An AI-Enabled Single-Cell Transcriptomic Analysis Pipeline for Gene Signature Discovery in Natural Killer Cells Linked to Remission Outcomes in Chronic Myeloid Leukemia

BACKGROUND: A major technical challenge in single-cell transcriptomics is the absence of an integrative analytic pipeline that can simultaneously leverage gene regulatory network (GRN) architecture, AI-assisted gene panel discovery, and functional relevance analyses to generate coherent biological insights. Existing approaches often treat these components independently, focusing on clusters, marker genes, or predictive features without integrating them into a mechanistically grounded framework. Consequently, comprehensive screening that links regulatory association, gene signature screening, and functional interpretation within single-cell datasets remains limited, underscoring the need for an integrated strategy. METHODS: We developed an integrative bioinformatics pipeline based on Gene regulatory network-AI-Functional Analysis (GAFA), combining latent-space integration, unsupervised clustering, diffusion pseudotime analysis, lineage-resolved generalized additive modeling, GRN inference, and machine learning-based gene panel discovery. This framework enables systematic mapping of cell-state structure, reconstruction of differentiation and effector trajectories, and identification of transcriptional and regulatory features strongly associated with clinical outcomes. As a case study, we applied the pipeline to NK cell transcriptomes from six CML patients (two early relapse, two late relapse, two durable treatment-free remission-TFR; 15 samples) collected at TKI discontinuation and 6-12 months after therapy cessation. RESULTS: regulons were enriched in relapse, and their downstream targets linked to IFN-γ signaling, metabolic reprogramming, and immunoregulatory feedback circuits. CONCLUSIONS: This AI-enabled single-cell analysis demonstrates how NK cell state composition, differentiation trajectories, and regulatory network rewiring collectively shape TFR versus relapse following TKI discontinuation in CML. The integrative pipeline provides a modular framework that could be extended to additional datasets for data-driven biomarker discovery and mechanistic stratification, and highlights candidate transcriptional regulators and NK cell programs that may be leveraged to improve remission durability, pending validation in larger patient cohorts.