Integrative single-cell and bulk transcriptomic analysis identifies a non-LSC17 companion program associated with LSC17-high acute myeloid leukemia

The LSC17 score reflects leukemia stem cell related transcriptional activity in acute myeloid leukemia (AML), but the single-cell basis and non-LSC17 programs accompanying bulk LSC17-high disease remain unclear. We integrated single-cell RNA-seq data from the 16 diagnosis-timepoint (D0) AML samples in GSE116256 with TCGA-LAML and two external AML datasets. LSC17 activity was mapped across the single-cell atlas. Primitive-like cells were stratified within each eligible patient, defining the upper and lower 20% of LSC17 scores as LSC17-high and LSC17-low states. Patient-aware pseudobulk analysis, enrichment analysis, and single-cell to bulk comparison were used to identify non-LSC17 genes associated with LSC17-high primitive-like cells. LSC17 activity was enriched in Primitive/Progenitor-like compartments, particularly HSC-like and primitive leukemic-like clusters. Among 5,124 primitive-like cells from 16 patients, 1,024 LSC17-high and 1,024 LSC17-low cells were identified; 14 evaluable patients contributed 1,018 cells per state to the paired pseudobulk analysis. Pseudobulk analysis detected 68 differentially expressed genes. LSC17-high cells were enriched for interferon alpha response, inflammatory response, and KRAS signaling, whereas LSC17-low cells were enriched for MYC targets, oxidative phosphorylation, and E2F targets. After excluding original LSC17 genes, 17 single-cell LSC17-high-upregulated genes overlapped with bulk LSC17-high-upregulated genes, from which an eight-gene high companion core (SPINK2, HOPX, MEF2C, MSI2, BCAT1, ANGPT1, NPR3, and CD52) was manually curated. The score was elevated in LSC17-high primitive-like cells, correlated with the LSC17 score in TCGA-LAML, associated with poorer overall survival, and reproduced in GSE37642-GPL96 and GSE12417-GPL96. However, prognostic discrimination in the external datasets was modest, and the companion score is not proposed as a standalone clinical predictor. This study identifies a single-cell-informed non-LSC17 companion program associated with LSC17-high primitive-like AML states and adverse survival.