Immunoglobulin Superfamily Protein BTNL9 Functions as a Non-Canonical Transcriptional Regulator to Suppress NSCLC Through Cell Cycle and p53 Pathways

Butyrophilin-like 9 (BTNL9), a member of the immunoglobulin superfamily containing a bZIP-like domain, has a poorly defined role in cancer. Here, we identify BTNL9 as a non-canonical transcriptional regulator and investigate its function in non-small cell lung cancer (NSCLC). Coiled-coil prediction, native PAGE, and co-immunoprecipitation demonstrated BTNL9 homodimerization via its bZIP-like region, while subcellular fractionation and immunofluorescence confirmed its presence in both the nucleus and cytoplasm. Chromatin immunoprecipitation sequencing (ChIP-seq) analysis identified 9709 BTNL9-associated genomic regions, including sites proximal to transcription start sites, with enrichment of a cytosine-rich motif. Whether chromatin association reflects direct DNA binding or indirect co-regulatory interaction remains to be experimentally confirmed. Integrated transcriptomic and protein analyses revealed that BTNL9 overexpression represses genes involved in cell cycle progression and DNA replication while activating a subset of p53-associated pathways. Consistently, functional assays showed that increased BTNL9 expression induces cell cycle arrest, suppresses proliferation and clonogenicity, and inhibits tumor growth in xenograft models. In addition, cytotoxicity assays demonstrated enhanced sensitivity to bortezomib, with context-dependent effects on etoposide response. Analysis of public clinical datasets further showed that low BTNL9 expression is associated with advanced tumor stage, reduced remission rates, and poorer survival outcomes in NSCLC. These findings identify BTNL9 as a non-canonical tumor-suppressive transcriptional regulator with potential biomarker relevance in NSCLC.