Characterisation of the Notch signalling small molecule inhibitor RIN1

master's thesis

• Title (croatian): Karakterizacija malomolekulskog inhibitora RIN1 signalnog puta Notch
• Author: Ivica Biljan
• Mentor: Franz Oswald (mentor)
• Mentor: Nada Oršolić (komentor)
• Committee member: Nada Oršolić (predsjednik povjerenstva)
• Committee member: Biljana Balen (član povjerenstva)
• Committee member: Ana Galov (član povjerenstva)
• Committee member: Jasna Lajtner (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2023-07-19, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

The Notch pathway is essential for embryonic development and tissue homeostasis, with
transcription factor RBPJ’s central dual repressor/activator role. Aberrant Notch signaling
contributes to the pathogenesis of many diseases and is consequently an important target of
therapeutic inhibition which particularly pertains to RBPJ that is targeted by novel small molecule
inhibitors like RIN1 and CB-103. To get more detailed insights into their molecular mechanisms
and potential off-target effects, luciferase assays were performed on various transcription factor
combinations. Firefly luciferase enzyme itself was inhibited by CB-103, while RIN1 conclusively
inhibited only Gal4 and HNF6 DNA binding. Moreover, to analyze potential nuclear export or
inhibition of nuclear import of RBPJ by inhibitors, immunofluorescence assay was performed
which showed no interference with RBPJ’s nuclear localization. Finally, Gene Set Enrichment
Analysis was carried out on publicly available RNA sequencing data from inhibitor treated Jurkat
T-cell line to get insights to in-depth genome-wide pattern in gene set expression changes. Both
RIN1 and CB-103 exhibited changes in regulating a much higher number of gene sets compared
to RBPJ knock-down by siRBPJ, and γ-secretase inhibitor DAPT, indicating less specific mode of
action and/or more elaborate signal integration pathways than what is currently known from the
literature.

Abstract (croatian)

Notch signalni put je ključan u embrionalnom razvoju i homeostazi tkiva, sa središnjom dvojnom
represorsko-aktivatorskom ulogom transkripcijskog faktora RBPJ. Aberantna signalizacija Notch
puta doprinosi patogenezi mnogih bolesti te je posljedično važna meta terapijske inhibicije što se
posebice odnosi na RBPJ na koji ciljano djeluju malomolekulski inhibitori poput RIN1 i CB-103.
Kako bi se dobio detaljniji uvid u njihove molekularne mehanizme kao i off-target učinke,
provedeni su luciferazni testovi na raznim kombinacijama transkripcijskih faktora. CB-103 doveo
je do inhibicije samog enzima Firefly luciferaze, dok je RIN1 doveo do definitivne inhibicije Gal4
i HNF6 DNA vezanja. Nadalje, kako bi se analiziralo potencijalno izbacivanje ili inhibicija
jezgrinog unosa RBPJ od strane inhibitora, proveden je test imunofluorescencije koji je pokazao
da nema interferencije sa lokalizacijom RBPJ u jezgri. Posljednje, provedena je analiza obogaćenja
genskih setova na javno dostupnim podacima RNA sekvenciranja iz T-stanične linije Jurkat
tretirane inhibitorima kako bi se dobio uvid u detaljne genomske obrasce promjena ekspresije
genskih setova. RIN1 i CB-103 pokazali su promjene u kontroli većeg broja genskih setova u
usporedbi sa RBPJ knock-down djelovanjem siRBPJ te γ-sekretaznim inhibitorom DAPT što
ukazuje na manje specifičan mehanizam djelovanja i/ili mnogo kompleksniju integraciju signalnih
puteva nego što je trenutno poznato iz literature.

• Keywords: Notch signaling pathway; RIN1; CB-103
• Keywords (croatian): signalni put Notch; RIN1; CB-103
• Language: english
• URN:NBN: urn:nbn:hr:217:324487
• Study programme: Title: Experimental Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra eksperimentalne biologije (magistar/magistra eksperimentalne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2023-07-21 09:47:10