophagy inhibition. 1) Under basal unstimulated conditions, turnover of important cellular factor is ensured by equilibrated proteolytic pathways 2) Inhibition of proteasome directs crucial cellular factors to ALS for degradation. 3) Autophagy inhibition drives the degradation of some cellular factors to proteasome-mediated degradation 4) when both proteolytic pathways are impaired, both UPS and ALS contribute to accumulate cellular factors and increase apoptosis in FLT3-ITD positive cells. Supporting Information Supplementary Fig 1. Cell death evaluation at 8h. Treatments using Bz 15 nM and BafA 10 nM with FBS 2% MOLM-14 before Annexin-V staining and flow cytometry, to validate Western blot conditions N= 24, 3 biological replicates. Statistical analyses were performed using unpaired two- tailed Student t-tests with Prism 6 software. P<0.05 was regarded as significant. *, **, ***,**** correspond to P<0.05, P<0.01, P<0.001, P<0.0001 respectively, the data are the s.e.m, n=3. Accepted Article

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ISSN: 1538-4101 (Print) 1551-4005 (Online) Journal homepage: https://www.tandfonline.com/loi/kccy20

Analysis of defective protein ubiquitylation associated to adriamycin resistant cells

Valérie Lang, Fabienne Aillet, Wendy Xolalpa, Sonia Serna, Laurie Ceccato, Rosa G. Lopez-Reyes, Maria Paz Lopez-Mato, Radosław Januchowski, Niels- Christian Reichardt & Manuel S. Rodriguez

To cite this article: Valérie Lang, Fabienne Aillet, Wendy Xolalpa, Sonia Serna, Laurie Ceccato, Rosa G. Lopez-Reyes, Maria Paz Lopez-Mato, Radosław Januchowski, Niels-Christian Reichardt & Manuel S. Rodriguez (2017) Analysis of defective protein ubiquitylation associated to adriamycin resistant cells, Cell Cycle, 16:24, 2337-2344, DOI: 10.1080/15384101.2017.1387694 To link to this article: https://doi.org/10.1080/15384101.2017.1387694

Accepted author version posted online: 03 Nov 2017. Published online: 20 Nov 2017.

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Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=kccy20 CELL CYCLE 2017, VOL. 16, NO. 24, 2337–2344 https://doi.org/10.1080/15384101.2017.1387694

REPORT Analysis of defective protein ubiquitylation associated to adriamycin resistant cells

Valerie Langa,†, Fabienne Ailleta,†, Wendy Xolalpaa, Sonia Sernab, Laurie Ceccatoc,d, Rosa G. Lopez-Reyesc,d, Maria Paz Lopez-Matoa, Rados»aw Januchowskie, Niels-Christian Reichardtb,f, and Manuel S. Rodrigueza,c,d aInbiomed, Mikeletegi Pasealekua, San Sebastian-Donostia, Spain; bGlycotechnology Laboratory, CIC biomaGUNE, Miramon Pasealekua, San Sebastian- Donostia, Spain; cInstitut des Technologies Avancees en sciences du Vivant (ITAV) 1 Place Pierre Potier, Universite de Toulouse, CNRS, UPS, Toulouse, France; dInstitut de Pharmacologie et de Biologie Structurale (IPBS), 205 Route de Narbonne, Universite de Toulouse, CNRS, UPS, Toulouse, France; eDepartment of Histology and Embryology, Poznan University of Medical Sciences, Swiecickiego 6 St., Poznan, Poland; fCIBER de Bioingenierıa, Biomateriales y Nanomedicina (CIBER-BBN), San Sebastian-Donostia, Spain

