Enhancement of antigen presentation restores immune recognition in rhabdomyosarcoma

Background : Immunotherapy offers a promising therapeutic option against pediatric tumors, but it is limited by suppression of the antigen processing machinery (APM), including major histocompatibility complex class I (MHC-I). In this study, we quantify antigen presentation in pediatric tumors and explore the role of transcriptional regulators and epigenetic-targeted therapies to restore antigen presentation and immune recognition in rhabdomyosarcoma (RMS). Methods : Expression of HLA-A/B/C was quantified by RNA sequencing (RNA-seq) in adult tumors (n=657) and pediatric cell lines (n=131). Surface MHC-I expression was quantified by flow cytometry in pediatric cell lines (n=76). RMS cell lines (n=9) were treated with IFN-gamma and clinically relevant drugs, decitabine (DAC), mocetinostat, and tazemetostat. Changes in MHC-I and APM gene expression were measured by RNA-seq and flow cytometry. NLR family CARD domain-containing 5 (NLRC5) was induced in RMS cell line and patient derived xenograft (PDX) models using lentiviral overexpression or CRISPR activation. MHC-I and APM expression were measured by RNA-seq, flow cytometry, and western blot. T cell cytotoxicity assays were performed with T cells expressing a PRAME-specific HLA-A*02:01 T cell receptor (TCR). Results : Pediatric tumors exhibited variable MHC-I expression, as determined by flow cytometry, and this expression was significantly lower than adult tumors, with RMS displaying low or absent expression. IFN-gamma and pharmacologic treatment increased MHC-I surface expression and immune gene signatures in RMS. NLRC5, a key immune regulator, was found to be most significantly correlated with MHC-I expression and induced by treatment with DAC. Epigenetic priming with DAC and upregulation of NLRC5 was sufficient to restore MHC-I expression and sensitized an RMS PDX to killing by engineered TCR-T cells targeting PRAME. Conclusions: Pediatric tumors show distinct patterns of antigen presentation, such as high MHC-I in alveolar soft part sarcoma and low expression in RMS, though individual tumor subtypes exhibit internal variability. IFN-gamma and epigenetic agents restore antigen presentation, including increased NLRC5 expression. Pharmacological treatment and NLRC5 restoration enhanced antigen presentation and sensitized RMS PDX to TCR mediated T-cell cytotoxicity. Compounds that reverse APM silencing will be systematically evaluated for their ability to enhance adoptive TCR therapies. This work will establish a foundation for overcoming immune resistance and expanding the impact of MHC-I dependent cancer immunotherapies.