Abstract B027: TNKS–NOTCH signaling regulates the Developmental state in glioblastoma stem cells

Abstract Glioblastoma (GBM) is the most common form of primary brain malignancy in adults. It is highly aggressive, and patients present a median survival of only 12 months. Treatment consists of surgery and chemotherapy, which are insufficient to prevent tumor recurrence. Resistance to therapy is largely attributed to the pronounced cellular heterogeneity of GBM. Glioblastoma stem cells (GSCs) exist along a transcriptional gradient between two cellular states: Developmental and Injury Response programs. Cells closer to the Developmental state rely primarily on neurodevelopmental transcription factors, while cells closer to the Injury Response state depend on genes related to inflammation, cholesterol metabolism, and integrin signaling. To study cellular heterogeneity and plasticity in GSCs, we are using precision genome-engineering approaches to develop reporter alleles for state-specific markers, and we are applying these alleles to perform phenotypic genome-wide CRISPR screens. Our goal is to identify the genetic networks that maintain each transcriptional state and to uncover vulnerabilities with therapeutic potential. Here, we used CRISPR/Cas homology-mediated end joining to tag OLIG2, a marker of the Developmental State, with a 3’ 2A-GFP cassette in a patient-derived GSC line. We focused on G523 cells, which correspond to a Developmental GSC subtype. G523-OLIG2-2A-GFP cells were subjected to a genome-scale phenotypic CRISPR screen, and populations with high and low GFP expression were isolated to identify positive and negative regulators of OLIG2 expression. Because TNKS influences cell proliferation, we designed an RNA-seq experiment that controlled for this effect by co-culturing G523 wild-type cells with G523 TNKS knockout (KO) mCherry-labeled cells. After 8 days, mCherry-positive and -negative populations were sorted and analyzed by RNA-seq. The results demonstrated upregulation of NOTCH target genes and downregulation of OLIG2 in TNKS KO cells. Interestingly, TNKS KO cells exhibited a decrease in the Developmental program and an increase in the Injury Response program. This preliminary data suggests that TNKS plays a role in GSC state transitions and may contribute to therapy evasion. Consistently, G523 cells treated with NOTCH inhibitor showed an increase in OLIG2 levels, supporting a NOTCH-dependent regulation of OLIG2. Ongoing studies aim to determine whether this regulation is direct or indirect. Elucidating how TNKS regulates OLIG2 will provide insight into novel therapeutic targets. In addition, since we are also investigating the Injury Response side of the gradient, the results generated here are going to help us to comprehend the GSC plasticity process, which can also be explored as a therapeutic strategy. Citation Format: Maira Almeida, Graham MacLeod, Stephane Angers. TNKS–NOTCH signaling regulates the Developmental state in glioblastoma stem cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Brain Cancer; 2026 Mar 23-25; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86(6_Suppl):Abstract nr B027.