ABSTRACT ARTICLE HISTORY DNA damage activated by Adriamycin (ADR) promotes ubiquitin–proteasome system-mediated Received 8 May 2017 proteolysis by stimulating both the activity of ubiquitylating enzymes and the proteasome. In ADR- Revised 20 September 2017 resistant breast cancer MCF7 (MCF7ADR) cells, protein ubiquitylation is signiﬁcantly reduced compared to Accepted 29 September 2017 the parental MCF7 cells. Here, we used tandem ubiquitin-binding entities (TUBEs) to analyze the KEYWORDS ADR ubiquitylation pattern observed in MCF7 or MCF7 cells. While in MCF7, the level of total ubiquitylation Ubiquitylation; ADR increased up to six-fold in response to ADR, in MCF7 cells only a two-fold response was found. To chemoresistance; further explore these differences, we looked for cellular factors presenting ubiquitylation defects in Adriamycin; proteasome; MCF7ADR cells. Among them, we found the tumor suppressor p53 and its ubiquitin ligase, Mdm2. We also degradation; TUBEs; p53; observed a drastic decrease of proteins known to integrate the TUBE-associated ubiquitin proteome after breast cancer; ovarian cancer ADR treatment of MCF7 cells, like histone H2AX, HMGB1 or b-tubulin. Only the proteasome inhibitor MG132, but not the autophagy inhibitor chloroquine partially recovers the levels of total protein ubiquitylation in MCF7ADR cells. p53 ubiquitylation is markedly increased in MCF7ADR cells after proteasome inhibition or a short treatment with the isopeptidase inhibitor PR619, suggesting an active role of these enzymes in the regulation of this tumor suppressor. Notably, MG132 alone increases apoptosis of MCF7ADR and multidrug resistant ovarian cancer A2780DR1 and A2780DR2 cells. Altogether, our results highlight the use of ubiquitylation defects to predict resistance to ADR and underline the potential of proteasome inhibitors to treat these chemoresistant cells.

Introduction multiple ubiquitin-binding domains (UBDs) that connect mod- Protein ubiquitylation regulates multiple essential cellular pro- ified substrates with effector functions.6 cesses such as proteolysis, DNA repair or transcription in Point mutations and chromosomal rearrangements affecting – response to diverse stimuli.1 3 Ubiquitin attachment to sub- substrates, enzymes or cofactors of the ubiquitylation/deubiqui- strate proteins is mediated by at least three categories of tylation cascade are known to alter ubiquitylation of multiple enzymes known as ubiquitin-activating enzymes (E1), ubiqui- cellular factors that have been proposed to be at the origin of tin-conjugating enzymes (E2), and ubiquitin-ligases (E3). Pro- pathologies such as cancer, inflammation, immunological dis- – tein substrates can be modified by one or more ubiquitin orders or neurodegeneration.8 10 Intracellular signaling path- moieties and the products are known as mono or multiple ways, including those activating DNA damage response, induce monoubiquitylated proteins, respectively. Ubiquitin chains protein ubiquitylation changes in stimulated cells. Therapeutic (known as polyubiquitylation) can also be formed using inter- doses of ADR promote UPS-mediated proteolysis by stimulat- nal lysine residues within ubiquitin (K6, K11, K27, K29, K33, ing both the activity of ubiquitylating enzymes and the protea- K48, K63). Furthermore, Met1-linked linear ubiquitin chains some.11,12 However, breast cancer MCF7 cells resistant to ADR are involved in the activation of signaling events.4 While K11 show low levels of ubiquitylation and fail to accumulate ubiqui- and K48 chains have been associated to proteasome-mediated tylated proteins after stimulation with ADR, compared to proteolysis, K63 chains have been linked to signaling pathways, parental ADR sensitive cells.13 The analysis of ubiquitylation intracellular traffic, DNA repair or autophagy among other defects after cell stimulation with ADR could help us to gain an – functions.5 7 The cleavage of ubiquitin moieties is regulated by improved understanding for the role of critical factors involved several families of isopeptidases known as deubiquitylating in the response to this drug, identify non-responding patients enzymes (DUBs).5 Ubiquitylated proteins are recognized by and ultimately, define alternative targets for intervention.

CONTACT Manuel S. Rodriguez [email protected] Institut des Technologies Avancées en sciences du Vivant (ITAV), Université de Toulouse, CNRS, UPS, 1 Place Pierre Potier, 31000 Toulouse, France; Niels-Christian Reichardt [email protected] Glycotechnology Laboratory, CIC biomaGUNE, Miramon Pasealekua, 182, 20014, San Sebastian-Donostia, Spain. y These authors have contributed equally. © 2017 Taylor & Francis 2338 V. LANG ET AL.

