Figure 4.
Coupling single cell RNA-sequencing (scRNA-seq) with cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) on AML PDX cells reveals an increased proportion of LSCs and identifies genes/pathways upregulated by GADD45A deletion at a single stem cell level. (A) t-distributed stochastic neighbor embedding (T-SNE) clustering of CITE-seq data showing human CD34+ LSC-enriched subpopulations (in red) with GADD45A KO (n = 1511 cells, 19.6%) vs CRISPR Ctr (n = 542 cells, 8.7%) in the bone marrow (BM) of PDX mice. Cutoff: minimum = q85 and maximum = q97 (q stands for quantile). (B) Heat map of integrated CITE-seq data analysis identifying 43 differentially expressed genes (DEGs) upregulated in GADD45A KO PDX LSCs (n = 233 cells), compared with CRISPR Ctr LSCs (n = 110 cells). The LSC compartment (CD34+CD38–CD45RA+CD90–) was defined by a stringent cutoff of CD34 > q85, CD38 < q50, CD45RA > q65, and CD90 < q65. Wilcoxon rank-sum test, fold change > 1.4, and adjusted P value with Benjamini-Hochberg method < 0.05. (C) Bar chart showing the top 9 enriched cancer-associated pathways from MSigDB oncogenic signatures, along with their corresponding P values and associated DEGs. (D) Scatter plot showing Gene Ontology (GO) function enrichments of the DEGs upregulated in GADD45A KO LSCs, compared with CRISPR Ctr LSCs. Clusters were computed using the Leiden algorithm, and similar gene sets were clustered together. Larger, black-outlined points represent significantly enriched terms. Points are plotted on the first 2 UMAP dimensions. The table lists enriched gene sets with P < 8.0e-04 and associated DEGs. (E) Fast preranked gene set enrichment analysis (FGSEA) of CITE-seq data (cutoff: adjusted P < 0.05 and NES > 1.8) identifying GADD45A loss-induced enrichment of gene sets associated with ROS sensing by NFE2L2, detoxification of ROS, cellular response to chemical stress, and iron uptake and transport, on GADD45A knockout in human AML PDX cells. (F) Kaplan-Meier curves of overall survival for 172 patients with AML, as stratified by expression levels of FTH1 (P = .0039) and PRDX1 (P = .019) in TCGA dataset.

Coupling single cell RNA-sequencing (scRNA-seq) with cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) on AML PDX cells reveals an increased proportion of LSCs and identifies genes/pathways upregulated by GADD45A deletion at a single stem cell level. (A) t-distributed stochastic neighbor embedding (T-SNE) clustering of CITE-seq data showing human CD34+ LSC-enriched subpopulations (in red) with GADD45A KO (n = 1511 cells, 19.6%) vs CRISPR Ctr (n = 542 cells, 8.7%) in the bone marrow (BM) of PDX mice. Cutoff: minimum = q85 and maximum = q97 (q stands for quantile). (B) Heat map of integrated CITE-seq data analysis identifying 43 differentially expressed genes (DEGs) upregulated in GADD45A KO PDX LSCs (n = 233 cells), compared with CRISPR Ctr LSCs (n = 110 cells). The LSC compartment (CD34+CD38–CD45RA+CD90–) was defined by a stringent cutoff of CD34 > q85, CD38 < q50, CD45RA > q65, and CD90 < q65. Wilcoxon rank-sum test, fold change > 1.4, and adjusted P value with Benjamini-Hochberg method < 0.05. (C) Bar chart showing the top 9 enriched cancer-associated pathways from MSigDB oncogenic signatures, along with their corresponding P values and associated DEGs. (D) Scatter plot showing Gene Ontology (GO) function enrichments of the DEGs upregulated in GADD45A KO LSCs, compared with CRISPR Ctr LSCs. Clusters were computed using the Leiden algorithm, and similar gene sets were clustered together. Larger, black-outlined points represent significantly enriched terms. Points are plotted on the first 2 UMAP dimensions. The table lists enriched gene sets with P < 8.0e-04 and associated DEGs. (E) Fast preranked gene set enrichment analysis (FGSEA) of CITE-seq data (cutoff: adjusted P < 0.05 and NES > 1.8) identifying GADD45A loss-induced enrichment of gene sets associated with ROS sensing by NFE2L2, detoxification of ROS, cellular response to chemical stress, and iron uptake and transport, on GADD45A knockout in human AML PDX cells. (F) Kaplan-Meier curves of overall survival for 172 patients with AML, as stratified by expression levels of FTH1 (P = .0039) and PRDX1 (P = .019) in TCGA dataset.

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