(« Stewart Parker (1941[START_REF]The Resurrection broke the iron connection between the past and the future and Pentecost brought joy beyond sorrow and beauty beyond horror[END_REF]et Anne Devlin (1951-) : Porte-parole d'une génération en péril », in Discours sur l'autre, discours sur soi. Constructions identitaires face à l'altérité. Flore Kimmel & Thomas Meszaros (dir.). Lyon : Publications de l'Université Jean Moulin-Lyon 3, 2007, pp. 73-89.), ses recherches se tournent désormais vers la re-définition de l'identité théâtrale en Irlande du Nord ("Monological Drama to re-define a Northern Irish Identity : A Night in November by Marie Jones", in Estudios Irlandeses n°5, mars 2010) et plus particulièrement sur les emprunts brechtiens par les dramaturges nord-irlandais.

Bien qu'écrite en 1987, la dernière pièce de théâtre de Stewart Parker , Pentecost, est un huis clos dramatique se déroulant sur toile de fond historique. Elle renvoie le spectateur treize ans en arrière afin que l'action couvre la première partie de l'année 1974. A cette date, une grève d'ouvriers loyalistes mettait un terme au pouvoir exécutif du gouvernement de partage du pouvoir entre nationalistes et unionistes en Irlande du Nord. Pour Parker, cette défaite politique représentait l'événement le plus décourageant dans le processus de réconciliation entre les communautés protestante et catholique en Irlande du Nord. Dans cette dernière oeuvre, le dramaturge relate les Troubles, censés être de nature religieuse, et, à travers cet épisode précis de l'histoire nord-irlandaise, il rappelle le traumatisme, autant physique que psychologique, qu'ils générèrent. Pourtant, il garde foi en l'être humain. L'homme, non la religion, est à l'origine de ce conflit, et il lui incombe d'y remédier.

Irlande du Nord, religion, théâtre, traumatisme, troubles

Irlande du Nord

Période contemporaine

Even if he wrote his last play, Pentecost, in 1987, Northern Irish playwright Stewart Parker (1941[START_REF]The Resurrection broke the iron connection between the past and the future and Pentecost brought joy beyond sorrow and beauty beyond horror[END_REF] was more interested in dealing with the events which had taken place in 1974 in his Province. Indeed, it was in this year, throughout Pentecost, that a loyalist workers' strike brought down the Executive of the power-sharing 1 government between the nationalists and the unionists. Parker considered this collapse to be "the most hopeless moment in the history of Northern Ireland". Throughout his last play Parker writes on the Troubles, which are said to stem from religious divisions; and through this particular episode, he recalls the trauma that they engendered, the disaster, physical and psychological, they led to. Yet, he still has faith in human beings. Man, not religion, is at the origin of the conflict, and he must solve it on his own. drama, Northern Ireland, religion, trauma, troubles Stewart Parker rédige Pentecost 1 un an avant son décès. L'oeuvre du jeune dramaturge s'achève sur cette pièce de théâtre qui, bien qu'écrite en 1987, retrace des événements passés. Elle renvoie le spectateur treize ans en arrière afin que l'action couvre les cinq premiers mois de l'année 1974, soit la période qui marqua la première tentative de mise en place d'un gouvernement de partage du pouvoir entre nationalistes et unionistes en Ulster 2 . Cet essai prit fin suite à une grève générale menée par des ouvriers loyalistes protestants. Bien que né dans une famille protestante, Stewart Parker se souvenait de cet épisode comme l'un des événements historiques les plus décourageants dans le processus de rapprochement entre les communautés protestante et catholique en Irlande du Nord 3 . Si dans sa pièce, il met en scène cinq personnages (Marianne, Ruth, Lily, Lenny et Peter) enfermés dans une demeure presbytérienne d'un quartier protestant de l'est de Belfast, c'est pour donner un peu plus de profondeur dramatique à cet épisode qu'il nomme « la grande insurrection loyaliste 4 », en écho à l'insurrection républicaine de Pâques 1916. Le passé compte en effet énormément pour les Nord-Irlandais. Pourtant le dramaturge ne se contente pas d'écrire les Troubles, de les représenter sur scène, il souligne également le profond traumatisme psychologique qu'ils engendrèrent. Malgré tout, il laisse transparaître un certain optimisme derrière cette ultime oeuvre. Il déplore que la religion ait été utilisée à mauvais escient par les hommes. Ils ont détourné le message central du Christ ; il convient de le retrouver. Pour le dramaturge, l'Homme a engendré l'état de guerre, il revient donc à l'Homme de le résoudre. Les Troubles ne sont pas une guerre de religion. Il expose cette problématique dans son ultime oeuvre théâtrale dont il se sert comme d'une arme pour reprendre le contrôle de la situation.

1 Afin de faciliter la lecture du présent article, toutes les citations sont extraites de la traduction française de Pentecost faite par Jérôme Hankins, Pentecôte, Paris, Éditions Théâtrales, 1996, SACD. Leurs versions originales, extraites de l'oeuvre de Parker, seront indiquées en notes de bas de pages (Parker Stewart, Plays 2: Northern Star, Heavenly Bodies, Pentecost [1989], Londres, Methuen Contemporary Dramatists, 2000).

2 Le second gouvernement de partage du pouvoir fut mis en place le 8 mai 2007, soit 33 ans plus tard. Le révérend Ian Paisley, fondateur du Democratic Unionist Party (DUP) ainsi que de la Free Presbyterian Church, fut nommé Premier ministre et Martin McGuinness, ancien dirigeant de l'IRA, membre du Sinn Fein, vice-Premier ministre.

3 Selon les propres termes de l'auteur : "One of the most hopeless moments". Cité in Marilynn Richtarik, "'Ireland, the continuous past': Stewart Parker's Belfast History Plays", in A Century of Irish Drama, Stephen Watt, Eileen Morgan et Shakir Mustafa (dir.), Bloomington, Indianapolis, Indiana University Press, 2000, p. 269.

4 "The Great Loyalist Insurrection". Stewart Parker, Plays 2: Northern Star, Heavenly Bodies, Pentecost [1989], Londres, Methuen Contemporary Dramatists, 2000, p. 200.

Contexte historique

Dès le premier acte, Stewart Parker évoque le lieu (Belfast) et la date (1974) de l'intrigue puis précise le temps qui passe à chaque changement de scène. Il se montre ainsi soucieux de retracer une partie de l'histoire des Troubles. Il est vrai que depuis la fin des années 1960, l'Irlande du Nord, territoire britannique, connaît un désordre politique, économique et social si grand que Londres en prend le contrôle direct en 19725 . Néanmoins, ne parvenant pas à maîtriser la violence qui y règne, le gouvernement national s'empresse de proposer une solution. L'offre de John Hume, membre fondateur du SDLP (parti social démocrate travailliste), vient ainsi à point nommé en juillet 1972. Il envisage une autorité conjointe de l'Irlande et de la Grande-Bretagne sur l'Irlande du Nord. Les termes de cette proposition sont rédigés au sein d'un Livre blanc que Westminster présente en mars 1973. Le Premier ministre britannique, Edward Heath, le Premier ministre irlandais, Liam Cosgrave, ainsi que des représentants de partis politiques nord-irlandais6 en faveur de l'accord se rencontrent à Sunningdale (Grande-Bretagne) du 6 au 9 décembre de la même année pour entériner la proposition. Cette dernière envisage également la mise en place d'un gouvernement de partage du pouvoir entre nationalistes (majoritairement catholiques) et unionistes (composés d'un fort nombre de protestants) en Irlande du Nord. Un pouvoir exécutif mixte se voit donc constitué et prend ses fonctions au château de Stormont (Belfast) le 1er janvier 1974. Ainsi, Brian Faulkner, Premier ministre en Irlande du Nord avant le contrôle direct, devient le président du pouvoir exécutif de ce nouveau gouvernement7 . Bien que cette décision semble apporter un espoir de réconciliation entre les deux communautés (catholiques et protestantes), elle ne satisfait pourtant pas certains unionistes qui se sentent trahis par Londres. Les désaccords autour de ce gouvernement atteignent leur apogée lorsqu'un groupe d'ouvriers loyalistes (protestants)8 mené par Glenn Barr, membre de l'Ulster Defense Association (UDA) et l'un des représentants unionistes à l'Assemblée nordirlandaise, appelle les ouvriers protestants d'industries phares de la Province à une grève générale en mai 1974. Cette grève paralyse l'Ulster pendant quatorze jours. La démission de Faulkner9 de son poste de président y met un terme le jour de la Pentecôte, faisant, par la même occasion, chuter le pouvoir exécutif. Stewart Parker retrace cet événement qui s'inscrit dans la période des Troubles nord-irlandais à travers une pièce de théâtre où trois langages se superposent de façon à représenter le conflit prétendument religieux au mieux. Celui des mots, au travers des dialogues des personnages et des didascalies de l'auteur, en sera le vecteur principal. Le corps de l'acteur se fera pareillement le gardien de la représentation ; enfin, les sons et l'éclairage seront utilisés dans le but d'illustrer la situation au mieux.

Représentation artistique

En 1974, l'Irlande du Nord est prise en otage par la grève loyaliste. La province britannique se voit ainsi privée d'électricité, de lait et de transport en commun principalement, ses habitants n'ont plus qu'une liberté de mouvements limitée. Stewart Parker relate cet état de paralysie à travers les expériences des quatre personnages principaux de Pentecost, Marianne, Ruth, Lenny et Peter, enfermés par sécurité dans la maison d'une bigote presbytérienne récemment décédée, Lily Matthews. Lenny rappelle cette situation carcérale à son ami d'enfance Peter, musicien belfastois exilé en Angleterre mais de retour dans sa province natale. Il en profite pour lui préciser qu'il ne pourra manger les céréales qu'il a rapportées puisque le lait est devenu une denrée rare :

Peter, fais marcher ta cafetière -grève de protestation des ouvriers loyalistes, o.k. ? Coupures d'électricité. Pas de ravitaillement d'essence. Pas de produits animaux. Des barricades partout dans la ville pour rendre la circulation impossible. Toutes les vaches sont aux trois-quarts protestantes. Tu crois vraiment que le lait a une chance?10 (34) Puisque sa pièce se veut avant tout réaliste (selon les propres mots de l'auteur), le dramaturge tente de recréer l'atmosphère tendue de l'époque sur scène, à travers les dialogues et les didascalies. Aussi chaque scène est-elle introduite par une date, suivie d'une fine description de la situation, comme la scène 3 de l'acte 1 : Le dramaturge n'omet pas non plus de mentionner l'origine du conflit nordirlandais : le sectarisme dû principalement aux différences de confessions religieuses. Si Lenny et Marianne sont tous deux catholiques, Peter, Ruth et Lily sont protestants. Néanmoins, Lenny, Marianne, Ruth et Peter ont su dépasser leur endogamie dans l'amitié qu'ils nourrissent et dans l'amour qu'ils portent ou ont pu porter les uns aux autres. Au contraire, Lily revient un instant sur terre pour hanter sa maison occupée et se faire le porte-parole du sectarisme nord-irlandais à l'origine des Troubles. Elle apparaît tout au long de la pièce afin d'en chasser ses occupants, et avant tout Marianne, son ennemie jurée. Elle a, à son égard, de violents propos tels que « Faites votre trafic entre vous et laissez-moi reposer en paix avec les miens 12 » (19). De manière générale, les échanges verbaux des personnages sont empreints d'agressivité car le Belfast dans lequel ils évoluent est une ville divisée, dont les tensions balayent les rues et affectent profondément ses habitants.