Specific anti-ubiquitin antibodies or the tandem ubiquitin- level and in response to ADR treatment in comparison to binding entities (TUBEs) are among the most popular alterna- MCF7 cells (Fig. 1A). We employed TUBEs-based microarrays tives used to analyze endogenous ubiquitylated proteins.14,15 In to quantify differences in ubiquitylation profiles.13 TUBEs- addition to their high affinity and specificity, TUBEs show two microarrays were incubated with known protein concentrations convenient properties to improve the purification yield of ubiq- of MCF7 and MCF7ADR cellular extracts stimulated or not with uitylated proteins: the protection from the action of DUBs and ADR (experiment shown in Fig. 1A). Ubiquitylated proteins – also from proteasome-mediated proteolysis.16 19 Here, we used bound to TUBEs-microarrays were detected with mouse anti- TUBEs to isolate and analyze ubiquitylated proteins from ubiquitin antibody (FK2) and secondary Alexa FluorÒ 647 rab- MCF7 cells responding or not to ADR treatment. Besides their bit anti-mouse antibody (Fig. 1B). The images of individual use as affinity probes for the enrichment of ubiquitylated pro- spots are shown to demonstrate the good spot morphology and teins, TUBEs were useful to quantify differences in ubiquityla- low background fluorescence obtained in the array experi- tion between MCF7 and MCF7ADR cells. Analysis of the ments. Average fluorescence values and the SD of the mean obtained ubiquitylation profiles indicated that UPS is active from five replicate spots were quantified from the original even in the case of resistant cells, where ADR treatment failed images scanned at 10 mm resolution and represented as histo- to induce the accumulation of ubiquitylated proteins. This grams above the images of individual spots for defined amounts implies that Ubiquitin Proteasome System (UPS) inhibitors of total protein (Fig. 1B). Basal levels of ubiquitylated proteins could have therapeutic value if used to treat ADR resistant cells. in unstimulated cells showed lower relative fluorescence units (RFU) values that were proportional to the total protein content employed in the microarray assay. Protein ubiquitylation Results was significantly increased in response to ADR treatment only in sensitive MCF7 cells but not in MCF7ADR cells (Fig. 1B). Analysis of protein ubiquitylation defects in adriamycin Interestingly, for higher protein concentrations between 50– resistant cells 100 mg, we observed only a modest 3-fold increase of ubiquity- To quantify ubiquitylated proteins in response to the genotoxic lation in MCF7 cells while for protein concentration below to agent ADR, MCF7 and MCF7ADR cells were stimulated (or 7 mg, ubiquitylation levels increased up to 6 fold, facilitating not) at a time point (1 h) where we had previously observed an the comparison of ubiquitylation profiles between MCF7 and accumulation of protein ubiquitylation.18 MCF7ADR cells MCF7ADR cells (Fig. 1C). This behavior might be explained by showed a strong decrease of total ubiquitylated proteins at basal a rapid saturation of TUBEs at high concentrations of

