High-Throughput Targeted Drug Screening for NF1-associated High-Grade Gliomas with ATRX Deficiency.

Neurofibromatosis type 1 (NF1)-associated high-grade gliomas (HGGs) harboring ATRX mutations exhibit an aggressive clinical phenotype, driven by heightened genomic instability and metabolic reprogramming. Existing therapies, including chemotherapy and radiotherapy, are limited by resistance mechanisms and formation of secondary malignancy, underscoring the need for novel therapeutic strategies. Here, we report the results of a high-throughput screening of 10,000 small molecules aimed at identifying compounds selectively targeting vulnerabilities associated with concurrent ATRX and NF1 loss. Among the screened compounds, K784-6195 (ChemDiv ID) emerged as a promising candidate, exhibiting marked selective cytotoxicity in NF1-associated glioma cell lines with ATRX deficiency (IC50 = 4.84 μM). In comparison, wild-type ATRX sporadic glioma cell lines (U251) exhibited significantly reduced sensitivity to K784-6195 (IC50 = 37.03 μM). However, ATRX knockout U251 glioma cells recapitulating concurrent ATRX and NF1 loss exhibited heightened susceptibility to K784-6195 (IC50 = 20-23 μM) compared to their wild-type counterpart. Metabolomic analysis revealed that K784-6195 treatment impairs metabolic pathways, including the pentose phosphate pathway, glutamine metabolism, and redox homeostasis, leading to oxidative stress and impaired cell survival. These findings highlight K784-6195 as a promising candidate for therapeutic development, offering a targeted approach for the treatment of NF-1 associated HGGs with ATRX deficiency.