Role of NFKBIA Deficiency in Subverting Neural Progenitor Commitment Toward Gliomagenesis - Gliomas arise from neural progenitor cells (NPCs) or their descendants that derive transcriptional identity from aberrant chromatin landscapes. Polycomb repressive complex 2 (PRC2) is a crucial chromatin modifier that orchestrates gene silencing and organizes genome architecture during lineage specification and commitment by depositing repressive histone H3K27 trimethylation marks. While mechanisms that alter PRC2 chromatin occu- pancy are understudied, they hold significant conceptual appeal due to the determinative role of PRC2 in gov- erning the fate of NPCs. We have shown that IκBα deletions frequently occur in diffuse gliomas, portend poor prognosis, reshape the DNA and histone methylome antipodal to the IDH mutation, and induce a transcriptome landscape partly resembling that caused by PRC2 loss-of-function in H3K27M mutant pediatric gliomas. IκBα is the main regulator of nuclear factor-κB (NF-κB), but we found phosphorylated-SUMOylated (ps-)IκBα exerts an alternative chromatin-regulatory function by dynamically binding histones H2A and H4, thereby recruiting PRC2 and priming lineage choice. The scientific premise of this proposal is grounded in our discovery that IκBα exerts a novel PRC2-regulatory role in glioma and progenitor cells, which is independent of NF-κB. Defining the PRC2 vs. NF-κB-dependent roles of IκBα is pivotal for understanding their malfunction during lineage specification and gliomagenesis. Our central hypothesis is that IκBα recruits PRC2 to active chromatin and thus controls critical lineage programs that are aberrant in gliomas. Our specific aims are: 1. Define the Contribution of Chromatin vs NF-κB Specific IκBα Functions in Neural Lineage Specification. We will use human induced pluripotent stem cell (iPSC)-derived IκBα-deficient (CRISPR/Cas9 RNP DIκBα)/Tet-On:IκBα-SOF (SOFDNF-κB vs. SOFDH2A/H4) tripotent wildtype iNPCs and their lineage-committed progeny to determine whether, independent of NF-κB, chromatin-bound ps-IκBα regulates chromatin architecture and modulates the transcription of developmental genes during neural lineage specification and commitment. 2. Define the Mechanism of Dynamic PRC2 Reg- ulation by IκBα. We will utilize wildtype iNPCs with DIκBα/Tet-On:SOFDNF-κB vs. DIκBα/Tet-On:SOFDH2A/H4 geno- types to define the dynamics of IκBα binding to PRC2-regulated chromatin regions and determine its impact on recruitment and assembly of PRC2 subcomplexes (PRC2.1/PRC2.2), characterized by distinct accessory pro- teins, which differ in their extent of poised lineage-specific gene silencing. 3. Define the Consequences of IκBα Editing on Fate-Switches and Gliomagenic Trajectories from NPC to GBM. We will use a gliomagenic (DCDKN2A/B-PTEN-TERTmut) derivative of the iNPC model genome-edited to carry mutations governing vari- ous iGBM subtypes (EGFRvIII/PDGFRAdel8/9/DNF1) and GBM PDXs, to define the impact of DIκBα/Tet-On: SOFDNF-κB vs. DIκBα/Tet-On:SOFDH2A/H4 on chromatin and PRC2 regulation, multi-step reprogramming, and fate- transitions driving gliomagenic trajectories from NPCs to GBM. Our studies will comprehensively elucidate the molecular dynamics of lineage-specific chromatin regulation by IκBα and its subversion during gliomagenesis.