Figure 1. Decreased of total protein ubiquitylation in MCF7ADR compared to MCF7 cells. (A) MCF7 and MCF7ADR cells were treated (C) or not for 1h with Adriamycin (1 mM). TUBE-capture fractions were analyzed by Western blot with the indicated antibodies. (B) Detection of ubiquitylated proteins on TUBEs microarrays. Analysis of ubiquitylated proteins in MCF7 or in MCF7ADR cell extracts at different concentrations of total protein (3, 7, 12, 25, 50, and 100 mg) with (white) or without (grey) ADR treatment (1h, 1 mM) is shown. Each histogram represents the average relative fluorescence units (RFU) value of five spots for each condition. (C) Fold of ubiquitylated proteins in MCF7 and MCF7ADR cell extracts at different concentrations of total protein (3, 7, 12, 25, 50, and 100 mg) comparing the level of total ubiquitylated proteins after ADR treatment to untreated cells. CELL CYCLE 2339 ubiquitylated proteins present in the cell extracts.13 Altogether, Inhibition of the ubiquitin-proteasome pathway recovers our results indicate that TUBEs microarrays are well suited for protein ubiquitylation and promote apoptosis in the quantification of ubiquitylated proteins in the low micro- Adriamycin resistant cells gram range. Intrigued by the low level of total protein ubiquitylation as well as the absence of p53 ubiquitylation in MCF7ADR cells, inhibi- Characterization of protein ubiquitylation profiles in tors of the proteasome and autophagy were used to accumulate response to adriamycin and capture ubiquitylated proteins from MCF7 and MCF7ADR cells. Overnight treatment with proteasome inhibitor MG132, To analyze the ubiquitylation profiles of MCF7 and ADR but not autophagy inhibitor chloroquine (CQ), partially recov- MCF7 cells in more detail, we tried to identify the key ered the total protein ubiquitylation in MCF7ADR cells cellular factors with major ubiquitylation defects after ADR (Fig. 3A). The ubiquitylation of p53 was also increased after treatment. To this end, we isolated ubiquitylated proteins proteasome inhibition as detected using a TUBE-IP p53 proce- from the corresponding cell lines via TUBEs based affinity 14,15 dure followed by Western blot with different ubiquitin antibod- capture. Input and captured fractions were analyzed by ies (Fig. 3B). These experiments were performed under Western blot using a panel of selected antibodies recogniz- conditions where most of the ubiquitylated proteins were cap- ing proteins that are ubiquitylated in response to ADR in fi fl 18 tured, since we could hardly observe speci c signal in the ow- MCF7 cells (Fig. 2). The most prominent ubiquitylation through 1 (FT1) or flow-through 2 (FT2) (Fig. 3C). Interest- differences were observed for the tumor suppressor p53 and ingly, the FK1 and K48 antibodies recognizing only polyubiqui- its ubiquitin E3 ligase Mdm2 (Fig. 2). Interestingly, p53 tylated proteins gave a stronger signal than the FK2 antibody protein level does not increase after ADR treatment of ADR that recognizes both mono- and polyubiquitylated species. MCF7 cells (Fig. 2, Input), explaining the low levels of These observations indicate that K48 polyubiquitylation of p53 expression of the p53-transcription dependent Mdm2 ligase is accumulated in both MCF7 and MCF7ADR cells in response (Fig. 2, input). Histone H2AX, HMGB1 or b-tubulin, that fi to MG132 treatment. In contrast, ADR treatment modestly but had been previously identi ed as part of the ADR-induced consistently reduced the p53 ubiquitylation in MCF7 cells ubiquitin proteome in MCF7 cells 18,alsoshowedlow ADR responding to this treatment. expression levels in resistant MCF7 cells, underscoring To investigate the role of isopeptidases in the defective ubiq- the differences observed in ubiquitylation profiles of MCF7 ADR ADR uitylation levels of MCF7 cells, the pan-inhibitor PR619 versus MCF7 cells after ADR treatment. These differen- was used. Since this inhibitor could produce side effects in pro- ces in ubiquitylation of these key cellular factors show longed treatments, cells were treated for no longer than 15– potential for stratifying good from bad responders to ADR 21,22 13,20 30 min. Under these experimental conditions, total ubiqui- treatment. tylation and p53 ubiquitylation were modestly but consistently increased after treatment of MCF7ADR cells, supporting an active role of isopeptidases in the regulation of this tumor suppressor in these chemoresistant cells (Fig. 3 and Supplementary Fig. 1). The combination of PR619 with overnight ADR or MG132 treatments did not result in an accumulation but rather a decrease of p53 ubiquitylation, suggesting that these forms might be controlled by alternative molecular mechanisms (Fig. 4A). Similar observations were obtained using 4h treatment with ADR and/or MG132 (Supplementary Fig. 1). Inter- estingly, MG132 alone induced apoptosis of MCF7ADR cells, highlighting the potential use of proteasome inhibitors to treat ADR resistant cells (Fig. 4B). In order to investigate if all these observations could be extrapolated to other ADR resistant cells, ovarian carcinoma A2780 cells resistant to this chemotherapy agent were used. TUBE capture experiments were performed using parental A2780 and resistant A2780DR1 and A2780DR2 cells stimulated or not during 1 or 4 hours with ADR. The pattern of total ubiquitylation detected in the TUBE-captured fraction was reduced in the ADR resistant cells at basal level and after ADR stimulation (Fig. 5A). Similarly, the pattern of ubiquitylated p53 was also reduced in ADR resistant cells at basal level but remain for longer after ADR treatment suggesting a delayed Figure 2. Ubiquitylation defects of specific proteins in MCF7ADR cells. MCF7 and cellular response. Interestingly these differences are less obvious ADR MCF7 cells were treated with ADR (1 mM) for the indicated times, ubiquitylated in the input fractions, underlining the benefit of using a TUBE- proteins were captured using TUBEs and analyzed by Western blot using the indicated antibodies. Input and TUBEs-capture fractions are shown. GAPDH was used capture approach to detect these differences. We have also con- as a loading control. firmed that MG132 alone could better induce the apoptosis of 2340 V. LANG ET AL.

