Figure 2.
Recurrent deletions of FTO intron 8 in patients with T-ALL impinge on a CTCF-binding site. (A) Focal heterozygous deletions identified in T-ALL genomes at FTO; 12 originate from primary patient samples and 1 from the ALL-SIL T-ALL cell line. Deletions identified in patient samples are from multiple cohorts including St. Jude (n = 264), UKALL2003 (n = 148), and ICGZ Poznan (n = 63). (B) IRX3 expression (FPKM) by RNA-seq from matched primary patient samples and the ALL-SIL T-ALL cell line harboring FTO intron 8 deletions. Allele-specific expression identified in patient 11 was determined by St. Jude’s previous analyses, and unbalanced promoter methylation for the ALL-SIL cell line was ascertained by analysis of the Cancer Cell Line Encyclopedia Promoter Methylation data set. (C) ChIP-seq for CTCF (NCBI GEO accession #GSE68976), MYB, and H3K27ac (NCBI GEO accession #GSE76783) in the Jurkat T-ALL cell line centered on the minimally deleted region within FTO intron 8. (D) Pie charts showing the frequency of FTO intron 8 deletions determined by digital droplet PCR in adult (n = 137) and pediatric T-ALL cohorts (n = 161). Stacked boxes summarize CTCF or MYB site-specific copy number calls for each patient identified with FTO intron 8 deletions and a bar chart showing IRX3 expression (FPKM) is shown for patient samples where matched RNA was available for sequencing. Matched IRX3 expression by RNA-seq for samples that exhibited normal copy number (n = 9, UKALL14 cohort) are also plotted. (E) Experimental outline for CRISPR/Cas9-mediated disruption of FTO intron 8 CTCF and MYB sites in the PF-382- (FTOwt) and IRX3-negative (FPKM <1) T-ALL cell line. (F) Bar chart showing IRX3 expression determined by quantitative PCR (qPCR) for PF-382 (FTOwt) and ALL-SIL (FTOint8del) T-ALL cell lines and unedited (wt) and edited (mut) clones. Data presented are 3 technical replicates ± standard deviation for each biological sample. (G) Bar chart showing IRX3 expression determined by qPCR for PF-382 polyclonal edited cells after CRISPR/Cas9-mediated disruption of the FTO intron 8 CTCF site. Technical replicates from 3 independent experiments are shown. P < .0001 from a 2-tailed t test. (H) Violin plot showing IRX3 expression (FPKM) of primary T-ALL samples with (n = 16) and without (n = 248) CTCF mutations from the St. Jude T-ALL cohort. P = .0007 from 2-tailed t test. (I) Bar chart showing CTCF and IRX3 expression by qPCR after CTCF knockdown in PF-382, PEER, Loucy, and HPB-ALL T-ALL cell lines. Final CTCF small interfering RNA (siRNA) pool concentrations used are shown on the x-axis, where NT is a negative control nontargeting siRNA pool. Technical replicates from 2 independent experiments are shown. Statistical comparisons were made to NT groups by a 2-tailed t test where ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; and ns, not significant. mut, mutant; sgRNA, single guide RNA; wt, wild-type.

Recurrent deletions of FTO intron 8 in patients with T-ALL impinge on a CTCF-binding site. (A) Focal heterozygous deletions identified in T-ALL genomes at FTO; 12 originate from primary patient samples and 1 from the ALL-SIL T-ALL cell line. Deletions identified in patient samples are from multiple cohorts including St. Jude (n = 264), UKALL2003 (n = 148), and ICGZ Poznan (n = 63). (B) IRX3 expression (FPKM) by RNA-seq from matched primary patient samples and the ALL-SIL T-ALL cell line harboring FTO intron 8 deletions. Allele-specific expression identified in patient 11 was determined by St. Jude’s previous analyses, and unbalanced promoter methylation for the ALL-SIL cell line was ascertained by analysis of the Cancer Cell Line Encyclopedia Promoter Methylation data set. (C) ChIP-seq for CTCF (NCBI GEO accession #GSE68976), MYB, and H3K27ac (NCBI GEO accession #GSE76783) in the Jurkat T-ALL cell line centered on the minimally deleted region within FTO intron 8. (D) Pie charts showing the frequency of FTO intron 8 deletions determined by digital droplet PCR in adult (n = 137) and pediatric T-ALL cohorts (n = 161). Stacked boxes summarize CTCF or MYB site-specific copy number calls for each patient identified with FTO intron 8 deletions and a bar chart showing IRX3 expression (FPKM) is shown for patient samples where matched RNA was available for sequencing. Matched IRX3 expression by RNA-seq for samples that exhibited normal copy number (n = 9, UKALL14 cohort) are also plotted. (E) Experimental outline for CRISPR/Cas9-mediated disruption of FTO intron 8 CTCF and MYB sites in the PF-382- (FTOwt) and IRX3-negative (FPKM <1) T-ALL cell line. (F) Bar chart showing IRX3 expression determined by quantitative PCR (qPCR) for PF-382 (FTOwt) and ALL-SIL (FTOint8del) T-ALL cell lines and unedited (wt) and edited (mut) clones. Data presented are 3 technical replicates ± standard deviation for each biological sample. (G) Bar chart showing IRX3 expression determined by qPCR for PF-382 polyclonal edited cells after CRISPR/Cas9-mediated disruption of the FTO intron 8 CTCF site. Technical replicates from 3 independent experiments are shown. P < .0001 from a 2-tailed t test. (H) Violin plot showing IRX3 expression (FPKM) of primary T-ALL samples with (n = 16) and without (n = 248) CTCF mutations from the St. Jude T-ALL cohort. P = .0007 from 2-tailed t test. (I) Bar chart showing CTCF and IRX3 expression by qPCR after CTCF knockdown in PF-382, PEER, Loucy, and HPB-ALL T-ALL cell lines. Final CTCF small interfering RNA (siRNA) pool concentrations used are shown on the x-axis, where NT is a negative control nontargeting siRNA pool. Technical replicates from 2 independent experiments are shown. Statistical comparisons were made to NT groups by a 2-tailed t test where ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; and ns, not significant. mut, mutant; sgRNA, single guide RNA; wt, wild-type.

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