Fig 2: RUNX1-ETO Induction Leads to Cell-Type and Dose-Dependent Changes in Gene Expression(A) Clustering of gene expression data for sorted populations of RUNX1C- and RUNX1C+ (CD45+CD34+) cells, both wild-type and after 24 h of RUNX1-ETO induction using 5 ng/mL Dox. The figure includes all genes that showed up-/downregulation after RUNX1-ETO induction in either the RUNX1C+ or the RUNX1C- cell populations.(B) Heatmap after KEGG pathway analysis depicting clustering of differentially expressed genes associated with KEGG terms upon RUNX1-ETO induction (5 and 10 ng/mL Dox) in sorted populations of both RUNX1C- and RUNX1C+ cells. Red intensity reflects the enrichment significance of the terms in -log10 (q value).(C) Gene set enrichment analysis (GSEA) correlating expression of genes differentially regulated during the cell cycle (G2/M in top panels and S phase in bottom panels) between induced (5 ng/mL Dox for 24 h) and uninduced conditions in both RUNX1C+ and RUNX1C- cell populations. ES, enrichment score; NES, normalized enrichment score; FDR, false discovery rate.(D) Bar graph depicting differentially expressed genes between uninduced and Dox-treated (3, 5, and 10 ng/mL Dox) sorted populations of CD45+CD34+RUNX1C+ cells.(E) Examples of individual genes differentially regulated in CD34+RUNX1C+ progenitors in response to RUNX1-ETO induction (3, 5, and 10 ng/mL Dox) for 24 h. n = 3. Each colored dot represents a distinct biological replicate.

Fig 3: Analysis of the Interplay between RUNX1 and RUNX1-ETO(A) Average profiles for RUNX1 ChIP-seq tag counts centered on all RUNX1 binding peaks (±1,000 bp from peak center) in the 0, 5, and 10 ng/mL Dox-treated samples.(B) Genome browser screenshot at the RASSF5 gene locus depicting RUNX1, HA-RUNX1-ETO, and H3K27ac ChIP-seq and ATAC-seq tracks for the indicated samples.(C) Comparison of chromatin accessibility in RUNX1C+ cells (0- and 5-Dox-treated samples) to myeloid progenitor cell types from Corces et al. (2016). Heatmaps show ATAC-seq tag counts ranked by fold difference between 0- and 5-Dox-treated RUNX1C+ samples. ATAC-seq tag counts from distinct myeloid progenitor cell types (Corces et al., 2016) are ranked along the same coordinates as the 0-Dox ATAC-seq peaks. Color intensity reflects tag counts per million, with light blue representing closed chromatin.(D) RUNX1-ETO-induced hESC-derived progenitors share a t(8;21) AML-specific gene expression profile with t(8;21) AML patients. GSEA correlating upregulated (left panel) and downregulated (right panel) RUNX1-ETO target genes between CD45+CD34+RUNX1C+ cells following 24-h RUNX1-ETO induction (5 ng/mL Dox) and the gene expression profile of the RUNX1-ETO targets in t(8;21) patients.

Fig 4: Immunohistological characteristics of case 1, a responder to ALK inhibitors(A) Immunohistochemical analysis of lung cancer tissue. a, Hematoxylin and eosin staining (100×); b, positive staining for p40 (100×); c, negative staining for TTF-1 (100×). (B) Diagnosis of ALK rearrangement. a, FISH analysis. Nuclei show a split positive pattern with separation between the 5' ALK green part (small arrow) and 3' ALK orange part (large arrow) of the FISH probe signal, indicating ALK rearrangement. Close apposition of the 3'/5' ALK parts (arrowhead) can be seen as a pseudo-colored (yellow) signal. b, Immunohistochemical analysis of ALK protein expression in tumor cells; ALK protein was overexpressed (IHC score 3) (100×). (C) Immunohistochemical analysis of off-target markers for ALK inhibitor resistance by rebiopsy of lung cancer tissue at the time of relapse after previous ALK inhibitor treatment. a, Positive staining for EGFR (150×); b, positive staining for vimentin in cancer cells surrounded by cancer stroma (arrowheads) (150×). (D) EMT-CTCs detected by liquid biopsy at the time of relapse after previous ALK inhibitor treatment. CTCs were positive for GFP using the telomerase-specific replication-selective adenovirus TelomeScan. False-positive cells were identified based on CD45 expression status. EMT CTCs were identified as GFP+/CD45-/EpCAM-/vimentin+. (E) ALK-rearranged CTCs identified by liquid biopsy at the time of relapse after previous ALK inhibitor treatment. CTCs (GFP+/CD45-) in blood samples with confirmed ALK rearrangement were subjected to FISH analysis. Close apposition of the 3' ALK orange part (arrow) and 5' ALK green part (arrow head) of the FISH probe signal indicates an intact wild-type copy of ALK in the cells.

Fig 5: Induction of RUNX1-ETO in the CD45+CD34+RUNX1C+ Population Results in the Emergence of a New Cell Population(A) Diagram of the sorting strategy for scRNA-seq performed following 24-h induction of RUNX1-ETO (5 ng/mL Dox at day 21).(B) Two-dimensional t-distributed stochastic neighbor embedding (t-SNE) maps displaying 3,321 (left) and 3,814 (right) sorted populations of CD45+CD34+RUNX1C+ single cells following 0 and 5 ng/mL Dox treatment, respectively. Colors represent the different clusters identified after RaceID analysis.(C) Pie charts displaying the proportion of cells in each cell-cycle phase (G1, G2-M, and S) within each cell cluster as identified by expression of cell-cycle-regulated genes, such as histone genes.(D) Expression of individual marker genes projected on the t-SNE maps of both untreated (0 Dox) and treated (5 Dox; 5 ng/mL for 24 h) scRNA populations. Color intensity represents number of transcripts sequenced in log2 of unique molecular identifier (UMI) counts +1.