Figure 3. Ubiquitin-chain proﬁles in ADR sensitive and resistant cell lines treated with proteasome and autophagy inhibitors. MCF7 and MCF7ADR cells were treated overnight with ADR (1 mM), MG132 (5 mM) or Chloroquine (CQ, 200 mM). Ubiquitylated forms were captured using TUBEs and bound material was eluted before being submitted to p53 immunoprecipitation as previously described 16. Input (A), TUBEs-IP p53 (B) and unbound fractions (FT1 and FT2) (C) were analyzed by Western blot using the indicated antibodies.

A2780DR1 and A2780DR2 cells (Fig. 5B and 5C), validating damage agents used in chemotherapy have the capacity to our previous observations in the breast cancer MCF7ADR cell simultaneously activate multiple signaling cascades altering the line. protein ubiquitylation status and thus, the stability and activity of multiple proteins.11 In this work, we show that ubiquitylation proﬁles of the totality of the proteins present in the cell Discussion can be used to distinguish between cells responding or not to Ubiquitylation defects of critical cellular factors including those the genotoxic agent ADR. Indeed, the results found by using involved in the activation of the p53 pathway have been associ- TUBEs-microarrays revealed that total protein ubiquitylation ated to distinct pathologies.23,10 Strong stimuli such as DNA increases up to 6 fold in ADR sensitive cells while in resistant CELL CYCLE 2341

their turnover by a proteasome dependent-ubiquitin independent mechanism.25,26 Thus, the fact that ubiquitylated p53 is partially accumulated in the presence of proteasome or DUBs inhibitors, indicates that ubiquitylation is mechanistically possible, however further investigations are required to better understand the origin of the reduced p53 ubiquitylation in MCF7ADR, A2780DR1 or A2780DR2 cells and its biological significance. Altogether, our characterization of ubiquitylation profiles indicated that even if ADR failed to accumulate ubiquitylated proteins in MCF7ADR, the UPS was active and could potentially be used to treat cancerous cells resistant to this chemotherapeutic agent. As matter of fact, the analysis of apoptosis using MG132 to treat ADR resistant cell lines supports this possibility (Fig. 4B and 5B). We have observed a highly altered protein expression profile in the ADR resistant MCF7 cell lines using proteomics approaches. These changes include components of the GSH pathway that were proposed to contribute in the development of chemoresistance.27,28 Interestingly, ADR significantly stimulated the formation of hydroxyl radical spin adducts [5,5- dimethyl-1-pyrroline N-oxide (DMPO)-OH] in the sensitive cells but not in the resistant cells.29 The role of UPS and regulation of redox processes have emerged as essential factors to control the fate of cells upon differentiation.30 This is coherent with the role of reactive oxygen species (ROS) affecting the conjugation of those ubiquitin family members that contribute to an appropriate response to chemotherapy.31 The analysis of ubiquitylated proteins associated to cellular events continues to be a difficult task partly due to the highly dynamic and reversible formation of ubiquitin chains. Multiple approaches for ubiquitin analysis have been developed mainly based on the use of specific antibodies that recognize populations of ubiquitylated proteins or specificubiquitinchains.14 We show here that TUBEs combined with selected antibodies can be used to analyze total Figure 4. Proteasome inhibition promotes efficient apoptosis in MCF7ADR cells. (A) MCF7 and MCF7ADR cells were treated overnight with ADR, MG132 or PR619 and individual ubiquitylated proteins from MCF7 and or (20 mM, 15 min) alone or in combination as indicated. Ubiquitylated proteins were A2780 cells in distinct protocols. This approach can be captured using TUBEs and analyzed by Western blot. Input and TUBEs-capture frac- adopted to investigate ubiquitylation in response to distinct tions were shown. GAPDH was used as a loading control. (B) To track MCF7 cells m stimuli and to identify high from low responders to undergoing apoptosis, cells were treated or not with ADR (1 M overnight), 13,20 MG132 (5 mM, overnight), or PR619 (20 mM, 15 min) alone or in combination as ADR. The further development of methods to improve indicated. Data represent the mean of three independent experiments done in the analysis of protein ubiquitylation will contribute to a triplicate. A total of 10.000 events were collected and analyzed for each sample. better understanding of the many cellular processes controlled by this post-translational modification and improve cells the increase was less than 2-fold. Altogether, these results the current chemotherapeutic treatment options. indicate that the analysis of protein ubiquitylation phenotypes using TUBEs-microarrays could be helpful to determine a per- sonalized treatment option for breast cancer patients. This Materials and methods holds also true for specific factors such as p53 and Mdm2 that Cell culture affected in their ubiquitylation levels in response to ADR. The concerted use of TUBEs with distinct antibodies could provide Breast cancer MCF7 and MCF7ADR cells (gifts from Dr. Gant, information on the degree and the type of accumulated protein MRC Toxicology 32,33) were grown in RPMI with 10% FBS and ubiquitylation in response to an individual treatment. antibiotics. MCF7ADR cell cultures were supplemented with The observed defects in p53 ubiquitylation in MCF7ADR cells Adriamycin (ADR; Sigma-Aldrich; 0.5 mM) and the drug was are most probably due to the absence or high turnover of removed 48 hours before performing experiments. Ovarian car- Mdm2 or other p53 ubiquitin ligases, or the hyperactivity of cinoma derived cell lines A2780, A2780DR1 and A2780DR2 DUBs 24 but not to a general lack of p53 ubiquitylation since were grown in DMEM supplemented with 10% FBS and antibi- DUBs inhibition revealed ubiquitylated forms of this tumor otics.34 A2780DR1 and A2780DR2 cells were grown in the suppressor. These results do not support the presence of non- presence of ADR (0.17 mM) and the drug was removed tetrameric p53 monomers in MCF7ADR cells that would favor 24 hours before doing any experimental procedure. 2342 V. LANG ET AL.

