HGG-09. TARGETING FACILITATES CHROMATIN TRANSCRIPTION (FACT) AS A NOVEL STRATEGY THAT ENHANCES RESPONSE TO HISTONE DEACETYLASE (HDAC) INHIBITION IN DIPG

DIPG is an aggressive and incurable childhood brain tumour for which new treatments are needed. A high throughput drug screen of 3500 pharmaceutical compounds identified anti-malarials, including quinacrine as having potent activity against DIPG neurospheres. CBL0137, a compound modelled on quinacrine, is a novel anti-cancer compound which targets Facilitates Chromatin Transcription (FACT), a chromatin remodelling complex involved in transcription, replication, and DNA repair. CBL0137 effectively crosses the blood-brain barrier and has recently completed Phase I testing in adult patients. CBL0137 induced apoptosis in DIPG neurospheres and had profound cytotoxic activity against a panel of DIPG cultures. In a DIPG orthotopic model, treatment with CBL0137 significantly improved survival. We found that treatment with CBL0137 up-regulated TP53 and increased histone H3.3 acetylation and tri-methylation in DIPG cells. We therefore examined the interaction between CBL0137 and the histone deacetylase (HDAC) inhibitor panobinostat. In vitro experiments showed that the two agents had profound synergistic activity against DIPG neurospheres in clonogenic assays and enhanced caspase activation and apoptosis. The FACT subunit SSRP1 was found to directly interact with H3.3K27M and treatment with CBL0137 targeted this epigenetic defect, restoring histone H3.3 trimethylation and leading to tumor cell death. Transcriptomic analysis and immunoblotting indicated that combination treatment activated signalling pathways controlled by Retinoblastoma (RB)/E2F1 and subsequently increased phosphorylation and enzymatic activity of enhancer of zeste homolog 2 (EZH2). Consistent with the in vitro results, the combination of CBL0137 and panobinostat significantly prolonged survival in two independent orthotopic models of DIPG, while histological analysis showed restoration of H3K27me3 and decreased Ki67 positive cells. In addition to panobinostat, CBL0137 has been found to combine synergistically in vitro and in vivo with PARP and BET inhibitors. Given these promising results, a paediatric trial of CBL0137 will open through the Children’s Oncology Group with an expansion cohort for DIPG patients.

DIPG-27. TARGETING FACILITATES CHROMATIN TRANSCRIPTION (FACT) AS A NOVEL STRATEGY FOR DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG) THAT ENHANCES RESPONSE TO HISTONE DEACETYLASE (HDAC) INHIBITION

Diffuse intrinsic pontine glioma (DIPG) is an aggressive and incurable childhood brain tumour for which new treatments are needed. A high throughput drug screen of 3500 pharmaceutical compounds identified anti-malarials, including quinacrine as having potent activity against DIPG neurospheres. CBL0137, a compound modelled on quinacrine, is an anti-cancer compound which targets Facilitates Chromatin Transcription (FACT), a chromatin remodelling complex involved in transcription, replication, and DNA repair. CBL0137 effectively crosses the blood-brain barrier and is currently in Phase I trials in adult cancer. CBL0137 induced apoptosis in DIPG neurospheres in vitro and had profound cytotoxic activity against a panel of DIPG cultures. In a DIPG orthotopic model, treatment with CBL0137 significantly improved survival. We found that treatment with CBL0137 up-regulated TP53 and increased histone H3.3 acetylation and tri-methylation in DIPG cells. We therefore examined the interaction between CBL0137 and the HDAC inhibitor, panobinostat. In vitro experiments showed that the two agents had profound synergistic activity against DIPG neurospheres in clonogenic assays and enhanced apoptosis. Transcriptomic analysis and immunoblotting indicated that combination treatment activated signalling pathways controlled by Retinoblastoma (RB)/E2F1 and subsequently increased phosphorylation and enzymatic activity of enhancer of zeste homolog 2 (EZH2). Consistent with the in vitro results, the combination of CBL0137 and panobinostat significantly prolonged the survival of two orthotopic models of DIPG, while histological analysis showed increased H3K27me3 and decreased Ki67 positive cells. Given these promising results, a paediatric trial of CBL0137 is planned to open through the Children’s Oncology Group with an expansion cohort for DIPG patients.