Outre les indications de l'auteur qui fournissent des précisions quant à la représentation de la ville ainsi que les dialogues qui reflètent les tensions dues aux Troubles et à l'ampleur de la grève en question, le corps de l'acteur, en tant que lieu hétérotopique, se fait lui aussi représentation de la situation. Le jeu de l'acteur dramatique a pour vocation de mettre en relation les sphères privée et publique, mais doit aussi permettre de lier les mondes intérieurs et extérieurs sur scène. Si les tensions des Troubles ne pénètrent dans la maison de Lily que par les dialogues puisqu'il n'est jamais question de violence physique sur la scène, celle-ci est tout de même évoquée dans l'oeuvre de Parker à travers le corps de l'acteur. En effet, il s'avère véritablement dangereux de quitter la maison de Lily puisque les personnages s'aventurant à l'extérieur n'en reviennent pas sans séquelles. Par exemple, Marianne, sortie un instant, revient chez Lily, blessée ; son agression est ainsi présentée : La musique de Lenny, qui descend ici aux oreilles des spectateurs comme si elle était céleste, est brutalement interrompue. Tout se passe comme si la réalité extérieure se rappelait à l'esprit de tous, y compris de ceux qui cherchent à s'en soustraire. Puisqu'il incarne les déictiques, l'acteur assure également la relation spatio-temporelle. Son occupation de l'espace scénique et le rapport qu'il entretient avec le temps sont des facteurs visant à refléter la situation. Dans le cas de Pentecost, les personnages sont confinés dans une maison, et, par conséquent, sont comme privés de liberté de mouvement. Lenny l'évoque ainsi sur le ton de la Contemporary Dramatists, 2000, p. 181).

13 "Lenny's trombone continues for a while upstairs. There is a sudden hammering from outside the backyard door. Marian's voice is heard shouting "Lenny! Lenny! Hello! Down here! Open up!" / The trombone music stops and Lenny is heard thundering down the stairs: he appears, rushing in from the hall carrying a torch, and continues straight through the kitchen to the scullery door, which he unlocks and opens; and thence down the yard to the yard door which he unbolts and flings backto reveal Marian, in the light of the torch, mud-spattered with her coat ripped, and scratch marks on her face. plaisanterie : « Plus rien ne me retient ici maintenant. Si ce n'est quelques trois cents barricades et des milliers d'hommes en cagoule armés de matraques 14 » (38). Lorsque les personnages tentent de s'affranchir des contraintes spatiales, c'est à leurs risques et périls ; ils sont donc contraints de se limiter à un espace clos. A travers cette situation, Parker évoque l'impasse dans laquelle le conflit s'est engagé. Alors, si sur scène, il ne peut maîtriser l'espace, il tente de le transgresser. Cette technique lui permet de reprendre le contrôle de la situation. Parker va même jusqu'à essayer de rééquilibrer l'occupation de l'espace nord-irlandais à travers sa pièce. Nous avons indiqué que Marianne, catholique, fait l'acquisition d'une maison presbytérienne dans un quartier protestant. Cette tentative de re-possession de l'espace est hautement symbolique dans cette partie de l'île où les catholiques se sont sentis dépouillés de leur terre depuis des siècles. Il serait donc juste que leur rendues leurs propriétés. En contrepartie, Marianne, qui possédait une boutique d'antiquités, s'en sépare et la vend à un galeriste protestant. Ce geste ressortit plus d'une décision personnelle qu'il ne résulte d'un acte de dépossession. Il est donc délibéré et légitime. Après ce jeu avec l'espace, l'auteur examine si le temps est lui aussi malléable. Dans sa pièce, de nombreux retours en arrière renouent avec le présent de l'action. Il existe dans Pentecost effacement des repères temporels, notamment par le truchement du retour sur terre de feu Lily Matthews. Le rôle de ce fantôme, qui rajeunit au fil de l'intrigue, est capital. En effet, Lily, dévote presbytérienne, a gardé toute sa vie durant un lourd secret qu'elle doit livrer si elle veut reposer en paix. Puisque son traumatisme remonte à plusieurs années, son discours est émaillé du récit des épreuves historiques qu'elle telles que la guerre civile irlandaise de 1922 ou encore les Première et Seconde Guerres mondiales. Sa maison recèle par ailleurs un véritable trésor constitué d'objets et de mobilier ancien qui rappellent le passé colonial de l'Irlande. Marianne, antiquaire de métier, s'émerveille de l'avoir découvert :

Regarde-moi ça. Soixantième anniversaire de la reine Victoria, noces de la reine Marie, couronnement de la petite Elizabeth II, 1953, ça doit être l'article le plus moderne dans la maison. Le plus gros du mobilier est de l'époque édouardienne, il y a une coiffeuse à l'étage qui a dû lui venir de ses grands-parents 15 (13).

L'auteur renvoie aussi subrepticement au siège de Londonderry en 1689, au cours duquel les protestants furent assiégés par les catholiques, lorsqu'il met dans la bouche de Peter la remarque suivante adressée à Lenny qui ne vit qu'avec Marianne et Ruth : « Te voilà donc assiégé depuis tout ce temps 16 (39). Fidèle à la tradition littéraire irlandaise, le dramaturge indique ainsi que le passé colonial occupe encore une place prépondérante dans le présent en Irlande du Nord, trop prépondérante 14 "Nothing to keep me here now. Apart from three hundred-odd barricades, and thousands of hooded men with clubs". (Stewart Parker, Plays 2: Northern Star, Heavenly Bodies, Pentecost [1989], Londres, Methuen Contemporary Dramatists, 2000, p. 202).

15 "Queen Victoria's Diamond Jubilee, the wedding of Queen Mary, the Coronation of Lizzie the Second, 1953 -that must be the most modern item in the house. Most of the furniture's Edwardian, there's a Regency dressing table upstairs that must have come down through her grand-parents." (Stewart Parker, 2: Northern Star, Heavenly Bodies, Pentecost [1989], Londres, Methuen Contemporary Dramatists, 2000, p. 175). 16 been besieged all that time." (Stewart Parker, Plays 2: Northern Star, Heavenly Bodies, Pentecost [1989], Londres, Methuen Contemporary Dramatists, 2000, p. 204). comme il le laissait entendre puisque les divisions sont nées de cet emprisonnement :

Quand les Irlandais disent « le passé », ils se rappellent en réalité les années de Cromwell ou du roi Guillaume, ce qui veut dire trois cents ans en arrière, voire plus.

[…] Si vous grandissez en Irlande du Nord aujourd'hui, chacun de vos pas est dans le sillon de n'importe lequel des deux camps dans lequel vous êtes né. Vous pouvez vous y résigner, réagir contre lui, ou le fuir mais vous ne pouvez pas l'ignorer. Le passé est en vie, se porte bien et tue des gens à Belfast 17 .

Le passé, à l'image de la maison où les personnages sont enfermés contre leur gré, est en effet une prison. Parker introduit des personnages historiques réels dans son oeuvre afin de rendre plus floues les frontières le passé et le présent. L'acte II s'ouvre sur la retransmission radiophonique du véritable discours prononcé le 25 mai 1974 sur les ondes de la BBC par Harold Wilson, Premier ministre britannique alors récemment réélu. Les paroles du chef du gouvernement britannique inondent le salon où se trouvent les personnages. Parker fait également référence au Révérend protestant Ian Paisley : Peter se met à imiter cet homme charismatique qui se positionnait contre le gouvernement de partage du pouvoir. Il adopte son ton et sa voix, comme le dramaturge le note en didascalie : « Imitant les inflexions du protestant extrémiste, le révérend Paisley 19 » (37). A travers cette intrusion de la réalité dans la fiction, l'auteur tente aussi d'abolir les frontières entre ces deux mondes, ce qui est confirmé par la présence du fantôme de Lily mais aussi le chant qu'elle entonne, Oh God, our Help in Ages past. Cet hymne protestant, que des techniciens de l'électricité et des politiciens loyalistes chantèrent à la lueur de chandelles 20 le 28 mai 1974 pour marquer le jour de leur victoire, permet à Lily de se faire l'écho sur scène de ce pouvoir loyaliste (c'est-à-dire protestant). Grâce au personnage de Lily Matthews, le dramaturge saisit l'occasion de rappeler certains éléments historiques et mythiques sur lesquels les idéologies protestantes sont fondées. Par exemple, lorsque le spectre se défend de n'avoir pas bougé d'un pouce malgré toutes les difficultés que la Province et lui traversèrent, il reprend à charge le slogan unioniste « pas d'un pouce » utilisé lors des élections de 1925 au sujet du rapprochement de l'Irlande du Sud et du Nord contre lesquelles se prononcèrent les unionistes. Pourtant, si Parker se réfère bien souvent à ces mythes, il ne les reprend pas tels quels. Il les transpose aux situations présentes ou les déconstruit. En effet, Lily, aussi puritaine fût-elle, eut une aventure extraconjugale dont naquit un enfant. Son secret est d'avoir eu puis abandonné ce fils illégitime qu'elle n'aurait jamais pu avoir avec son mari très gravement blessé lors de la Première Guerre mondiale à laquelle il avait pris part, comme de nombreux soldats nord-irlandais. 17 "When the Irish say 'the past', they're gesturing back at least three hundred years to Cromwell and King Billy, and often beyond…. Grow up in Northern Ireland today, and your every step is dogged by whichever of the two camps you were born into. You can surrender to it, react against it, run away from it…you can't ignore it. The past is alive and well and killing people in Belfast." Marilynn Richtarik,"'Ireland, the continuous past': Stewart Parker's Belfast History Plays", in A Century of Irish Drama, Stephen Watt, Eileen Morgan et Shakir Mustafa (dir.), Bloomington, Indianapolis, Indiana University Press, 2000, p. 256. 18 Discours dans lequel il condamnait les protestants nord-irlandais. "Paisleyite voice" (Stewart Parker, 2: Northern Star, Heavenly Bodies, Pentecost [1989], Londres, Methuen Contemporary Dramatists, 2000, p. 201). 20 Jonathan Bardon, A History of Ulster, Belfast, The Blackstaff Press, 2001, p. 711.

Enfin, l'éclairage et les bruits viennent compléter la description de la situation. Les jeux de son et de lumière sont en effet des outils efficaces pour refléter le monde extérieur au théâtre. Les sons relatifs à l'époque tels que celui du vol d'un hélicoptère qui plane au dessus de Belfast pour en assurer la sécurité visent à recréer l'ambiance de l'époque. De la même façon, le son des tambours de procession orangiste (protestante) en provenance de l'extérieur résonne à l'intérieur comme pour marquer le territoire mais aussi et surtout célébrer la victoire des grévistes. A cette représentation sonore s'ajoute une représentation visuelle, notamment à travers l'absence d'électricité due à la grève des ouvriers loyalistes. Celle-ci se fait absolument ressentir sur scène et plonge la pièce dans une pénombre à double sens. Cette obscurité qui n'est pas rassurante, peut, par la même occasion, protéger du monde extérieur puisqu'elle permet la discrétion. En temps de conflit, mieux vaut ne pas attirer l'attention sur soi de peur de devenir une cible potentielle. Privés de lumière, les personnages se voient contraints d'allumer des bougies et autres lampes pétroles. Cette technique visuelle renforce le sens du titre de la pièce, puisqu'il renvoie à la Pentecôte, épisode biblique qui relate comment des langues de feu descendirent sur les apôtres. Ainsi la situation extérieure chaotique, le clivage entre les deux communautés religieuses, se reflètent-ils dans toutes ces tensions qui règnent à l'intérieur de la maison et font de celle-ci un refuge bien précaire. L'auteur dévoile en effet que le traumatisme va bien au-delà puisqu'il est aussi ancré au plus de chacune des âmes des personnages de Parker.

