OP19. MANIPULATING GLIOBLASTOMA INVASION USING LIGHT-INDUCIBLE GENOME ENGINEERING
Authors: Ms Pamela Dominguez Perez, Mr Jamie Wood, Dr Vinoj George, Dr Ruman Rahman
Journal: Neuro-Oncology (2017)

BACKGROUND: Glioblastoma Multiforme (GBM) is the most common and malignant primary tumour of the Central Nervous System (CNS), with no effective current treatment options available. Median overall survival after radical surgical resection with intensive adjuvant radiotherapy and temozolomide is just 15 months. As a result, there remains a critical need for the development of new treatment strategies. Tumour invasion is a characteristic GBM phenotype amenable for therapeutic targeting, as infiltrative residual disease ultimately results in tumour recurrence. However the molecular factors and signalling pathways that control and regulate the invasive phenotype are poorly understood. In considerable part, this has been due to an absence of precision genetic tools to modulate that transcription of endogenous genes and emergent phenotypes. Recent studies have linked the invasive and migratory capacity of GBM to the repression of PIM1 kinase. Here we manipulate PIM1 through an optogenetic system using a combination of CRISPR-dCas9 technology and light-inducible heterodimerizing proteins CRY2 and CIB1. OBJECTIVE: To assess GBM invasiveness in vitro through optogenetic transcriptional repression of PIM1. MATERIALS AND METHODS: In order to regulate PIM1 expression, LITEs (Light-inducible transcriptional effectors) were used by combining two modular components: (1) a genomic anchor (a guide RNA based on dCas9) fused to CIB1 (interacting partner of CRY2), and (2) the light-sensitive cryptochrome 2 (CRY2) protein fused to the repression domain CRAB. Guide RNAs were tested for optimal efficiencies using the NanoLuc® luciferase reporter assay. The technology involves exposure of optogenetically-modified U87 GBM cells to an LED light source, with a 466nm blue light filter at an intensity of 5mW/cm2 at 0.016 Hz for up to 24h in an incubator at 37ºC and 5% CO2. Once PIM1 activity was conditionally repressed via read-out of GFP, tests for migratory and invasion capacity were conducted through invasion assays based on the Boyden chamber principle. RESULTS: Induction or repression of PIM1 endogenous transcription in U87 cells occurred within minutes of light exposure and the response could be theoretically graded with light dose, with the induction being fully reversible after light retraction. The effects on U87 invasive and migratory capacity upon transcriptional repression of PIM1 will be discussed. CONCLUSIONS: The coordination of dCas9 efficiency and LITE system benevolence, proved to be a very successful and manageable combination, creating a system with multiple usages that will have an impact, not only on scientific research, but also in clinical work. In this project we focused on the study of PIM1’s role in GBM invasion and migration in vitro. Further bioinformatic and molecular research concerning PIM1 co-expression networks might help to elucidate invasion pathways, that will ultimately help to discover putative treatment targets and/or genetic biomarkers that predispose for GBM invasiveness.