In vitro lymphoid transformation and in vivo leukemogenesis evoked by BCR-ABL1 are dependent on GAB2. (A) Agarose colony assays. BM from the indicated Balb/c donor (blue, Gab2^+/+; red, Gab2^−/−) mice was transduced with retrovirus coexpressing p210 BCR-ABL1 and either GFP or the indicated GAB2 variant. Transduced cells were plated directly in agarose as described in Methods, and transformed pre-B-lymphoid colonies were counted at day 10. The differences in colony numbers between p210-GFP–transduced Gab2^+/+ and Gab2^−/− BM and between p210-GFP–transduced and p210-Gab2WT–transduced Gab2^−/− BM, were significant (P < .0001 and P = .001, respectively, unpaired Student t tests), whereas there was no difference between p210-GFP–transduced Gab2^+/+ and p210-Gab2WT–transduced Gab2^−/− BM (P = .224). The difference in colony numbers between Gab2^−/− BM transduced with p210-Gab2WT retrovirus and either p210-Gab2Δp85 or p210-Gab2ΔShp2 retrovirus was significant (P = .0004 and P = .0002, respectively, unpaired Student t tests), whereas the difference between Gab2^−/− BM transduced with p210-Gab2Δp85 and p210-Gab2ΔShp2 retrovirus did not reach significance (P = .0647, unpaired Student t test). (B) Whitlock-Witte assays. Freshly harvested Balb/c BM from Gab2^+/+ (top) and Gab2^−/− (bottom) donors was transduced with retrovirus coexpressing p210 BCR-ABL1 and either GFP or the indicated GAB2 variant, and plated in triplicate at the indicated cell numbers per well. Nontransduced cells were added to provide 10⁶ total cells for stromal support. Wells were scored as positive when cell number reached 10⁶ viable nonadherent cells per well, as described in Methods. (C) Extracts from immortalized BCR-ABL1–transformed B-lymphoid cell lines (from Figure 5B) from Gab2^+/+ (WT) or Gab2^−/− (knockout) donors were analyzed for GAB2 protein expression and phosphorylation, as described in Methods. (D) K-M curves of recipients of BM from Gab2^+/+ littermates transduced with p210MIGFP retrovirus (blue solid line, n = 8), and of Gab2^−/− donors (red lines) transduced with p210-IRES_wt-Gab2WT retrovirus (dotted line, n = 8) or the indicated Gab2 mutant retrovirus (dashed lines, n = 8 for each cohort). All deaths were due to B-ALL. There was no significant difference in survival between recipients of p210MIGFP-transduced Gab2^+/+ BM and p210-IRES_wt-Gab2WT–transduced Gab2^−/− BM (P = .493) or between recipients of p210MIGFP-transduced Gab2^−/− BM and p210-IRES_wt-Gab2ΔShp2–transduced Gab2^−/− BM (P = .734). By contrast, the survival of recipients of p210MIGFP-transduced Gab2^+/+ BM and p210MIGFP-transduced Gab2^−/− BM, and of recipients p210-IRES_wt-Gab2WT–transduced and p210-IRES_wt-Gab2Δp85–transduced Gab2^−/− BM, was different (P < .0001 and P = .001, respectively, Mantel-Cox tests).