Figure 5. Proteasome inhibition promotes efﬁcient apoptosis in A2780DR1 and A2780DR2 cells. (A) A2780, A2780DR1 and A2780DR2 cells were treated with ADR (2 mM) during 1 or 4 hours. Ubiquitylated proteins were captured using TUBEs and analyzed by Western blot with the indicated antibodies. Input and TUBEs-capture fractions were shown. b-actin was used as a loading control. (B) To track A2780DR1 and A2780DR2 cells undergoing apoptosis, these were treated or not during 24hrs with ADR (5 mM) or MG132 (2.5 mM) or in combination as indicated. Data represent the mean of three independent experiments.

Analysis of ubiquitylated proteins Protein arrays MCF7 cells were treated or not with MG132 (Sigma-Aldrich, cat Detection of ubiquitylated proteins on TUBEs microarray was 6¼C2211, 5 mM, overnight), ADR (Sigma-Aldrich, cat 6¼44583, performed as previously described.13 Cells were treated or not as indicated), Chloroquine (Sigma-Aldrich, 200 mM, overnight) for 1h with ADR 1 mM and lysed for 30 min on ice. Cell lysate or PR619 (Merck, 20 mM, 15 min) alone or in combination. dilutions (100 mL) were incubated for 2 hours at room temper- A2780 cells were treated with ADR (2 mM) during 1 or 4 hours. ature on the TUBEs microarrays. Under these conditions To capture low abundant ubiquitylated substrates and ubiquitin TUBEs become saturated at high cell extract concentrations. chains, saturating conditions were used during the TUBE-cap- Detection of ubiquitylated proteins was performed by incuba- ture experiments. To proceed, the lysis buffer was supplemented tion with anti-ubiquitin mouse monoclonal antibody (FK2) fol- with 3.5 mM of TUBEs hHR23A as previously described.14,16 lowed by Alexa FluorÒ647 rabbit anti-mouse IgG HCL (Life Lysates were clarified by cold centrifugation, and added to gluta- Technologies). The slides were washed with TBS, water and thione agarose beads (Biontex, cat 6¼R030). When indicated, dried in a slide spinner. Fluorescence measurements were per- glutathione beads were washed after ubiquitin capture (PBS- formed on a microarray scanner (Agilent G2565BA, Agilent tween 0.05%), and the bound material was eluted before being Technologies). Quantification of fluorescence was performed submitted to p53 (DO.1 p53 antibody) immunoprecipitation as by ProScanArrayÒ Express software (Perkin Elmer). Average previously described.16,35 Input, TUBEs-IP p53 and unbound RFU values with local background subtraction of five spots and fractions (FT1: obtained after the first capture with glutathione standard deviation of the mean were reported using GraphPad beads and FT2: obtained after TUBEs-IP p53) were analyzed PrismÒ software. using the corresponding antibodies.

