SLFN5 restrains type I interferon responses and promotes glioblastoma via its N-terminal Schlafen core domain.

Schlafen 5 (SLFN5) is an important regulator of Type I interferon (IFN-I) signaling and tumorigenesis, yet the structural basis for its biologic activity remains unclear. Here, we dissect functional domains of SLFN5 and uncover a key role for its N-terminal Schlafen core domain in IFN-stimulated gene (ISG) expression and glioblastoma proliferation, independently of the SWAVDL motif and the C-terminal Walker A/B motifs. Partial deletions within the core domain or substitution of two arginine residues (R271E/R326E) near the zinc finger motif disrupt chromatin association and phenocopy SLFN5 loss, including de-repression of ISGs that act as immune checkpoints, such as PD-L1 and PD-L2. In contrast, the C-terminal ATPase/helicase activity is dispensable for transcriptional repression and tumor-promoting functions. Structural comparison of human and mouse SLFN5 revealed a conserved architecture, and experimental evidence demonstrates that murine SLFN5 is a functional ortholog of human SLFN5. These findings establish SLFN5 as a key transcriptional modulator of malignant glioblastoma growth by dampening IFN-I responses and have implications for the development of future immunotherapy approaches.