The role of NSD2-NEO1 axis in CNS infiltration of acute lymphoblastic leukemia - PROJECT SUMMARY/ABSTRACT Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer and a leading cause of cancer-related mortality in children due to relapse, particularly involving central nervous system (CNS) infiltration. CNS leukemia presents significant challenges, as leukemic cells migrate to the brain and evade treatment, leading to relapse. Current CNS-directed treatments, including prophylactic chemo- and radiotherapy, are associated with limited efficacy and substantial long-term toxicities. Thus, there is an urgent need to understand the mechanisms driving CNS infiltration in ALL and develop targeted therapies with reduced toxicity. Histone methyltransferase NSD2 is frequently mutated in relapsed pediatric ALL and is associated with poor outcomes. Our preliminary data suggest that the most prevalent NSD2 mutation, p.E1099K, is strongly correlated with CNS infiltration in ALL, yet the mechanisms remain poorly understood. NSD2 catalyzes histone modification H3K36me2, leading to widespread epigenetic reprogramming. The NSD2 p.E1099K mutation, which occurs in the catalytic domain, drives aberrant chromatin remodeling, gene expression, and leukemic transformation. NSD2 mutations increase leukemia cell adhesion and migration and promote neurotropism and relapse. Notably, multi-omics analyses have identified neogenin-1 (NEO1) as a critical downstream target of NSD2 mutations. NEO1, a cell adhesion and neuron related molecule, is significantly overexpressed in NSD2 mutant ALL cells and contributes to CNS infiltration. Knockdown of NEO1 reduces CNS infiltration, paralysis, and leukemia burden, underscoring its critical role in NSD2 mutation-driven CNS infiltration. Importantly, a novel NSD2 inhibitor (EX-A5782) reverses NSD2 mutation driven epigenetic reprogramming, reduces NEO1 expression, and significantly delays CNS infiltration in preliminary studies. Here, we hypothesize that NSD2 mutations promote CNS infiltration and relapse in ALL by inducing epigenetic reprogramming that enhances adhesion, migration, and neurotropism through the NSD2- NEO1 axis. Targeting this pathway will prevent CNS infiltration and improve outcomes. We propose three aims to test our hypothesis in cell lines and mouse models (CD45.1 mice for mouse cell transplantation and immunodeficient mice for xenograft experiments): 1) Elucidate the cellular and molecular mechanisms underlying NSD2 mutation-driven CNS infiltration. Using CRISPR/Cas9-edited isogenic cell lines, cell-derived xenografts (CDX), patient-derived xenografts (PDX), and conditional NSD2 p.E1099K mouse models, we will define how NSD2 mutations alter chromatin architecture, gene expression, and cellular behavior to drive CNS infiltration. 2) Determine the role of NEO1 in leukemia cell growth and CNS infiltration. By manipulating NEO1 expression in NSD2 mutant and wild-type cells, we will evaluate its function and NEO1-mediated interactions with the Blood- Brain Barrier in CNS infiltration. 3) Evaluate the therapeutic potential of targeting the NSD2-NEO1 axis to prevent CNS infiltration in established models. By targeting the NSD2-NEO1 axis, this study addresses a critical unmet need in relapsed ALL therapy, offering a novel strategy to prevent CNS infiltration and improve patient outcomes