Traumatisme

Lorsque Stewart Parker écrit sa pièce en 1987, plusieurs années se sont écoulées, ce qui lui permet de prendre assez de distance et d'adopter un regard critique sur la grève loyaliste. Or, pour ce faire, il préfère d'emblée traiter de la question indirectement puisque aucune scène de violence liée aux Troubles ou à la grève n'est jouée sur scène, le lieu de l'action étant exclusivement l'intérieur de la maison. Alors, si Pentecost des allures de huis clos, ce n'est que pour renforcer le traumatisme de cette crise : le conflit devient psychologique. Ce n'est pas uniquement la violence des Troubles qui conduit les personnages à communiquer dans la violence; ils cachent tous des secrets, des souffrances, dont ils n'osent pas parler lorsque la pièce débute. Ces souffrances se retrouvent dans leurs échanges verbaux. Le spectateur peut en effet d'emblée percevoir le malaise régnant entre les deux premiers personnages sur scène, Lenny et Marianne. La violence de leur dialogue reflète alors à quel point le monde peut être disloqué et divisé, véritable Tour de Babel où la communication menace de se rompre à tout instant. Lenny et Marianne tentent bien de communiquer mais leur dialogue n'est en réalité que la juxtaposition de deux monologues. L'échange suivant, ponctué de phrases courtes, en donne un exemple : Plus loin, le spectateur se voit confié la raison de cette séparation. Lenny et Marianne eurent un enfant, mais ce dernier mourut à l'âge de cinq mois :

Marianne :

[…] J'ai eu un enfant une fois. Lenny :

Arrête. Marianne… Marianne : Je l'appelais Christophe. Parce qu'il était une sorte de Christ pour moi, il apportait l'amour avec lui… la vérité et la vie 23 (76).

Il est possible de constater ici à quel point cette expérience personnelle fur traumatisante pour Marianne qui rend les Troubles nord-irlandais responsables de la de son enfant. Il existe un lien fort entre l'événement public et la vie privée de cette Nord Irlandaise catholique. Pour corroborer cet argument, notons que la naissance de Christophe (dont le prénom est assez évocateur puisqu'il rappelle celui du Christ) coïncide avec une date significative des Troubles nord-irlandais : « Christophe aurait eu cinq ans en août. Commencé l'école. S'il ne m'avait pas quitté. Abandonné. Rendu l'esprit qui était en moi. Ma propre âme laissée pour 21 "Marian: What's this? /Lenny: That's old. So. What do you reckon?/ Marian: There's a cup sitting here with milk in it and sugar in it. Christ. / Lenny: I told you. I haven't touched a thing./ Marian: She must have brewed this up just before it happened. / Lenny: No such thing. / Marian: She never got a chance to pour it out. / Lenny: The ambulancemen, is all it would have been. / Marian: It's stone cold. / Lenny: The ambulancemen at the door, is all it possibly could have been. / Marian turns her attention on him for the first time. / Marian: I see. They prepared a nice pot of tea, prior to removing the corpse. / Lenny: She walked out of here, in her Sunday hat and coat and best handbag, is what I'm saying, under her own steam, into the ambulance, it was in the hospital she died…" (Stewart Parker, Plays 2: Northern Star, Heavenly Bodies, Pentecost [1989], Londres, Methuen Contemporary Dramatists, 2000, p. 172).

22 "Lenny: The house is yours as stated, terms agreed. In return for a divorce. / Marian: We are divorced, as good as. / Lenny: I'm talking about your signature on a petition. / Marian: What difference does it make? / Lenny: It makes it official, it makes it binding, it makes it definite." (Stewart Parker, Plays 2: Northern Star, Heavenly Bodies, Pentecost [1989], Londres, Methuen Contemporary Dramatists, 2000, p. 178).

23 "Marian: […] I had a child once./ Lenny: No. Marian… / Marian: I called him Christopher. Because he was a kind of Christ to me, he brought love with him… the truth and the life. He was a future. Until one day I found him dead. I thought like you for a long time. He chose death in the cot rather than life in this town, in these times, it was their fault, they had done it to me […]" (Stewart Parker, Plays 2: Northern Star, Heavenly Bodies, Pentecost [1989], Londres, Methuen Contemporary Dramatists, 2000, p. 244).

morte 24 » (47). Le mois d'août 1969 fut en effet chargé en événements tels que le renfort des troupes paramilitaires en Irlande du Nord et les émeutes éclaboussant la province. Suite à ce traumatisme, cette perte de l'enfant tant attendu et qui fut comme sacrifié, Lenny indique au spectateur combien Marianne fut bouleversée, et invite son épouse à prendre conscience de sa perte de lien avec la réalité : Marianne :

A quelle réalité fais-tu référence ? Lenny : La tienne, Marianne, ta réalité à toi, tu n'as pas cessé de parler toute seule, de compter les cuillères, de délirer en trente-six langues 25 […] Lily : J'ai péché contre ma propre chair dans la luxure et la fornication, j'ai été obligée d'abandonner mon bébé, personne n'a su, seul le Seigneur notre Dieu l'a su et Son oeil était rivé sur moi, brûlant jusqu'au fond de mon âme, Lui seul fut témoin du tourment que j'ai enduré chaque heure de ma vie dans cette maison où même les murs et les portes me hurlent leur accusation, avec personne à qui parler du poignard me transperçait mille fois par jour, rappelant comment j'avais abandonné mon enfant, tranquillement, le laissant comme un paquet, là dans une caisse en bois, personne pour m'aider, seulement moi ici, dans cette maison, me rongeant et me déchirant sans arrêt le coeur et la lumière, jour après jour… jusqu'à ce que je sois entièrement dévorée par ma propre méchanceté, à l'intérieur, sans plus rien qui reste de moi, que l'écorce, pour le bien des apparences… 27 (63-64).

24 "Christopher would have been five in August. Starting school. If he hadn't gone. Left me. Given up the ghost in me. My own soul, left for dead." (Stewart Parker, Plays 2: Northern Star, Heavenly Bodies, Pentecost Londres, Methuen Contemporary Dramatists, 2000, p. 212). 25 On peut ici noter un nouveau renvoi à l'épisode biblique de Babel.

26 "Marian: Which reality did you have in mind? / Lenny: Your own, Marian, your own reality, you've been talking to yourself, you've been counting spoons, you've been babbling in tongues in the middle of the night." (Stewart Parker, 2: Northern Star, Heavenly Bodies, Pentecost [1989], Londres, Methuen, Contemporary Dramatists, 2000, p. 226).

27 "Marian: What about the life of your baby? Lily: My baby was strong … he was well happed-up… / Marian: You abandoned him. / […] Marian: You and Alan Ferris. On the front parlour sofa. He'd no inkling of that? / Lily: Oh, sweet God in heaven forgive me. / Marian: Alfie was impotent, wasn't he. […] / Lily: I sinned against my own flesh in lust and fornication, I had to desert my own baby, nobody ever knew only the Lord and His eye was on me all right, burning into the very soul of me, He alone was witness to the torment I've suffered every living hour in this house where the very walls and doors cry out against me, there was never anybody to tell the knife that went through me a dozen times a day, minding how I left my child, walking away from him, leaving him bundled up there in that wooden box, nobody to help me, only me there in this house, gnawing and tearing away at my own heart and lights, day in day out…until I was all consumed by my own wickedness, on the inside, nothing left but the shell of me, for appearance's sake…" (Stewart Parker, Plays 2: Northern Cette révélation de Lily, véritable rédemption, va faciliter le travail de deuil de Marianne, qui en parle librement alors que la pièce touche à sa fin. C'est en soulageant leur conscience que Marianne pourra continuer à vivre, et que Lily reposera en paix.

Une leçon d'humanité

Néanmoins, malgré les traumatismes physiques et psychiques que les Troubles générèrent, le dramaturge reste optimiste. Ce n'est pas seulement pour des raisons politiques qu'il choisit d'intituler sa pièce Pentecost, c'est aussi pour des raisons religieuses. La Pentecôte, est, en effet, l'épisode biblique qui explique comment les hommes, en recevant le don de l'Esprit saint, purent communiquer entre eux à nouveau malgré différences. Cet épisode vint remédier à celui de Babel où Dieu avait puni les en leur ôtant le pouvoir de se comprendre. Ainsi, à travers le langage, les dialogues mais aussi la gestuelle et les transgressions spatiotemporelles, Parker tente d'abolir les différences de religion et de transcender l'état de guerre pour revenir à une certaine humanité et réconcilier avec l'Autre. Son ultime, comme il se plaisait à le répéter, notamment dans son introduction à la pièce de théâtre Over the Bridge de Sam Thompson, était de jeter des ponts les deux communautés en Irlande du Nord. Le dramaturge déplorait la situation de crise que l'Ulster traversait. C'est pourquoi, il choisit l'époque de la fête religieuse de la Pentecôte comme temps de sa pièce. C'est un instant privilégié dans la recherche de l'intégrité comme le stipule le Révérend John Dunlop dans A Precarious Belonging, Presbyterians and the Conflict in Ireland : « La Résurrection rompit les liens de fer entre le passé et le futur et la Pentecôte apporta joie et beauté en lieu et place de la tristesse et de l'horreur 28 . » A travers des expressions telles que « Quand arriva la Pentecôte (le cinquantième jour après Pâques), [les apôtres] se trouvaient réunis tous ensemble » 29 , le texte de cet épisode biblique, raconté dans les Actes des Apôtres, a pour objet « de montrer comment la mosaïque des peuples païens se transforme en une collectivité cohérente et unifiée, à partir de l'audition de la prédication des apôtres 30 . Dans son oeuvre, Parker se rapproche de ce message chrétien puisque Pentecost, comme la Bible, commence par une mise en perspective du désordre et de la confusion, pour se terminer sur une note positive. Si Marianne explique que la maison doit être aérée, Ruth ouvre la fenêtre, image qui clôt la pièce sur l'espoir de voir se lever des jours meilleurs en Irlande du Nord. Lenny et Peter, quant à eux, s'emparent de leur instrument de musique respectif et s'accordent pour jouer ensemble Just a Closer walk with Thee, cantique chrétien. Tout se passe comme si le Saint-Esprit était descendu sur ces cinq personnages confinés dans cette maison, et ce, indépendamment du désordre qui règne au dehors et de leur Marilynn, " 'Ireland, the continuous past': Stewart 's Belfast History Plays", in A Century of Irish Drama, Stephen Watt, Eileen Morgan et Shakir Mustafa (dir.), Bloomington, Indianapolis, Indiana University Press, 2000

Traduction oecuménique de la Bible, Textes traduits sur les textes originaux hébreu et grec, Paris, Alliance Biblique Universelle, 1988

  [… ]. / Lenny: Are you hurt bad? / Marian: Scratched a bit, but not as much as my car is. / Lenny: You found it? / Marian: It's the centrepiece of the barricade at the entrance of the estate up there. […] It was all women -shrieking and squealing and scrabbing at me[…]. (Stewart Parker, Plays 2: NorthernStar, Heavenly Bodies, Pentecost [1989], Londres, Methuen Contemporary Dramatists, 2000, pp. 222-223).