Apoptosis analysis Immunoblotting To track apoptosis, MCF7 cells were treated or not with MG132 Western blots were performed using the following primary (5 mM, overnight), ADR (1 mM, overnight), Chloroquine antibodies: anti-p53 (clone DO1, Santa-Cruz, cat 6¼SC-126); (200 mM, overnight) or PR619 (Merck, 20 mM, 15 min) either anti-Mdm2 (Calbiochem, EMD Chemicals); anti-ubiquitin alone or in combination as indicated. A2780 cells were treated P4D1 (Santa Cruz Technology, cat 6¼SC-8017); anti-Histone or not as indicated with MG132 (2.5 mM, 24 hours), ADR H2AX (Abcam, cat 6¼Ab124781); anti-HMGMB1 (Abcam, cat (5 mM, 24 hours), either alone or in combination. Co-staining 6¼Ab79823); anti-b Tubulin (Abcam, cat 6¼Ab6046); anti- with Annexin-V-DY634 (Immunostep, cat 6¼ANXVKDY- GAPDH antibody (Sigma-Aldrich, cat 6¼A5441); anti-ubiqui- 100T) and dead cell stain Sytox green (Fischer Bioblock Scien- tin lys-K48 (Millipore, cat 6¼05–1307), anti-ubiquitin lys-K63 tific, cat 6¼05BR080) was performed to differentiate: early and specific (Enzo Life Sciences, cat 6¼PW0600-010), anti-ubiquitin late apoptosis as well as necrotic cells. The percentage of FK1 (Enzo Life Sciences, cat 6¼BML-PW88) and anti-ubiquitin Annexin VC/sytox green- was analyzed by flow cytometry FK2 (Enzo Life Sciences, cat 6¼PW8810-0500). excluding doublets. Appropriate single staining controls were CELL CYCLE 2343 used to set voltage and compensation values. Data were col- [10] Gallo LH, Ko J, Donoghue DJ. The importance of regulatory ubiqui- lected on a FACSCanto (BD Biosciences) and were analyzed tination in cancer and metastasis. Cell Cycle. 2017;16:634–648. using FlowJo software (www.flowjo.com). doi:10.1080/15384101.2017.1288326. PMID:28166483. [11] Liu J, Zheng H, Tang M, Ryu YC, Wang X. A therapeutic dose of doxorubicin activates ubiquitin-proteasome system-mediated prote- List of abbreviations olysis by acting on both the ubiquitination apparatus and proteasome. Am J Physiol Heart Circ Physiol. 2008;295:H2541–H2550. ADR Adriamycin doi:10.1152/ajpheart.01052.2008. PMID:18978187. CQ Chloroquine [12] Ranek MJ, Wang X. Activation of the ubiquitin-proteasome system in doxorubicin cardiomyopathy. Curr Hypertens Rep. 2009;11:389– DUBs Deubiquitylating enzymes 395. doi:10.1007/s11906-009-0068-8. PMID:19895749. TUBEs Tandem ubiquitin-binding entities [13] Serna S, Xolalpa W, Lang V, Aillet F, England P, Reichardt N, Rodri- UPS Ubiquitin–proteasome system guez MS. Efficient monitoring of protein ubiquitylation levels using UBDs ubiquitin-binding domains TUBEs-based microarrays. FEBS Lett. 2016;590:2748–2756. doi:10.1002/1873-3468.12289. PMID:27410252. [14] Hjerpe R, Aillet F, Lopitz-Otsoa F, Lang V, England P, Rodriguez Disclosure of potential conflicts of interests MS. Efficient protection and isolation of ubiquitylated proteins using tandem ubiquitin-binding entities. EMBO Rep. 2009;10:1250–1258. The authors declare that they have no conflicts interests. doi:10.1038/embor.2009.192. PMID:19798103. [15] Hjerpe R, Rodriguez MS. Efficient approaches for characterizing ubiquitinated proteins. Biochem Soc Trans. 2008;36:823–827. Acknowledgments doi:10.1042/BST0360823. PMID:18793144. [16] Aillet F, Lopitz-Otsoa F, Hjerpe R, Torres-Ramos M, Lang V, We thank Dr. Gant for providing the cell lines used in this study and labo- Rodriguez MS. Isolation of ubiquitylated proteins using tandem ratory members for technical support and helpful suggestions. We also ubiquitin-binding entities. Methods Mol Biol. 2012;832:173–183. thank Dr. J.E. 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