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Letters to the Editor
locyte-macrophage colony-stimulating factor (CM-CSF) and stem cell factor constitutively (NSG-3GS mice). In these experiments, equal amounts of the NA9- and con- trol-transduced cells were combined immediately after transduction and injected together into ten of these mice. Follow-up of these mice for 7 months showed that all the human cells subsequently detected in them were exclu- sively myeloid (CD33+) (Online Supplementary Figure S3), as is typical of CP-CML xenografts.10 In nine of the ten mice, bone marrow aspirates showed competitive over- growth by the NA9-transduced (GFP+) cells already evi- dent within 3 weeks after transplantation and increasing thereafter. In five mice, this led to death or symptoms requiring euthanasia (Figure 1A-D, Online Supplementary Table S2).
RNA-sequencing analysis of extracts of NA9+ (GFP+)- and control (YFP+)-transduced cells obtained 2 days after exposure to virus revealed transcripts of 54 coding genes to be already upregulated in all three NA9+ CD34+ cell samples (Figure 2A, Online Supplementary Datasets S1-5). These upregulated transcripts allowed the NA9+ CML cells to be separated from the control-transduced cells in an unsupervised clustering analysis (Figure 2B). Functional annotation of this NA9 signature showed an overrepresentation of transcription factors (TF) with DNA binding and RNA pol II distal enhancer activity. Many of these are the same as those found to be rapidly deregulated in normal CD34+ cells transduced with a NA9-encoding retroviral vector,2,9 consistent with the similarity in the early in vitro biological responses exhibit- ed by NA9-transduced CP CML and normal CD34+ cells (Online Supplementary Figure S3A-C).2,9 However, compar- ison of our transcriptome data for NA9-transduced CP CML cells with those published for NA9-transduced CD34+ cells isolated from normal mobilized blood sam- ples also indicated major differences, with 60% of the transcripts found to be upregulated in the CML NA9 sig- nature and not similarly affected in NA9+ normal cells (Online Supplementary Dataset S6).
We also performed an unsupervised clustering analysis of the GSE4170 microarray dataset for cells from 42 CD34+ CP-CML and 28 BP-CML patients using the 54 transcripts that constituted the NA9-CP CML signature identified here. This analysis showed that many of the transcripts in the NA9 signature were among the tran- scripts with the most highly upregulated expression in the leukemic CD34+ cells from BP patients compared with those from patients with accelerated phase or CP disease (including ARID5B, STARD9, AHR, TOX and FOXP1, Fisher one-way analysis of variance; P<5x10-9) (Online Supplementary Figures S4 and S5). These results suggest that increased expression of NA9 targets may be a frequent hallmark, and hence an important element, in the creation of a BP disease program. It was therefore of interest that the transcripts comprising the NA9 signature were also deregulated in the transcriptomes of intermedi- ate and high-risk acute myeloid leukemia patients carry- ing a variety of driver mutations (Online Supplementary Figure S6 and S7). Notably, the NA9 signature was also predictive of a significantly reduced overall survival in The Cancer Genome Atlas acute myeloid leukemia cohort11 (Online Supplementary Figure S8).
Gene set enrichment analysis (GSEA) of the NA9- upregulated transcripts in the transduced CML samples also showed a significant enrichment in transcripts found at higher levels in the CD34+CD38– fraction of normal bone marrow, cord blood or mobilized blood cells in comparison to the more predominant, and more differen- tiated, CD34+CD38+ cells present in the same normal
samples12 (Figure 2C). Specific examples include genes in the HOXA and HOXB clusters and their cofactors, MEIS-1 and PBX3, MECOM, AHR, BMP6 and GDF10. GSEA also revealed a high enrichment in the NA9-trans- duced CML cells of previously reported upregulated NA9 targets, particularly TF transcripts. These findings also extended to differential transcriptome data derived from a comparison of CD34+CD38–CD90+ cells CP-CML cells and their more differentiated progeny as well as their normal counterparts (Figure 2D-G) (GSE47927). Taken together, these results all point to a rapid and selective upregulation of a “stem cell” program in NA9-transduced CD34+ CP-CML cells.
We also compared the frequency and sites of H3K27ac marks in the same paired sets of NA9- and control-trans- duced cells. NA9-specific de novo H3K27ac marked regions were found near (±2 kb) the transcriptional start sites, or within intragenic regions of HOXA9, HOXA7, MEIS1 and PBX3, but not in HOXC or HOXD clusters, or in PBX1 (Figure 3A, Online Supplementary Dataset S7). These NA9-specific de novo H3K27ac regions overlap with previously identified NA9 binding sites in the HEK293FT cell line (GSE62587).3 Additional NA9-specif- ic de novo H3K27ac marked regions that were identified are known to drive expression of genes previously report- ed as upregulated in normal CD34+ cells expressing a NA9 transgene, in the GM-CSF signaling pathway, and in myeloid cells exposed to lipopolysaccharide (Figure 3 B and C, Online Supplementary Dataset S8). De novo H3K27ac marks in NA9+ CML CD34+ cells also showed a significant enrichment of TF binding sites in GATA1, RELA, MEF2A and IKZF1, previously mapped by chro- matin immunoprecipitation sequencing experiments (adjusted P-value <0.05) (Figure 3D). REL is of interest as it belongs to the set of genes (i.e., GATA1, RELA, MEF2A and IKZF1) whose expression was altered by forced NA9 expression in the CML CD34+ cells with concomitant acquisition of H3K27ac marks that overlap with previ- ously identified NA9 binding sites (GSE62587) (Online Supplementary Figure S9).3
We also identified ten super enhancers consistently present in the NA9-transduced CD34+ CP CML cells and absent in the control cells (Figure 3A, E and F). These super enhancers are predicted to regulate ten genes, including PBX3, ANGPT1 and MBNL1 with the PBX3 super enhancer again overlapping extensively with NA9 binding sites previously identified (GSE62587).3 The MBNL1 super enhancers are part of a previously identi- fied short list of common binding sites of NA9, NUP98- HOXD13 and MLL-AF9 fusions and ANGPT1 is one of the rare genes with NA9 binding sites in its enhancers and increased expression in NA9-expressing cells.13 Another notable finding was an increase in H3K27ac marks forming an intronic super enhancer 193 kb from the FOXP1 transcriptional start site enriched in NA9- binding sites and an associated upregulated expression of FOXP1 (Online Supplementary Figure 9D). FOXP1 has recently been found to enhance normal and leukemic stem/progenitor cell expansion by downregulating expression of p21-CIP1 and p27-KIP1, two cell-cycle inhibitors.14
Taken together, these findings suggest that in spite of the somewhat variable biological changes induced by NA9 expression in the total CD34+ CP CML populations from different patients, the transcriptional program is coherently altered to activate key elements of an enhanced self-renewal program. Given previous evidence that the blasts in BP CML have features of self-renewing
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