Fig. 5: Single-cell RNA sequencing of xenografts reveals persistance of miR-126^high LSCs with enhanced transcriptional features of stemness upon in vivo chemotherapy.

A UMAP plot of day 8 BM AML blasts from treated or control PDX, colored by unsupervised clustering at resolution = 0.6. Dashed lines show manual annotation of clusters. n = 96,931 cells from 35 PDX from 4 patients. B UMAP density plots of AML blasts from control (top, black) or treated (bottom, red) xenografts. C UMAP density plots of sorted GFP^low/miR-126^high blasts from control (top) or treated (bottom) xenografts. D 126High signature expression in scRNAseq AML blasts from control (top) or treated (bottom) xenografts. E Enrichment (right) or depletion (left) of scRNAseq cluster abundance in treated AML compared to the average abundance of each cluster in control xenografts from the same donor. n = 35 PDX from 4 patients over 4 independent experiments. Data are presented as standard Tukey boxplots (center line: median, box: interquartile range, whiskers: IQR*1.5). F Ridge plot of 126High signature expression across cells in scRNAseq from treated (red) or control (grey) xenografts. Single-cell events are displayed in the underlying rug plots. G Tile plot of normalized enrichment scores (NES) from GSEA of hallmark terms on DEGs between blasts from treated vs. control xenografts within the indicated clusters (see Fig. 5A for unsupervised clustering). Hallmarks (rows) are grouped by semantic similarity. Columns are ordered by decreasing 126High expression. Non-significant enrichment results are plotted in light grey (Benjamini–Hochberg adjusted p-value > 0.1). H Same as G but for senescence-associated gene signatures. I Percent of day 8 EdU-incorporating BM blasts within sorted GFP^high or GFP^low AML cells from treated PDX. n = 16 PDX from 3 patients over 3 independent experiments. Comparisons between paired groups were performed with the two-sided paired Wilcoxon test. For PT01 and PT03 (n ≤ 5 with a minimum two-sided p-value=0.0625), a bootstrap sampling-based nonparametric test was employed and p-values are marked in italics. n = 16 PDX from 3 patients over 3 independent experiments. Data are presented as mean ± SD. J Percent of G0 GFP^low/miR-126^high blasts at baseline (B/L) or after in vivo chemotherapy (Treat). n and statistical comparisons and data presentation as per panel I. K Volcano plot of DEGs between treated vs. control in cluster 1 AML blasts. Top 12 genes by log2FC with increased expression in treated blasts are labelled (red). L Enrichment (right) or depletion (left) of LSC subclusters in treated AML compared to the average abundance of each cluster in control xenografts from the same donor. n = 35 PDX from 4 patients over 4 independent experiments. Standard Tukey boxplots: center line: median, box: interquartile range, whiskers: IQR*1.5. M Expression of top 10 marker genes for LSC subclusters 5′, 6′, 19′ and of HSC-latency-associated genes in LSC subclusters. Dot size reflects percentage of cells expressing the queried gene (columns), color scale represents relative gene expression within the subclusters (rows). N Longitudinal evaluation of AML engraftment within mice transplanted with PT16 AML blasts treated ex-vivo with an OxPhos inhibitor (IACS-010759) or solvent (Mock-treated). n = 6 PDX from 1 patient over 1 experiment. Source data are provided as a Source Data file.