Physiologically Significant Genetic Interaction between STAT5 and Gab2 during Normal and Pathologic Hematopoiesis.

Signal transducer and activator of transcription-5 (STAT5) is critical for normal hematopoiesis and its dysregulated activation is associated with hematologic disease in mice and humans. Recently, a constitutively active mutant of STAT5 (STAT5a^S711F, also called STAT5a*6) was shown to bind a cytosolic Gab2 complex leading to increased Akt activation. We have also found that co-transfection of erythropoietin (EPO) receptor along with STAT5a or STAT5a*6 in 293T cells leads to STAT5/Gab2 physical interaction in response to EPO. However, the physiological significance in vivo is not known. Therefore, we asked whether STAT5 and Gab2 provide overlapping/non-overlapping, or cooperative/additive functions in fetal and adult hematopoiesis. To do this, we generated and crossed STAT5ab^+/nullGab2^+/− mice to yield STAT5ab^null/nullGab2^{−/ −} double knockout embryos. At E14.5, a normal Mendelian ratio (6/94) of double mutant was obtained. So far no double mutant embryos at E18.5 (0/16) have been detected. Histology analyses of mutant embryos are pending. At E14.5 STAT5ab^null/nullGab2^{−/ −} total CFU-C frequency was reduced to 11–29% of normal (N=3) compared with STAT5 or Gab2 single mutants which ranged 63–100%. We also tested for interaction between adult STAT5ab^+/null and Gab2^{−/ −} genotype. Both STAT5ab^+/null and Gab2^{−/ −} mice and their STAT5ab^+/nullGab2^{−/ −} cross were born at normal Mendelian ratio. STAT5ab^+/nullGab2^{−/ −} mice had 57% of normal absolute numbers of IgM⁺/B220⁺ B cells but all other lineages were comparable to the single mutants. However, in two independent transplant experiments, STAT5ab^+/null and Gab2^{−/ −} bone marrow (BM) had competitive multilineage hematopoietic stem cell repopulating defects averaging 36% and 18% of wild-type respectively. Notably, STAT5ab^+/nullGab2^{−/ −} BM cells had an average 6% of wild-type engraftment, which is exactly as predicted for additive non-redundant defects (0.36 x 0.18). Next, we performed experiments using persistent high level STAT5 activation using retroviral-mediated expression of MSCV/STAT5a*6-IRES-GFP. To determine whether myeloproliferative disease (MPD) driven by constitutively active STAT5 utilizes Gab2 interactions, wild-type or Gab2^{−/ −} BM cells were transduced and transplanted into recipient mice followed by analysis of MPD. We observed good levels of gene transfer (GFP⁺ cells) using the IRES-GFP control or STAT5a*6 vector for either wild-type or Gab2^{−/ −} BM (33–70%), indicating no adverse effects of Gab2 deficiency on retroviral transduction and transplantation. STAT5a*6 vector transduced into wild-type BM increased the frequency of Gr-1⁺Mac-1⁺ cells (8-fold) in peripheral blood and disrupted the splenic architecture of transplanted mice. In the liver the architecture remained intact despite perivascular infiltration of the portal track, central vein, and lobules with granulocytic lineage cells in different stages of differentiation. Interestingly, the expansion of the Gr-1⁺Mac-1⁺ population was 5- to 6-fold reduced in the absence of Gab2 and led to 2- to 3-fold reduced spleen weights. This attenuation modestly improved survival, although both groups of mice died of MPD. A second independent cohort of mice has been transplanted. Overall, these data indicate that STAT5 and Gab2 play critical non-redundant functions in normal and pathologic hematopoiesis and suggest that combined STAT5/Gab2 inhibition strategies might lead to at least an additive therapeutic benefit.