High-risk comutations expand distinct SF3B1-mutant HSPC compartments. (A) Representative flow plots and gating strategy used to measure the frequency of CD34⁺CD38^– and CD34⁺CD38^–CD133^+/− HSPCs during in vitro culture. (B-C) Frequency of SF3B1-mutant double-edited RUNX1 mutant (S-R) or STAG2 mutant (S-S) vs WT (S-A) CD34⁺CD38^–CD133⁺ (B) or CD34⁺CD38^–CD133^– HSPCs (C) for 14 days of in vitro culture. Data are presented as mean ± SD from 5 independent CB donor experiments (n = 3 with S-A, S-R, and S-S groups; n = 2 with S-A and S-R groups); 2-way ANOVA. (D) Representative flow plots and gating strategy to measure the frequency of HSCs, MPPs, and LMPPs during in vitro culture. (E) FC in the frequency of S-R or S-S phenotypic HSCs, MPPs, and LMPP relative to S-A at day 7 of in vitro culture. (F) Frequency of S-R or S-S vs S-A HSCs, MPPs, and LMPPs for 7 days of in vitro culture. Data are presented as mean ± SD from 2 independent CB donor experiments; 2-way ANOVA. (G) Frequency of SF3B1-mutant double-edited TET2 mutant (S-T) vs WT (S-A) CD34⁺CD38^–CD133⁺ (left) or CD34⁺CD38^–CD133^– (right) HSPCs for 14 days of in vitro culture. Data are presented as mean ± SD from 5 independent CB donor experiments; 2-way ANOVA. (H) Frequency of SF3B1-mutant triple-edited TET2 (S-T-A), RUNX1 (S-R-A), or TET2 + RUNX1 mutant (S-R-T) vs WT (S-A) CD34⁺CD133⁺ phenotypic HSCs during in vitro culture. Data are presented as mean ± SD from 2 independent CB donor experiments; 2-way ANOVA. ns, not significant.