Figure 2.
High-risk comutations induce divergent transcriptional changes. (A) Outline of the experimental approach for CRISPR/Cas9 editing of 5F-HPCs. (B) Proportion of frameshift mutations in AAVS1 (A), RUNX1 (R), and STAG2 (S) in SF3B1-mutant (S-A, S-R, and S-S) and WT (A, R, and S) patient-derived isogenic 5F-HPCs. Genotyping was performed by Sanger sequencing and ICE analysis. Data are presented as mean ± standard deviation (SD) from 3 independent experiments. (C) Normalized enrichment score of Hallmark gene sets (left) and human hematopoietic cell gene signatures from Laurenti et al39 and Hay et al40 (right) in gene set enrichment analysis (GSEA) analysis of S-R vs S-A and S-S vs S-A 5F-HPCs. FDR q < 0.05. (D) Relative expression of genes with divergent dysregulation in S-R and S-S 5F-HPCs. Values are shown as log2FC relative to S-A 5F-HPCs; P < .05. (E) STRING protein–protein interaction network of genes with divergent dysregulation in S-R and S-S 5F-HPCs. Disconnected nodes were removed; line thickness proportional to interaction score of >0.40. (F) PU.1 protein level in CD34+ 5F-HPCs measured by intracellular flow cytometry. Representative flow plot (left). FC of MFI in S-R or S-S relative to S-A (right). Data are presented as mean ± SD from 4 independent experiments; 1-sample t test. (G) GSEA analysis of TRRUST PU.1 target genes in S-R vs S-A and S-S vs S-A 5F-HPCs. FDR q < 0.05. (H) Proportion of mis-spliced isoforms by category in SF3B1-mutant K562 cells edited for RUNX1 (S-R), STAG2 (S-S), or AAVS1 control (S-A). Mis-spliced events were categorized as tandem 3′ untranslated regions (tutr), cassette or skipped exons (se), retained introns (ri), mutually exclusive exons (mxe), alternative usage of normally constitutively spliced junctions (cj), alternative retention of normally constitutively spliced introns (ci), alternative 5′ss (a5ss), or alternative 3′ss (a3′ss). Events were restricted to ≥10% mis-splicing and Bayes factor of ≥5. (I) Spearman correlation matrix of the level of mis-splicing between S-A, S-R, and S-S. ∗∗∗∗P < .0001. (J) Proportion of a3′ss mis-spliced events shared between low-risk (S-A) and high-risk (S-R or S-S) genotypes (red), shared by high-risk (S-R and S-S) but not S-A (yellow), and unique to high-risk genotypes (blue). FC, fold change; FDR, false discovery rate; MFI, mean fluorescence intensity.

High-risk comutations induce divergent transcriptional changes. (A) Outline of the experimental approach for CRISPR/Cas9 editing of 5F-HPCs. (B) Proportion of frameshift mutations in AAVS1 (A), RUNX1 (R), and STAG2 (S) in SF3B1-mutant (S-A, S-R, and S-S) and WT (A, R, and S) patient-derived isogenic 5F-HPCs. Genotyping was performed by Sanger sequencing and ICE analysis. Data are presented as mean ± standard deviation (SD) from 3 independent experiments. (C) Normalized enrichment score of Hallmark gene sets (left) and human hematopoietic cell gene signatures from Laurenti et al39 and Hay et al40 (right) in gene set enrichment analysis (GSEA) analysis of S-R vs S-A and S-S vs S-A 5F-HPCs. FDR q < 0.05. (D) Relative expression of genes with divergent dysregulation in S-R and S-S 5F-HPCs. Values are shown as log2FC relative to S-A 5F-HPCs; P < .05. (E) STRING protein–protein interaction network of genes with divergent dysregulation in S-R and S-S 5F-HPCs. Disconnected nodes were removed; line thickness proportional to interaction score of >0.40. (F) PU.1 protein level in CD34+ 5F-HPCs measured by intracellular flow cytometry. Representative flow plot (left). FC of MFI in S-R or S-S relative to S-A (right). Data are presented as mean ± SD from 4 independent experiments; 1-sample t test. (G) GSEA analysis of TRRUST PU.1 target genes in S-R vs S-A and S-S vs S-A 5F-HPCs. FDR q < 0.05. (H) Proportion of mis-spliced isoforms by category in SF3B1-mutant K562 cells edited for RUNX1 (S-R), STAG2 (S-S), or AAVS1 control (S-A). Mis-spliced events were categorized as tandem 3′ untranslated regions (tutr), cassette or skipped exons (se), retained introns (ri), mutually exclusive exons (mxe), alternative usage of normally constitutively spliced junctions (cj), alternative retention of normally constitutively spliced introns (ci), alternative 5′ss (a5ss), or alternative 3′ss (a3′ss). Events were restricted to ≥10% mis-splicing and Bayes factor of ≥5. (I) Spearman correlation matrix of the level of mis-splicing between S-A, S-R, and S-S. ∗∗∗∗P < .0001. (J) Proportion of a3′ss mis-spliced events shared between low-risk (S-A) and high-risk (S-R or S-S) genotypes (red), shared by high-risk (S-R and S-S) but not S-A (yellow), and unique to high-risk genotypes (blue). FC, fold change; FDR, false discovery rate; MFI, mean fluorescence intensity.

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