  Une lueur d'espoir au coeur des Troubles nordirlandais : Pentecost de Stewart Parker

	Virginie Privas-Bréauté
	Virginie Privas-Bréauté est professeure certifiée en
	anglais et enseigne à l'université Jean Moulin -Lyon 3
	depuis 2002. En 2007, elle a soutenu un doctorat de
	troisième cycle dont le sujet explorait le rôle de la
	religion dans le conflit nord-irlandais filtré par deux
	dramaturges belfastois, Anne Devlin et Stewart Parker.
	Après avoir rédigé de nombreux articles sur la vision
	dramaturgique	et	la	création	d'une	identité	nord-
	irlandaise par des artistes ulstériens contemporains

  Le trombone de Lenny continue à jouer pendant un moment à l'étage. Dehors, on entend soudain frapper à grands coups contre la porte de la cour. La musique du trombone s'interrompt et on entend Lenny descendre en trombe les escaliers : il apparaît en courant venant du hall d'entrée une torche à la main, et traverse directement la cuisine jusqu'à la porte de l'office, tire le verrou et l'ouvre ; il traverse la cour jusqu'à la porte de derrière qu'il déverrouille et ouvre d'un coup -révélant Marianne à la lumière de sa torche, éclaboussée de boue, le manteau déchiré, et des

							Voix de Marianne
	criant :	« Lenny !	Lenny !	Tu	m'entends !	Ici	bas !	Ouvre-moi ! »
			au			visage.
	Lenny :		Tu		es			blessée ?
	Marianne : un peu égratignée, mais pas autant que ma voiture.
	Lenny :		tu		l'as			retrouvée ?
	Marianne : ils l'ont utilisée comme pièce centrale dans la barricade à l'entrée du
	quartier							là-haut.
	[…]
	Rien que des femmes -couinant et hurlant après moi toutes griffes dehors […] (56).

  Les ambulanciers qui ont sonné à la porte, ça doit forcément être eux. (Marianne lui accorde son entière attention pour la première fois.) Marianne : C'est ça. Ils se sont fait une bonne tasse de thé, juste avant de déménager le corps. Lenny : Elle est sortie d'ici à pied, portant son manteau et son chapeau du dimanche, son meilleur sac à main, tu m'entends, toute seule comme une grande, droit dans l'ambulance, c'est à l'hôpital qu'elle est morte… 21 (11).Puis, au fil de l'intrigue, des bribes d'informations à leur sujet seront données au spectateur. Ainsi apprend-il que Marianne et Lenny, autrefois mariés, vivent désormais séparés :

	Lenny :	Les	ambulanciers,	ça		doit	être		eux.
	Marianne :				Complètement				froid;
	Lenny : Lenny : […] La maison t'appartient comme indiqué, marché conclu. En échange d'un
	divorce.
	Marianne :	Nous	sommes	divorcés	-	dans	les	faits.
	Lenny :	Je	veux	parler	d'un	document	signé	de	ta	main.
	Marianne :		Qu'est-ce		que		ça			change ?
	: Ça devient officiel, obligatoire, et irrévocable (16).
	Marianne :				C'est					quoi ?
	Lenny :		C'est	vieux.	Alors.		Ton		estimation ?
	Marianne : Il y a une tasse toute prête avec du lait et du sucre dedans. Merde.
	Lenny :	Je	t'ai	dit.	Je	n'ai	touché		à	rien.
	Marianne :		Elle	allait	le	boire		quand	c'est		arrivé.
	Lenny :			Pas				du			tout.
	Marianne :		Même	pas	eu	le		temps	d'y		toucher.

  comme une illuminée au milieu de la nuit26 (40).Une situation qui le poussa à envisager une psychothérapie pour elle. Il semble que l'emménagement dans cette maison en particulier et à ce moment très précis, va lui épargner une visite chez un psychothérapeute. En effet, le fantôme de feu Lily Matthews apparaît à Marianne lorsqu'elle est seule pour jouer le rôle d'alter ego.apparition, dont elle est parfaitement consciente, vise ultimement à la ramener la réalité et à l'aider à exorciser ses douleurs. Marianne découvre progressivement que Lily eut elle aussi une expérience traumatisante quelque peu similaire à la celle de l'abandon de son fils illégitime :

	Marianne :	Et	la	vie	de		ton	enfant,	qu'en	fais-tu ?
	Lily :	Mon	enfant	était	robuste,	solide…il	était	né	coiffé…
	Marianne :		Tu		l'as		abandonné.	[…]
	Marianne : Toi et Alan Ferris. Sur le canapé du salon. Pas le moindre soupçon de ça ?
	:	O	Dieu		du	ciel	pardonne-moi	dans	ta	bonté !
	Marianne :	Alfie		était	impuissant,	n'est-ce	pas ?

Cf. Henry Patterson, Ireland since 1939, Oxford, Oxford University Press, 2002.

Ulster Unionist Party (UUP), Social Democratic and Labour Party (SDLP), et Alliance Party of Northern Ireland (APNI).

Gordon Gillepsie, "The Origins of the Ulster Workers Council Strike", Études irlandaises, vol. 29 n°1, 2004, pp. 129-144.

Ils avaient créé le Ulster Workers Council suite à une grève menée en mars 1973 pour protester contre les internements des loyalistes. James Loughlin, The UlsterQuestion since 1945, Londres, MacMillan Press, 1998, p. 74.

Il démissionna car Merlyn Rees, ministre de l'Irlande du Nord, ne souhaitait pas s'entretenir avec les grévistes.

"Peter, use the loaf -protest strike by Loyalist workers, right? Electricity cuts. No petrol supplies. No animal foodstuffs. Barricades all over the city turning back the traffic. Three quarters of all cows are Protestant. What chance has the milk got?" Stewart Parker, Plays 2: NorthernStar, Heavenly Bodies, Pentecost [1989], Londres, Methuen Contemporary Dramatists, 2000, p. 198.

The night of Sunday, 19 May. The kitchen and scullery are dark and deserted. The sound of half a dozen drunken youths running up the back entry, shouting and whistling, is heard from off. A beer bottle sails over the yard wall and smashes harmlessly on the floor of the yard. Then silence returns. Until the front door can be heard opening and two slightly drunk men (Lenny and Peter) coming into the hall. (Stewart Parker, Plays 2: NorthernStar, Heavenly Bodies, Pentecost [1989],Londres, Methuen Contemporary Dramatists, 2000, p. 196).

"I want no truck with any of you, stay you with your own and let me rest easy with mine." (Stewart Parker, Plays 2: NorthernStar, Heavenly Bodies, Pentecost [1989], Londres, Methuen

Image évoquée par Jérôme Hankins, Pentecôte, Paris, Éditions Théâtrales,SACD, 1996, p. 82.


Sarah Figarol

Célia Delahaye

Rémi Gence

Raghda Asslan

Sandra Pagano

Claudine Tardy

Jacques Colinge

Jean-Philippe Villemin

Antonio Maraver

Irene Ferrer

Luis Paz-Ares

Isabelle Lajoie-Mazenc

Estelle Clermont

Anne Casanova

Anne Pradines

Julien Mazières

Olivier Calvayrac
email: olivier.calvayrac@inserm.fr

Gilles Favre
email: favre.gilles@iuct-oncopole.fr

Farnesyltransferase inhibition overcomes the adaptive resistance to osimertinib in EGFR-mutant NSCLC

RESULTS

Drug-tolerance is a dynamic rather than a dormant state Although an increasing number of studies have focused on characterization of drug-tolerant "dormant" cells (DTC) and/or fully resistant proliferative clones (RPC) [START_REF] Sharma | A chromatin-mediated reversible drug-tolerant state in cancer cell subpopulations[END_REF][START_REF] Hata | Tumor cells can follow distinct evolutionary paths to become resistant to epidermal growth factor receptor inhibition[END_REF][START_REF] Ramirez | Diverse drug-resistance mechanisms can emerge from drug-tolerant cancer persister cells[END_REF][START_REF] Kurppa | Treatment-Induced Tumor Dormancy through YAP-Mediated Transcriptional Reprogramming of the Apoptotic Pathway[END_REF][START_REF] Guler | Repression of Stress-Induced LINE-1 Expression Protects Cancer Cell Subpopulations from Lethal Drug Exposure[END_REF][START_REF] Vallette | Dormant, quiescent, tolerant and persister cells: Four synonyms for the same target in cancer[END_REF] , the kinetics of evolution throughout the different states is largely unknown. We used the FUCCI (fluorescence ubiquitination cell cycle indicator) system [START_REF] Sakaue-Sawano | Visualizing spatiotemporal dynamics of multicellular cell-cycle progression[END_REF] to perform a real time monitoring of cell cycle dynamics in response to 1µM EGFR-TKi erlotinib or osimertinib in a panel of EGFR-mutated cell lines. Importantly, cell lines were previously subcloned to minimize/avoid the presence of potential pre-existing resistant cells [START_REF] Hata | Tumor cells can follow distinct evolutionary paths to become resistant to epidermal growth factor receptor inhibition[END_REF][START_REF] Turke | Preexistence and clonal selection of MET amplification in EGFR mutant NSCLC[END_REF] (Supp Fig. 1A andB). For all cell lines, we observed a common pattern of G1 accumulation within the first 48 hours of treatment (Fig. 1a, Supp Fig. 2A, videos), which was invariably associated with p27 Kip1 overexpression and Retinoblastoma (Rb) protein dephosphorylation (Supp Fig. 2B). While most cells remained in G1 and progressively died resulting in bulk population decrease, a subset of cells (referred to as early escapers) rapidly progressed though S/G2 (Fig. 1a-c, Supp. Fig. 2A, videos), an observation consistent with a rare, drug-tolerant cycling subpopulation as recently described [START_REF] Oren | Cycling cancer persister cells arise from lineages with distinct programs[END_REF] . Interestingly, although DTC and early escapers could be observed in H3255and HCC2935-treated cells, they failed to form resistant proliferative clones in these conditions (Supp Fig 3A-D) but retained the capacity to resume proliferation after drug withdraw (Supp Fig. 3E).

These results suggest that the establishment of an adaptive resistance mechanism through a drug-tolerant state is a dynamic and progressive process and is highly variable even among genetically homogeneous cellular models.

