Figure 3
Figure 3. GAB2 structural domains required for induction of CML-like MPN by BCR-ABL1. (A) Scheme showing GAB2 mutants used in this experiment. PH, pleckstrin homology domain; HA, hemagglutinin epitope tag. Binding sites for the GRB2 SH3 domains and SH2 domains of p85 PI3K and SHP2 are indicated. (B) GAB2 mutants fail to rescue myeloid cell transformation in vitro. BM from the indicated donor mice (blue, Gab2+/+; red, Gab2−/−) was transduced with retrovirus coexpressing BCR-ABL1 and the different GAB2 variants depicted in A and plated in methylcellulose without exogenous cytokines. The difference in colony numbers between Gab2−/− BM transduced with p210-IRESwt-Gab2WT virus, compared with all Gab2 mutant viruses, was significant (P ≤ .004, Student t tests). (C) K-M curves of recipients of BM from Gab2+/+ littermates (blue solid line, n = 5) transduced with p210MIGFP retrovirus and of Gab2−/− donors (red lines), transduced with p210-IRESwt-Gab2WT retrovirus (dotted line, n = 9) or the indicated Gab2 mutant retrovirus (dashed lines, n = 8 for each cohort). There was no difference in survival between recipients of p210MIGFP-transduced Gab2+/+ BM and p210-IRESwt-Gab2WT–transduced Gab2−/− BM. All recipients of Gab2 mutant-transduced BM showed prolonged survival (P < .0001, Mantel-Cox tests). (D) Survival curve as in C, except that the transduced BM was lineage depleted prior to transplantation, as described in Methods. (E) Scatter plot of peripheral blood (PB) leukocyte counts in the 4 cohorts from the transplant in D vs time after transplantation. (F) Flow cytometric detection of the retrovirally expressed HA-tagged GAB2 proteins in PBLs from the indicated recipients of transduced Gab2−/− BM. Note the discrete populations of Mac-1+HA+ myeloid cells. Leukocytes from nontransduced Gab2−/− donors had no detectable fluorescein isothiocyanate fluorescence (data not shown). Detection of the HA epitope tag on the GAB2ΔPH protein by intracellular antibody staining was consistently less efficient than for the other GAB2 proteins, despite equivalent protein expression (Figure 5C).

GAB2 structural domains required for induction of CML-like MPN by BCR-ABL1. (A) Scheme showing GAB2 mutants used in this experiment. PH, pleckstrin homology domain; HA, hemagglutinin epitope tag. Binding sites for the GRB2 SH3 domains and SH2 domains of p85 PI3K and SHP2 are indicated. (B) GAB2 mutants fail to rescue myeloid cell transformation in vitro. BM from the indicated donor mice (blue, Gab2+/+; red, Gab2−/−) was transduced with retrovirus coexpressing BCR-ABL1 and the different GAB2 variants depicted in A and plated in methylcellulose without exogenous cytokines. The difference in colony numbers between Gab2−/− BM transduced with p210-IRESwt-Gab2WT virus, compared with all Gab2 mutant viruses, was significant (P ≤ .004, Student t tests). (C) K-M curves of recipients of BM from Gab2+/+ littermates (blue solid line, n = 5) transduced with p210MIGFP retrovirus and of Gab2−/− donors (red lines), transduced with p210-IRESwt-Gab2WT retrovirus (dotted line, n = 9) or the indicated Gab2 mutant retrovirus (dashed lines, n = 8 for each cohort). There was no difference in survival between recipients of p210MIGFP-transduced Gab2+/+ BM and p210-IRESwt-Gab2WT–transduced Gab2−/− BM. All recipients of Gab2 mutant-transduced BM showed prolonged survival (P < .0001, Mantel-Cox tests). (D) Survival curve as in C, except that the transduced BM was lineage depleted prior to transplantation, as described in Methods. (E) Scatter plot of peripheral blood (PB) leukocyte counts in the 4 cohorts from the transplant in D vs time after transplantation. (F) Flow cytometric detection of the retrovirally expressed HA-tagged GAB2 proteins in PBLs from the indicated recipients of transduced Gab2−/− BM. Note the discrete populations of Mac-1+HA+ myeloid cells. Leukocytes from nontransduced Gab2−/− donors had no detectable fluorescein isothiocyanate fluorescence (data not shown). Detection of the HA epitope tag on the GAB2ΔPH protein by intracellular antibody staining was consistently less efficient than for the other GAB2 proteins, despite equivalent protein expression (Figure 5C).

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