Figure 5.
Repression of the RNA splicing factor Rbm25 accelerates the expansion of Tet2 KO HSPCs. (A) Rbm25 expression in AML with preleukemic mutations from patients. Controls are CD34+ BM cells from healthy individuals. Healthy CD34+ n = 16, TET2mut n = 38, DNMT3Amut n = 72, ASXL1mut n = 29, IDH2mut n = 41, and NPM1mut n = 93. Two-tailed t test. (B) Spearman correlation (rs) of WBC count with Rbm25 expression (left) or with overall survival (right) of patients with AML carrying preleukemic mutations as shown in panel A. (A-B) Data derived from BEAT AML ELN2017 cohort.47,48 (C) Workflow for engineering leukemic cell lines to carry Tet2 KO and Rbm25 expression knockdown (CRISPRi) or Rbm25 expression activation (CRISPRa). Transduced cells were sorted after each transduction. (D) 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell proliferation assay of Tet2 KO leukemic cell lines with CRISPRi of Rbm25. (E) MTS cell proliferation assay of Tet2 KO leukemic cell lines with CRISPRa of Rbm25. (D-E) Cells were transduced with either nongenomic targeting (NT; gray) or Rbm25-targeting (red) sgRNAs. Error bars represent SEM. Two-tailed t test. (F) Experimental workflow to determine the effect of Rbm25 expression on the expansion of Tet2 KO HSPCs. HSCs carrying heterozygous Tet2 KO and dCas9-KRAB were transduced with either NT sgRNAs or Rbm25-targeting sgRNAs. (G) Fraction of various types of HSPCs among all red fluorescent protein (RFP)+ MNCs in the BM 3 months after transplantation. RFP indicates successful transduction of sgRNAs. The experiment was performed twice. The combined results of 8 mice in the NT sgRNA group and 7 mice in the Rbm25 sgRNA group are shown. (H) Fraction of various types of blood cells among all RFP+ MNCs in the peripheral blood 6 months after the transplantation. The experiment was performed twice. The combined results of 7 mice in the NT sgRNA group and 5 mice in the Rbm25 sgRNA group are shown. (G-H) Each dot depicts data from 1 mouse. One-tailed t test. (I) Rbm25 expression in control and Tet2 KO HSCs. (J) Spearman correlation (rs) of clonal abundance and averaged expression level of Rbm25 in each myeloid-biased clone. Clonal abundance ranked from least to greatest, with rank 1 being the least abundant. (I-J) Data derived from the scRNA-seq experiment is shown in Figure 4. (K) Bcl2l1 mRNA isoform abundance in Tet2+/− Cas9-KRAB+/− HSPCs transduced with NT sgRNAs or Rbm25-targeting sgRNAs. One-tailed t test. (L) Model depicting how Tet2 KO and Rbm25 repression drive hematopoietic expansion. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .001.

Repression of the RNA splicing factor Rbm25 accelerates the expansion of Tet2 KO HSPCs. (A) Rbm25 expression in AML with preleukemic mutations from patients. Controls are CD34+ BM cells from healthy individuals. Healthy CD34+ n = 16, TET2mut n = 38, DNMT3Amut n = 72, ASXL1mut n = 29, IDH2mut n = 41, and NPM1mut n = 93. Two-tailed t test. (B) Spearman correlation (rs) of WBC count with Rbm25 expression (left) or with overall survival (right) of patients with AML carrying preleukemic mutations as shown in panel A. (A-B) Data derived from BEAT AML ELN2017 cohort.47,48 (C) Workflow for engineering leukemic cell lines to carry Tet2 KO and Rbm25 expression knockdown (CRISPRi) or Rbm25 expression activation (CRISPRa). Transduced cells were sorted after each transduction. (D) 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell proliferation assay of Tet2 KO leukemic cell lines with CRISPRi of Rbm25. (E) MTS cell proliferation assay of Tet2 KO leukemic cell lines with CRISPRa of Rbm25. (D-E) Cells were transduced with either nongenomic targeting (NT; gray) or Rbm25-targeting (red) sgRNAs. Error bars represent SEM. Two-tailed t test. (F) Experimental workflow to determine the effect of Rbm25 expression on the expansion of Tet2 KO HSPCs. HSCs carrying heterozygous Tet2 KO and dCas9-KRAB were transduced with either NT sgRNAs or Rbm25-targeting sgRNAs. (G) Fraction of various types of HSPCs among all red fluorescent protein (RFP)+ MNCs in the BM 3 months after transplantation. RFP indicates successful transduction of sgRNAs. The experiment was performed twice. The combined results of 8 mice in the NT sgRNA group and 7 mice in the Rbm25 sgRNA group are shown. (H) Fraction of various types of blood cells among all RFP+ MNCs in the peripheral blood 6 months after the transplantation. The experiment was performed twice. The combined results of 7 mice in the NT sgRNA group and 5 mice in the Rbm25 sgRNA group are shown. (G-H) Each dot depicts data from 1 mouse. One-tailed t test. (I) Rbm25 expression in control and Tet2 KO HSCs. (J) Spearman correlation (rs) of clonal abundance and averaged expression level of Rbm25 in each myeloid-biased clone. Clonal abundance ranked from least to greatest, with rank 1 being the least abundant. (I-J) Data derived from the scRNA-seq experiment is shown in Figure 4. (K) Bcl2l1 mRNA isoform abundance in Tet2+/− Cas9-KRAB+/− HSPCs transduced with NT sgRNAs or Rbm25-targeting sgRNAs. One-tailed t test. (L) Model depicting how Tet2 KO and Rbm25 repression drive hematopoietic expansion. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .001.

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