Complementation of Gab2 deficiency by retroviral GAB2 expression. (A) Schematic of retroviral constructs coexpressing BCR-ABL1 and GAB2 or a reporter gene via IRES sequences. A mouse Gab2 cDNA (blue box) was cloned either 5′ or 3′ of a mutant form of the encephalomyocarditis virus IRES (IRES_mut, orange box) in the retroviral vector Minnal virus (MINV)³³  or 3′ of the wild-type IRES (IRES_wt, yellow box) in murine stem cell virus (MSCV)-IRES-GFP (MIGR1)³² ; the BCR-ABL1 cDNA was placed in the other position. As a control vector, either GFP (for IRES_wt) or the neomycin resistance gene (for IRES_mut) was cloned in place of Gab2. (B) Coexpression of GAB2 and BCR-ABL1 restores efficient myeloid cell transformation in vitro. BM from the indicated Balb/c donor mice (blue, Gab2^+/+; red, Gab2^−/−) was transduced with the indicated viral stock from A and plated in methylcellulose without cytokines. The number of cytokine-independent mixed myeloid colonies (CFU-G, -M, -GM, and -GEMM) from Gab2^−/− BM was increased by transduction with viruses 2 or 4, relative to viruses 3 or 5 (P < .0001 and P = .0005, respectively, Student t tests), whereas there was no significant difference in the efficiency of transformation of Gab2^+/+ BM with viruses 2 to 5. Virus 2 (p210-IRES_wt-Gab2) induced significantly more colonies from Gab2^−/− BM than virus 4 (p210-IRES_mut-Gab2; P = .0082, Student t test), whereas the difference in transformation of Gab2^+/+ and Gab2^−/− BM by virus 2 was not significant (P = .167, Student t test). In these experiments, the number of cytokine-independent colonies induced in Gab2-deficient BM was somewhat higher than previously reported,¹⁶  possibly due to lower retroviral titers or the different genetic background (129;B6) in our earlier study. (C) Rescue of CML-like MPN by coexpression of GAB2 and BCR-ABL1 in BM. K-M curves for recipients of BM from Gab2^+/+ (blue lines) or Gab2^−/− (red lines) donors, transduced with p210-IRES_wt-Gab2 or p210-IRES_wt-GFP retroviruses (viruses 2 and 3 from A, respectively; left) or with p210-IRES_mut-Gab2 or p210-IRES_mut-neo retroviruses (viruses 4 and 5 from A, respectively; right). Symbol nomenclature is as in Figure 1A. Histiocytic sarcoma is a BCR-ABL1–induced disease of monocyte/macrophage proliferation that can be seen in recipients of BCR-ABL1–transduced BM from WT donors that have not been treated with 5-FU.⁹  (D) Immunoblot of protein extracts from primary MPN cells from the transplants in C probed with anti-ABL1 (upper) and anti-GAB2 (lower) antibodies.