Early escapers emerge from a differentiated alveolar-like phenotype with contractile features

To characterize the molecular mechanisms underlying evolution from G1-arrest to early escape, we performed scRNAseq on ~3000 G1 (red) and S/G2 (green) cells sorted from both untreated and osimertinib-treated HCC4006 cells, which enabled enrichment of the rare population of early escapers (Fig. 1d, Supp Fig. 4A andB). In line with previous reports [START_REF] Hata | Tumor cells can follow distinct evolutionary paths to become resistant to epidermal growth factor receptor inhibition[END_REF][START_REF] Kurppa | Treatment-Induced Tumor Dormancy through YAP-Mediated Transcriptional Reprogramming of the Apoptotic Pathway[END_REF] , Myogenesis and Epithelial-to-Mesenchymal (EMT) signatures were strongly upregulated in both G1-and S/G2-treated cells, while cell cycle-related gene signatures such as E2F_targets or G2M_checkpoint were profoundly downregulated in G1 and fully restored in early escapers (Supp Fig. 4C). We observed that the most deeply cell cycle-repressed cluster (Osi G1 cluster #4 , Supp Fig. 4B) was highly enriched in alveolar type 1 (AT1)-related genes such as AQP4, CYP4B1, CLIC5, AGER or TNNC1, but also in alveolar type 2 (AT2)-specific marker SFTPD (Surfactant Protein D) (Fig. 1e, Supp Fig. 4D), consistent with recent data reported in minimal residual disease (MRD) of osimertinib-treated NSCLC patients [START_REF] Maynard | Therapy-Induced Evolution of Human Lung Cancer Revealed by Single-Cell RNA Sequencing[END_REF] . Interestingly, these genes were among the most overexpressed in healthy lungs compared with adenocarcinomas as revealed by a differential expression analysis using the TCGA database (Supp Fig. 4E). Pseudotime analysis revealed a progressive switch from an alveolar to a mesenchymal transcriptomic profile in the Osi G1 cluster #5 (Fig. 1e andf, Supp Fig. 5A-C) which was further enhanced in early escapers (Fig. 1g, Supp Fig. 5D) strongly suggesting that the EMT process could have been initiated from the alveolar-like sub-population.

Using recently published gene signatures of the different lung cell types [START_REF] Travaglini | A molecular cell atlas of the human lung from single-cell RNA sequencing[END_REF] , we observed that lineage switching involved a robust downregulation of mucous/goblet-related genes such as MUC5B, BPIFB2, SCGB3A1 or AZGP1 as well as a strong overexpression of fibroblast and muscle-specific genes (Supp Fig. 5E, Supp Fig. 6A-C). This process was strongly associated with extracellular matrix-and muscle-specific gene signatures (Supp Fig. 6D) and correlated with a strong enrichment in cytoskeletal-and extracellular matrix-coding genes (Fig. 1h), with a different pattern of (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

expression during the phenotypic switch (Supp Fig. 6E). Since these features could be specific to the HCC4006 model, we performed RNAseq experiments in drug-tolerant cells generated from other EGFR-mutated cell lines treated with either erlotinib or osimertinib at 1µM. Although each cell line seemed to follow a different path of differentiation (Supp Fig. 7A-C), all cell lines consistently overexpressed alveolar type 1 and contractile-related gene signatures (Supp Fig. 7D andE), which were the unique common gene signatures among the models tested (Fig. 1i). Interestingly, gene signatures related to cellular contractility were highly specific to the drug-tolerant state and did not necessarily evolve toward a mesenchymal phenotype (Supp Fig. 8). Indeed, H3255-derived DTC strongly overexpressed alveolar and muscle-specific genes but were not associated with EMT signatures (Supp Fig. 9A) and did not express EMTassociated genes such as SERPINE1, AXL, SPOCK1, or EMT-TF (Supp Fig. 9B), which could explain their inability to form fully resistant proliferative clones. Strikingly, a similar enrichment in contraction-associated cytoskeletal genes and muscle specific signatures was observed in healthy lungs compared to lung adenocarcinomas (Fig. 1j andk, Supp Fig. 10A-D), strongly reinforcing the evidence that the DTC subtype shared phenotypic resemblance with normal epithelial lung cells. Using our transcriptomic data, we built a new signature of drug tolerance composed of 350 genes that were commonly upregulated in our models (i.e. log2FC>1 in at least 4 out of 5 cell lines) (Supp Fig. 11A). This signature was positively associated with osimertinib-induced drug-tolerance in a model of osimertinib-treated EGFRmutated lung PDX (see Fig. 4) and in residual disease in patients (Supp Fig. 11B) but also with healthy lungs compared to lung adenocarcinomas (LUAD) (Supp Fig. 11C andD). Finally, we aimed to assess the physiological consequences of this increased contractile signatures. We observed a highly reorganized cytoskeleton in all the DTC as revealed by F-actin staining, with the presence of actin stress fibers and/or lamelipodia, depending on the cell type (Fig. 1l, Supp Fig. 12 and13). Interestingly, stress fibers were not detected in the H3255 cell line, which was in line with the absence of EMT signatures, however increased cortical F-actin was observed.

Taken together, our data suggest that osimertinib-resistant cells emerge from a differentiated drug-tolerant phenotype that shares similarities with an alveolar type 1 phenotype and involves cytoskeletal remodeling and contractile features.

Farnesyltransferase inhibitors prevent relapse to EGFR-TKi in vitro

Given that the presence of F-actin often correlates with activation of one or more Rho-GTPases [START_REF] Rottner | Actin assembly mechanisms at a glance[END_REF][START_REF] Sit | Rho GTPases and their role in organizing the actin cytoskeleton[END_REF] , we hypothesize that Rho-proteins could be involved in the establishment of the drug-tolerant contractile state. Indeed, RNAseq data revealed that expression of RHOB and RND3 (RHOE) was consistently increased in EGFR-TKi-derived DTC in vitro as well as in osimertinib-treated PDX (Fig. 2a). Using available in vivo scRNAseq data from Pasi Janne's lab [START_REF] Kurppa | Treatment-Induced Tumor Dormancy through YAP-Mediated Transcriptional Reprogramming of the Apoptotic Pathway[END_REF] , we observed that RHOB and RND3 were among the most highly upregulated genes in PC9 xenografts treated with osimertinib or with osimertinib + trametinib (Fig. 2b). RHOB and RHOE were also increased at the protein level and correlated with a transient increase in phospho-MLC2 and phospho-cofilin, suggesting activation of the Rho/ROCK pathway during an early stage of drug-tolerance (Fig. 2c, Supp Fig. 14A). Interestingly, RHOB overexpression appeared to be highly associated with cell cycle arrest (Fig. 1c, Supp Fig. 14A-D) and its expression was also correlated with an AT1 signature in lung cancer patients as revealed by a TCGA analysis (Supp Fig. 14C). By contrast, RHOE expression was correlated with EMT and fibroblastic features (Supp Fig. 14B andE), suggesting different functions for the two GTPases during the adaptive response. These data are in agreement with our findings describing a role for RHOB in resistance to (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

targeted therapy in EGFR-mutated lung cancer patients [START_REF] Calvayrac | The RAS-related GTPase RHOB confers resistance to EGFR-tyrosine kinase inhibitors in non-small-cell lung cancer via an AKT-dependent mechanism[END_REF] and BRAF-mutated melanoma [START_REF] Delmas | The c-Jun/RHOB/AKT pathway confers resistance of BRAF-mutant melanoma cells to MAPK inhibitors[END_REF] , although implication of RHOB or RHOE in the establishment of a drug-tolerant state has never been assessed. These observations prompted us to assess the efficacy of a panel of inhibitors of the Rho/ROCK pathway to prevent relapse to EGFR-TKi, such as ROCK inhibitors (Y27632, GSK269962A, RKI1447), the RhoA/B/C inhibitor C3-exoenzyme (tatC3), AKT inhibitor (G594), farnesyltransferase inhibitor (FTI, tipifarnib) and geranylgeranyltransferase inhibitor (GGTi, GGTi-298).

Strikingly, all the inhibitors, excepted RKI1447, displayed cytotoxic or cytostatic activity on DTC and strongly delayed the emergence of resistant clones (Fig. 2d ande), however only tipifarnib fully prevented the development of resistances to osimertinib or erlotinib in all the cell line tested (Fig. 2d ande, Supp Fig. 15A), with no significant effect when used alone (Supp Fig. 15B). Combination with other FTIs such as CP-609754, lonafarnib or FTI-2153 showed similar effect, leading to cell death (Fig. 2f, Supp Fig. 15C). Interestingly, tipifarnib could also prevent the emergence of resistance to EGFR-TKi in both subclonal and parental cell lines (Fig. 2d-f), suggesting that inhibition of farnesyltransferase could also affect potentially pre-existing resistant subclones (Supp. Fig. 1A). Unexpectedly, treatment of early-emerging osimertinib-resistant proliferative clones with tipifarnib also lead to tumor regression, suggesting that these cells still displayed vulnerability to inhibition of farnesyltransferase (Fig. 2g andh). However, complete clearance of tumor cells was not often achieved when tipifarnib was used after relapse, suggesting that the cells that had acquired resistance to osimertinib displayed reduced sensitivity to farnesyltransferase inhibition (Fig. 2h, Supp Fig. 16).

Our data show that Rho/ROCK pathway inhibitors efficiently prevent relapse to EGFR-TKi, among which tipifarnib displayed the most efficient and profound anti-tumor effect on both DTC and early-emerging clones.

Osimertinib+Tipifarnib treatment induces mitotic defects and integrated stress response (ISR)-mediated apoptotic pathway

To decipher the molecular mechanisms underlying tipifarnib's ability to prevent relapse to osimertinib, we performed scRNAseq on osimertinib + tipifarnib (OT)-treated G1 and S/G2 sorted cells and compared transcriptomes with those from cells treated with osimertinib. Strikingly, osimertinib-induced genes associated with our DRUG_TOLERANT_UP signature were drastically downregulated by tipifarnib as were many of the gene signatures related to EMT, myogenesis and cell-cycle (Fig. 3a andb, Supp Fig. 17A-D). Unexpectedly, tipifarnib prevented the upregulation of alveolar type 1 and fibroblast-specific genes but maintained muscle-associated genes such as MYL7, ACTC1 or ACTA1, specifically in the S/G2 population (Fig. 3c, Supp Fig. 18A-C). We aimed to decipher the physiological consequence of tipifarnib treatment on DTC. Time-lapse imaging of FUCCI-transduced cells revealed that, although tipifarnib did not prevent the emergence of osimertinib-derived early escapers, escaping cells failed to undergo mitosis and ultimately died (Fig. 3d), suggesting that tipifarnib may interfere with cell division in these cells. We also observed that a high percentage of OT-treated cells that progressed to S/G2 returned to G1 without dividing, consistent with a process of mitotic slippage or endoreplication [START_REF] Shu | Endoreplication: The Good, the Bad, and the Ugly[END_REF][START_REF] Gandarillas | Mammalian endoreplication emerges to reveal a potential developmental timer[END_REF] , which was also observable at lower frequency in osimertinib-treated cells (Fig. 3d, Supp Fig. 19). Although this process is often used by cells as a mechanism of drug-resistance [START_REF] Shu | Endoreplication: The Good, the Bad, and the Ugly[END_REF][START_REF] Gandarillas | Mammalian endoreplication emerges to reveal a potential developmental timer[END_REF] , none of the cells that underwent this process were able to divide and ultimately died (Fig. 3d, videos). We also observed that OTtreatment strongly upregulated ATF4-regulated genes [START_REF] Torrence | The mTORC1-mediated activation of ATF4 promotes protein and glutathione synthesis downstream of growth signals[END_REF] such as DDIT3/CHOP, ATF3, CHAC1, PSAT1, FAM129A/NIBAN1 and the pro-apoptotic BBC3/PUMA (Fig. 3e andf). ATF4 can be induced by several processes (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

including the Unfolded Protein Response (UPR) or the Integrated Stress Response (ISR) [START_REF] Pakos-Zebrucka | The integrated stress response[END_REF] . Upregulation of ATF4 and CHOP by OT treatment was confirmed at the protein level and correlated with PARP and Caspase 3 cleavage (Fig. 3g), strongly suggesting that activation of this pathway was responsible for OT-induced cell death.