LEDGF and HDGF2 relieve the nucleosome-induced barrier to transcription

FACT (facilitates chromatin transcription) is a protein complex that allows RNAPII to overcome the nucleosome-induced barrier to transcription. While abundant in undifferentiated cells and many cancers, FACT is not abundant or is absent in most tissues. Therefore, we screened for additional proteins that might replace FACT upon differentiation. Here we report the identification of two such proteins, LEDGF and HDGF2, each containing two HMGA-like AT-hooks and a PWWP methyl-lysine reading domain known to bind to H3K36me2 and H3K36me3. LEDGF and HDGF2 localize with H3K36me2/3 at genomic regions containing active genes, usually adjacent to H3K27me3 domains. In myoblasts, where FACT expression is low, LEDGF and HDGF2 are enriched on most active genes. Upon differentiation to myotubes, LEDGF levels decrease across the genome while HDGF2 levels are maintained. Moreover, HDGF2 is recruited to the majority of myotube up-regulated genes and is required for their proper expression. HDGF2 knockout myoblasts exhibit an accumulation of paused RNAPII proximal to the first nucleosome within the transcribed region of many HDGF2 target genes, indicating a defect in early elongation. We propose that LEDGF and HDGF2 substitute for FACT in differentiated tissues and that their distribution on the genome helps maintain transcriptional programs unique to particular cell types.

Mechanism of FACT removal from transcribed genes by anticancer drugs curaxins

Human FACT (facilitates chromatin transcription) is a multifunctional protein complex that has histone chaperone activity and facilitates nucleosome survival and transcription through chromatin. Anticancer drugs curaxins induce FACT trapping on chromatin of cancer cells (c-trapping), but the mechanism of c-trapping is not fully understood. Here, we show that in cancer cells, FACT is highly enriched within the bodies of actively transcribed genes. Curaxin-dependent c-trapping results in redistribution of FACT from the transcribed chromatin regions to other genomic loci. Using a combination of biochemical and biophysical approaches, we have demonstrated that FACT is bound to and unfolds nucleosomes in the presence of curaxins. This tight binding to the nucleosome results in inhibition of FACT-dependent transcription in vitro in the presence of both curaxins and competitor chromatin, suggesting a mechanism of FACT trapping on bulk nucleosomes (n-trapping).

Ο ρόλος των μεταγραφικών μηχανισμών και της επιγενετικής στην ανθρώπινη παθογένεια

Σκοπός της διατριβής αυτής είναι η διασύνδεση των επιδιορθωτικών μηχανισμών του κυττάρου με τη λειτουργία της μεταγραφής. Εστιάζουμε στη μελέτη του ρόλου της μεταγραφής στη διασφάλιση της πιστότητας της γενετικής πληροφορίας σε περιπτώσεις βλαβών στο DNA τόσο σε φυσιολογικές όσο και σε παθολογικές καταστάσεις. Απώτερος σκοπός μας είναι η κατανόηση σε μοριακό επίπεδο της κλινικής εικόνας όπως απεικονίζεται σε σοβαρές εγγενείς διαταραχές και σύνδρομα πρόωρης γήρανσης.Στις επόμενες σελίδες θα παρουσιαστούν δυο εγχειρήματα τα οποία εκπονήθηκαν στα πλαίσια της διατριβής αυτής και που εστίασαν σε μηχανισμούς επιδιόρθωσης του DNA μετά από επαγωγή βλαβών. Και για τα δυο κοινός σκοπός είναι η μελέτη και αποσαφήνιση των μηχανισμών που είναι υπεύθυνοι για την επιδιόρθωση βλαβών στο DNA και τη διατήρηση της ομοιόστασης του κυττάρου.Στο πρώτο εγχείρημα, επικεντρωθήκαμε στον ρόλο της πρωτεΐνης TOP1, τοποϊσομεράσης I, στη ρύθμιση της μεταγραφής κάνοντας χρήση του χημικού CPT, καμπτοθεσίνη, το οποίο δεν επιτρέπει την απελευθέρωση της TOP1 από το DNA αφού επιτελέσει το ρόλο της. Η δημιουργία συμπλόκων TOP1cc με το DNA αποτελεί φυσικό εμπόδιο για τις κυτταρικές λειτουργίες της αντιγραφής και της μεταγραφής. Επίσης, ο ρόλος της TOP1 μελετήθηκε και σε κύτταρα ασθενών SCAN1 με μεταλλαγμένη κ μη λειτουργική TDP1 πρωτεϊνη, τυροσινική φωσφοδιεστεράση, η οποία αποτελεί μέρος του μηχανισμού απομάκρυνσης των TOP1cc. Στo δεύτερο εγχείρημα εστιάσαμε στον παράγοντα FACT (FAcilitates Chromatin Transcription) και στον ρόλο του στην επανάκαμψη της μεταγραφής μετά από βλάβη που έχει προκληθεί από υπεριώδη (UltraViolet- UV) ακτινοβολία.