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Letters to the Editor
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Figure 2. RNA-sequencing analysis of NA9+ chronic myeloid leukemia CD34+ cells. Libraries were prepared from 10 ng aliquots of total RNA extracted from FACS-sorted GFP+ (NA9+) and YFP+ (control-transduced) CD34+ cells cultured for 2 days after transduction in serum-free medium plus five growth factors. Differential expression analysis was performed with DEfine v.1 (unpublished), with a false discovery rate of 0.05 and a minimum reads per kilobase per million mapped reads of 0.01. (A) Venn diagram showing the distribution of upregulated genes in the three tested NA9-transduced samples from patients (Venny 2.1.0). (B) Heatmap of genes upregulated in all NA9-transduced CD34+ chronic myeloid leukemia cell samples compared to control-transduced cells. An unsupervised clustering analysis using Euclidean distance was performed on log2-transformed expression data for genes referenced in HUGO with increased transcript levels in all three samples (using Gplots package in R version 3.2.3). (C) Gene set enrichment analysis of whole transcriptomes showing significant enrichment of a previously reported set of transcripts found to be present at increased levels in lin–CD34+CD38– cells compared with lin+CD34+CD38+ cells from normal cord blood, adult bone marrow and mobilized peripheral blood cells.12 (D) Principal component analysis (PCA) of the NA9 gene signature identified in Figure 2B exam- ined in normal lin–CD34+CD38–CD45RA–CD90+ cells (HSC), lin–CD34+CD38+CD123+CD45RA–(CMP) and lin–CD34+CD38+CD123+CD45RA+ (GMP) (GSE47927.). (E) PCA x-axis coordinates of transcripts present at levels that distinguish HSC from GMP and CMP from healthy donors. (F) PCA of the NA9 gene signature in lin–CD34+CD38–CD45RA–CD90+ leukemic stem cells (LSC), and CMP and GMP from CML patients (GSE47927). (G) PCA x-axis coordinates of transcript levels separating LSC from GMP and CMP isolated from CML patients. NES: normalized enrichment score; FDR: false discovery rate; Ctrl: control; CMP: common myeloid progenitors; GFP: green fluorescent protein; GMP: granulocyte-monocyte progenitors; HSC: hematopoietic stem cells; NA9: NUP98-HOXA9; YFP: yellow fluorescent protein.
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haematologica | 2021; 106(3)