Identification of potential pharmacological targets of tipifarnib

We sought to identify farnesylated proteins that might explain tipifarnib's ability to prevent relapse to EGFR-TKI.

Consistent with previous findings [START_REF] Calvayrac | The RAS-related GTPase RHOB confers resistance to EGFR-tyrosine kinase inhibitors in non-small-cell lung cancer via an AKT-dependent mechanism[END_REF] , siRNA-mediated inhibition of RHOB, but also RHOE, increased initial sensitivity to osimertinib (Supp Fig. 20A andB), although it failed to fully prevent relapse (Supp Fig. 20B, data not shown), suggesting that inhibition of these GTPases could not fully recapitulate tipifarnib's effect. Using a recently published list of farnesylated proteins [START_REF] Storck | Dual chemical probes enable quantitative system-wide analysis of protein prenylation and prenylation dynamics[END_REF] we observed in our scRNAseq data that several GTPases including RHOB and RND3 were overexpressed by osimertinib in G1-treated cells, whereas the expression of cell-division-related genes such as LMNB1, CENPF or SPDL1 was highly repressed during G1 but fully restored in S/G2 (Fig. 3h). Although individual inhibition of these genes increased sensitivity to osimertinib (Fig. 3i, Supp Fig. 20A andB), only inhibition of both RHOE and LaminB1 induced a complete clearance of osimertinib-treated cells, thus recapitulating tipifarnib's effect (Fig. 3j), suggesting inhibition of both proteins was needed to fully prevent relapse to osimertinib.

Collectively, our data suggest that tipifarnib prevents relapse to EGFR-TKi through a multi-targeted effect involving GTPases and cell-cycle regulators, resulting in impaired phenotypic differentiation, mitotic defaults and subsequent cell death through an ATF4-CHOP-dependent apoptotic pathway.

Tipifarnib prevents relapse to EGFR-TKI in vivo

Finally, we aimed to test the combination of osimertinib and tipifarnib (OT) in vivo using several models of EGFRmutated lung tumors. In a PC9 xenograft model, combination of osimertinib (5 mg/Kg, q.d., 5d/w) and tipifarnib (80 mg/Kg, b.i.d., 5d/w) induced a strong and stable response for up to 6 months with an almost complete response in all animals, whereas osimertinib induced a less potent effect and relapse was observed in 4/10 tumors (Fig. 4a-c, Supp Fig. 21A). OT treatment was well tolerated by the mice as indicated by a stable body weight, with no discernible effect on the general appearance of the mice (Supp Fig. 21B). We used the same treatment protocol in a PDX model of EGFR L858R/T790M lung adenocarcinoma (TP103 [START_REF] Quintanal-Villalonga | FGFR1 Cooperates with EGFR in Lung Cancer Oncogenesis, and Their Combined[END_REF] ). Osimertinib induced a moderate response with relapse of all (10/10) mice within 2 months, whereas OT-treatment induced significantly higher tumor regression in the first months with an almost stable disease lasting up to 5 months (Fig. 4d-g, Supp Fig. 22A-H), at which time OT-treated mice were sacrificed for subsequent molecular analysis of their tumors. As before, OT treatment was well tolerated by the mice as indicated by stable body weight (Supp Fig. 21B andF) and a good general aspect, thus confirming a good long-term tolerability of the treatment. Consistently with in vitro data, OT-treated tumors showed a strongly reduced proliferative state even after 5 months of treatment as highlighted by Ki67 staining (Fig. 4h). This correlated with increased p27 Kip1 levels and reduced phospho-Rb as well as increased PARP and Caspase 3 cleavage, suggesting that non-cycling OT-treated cells were undergoing apoptosis (Fig. 4i). Low proliferation was confirmed by scRNAseq analysis (Fig. 4j-l, Supp Fig. 23), which also revealed an increased expression of ATF4-regulated genes (Fig 4m), which could suggest that the ISR pathway may also contribute to apoptosis in vivo.

(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Here, we report that adaptive response to osimertinib is a highly dynamic process which invariably involves differentiation through an alveolar type-1 phenotype characteristically expressing cytoskeletal proteins involved in cellular contraction. Using a panel of Rho/ROCK pathway inhibitors, we found that tipifarnib, a clinically active farnesyltransferase inhibitor, efficiently and durably prevented relapse to osimertinib in vitro and in vivo by inducing an ATF4-dependent apoptotic response, with no evidence of toxicity in mice. Initially designed to inhibit RAS-driven cancer, FTIs did not achieve the expected clinical success, due to the lack of clarity of the clinical indication and the farnesylated proteins targeted for their pharmacological effects. Here we propose tipifarnib prevents the appearance of DTC and limit resistance to EGFR TKIs in EGFR-mutated lung cancer. In addition, we propose that concomitant inhibition of at least two farnesylated proteins RHOE and LaminB1 is sufficient to phenocopy tipifarnib's effects suggesting that tipifarnib act on multiple targets. Our work opens the way to a new therapeutic combination to increase the efficacy of EGFR-targeted therapies.

(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

MATERIALS AND METHODS

Cell culture

The human NSCLC cell-lines HCC4006 (CRL-2871, EGFR del L747-E749, A750P), HCC827 (CRL-2868, EGFR del E749-A750), HCC2935 (CRL-2869, EGFR del E746-T751, S752I) were obtained from the American Type Culture Collection (Manassas, VA, USA). The H3255 NSCLC cell line (EGFR L858R) and PC9 NSCLC cell-line (EGFR del E746-A750) were a kind gift from Helene Blons (APHP, Paris, France) and Antonio Maraver (IRCM, Montpellier, France) respectively. and were a kind gift from. For each cell-line, subclones were generated by limiting dilution. Moreover, subcultures were conducted for a limited period of time in order to limit cell deviation.

NSCLC cell-lines were cultured in RPMI (Roswell Park Memorial Institute) 1640 medium containing 10% fetal bovine serum (FBS) and were maintained at 37°C in a humidified chamber containing 5% CO2. Cell lines were authenticated and tested for mycoplasma contamination within the experimental time frame.

Cells were transduced with the Incucyte® Cell Cycle Green/Red lentivirus based on the FUCCI system, and selected by puromycin treatment for one week.

SiRNA targeting RHOB, RHOE, LaminB1.

Twenty-four hours after seeding, cells were transfected for 4h with siRNA (SMARTpool Dharmacon™) at a final concentration of 10nM, and 1µM osimertinib was added 24h hours after transfection. Transfection was repeated twice a week until relapse while maintaining osimertinib treatment. For combo transfection with two siRNAs, each siRNA was transfected at a final concentration of 5 nM and response to osimertinib was compared to cells transfected with siRandom at a final concentration of 10 nM.

Inhibitors

Cells were treated with the following inhibitors at the indicated concentrations two to three days after seeding, with medium changes twice a week for the whole duration of the experiment.

Cell growth and viability assays on GFP-positives cells

Cells were transduced with a lentiviral vector (pLenti CMV GFP DEST (736-1), 9216bp) to obtain a constitutive expression of GFP protein. GFP-positives cells were selected by cell sorting using FACS Melody (BD Biosciences).

Fluorescence intensity in each well of 96-well plate was evaluated twice a week using Synergy™ 2 Multi-Detection Microplate Reader. Relative cell survival in the presence of inhibitors which is proportional to fluorescence intensity was normalized to untreated cells after background corrections. Images of GFP-positive cells were obtained with Incucyte® S3 Live-Cell Analysis software (Sartorius).

Crystal violet staining

Untreated control cells and treated cells in culture plates were wash with PBS, fixed with paraformaldehyde 4 % solution for 10 min and stained with a solution containing PBS -0.5% crystal violet (Sigma-Aldrich/Merck, ref: C3886) -25% methanol for 10 minutes. After 3 washes and drying, cell staining was imaged with a ChemiDoc™ MP Imaging system (Bio-Rad).

Cytotoxicity assay

Viability of untreated cells or treated cells for 5 days was assessed using CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) (Promega®, ref:G3580) according to manufacturer's instructions.

Western blot analyses

For in vitro experiments, cells were lysed with RIPA buffer complemented with proteases-and phosphatasesinhibitors. For animal experiments, frozen tumors were ground and lysed with Tris-SDS 1% buffer complemented with proteases-and phosphatases-inhibitors. After sonication, protein content was quantified using Bradford Detection was performed using peroxydase-conjugated secondary antibodies and chemiluminescent detection kit (Clarity™ Western ECL, Bio-Rad) with a ChemiDoc™ MP Imaging system (Bio-Rad).

Immunoprecipitation

Cells protein lysates were immunoprecipitated with a specific antibody for the GTP-bound active form of RHOA, RHOB and RHOC GTPases (RH12) designed in the laboratory 25,26 . Then RHOA-, RHOB-and RHOC-active form expression was revealed by western blot.

Phase contrast microscopy

Cells were imaged with ZEISS Axio Vert.A1 microscope and analysed with ZEN software (ZEISS).

Phalloidin staining

(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Cells were first fixed with paraformaldehyde 4 % solution, permeabilized with a PBS-BSA 0.1 %-Triton X-100 0.5 % solution, blocked with a PBS-BSA 1 %-Triton X-100 0.1 % solution and stained using Alexa Fluor™ 594 phalloidin (ThermoFisher Scientific, #A12381) diluted 50 times in a PBS-BSA 0.1 %-Triton X-100 0.1 % solution. Then, nuclei were stained with DAPI (4', 6-diamidino-2-phenylindole, dihydrochloride, ThermoFisherScientific, #D1306). Images were acquired with ZEISS Axio Vert.A1 microscope and analysed with ZEN software (ZEISS). Quantification of F-actin intensity was performed by ImageJ software

Cell cycle analysis

Cells were transduced with Incucyte® Cell Cycle Green/Red Lentivirus Reagent (EF1α-Puro) (Sartorius, Cat. No. 4779) as recommended by the manufacturer and transduced cells were selected by puromycin treatment. Cells were imaged every hour with Incucyte® S3 Live-Cell Analysis System (Sartorius). G1-phase, S/G2/M-phase and G1/S-phase cells were quantified using Incucyte® S3 Live-Cell Analysis software (Sartorius).

RNA sequencing

Subclones generated from HCC4006, PC9 and H3255 cell lines were treated with 1µM erlotinib for 24h, 21 days (DTC state) and cultured for more than 3 months in the presence of the drug (RPC state, Resistant Proliferative Clones) for HCC4006 and PC9. HCC827 were treated until reaching the drug-tolerant state (11 days) with 1 µM osimertinib. RNA extraction was performed using AllPrep DNA/RNA Mini kit (Qiagen, #80204) according to manufacturer's protocol.

RNA quality was assessed using Fragment Analyzer (Agilent technologies) and the RQN values were provided to confirm the integrity of total RNA. RNA concentration was determined by fluorescent method using Quant-iT™ RNA Assay Kit, Broad Range (ThermoFisher Scientific). RNA samples were processed with Illumina TruSeq® Stranded mRNA Library Preparation Kit following the manufacturer's protocol. Library size and quality were confirmed on Fragment analyzer (Agilent Technologies). KAPA quantification kit for Illumina platforms (KAPA Biosystems, Roche)

was used to quantify library by qPCR. Indexed libraries were pooled and sequenced on an Illumina NextSeq 550 (2x75 bp paired-end reads).

