PRC2 mutations co-occur with JAK3 mutations and Suz12 loss cooperates with JAK3(M511I) in vitro. (A) Pie charts comparing the frequency of JAK3 mutations (orange) and IL7R-JAK1-STAT5 mutations (brown) within patients with PRC2 mutant T-ALL vs patients with PRC2 wild-type. Similar pie charts for JAK/STAT mutation frequencies for patients with T-ALL carrying mutations in the 3 different PRC2 components SUZ12, EED, and EZH are shown aside. Using Pearson’s χ-squared test (*) or the Fisher’s exact test, P values were calculated for testing significance of positive association between JAK3 mutations and mutations in PRC2, SUZ12, EZH2, or EED. T-ALL patient data (n = 419). (B) Scheme of ex vivo pro-T-cell culture requiring interleukin 7 (IL7), stem cell factor (SCF), and immobilized Δ-like ligand 4 (DLL4). (C) Cell densities (mean with standard deviation) over time for different IL7-deprived pro-T-cell conditions: JAK3(M511I)+Suz12gRNA TS1, TS5, and TS6 (J+S1, J+S5, J+S6); Suz12 gRNA TS1, TS5, and TS6, always in combination with green fluorescent protein (GFP) empty vector (S1+EV, S5+EV, S6+EV) controls; JAK3(M511I) in combination with blue fluorescent protein (BFP) empty vector (J+EV); and BFP empty+GFP empty vector (EV) controls. (D) Western blot on EV pro-T cells and IL7-independent JAK3(M511I)+Suz12gRNA pro-T cells (J+S1, J+S5, J+S6). β-actin was used as loading control. (E-F) Suz12 protein (E) and H3K27me3 (F) levels were measured by intracellular flow cytometry in IL7-independent JAK3(M511I)+Suz12gRNA pro-T-cells (J+S1, J+S5, J+S6) and JAK3(M511I) (J) pro-T-cells. MFIs were calculated for APC emission. (G-H) Suz12 protein (G) and H3K27me3 (H) levels were measured by intracellular flow cytometry. MFIs were calculated for APC emission. (I) Percentage mCherry relative to d0 was measured over time in IL7-independent J+S1 and J+S5 pro-T-cells that were transduced with mCherry EV or mCherry Suz12 cDNA overexpression (Suz12 OE).