Reads were mapped and counted using the RNA-Seq by Expectation Maximization (RSEM) software v. 1.3 (with bowtie2-2.3.5.1) based on the human reference genome UCSC hg38. Differential expression was analysed with DESeq2.

Single-cell capture

HCC4006-treated cells

HCC4006 subcloned cells were expanded in untreated, osimertinib-or osimertinib + tipifarnib-containing medium for 4, 20 and 12 days respectively. Medium was changed 24h before sorting. Cells were dissociated by trypsinisation, recovered in FACS buffer (0,04% BSA in PBS) and kept on ice. G1 (red) and S/G2 (green) cells were sorted at 4°C using FACS Melody (BD Biosciences). Cells were spun down and resuspended in appropriate volume for a final concentration of 500 to 1500 cells / µl, with a viability above 85%. 6000 cells per sample were loaded to the Chromium Controller (10X Genomics) and 1900-3300 cells were recovered depending on the sample. scRNA-seq (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

libraries were generated using the 10X Genomics Chromium Single Cell 3' Kit v2 according to manufacturer's instructions. The libraries were profiled with the HS NGS kit for the Fragment Analyzer (Agilent Technologies) and quantified using the KAPA library quantification kit (Roche Diagnostics). The libraries were pooled and sequenced on the Illumina NextSeq550 instrument by the CRCT Genomics platform using the High Output 150 cycles kit. The singleindexed sequencing parameters were 28, 8, 0, 91 cycles (read 1, index 1, index 2, read 2). The dual-indexed sequencing parameters were 28, 10, 10, 90 cycles (read 1, index 1, index 2, read 2). An average depth of ~46000 reads/cell was obtained.

Patient-Derived Xenograft

The PDX model (EGFR T790M/L858R , TP103 [START_REF] Quintanal-Villalonga | FGFR1 Cooperates with EGFR in Lung Cancer Oncogenesis, and Their Combined[END_REF] ) was generated in the Paz-Ares laboratory at the Instituto de Biomedicina de Sevilla (IBIS). Tumours were collected, cut into 2-4 mm pieces on ice and dissociated by mechanical and enzymatic reaction following the Miltenyi Tumour Dissociation Kit instructions (GentleMACS program h_tumor_02). Samples were filtered through a MACS SmartStrainer 70 µm. Erythrocytes were lysed following the Red Blood Cell Lysis Solution protocol (Miltenyi Biotec). Samples were frozen in RPMI 10% DMSO 20% FBS until the day of single-cell capture. Samples from all conditions were thawed and processed on the same day following the 10X Genomics Thawing Dissociated Tumor Cells for Single Cell RNA Sequencing protocol. Cells were recovered in appropriate volume for a final concentration of 500 to 1000 cells / µl, with a viability above 70%. 4500 cells per sample were loaded to the Chromium Controller (10X Genomics) and scRNA-seq libraries were generated using the 10X Genomics Chromium Single Cell 3' Kit v2 according to manufacturer's instructions. The libraries were sequenced with a NextSeq 550 (Illumina) by the CRCT Genomics platform. An average depth of ~27000 reads/cell was obtained.

Single cell RNA sequencing analysis

The single cell transcriptomic data was loaded from Kurppa et al. [START_REF] Kurppa | Treatment-Induced Tumor Dormancy through YAP-Mediated Transcriptional Reprogramming of the Apoptotic Pathway[END_REF] on GEO under accession number GSE131604. The data from the different conditions were pulled together to create a global counts matrix. A dimension reduction by principal component analysis (PCA), a t-SNE projection and a k-means clustering were done using the clustering function of the SingleCellSignalR R package. Two differential gene expression analyses were done using the cluster_analysis function of SingleCellSignalR using respectively the cluster vector from the clustering function cited above and a cluster vector created to correspond to the different conditions. The p-value threshold was set by default at 5%. A pathway signature genes table was retrieved from Reactome and GO. The mean expression of the genes in each pathway was calculated in each cell to create a mean pathway expression matrix. Violin plots of the mean pathway expression were computed and displayed using the ggplot2 package. The heatmaps were generated using the ComplexHeatmap R package. The t-SNE expression plots were done using the expression_plot function of SingleCellSignalR. All the analyses were carried out with RStudio.

Mouse xenograft studies

(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Cell line xenograft experiments were performed in 6 to 8-week old female NMRI mice (Charles River Laboratories) by injecting 10 million PC9 cells in 50% matrigel subcutaneously on each flank of the mouse. The PDX model (EGFR T790M/L858R , TP103 [START_REF] Quintanal-Villalonga | FGFR1 Cooperates with EGFR in Lung Cancer Oncogenesis, and Their Combined[END_REF] ) was cut and engrafted subcutaneously into the flank of NSG mice.

When tumors reached on average 200 to 300 mm 3 (calculated as [length x width 2 x 3.14/6]), mice were randomized and treated by oral gavage 5 days a week with 100µl of vehicle, osimertinib (5 mg/kg q.d), tipifarnib (80 mg/kg b.i.d) or a combination of osimertinib and tipifarnib. Osimertinib was diluted in 0,5% carboxymethylcellulose (Sigma, M0512) + 0,1% Tween80 (Sigma, P8074) and Tipifarnib in 2-hydroxypropryl-cyclodextrin 20% (Sigma, H107). Tumors were measured twice a week. Mice were weighted twice a week to monitor toxicity and humanely killed at indicated times and tumors harvested.

Immunohistochemistry

Tissues were fixed in 10% formalin overnight at room temperature, stored in 70% ethanol at 4°C and embedded in paraffin. Four-micrometer paraffin sections were used for hematoxylin and eosin staining followed by immunohistochemistry using standard procedures with Ki67 antibody (SP6; Thermo Scientific). Quantification of Ki67 IHC scores for each treatment was blind evaluated by two operators.

TCGA and GSEA analysis

RSEM-normalized expression data of human lung adenocarcinomas (517 samples) and healthy lungs (59 samples)

from The Cancer Genome Atlas (TCGA) database were downloaded from http://firebrowse.org. Spearman's correlation and statistical analysis of mRNA expression in LUAD was performed using the cbioportal co-expression tool (www.cbioportal.org). Pathway Gene Set Enrichment Analysis (GSEA) was performed with dedicated software (https://www.gsea-msigdb.org/gsea). (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figarol et al. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figarol et al. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figarol et al.

  Figarol et al.

Figure 1 :

 1 Figure 1: Drug-tolerance is a dynamic state which invariably implicates alveolar differentiation and cytoskeletal remodelinga: Percentage of total cells (blue), S/G2 (green) or G1 (red) population of HCC4006 subclonal cells during osimertinib treatment (1µM). b: Representative fluorescence images of S/G2 (green) or G1 (red) HCC4006 subclonal cells during osimertinib treatment (1µM). c: S/G2 (green) or G1 (red) fractions (%) of PC9, HCC4006, HCC827, HCC2935, H3255 subclonal cells during osimertinib treatment (1µM). d: Flow cytometry charts of red cells (G1) or green cells (S/G2 or G1-S) of untreated or osimertinib treated (1µM, 20 days) HCC4006 subclonal cells sorted for single-cell RNA sequencing experiment. Percentages of each fractions according to total population are specified. e: Osimertinib-induced genes overexpressed specifically in G1 cluster 4 vs cluster 5 (left) or in cluster 5 vs cluster 4 (right). AT1 (blue), AT2 (green) or EMT (red)-specific genes are highlighted. f: Mean z-score evolution of alveolar (blue), mesenchymal (red) and E2F_target (green) signatures according to latent time cell repartition. Osi-G1 cluster 4 (orange) and cluster 5 (blue) are specified. g: Violin plots representing the mean z-score values of alveolar type 1 (AT1), EMT-specific and E2F_target signatures in the different clusters. h: Top significantly overrepresented protein classes (pantherbd.org) in osimertinib-treated HCC4006 G1 (red bars) and S/G2 cells (green bars) compared to untreated-G1 and S/G2 populations, respectively (log2FC>1, p-value<0.05) i: Left: Venn diagram comparing the significantly enriched pathways (p<0.001) in erlotinib-or osimertinib-derived drug-tolerant cells. GSEA analysis was performed on differentially expressed genes (p<0.05) obtained by RNAseq in PC9 and H3255 cells were treated with 1µM erlotinib for 21 days, HCC827 cells treated with 1µM osimertinib for 11 days, and HCC4006 G1-cells treated with osimertinib for 21 days, compared to respective control cells. Right: Volcano plots of the pathways enriched in indicated EGFR-TKi-treated DTC. j: Differential expression of the contraction-associated cytoskeletal genes in healthy lungs compared to lung adenocarcinoma using TCGA data. k: GSEA analysis of alveolar type 1, muscle-contraction and E2F_targets signatures in healthy lungs compared to lung adenocarcinoma using TCGA data. l: Phalloidin F-actin staining of HCC827, H3255, HCC2935, HCC4006 and PC9 control cells or DTC upon osimertinib treatment (1µM) (X20 magnification, scale bar = 50µM). Images were analyzed by Image J software (filter: 16 colors).

(

  Figarol et al.

Figure 2 :

 2 Figure 2: Farnesyltransferase inhibitors prevent resistance to EGFR-TKi in vitro a: Differential mRNA expression of the 21 known Rho-GTPases in DTC generated from erlotinib-treated (HCC4006, PC9, H3255) or osimertinibtreated (HCC827, HCC4006) cells or in a PDX model of EGFR-mutated lung adenocarcinoma (described in Fig. 4). b: Volcano plot of the differentially expressed genes in osimertinib-(up) or osimertinib+trametinib-(down) treated PC9 xenografts. Data were obtained from a scRNAseq published by Kurppa et al. Cancer Cell 2020). c: Representative Western Blot of proteins related to EGFR pathway (phospho-EGFR, EGFR, phospho-ERK, ERK), cell contractility (phospho-MLC2, phospho-cofilin), EMT (vimentin, N-cadherin, SNAIL, SLUG), cell cycle (p27Kip1, phospho-RB, RB) and RhoGTPases (RHOB, RHOE) during osimertinib treatment (1µM) in HCC4006, PC9 and HCC827 subclonal cells. RPC= resistant proliferative clone. d: Cell survival (%) of HCC4006 parental cells during osimertinib treatment (1µM) alone or in combination with ROCK inhibitors (RKI, RKI1474 and Y27, Y27632), RHOA/B/C inhibitor (TatC3) or farnesyl-transferase inhibitor (Tipi, tipifarnib). e: Cell survival of PC9, HCC4006, HCC827 parental and subclonal cells upon osimertinib treatment (1µM) alone or in combination with ROCK inhibitors (Y27632, GSK269962A, RKI1447), RHOA/B/C inhibitor (TatC3), AKT inhibitor (G594), actin inhibitor (LatB, Latruncunlin B), geranylgeranyltransferase inhibitor (GGTI) or farnesyltransferase inhibitor (Tipi, tipifarnib). Deep blue round refers to no response or relapse, light blue round to partial or delayed response and white round to complete response. f: Crystal violet staining of clonal and parental PC9, HCC4006 and HCC827 cells treated for 5 days, at DTC state or after relapse with osimertinib (1μM) alone or in combination with farnesyltransferase inhibitors (tipifarnib 1µM, Tipi; CP-609.754 1µM, CP; lonafarnib 10µM, Lona). g: Cell survival (%) of HCC4006 and PC9 cells during osimertinib treatment (1µM) with or without tipifarnib (1µM) added at indicated timepoints. h: Images of HCC827 clonal cells upon osimertinib (1µM) +/-tipifarnib (1µM). G1 cells are red-fluorescent, S/G2 cells are green-fluorescent and G1-S cells are in yellow. Blue and red arrows represent osimertinib treatment and tipifarnib treatment beginning respectively.

Figure 3 :

 3 Figure 3: Tipifarnib causes multiple interferences in the adaptive response to osimertinib a: GSEA analysis of Drug-Tolerant_Up and EMT signatures comparing Osimertinib+Tipifanib-versus Osimertinib-treated HCC4006 G1 and S/G2 cells. b: Violin plots representing the Log2 mRNA expression of drug-tolerant and EMT-related genes significantly altered by osimertinib+tipifarnib vs osimertinib treatment. c: Mean z-score of normal cell type-associated signatures (from Maynard, Nature 202010) significantly regulated by osimertinib and/or osimertinib+tipifarnib treatments. d: Cell fate after entry to S/G2 of HCC4006 clonal cells treated or not with osimertinib (1µM), tipifarnib (1µM) or the combo. S/G2 cells are in green, G1 cells are in red. Mitosis event are represented in blue, endoreplication in orange and death in black. e: GSEA analysis of ATF4-dependent Integrated Stress Response (ISR) signature (Torrence, elife 202118) significantly modulated by Osimertinib+Tipifarnib versus Osimertinib treatment f: Violin plots representing the Log2 mRNA expression of ATF4-regulated genes significantly upregulated by Osimertinib+tipifarnib treatment g: Protein expression by western blot of proteins related to EGFR pathway (phospho-EGFR, EGFR), farnesyltransferase inhibitor efficacy (HRAS), apoptosis (PARP and caspase-3) and ISR (ATF4, CHOP) on HCC4006 clonal cells treated with osimertinib (1µM) alone or in combination with tipifarnib (1µM). h: Differential expression of genes coding for farnesylated proteins (Stock et al, Nat Chem, 2019) in osimertinib-treated G1 versus G1-untreated HCC4006 cells or osimertinib-treated S/G2 versus osimertinib-treated G1 cells i: Cell survival (%) of HCC4006 clonal cells during osimertinib treatment (1µM) and transfected with siRNA control (Neg) or targeting RHOE or LMNB1. j: Cell survival (%) of HCC4006 clonal cells during osimertinib treatment (1µM) and transfected with siRNA control (Neg) or targeting RHOE and LMNB1.

Figure 4 :

 4 Figure 4: Tipifarnib prevents relapse to osimertinib in vivo a: Mean tumor volume (mm3) of PC9 xenografts treated 5 days/week with vehicle, Tipifarnib (Tipi, 80mg/kg, b.i.d.), Osimertinib (Osi, 5 mg/kg, q.d), or by the combo (Osi + Tipi). n = 6 tumors in the vehicle and Tipifarnib arms, and n = 10 in the Osimertinib arm and n=12 in the combination arms. Mean ± SEM are shown. b: Change in tumor volume versus baseline of PC9 xenografts after 6 months of treatment with Osimertinib or a combination of Osimertinib and Tipifarnib. c: Progression-free survival of mice with PC9 xenografts and treated with Osimertinib or a combination of Osimertinib and Tipifarnib. P-value was determined by log-rank Mantel-Cox test. d: Mean tumor volume of an PDX model of EGFRL858R/T790M lung adenocarcinoma treated 5 days/week with vehicle, Tipifarnib (Tipi, 80mg/kg, b.i.d.), Osimertinib (Osi, 5 mg/kg, q.d), or by the combo (Osi + Tipi). The graph is the result of two independent cohorts of mice with a total of n = 4 tumors in the vehicle arm, n = 5 tumors in the Tipifarnib arm and n = 10 tumors in the Osimertinib and combination arms. Mean ± SEM are shown. e: Log2 fold change of the PDX growth compared to baseline after 60 days of treatment with Osimertinib or a combination of Osimertinib and Tipifarnib. f: Log2 fold change of the PDX growth compared to baseline at the best response from 4 weeks after treatment with Osimertinib or a combination of Osimertinib and Tipifarnib. g: Overall survival of EGFR-L858R-T790M PDX mice treated with Osimertinib or the combination of Osimertinib and Tipifarnib. The graph is the result of one cohort of mice with n = 6 mice in both arms. P-value was determined by log-rank Mantel-Cox test. h: Representative images of Hematoxylin and Eosin (H&E) stainings (top) and Ki67 IHC (bottom) from PDX tumors collected after 2 weeks, 2 months and 5 months of treatment with Tipifarnib, Osimertinib or the combination of Osimertinib and Tipifarnib, respectively. Quantification of Ki67 IHC scores for each treatment. Data are representative of 3 to 6 independent tumors. Scale bar = 50µm i: Immunoblot of lysates from individual PDX harvested after 2 weeks, 2 months and 5 months of treatment with Tipifarnib, Osimertinib or the combination of Osimertinib and Tipifarnib, respectively. Quantification of protein levels of HRAS, p27Kip1 and phospho-Rb normalized with actin level. Images represent n = 3-4 independent tumors. Immunoblot was performed once.

(

  which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. j: Proportion of G1, S and G2/M populations determined by scRNAseq Seurat clustering in PDX tumors collected after 2 weeks, 2 months and 5 months of treatment with Tipifarnib, Osimertinib or the combination of Osimertinib and Tipifarnib, respectively. k: GSEA analysis of the E2F_target signature in Osimertinib+Tipifarnib versus Osimertinib-treated EGFR-L858R-T790M PDX in the Ki67-negative (top) or positive (bottom) cell populations. l: Mean mRNA expression levels of genes implicated in replication or (m) in ISR, in the different cell populations, determined by scRNAseq. Mean ± SD are shown.

  which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

		FTi-2153	Kindly provided by Saïd Sebti	1µM
			and Andrew D. Hamilton
	Géranylgeranyl-transferase I	GGTI-298	Selleckchem	S7466	1µM
		GGTi-2166	Kindly provided by Saïd Sebti	1µM
			and Andrew D. Hamilton
	ROCK	Y27632	Selleckchem	S1049	10µM
		GSK269962A	Selleckchem	S7687	0.3µM
		RKI-1447	Selleckchem	S7195	1µM
	RHOA, B, C	TAT-C3	C3 exoenzyme coupled to	2 à 5 µg/ml
			permeant peptide TAT (1 or 10
			µg/ml) were produced and
			purified in our laboratory using
			an Akta purifier (GE Healthcare)
			as previously described (réf:
			Sahai, E. & Olson, M. F.
			Purification	of	TAT-C3
			exoenzyme. Meth. Enzymol.
			406, 128-140 (2006)).
	Actin	Latrunculin B	Sigma-Aldrich	428020	0.3µM
	CDK4/6	Palbociclib	Selleckchem	S1579	1µM
						Concentrations
	Target	Drug	Provider	Reference	used for in vitro
						experiments
	EGFR	Erlotinib (OSI-744, CP-358774,	LC Laboratories	E4997	1µM
		NSC 718781)
		Osimertinib (AZD9291)	LC Laboratories	0-7200	1µM
	Farnesyl-transferase	Tipifarnib (R115777, IND 58359)	Kura Oncology			0.1 -1µM
		Lonafarnib	Selleckchem	S2797	0.1 -1µM
		CP-609754	Kura Oncology			0.1 -1µM

(

  (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Protein extracts were separated on SDS-PAGE and electrotransferred on polyvinylidene difluoride membranes. Blots were probed with primary antibodies reported in the following table:

	bioRxiv preprint	doi:	; https://doi.org/10.1101/2022.04.01.486707	this version posted April 3, 2022.	The copyright holder for this preprint
	method. Target		Provider	Reference
	RhoB		Santa Cruz Biotechnology Sc-180
	RhoA		Santa Cruz Biotechnology Sc-418
	RhoC		Cell Signaling	#3430
	RhoE		Cell Signaling	#3664
	p-ERK1/2 Thr202/Tyr204 Cell Signaling	#4377
	ERK		Santa Cruz Biotechnology Sc-93
	p-EGFR Tyr1068	Cell Signaling	#2234
	EGFR		Cell Signaling	#4267
	Vimentin		Cell Signaling	#3932
	E-cadherin		Cell Signaling	#3195
	N-cadherin		Cell Signaling	#4061
	SLUG		Cell Signaling	#9585
	SNAIL		Cell Signaling	#3879
	p-RB Ser807/811	Cell Signaling	#9308
	RB		Cell Signaling	#9309
	p27		Cell Signaling	#3686
	p53		Cell Signaling	#48818
	Cyclin D1		Cell Signaling	#2978
	Cyclin E1		Cell Signaling	#20808
	p-MLC2 Ser19		Cell Signaling	#3671
	MLC2		Cell Signaling	#8505
	p-Cofilin Ser3		Cell Signaling	#3313
	Cofilin		Cell Signaling	#5175
	PARP		Cell Signaling	#9542
	Caspase-3 cleaved	Cell Signaling	#9661
	Caspase-3		Cell Signaling	#9662
	Lamin B1		Abcam	Ab16048
	Actin		Merck Millipore	MAB1501

ACKNOWLEDGMENTS

We thank all members of the SIGNATHER team at CRCT (Cancer Research Center of Toulouse, INSERM U1037, CNRS ERL5294, UPS, Toulouse, France) for stimulating and thoughtful discussions. We thank David Santamaría, John Hickman, Agnese Cristini and Olivier Sordet for critically reading the manuscript. We thank all members of mice core facilities (CREFRE, UMS006 Inserm, Toulouse) in particular Cédric Baudelin, Amanda Corini, Emilie Sinhlivong, Tristan Chapon, Charlotte Delos, Thi-Lan Huong Huynh and Charlène Lopez for their support and technical assistance. We acknowledge the cytometry/cell-sorting and imaging facilities of the CRCT, in particular Manon Farcé and Laetitia Ligat for their assistance with flow cytometry and microscopy, respectively. We thank Carine Valle and Emeline Sarot from the Genomics and Transcritomics platform of the CRCT for their support and technical assistance, as well as Marie Tosolini from the Bioinformatics platform of the CRCT for the bioinformatics analysis. We thank Géraldine Touriol for the administrative and financial management. AUTHOR CONTRIBUTIONS Study design, data interpretation and preparation of the manuscript: SF, CD, RG, AP, JM, OC, GF; Execution of experiments: SF, CD, RG, RA, SP, CT, EC, AC, OC; Computational and statistical analysis: JC, JPV; Writing, review, and/or revision of the manuscript: SF, CD, RG, JPV, AM, AP, JM, OC, GF; Material support: ILM, IF, LPA. FUNDINGS This work was supported in part by institutional grants from INSERM, Fondation pour la Recherche Médicale (FRM, équipe labellisée [DEQ20170839117]), Programme de Recherche Translationnelle en Cancérologie (INCa-DGOS, (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.